食品科学

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双纤维素酶基因在乳酸杆菌中的融合分泌表达

丁 轲,罗伟光,丁盼盼,李 旺,李元晓,余祖华,恒子钤,贾艳艳,程相朝   

  1. 1.河南科技大学宏翔发酵饲料实验室,河南 洛阳 471003;2.河南省动物疫病与公共安全院士工作站,
    河南 洛阳 471003;3.河南省高等学校环境与畜产品安全重点学科开放实验室,河南 洛阳 471003
  • 出版日期:2014-08-15 发布日期:2014-08-25

Fusion Design and Secretory Expression in Lactobacillus casei of Two Cellulase Genes

DING Ke, LUO Wei-guang, DING Pan-pan, LI Wang, LI Yuan-xiao, YU Zu-hua, HENG Zi-qin, JIA Yan-yan, CHENG Xiang-chao   

  1. 1. Hongxiang Biological Feed Laboratory of Henan University of Science and Technology, Luoyang 471003, China; 2. Animal
    Disease and Public Safety Academician Workstation of Henan Province, Luoyang 471003, China; 3. Open Laboratory of Key
    Discipline for Enviroment and Animal Products Safety of Henan Institute of Higher Learning, Luoyang 471003, China
  • Online:2014-08-15 Published:2014-08-25

摘要:

为提高纤维素的降解效率,将两种不同的纤维素酶基因在乳酸杆菌中融合表达,实现双纤维素酶在乳酸杆菌中的高效表达。根据两纤维素酶的基因序列设计两对引物,并在两基因间引入一段连接肽。通过聚合酶链式反应(polymerase chain reaction,PCR)分别扩增得到两纤维素酶基因CelL15和CelL73,将其融合后连接到乳酸杆菌分泌性表达载体pMJ67中,构建重组表达质粒pMJ-CelL15-CelL73,电转化入乳酸杆菌Lactobacillus casei,利用红霉素MRS平板筛选阳性克隆,通过PCR验证获得了分泌表达双纤维素酶的重组乳酸杆菌。电泳实验结果表明重组乳酸杆菌成功表达了1 个约为83 kD的融合蛋白。平板酶活力检测发现重组乳酸杆菌能明显降解底物中的羧甲基纤维素钠,培养液上清中纤维素酶酶活力可达1.25 U/mL。

关键词: 纤维素酶, 重组乳酸杆菌, 融合, 分泌

Abstract:

Cellulase is a key enzyme for cellulose degradation. In order to enhance the efficiency of cellulose degradation,
two different cellulase genes were fused and integrated into Lactobacillus genome, and efficiently expressed. Two pairs
of primes were designed according to two cellulose genes in GenBank, a peptide containing 10 amid acids was inserted
between the two genes. CelL15 and CelL73 were amplified by PCR method, and then fused and cloned into the expression
vector pMJ67. The recombinant vector was electrotransferred into Lactobacillus casei. SDS-PAGE analysis showed that the
fusion cellulase protein was successfully expressed and secreted into the cell culture supernatant, which was approximately
83 kD. Enzyme activity detection on solid medium indicated that the recombinant Lactobacillus could obviously degrade
sodium carboxymethylcellulose in the medium, and the cellulase activity in the supernatant was up to 1.25 U/mL after
culture for 36 h.

Key words: cellulase, recombinant Lactobacillus, fusion, secretion

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