Determination of factors limiting enzymatic hydrolysis of the "Chlorella sorokiniana" cell wall

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Material Information

Title:
Determination of factors limiting enzymatic hydrolysis of the "Chlorella sorokiniana" cell wall
Physical Description:
xii, 153 leaves : ill. ; 29 cm.
Language:
English
Creator:
Russell, Brenda Lurline, 1961-
Publication Date:

Subjects

Subjects / Keywords:
Chlorella sorokiniana   ( lcsh )
Chlorella sorokiniana -- Cytology   ( lcsh )
Chlorella sorokiniana -- Physiology   ( lcsh )
Microbiology and Cell Science thesis, Ph. D
Dissertations, Academic -- Microbiology and Cell Science -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 144-152).
Statement of Responsibility:
by Brenda Lurline Russell.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 002070023
oclc - 34360432
notis - AKQ8284
System ID:
AA00002047:00001

Full Text













DETERMINATION


OF FACTORS
CHLORELLA


LIMITING ENZYMATIC HYDROLYSIS
SOROKINIANA CELL WALL


OF THE


BRENDA


LURLINE


RUSSELL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA

1995

































So much


of what


have


learned


not


been


from


books


or in


classroom,


through


special


people


who


were


willing


share


their


experience.


teachers


















ACKNOWLEDGMENTS


am grateful


to Dr.


Robert


Schmidt


providing


opportunity


to conduct


this


res


earch


proj ect


in his


laboratory,


having


patience


commitment


to guide


project


through


to its


completion.


thank


supervisory


committee


members


, Dr. Henry


Aldrich


, Dr.


Robert


. Ferl,


John


Gander


, and


Curtis


Hannah,


their


important


contributions


towards


development


execution


this


research


project.


Dr. Henry


Aldrich,


Donna


Williams


Lorraine


McDowell


provided


expertise,


supplies


, and


undying


patience


contributing


towards


"just


one


more"


electron


microscopy


experiment.


Dave


Powell


provided


generous


help


with


elemental


analysis


, infrared


spectroscopy


interpretation


of data.


Marian


Busko


provided


assistance


with


experiments


, and


Dr. Chris


West


was


helpful


with


carbohydrate


anal


data.


This


research


was


enhanced


generosity


of Dr.


Ryzard


Brzezinski


Univ


ers


de Sherbrooke











Assistance


in the


preparation


this


manuscript


was


kindly


supplied


Marc


Adkins


Jill


Cocanougher


, and


John


van


Hooke.


thank


my colleagues


, Waltraud


Dunn,


Dr. Philip


Miller

friend


Richard


ship.


wish


Hutson,


thank


their a

SJames


assistance


Maruniak


always


being


there


with


helpful


suggestions


support.


thank


friends


Peter


Gomez,


Lorraine


Yomano,


Louise


Monroe


Doran


valuable


encouragement.


Completion


of this


degree


would


have


been


possible


without t


thank


support


mother


love


Barbara


provided


Phipps


my family.


support


wish


understanding


father


Gerry


Russell


"fatherly"


advice.


thank


sisters


, Barbara


Campbell,


Donna


Cooper,


Gwen


Trufant,


Mary


Dougherty-Hunt,


(who


may


as well


my sister)


always


being


there


me.
















TABLE OF CONTENTS


ACKNOWLEDGMENTS .... .. .. .. .. . .. .iii

LIST OF TABLES .. . .. .... .. .... .... .... .. ... ...vil

LIST OF FIGURES ......... ... ... .... .. .... .... ....vi

ELIST OF ABBREVI ORATIONS .. . . .... .. . ... ......x

ABSTRACT ... ... .. .. .. . . .X

INTRODUCTION .. ... .. .......... .. ... ... .. .. ..... 1

LITERATURE REVIEW ............ .. .... .... .... .. ... ..... 4

MATERIALS AND METHODS ... .. ...... .. .. ... .. ...27
Culture Conditions ..... ... ... .. .. ... ... ........ 27
Source of Chlorella Cultures ... ......................28
Preparation of Samples for Transmission Electron
Microscope .. ...... .. ..... ..........28
Preparation of Samples for Scanning Electron
Microscope .. .. .. . . .. . .. 29
Determination of Cell Wall Thickness .... ..............30
Purification of Chlorella Cell Walls .... ...... .. ......30
Dry Weight Determinations ..............................31
Instruments Used for Spectroscopic Analyses .........31
Extraction of Hydrolyzable Carbohydrates from Chlorella
Acid Hydrolysis of Cell Walls. .. .. .. ... .. .... .. .. .. ...32
Alkaine Hydrolysis of Cell Walls .... ................32
Alkaline Hydrolysis of Cell Walls .. ..............34
Protein and Amino Acid Analysis of Purified
Cell Walls .. .. ... .. .. ...... .... .. . ....... .35
Acetolysis . . . . .... . . . ..... ... .35
Assessment of Cell Wall Autofluorescence ........ ......36
Determination of Cell Wall Solubility in Phosphoric
Acid .. 4 4 4. .. . . 4 . . .. .*. ... 37
Phloroglucinol Assay for Lignin .....................37
Analysis of Alkaline Extracts for the Presence of












Chitosanase


S. . .. .. .. 4


Product
for C
atment o
Degradi
atment o
Degradi
atment o
Degradi
empts to
in C. so
erminati
Growth R
Acetolys
C. sorok
erminati'
Chlorell
duction
Defectiv


0
O
e
f
n
f
rn
f
r

r
o


I

L
I



I


n of C. sorokiniana Ce]
11 Wall Degradation .
Whole Cells with Cell
g Enzymes ............
Purified Substrates w:
g Enzymes .. .. .......
Thin Sections with Po
g Enzymes ...... ......


Indu
okin
n of
,te,
s Re
nian
n of


.C
I


Accm


Tre

Tre

Tre

Att

Det



Det

Pro


Mutants


IL1


Homogenate


..... ........ ....... 41
Wall


.....
* 4 4*
ith C

lysac
.* *


emulation of
fusca Cell;
s of Norflu.
Content, an
.1 Wall Frac


.......... .... ...42
ell Wall
. ............. 44
charide
.... . . .. 45
Ketocarotenoids


s.
ra
d
ti


zon on
Percentage
on of


. .45

of


.. .. 46


Growth


MON


-20763


* *Wall
Wall


. ... . . . 47


RESULTS


. .. ....... 51


sorokiniana


Cre


Polysac
Protein
Analyse
Spor
Phen
Acet
sor
ect of I
Wall .
Effect
Effect
Effect <
empts t<
Biosynt
Attempt;
sorok.
Effect <
Effect
ation, ;


Cell


charide


and
for
poll
lic


olvsi


Wall


Analys


Amino Aci
SOther Ce
enin .
compounds
s-resistar


okiniana
ivdrolvt


-i -'


i


(
C


* n z y
f Enzyme
f Enzyme
f Enzyme
Disrupt
esis ...
to Indu


cell
; En
4 S
s or

s or
s on


Characterization
is .... .. .. ...
d Analyses ......
11 Wall Compounds


it portion of the


wall
zymes


.. .. ... .51
. ... .. .. 51
. . .. .. .65
. ......... .67
......... .. 67
S. .. .76


sorokiniana


Whole Cells ...
Purified Walls
Thin Sections o


sorokiniana C'

Ketocarotenoid


ell


S* 44 S S S

f Cells
Wall


Synthesi


niana and C. fusca Cells............
f Norflurazon on C. sorokiniana Cell
'f MON-20763 on Cell Wall Synthesis..
election, and Characterization of Ce


S. ...79
11


.. . ..80
S. . .. .80
S. .. .86
. .. .92


. . 92
C.
S. .. 92
alls .97
*......101


Wall


Defective


Mutants


DISCUSSION


iana ana c.
the Effect
Carotenoid
sistant Cel
a . .
the Effect
Is ... .
screening of


As s ay


:e


.......,......,....47


m



















LIST


OF TABLES


GC-MS


determination


monosaccharide


constituent


composition


sorokiniana


cell


wall


. . .55


Colorimetric


cell


analyses


of acid


walls


hydrolysates of
. ... .. ..... 57


Elemental


analysis


of chitosan


sorokiniana


cell


wall


res


idues


S S S SS SS SS 5.64


Amino


acid


compos


ition


sorokiniana


cell


wall


..... .... 66


Acetolys


cell


and phosphorolysis
walls .. ... .


purifi


. . .68


Percent


subjected


degradation


to enzymatic


of purified
digestion ..


cell


walls


S SS SS S588


Effect


of norflurazon


on growth


rate,


total


carotenoid


content,


percentage


of acetolysis-


resistant


wall


fraction


of C.


sorokiniana


cells


.. .100


Identification


carotenoids


from


sorokiniana
absorption


using


thin


spectrum


layer


chromatography


analysis


and


......102


Comparison


of C.


sorokiniana


wild


type


and


mutant


TFA-extractable


cell


wall


monosa


ccharides


Comparison


mutant


HCl-extractable


sorokiniana


cell


wall


wild


monosa


type and
ccharides


Comparison


of C.


sorokiniana


wild


type


mutant


total


extractable


cell


wall


monosaccharides


S.125


Chlorella


Chlorella


proteins


Table


oa se


w F


















LIST


OF FIGURES


Figure


Identification


monosaccharides


released


through
wall ..


acid


hydrolysis


sorokiniana


cell


. ... 54


Proton-decoupled


ethanol


precipitate c
. sorokiniana


[13C] NMR profile of
f an alkaline-soluble


cell


the
fraction


wall....


IR spectra


remaining after
sorokiniana cell


of chitosan


alkaline
wall .


residue


extraction


. . .... .. .63


4. Acetolysis
sorokiniana an


Transmission
. sorokiniana a:


Autofluore


resistant


fusca


electron


fusc4


science


res


cell


idues


of C


walls.


micrographs
a cell walls


of purified


of
..... . ..73


Chlorella


cell


walls


7. IR spectra
walls . ....


of purified


sorokiniana


cell


IR spectra


of C.


sorokiniana


alkaline


extracted


cell
cell


wall
wall


residue
residue


and a
after


Ikaline


extracted


subsequent


treatment


acetolysis
... ... .... . .82


TEM


and


SEM


treated


sorokiniana


16 h with


CHP


cells


untreated


Kinetics


of degradation


of substrates


chitosanase


from


lividans


pRL207


Measurement


inzvmatic


ree


.ase


Da9e


-r.


VL


r


V











Growth


containing


sorokiniana


cells


norflurazon


cultures


... .. .... ..99


Growth


of C.


sorokiniana


fusca


cells


in cultures


containing


MON-20763


Morphology


sorokiniana


fusca


cells


grown


in cultures


containing


MON-20763


.. .. ..106


Wall


morphology


sorokiniana


fusca


cells


grown


in MON-20763


with


without


subsequent


CHP


treatment


Kill


curve


sorokiniana


cells


expos


ed to


UV light


Growth


wild


type


mutant


sorokiniana


cells


. .. 114


Compari


son


of monosaccharide


composition


TFA hydrolysates
sorokiniana cell


Comparison
hydrolysates


of wild
walls .


type


mutant


. . ... .118


of monosaccharide


of wild


type


composition


mutant


sorokiniana


cell


walls


.... .120


21. Compare
hydrolysates


son


of monosaccharides


of wild


type


in total


mutant


acid


sorokiniana


cells


walls


S.... .. .124
















LIST


OF ABBREVIATIONS


CaMV


cauliflower


mosaic


virus


cellulase,


hemicellulase,


pectinase


glutamate


dehydrogenase


ICBR


Interdisciplinary


Center


Biotechnology


Research


MWCO


molecular


weight


not


determined


NOS ... .

NP-40 ......... .... .. .. ..


nopaline

Nonidet


synthase

P-40


parts


million


RG-I

RG-II


* a a a a a a a a a .' a .
.. . a. .a. a.aa a. a

.. .. .. .. .


rhamnogalac turonan

rhamnogalacturonan


scanning


electron


microscopy


transmission
microscopy


. ...a.a... .. *. .. a. a.

.a .a. ..a. a. a. a a. a


electron


tetramethylsilane

3-(trimethylsiayl)-


propionate


tryptic


broth



















Abstract


Dissertation


Presented


Graduate


School


the University
Requirements i


of Florida


Partial


Degree


Doctor


Fulfillment


of Philosophy


DETERMINATION


OF FACTORS
CHLORELLA


LIMITING


SOROKINIANA


ENZYMATIC


CELL


HYDROLYSIS


OF THE


WALL


Brenda


December,


Russell

S1995


Chairman:


Robert


Schmidt


Major


Department


nature


Microbiology


of resistance


and


the


Cell


Chi o


Science


'rella sorokiniana


cell


wall


to enzymatic


degradation


preventing


protoplast


formation


was


evaluated.


cell


wall


of this


alga


was


analyzed


using


biochemical,


microscopic,


mutagenic


techniques.


