一种耐低温α-乙酰乳酸脱羧酶在枯草芽孢杆菌中的高效表达

High-Level Expression of Cold-Adapted α-acetolactate Decarboxylase in Bacillus subtilis

DOI：10.3969/j.issn.1673-1689.2013.05.011

中文关键词: 枯草芽孢杆菌 α-乙酰乳酸脱羧酶 耐低温 双乙酰 Ni柱亲和层析 酶学性质

英文关键词: Bacillus subtilis α-acetolactate decarboxylase(ALDC) cold-adapted diacetyl Ni column affinity chromatography characterization

基金项目:国家973计划项目(2012CB725202);国家863计划项目(2011AA02A211);国家自然科学基金项目(21276110,30970056);教育部新世纪优秀人才计划项目(NCET-10-0459);高等学校博士学科点科研基金专项(20110093120001);中央高校基本科研资金专项(JUSRP51306A);江苏高校优势学科建设工程项目

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中文摘要:

从Bacillus subtilis L5-20染色体上克隆得到ALDC基因alsD,构建重组表达载体pMA5-alsD-His,将其转化到B.subtilis WB600中。SDS-PAGE结果显示ALDC在重组B.subtilisWB600中成功表达,其相对分子质量(Mr)为32×103。采用Ni柱亲和层析纯化重组ALDC,所得纯酶的比酶活为272.3 U/mg,总回收率为89.8%。该重组ALDC最适反应温度仅为25℃,在20～35℃范围内均表现出较高的活性。Ni2+对ALDC有一定的激活作用,Fe3+使ALDC活性完全丧失。经摇瓶培养,重组菌的ALDC酶活为27.0 U/mL,约为出发菌酶活的70倍。经5 L的发酵罐发酵放大试验,ALDC酶活最高可达75.9 U/mL。

英文摘要:

α-Acetolactate decarboxylase(ALDC) gene alsD was cloned from Bacillus subtilis L520 and constructed into pMA5.The recombinant plasmid pMA5-alsD-His was transformed into B.subtilis WB600.SDS-PAGE indicated that alsD gene was successfully expressed in recombinant B.subtilis and the molecular mass of ALDC was 32×103.Recombinant protein was purified by Ni-NTA affinity chromatography.The specific activity and total yield of the purified enzyme were 272.3 U/ mg and 89.8%,respectively.The optimum temperature of purified ALDC was 25 ℃.And the enzyme showed high activity between 20 ℃ and 35 ℃.The enzyme activity was activated by Ni2+ and totally inhibited by Fe3+.When cultured in shake-flask,the ALDC activity of the recombinant strain was approximately 70-fold higher than that of original strain.The result of scaled-up experiment showed that the enzyme activity was 75.9 U/mL in 5 L fermentor.

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