Biosynthesis of Deoxynojirimycin in Bacteria

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Material Information

Title:
Biosynthesis of Deoxynojirimycin in Bacteria
Physical Description:
1 online resource (84 p.)
Language:
english
Creator:
Wu, Yanbin
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
HORENSTEIN,NICOLE ALANA
Committee Co-Chair:
SMITH,BEN W
Committee Members:
BRUNER,STEVEN DOUGLAS

Subjects

Subjects / Keywords:
biosynthesis -- deoxynojirimycin -- gutb1 -- yktc1
Chemistry -- Dissertations, Academic -- UF
Genre:
Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Certain species of soil bacteria, such as Bacillus amyloliquefaciens and Bacillus atrophaeus, produce the alpha-glycosidase inhibitor1-deoxynojirimycin (DNJ).  A cluster of three genes, gabT1, yktC1, and gutB1, were identified as part of the DNJ biosynthetic pathway in previous work in the Horenstein group. In this thesis, I present study and characterization of YktC1 and GutB1 enzymes. The first project focused on characterization of GutB1enzyme. The genes were synthesized and codon-optimized, incorporated into a T7vector to be expressed in E.coli,followed by purification of the enzyme and kinetic characterization. Kinetic parameters were also obtained for GutB1 from both P.polymyxa and B.amyloliquefaciens using synthetic 2-amino-2-deoxy-mannitol (2AM) as the substrate. The GutB1enzyme from P.polymyxa had higher activity (kcat = 4.1 min-1, Km for 2AM was1.7 ± 0.1 mM, Km for NAD+ was 1.1±0.1 mM), the turnover is approximately 60-fold higher than the B.amyloliquefaciensenzyme (kcat = 0.07 min-1, the Km for 2AMwas 5.5 ± 1.6 mM, the Km for NAD+ was 105 ± 37 µ?). The P.polymyxa enzyme also accepts aminoalcohols such as 1-amino-4-butanol and 1-amino-6-hexanol as alternate substrates. The second project focused on crystallization of GutB1using GutB1 from different species for initial crystallization screening.N-terminal histidine tag enzymes and C-terminal his-tag enzymes with very short tags were constructed, overexpressed and purified for crystallization screening. Current studies of the crystallization of GutB1 enzymes studies have not yet led to successful crystallization conditions. The third project addressed the characterization of Yktc1enzyme. This gene was cloned from B.amyloliquefaciens,and incorporated into pETBlue2 vector for expressing in E.coli, followed by purification of the enzyme. Initial kinetic assays were performed confirming that YktC1 is a phosphatase with activity against fructose-6-phosphate as a substrate analogue for the putative substrate, 6-phospho-2-deoxy-2-aminomannitol. The fourth project involved studies of two putative genes (ppsc2_c2587and ppsc2_c2588) from P.polymyxa that may be part of the biosynthesis pathway from mannojirimycin to deoxynojirimycin.Annotations of these two genes were suggested that they had reductase and epimerase activity, respectively. They were codon-optimized and cloned into the pET30aexpression vector. For initial results, epimerase was successfully overexpressed and purified.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Yanbin Wu.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: HORENSTEIN,NICOLE ALANA.
Local:
Co-adviser: SMITH,BEN W.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046065:00001