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The Improvement of Quantitative Detection of γ-PGA by Hydrolysis and the Research of Biosynthetic γ-PGA’s Molecular Weight Distribution and YWTD

Author: WuJianGuang
Tutor: ZhongWeiHong
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: poly-γ-gutamic acid Bacillus subtilis SDS-PAGE ywtD
CLC: TQ920.1
Type: Master's thesis
Year: 2011
Downloads: 12
Quote: 0
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Abstract


Poly (γ-glutamic acid) (γ-PGA) is an unusual anionic, water-solubleand high viscous polymer in which glutamate is polymerized by amidelinkages between a-amino andγ-carboxylic acid groups. For its excellentcharacters, potential applications ofγ-PGA have been of interest in the pastfew years in a broad range of industrial ?elds. It has been used asthickeners, humectant and drug carrier.Bacillus subtilis ZJUTZY, which was aγ-PGA producer, was isolatedfrom natto. And the optimum condition for producingγ-PGA was studiedthrough with immobilized Bacillus subtilis ZJUTZY. The betterimmobilizing material was calcium-alginate and the optimum conditionwas as follows: temperature 30℃, initial pH7, rotation speed 200 rpm,inoculum size 12%，beads to liquid 1 : 3, and CaCl2 concentration 4%.We improved the method of mearing the yield ofγ-PGA by two steps.One is adding a meaturement of the concentration of glutamic acid in thedeposit and the other is the confirmation of the dosage of NaOH in the step of neutralization. And in this study, it shows SBA is more convenientthan HPLC in detecting theγ-PGA’s yield. Then on the based, we detectedthe influence of Bacillus subtilis ZJUTZY toγ-PGA’s degradation. Anddetected by SDS-PAGE, we also found that the different carbon sourcescould do affect the Molecular weight distribution ofγ-PGA.Two pairs of degenerate primers were designed to be used to amplifythe partial fragment of ywtD from Bacillus subtilis ZJUTZY according tothe conservative regions of ywtD and the sequence of ywtD from Bacillussubtilis ZJUZY. A DNA band of 1370bp was obtained when the latter onewas used. The DNA fragment had 99% identity with the ywtD from manyBacillus subtilis. Based on these results, it was confirmed that partial ofywtD gene was successfully cloned from the Bacillus subtilis ZJUTZY.Repeat the operation, it was also confirmed that partial of ywtD gene wassuccessfully cloned from the Bacillus subtilis ZJUTZY.

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