Subsidiary Hydrolases In Industrial Glucoamylase Producer

Glucoamylase is one of the important industrial enzymes. It has been widely applied in fermentation industry that need glycosylated starch as a raw material. However, when microorganism produces glucoamylase, a kind of hydrolase—α-glucosidase is produced as a by-product, which would decrease glucoamylase efficiency in fermentation process. Theα-glucosidase can catalyze gulcose from the non-reducing ends of oligosaccharides or transfer the glucose residue to another carbohydrate substrate to formα-1, 6 glycosidic bonds which belongs to the oligosaccharides of non-fermentation. Therefore, theα-glucosidase is paid more attention by many researchers.CICIM F0410 is an industrial producer of glucoamylase. The time course of the enzyme production and the properties were investigated in this study. The maximum glucosidase activity was obtained in 96 h of the fermentation, at 55℃and pH 5.0. Furthermore, 0.5 mM Mg2+ promoted the enzyme activity and increased the heat stability of glucosidase.The optimization of medium and fermentation conditions for F0410 was conducted. We tried to decrease the relativeα-glucosidase activity per 1000 U glucoamylase as much as possible Get the optimal fermentation medium (g/L): glucose 109, soybean powder 25 (add a final concentration of 0.01 M of NH4Cl), corn steep liquor 31; The optimal fermentation conditions were 50 mL broth with initial pH 5.5 in 250 mL flask, inoculated 96 h at 34℃on rotary shaker, the rotational speed was 220 r/min, inoculum concentration was 9%, fermentation period was 144 h. Under the optimized condition, the content ofα-glucosidase per 1000U glucoamylase decreased to 51.36%.A 3124 bp nucleic acid fragment containingα-glucosidase gene was amplified by PCR. The coding sequences were 2973 bp, containing three introns. The deduced protein was 990 amino acids containing a signal peptide consisting of 57 amino acid residues. Sequence alignment with theα-glucosidase sequence of another A. niger.(GenBank accession number XP₀01402053) showed that the two nucleotide sequences' similarity reached to 99.94%. Meanwhile, a recombinant plasmid pMD19-aglU::G418 was constructed which would be used to knockout theα-glucosidase gene in the further study.