Improving The Yield Of Recombinant Alkaline Amylase In Bacillus Subtilis By ARTP Mutagenesis, Directed Modification Of Expression Elements And Fermentation Optimization

Alkaline amylase is an important industrial enzyme characterized by its catalytic efficiency and stability under alkaline conditions. The enzyme has been widely applied in the textile, paper, and detergent industries, as well as in many other fields, and possesses excellent market prospects. Current production strategies for alkaline amylase mainly focus on approaches such as screening wild-type alkaliphilic microorganisms, mutation breeding of the dominant strains, and heterologous expression of the alkaline amylase gene. Bacillus subtilis is a gram-positive species with a well-defined genetic background. Furthermore, it is easy to be cultured and can secrete large quantities of desired proteins. Therefore, it serves as an efficient well-characterized expression host for industrial enzymes. In this study, atmospheric and room temperature plasma(ARTP) mutagenesis and high throughput screening were used to create and isolate recombinant B. subtilis strains that could serve as more efficient hosts for alkaline amylase production. To improve alkaline amylase yield, the selected mutant strain underwent fermentation optimization. In addition, the signal peptide and promoter were selected and directionally modified, and alkaline amylase production was improved eventually based on optimized conditions. More analytically, the research steps undertaken were the following:(1) The B. subtilis WB600 strain, regarded as the wild-type strain is this study, was subjected to ARTP mutagenesis. A library of mutated hosts was created, and the mutants were screened for their efficiency in producing recombinant alkaline amylase. A genetically stable, alkaline amylase overexpressing mutant, displaying 35.0% higher alkaline amylase activity than the wild type, was selected and was named WB600 M.(2) After further host screening, B. subtilis 168 was identified as the most suitable expression host for the plasmid encoding recombinant alkaline amylase and was used as the initial strain in a directed mutagenesis screen. The mutant B. subtilis 168 mut-16# was quickly acquired, which provided a 31.4% higher alkaline amylase yield than the initial strain.(3) Using a single factor experiment method and an orthogonal test, the culture conditions and medium were optimized for B. subtilis 168 mut-16#. The alkaline amylase production level reached 369.3 U·m L-1 after optimization, that is, 128.8% higher than production levels under non-optimized conditions. For production in 3 L fermentors, the stirring speed, initial medium concentration, and feeding strategy were optimized separately. The highest alkaline amylase activity achieved was 591.5 U·m L-1 at the following fermentation conditions: speed, 550 r·min-1; double the initial concentration of carbon and nitrogen sources; feeding with liquefied starch at 2.0% final concentration after 10 h.(4) An optimal signal peptide, YwbN(M1), was determined. The improved signal peptide, M11, was created by deleting a 57-bp nucleotide fragment from the sequence of the twinarginine signal peptide, M1. The alkaline amylase activity achieved reached 538.1 U·mL-1, which was 1.5 times higher than the level achieved using the unmodified YwbN sequence. Considering the data regarding the modified signal peptide M11, the YwbN sequence was edited to increase the number of positively charged amino acid residues in the N-region of YwbN. This change increased alkaline amylase yield to 624.3 U·m L-1. An even higher level, up to 983.8 U·mL-1, was reached when the nonpolar amino acids I, L, and W of the H domain were replaced with the polar amino acids N, S, and S.(5) Single PHpaII and P43 promoters, as well as dual PHpa II-PHpa II and P43-PHpaII promoters, were compared with respect to expression of the gene encoding alkaline amylase(amyK), and PHpaII proved the most suitable promoter. In addition, the effect of modifying the-10 to-35 region of the PHpaII promoter was examined. After deleting the deoxythymidine at position-27, alkaline amylase production reached 1213.1 U·mL-1(i.e., 23.3% increase compared to the unmodified promoter), while deleting the deoxyadenosine at position-31 led to a production level of 1131.2 U·mL-1(i.e., 15.0% increase). It was confirmed that the increases in alkaline amylase production were attributable to elevated mRNA levels by semi-quantitative RT-PCR. Under the optimized fermentation conditions, the alkaline amylase yield of mutant B. subtilis PM2 reached a maximum of 1361.0 U·m L-1.