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(p KLAC1-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/p KLAC1-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NH_4NO_3可提高酶比活性,最适表达p H为7.0~7.5,最适反应p H为7.0~8.0,最适反应温度为37℃。实验表明,低于40℃,p H 3~6之间时,重组溶葡球菌酶较稳定。Sr^(2+)对其酶活性有明显的促进作用,Ba^(2+)、Ca^(2+)、Zn^(2+)、Cu^(2+)、Mn^(2+)、Mg^(2+)对其有明显的抑制作用。
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 pKLACl, and transformed to K. lactis GG799. The K. lactis GG799/pKLACl- Lys was cultivated to express Lys. A high expression strain （mu4#） were abtained by using powerful mutagen （ N-methyl- 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 ＋ , Caz ＋ , 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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第12期49-58,共10页
China Biotechnology
基金
国家自然科学基金(31400437)
甘肃国际合作项目(1504WKCA097)资助项目
