| Alkaline amylases can hydrolyze starch efficiently by cleaving α-1,4-glucosidic and keep stable under alkaline conditions(p H 9.0-11.0), they have been widely used in the textile, detergents and papermaking industries and have a great market demand. As low enzyme production by wide type strain, constructing recombinant strain which can produce high-level enzyme becomes a hot research at home and abroad. The current recombinant strains such as Escherichia coli and Pichia pastoris exist a series of questions, like low production, secretion barrier or needs induction, spends a long period. In this study, alkaline amylase from Alkalimonas amylolytica N10(Amy K) was expressed in Bacillus subtilis, a recombinant strain with high-level Amy K production was achieved through screening of signal peptides and Atmospheric and Room Temperature Plasma(ARTP) mutagenesis. After optimization of fermentation medium and conditions, high-level Amy K production was achieved, which laid a solid foundation for industrial production of Amy K.The main research results are as follows:(1) Expression of A. amylolytica Amy K in B. subtilis WB600The Amy K gene from A. amylolytica N10(1674 bp, without signal peptide) was cloned and subsequently ligated into nine p MA0911 derivatives with different signal peptides. Recombinant plasmids constructed were transformed into the expression host strain B. subtilis WB600. The recombinant expression rusult shows that recombinant strain with signal peptide Vpr produces the lowest extracellular amylase activity, which was only 1.72 U·m L-1. With Ywb N directing, the highest extracellular Amy K activity of recombinant strain WB11M1 reached 146.86 U·m L-1, with a 95% secretion efficiency, which achieved high-level Amy K secretory expression in B. subtilis firstly.(2) Increasing enzyme production of WB11M1 by ARTP mutagenesisTo improve the Amy K production further, ARTP mutagenesis was conducted with WB11M1 as the original strain. Based on lethal rate, the mutagenesis time was defined as 30 s. Screening the positive mutants using high-throughput method, a mutant named A11 with amylase activity increased by 24% was finally achieved. However, through analysis of genetic stability, amylase activity of A11 decreased to the level of the original strain after the fourth generation, so the high production characteristic of A11 could not inherit stably.(3) Fermentation optimization of WB11M1 in flasksThrough single factor test, fermentation medium was optimized from aspects of initial fermentation medium, carbon source, nitrogen source, Na2CO3 addition and inoculum size, the optimal medium contains(g·L-1):dextrin 32, tryptone 12, yeast extract 24, KH2PO4 2.32,K2HPO4·3H2O 16.43, 0.2% Na2CO3(w·v-1) was added after 24 h fermentation to regulate the p H value of the medium. Fermentation conditions were as follows: 37°C,200 r·min-1, 12% inoculum size, initial p H 7.0, a period of 72 h. Under the optimized medium and conditions, the highest extracellular Amy K activity reached 640.33 U·m L-1 with an increase of 3.36 times compared with unoptimized medium and conditions.(4) Fermentation optimization of WB11M1 in 3 L fermenterPreliminary exploration of fermentation conditions and feeding strategy of recombinant WB11M1 in a 3 L fermenter were proceeded. Firstly, dissolved oxygen and p H were optimized, and a two-stage p H control strategy was raised in which p H was controlled at 7.0 in the first 9 hours and 8.0 in the remaining time. Then the influences of feeding dextrin, mixture of dextrin and tryptone on recombinant strain growth and amylase production were investigated. Finally, the highest extracellular amylase activity reached 809.03 U·m L-1 with an increase of 3.32 times compared with the batch fermentation. |
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