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通过构建植物乳杆菌属（Lactobacillus sp. LMY-20）中亚硝酸盐还原酶（nitrite reductase,NiR）的重组乳酸克鲁维酵母菌株、纯化重组蛋白并对其进行酶学性质表征。采用密码子优化后的亚硝酸盐还原酶基因，将其亚克隆至pKLAC2载体，构建成重组表达质粒pKLAC2-nir，并转化至乳酸克鲁维酵母（K. lactis）中实现表达；再利用镍柱亲和层析纯化得到重组亚硝酸盐还原酶。SDS-PAGE分析结果显示，在约45 kDa位置出现与预期大小相符的蛋白质条带。酶活检测结果显示，发酵48小时后重组NiR达到最高酶活，酶活性约为34 U/mg。纯化后的重组NiR的最适pH为6.5，最适反应温度为37℃。该酶稳定性及酶活性较好，酶活力保持在较高水平，即90%左右。本研究成功构建了产亚硝酸盐还原酶的重组乳酸克鲁维酵母菌株并纯化获得重组亚硝酸盐还原酶，重组NiR具有良好的稳定性。
Abstract：A recombinant Kluyveromyces lactis strain expressing nitrite reductase(NiR) from Lactobacillus sp. LMY20 was constructed,the recombinant protein was purified,and its enzymatic properties were characterized. The codon-optimized nitrite reductase gene was subcloned into the vector pKLAC2,and the recombinant expression plasmid pKLAC2-nir was transformed into K. lactis for heterologous expression. Recombinant nitrite reductase was purified by nickel column affinity chromatography. SDS-PAGE analysis revealed the presence of protein bands with the expected molecular weight of approximately 45 kDa. The optimal fermentation time for recombinant NiR achieving peak enzyme activity was determined to be 48 hours,corresponding to an activity level of approximately 34 U/mg. The purified recombinant NiR exhibited optimal activity at a pH of 6.5 and a temperature of 37℃. The enzyme demonstrated good stability and maintained high activity levels,that is,about 90%. It was concluded that a recombinant K. lactis strain producing nitrite reductase was successfully constructed,the recombinant NiR was purified,and the enzyme displayed favorable stability.
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基本信息:
DOI：10.19804/j.issn1006-2513.2026.3.004
中图分类号:TS201.3
引用信息:
[1]刘淼,李美玉,张庆芳,等.植物乳杆菌亚硝酸盐还原酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征[J].中国食品添加剂,2026,37(03):33-39.DOI:10.19804/j.issn1006-2513.2026.3.004.
基金信息:
国家重点研发计划项目(2022YFC2805105); 大连市科技创新基金项目(2020JJ26SN048)