绿色木霉纤维二糖水解酶基因的克隆及其在乳酸菌中的表达与鉴定  

Cloning,Expression and Identification of Trichoderma viride Cellobiohydrolase Gene in Lactic Acid Bacteria

作　　者：王翠艳[1] 王玉华[1] 朴春红[1] 刘俊梅[1,2] 胡耀辉[1] 任大勇[1] 于寒松[1] WANG Cuiyan WANG Yuhua PIAO Chunhong LIU Junmei HU Yaohui REN Dayong YU Hansong(College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China Division of Soybean Processing, Soybean Research ＆ Development Center, Changchun 130118, China)

机构地区：[1]吉林农业大学食品科学与工程学院,吉林长春130118 [2]国家大豆产业技术研发中心加工研究室,吉林长春130118

出　　处：《食品科学》2016年第19期141-146,共6页Food Science

基　　金：国家现代农业(大豆)产业技术体系建设专项(CARS-04);吉林省重点科技攻关项目(20150204036NY)

摘　　要：采用乳酸菌表达系统进行绿色木霉纤维二糖水解酶基因cbhⅡ的表达研究。参照Gen Bank上发表的绿色木霉(Trichoderma viride)cbhⅡ基因(Gen Bank登录号:M55080)设计引物并通过聚合酶链式反应克隆获得其c DNA序列长1 441 bp。为了达到表达分泌该酶的目的,在其基因上游序列前添加了大小为80 bp的信号肽序列。进一步通过生物信息学方法将密码子按照乳酸菌的偏好性进行了优化,通过全基因合成获得了优化后的cbhⅡ基因。将该基因与穿梭表达载体p MG36e连接,构建原核表达载体p MG36e-S-cbhⅡ;利用电转化方法将其转入乳酸乳球菌NZ3900感受态细胞中构建重组乳酸菌,纯化的重组菌蛋白通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳检测,得到一条约为53 k D的目的蛋白条带,与预期大小相符;利用3,5-二硝基水杨酸法测定培养基上清液中水解纤维素酶的活力达到16.7 U/m L,菌体裂解液上清液和菌体沉淀几乎没有酶活力。结果表明,纤维二糖水解酶基因信号肽序列被乳酸乳球菌正确识别,并成功实现了胞外表达。The expression of the cbhⅡ （cellobiohydrolase） gene from Trichoderma viride was studied by using Lactobacillusexpression system. Primers were designed according to T. viride cbhⅡ gene （GenBank accession number： M55080） andthe cbhⅡ gene cDNA was obtained by PCR method with a sequence length of approximately 1 441 bp. An 80 bp signalpeptide sequence was inserted into the upstream gene for the secretory expression of the target protein. Then, the gene wasoptimized according to the codon usage of lactic acid bacteria for the synthesis of the whole cbhⅡ gene. The prokaryoticexpression vector pMG36e-S-cbhⅡ was constructed through connection between the target gene and the shuttle vectorpMG36e. Electrotransformation was used to obtain recombinant lactic acid bacteria in competent cells of Lactococcus lactisNZ3900. The recombinant protein displayed a molecular weight of approximately 53 kD as determined by sodium dodecyl sulfatepolyacrylamidegel electrophoresis, which is consistent with the expected size. The cellulase activity was 16.7 U/mL in culturesupernatants by 3,5-dinitrosalicylic acid assay, and there was almost no activity in cell lysate precipitates or supernatants.These results showed that the signal peptide sequence was correctly identified by Lactococcus lactis and extracellularexpression was achieved successfully.

关 键 词：绿色木霉 纤维素酶 cbhⅡ基因 乳酸乳球菌 酶活力 

分 类 号：Q7[生物学—分子生物学]