The cell


wall


was


shown


to be composed


approximately


carbohydrate


protein.


Using


GC/MS,


major


monosaccharides


identified


cell


wall


N TFA


N HC1


hydrolysates


were


rhamnose,


glucuronic


acid,


galactose,


xylitol,


mannose.


Using


NMR


spectroscopy


trn~


- -1


.T ht'~' iLi fin I


,1 ,


mt a 1. -. I a aa fi a aC ,, a n n a


E


1


k


L











hydroxymethyl


groups.


Cell


wall


proteins


contained


only


0.02


mol%


of hydroxyproline.


Glycine


alanine


were


at slightly


higher


concentrations


than


other


amino


acids.


Glucosamine


was


present


in the


N HC1


hydrolysate


wall


indicating


presence


chitosan.


Compounds


such


as lignin,


other


phenolic


compounds,


sporopollenin,


that


give


some


cell


walls


resistance


to enzymatic


degradation,


were


found


sorokiniana


wall.


Much


resistance


cell wall


to acetolysis


high


concentration


polysaccharides


containing


rhamnose.


Despite


severe


disruption


sorokiniana


mother


cell


wall


growth


cells


MON-20763,


a pollen


biosynthesis


inhibitor,


cell


wall


was


not


degraded


a solution


containing


cellulase,


hemicellulase,


pectinase.


Three


cell


wall


defective


mutants


were


isolated.


concentration


total


cell


wall


protein


certain


monosaccharides


in cell


wall


acid-hydrolysates


varied


among


mutant


wild


type


strains.


In cell


walls


each


mutant,


glucosamine


concentration


was


similar


that


wild


type.


Using


a mixture


cellulase,


hemicellulase,


pectinase,


osmotically


labile


cells


could


be prepared


from


mutant


strains.

















INTRODUCTION


eucaryotic


microalga,


Chlorella,


long


been


studied


events


as a model


such


organism


as photosynthesis,


elucidation


transport,


biochemical


nutrient


metabolism.


organism.


For


Large


several r

quantities


seasons,


Chlore


Chlorella


hlla is

cells


an ideal


can


model


be cultured


days


rather


than


weeks


or months


required


higher


plants.

complicat


Biochemical


studies


presence


with


multiple


cell


are


not


types


as in


higher


plants.


Many


species


Chlorella


can


be cultured


synchronous,


biochemically


homogeneous,


cells.


Some


these


same


characteristics


make


Chiorella


useful


organism


industrial


production


of metabolites


(Millis


et al.,


1988;


Running


et al.,


1994).


scope


industrial


manipulation


value


this


limited


asexual


difficulty


microalga.


of genetic


Stable


trans formation


Transient


in Chlorella


expression


cells


firefly


been


luciferase


achieved.


been


demonstrated


ellipsoidea


protoplasts.


Transformation


ellipsoi dea


was


achieved


using


PEG/CaC12-mediated


Chlorella











Research on


sorokiniana would be enhanced by


development


a transient expression assay.


this


laboratory,


the C.


sorokiniana gene


encoding


an NADP-specific


glutamate dehydrogenase has


been studied


extensively.


promoter


this


gene may


be regulated,


either


directly or


indirectly,


concentration of


ammonium


(Cock et al.,


1991).


Plasmids


could be constructed


test


the effect of


different promoter regions on reporter gene


transcription.


The level


using


reporter


Chlorella cells


gene expression

transformed with


could be determined

the plasmid


constructs.


Many DNA transformation methods


are designed


transfer


DNA into a


low percentage of


total


cell


population.


transformation


frequency is useful


selection of


stable


trans formants,


expression.


but not


Transfer of


practical


plasmids


assessment


into protoplasts


transient


using


electroporation or


PEG/CaCI


results


relatively high


transformation


frequency.


In C.


sorokiniana


cells,


development of


a transient


expression assay


is limited by


inability to prepare protoplasts


from


this alga.


Production


of protoplasts would also allow


for more efficient


isolation


of its organelles


This study was


other types of


designed


experiments.


to increase an understanding of











effects of metabolic


inhibitors


on cell


wall


synthesis,


(iii)


isolation and characterization of


cell


wall


defective


mutants.

















LITERATURE REVIEW


General 1


Cell


Wall


Composition


Four of


five kingdoms


life,


Monera,


Protista,


Fungi,


and Plantae,


are dominated by organisms with cell


walls.


Only


members.


the kingdom Animalia


Since most


is dominated by walless


eucaryote walls are


fibrillar and


polysaccharide-based and most procaryote walls


are non-


fibrillar and peptidoglycan-based,


likely


eucaryotic


and procaryotic cell


walls


walls appeared early in


have evolved independently.


evolution,


most likely


Cell


to enable


protoplast


to maintain


turgor pressure


to allow for


increased


metabolic activity.

walls developed to

environmental damage


In more recent


provide protection


evolutionary time,

to the protoplast


e and attack by pathogens.


Cell


cell

from


walls


have also enabled some single-celled organisms


to occupy


novel


ecological niches


evolving into


large multicellular


forms


(Ruiz-Herrera,


1992).


Most


eucaryotic cell


walls


are composed primarily of


polysaccharides,


but also


possess


significant


concentrations











components


cell


wall,


some


wall


polysaccharides


are


involved


in cell/cell


recognition


gene


regulation


(McNeil


et al.,


1984) .


Protein,


usually


present


as glycoprotein,


second


many


most


functions


common


of cell


cell


wall


wall


constituent.


glycoproteins


Some


include


cell


structure,


protein


growth,


replaces


recognition,


polysaccharide


defense.


primary


rare


cell


cases,


wall


constituent.


Despite


polysaccharide


protein


making


bulk


of all


cell


walls,


additional


compounds


, such


lignin


pigment


other

s such


phenolic co

as melanin


pounds

and ca


esters


rotenoid:


, lipids

s add to


, silica,


variation


cell


wall


structure


function


(Bartnicki-Garcia,


1984


, 1986).


Plant


Cell


Wall


Composition


The

thick.


cellulose.


world,


primary


wall


fibrillar


Cellulose,


comprises


of a higher


or rigid


most


to 30%


plant


cell


portion


abundant


a primary


is about


of a wall


organic


wall


consists


compound


It is


unbranched


polymer


of J


,4-glucose


consis


ting


of between


several


have


thousand


a parallel


glucose


arrangement


units.


are


glucose


specifically


polymers


hydrogen


bonded


to each


other


, forming


microfibril


Microfibrils


are











Hemicellulose


pectin


constitute


to 60%


most


walls


compose


a nonfibrillar


or gelatinous


matrix


which


cellulose


microfibrils


are


embedded.


Matrix


polysaccharides


are


synthesized


intracellularly


are


exported


to the


wall


in Golgi-derived


vesicles.


Cellul


ose


microfibrils


are


subsequently


deposited


into


-like


matrix


(Northcote,


1991).


Depending


upon


composition


polysaccharide


backbone,


most


hemicelluloses


are


grouped


as xylans,


xyloglucans


, or glucans.


Xylans,


polymers


of B-1


,4-xylose


with


branches


of arabinose


glucuronic


acid


molecules,


are


more


prevalent


in grasses


than


they


are


in dicot


plants.


Also


in grasses,


ester


groups


ferulic,


coumaric,


hydroxybenzoic


acids


may


linked


to xylan


monosaccharides.


number


of side


chains


varies


among


xylans.


Xyloglucans


are


more


prevalent


dicots


than


in grasses.


They


have


,4-glucose


(cellulose)


backbone;


most


side


chains


consist


a single


xylose


residue


In addition


to the


xylose


residue,


other


xyloglucan


galactose


side


fucose


chains


have


residues.


Sic


galactose,

de chains


arabinose,


occur


different


frequencies


among


xyloglucans


Glucans


are


unbranched


polymers


of both


,4-glucose.


They


are


abundant


in grasses


are


found


infrequently,


a P-











Pectin


is a polymer


of polyuronic


acid


and


can


present

hairy (


in a single


branched)


cell


forms.


both


Smooth


i smooth

pectins


unbranchedd)


consist


of stretches


of galacturonic


acid


residues


occasionally


interrupted


rhamnos e


residues.


Hairy


pectins


have


backbones


rich


rhamnose


galacturonic


acid


side


chains


that


vary


considerably


among


pectins.


Side


chains


hairy


pectins


rhamnogalacturonans


RG-I


RG-II


can


be composed


twelve


different


sugars,


including


rhamnose,


arabinose,


fucose,


galactose,


methylfucose,


glucuronic


acid.


pectic


polysaccharides


vary


in their


extractability


from


walls.


Hairy


polysaccharides


are


more


resistant


to enzymatic


degradation


pectinase


than


are


smooth


pectins.


Pectins


help


maintain


functions


Joseleau


structure


within


, 1980


growing


Blaschek


cell


plant


et al.,


wall

cell


1981;


and

wall


possibly

(Chambat


McNeil


et al.,


other

and


1984


Fry,


1988).


Plant


cell


wall


proteins


constitute


about


wall


dry-weight


associated


can

cell


with


be either


wall


tightly


or loosely


polysaccharides.


Loosely


associated


cell


wall


proteins


can


be extracted


with


detergents,


salts


, or cold


aqueous


acids


alkalis.


Tightly


associated


proteins


become


insoluble


wall


and











arabinogalactan


proteins.


these


protein


classes,


only


glycine-rich


proteins


are


not


hydroxyproline-rich


glycoproteins.


functions


many


these


structurally


characterized


proteins


are


unknown.


There


evidence


some


functions


healing,


plant


such


as maintenance


defense,


pathogen


of wall


structure,


immobilization,


wound

cell-


cell


interactions.


Finally,


there


are


some


cell


wall


proteins


that


into


any


five


classes


; many


these


proteins


are


enzymes


that


degrade


cell


walls


(Showalter,


1993) .


Other


plant


cell


wall


components


, most


notably


lignin


cutin,


render


cells


with


unique


property


es.


Because


high


concentration


secondary


walls,


lignin


second


most


abundant


organic


molecule


on earth.


Lignin,


polymer


of phenylpropanoid


units,


also


found,


to a 1


esser


extent, i

structure


n primary


and


cell


defense


walls.


against


functions


pathogen


in both


invasion.


cell


Cutin,


polyester


of hydroxy


fatty


acids,


is associated


with


some


epidermal


cells


protects


plant


from


water


loss


other


environmental


stresses


(Fry,


1988) .


Funral


Cell


Wall


Composition


fibrillar


portion


fungal


wall


also











species,


particularly


Chithridiomycetes,


have


substantial


amounts


of both


cellulose


(P-1


,4-glucose)


chitin


wall.


Chitin,


which


not


found


higher


plant


walls,


composes


some


fungal


walls.


Chitosan,


af3-


,4-glucosamine


polymer


with


varying


degrees


N-acetylation,


also


associated


with


some


fungal


rigid


walls.


that


Ruiz-Herrera


chitosan


(1992)


is present


speculates


these


that


walls


likely


crystalline


form


wall


chitin


structure


glucan.


unclear,


role


although


chitosan


contribute


in rigid


to the


resistance


wall


to degradation


pathogens.


Saccharomyces


cerevisiae


spore


wall


about


weight


chitosan.


Chitosan


is located


in a layer


internal


prot


wall

al.,


layer


highly


1988).


external


resistant


Chitosan


to a glucan/mannan


to enzymatic


is polycationic


layer.


degradation


under


This


(Briza


physiological


conditions


may


associate


with


polyanionic


glucuronic


acid


wall.


Many


fungal


wall


polysaccharides


are


characteristic


of specific


fungal


groups.


Polyuronides


are


common


in fungi


consist


mostly


glucuronic


acid


opposed


to galacturonic


acid


higher


plants.


Different


heteropolysaccharides


have


been


described


fungi.


Many


polysaccharides


are


rich


in glucose,


mannose,


galactose,











walls


consist


of 3%-20%


protein.


Hydroxyproline


not


typically


found


in fungal


walls.


Protein


profiles


fungi


are


extremely


complex;


attempts


are


being


made


group


fungal


wall


proteins


into


families


of similar


proteins.


Other


components


some


fungal


walls


include


lipids


melanins


to 21%


in some


spore


walls


sporopollenin


(Ruiz-Herrera


, 1991).


Cell


Wall


Composition


Green


Alcae


Algal


cell


walls


have


basic


wall


structure


shared


plant


fungal


walls;


they


are


compos


ed of microfibrillar


polysaccharides


embedd


ed in


a nonfibrillar


polysaccharide


matrix.


