Enhancing Expression Of ?-Amylase In Pichia Pastoris By Combined Strategies

Amylase is a widely used industrial enzyme, and studies on amylase capable of hydrolyzing raw starch is hot in recent years. This work aims at expression promotion of an a-amylase Gs4j-amyA from deep sea Geobacillus sp 4j that hydrolyzes raw starch ?-amylase by Pichia pastoris. Our previous study proved that the yield of Gs4j-amyA was low when expressed in E. coli and its host strain, thus P. pastoris was used in this study, and a series of strategies were utilized to promote the production.Firstly, amyA gene from the deep-sea thermaphile Geobacillus sp.4j was optimized for expression under the control of VAOXI in P. pastoris. The codon adaption index (CAI) was increased from 0.63 to 0.91 and the GC content was adjusted from 52% to 42%. Then we analysed the minimus mRNA free energy, which increased from-552 kcal/mol to -408.6 kcal/mol. The enzyme activity reached 141 U/ml after 1% methanol induction for 120 h, which was enhanced by 40% by codon optimization.Furthermore, we set up a series of standard curves to determine the gene dosage inserted into the P. pastoris genome. Based on the optimized gene opt-amyA, we screened strains with different copies of opt-amyA and the effect of gene dosage was investigated by increasing the copy number of opt-amyA, which resulted in approximately 2.2 fold enhancement of amylase production by the strain with 12 copies of opt-amyA. In 5 L bioreactor, the enzyme activity reached 1.8 × 103 U/ml,5.1 folds higher than that in shake flaks; and the productivity reached 3.3 X 104 U/(L·h),13.2 fold higher than that in shake flask.By co-expressing the spliced form of HAC1 encoding the unfolded protein response activator HAC1p, the expression of amylase was further enhanced by 6.2 fold by the strain with 6 copies of HAC1 under the promter VAOXI In 5 L bioreactor, the protein activity reached 1.4 ×104 U/ml after 56 h methanol induction. The enzyme productivity in 5 L bioreactor reached 2.7 × 105 U/(L·h),13.8 fold higher than that in flask culture. Then we found that the promoter titration effect decresed the activity in strains with more copies of HAC1 under the same PAOXI. We overexpressed the HAClp under PGAp to avoid the promoter titration effect, and the enzyme acitivity finally reached 3.7×103 U/ml, which further increased protein activity by 68% than strains expressing HAC1p under PAOXI only.After Ni-NTA His purification of the protein, the optimum temperature of the purified enzyme became 75?, different from 60?65? in E. coli. By analyzing the amino acid sequence of the protein, it was found that protein epxressed in P. pastoris was glycosylated, which increased the molecular weight by of 8?10 kDa. Glycosylation in this protein enhanced thermal stability under higher temperature.