Genetic Improvement Of Glucoamylase Industrial Producer

Glucoamylase, also known as amyloglucosidase, is an extremely important enzyme for starch hydrolysis processes. It catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and poly-saccharide molecules. At present, Aspergillus niger and Aspergillus awamori are commonly used to produce glucoamylase in the industry.Fristly, the conserved sequence including partial ITS1, ITS2 and entire 5.8S rDNA was amplified from the chromosomal DNA of F0410, a glucoamylase industrial producer, and sequenced. The strain was confermed as A. niger by the BLAST analysis of network service. A set of primer was designed according to the public glucoamylase gene in GenBank. A 3.3 kb fragment containing complete glaA was amplified from A. niger F0410 chromosomal DNA by PCR. Sequencing the PCR product showed that the nucleotide sequence of glaA was 2167 bp located in the middle of the amplified 3327 bp DNA fragment. The deduced protein was 640 amino acids. The PCR product was purified and subcloned into the Sma I site of pBC-Hygro, creating pBC-Hygro-glaA.The recombinant plasmid was transformed into A. niger F0410 by PEG-mediated protoplast transformation which was constructed in the research. One hundred and twenty three transformants were obtained on the selective plates containing hygromycin B. Six of the transformants were subjected to characterize extensively the glucoamylase production in shake flask fermentations and glaA copy numbers. The results revealed an optimal integrated copy number of glaA for the maximum yield of glucoamylase. It was found that 2-3 multiples of glaA were optimal for the highest expression of glucoamylase. The transformants carrying more than 8 multiples of glaA were found to have slightly elevated glucoamylase activity. After several times of screening, transformant GB0506 was selected for the further research.Several culture media were tested at different conditions to optimize glucoamylase production with recombinant A. niger GB0506. The optimal shake-flask culture turned out to be 30 g/L yeast extract, 20 g/L cottonseed, 2 g/L ammonium nitrate and 80 g/L glucose at initial pH 5.5 with 8% inoculation. Time course of glucoamylase production with A. niger GB0506 was carried out in shake-flask fermentation at optimal conditions. The glucoamylase yield with the transformant GB0506 was steadily higher than parental strain F0410 during the process. The enzyme secreted by transformant GB0506 was 17.5% higher than parental strain F0410 at the end of fermentation, which was...