Microfibrils


often


consist


of cellulose


, although,


some


algae


have


-1,4-mannans,


p-1,3-xylans,


chitosan


instead


cellulose


(Takeda


Hirokawa,


1984;


Vian


Reis,


1991) .


Like


fungi,


algal


walls


have


only


a primary


wall.


Generalizations


about


hemicellulose


structure


in algae


have


been


made


they


have


plants.


Green


algae


commonly


possess


glycoprotein-rich


cell


walls.


Cell


wall


proteins


some


green


algae


are


particularly


rich


in the


amino


acids


hydroxyproline,


glycine,


alanine


(Voigt


et al.


, 1994)


Chl amydomonas


reinhardtii


vegetative


cell wall


unique


that


lacks











ellipsoidea


C-87


mol%


hydroxyproline


and


three


other


strains


of C.


ellipsoidea


(one


later


reclassified


vulgar s)


have


no hydroxyproline


(Takeda


and


Hirokawa,


1984).


Some


algae


possess


additional


cell


wall


compounds


such


as ketocarotenoids,


sporopollenin


, hydrocarbons,


lignin-


like


compounds


Alexander,


typically


1975;


(Atkinson

; Berkalo


associated


with


et al., 1

ff et al.


algae,


972


Gunnison


, 1983).


although


and


Lignin


lignin-like


compounds


were


identified


in Staurastrum


Coleochaete


(Chlorophyta,


class


Charophyceae),


where


they


are


likely


function


as an antimicrobial


agents


(Alexander


Gunnison,


1975


Delwiche


et al.,


1989).


Taxonomy


Chlorella


Chlorella,


a eucaryotic


microalga,


classified


with


Protista


and,


since


chlorophyll


a and


b and


stores


carbon


It divides


form


with


of starch,


a member


of a phycoplast


Chlorophyta.


thus


further


classified


class


Chlorophyceae.


Chlorella


unicellular


nonmotile,


possesses


a single


cup-shaped


chloroplast.


Diameter


an average


Chiorella


cell


about


Chl orel la


followed


cells


divide


chloroplastic


asexually


nuclear


enlargement


divisions.


Depending











cells,


are


released


from


surrounding


mother


wall


most


likely


both


enzymatic


mechanical


means.


Cell


wall


autolytic


enzymes


have


been


characterized


from


several


species


of Chlorella.


Because


morphological


simplicity


algae,


taxonomi c


classification


Chlorella


species


difficult.


This


point


well


illustrated


in a study


ch the


biochemical


characteristics


were


tested


on 58


strains


Chlorella


from


Culture


Collection


University


Texas


at Austin


(Kessler


Huss


, 1992).


Only


17 of


strains


been


properly


classified.


earliest


attempts


group


Chl orel la


species


were


made


using


approximately


biochemical


characteristics.


Chemical


characterization


traits


including


hydrogenase


activity,


synthesis


of secondary


carotenoids,


salt,


temperature


limitations


growth


been


used


identify


Chlorella


species.


Despite


large


number


of biochemical


tests,


some


strains


still


can


unambiguously


characterization


classified.


limited


Usefulness


large


of chemical


number


characteristics


that


need


to be analyzed.


sorokiniana


characterized


biochemically


hydrogenase


activity


during


anaerobiosis,


nitrate


reduction,


lack


of secondary


carotenoids


produced


under


nitrogen


defi


clency,


growth











throughout


cell


cycle


organism.


ellipsoidea


C-27


maintains


a constant


qualitative


cell


wall


composition


throughout


cell


cycle.


Rigid-wall


carbohydrate


concentration


also


remains


constant


throughout


cell


cycle;


matrix


carbohydrate


concentration


increases


proportion


to the


growing


cell


surface


(Takeda


Hirokawa,


1978).


Based


upon


cell


wall


morphology,


Chlorella


species


have


been


divided


into


three


groups.


Type-i


walls


have


two


layers,


a thick


inner


cellulosic


layer


thin


outer


trilaminar


layer


containing


sporopollenin.


fusca


typical


type-1


wall


Type-


walls


have


layers,


thick


cellulosic


inner


layer


thin,


pectinase-sensitive


outer


layer.


Protoplasts


are


formed


enzymatic


digestion


some


type-2


walls.


The


type-3


wall


consists


of a single


homogeneous


layer


that


resists


degradation


polysaccharide-


degrading


enzymes


(Yamada


Sakaguchi,


1982).


sorokiniana


was


examined


this


study,


a TEM


photograph


a C.


sorokiniana


cell


appears


to have


type-3


wall


(Biedlingmaier


et al .,


1986).


Three


additional


cell


wall


characteristics,


rigid


wall


polysaccharide,


ruthenium


stainability,


anisotropy


measure


of degree


of wall


crystallinity)


provide


most











mannose


tested


or glucosamine.


a glucosamine


four


rigid


strains


walls


of C.


, positive


sorokiniana


anisotropy


(indicating


a crystalline


wall)


could


be stained


with


tested


ruthenium


that


red.


sorokiniana


a glucosamine


rigid


only


wall


species


positive


anisotropy


(Takeda,


1988a,


1988b,


1991,


1993) .


Based


upon


both


biochemical


cell


wall


characteristics,


sorokiniana


kessleri


most


(Kessler


closely


Huss


related


, 1992


to C.


Takeda,


vulgaris


1993).


Chlorella


Cell


Wall


Composition


There


are


several


reports


on monosaccharide


composition


of polysaccharides


protein


concentration


in Chlorella


cell walls.


Structure


of wall


polysaccharides,


whole


wall


, have


not


been


studied.


only


compound


identified


Chlorella


wall


other


than


carbohydrate


or protein


sporopollenin


discussed


in another


section)


in most


cell


walls,


carbohydrate


major


cell


wall


constituent


Chlorella.


As discussed


briefly


previous


section,


Chlorella


rigid


wall


consists


polymers


of either


glucose


mannose


or glucosamine.


ecies


with


a glu


cose-mannose


rigid


wall


have


matrix


polysaccharides


rich


mannose


glucose


and,


in some











galactose


was


identified


a cell wall


constituent


of C


vulgaris


K-22


(Ogawa


et al., 1994).


sorokiniana


its relative


vulgaris


kessleri


have,


order


decreasing


concentration,


rhamnose


, xy


ose


, mannose


, galactose


glucose


comp


matrix


polysaccharides.


In addition


, C.


kessl er-i


relatively


Three


high


symbiotic


percentage


strains


fucose


Chlorella


Takeda,


also have


1991


cell


1993


wall


compos


itions


similar


. sorokiniana


group


Kapaun


, 1992


following


are


generalizations


about


quantity


monosa


ccharide


present


in cell


wall


polysaccharides


Chlorella


species


of wall


weight).


sorokiniana


Three


wall


strains


was


analyzed


ellipsoidea


in any


have


15-39%


these


glucose


studi


in the


alkalin


Neutral


e-resistant


sugars


wall


make


fraction


from


(Takeda


-44%


Hirokawa,


cell


1978).


wall


polysaccharides


wall


of Chlorell


to 80%


spec


wall


with


those


a glucosamine


species


rigid


with


glucose/mannose


rigid


wall


these


studi


, neutral


sugars


were


are


analyzed


unstable


H2S04


in H2S04


hydrolysat


resulting


es.


Some


a possible


monosaccharides


underestimation


of neutral


sugar


concentration


(Fry


, 1988)


For


those











1958;


Loos


Meindl,


1982;


Blumreisinger


et al.,


1983


Kapaun


et al.,


1992).


Protein


concentration


in Chlorella


walls


ranges


from


.7%-17%


cell


wall


weight.


Cell


wall


protein


amino


acid


composition


been


analyzed


only


alkaline-extracted


cell wall


residue


several


strains


of C.


ellipsoidea.


Since


alkaline-extraction


removes


most


cell


wall


protein,


this


report


is not


representative


wall


of a whole


Chiorella


cell.


Hydroxyproline


was


present


in one


four


strains


of C.


ellipsoidea


tested


(Takeda


and


Hirokawa,


1984).


Enzymatic


Degradation


of Cell


Walls


Autolvtic


Enzyme


Activities


Cells


protected


cell


walls


must


have


autolytic


enzymes


to degrade


that


wall


to allow


cell


expansion


division.


Chlorella


mother


wall


is not


completely


digested


during


cell


division,


as evidenced


accumulation


empty


likely


walls


that


medium


a combination


growing


precise


cultures.


enzymatic


It is


mechanical


mechanisms


involved


in release


daughters


from


surrounding


mother


wall.


Enzymes


with


cell


wall


utolytic


%" p











Enzymes


that


degrade


polysaccharides


into


units


of both


high


molecular


weights


have


optima


from


are


typical


Chlorella


species


having


mannose


glucose


in the


rigid


wall


(Araki


and


Takeda,


1992).


endoenzymes


,4-mannanase,


carboxymethyl


cellulase,


j-D-fucosidase


are


ass


ociated


with


fusca


cell


wall


(Loos


and


Meindl,


1984;


1985) .


polysaccharides


degrade


endoenzymes


into


use


oligomers,


daughter


selectively


which


wall


degrade


cytoplasmic


synthesis.


wall


enzymes


exoenzymes


p-D-mannosidase,


B-D-fucosidase,


- D-glucosidase


are


found


in supernatants


fusca


cell


extracts


degrade


mother


wall


(Walter


Aach,


1987;


Araki


Takeda,


1992


Cell


wall


lytic


enzymes


that


degrade


walls


into


mainly


high


molecular


weight


oligosaccharides


have


optima


around


are


more


typical


those


species


of Chlorella


with


a glucosamine


rigid


wall


(Araki


Takeda,


1992) .


protease


was


identified


ellipsoidea


C-27


(reclassified


as C.


vulgar s


that


cleaves


peptide


bonds


of cell


wall


proteins


(Takeda,


1991) .


Characterization


this


enzyme


activity

maximum


as a prot


ease


inhibition


supported

a protease


alkaline


inhibitor


(Hatano


al.,


1992


Cell


wall


autolytic


activity


of C.


sorokiniana












Protoplast


Production


Protoplasts


have been produced


from many types


of cells


of both monocot and dicot plants.


procedure for preparation of


There is no


protoplasts,


single


but some


generalizations


can be made.


Different


tissues within a


single plant and cells


different ages yield much different


quantities of viable protoplasts.


the amount of


Growth


light and nitrogen source,


conditions,

also have an


such a

effect


on protoplast


frequency.


In a


typical


procedure


the preparation of


protoplasts,


plant


cells are


treated


to 24 h with


cellulase,


hemicellulase,


and/or pectinase.


enzyme


solution is


buffered


to pH


contains


0.2-4% of


each


enzyme and an


osmoticum.


An osmoticum usually consists of


0.3-0.7


mannitol,


sorbitol,


sucrose,


or glucose


(Evans and


Bravo,


1983;


Eriksson,


1985).


Protoplasts


have been prepared successfully from several


species of


Chl orel la.


Incubation


of C.


eli ipsoi dea


211-lb


and C.


saccharophila 211-9a


for 90


h in a mixture of


cellulase


protoplasts,


, hemicellulase,


respectively.


and pectinase produced 20%


Protoplasts were produced from


cells


only when


they were


treated with


enzymes while being


-. -l I it, .-. A 1. I AnEA


n~ r.r: C~ n


L,,


Inl rr\


4-











and C.


saccharophila 211-la


produced a


large percentage of


protoplasts.


Protoplasts were produced within 24


h after


treatment of


enzyme,


cells with cellul


Patnaik and Cocking,


ase,


1982


Macerozyme

, and pectinr


(a pectinolytic

ase (Yamada and


Sakaguchi,


1981)


Using cellulase


percentage of


, Macerozyme


protoplasts was made


, and pectinase,


from C.


a low


vulgaris C-135 and


C-169


(Yamada and Sakaguchi,


1982) .


Using


cellulase alone,


higher percentage of protoplasts was prepared


from an


unidentified


vulgaris strain


from Carolina Biological


(Berliner,


1977) .


Since publication of


these data,


many


strains of Ch

is unclear if


lorella hav

the strains


,e been reclassified.


Therefore,


vulgaris tested have


glucosamine rigid wall


typical


the species


(Kessler


Huss,


1992).


those species


glucosamine rigid wall,


Chlorella known


protoplasts


to have


have been produced only


from C.


ellipsoidea C


(reclassified as


vulgar-is


30.80).


treatment of


ellipsoidea C-27 with a


combination of


glycosidase mixture,


resulted in


chitosanase


osmotically


, and Macerozyme


labile cells.


