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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (12): 49-58    DOI: 10.13523/j.cb.20171208
研究报告     
溶葡球菌酶在乳酸克鲁维酵母中重组表达、诱变、优化及酶学研究*
王曦1,张光德1,陈熙明2,浦铜良1*()
1 兰州大学生命科学学院 兰州 730000
2 中国科学院西北生态环境资源研究院 兰州 730000
Heterologous Expression, Mutation, Optimizing the Expression Condition and Characterization of Lysostaphin in Kluyveromyces lactis
Xi WANG1,Guang-de ZHANG1,Xi-ming CHEN2,Tong-liang PU1*()
1 School of Life Sciences,LanZhou University, Lanzhou 730000, China
2 Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
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摘要：

根据模仿葡萄球菌(Staphylococcus simulans)的溶葡球菌酶基因序列以及乳酸克鲁维酵母密码子偏好性设计引物扩增溶葡球菌酶基因表达片段,构建溶葡球菌酶(lysostaphin,Lys)基因表达载体(pKLAC1-Lys),转化乳酸克鲁维酵母(K. lactis GG799),实现了Lys基因的分泌表达。对重组菌株(K. lactis GG799/pKLAC1- Lys)进行NTG随机化学诱变,优化表达条件,筛选获得高表达菌株,并通过Ni-NTA亲和层析纯化蛋白并研究其酶学性质。结果表明:通过诱变重组溶葡球菌酶乳酸克鲁维菌株,Lys酶比活性提高了约5.2倍(约8 000U/L)。最适接种量为40g/L,诱导过程中每24h添加一次终浓度为20g/L的半乳糖和NH4NO3可提高酶比活性,最适表达pH为7.0~7.5,最适反应pH为7.0~8.0,最适反应温度为37℃。实验表明,低于40℃,pH 3~6之间时,重组溶葡球菌酶较稳定。Sr2+对其酶活性有明显的促进作用,Ba2+、Ca2+、Zn2+、Cu2+、Mn2+、Mg2+对其有明显的抑制作用。
关键词： 乳酸克鲁维酵母;  溶葡球菌酶;  重组表达;  诱变    
Abstract:

According to the sequence of lysostaphin gene from Staphylococcus simulans and codon bias of Kluyveromyces lactis, the PCR primers were designed to amplify the fragment of lysostaphin gene. The fragment was inserted in pKLAC1, and transformed to K. lactis GG799. The K. lactis GG799/pKLAC1- Lys was cultivated to express Lys. A high expression strain (mu4#) were abtained by using powerful mutagen (N-methy1-N-nitro-N-nitrosoguanidine,NTG) on the recombinant and optimized the expression condition .The fermentation broth of mu4# was purified by Ni-NTA agarose and the enzyme characterization was studied. The result showed that the activity of Lys was approximately 5.2 times (8 000U/L) higher in the mutation. The optimal inoculum dose of the mutant (mu4#) was 40g/L; Galactose and NH4NO3 (20g/L) were added in every 24 hours, Lys exhibited optimal expression at pH 7.0~7.5; Furthermore, the Lys enzyme optimal reaction performed at pH 7.0~8.0 and temperature at 37℃. The recombinant Lys was stable below 40℃ and pH between 3.0 and 6.0. Sr2+ stimulated its activity whereas Ba2+、Ca2+、Zn2+、Cu2+、Mn2+、Mg2+ inhibited the activities. This research accomplished Lys recombinant expression, yield improvement by chemical mutagenesis in K. lactis and characterization of lysostaphin. These research results provide profound guiding significance for the large-scale production and application of recombinant lysostaphin.
Key words: Kluyveromyces lactis    Lysostaphin    Recombinant    Mutation
收稿日期: 2017-06-12 出版日期: 2017-12-16
ZTFLH:  Q819  
基金资助: * 国家自然科学基金 (31400437)、甘肃国际合作项目 (1504WKCA097)资助项目
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引用本文:

王曦,张光德,陈熙明,浦铜良. 溶葡球菌酶在乳酸克鲁维酵母中重组表达、诱变、优化及酶学研究*[J]. 中国生物工程杂志, 2017, 37(12): 49-58.

Xi WANG,Guang-de ZHANG,Xi-ming CHEN,Tong-liang PU. Heterologous Expression, Mutation, Optimizing the Expression Condition and Characterization of Lysostaphin in Kluyveromyces lactis. China Biotechnology, 2017, 37(12): 49-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171208    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I12/49

ddH2O10 × Buffer
(含Mg2+)		dNTPs
(2.5mmol/L)		P1P2Taq
酶
37.555110.5
表1  PCR反应体系
图1  PCR 扩增Lys基因琼脂糖凝胶电泳
图2  表达载体pKLAC1-Lys结构
图3  表达载体pKLAC1-Lys酶切鉴定
图4  表达载体pKLAC1-Lys单酶切
图5  K. lactis GG799/pKLAC1- Lys 8#不同诱导时间溶葡球菌酶酶活
图6  mu4#不同诱导时间溶葡球菌酶的酶活
图7  mu4#生长曲线
图8  mu4# 不同起始量对酶活的影响
图9  mu4#不同时间半乳糖添加量对酶活的影响
图10  mu4#不同时间氮源添加对酶活的影响
图11  mu4# 不同pH对酶活的影响
图12  mu4#发酵液SDS-PAGE分析
图13  溶葡球菌酶反应最适pH
图14  溶葡球菌酶pH稳定性
图15  溶葡球菌酶反应最适温度
图16  溶葡球菌酶热稳定性
图17  不同二价金属阳离子对溶葡球菌酶酶活的影响
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