Bacillius naganoensis普鲁兰酶基因的分子改造及酶学性质研究  

Molecular Evolution of Pullulanase from Bacillius naganoensis and Its Enzymatic Characterization

作　　者：秦艳[1,2] 谢能中[2] 曹薇[1] 王青艳[2] 米慧芝[2] 申乃坤[2] 黄日波[1,2] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530005 [2]广西科学院国家非粮生物质能源工程技术研究中心,广西南宁530007

出　　处：《生物技术》2014年第2期53-57,共5页Biotechnology

基　　金：广西自然科学基金项目("普鲁兰酶的分子改造与高效表达研究";编号:2012GXNSFBA053063);广西科学研究与技术开发计划项目("木薯黄浆废水混合发酵高效;清洁生产技术研发与示范";编号:桂科重12118004-3;"利用整合型重组枯草芽孢杆菌高效生产普鲁兰酶";编号:桂科重1348004-2);广西科学院基本科研业务费资助项目("耐酸耐热普鲁兰酶的分子改造与高效分泌表达";编号:12YJ25SW01;"高产酒精酿酒酵母菌株的选育";编号:12YJ25SW02)资助

摘　　要：目的：通过缺失原酶上氨基酸残基，提高普鲁兰酶的酶活，并对其突变体重组酶进行酶学特性研究。方法：以Bacillius naganoensis基因组DNA为模板，PCR扩增大小为2781bp的普鲁兰酶编码基因，并通过PCR方法缺失原酶上的前78个氨基酸残基，获得突变体PulA234，将编码基因酶切连接到表达载体pET-22b（＋），转化Escherichia coli BL21（DE3）。IPTG诱导表达后对表达产物进行SDS—PAGE和酶学特性研究。结果：突变体发酵液的酶活是原酶的19倍，SDS—PAGE电泳结果显示有明显特异性条带，分子质量约为100kDa。该突变酶的最适反应温度为60℃，最适pH为4．75，在pH3．8～6．6范围内活性稳定，cu2＋、ca2＋对酶活有激活作用。结论：经改造的重组酶酶活力有所提高，具有良好的pH和酸稳定性，为普鲁兰酶酶制剂工业化生产及应用奠定基础。Objective：The activity of pullulanase is enhanced by lacking amino acid residues. Then the characterization of mutant recombi- nant pullulanase was studied. Method：The gene of pullulanase with an open reading frame （ORF） of 2 781 bp in length was amplified by PCR with the template of the genomic DNA of Bacillius naganoensis, mutant pulA234 was obtained by the PCR by lacking 78 amino acid residues, and was cloned into the expression vector pET -22b （ ＋ ） , which was transformed into the E. coli BL21 （DE3）. Result：The to- tal soluble activity of pullulanase induced with IPTG was 19 - fold higher than that of wild - type enzyme. The SDS - PAGE analysis showed a band with apparent molecular weight of 100 kDa. The optimum temperature and pH for the mutant were determined as 60℃ and 4. 75, respectively, and the pH range were 3.8 - 6. 6. The mutant could be stimulated by Ca2 ＋ , Cu2 ＋. Conclusion： Because the mutant recombinant pullulanase activity was higher than that of wild - type enzyme, also good pH and acid stability, so the recombinant pullula- nase had laied the foundation for industrialized production and application.

关 键 词：普鲁兰酶 长野芽孢杆菌 酶分子改造 酶学性质 

分 类 号：Q784[生物学—分子生物学] Q786