24 h


Microscopic


observation of


osmotically


labile


cells


revealed


that


they


still


retained an intact


cell


wall


(Yamada


et al.,


1987).











demon


strated


ability


to solubilize


to 30%


(14(2)


-labeled


cell


wall


a high


molecular


weight


polysaccharide.


lytic


enzyme


a pH


optimum


of 8


This


enzyme


was


a protease


tha t


cleaved


peptide


bonds


proteins


that


bridged


cell


wall


polys


accharides.


Chitosanase


from


Bacillus


partially


solubilized


wall


at pH


chitosanase,


as prepared,


known


to have


contaminating


protease


which


be responsible


cell


wall


lytic


activity.


cell


wall


was


also


partially


solubili


treatment


with


Pronase


or trypsin


(Satoh


Takeda,


1989) .


was


released


from


wall


when


treated


with


other


polysaccharide-degrading


enzymes.


Pro toplasts


were


ultimately


produced


from


ellipsoidea


(2-27


using


mixed


glucanases,


chitosanase


from


Bacillus


R-4,


algal


cell


homogenate.


Each


three


enzyme


components


alone


produced


less


than


osmotically


labile


cells.

labile


Chitosanase


cells.


plus


In a mixtu


homogenate

re of all


produced


three


enzyme


osmotically

components,


approximately


cells


were


osmotically


labile


within


at pH


essential


Glucanas


efficient


chitosanase,


protoplas t


homogenate


formation.


were


Greatest


protoplas t


frequency


was


obtained


from


cells


harvested


eas t


half


way


through


cell


cycle.


protease


[14C]











Cell


Wall


Factors


that


Decrease


Enzymatic


Wall


Diqestion


Licnin


Licrnin-Like


Compounds


Lignin


lignin-like


compounds


, such


as phenolic


acids


other


phenolic


biopolymers,


are


very


common


in walls


higher


plants


occur


only


rarely


in algal


walls.


presence


lignin


some


phenolic


compounds


correlates


with


poor


prevents

probably


wall


wall


digestibility.


digestion


prevents


access


mechanism


completely


enzymes


which


clear.


to other


cell


lignin


Lignin

wall


substrates


decreasing


cell wall


pore


size


or adsorbing


enzymes


(Hartley


et al.


, 1990;


Jung


et al.,


1992


Converse


1993


, Besle


et al.


, 1994) .


Several


methods


of chemical


delignification


forage


stems,


in some


not


cases,


resulted


an increase


in digestibility


of substrate


rumen


microorganisms


It is possible


that


disruption


of the


wall


delignification


contributed


to increased


digestion


(Jung


et al.,


1992).


increase


in lignin


content


in sorghum


cell


walls


plants


aged


was


paralleled


increase


p-coumaric


acid


a decrease


in cellulose


degradability.


of phenolic


was


compounds


proposed


with


tha t


cellulose


an increased


response


association


ible











Resistance of


two algal


cell


walls


to microbial


digestion


to the discovery of


lignin-like compounds.


wall


of Staurastrum sp.


subjected


to microbial


was


fractionated,


degradation.


and fractions were


A fraction


that was not


degraded by a mixed microbial


culture contained a


lignin-like


compound


(Gunnison


al.,


1975) .


Resistance of


Coleochaete wall


to microbial


degradation is also


believed


result


from


the presence of


a phenolic biopolymer


(Delwiche


et al.,


1989) .


SnoroDollenin


Sporopollenin was


first


described


as a highly resistant


compound present


in outer


layers


of pollen and


fungal


spores.


It has since been

(Atkinson et al.,


found in

1972; B


cell


walls of many green algae


runner and Honegger,


1985) .


Because of resistance of


sporopollenin


to chemical


solubilization and


enzymatic


degradation,


the chemical


structure has


not


yet been


elucidated.


Sporopollenin is


defined based upon


solubility


characteristics .


It is


insoluble in most acids,


bases,


lipid solvents,


detergents.


It is


also


resistant


to acetolysis


(boiling


acetic anhydride


and concentrated sulfuric acid).


Lignin,


carotenoids,


6-deoxysugars,


such


as rhamnose and











Sporopollenin


was


first


proposed


to be a polymer


carotenoids


and/or


carotenoid


esters.


Plants


fungi


grown


with


[14C] -acetate,


[14C]-mevalonate


(carotenoid


precursors),


[14C]


- -carotene


incorporated


[14C]


into


pollen


spore


sporopollenin.


Sporopollen i n


some


chemical


characteristics


in common


with


carotenoids


such


as pyrolysis-


chromatograms,


elemental


analysis,


x-ray


diffraction


patterns.


Also


like


carotenoids,


sporopollenin


absorbs


light


is autofluorescent


(Brooks


Shaw,


1978;


Singh


Devi,


1992).


"three-way


correlation"


(Atkinson


et al.,


1972


predicts


that


those


cells,


which


have


a cell


wall


with


outer


trilaminar


layer


produce


ketocarotenoids,


also


have


sporopollenin.


This


hypothesis


is supported


mutant


studies


on Chlorella


fusca,


an alga


whose


phenotype


consistent

selected b


with


ased


the

upon


"three-way

carotenoid


correlation


deficiency.


Mutants


were


mutants


lacked


trilaminar


layer


sporopollenin


(Burczyk


Hesse


, 1981


Burczyk


Czygan,


1983).


parasitic


alga


Prototheca ,


possibly


an apochlorotic


form


Chlorella,


does


"three


way


correlation.


Pro to theca


an outer


trilaminar


wall


layer


sporopollenin,


lacks


carotenoid


pigments


colorless


carotenoid


precursors


phytoene











called


Sandoz


SAN


9789


competitively


inhibits


phytoene


desaturase


the


enzyme


first


committed


step


carotenoid


biosynthesis


(Sandmann


et al.,


1991) .


Norflurazon-treated


cultures


green


alga


Scenedesmus


obliquus


decrease


a 87%


in sporo


decrease


pollenin


ketocarotenoid


concentration


content


cell wall


weight)


(Burczyk


, 1987).


Pollen


extracts


from


squash


plants


Cucurbi ta


pepo)


treated


with


norflurazon


had


decreased


carotenoid


concentration


accumulation


nonpigmented


carotenoid


precursors


phytoene


phytofluene.


decrease


in carotenoid


concentration


was


reflected


a decrease


sporopollenin


concentration


(Prahl


et al.,


1985,


1986).


Inhibition


of sporopollenin


synthesis


termination


pollen


development


was


observed


in plants


treated


with


MON-


20763


(also


called


RH-0007


fenridazon),


a phenyl


pyridazone


Cross


Ladyman


, 1991)


phenyl-cinnoline


carboxylate


chemical


compounds,


structures


SC-1058


effects


SC-1271,


on pollen


have


similar


development


as does


MON-20763.


Abnormal


vacuolation,


possibly


a result


coal


escence


secretary


vesicles


, was


observed


in SC-1058-


SC-1271-treated


pollen.


El-Ghazaly


Jensen


(1990)


proposed


that


MON-20763


interferes


with


pollen


development


preventing


polymerization


of sporopollenin


monomers.


Other











compounds


have


not


been


tested


on sporopollenin


synthesis


lower


eucaryotes.


Solid


state


NMJR


spectroscopy


results


showed


that


sporopollenin


from


algae


(including


fusca)


higher


plants


have


structural


characteristics


of carotenoid


polymers.


Sporopollenin


is a polymethylenic


carbon


chain


with


a high


degree


saturation.


Sporopollenin


can


vary


numbers


types


of oxygenated


compounds


such


as ether,


hydroxyl,


ketone,


ester


carboxylic


acids.


Sporopollenins


should


considered


as a group


of related


compounds


(Guilford


et al. ,


1988


Espelie


et al.,


1989


Derenne


al.,


1992).


Pyrolysis


GC-MS


of sporopollenin


from


Chlorella


fus ca


Nanochl orum


eucaryotum


detect


isoprenoid


carbons.


Polymethylenic


chains


sporopollenin


from


these


algae


to 30


mass


carbons


spectroscopy


(Derenne


et al.,


of sporopollenin


1992).


from


Pinus


Using


mugo


pyrolysis


pollen,


coumaric


acid


was


detected.


It is


likely


that


p-coumaric


acid


is a structural


unit


in some


sporopollenins,


and may


contribute


to sporopollenin


autofluorescence


(Wehling


et al.,


1989).


Cell


Wall


Structure











polysaccharides.


Strong


negative


correlation


between


degree


crystallinity


demonstrated.


rate


Incomplete


of wall


enzymatic


hydrolysis


hydrolysis


been


of highly


crystalline


substrates


often


to low


surface


area


substrate


(Converse,


1993).


Pectins


vary


their


sensitivity


to pectinases.


Branched


'hairy'


pectins


are


often


resistant


to pectinase


whereas


unbranched


'smooth


' pectins


are


more


pectinase


sensitive


(Fry,


galacturonic


1988) .


acid


A heteropolymer


wall


alga


containing


Fi scherella


muscicol a


probably


component


rendering


this


wall


highly


resistant


to microbial


decomposition


(Gunnison


Alexander,


1975) .


















MATERIALS


AND


METHODS


Culture


Conditions


Autotrophic


growth


Chlorella


cells


was


performed


light


in SUN


medium


as described


Prunkard


et al.


(1986)


This


basal


salts medium


contained


mM concentration:


KNO3,


CaCI


.340;


K2SO4,


.00;


KH2PO4,


18.4


; MgC12


concentration


CoCI


.189


CuCl2,


.352


EDTA


FeC13


; H3B03,


38.8


MnC1


, 10


; NH4VO4,


.200;


(NH4


6Mo7024,


.19; NiC12,


.190


; SnCl2,


.190


ZnC1


734.


After


KOH.


autoclaving,


sorokiniana


medium


cell


was


were


adjusted


cultured


to pH


.8 with


in a 38.50C


water


bath


with


mirrors


behind


fluorescent


lamps.


fusca


, C.


ellipsoidea,


saccharophila


were


grown


in a 250C


lighted


water


bath


without


mirrors.


cultures


were


bubbled


with


a 2%


(v/v


CO2-air


mixture.


For


cultured


heterotrophic


medium


growth,


supplemented


sorokiniana


with


cells


mM glu


were


cose.


Cultures


were


grown


37C


L Erlenmeyer


flasks


darkness


while


shaking


at 2


' w-


rmm.











Source


Chlorella


Cultures


sorokiniana


strain


IIIB2NA


was


derived


from


. C.


Sorokin


s original


environmental


isolate


from


warm


surface


waters


in Austin,


Texas.


It has


been


maintained


laboratory


about


under


years.


auto trophi c


sorokinia


conditions

na strain


on aga

211-8k


media


is also


for

derived


from


Sorokin


original


culture


(Starr


Zeikus


, 1993)


Prior


to initiation


of experiments


presented


herein,


sorokiniana


culture


was


streaked


isolation


on agar


medium.


experiments


were


conducted


on cultures


derived


from


one


colony.


ellipsoidea


C-87


saccharophila


C-211


were


obtained


from


culture


collection


Institute


Applied


Microbiology


University


Tokyo.


fusca


was


obtained


from


University


Texas


culture


collection


of algae.


Preparation


Samples


Transmission


Electron


MicroscoDy


Samples


were


harvested


centrifugation


fixed


room


temperature


in microfuge


tubes


containing


v/v)


acrolein


Sigma)


(v/v)


glutaraldehyde


(Tousimis


M cacodylate


buffer


(Sigma),


After


m











were washed 2-times with water


and dehydrated sequentially


10 min


in each of


solutions containing


25%,


50%,


75%,


95%,


and 100% ethanol


followed by


15 and 30 min


in acetone.


Dehydrated sample pellets were embedded at


room


temperature


in Spurr's


Low Viscosity


Resin


incubating pellets


in 30%


plastic


in acetone


followed by


70% plastic


and 100% plastic


Cells


in 100% plastic were


incubated at


60 CC


approximately


12 h or until


hardened.


Thin sections of


embedded samples were cut


on a


microtome


(RMC)


and placed on


formvar-coated copper grids.


Thin sections were post-stained with


freshly


filtered 2%


KMn04


mm,


rinsed with water,


bleached


30 s with a


solution containing


drops of


drops of


1% oxalic acid in


1.2%


sodium sulfite and


10 mL of water,


rinsed with water,


stained with Reynold's


lead


citrate


30-60


and rinsed


with water


(Atkinson,


1972).


Samples were observed using a


Zeiss


electron microscope.


Preparation of


Samples


ScanninO


Electron MicroscoDp


Samples


for SEM were


fixed in


the same manner


those


for TEM.


After


dehydration


of samples


100% ethanol,


samples were placed


in HMDS


20 min,


centrifuged,


and HMDS


was


removed and samples were air


dried.


