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Genetic Improvement of Glucoamylase Industrial Producer

Author: YaoZuoZuo
Tutor: WangZhengXiang
School: Jiangnan University
Course: Biochemistry and Molecular Biology
Keywords: Aspergillus niger Glucoamylase Clone Chromosomal integration Flask fermentation
CLC: Q933
Type: Master's thesis
Year: 2006
Downloads: 429
Quote: 3
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Abstract

Glucoamylase, partial glucoamylase (glucoamylase EC.3.2.1.3), also known as amyloglucosidase, function is the release of β-D-glucose from the non-reducing end of the carbohydrate of the starch, dextrin or glycogen. Glucoamylase hydrolysis of starch to glucose enzymes, and therefore is widely used in food, pharmaceutical, fermentation industry, with a high commercial value, is one of the China's largest production enzyme product. Currently, the industry is widely niger, A. awamori, etc. to extract the glucoamylase for the production of strains. Firstly conservative family including ITS1 partial sequence of ITS2 and 5.8S rDNA complete sequence by PCR amplified from the glucoamylase industrial production strains F0410 chromosome sequencing and alignment analysis confirmed that the strain of Aspergillus niger. Published in the GenBank, Aspergillus niger glucoamylase gene primers were designed, obtained by PCR amplification of the glucoamylase gene of Aspergillus niger F0410, amplification product was 3327 bp, wherein the glucoamylase encoding gene is 2167 bp, inferring the amino acid sequence 640 amino acid residues. PCR amplified the glaA gene fragments purified by the kit and inserted into the vector pBC-Hygro the Sma Ⅰ sites, construct the recombinant plasmid pBC-Hygro-glaA. Construction of Aspergillus niger F0410 of PEG-protoplast transformation system. Protoplast transformation with the recombinant plasmids pBC-Hygro-glaA. Several transformed hygromycin B resistance plate 123 transformants were obtained, the enzyme activity increased by 6 transformants pick flask fermentation and glucoamylase gene copy number. Glucoamylase gene in the chromosomal integration 2-3 times the excess synthesis of glucoamylase appropriate the glucoamylase vitality than the original strain increased from 12.7% to 23%, when the glucoamylase gene integrated in the chromosome copy number of the original strain 8 times and glucoamylase activity increased only 6%. Screening and rescreening, ultimately, the enzyme activity was significantly increased conversion sub-GB0506 for further research. Initially identified a recombinant strain GB0506 suitable for medium and fermentation conditions, the optimal fermentation condition of 30 g / L yeast extract, 20 g / L, cottonseed flour, 2 g / L ammonium nitrate and 80 g / L of industrial glucose medium pH 5.5, inoculum size 8%. The flask fermentation under optimized conditions the recombinant bacteria GB0506 and the original strain F0410 throughout the fermentation process, the GB0506 glucoamylase activity stable than F0410 end of the fermentation at 144 h, the transformants GB0506 glucoamylase activity than the original strain. F0410 increased 17.5%.

Genetic Improvement of Glucoamylase Industrial Producer

Abstract

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