| Lactase, also referred to as β-D-galactosidase, hydrolyzes the disaccharide lactose to glucose and galactose. Lactase can be used for the treatment of "lactose intolerance" and production of low-lactose milk, whey, oligomeric galactose and other dairy products. Although lactase has important applications in the fields of medicine, dairy industry, some problems obstruct large-scale industrial production of lactase. The main problems are as follows:(i) low production and enzymatic activity of lactase;(ii) most commercial lactases are intracellular enzyme, which results to the high cost due to the extraction and purification processes;(ⅲ) most commercial lactases are derived from yeasts, which have low thermostability and narrow pH range.In this study, the lactase gene from Aspergillus oryzae was inserted into pGAPZaA and transformed into Pichia pastoris in order to achieve efficient secretion and high level expression. The lactase gene (length of3kb) from A. oryzae was cloned by PCR using the cDNA library as template. Then the codon usage of the mature peptide coding sequence was analyzed using the DNAMAN software, and the rare codons were replaced according to P. pastoris preference without any amino acid change. The optimized sequence was inserted into plasmid pGAPZaA digested by XhoI and XbaI, and transformed in P. pastoris expression system by electrotransformation. The recombinants yeast transformants were screened by color changes and enzyme activity analysis, then the GalC131strain with highest lactase expression level was selected. After fermentation in YPD medium, high expression level of secreted lactase protein was obtained in the culture medium without methanol induction. It was easy to be purified, which would be beneficial to industrial application.In this study, based on industrialization, the fermentation medium was optimized as follows:glucose45g/L, glycerol15g/L, tryptone20g/L, yeast extract20g/L, MgSO4·7H2O9g/L, FeSO4·7H2O60mg/L, CuSO4·5H2O6mg/L, K2HPO44g/L, CaSO40.93g/L, K2SO418.2g/L. The expression conditions were also optimized as pH7,30℃, and300r/min with inoculum concentration6%. After fermentation for72 h using the conditions above, the enzymatic activity reached672U/mL, which increased by30.7%than before. The feeding carbon speed (15mL/h-L after16h) could also increase the enzymatic activity. Finally, after fermentation for96h in a50L fermentor, the recombinant enzyme in the culture supernatant had an activity of4312U/mL.The enzymatic character study of the recombinant lactase showed that the optimum pH was pH5.2, the optimum temperature was60℃, and the enzyme had good pH stability in the pH range from4.6to8.0. The recombinant lactase had better enzymatic characteristics than that of wild-type A.oryzae lactase including the thermostability, and metal ion stability.To study the application of the recombinant lactase in the production of low-lactose milk, the hydrolysis rate of lactose was detected by HPLC under different conditions. The lactase was added into milk with different enzyme/milk ratio and treated at10℃\25℃and37℃using water bath. The hydrolysis rate had a good performance at low temperatures, reached a value which was more than83%with the enzyme/milk ratio of0.9%at10℃, more than93%with the enzyme/milk ratio of0.9%at25℃, and more than94%with the enzyme/milk ratio of0.3%at37℃.In conclusion, we exploited a new lactase with high enzymatic activity, easy purification, good enzymatic characteristics and safe production process. It was suitable for the production of low-lactose milk at low temperatures and has broad application prospects in the industry. |
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