Dry samples were











Determination


of Cell


Wall


Thickness


Images


from


.25"


negatives


were


enlarged


to 8"


printed


on photographic


paper.


Cell


wall


image


widths


in mm were


measured.


To determine


actual


wall


thickness,


width


wall


obtained


from


each


photograph


was


divided


magnification


microscope


also


amount


enlargement


from


negative


to print


times


x 11"


print).


final


value


was


cell


wall


thickness


micrometers.


Reported


cell wall


thickness


resulted


from


calculations


compiled


from


least


photographs.


Purification


of Chlorella


Cell


Walls


Cell


walls


were


puri field


from


cells


cultured


heterotrophically


in darkness


following


experiments


carbohydrate,


protein,


amino


acid


analyses;


NMR


analysis;


acetolysis


of walls


from


norflurazon-treated


cells.


remaining


experiments


, walls


were


purified


from


cells


cultured


autotrophically


light.


wall


purifications


approximately


L of


logarithmically


growing


Chlorel la

4C. Cell


cells


were


pellets


centrifuged


were


washed


at 20


once


OO00g


with


dH9O and


min a

stored


-20C


until


needed


cell


wall


isolation.


Breaking,











fraction


was


removed


centrifugation


in 30 mL Corex


centrifuge


tubes


at 20


,000g


pellets


were


washed


with


water


repeatedly


until


supernatant


was


colorless.


After


each


leaving


centrifugation


behind


step,


a dense


pellets


, white,


were


stare


suspended

containing


in water

portion


llet.


Remaining


starch


or whole


cells


were


removed


centrifugation


3-times


at 1,000g


15 min.


Removal


whole


cells


starch


was


confirmed


microscopically.


Partially


supernatant


purified


was


walls


were


colorless


extracted


Four


in acetone


additional


until


extractions


were


performed


10 min


at 50 C


before


centrifugation;


acetone


dH20


1985).


and

and

Cell


two


in methanol.


lyophilized


walls


over


Purified


night


purified


walls


(Brunner


carbohydrate


were

and H


suspended


onegger,


analysis


were


treated


with


amylase


(Sigma).


Weight


Determinations


Prior


to weighing,


aluminum


pans


were


dried


in an oven


at 100C:


at least


Preweighed


pans


containing


samples


were


incubated


100C


24 h and


weighed


again


Sample


weights


were


determined


subtracting


weight


a pan


from


weight


sample.











Department


of Chemistry,


Spectroscopic


Services


Laboratory.


GC-MS of


carbohydrates


was


performed at


the University of


Florida,


ICBR,


Glycobiology


Core


Laboratory


on a Shimadzu QP-


5000 GC-MS work station.


Amino acid analyses were performed


the University of


Florida,


ICBR,


Protein Core Laboratory


on a


Beckman


6300


Amino Acid Analyzer.


Elemental


analysis


was


performed at


the University of Florida,


Department


Chemistry,


Spectroscopic


Services


on a


Fisons


1108 CHN


Elemental Analyzer.


Extraction


of Hvdrolvzabl


Carbohydrates


from


Chlorella


Acid Hydrolysis of Cell


Walls


Walls


Hydrolysis


cell


wall


samples


GC-MS analyses was


performed at


the Glycobiology Core Laboratory by


laboratory


staff.


Hydrolysates used in spectrophotometric


assays


were


prepared by the author using different


chemical stocks and


different


Heating


facilities


of cell


than

wall


those at

samples


the Glycobiology Core.

in acid solutions was


conducted under nitrogen


in 3


or 5


thick-walled V-


vials


(Wheaton).


Samples


were


hydrolyzed at


100OC and 110C


in a heating block or


an autoclave.


TFA hydrolysates were


prepared by incubating


2-10 mg of


cell


walls


in 1-2 mL of











were prepared by soaking residue


from TFA hydrolysis


mL of


(w/v)


h at room


temperature.


The H2S04


was


diluted

at 100C


to 4% with dH20 and


further hydrolysis was


performed


present after


centrifugation of


the H2SO4


treated walls,


was designated


H2S04


hydrolysate.


The H2S04


hydrolysate was neutralized


with solid barium carbonate.


to remove


After


solid barium carbonate,


centrifugation at 15,OOOg


the supernatant was dried


in a rotary


evaporator.


In another sample,


residue remaining


after TFA hydrolysis was


hydrolyzed


further


in 1-2


mL of


18 h at


110 cc.


Unhydrolyzed cell


wall


residue was


removed by

stream of


centrifugation and HC1


nitrogen or


was


in a Speed-Vac.


evaporated under a

Residues remaining


after each acid hydrolysis procedure were dissolved in dH20


prior to analysis


(Takeda,


1991).


Monosaccharide composition


of TFA and HCI hydrolysates


was determined using GC-MS.


Prior


to gas


chromatography,


monosaccharides were converted


their


tetramethylsilane


(TMS)


derivatives


and amino


sugars were acetylated.


Monosaccharides were identified based upon retention


time


(gas chromatography)


and mass


to charge ratio


(mass


spectrometry). An external

concentrations of inositol


standard


consisted of


each monosaccharide


known molar

in the


The supernatant,











Hydrolysates


were


also


analyzed


using


spectrophotometric


assays.


using


1986) .


TFA


hydrolysates


metahydroxy


H2S04


were


diphenyl


hydrolysates


analyzed for

assay (Chapli


were


uronic

n and


analyzed


acids

Kennedy,


using


phenol


sulfuric


assay


total


carbohydrate,


Elson-


Morgan


assay


amino


sugars


(Chaplin


Kennedy


, 1986),


glucose


oxidase


assay


Sigma,


Procedure


510)


Alkaline


Hvdrolvsis


of Cell


Walls


Alkaline


hydrolysis


was


formed


on purified


sorokiniana


N NaOH


cell walls


hydrolysis,


in both


of walls


N and


were


N NaOH.


incubated


a 0.4


ml


NaOH


solution


under


nitrogen


20 h at 30C


(Takeda


and


Hirokawa,


were

times


1978).


extracted


with


h each


Hydrolysate


was


N NaOH


mL of NaOH


at 30C


recovered


hydrolysis

solution u


(Morrison


of walls


Inde


al.,


precipitation


nitrogen


1993).


in 2


volumes


ethanol


at -20 C


over


night.


Hydrolysates


were


dried


under


nitrogen


prior


to resuspending


@1-120.


Alkaline-soluble


cell


wall


fraction


was


analyzed


using


proton-decoupled


NMR


spectroscopy.


Alkaline-insoluble


wall


residue


was


analyzed


using


IR spectrometry


elemental


analysis.


[13C]











Protein and Amino Acid Analyses of


Purified Cell


Walls


Cell


wall


protein was


extracted from approximately


15 mg


of purified cell


walls


mL of


N NaOH.


Samples were


incubated while shaking


at 379C


20 h


(Loos


and Meindl,


1982) .


Extracts were analyzed using


(1951)


protein


BioRad and Lowry


assays.


Amino acids were extracted from cell


walls by


incubating


approximately


20 mg of walls


mL of


w Hel


containing


1% phenol


and 0


02% mercaptoethanol


at 110C


20 h


(Loos


and Meindl,


1982).


Supernatants were recovered by


centrifugation in a microfuge and


evaporated using a Speed


Vac.


Residues were


solubilized


in dH20 and subj


ected


automated analysis of


amino acid


composition.


Total


wall


protein was calculated


from amino acid


composition data.


average amino acid molecular weight of


125 was calculated


based upon


mol% of


constituent


amino acids


final


value


was


corrected


for weight


of a water molecule lost upon


formation of


a peptide bond between


each of


two amino acids


the peptide.


Acetolvsis


Whole cells,


purified


cell


walls,


chitin


(Sigma),


- a


LZ A-.--sr9, ,


1 *


- <


1











20,000g at


4 C.


Prior


acetolysis,


purified cell


walls


were washed sequentially in methanol


5 min at


50C,


methanol:chloroform


min at


50 C,


and in


N NaOH and


acetic acid


each for


10 min.


Acetolysis was performed by


suspending washed walls


(v/v)


acid:acetic anhydride and placing


concentrated sulfuric


tubes


in a boiling water


bath

aceti


for up


acid


30 min.


Residues were washed sequentially in


, 0.1 M sodium acetate,


0.025 M phosphate buffer


(PH 7),


dH20,


and methanol


and dried in a


60 C oven.


TFA


treatment


of cell


walls prior to acetolysis was


performed by heating


approximately 50 mg


of walls


in 1.5 mL


of 1 M TFA in a


3 mL


V-vial


to 100C


(Fry,


1988).


Residues were washed with dH20O and subjected


to acetolysis


described above.


Alkaline extraction,


prior


to acetolysis,


was performed on approximately 50 mg


dried walls


successive


100C


dH20


extraction


(v/w


10 min at


in 10%


ethanolic


100 Cc,


(v/w)


aqueous


(95% ethanol


and dH20 until


h at


h at


neutralization


75C,


(Burczyk,


1987).


Assessment


of Cell


Wall Autofluorescence


Autofluorescence of


purified substrates was determined


observing


a wet mount with


a Nikon Labphot


fluorescence












Determination


of Cell


Wall


Solubility


in Phosphoric Acid


Approximately


80 mg


of dried,


alkaline-extracted


(in 10%


(w/v)


as described above)


walls were incubated in


20 mL


of 85% phosphoric acid in


Prior


a 55 C water bath for up


to dry weight determination,


to 30 d.


insoluble residues were


washed with dH20 until

washing with methanol


a neutral

3-times at


pH was

65 ,


obtained,

acetone 3-


followed by


times


room


temperature,


and once with ethyl


ether


(Burczyk,


1987).


Phlorocqlucinol Assay for Licnin


Using the phloroglucinol assay


(Berkaloff


et al.,


1983),


purified C.


sorokiniana,


fusca,


and C.


ellipsoidea cell


walls were


tested


lignin content.


Pine saw dust was used


as a positive control.


A drop of


a saturated phloroglucinol


(Sigma)


solution,


prepared in 20% HC1


(v/v) ,


was placed on a


sample of


saw dust


or cell


walls


on a glass


slide.


positive reaction


lignin was


indicated by


formation of


magenta


color.


Analysis of Alkaline


Extracts


Presence of


Phenolic


Compounds


Approximately 0


g of purified


sorokiniana


cell


-


I Ir -- -7.- !r I ~ n y t-


n _.r 1


r


J


A












3-times with 1-butanol


(Morrison


et al.,


1993).


A UV


spectrum was


taken of


combined butanol


extracts


(Beckman,


model DU


640)


Butanol


was


evaporated under a stream of


nitrogen.

analysis


Residue was dissolved in

by proton-decoupled [3C]


mL of water before


NMR spectroscopy.


Determination of


Cell


Wall


Solubility in Chromium Trioxide


Approximately


purified walls


were incubated in


a solution


containing


g CrO3


in 2 mL of


dH20O and 2 mL of


acetic acid.


Solubility of


cell


walls


the Cr03


solution


was


assessed within


d incubation at room


temperature


(Berkaloff


et al.,


1983) .


Preparation of


Cell


Wall


Degradinq


Enzymes


Enzymes Tested


Enzymes


tested


to degrade purified walls and walls


intact


cells were:


hemicellulase


(w/v)


(Sigma),


Cellulysin


(w/v)


pectinase


(Calbiochem),


(Sigma)


(w/v)


(each


singly and


in a mixture of


three enzymes


referred


CHP),


a mixture of


U/mL chitinase


(Sigma),


U/mL N-


acetylglucosaminidase


(Sigma),


(w/v)


lysozyme


(Sigma),


I' l. l -NU -r .


n nrr


* _


I


i I


m


r











123,


hemicellulase


pectolyase


Y23,


HP 150,


cytolase


cellulase


(Genecore),


Macerozyme


Karlan


Research


Products).


Growth


lividans


DRL207


Chitosanase


Production


A S.

generous a


lividans


mount


culture was

culture from


started

a slant


transferring


(provided


Brzezinski)


of TSB


into


(Gibco


a 125


plus


mL Erlenmeyer


pg/mL


flask


containing


filter-sterilized


kanamycin.


culture


was


incubated


30C


while


shaking


at 250


until


dense


growth


was


observed


Sporulation


agar


plates


(SLM3


medium)


(DeWitt,


1985)


were


inoculated


with


mL of TSB


culture.


Plates


were


incubated


at 3092


about


10 d.


Spores


grown


on SLM3


medium


served


inoculum


precultu


res.


