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纳豆激酶第36位氨基酸突变对其活性及热稳定性的影响

DOI: 10.7685/j.issn.1000-2030.2008.03.026, PP. 130-136

Keywords: 纳豆激酶,nk基因,序列分析,毕赤酵母,热稳定性

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Abstract:

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15min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。

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