为提高纳豆激酶的表达量,在不改变编码纳豆激酶的氨基酸序列前提下,将aprN基因的N端编码序列(N-terminal coding sequences,NCSs)进行同义突变,构建包含突变序列的pHY-P43质粒,经转化和测序分别构建4株突变菌株NK1、NK2、NK3和NK4,纤维蛋白板检测纳豆激酶活力,利用SDS-PAGE验证,通过响应面优化发酵条件。结果表明,NK1、NK2、NK3、NK4菌株纳豆激酶活力分别为94.8、101.9、124.2、69.4 IU/mL。其中NK3菌株的酶活力最高,较原始菌株提高了50%;NK4菌株酶活力最低仅为原始菌株酶活力的83%。SDS-PAGE显示重组酶生产纳豆激酶的分子质量约为28 kDa,与理论的分子质量27 kDa相符。通过响应面分析得出优化后重组菌株NK3产纳豆激酶的最佳条件为:初始pH 7.56、发酵温度35 ℃、发酵时间38 h。纳豆激酶酶活力138.1 IU/mL,较未优化前提高11.2%。该研究为提高纳豆激酶活力提供一种新的策略,且不改变纳豆激酶本身的性质。
In order to improve the expression level of nattokinase, the N-terminal coding sequences (NCSs) of the aprN were synonymously mutated without changing the amino acid sequence encoding nattokinase.Four mutant strains, NK1, NK2, NK3 and NK4, were constructed by transforming recombinant pHY-P43 plasmid which containing the mutated sequences into Bacillus subtilis.The enzyme activity of nattokinase was detected by fibrin plate.The expression of recombinant protein was verified by SDS-PAGE.The fermentation conditions of mutant strains were optimized by response surface.The results showed that the nattokinase activity of NK1, NK2, NK3 and NK4 strains were 94.8 IU/mL, 101.9 IU/mL, 124.2 IU/mL and 69.4 IU/mL, respectively.The NK3 strain had the highest enzyme activity, which was 50% higher than that of the original strain.The NK3 strain had the lowest enzyme activity, which was 83% of that of the original strain.The results of SDS-PAGE showed that the molecular weight of recombinant nattokinase was about 28 kDa, which was consistent with the theoretical molecular weight of 27 kDa.Response surface analysis showed that initial pH 7.56, fermentation temperature 35 ℃, and fermentation time 38 h were the optimal conditions for production of nattokinase in NK3 strain.The enzyme activity of nattokinase was 138.1 IU/mL, which was 11.2% higher than before the optimization.This study provides a new strategy to enhance the activity of nattokinase without altering the properties of nattokinase itself.
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