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2025年4月4日 星期五
  2018, Vol. 26 Issue (8): 1275-1287    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
黑胸散白蚁体内产纤维素酶细菌的筛选及其纤维素酶基因的鉴定与原核表达
王智伟1,周小敏1,2,杨雨鑫3,陈玉林3
1. 西北农林科技大学动物科技学院
2.
3. 西北农林科技大学
Screening of Cellulolytic Microorganisms from the Reticulitermes chinensis and Identification of Cellulase Gene and Its Prokaryotic Expression
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摘要 摘 要 白蚁(Isoptera)常以富含纤维素的木材为食,体内存在纤维素酶产生菌。从源于陕西佛坪的白蚁(经鉴定为黑胸散白蚁(Reticulitermes chinensis))体内筛选出了4株产纤维素酶菌株,采用单因素实验设计对酶活最高的菌株进行了产酶条件优化,并对其所产纤维素酶进行了酶学性质研究;同时,依据NCBI数据库设计的引物扩增出了该菌株的内切-β-1,4-葡聚糖酶基因Cbs和β-葡萄糖苷酶基因BaG,之后在大肠杆菌(Escherichia coli)中进行了表达实验。结果表明:从黑胸散白蚁体内筛选出4株菌分别是阿氏肠杆菌(Enterobacter asburiae)、炭疽芽胞杆菌(Bacillus cereus)、枯草芽胞杆菌(Bacillus subtilis)、解淀粉芽胞杆菌(Bacillus amyloliquefaciens),其中枯草芽胞杆菌所产纤维素酶的活力最高,该菌发酵产酶的最适温度和pH分别是42 ℃、6.5,酶促反应的最适温度和pH分别是70 ℃、4.5,在此条件下该菌株酶活力为2.36 U/mL。该菌株所产的纤维素酶在50 ℃、pH 6.5的条件下热稳定性和pH稳定性最佳,使用该菌株所产的粗酶液发酵粗饲料,结果发现其对玉米(Zea mays)秸秆、小麦(Triticum aestivum)秸秆、苜蓿(Medicago polymorpha)干草和青贮饲料的粗纤维降解率分别为12.21%、1.3%、3.24%和17.42%。基因克隆结果表明该菌株具有BaG、Cbs 2个纤维素酶基因,且大肠杆菌表达结果显示BaG基因的粗酶液proBaG无纤维素酶活性,而Cbs基因的粗酶液proCbs在60 ℃、pH 5.0时酶活可达4.14 U/mL,将BaG和Cbs基因进行原核融合表达,产生的蛋白约35 kD,其在最适条件70 ℃、pH 4.5时的酶活为4.57 U/mL,说明无纤维素酶活性的BaG基因与Cbs基因融合后可能表达出了一个活性更高的纤维素酶蛋白。本研究对后期构建可降解木质纤维素的人工重组菌具有重要的理论价值。
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王智伟
周小敏
杨雨鑫
陈玉林
关键词 白蚁枯草芽胞杆菌纤维素酶内切-β-1,4葡聚糖酶β-葡萄糖苷酶    
Abstract:Abstract Termites (Isoptera) often eat wood rich in cellulose, and there are much bacteria producing cellulase in vivo. The aim of this study was to screen a strain of producing highly active cellulase from termites to amplify the cellulase gene of this strain and express it in Escherichia coli, which had a potential basis for the the construction of cellulase gene recombinant bacteria. Collecting termites samples from Foping County of Shaanxi province, this study screened 4 strains of producing cellulase from termites (Reticulitermes chinensis) in vivo by cellulase screening medium and measurements of cellulase activity; then optimized the fermentation conditions were conducted by single factor experiment on the strain which producing the highest active cellulase, and studied the enzymatic properties of cellulase. Finally, according to the data of NCBI primers which amplified the cellulase genes were designed and prokaryotic expression was completed. The results showed that the 4 cellulase producing strains screening from termites were Enterobacter asburiae, Bacillus cereus, Bacillus subtilis and Bacillus amyloliquefaciens, respectively. The cellulase activity of the Bacillus subtilis was the highest, and the optimum fermentation temperature and pH of the strain was 42 ℃ and 6.5. The optimum temperature and pH of enzymatic reaction was 70 ℃ and 4.5, under this condition, the cellulase activity reached 2.36 U/mL. Under the condition of 50 ℃ and pH 6.5, the thermal stability and pH stability of the cellulase produced by the strain were optimum. Using crude enzyme solution to ferment crude fiber, it was found that the degradation rate of crude enzyme solution to four kinds of feed, corn (Zea mays) stalk , wheat (Triticum aestivum) straw, alfalfa (Medicago polymorpha) hay and silage corn stalk was 12.21%, 1.30%, 3.24% and 17.42%, respectively. Gene cloning results showed that the Bacillus cereus strain contained two cellulase genes BaG and Cbs. Under prokaryotic expression of the cellulase gene, the proBaG didn't show any cellulase activity, while the proCbs showed the optimum conditions of 60 ℃, pH 5.0, cellulase activity reached 4.14 U/mL. Under the condition of 50 ℃ and pH 5.0, the thermal stability and pH stability of the cellulase were optimum. Finally, the fusion expression of BaG and Cbs gene showed a new protein of about 35 kD while BaG and Cbs gene expressed the protein with a molecular weight of 27.4 kD and 55.2 kD, respectively. In the optimum conditions of 70 ℃ and pH 4.5, the cellulase activity of the fusion product expression reached 4.57 U/mL. Under the condition of 50 ℃ and pH 4.5, the thermal stability and pH stability of the cellulase were optimum. This study indicated that the BaG gene without cellulase activity could interact with the Cbs gene and may produce a new protein with higher cellulase activity and smaller molecular weight. This study should have important theoretical value for the construction of recombinant bacteria which could degradate lignocellulose.
Key wordsTermites    Bacillus cereus    Cellulase    Endo-β-1,4-glucanase    β-glucosidase
收稿日期: 2017-11-29      出版日期: 2018-07-20
基金资助:国家绒毛用羊产业技术体系项目
通讯作者: 王智伟     E-mail: ZwWang99@163.com
引用本文:   
王智伟 周小敏 杨雨鑫 陈玉林. 黑胸散白蚁体内产纤维素酶细菌的筛选及其纤维素酶基因的鉴定与原核表达[J]. , 2018, 26(8): 1275-1287.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I8/1275
 
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