食品科学

• 生物工程 • 上一篇    下一篇

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

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,*   

  1. 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
  • 出版日期:2016-10-15 发布日期:2016-12-01

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

WANG Cuiyan1, WANG Yuhua1, PIAO Chunhong1, LIU Junmei1,2, HU Yaohui1, REN Dayong1,*, YU Hansong1,*   

  1. 1. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China;
    2. Division of Soybean Processing, Soybean Research & Development Center, Changchun 130118, China
  • Online:2016-10-15 Published:2016-12-01

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GenBank上发表的绿色木霉(Trichoderma viride)cbhⅡ 基因(GenBank登录号:M55080)设计引物并通过聚合酶链式反应克隆获得其cDNA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pMG36e连接, 构建原核表达载体pMG36e-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 kD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/mL,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。

关键词: 绿色木霉, 纤维素酶, cbhⅡ 基因, 乳酸乳球菌, 酶活力

Abstract:

The expression of the cbhⅡ (cellobiohydrolase) gene from Trichoderma viride was studied by using Lactobacillus
expression system. Primers were designed according to T. viride cbhⅡ gene (GenBank accession number: M55080) and
the cbhⅡ gene cDNA was obtained by PCR method with a sequence length of approximately 1 441 bp. An 80 bp signal
peptide sequence was inserted into the upstream gene for the secretory expression of the target protein. Then, the gene was
optimized according to the codon usage of lactic acid bacteria for the synthesis of the whole cbhⅡ gene. The prokaryotic
expression vector pMG36e-S-cbhⅡ was constructed through connection between the target gene and the shuttle vector
pMG36e. Electrotransformation was used to obtain recombinant lactic acid bacteria in competent cells of Lactococcus lactis
NZ3900. The recombinant protein displayed a molecular weight of approximately 53 kD as determined by sodium dodecyl sulfatepolyacrylamide
gel electrophoresis, which is consistent with the expected size. The cellulase activity was 16.7 U/mL in culture
supernatants 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 extracellular
expression was achieved successfully.

Key words: Trichoderma viride, cellulase, cbhⅡ gene, Lactococcus lactis, cellubiohydrolase activity

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