SLM3


agar


contain


d in g/L:


starch,


corn


steep


liquor,


CaCO3,


.00; FeSO4-7H20,


agar


,20.0


was


adjusted


to 5


with


medium


was


sterilized


autoclaving.


preculture


was


grown


in 50 mL of


plus


pg/mL


kanamycin


in a 250


mL Erlenmeyer


flask.


was


inoculated


heavily


with


gray


spores


that


formed


surface


SLM3


plate.


culture


was


incubated


48 h


at 30C


while


shaking.











hundred milliliters of


chitosanase-production medium


(in a


L Erlenmeyer


flask)


were


inoculated with


mL of bacterial


suspension.


Chitosanase-production medium contained in g/L:


K2HPO4,


0.5;


MgSO4 -7H20,


(NH4)2S04,


2.0;


FeSO4


*7H20,


0.01;


CaC12 2H20,


grinder),


15.0;


0.01;


starch,


chitosan


(milled in


and in mL:


a coffee-bean


olive oil,


2.0.


was adjusted


autoclaving.


to 7


After


with HC1


the medium was


sterilization,


following


sterilized by


filter-


sterilized components were added


M MOPS buffer,


L of medium:


trace elements solution


100 mL


(which


contained in mg/L:


ZnCl2,


CuC12,


boric


acid,


(NH4


6Mo7024,


10) ,


and 1


mL 10 mg/mL riboflavin.


culture was


incubated


72 h at


30 C while shaking at


rpm.


Culture supernatant was used as


starting material


chitosanase purification


(Masson


et al.,


1993;


Boucher and


Brzezinski,


personal


communication).


Purification of Chitosanase


Supernatant


from


the chitosanase production medium was


harvested by


centrifugation at


4C.


EDTA


was added at a


final


concentration of


The supernatant was


acidified


to pH


4.5 with 5 M acetic


acid.


A 2%


solution


of polyacrylic


acid


250,000,


Aldrich Chemical


was added dropwise











300 mL of


dH20 and brought


to pH


8.5 with


M NaOH.


Residual


polyacrylic acid was precipitated by addition of


M calcium


acetate dropwise


to a


final


concentration of


35 mM


Precipitate was removed by


centrifugation and discarded.


supernatant,


which


contained chitosanase,


was


stored at


or diluted with an


equal


volume of


sterile 20% glycerol and


stored at


-20 C.


centrifugation


steps were performed at 11,000g for


15 min.


A temperature of


4cC was maintained


throughout


purification procedure.


Chitosanase Assay


Chitosanase activity was


tested on soluble chitosan


(solubilized


as a 10 mg/mL stock in


N HC1)


and insoluble


chitosan which were diluted


to prepare a


0.1% chitosan


solution


(v/v)


or suspension


(w/v)


in 50 mM sodium acetate,


pH 5


Assay mixtures


950 pL of


chitosan solution and 20


mU of


chitosanase were


incubated


10 min at


37C


(Boucher


et al.,


1992)


The supernatants were assayed


for reducing


sugars using


the neocuproine


assay


(Chaplin and Kennedy,


1986) .


To prevent precipitation


samples


prior to


reading


on the spectrophotometer,


they were maintained at


37 C.











light:dark:light


cycle.


starting


culture


absorbance


(640


After


dark


period,


cultures


were


again


diluted to


an absorbance


final


light


period


(i.e.,


cells


were


washed


in water,


suspended


water,


broken


during


one


pass


through


a French


pressure


cell.


remove


cell


debris,


homogenate


was


centrifuged


at 4 C:


20,000g


30 min.


Supernatants


were


lyophilized


stored


at -20QC


(Hatano


et al.,


preparation


was


1992) .


Protein


determined


concentration


Bradford,


1976).


dried


Immediately


before


use,


dried


supernatant


was


prepared


solution


(w/v)


buffer


resulting


final


concentration


approximately


mg protein/mL.


Trea tmen t


of Whole


Cells


wi t~h


Cell


Wall


Degradinq


Enzymes


Chlorella


cells


were


grown


autotrophically


light,


harvested


during


logarithmic


growth,


placed


in an


Erlenmeyer


flask


overnight


under


light


to allow


division


most


cells


into


daughter


cell


Before


cells


were


harvested


treatment


with


cell


wall


degrading


enzymes,


culture


was


grown


autotrophically


an additional


For


treatment


cells


with


polysaccharide-degrading


enzymes


, cells


were


handled


aseptically


1 buffers a












same


buffer


counted


using


a hemocytometer.


Cells


were


centrifuged


or buffer


plus


microfuge


enzymes


tubes


final


resus


volume


pended


mL of


in buffer


x 107


cells/mL


50 mL Erlenmeyer


flasks.


Flasks


were


incubated


dark


at 35 C


to 16 h while


shaking


at 75


rpm.


Enzyme-free


controls


were


included


with


each


experiment.


For


experiments


in which


a protease


and/or


cell


homogenate


in conjunction


with


polysaccharide-degrading


enzymes


were


tested


their


ability


to induce


formation


protoplasts,


following


conditions


were


applied.


Daughter


cells


were


cultured


autotrophically


in light


Approximately


cells/mL


were


suspended


reaction


buffer


mM sodium


phosphate


M mannitol,


Enzyme


solutions


prepared


in reaction


buffer


contained


a subset


of CHP,


Pronase,


chitosanase,


homogenate.


solution


containing


part


cells,


parts


enzyme


mixture,


parts


mM CaCl


were


incubated in


mL microfuge


tubes


at 300C,


while


shaking


dark


(Hatano


et al.,


1992).


After


least


h of


enzyme


treatment


, formation


osmotically-labile


cells


was


assessed.


Cell


lysis


aia


hypo tonic


solution


was


indicative


of either


a weakening


complete


removal


cell


wall.


Water


was


drawn


under












Using ultrafiltration


in a stirred cell


with a Diaflow


PM-10


filter


(10,000 mwco,


Amicon),


reducing


sugars were


removed from commercial


enzyme preparations


Enzymes were


diluted


to working


concentrations


in 0.1


M citrate and


60 mM


EDTA,


5.5.


Solutions were concentrated at


approximately


10% of


the original


sample volume.


Buffer was


added


to return


the concentrated sample


its original


volume.


final


This procedure was


concentrate was


repeated a


returned


total


the original


3-times.


volume with


buffer


Brown,


personal


communication).


Treatment


Purified Substrates with Cell


Wall


Dearadinc


Enzymes


Ten milligrams


cell


walls were incubated at


30 Cc


16 h while shaking at


75 rpm in 0.1 M citrate buffer adjusted


to an appropriate pH


each


enzyme.


Supernatants were


assayed


for reducing


sugars


(dinitrosalicylic acid assay),


total


carbohydrate


(phenol


sulfuric acid assay),


amlno sugars


(Elson Morgan assay)


, uronic


acids


(metahydroxy diphenyl


assay)


(Chaplin and Kennedy,


1986),


and glucose


(glucose


oxidase assay,


Sigma.


Procedure No.


510)


fluorescamine


assay


(Udenfriend et al.,


1972)


was


used


assess


enzymatic release of primary amines


(amino











In each


reaction


mixture,


of substrate


in 25


mM sodium


phosphate


buffer,


or pH


were


mixed


with


50 pL


enzyme


dH20


incubated


at 37C


Three-hundred


microliters


supernatant


diluted


mO


M sodium


borate


were


mixed


with


0.01%


(v/v)


fluorescamine


(Sigma)


in acetone.

excitation


Using a

wavelength


Shimadzu DR-15

of 390 nm and


fluorimeter

an emission


with


wavelength


fluorescence


emission


was


determined.


Treatment


Thin


Sections


with


Polvsaccharide-Deqradina


Enzymes


Logarithmically


growing


sorokiniana


fusca


cells


were


prepared


TEM


as described


previously


except


osmium


was


omitted


thin


sections


were


placed


on formvar-


coated


ckel


grids.


Before


post-staining,


grids


were


incubated


on drops


CHP


or a mixture


of CHP,


w/v


homogenate,


pronase,


mU chitosanase


room


temperature.


Prior


to microscopic


observation,


grids


were


washed


with


water


post-stained


as described


previous


section.


At temD t.s


to Induce


Accumulation


of Ketocarotenoids


sorokiniana


sca


Cells


-~' I* -


I'


'


*


..












using


carotene


(Kessler,


(Sigma)


1978


were


Canthaxanthin


used


(Fluka


standards.


Determination


of Effects


of Norflurazon


on Growth


Carotenoid


Content


, and


Percentage


Acetolvsis


Resistant


Cell


Wall


Fraction


. sorokiniana


Norflurazon


was


provided


Robert


Lamoreaux,


Sandoz


Agro


, Inc..


To determine


concentration


of norflurazon


required


to decrease


carotenoid


content


, while


also


having


minimal


affect


on growth


rate,


sorokiniana


cultures


were


grown


heterotrophically


in medium


supplemented


with


several


different


concentrations


of norflurazon.


For


carotenoid


wall


analyses,


cells


were


grown


ml


norflurazon


doublings.


Total


carotenoid


content


cells


was


determined


(Liaaen-Jensen


Jensen


, 1971) .


identification


individual


carotenoids,


pigments


were


separated


using


TLC


(Kessler,


1978)


Pigment


ed bands


from


TLC


plates


were


dissolved


in both


ethanol


chloroform.


Using


a Beckman


spectrophotometer,


absorption


maxima


were


measured


two


solvents


and


compared


to published


data


(Britton,


1985).


weight


of acetolysis-resistant


residues


of whole


Chlorella


cells


was


determined


(methods


described


previous


sections).


Rate,












Determination


Effect


of Growth


MON-20763


Chlorella


Cells


MON-20763


was


supplied


Dennis


Dunphy


, HybriTech


Seed


International


, Inc.


Cells


were


cultured


autotrophically


light


medium


supplemented


with


various


concentrations


of MON-20763


Cells


were


grown


in cultures


supplemented


would


with


allow


highest


OD640


concentration


culture


of MON-20763


to double


that


twice.


Before


after


treatment


with


CHP


, C.


fusca


sorokini ana


cells,


cultured


MON-20763,


were


observed


under


light


microscopy


TEM.


Whole


cells


were


also


subjected


acetolysis.


Production


Screening


of Cell


Wall


Defective


Mutants


Logarithmically


transferred


growing


an Erlenmeyer


sorokiniana


flask


cells


left


were


light


without t


agitation


This


treatment


allowed


most


cells


to divide


into


daughter


cells,


to decrease


their


starch


content.


Cells


were


counted


using


hemocytometer.


To construct


a kill


curve,


Chlorella


cells


were


centrifuged


a 50


mL polypropylene


centrifuge


tube


rP~ C1 Crncnn qc,


tt?~ f-h I I I'* 4. ~rnr~r%.-lI -.,1-


mrna 4. in,


t -in9


rt; th


,,1


E I f f|


L-


I











bulb of a


UV lamp


(Model MR-4,


George Gates Co.,


Inc.,


with a


Silvania


G8T5,


8W bulb)


Upon


exposure


to UV light,


cells


the Petri


plate were stirred


slowly


as possible with


stir


bar made by


heat sealing


a straightened paper


clip


inside a piece of Tygon

photo-repair mechanisms,


tubing.

efforts


To prevent

were made


induction of

to minimize


exposure of mutagenized cells


to ambient


light.


At 1 min


intervals


(there was no


UV exposure at


zero


time),


for a


total


of 12 min,


100 uL of


cell suspension were removed from


Petri


dish and diluted


in SUN medium


to a


final


concentration


of 1.8


x 103


cells/mL.


00 pL aliquot


this


final


dilution


(360


cells)


was


spread onto each of


four


SUN plus


50 mM glucose plates


the dark at 37 C.


After


(1.5% agar)


and incubated in


a one week incubation,


colonies on


each plate were counted and plating efficiency and percent


kill


were calculated.


For production and screening of


sorokiniana mutants,


cells were prepared for


UV mutagenesis


as described for the


kill


curve


Cells were exposed


to UV light


resulting


97%-98%


kill.


UV-exposed cells were diluted


cells/300


pL of


SUN-glucose medium and 300 pL were


aliquotted into each well


of a 96-well microtiter plate


(Sarstadt


, uncoated,


flat


bottom)


After


4 d incubation


mm











first plate


to SUN-glucose medium in


the wells


second plate.


Since cells


in the


first


plate were killed by


the screening procedure,


the second 96-well


plate was used


for retrieval


of possible cell


wall


defective mutants.


first plate was


centrifuged


(5 min,


i, 000g,


4C)


in a Beckman


DU 640


centrifuge


(adapted


for 96-well


plates),


supernatants were discarded.


Cells


each well


were


resuspended in 300


incubated in


pL of


in buffer.


the dark while shaking at


first plate was


rpm at


35C


first


removed,


(Sigma).


plate was


and pellets


first


centrifuged


resuspended in


plate was


for 5 mm


pL of


centrifuged


, supernatants


1% NP-40


10 min and


wells were observed


for green supernatants


(putative cell


wall


defective mutants).


