纳豆激酶第36位氨基酸突变对其活性及热稳定性的影响

Effect of the 36th amino acid site mutagenesis on the activity and thermostability of nattokinase

为了解纳豆激酶分子结构与其功能的关系,研究了纳豆菌诱变株DU115的纳豆激酶基因突变对其酶活性和热稳定性的影响。从原生质体紫外诱变纳豆菌DU115和野生纳豆菌BN10基因组DNA中扩增纳豆激酶成熟肽基因nkD和nkB,构建重组表达载体pPICZα-A-nkD和pPICZα-A-nkB,在毕赤酵母X-33中实现了表达,并对表达产物进行分离纯化、酶活性测定及热稳定性检测。结果表明,与nkB基因序列相比,nkD基因有两处的核苷酸发生了突变(A107G和C396T),A107G碱基突变导致氨基酸的替代D36G(Asp→Gly);诱变菌株纳豆激酶DNK与野生菌的BNK在65℃处理15 min后,前者的热稳定性提高20%,比活力提高16.6%。由此可推断,第36位氨基酸残基的突变可能与其酶热稳定性及活性提高密切相关。

The effect of mutational nattokinase （NK） gene on the activity and thermostability in Bacillus natto mutant induced mutagenesis by UV on protoplasts was researched to reveal the relation between their molecular structure and function. The mature peptide gene of NK was obtained by PCR amplification from Bacillus natto mutant strain DU115 which was protoplast mutagenesis with UV and wild-type strain BN10 genomic DNA, and sub-cloned into pPICZα-A. The recombinants pPICZα-A-nkD and pPICZα-A- nkB were expressed in Pichia pastoris X-33. The results showed that sequencing of the amplified fragment revealed a synonymous mutation C396T and a nonsynonymous mutation A107G leading to amino-acid substitution D36G （Asp→Gly）. The comparison experiments of purified mutant enzymes DNK with natural BNK showed that the thermostability of the former increased by 20% , when it was exposed for 15 min at 65 ℃ and specific activity increased by 16. 6%. The mutation of the 36th amino acid of nattokinase was positive for the thermostability and activity.