几丁质裂解性多糖单加氧酶的表达及应用研究  

Expression and application of chitinolytic polysaccharide monooxygenase

作　　者：徐倩倩 马春桐 姚莹莹 徐伟[1] 周秀玲[1] 张扬[1] XU Qianqian;MA Chuntong;YAO Yingying;XU Wei;ZHOU Xiuling;ZHANG Yang(School of Life Science,Liaocheng University,Liaocheng 252059,China)

机构地区：[1]聊城大学生命科学学院,山东聊城252059

出　　处：《中国酿造》2022年第8期97-104,共8页China Brewing

基　　金：国家自然科学基金资助项目(32100065);山东省自然科学基金资助项目(ZR2018QC002)。

摘　　要：以具有几丁质降解能力菌株Chitiniphilus sp. LY72的裂解多糖单加氧酶(Cs LPMO)为研究对象,在克隆Cs LPMO全长基因的基础上对其进行生物信息学分析,利用3种表达质粒p ET-22b、pET-28a和pCold,构建Cs LPMO异源表达菌株EBL-22、EBL-28和EBL-Co。重组Cs LPMO蛋白经Ni-NTA亲和层析柱纯化及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,筛选最佳表达质粒,对其表达条件进行优化。结果表明,Cs LPMO是一种裂解性多糖单加氧酶,隶属于AA10家族。3株重组菌中,纯化重组酶蛋白相对含量分别为5.9%、4.1%、5.4%,比酶活力分别为155.42 U/mg、80.26 U/mg、148.29 U/mg,因此,确定最佳质粒为pET-22b,菌株EBL-22表达条件为:OD600 nm值至0.4时,添加异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度为0.4 mmol/L后,在25℃下诱导表达16 h。在优化条件下,其可溶性蛋白含量为928.32 mg/L,比酶活力为3.34 U/m L。Cs LPMO的加入可使还原糖的产量提高1.67倍。Using lytic polysaccharide monooxygenase of Chitiniphilus sp.(CsLPMO)with chitin degradation ability as research object,bioinformatics analysis was carried out on the basis of cloning CsLPMO full-length gene.CsLPMO heterologous expression strains EBL-22,EBL-28 and EBL-Co were constructed using 3 kinds of expression plasmids pET-22b,pET-28a,and pCold.The recombinant CsLPMO protein was purified by Ni-NTA affinity chromatography column and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the optimal expression plasmids were screened,and their expression conditions were optimized.The results showed that CsLPMO was a lytic polysaccharide monooxygenase belonging to the AA10 family.In the 3 recombinant strains,the relative contents of purified ecombinase protein were 5.9%,4.1%,and 5.4%,and the specific enzyme activities were 155.42 U/mg,80.26 U/mg,and 148.29 U/mg,respectively.Therefore,the optimal plasmid was pET-22b.The expression conditions of strain EBL-22 were as follows:when OD600 nm value was 0.4,IPTG was added to the final concentration of 0.4 mmol/L and the expression was induced at 25℃for 16 h.Under the optimized conditions,the soluble protein content was 928.32 mg/L,and the specific enzyme activity was 3.34 U/ml.The addition of CsLPMO increased the yield of reducing sugar by 1.67 times.

关 键 词：几丁质 裂解性多糖单加氧酶 蛋白质表达 表达条件优化 

分 类 号：Q55[生物学—生物化学]