Putative mutant


cells,


maintained in


the second plate,


were diluted and distributed


so there was approximately


cell/well


in a


third microtiter plate.


The cells


from the


third plate were grown and screened as


first plate.


they were


A fourth plate was maintained as a


for the


copy of


third plate.


Putative mutants


retrieved


from


fourth


plate were again


diluted


to 1


cell/well


into a


fifth plate


and grown and screened


as described for


first and


third


plates.


Each


final


mutant


culture was derived


from a


colony


* I J- '-.










plates


Mutant


cultures


were


maintained


on SUN


SUN-glucose


slants


under


fluorescent


light.


Using


methods


described


previous


sections


, putative


mutants


were


analyzed


their


growth


characteristic


microscopic


treatment


appearance,


with


susceptibility


polysaccharide-degrading


of whole


enzymes,


cells


cell


wall


composition.



















RESULTS


sorokiniana


Cell


Wall


Characterization


Polvsaccharide


Analysis


Qualitative


data


on C.


sorokiniana


cell


wall


polysaccharide


composition


have


been


reported


previously.


strain


of C.


sorokiniana


used


this


laboratory


been


maintained


about


years.


culture


been


periodically


streaked


isolation


colonies


have


been


selected


future


cultures


based


upon


fast


growth


rate.


ensure


cell


wall


composition


was


still


similar


that


reported


other


strains


sorokiniana,


a complete


analysis


sorokiniana


IIIB2NA


wall


was


conducted.


Purified


sorokiniana


cell


walls


were


acid


hydrolyzed


to allow


ease


and


subsequent


identification


of cell


wall


monos


accharides.


Mild


acid


treatment


in TFA


hydrolyzes


matrix


polysaccharides


which


can


be destroyed


under


stronger


acid


conditions.


Stronger


acids


, HC1


H2S04,


are


used


S -I -I 4 .. -- A-1-~- A- ~3


I


m


-1












Approximately


weight


sorokiniana


cell


wall


was


released


as monosaccharides


analyzed


GC-MS)


after


TFA


hydrolysis.


primary


monosaccharide


released,


rhamnose,


contributed


to 72%


TFA


hydrolysate


or 38%


cell


wall


weight.


Galactose


made


TFA


hydrolysate


or 6


7% of


cell wall


weight.


Monosaccharides


that


each


contributed


to at least


hydrolysate


were


glucuronic


acid,


mannose,


xylitol.


Trace


amounts


of xylose,


glucose,


and


glucosamine


were


also


identified


Figure


Table


Hydrolysis


of TFA-extracted


walls


with


release


additional


of wall


weight.


Glucuronic


acid,


a major


component


pectin,


made


hydrolysate


of cell


wall


weight.


Galactose,


rhamnose


xylitol


making


contributed


up 22%,


significantly


, and


hydrolysate


hydrolysate,


respectively.

as mannose, g


remaining


glucose,


monosaccharides


glucosamine,


and


were


galac tosamin


identified

e. Each


contributed


less


than


4% of


monosaccharides


released


hydrolys


An additional


peak


was


present


on the


chromatogram


hydrolysate,


was


identified


as an


amino


sugar


using


mass


spectrometry.


concentration


unidentifi


amino


sugar


could


be determined


without
























Figure 1. Identification of the m
through acid hydrolysis of the C.
Purified cell walls were hydrolyze4
100C followed by hydrolysis in 6
Percentage of hydrolysate represent
monosaccharide in TFA hydrolysate,
the two acid hydrolysates combined


onosaccharides re
sorokiniana cell
d in 2 N TFA for
N HCI for 18 h at
ted by each
HCl hydrolysate,
, is presented.


leased
wall.
6 h at
110.





























Total




TFA





6 N HC1


25 50 75


-- [ Rhamnose

;| [ Xylose

[] Xylitol

07 Mannose
4,
/ U Galactose

H Glucose

E3 Glucuronic Acid

: Glucosamine

1 Galactosamine
100


Percentage of Wall Hydrolysate














Table


GC-MS determination of


composition of


constituent monosaccharide


wall


Percent


of Cell


Wall


Dry Weight


TFA


Total


Monosaccharide


Hydrolysate


Hydrolysate


Hydrolysate


Rhamnose
Xylose
Xylitol


0.75


0.70


Mannose
Galactose
Glucose
Glucuronic Acid
Glucosamine
Galactosamine


0.92
0.68


Total
Polysaccharidea


aTotal


polysaccharide was


estimated by calculating mol


monosaccharide and subtracting weight


of one mol


of water


lost upon formation of


a glycosidic bond


for every two mol


monosaccharide.


sorokiniana cell











cell


wall


weight.


Other


ma3or


monosaccharides


were


glucuronic


acid


galac those


making


wall


weight


at least


3% of


galactosamine


weight


using


, respectively.


wall


each


(Table


Xylitol


xylose,


contributed


Results


spectrophotometric


assays


to 1%


analyses


are


mannose


glucosamine


or less


of acid


presented


made


, and


wall


hydrolysates


in Table


amount


uronic


acid


amino


sugar


was


identified


acid


hydrolysates


sorokiniana.


Glucose


was


identified


H2S04


hydrolysate


fusca


that


sorokiniana.


As determined


phenol


sulfuric


acid


assay,


sorokiniana


cell


wall


dry


weight


was


extracted


carbohydrate


with


N NaOH.


Glycosidic


bonds


are


labile


alkali


they


are


in acid.


As a result,


oligosaccharides


as opposed


to monosaccharides


are


often


released


alkaline


hydrolysis.


Proton


decoupled


[13C]


NI4R


useful


determination


characteristics


polysaccharides.


spectrum


an ethanol


precipitate


alkaline


extract


sorokiniana


wall


about


signals


after


Figure


dialysis


A similar


sample


spectrum


in 1,000


was


mwco


obtained


dialysis


even


tubing


A [13C]

















Table


Colorimetric


analyses


of acid


hydrolysates


Chlorella


cell


walls


Percentage


of Cell


Wall


Weight


Acid


Species


Total


Hydrolysate


Glucoseb


Carbohydratea


Amino
Sugarc


Uronic
Acidd


C. sorokiniana


TFA


H2S04

HC1


0.06


0.07

0.30


.70 0.01


H2S04

HCI1


8.4 0


0.02


aTotal
assay


carbohydrate


was measured using


the phenol


sulfuric


acid


bGlucose
CAmino s
duronic
eND not


was measured


ugars
acids


using


were measured


were


measured


the glucose


using
using


oxidase


assay


the Elson Morgan
the metahydroxy


assay


diphenyl


assay


determined


fusca


TFA
























Figure
ethanol


Proton-decoupled


precipitate


[13C]


NMR


profile


an alkaline-soluble


fraction


sorokiniana


cell


wall




































5* I I I I I 7r r I I I I I I 7


SI I I p I p I I I p I i T


PPM


PPM











complex


portion


[13C]


NMR


spectrum


of polysaccharides


from


approximately


60-90


ppm.


Two


ma3 or


signals


at 105


and


represent


anomeric


carbons


saccharides.


signal


at 103


represents


a lower


concentration


third


saccharide.


likely


that


secondary


hydroxyl


groups


of xylitol


are


represented


maj or


signal


at 75


ppm.


Characteristics


other


than


saccharide


identification


can


be determined


using


NMR.


Hydroxy


methyl


groups


on C6


resonate


65-69


ppm.


Two


signals


at approximately


66.0


represent


hydroxy


methyl


groups.


is not


known


with


which


saccharides


they


are


associated.


Carbons


glycosidic


bonds


can


have


characteristic


chemical


shifts.


Signals


at 68


represent


residues


coupled


through


a glycosidic


,6-linkage.


signal


at 88


also


represents


a carbon


involved


in a glycosidic


linkage.


In Figure


signals


at 60


ppm


represent


carbons


from


ethanol


that


was


completely


removed


during


sample


preparation.


In other


samples


where


ethanol


was


completely


removed


(data


not


presented),


only


a small


signal


at 20


persisted.


Relatively


minor


signals


at 20


ppm


and


175-


represent


methyl


carbonyl


groups,


13C]











Like


TFA


hydrolysis


, alkaline


hydrolysis


solubilizes


matrix


polysaccharides


leaves


rigid


portion


wall


unaffected.


Because


of harsh


conditions


under


which


glucosamine


polymers


are


acid


hydrolyzed,


some


destruction


glucosamine


can


occur


during


hydrolysis.


Destruction


glucosamine


results


in an underestimation


of glucosamine


concentration.


Alkaline-insoluble


wall


residue


was


analyzed


IR and


elemental


analysis


see


how


closely


this


cell


wall


fraction


resembles


an authentic


glucosamine-containing


polymer,


chitosan.


An IR spectrum


of a chitosan


positive


control


was


identical


to a published


spectrum


of chitosan


(Muzzarelli,


1973) .


Despite


chitosan


spectrum


having


more


dominant


peaks


than


cell


wall


sample


at 1597,


1422


, and


1154


spectra


are


very


similar


represent


similar


compounds


(Figure


Because


there


are


many


types


chemical


bonds


that

types


absorb in these

of bonds causing


regions,

c the di


is not


-fferences


possible

in the IR


to identify


spectra.


Elemental


analysis


alkaline-insoluble


portion


the wall


differs


slightly


from


that


of a chito


san


positive


control


(Table


Chitosan


a C:N


ratio


as expected.


C:N ratio


cell


wall


sample


three


elements


combined


account


about


H,

























Figure 3.
remaining
cell wall


IR spectra


after


alkaline


of chitosan


extraction


residue


sorokiniana
















t118.86
XT


0'\'

x 'vi
/
/
Kr,


I
A S..
6
V
0
'A


32.35


4000 3500 3000 2500


2000 1500


1000 cm














Table


Elemental


analysis


of chitosan


and


sorokiniana


cell


wall


residues


Percentage


of Sample


Weight


Total


Sample


Chitosan
(expected)


Chitosan
(experimental)


sorokiniana


Alkaline-
extracted


wall


C. soroK
Alkaline
wall fol


iniana
-extracted
lowed by


ace


toly


S'S


aNumbers


in parentheses


represent


of C,


or N


per


of N in each


sample











wall


weights,


chitosan and


88% of


alkaline-extracted


wall


weights are accounted


as C,


and 0.


When


5 mol


of oxygen are calculated


into weight of


alkaline-extracted


wall,


97% of


the wall


dry weight


is accounted for as


and 0.


Protein and Amino Acid Analyses


Total


cell


wall


protein was


estimated


from analyses of


alkaline extracts of purified cell


walls.


Lowry protein assays were performed on


The Bradford and


extracts.


Protein


concentrations of


the alkaline extracts were compared with


those estimated from automated amino acid analysis of


an HC1


hydrolysate of


cell


wall


proteins.


Lowry and automated


analyses yielded similar cell


wall


protein


concentrations of


17%


and 17% of


the cell


wall


dry weight,


respectively.


The Bradford assay,


in which protein was


calculated


as 9


0.72% of


the cell


wall


dry weight,


had a consistent


underestimation of protein


concentration


for C.


sorokiniana


cell


wall


proteins.


Amino acid analysis


of a 6 N


extract of


the wall


revealed


that


glycine and alanine


concentrations were slightly


higher


than


that of


other amino


acids


(Table 4


Estimations


of lysine concentration were


not repeatable between


cell


wall


samples


from same or













Table 4.


Amino acid composition of


sorokiniana


cell


wall


proteins


Amino acid


mol%


.020


Asx
Thr


7.90


Leu














omitted


from


final


amino


acid


data


presented.


Hydroxyproline


was


present


in a


very


concentration


cysteine


tryptophan


concentrations


were


estimated.


Analyses


Other


Cell


Wall


Compounds


SDoroDollenin


Because


presence


an acetolysis-resistant


fraction


both


whole


sorokini ana


cells


purified


cell


walls


, the


cell


wall


was


suspected


to contain


sporopollenin.


SCa


wall


, whi


contain


sporopollenin,


also


had


acetolysis-resistant


residue


whereas


walls


species


without


sporopollenin,


ellipsoidea


sa ccharophi a


dissolved


even


before


acid


solution


was


heated


(Table


Using


TEM


, it


was


confirmed


that


portion


sorokiniana


cell


resistant


to acetol


ysis


was


wall,


or a


portion


thereof.


Only


trilaminar


sporopollenin-


containing


layer


remained


after


acetolysis


.The


. sorokiniana


wall


exhibi t


a decrease


cell wall


thickness


following


acetolysis


treatment


(Figure


sorokiniana


cells


do not


possess


an outer


trilaminar


wall


layer


seen


in algae


that


have


sporopollenin


e.g.,


fusca).


