基因突变提高毕赤酵母表达内切β-1,3-葡聚糖酶活性  

Endo-β-1,3-Glucanase Expression in P.pastoris Improved by Gene Mutation

作　　者：金树霞 王力 朱莉 李菲菲 刘丽萍 詹晓北[1] 郑志永[1,4] 高敏杰 JIN Shuxia;WANG Li;ZHU Li;LI Feifei;LIU Liping;ZHAN Xiaobei;ZHENG Zhiyong;GAO Minjie(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Jiangsu Aijin Agrochemical Co.Ltd.,Nanjing 211511,China;Wuxi Glyco Biotechnology Co,Ltd.,Wuxi 214125,China;School of Environmental and Civil Engineering,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江苏艾津农化有限责任公司,江苏南京211511 [3]无锡格莱克斯生物科技有限公司,江苏无锡2141254 [4]江南大学环境与土木工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2021年第2期57-64,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31301408);江南大学自主科研计划重点项目(JUSRP51632A);江苏省重点研发计划(现代农业)项目(BE2018367)。

摘　　要：为构建一株高效表达内切β-1,3-葡聚糖酶的毕赤酵母菌株,并用于水解热凝胶生产β-1,3-葡寡糖。作者首先对哈茨木霉来源内切β-1,3-葡聚糖酶基因进行随机突变,并构建BGN13.1a突变库;然后采用高通量技术及刚果红平板筛选高表达菌株;最后采用TLC薄层色谱和MALDI-TOF MS分析热凝胶水解产物,并分析了酶学性质。结果表明,本研究成功筛选得到毕赤酵母突变株K827,酶活较初始菌株提高了25%;其水解产物聚合度为2~10,且寡糖产量为1.492 g/L;其最适pH与最适温度分别为5.5和50℃;相较亲本酶,突变株K827的pH稳定性与温度稳定性都有明显提高。本研究为内切β-1,3-葡聚糖酶的工业化生产及应用提供依据。A P.pastoris strain for efficient endo-β-1,3-glucanase expression was constructed to hydrolyze curdlan and produce curdlan oligosaccarides.Firstly,error-prone PCR technology was implemented on endo-β-1,3-glucanase gene from Trichoderma harzianum to construct a mutation library of BGN13.1a.High-throughput screening technology and Congo red plate screening were then employed to obtain high expression P.pastoris strains.Finally,TLC and MALDI-TOF MS were used to analyze the curdlan hydrolysate and their enzymatic properties.The results showed that the activity of endo-β-1,3-glucanase from the successfully screened mutant P.pastoris K827 was improved by 25%compared with that of the original enzyme.The expressed endo-β-1,3-glucanase hydrolyzed curdlan to produce oligosaccharides at 1.492 g/L with a polymerization degree of 2~10.The optimal reaction temperature and pH for endo-β-1,3-glucanase were 50℃and pH 5.5,respectively.The pH stability and temperature stability of mutant K827 were significantly improved compared with the original enzyme.This study could provide experimental basis for the industrialized production and application of endo-β-1,3-glucanase.

关 键 词：内切β-1 3-葡聚糖酶 毕赤酵母 易错PCR 酶学性质 

分 类 号：Q815[生物学—生物工程]