. sorokiniana


wall


was


somewhat


*












Table


Acetolysis and phosphorolysis of


purified


Chlorella


cell


walls


Cells


Conditions


% Cell


Wall


Dry Weight


Remaining


ellipsoidea


saccharophila


acetolysis


acetolysis


fusca


acetolysis


34.8


3.6


TFA hydrolysis
followed


acetolysis


14.4


14.6


alkaline extraction
followed by


phosphorolysis


5.25


0.66


sorokiniana


acetolysis


21.5


3.0


TFA hydrolysis
followed
by acetolysis


8.43


+ 5.4


alkaline extraction
followed by


acetolysis


0.71


+ 0.69


alkaline extraction

























Figure


Acetolysis


resistant


residues


sorokiniana


fusca


cell


walls.


Enlargement


X50,000.







70




















a






w
|-'















1. *x











seen


as having


a single


outer


monolayer


a thicker


inner


layer.


Despite


convincing


evidence


two-layered


wall,


single


homogeneous


wall


layer


was


observed


more


frequently,


electron


micrographs


(Figure


Cell


wall


auto fluorescence


is another


characteristic


that


indicated


presence


of sporopollenin


sorokiniana


wall.


Yellow


auto fluorescence


upon


excitation


with


blue


light


present


in both


sorokiniana


fusca


walls


present


sporopollenin-lacking


ellipsoidea


wall


(Figure


.The


acetolysis-resistant


residues


sorokiniana


fusca


are


autofluore


scent.


There


some


indication


that


autofluorescence


sorokiniana


wall


be due


to a compound


other


than


sporopollenin.


alkaline


resistant


cell


wall


residue


faint


auto fluorescence;


whereas


an ethanol


precipitate


alkaline


solubilized


portion


wall


had


stronger


autofluorescence.


Autofluorescence


was


also


observed


commercially


prepared


chitin


chitosan.


A definitive


test


sporopollenin


insolubility


when


incubated


alkali


followed


incubation


in concentrated


phosphoric


acid


(i.e.,


phosphorolysis).


Unlike


fusca


cell


wall


sorokiniana


cell


walls


are


completely


solubilized


phosphorolysis


and,


therefore,


do not


contain


sporopollenin


























Figure


soro
cell
as s
prep
dist
fusc
tril


kinian
wall
hown i
aratio
inct 1
a wall
aminar


Transmi


a and
often
n the
ns, t
ayers
has
spor


Enlargement


L

h


ssion


electron


C. fusca cel
has a sing
daughter wal
e wall appeal
as shown in I
f a thick in


ne


crographs


walls. Th
homogenec
(a). In
to be co
e mother
.r layer


ollenin-containing


Le


)us ce
some
nstru
wall
md th


orokin
1 wall
ell
ted of
a). T


inner


o


lana
layer


two
he C.
uter-


layer


X40,000.








73















:7
I. *iH~.



















a



























4. *t,..-.t

























Figur
walls
micro
micro
fusca
under
fluor


-.- ..
. Cel
scopy
scopy
under
light
escenc


Autofluorescence of pi
1 wall samples are C.
(a), C. sorokiniana u
(b), C. fusca under 1
fluorescence microsc
microscopy (e), and
e microscopy (f). En


purified Chlorella cell
sorokiniana under light
nder fluorescence
ight microscopy (c), C.
opy (d), C. ellipsoidea
C. ellipsoidea under
largement X1200.






75











a b








C d








e fZ














Phenolic


compounds


Purified


cell walls


from


ell ipsoi dea,


saccharophila,


to a qualitative


fusca,


phloroglucinol


sorokiniana


test


were


lignin.


subjected


Pine


sawdust


was


used


a positive


control.


Pine


sawdust


mixed


with


phloroglucinol


reagent


immediately


produced


a dark


magenta


color,


a positive


test


lignin.


Chlorella


walls


gave


colorless


, negative


results.


Treatment


of C.


sorokiniana


cell


walls


with


N NaOH


was


rigorous


enough


to extract


most


phenolic


compounds.


A UV


absorption


spectrum


analysis


were


performed


on a


butanol


extract


spectrum


indicating


alkaline


absorption


presence


extract.


maxima,


protein.


at 280


absorption


nm and


Absorption


peaks


representing


other


aromatic


compounds


, were


present.


NMR


spectrum


no major


peaks


that


indicated


presence


of phenolic


or related


substances


in a concentration


greater


than


approximately


absorption


spectrum)


pmol

was


mL-1.

present


Protein


(identified


at a quantity


insuffi


cient


measurement


using


:130]


NMR.


An infrared


spectrum


of purified


cell


walls


was


examined


presence


of phenolic


compounds


(Figure


Skeletal

























Figure
walls.


IR spectra


Alkaline


of purified


resistant


cell


wall


sorokiniana


residue


cell


total


cell


wall


(b)























i~5KN


wEUE


117.00-
IT






















48.60


/


4000 3500 3000 2500 2000 1500 1000 c-I











aromatic


compounds.


Aromatics


also


have strong absorption


from 900


to 650


Since


the C.


sorokini ana


wall


was


void of absorption peaks


did not provide evidence


this region,


phenolic compou


an IR spectrum

nds in the cell


wall.


Acetolvsis-resistant portion of


sorokiniana


cell


wall


Polysaccharides of


the C.


sorokiniana wall


contain a


relatively high concentration of


rhamnose.


Glycosidic bonds


involving rhamnose molecules are sensitive


to TFA hydrolysis,


but are


fairly


resistant


to acetolysis.


To determine if


TFA-hydrolyzable


cell wall


fraction contributes


to cell


wall


resistance


to acetolysis,


TFA hydrolysis of


the cell


wall


was


performed prior to acetolysis.


TFA hydrolysis


of C.


sorokiniana


cell


walls prior


ace


tolysis decreased


amount of acetolysis


resistant wall


fraction


from 22%


acetolysis alone


8.4%


for TFA hydrolysis plus acetolysis.


Alkaline extraction of


the wall,


which removed many


polysaccharides,


proteins


, and possibly other


compounds


prior


to acetolysis,


decreased


the acetolysis-resistant residue


less


than


1% of


the cell


wall


dry weight


(Table 5).


To determine whether chitin and chitosan


are


solubilized


by acetolysis,


acetolysis of


these compounds


was performed.


Resistance of


these polymers


to acetolysis would leave open a











acetolysis


solution,


was


solubilized


during


subsequent


sodium


acetate


wash


step.


an attempt


acetolysis


elemental


gain


resistant


analysis


were


additional


residue,

performed


evidence


an infrared


as to identity


spectrum


residue.


An IR


spectrum


an acetolyzed


cell


wall


resembled


that


alkaline


resistant


wall,


with


some


differences


in peak


location


intensity


(Figure


increase


absorbance


around


1720


indicated


presence


carbonyl


groups


formed


acetolysis.


Other


changes


could


not be characterize

Elemental analysis


to complexity


of acetolysis


resistant


spectrum.


residue


showed


tha t


a higher


C:N


ratio


than


either


chitosan


alkaline

molecules


resistant


oxygen


wall

i are


residue


(Table


one


calculated


or two


nitrogen,


wall,


respectively,


accounted


for.


Effect


Hvdrolvtic


Enzymes


on the


b(JZVAJALaII8


Cell


Wall


Effect


Enzvmes


on Whole


Cells


Protoplasts


were


produced


polysaccharide-


degrading


enzymes


either


alone


in combinations.


When


cells


were


treated


with


CHP,


osmotic


ability


of cells


was
























Figure 8.
extracted
cell wall
(b).


IR spectra of the
cell wall residue
residue after subs


C. sorokiniana
(a) and alkaline
equent acetolysis


alkaline
extracted
treatment






82





r--- IN BIWn
a
120.95 --
IT
V / \
1 /
b> / l <

IS'H (I A J
1y1 rx,







39.40- .-.


4000 3500 3000


2500


2000 1500


1000











about


5-10%


of cells


ruptured


cell


contents


leaked


partially


or entirely


out


one


spot


cell


wall.


cell wall,


void


contents


, was


still


sible


light


microscopy.


These


cell


had


no apparent


change


in cell


wall


morphology.


However,


there


appeared


to be


an increase


internal


disorganization


cells


such


as cytoplasmic


thylakoid


membrane


disruption.


Therefore


, it


likely


that


these


cells


were


displaying


signs


of cell


death


rather


than


enzymatic


cell


wall


degradation


(Figure


From


attempts


to make


protoplasts


treatment


of cells


with


a mixture


of polysaccharide-degrading


enzymes,


cell


homogenate,

obtained.


a protease,


Partially


inconsistent


synchronized


cells


results were

(harvested 3


h and


into


cell


cycle)


were


treated


with


chi tosanase


, cell


wall


homogenate,


pronase.


After


h of


enzyme


treatment


buffer,


a low


percentage


cells


(<5%)


lysed


water.


Unlike


cells


treated


with


carbohydrate-degrading


enzymes


that


leaked


out


of a single


spot


in an otherwise


intact


cell


wall,


a cell


wall


was


apparent


light


microscopy)


these


cells


lysed


in water.


When


cells


were


harvested


h into


cell


cycle


treated


with


cell


homogenate,


CHP,


chitosanase


, and


protease,


approxima t


of cells


lysed


they


were


diluted


with


dH20


on a slide.


mmn


























Figure


TEM


treated


(al,


TEM


SEM


of C.


16 h with


of enzyme-treated


sorokiniana


CiP.


TEM


cells


cells


untreate


SEM


untreated
d cells


untreated


cells


, and


of enzyme-treated


cells


Enlargement


X12,500







85








SI.






a b



C'
1 ..






4|


m




a













sodium phosphate buffers.


Cell


lysis was


still


obtained when


either homogenate or protease was omitted from the enzyme


solution.


These results represent data


from


three


experiments performed on


the same starting


culture of


daughter


cells.


However,


in six subsequent


experiments


(one


in which


the same starting


culture was used as


in the


first


three experiments)


success


cell


lysis,


and possible


protoplast


Effect of


formation was not


Enzvmes


repeated.


on Purified Walls


Although walls of


intact


sorokiniana cells were


resistant


to enzymatic


degradation,


purified cell


walls were


also


tested for their


for several


wall


reasons:


degradation


sensitivity to enzymatic degradation


a quantitative assessment of cell


could be made on purified walls without a


possibility of


cell metabolites


interfering with assay


results,


(ii)


induced metabolic responses,


such as wall


protein cross-linking and


production of


compounds


decrease cell


wall


degrading enzyme activities could be ruled


out


as a sole


reason


lack of


cell


wall


enzymatic


hydrolysis,


(iii)


could be determined whether or not


an outer


resistant


cell


wall


layer protected an inner,











sufficient


to interfere


with


phenol


sulfuric


acid


assay


Soluble


sugars


were


removed


(90-100%


decrease)


ultrafiltration.


Reducing-sugar


-free


enzyme


preparations


retained


activity


as demonstrated


their


ability


to produce


protopl


asts


from


eli ipsoi dea


cell


ellipsoidea


walls


, treated


with


, were


used


as a


positive


wall


control.


as carbohydrate


Combined

e (as me


enzymes


assured


released


about


phenol


sulfuric


the

acid


assay)


, and


each


enzyme


alone


eased


slightly


less


When


sorokiniana


wall


were


treated


with


CHP


, approximately


wall


was


released


as carbohydrate.


This


carbohydrate


was


equivalent


amount


of carbohydrate


released


from


walls


treated


with


pectinase


alone


Cellulase


and


hemi


cellulase


each


eased


slightly


ess


carbohydrate


from


purified


wall


s (Table


phenol


sulfuric


acid


assay


does


measure


amino


sugars;


Elson-Morgan


assay


does


measure


amino


sugars


, but


insensitive


this


application.


Therefore,


neocuproine


assay


reducing


sugars


was


used


measure


amount


of reducing


sugar


released


treatment


of cell


wall


with


chitosanase.


Chitosanase


, purified


from


i vidans


pRL207


, degraded


soluble


chitosan


more


readily


and


r


more er


ficiently


than













Table


Percent


degradation of purified cell


walls


subj


ected


to enzymatic digestion


Substrate


Enzyme


Percent Dry Weight of
Substrate Releaseda


ellipsoidea


walls


CHPb


28 +


cellulase


hemicellulase


pectinase


19 1.4


18


sorokini ana


walls


CHP


S3.9


cellulase


hemicellulase


pectinase


a 0.35


+ 0.070


+ 0.28


chitosanase


0.65c


+ chitosanase


Phenol-sulfuric


acid assay


for total


carbohydrate was


ica vinl ncc


enP r\ f; Pa