纤维素降解菌中葡聚糖酶基因的克隆表达及活性
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摘要
为探明约氏梭菌(Clostridium josui)中纤维小体内某个酶的功能性,从该菌属CJ-1菌株中克隆出基因片段Cel9C,大小为1943 bp,编码647个氨基酸,将该基因构建于大肠杆菌表达载体p ET28a(+)中,获得重组表达载体p ET28a-Cel9C,转化至大肠杆菌菌株BL21(DE3)中进行表达。结果表明,该酶的最适反应温度为45℃,最适反应p H为p H6.0;对大麦-β-葡聚糖(Barley-β-glucan)和魔芋葡甘聚糖(Konjac glucomannan)具有较高的反应活性,比活值分别达到114和57 U·mg-1;几乎不能以木聚糖(Brichwood xylan)和半乳甘露寡糖(Galacto mannan)为底物。说明该酶对葡聚糖类物质时有较高的降解能力,应为葡聚糖酶。
With the purpose of detecting enzyme property of cellulosome in Clostridium josui, cel9 C from Clostridium josui CJ-1 was cloned into the vector p ET28 a to construct the recombinant plasmid p ET28a-Cel9 C which was further transformed into E coli. strain BL21(DE3). The results showed that the Cel9 C gene(1943 bp with 647 amino acids) was effectively expressed in E.coli. The optimum condition of the enzyme was 45℃ and p H 6.0. The enzyme showed a strong activity towards Barley-β-glucan(114 U/mg) and Konjac glucomannan(57 U/mg), while it showed an extremely low activity towards brichwood xylan and galacto mannan, suggesting that this enzyme should be a kind of glucanase.
With the purpose of detecting enzyme property of cellulosome in Clostridium josui, cel9 C from Clostridium josui CJ-1 was cloned into the vector p ET28 a to construct the recombinant plasmid p ET28a-Cel9 C which was further transformed into E coli. strain BL21(DE3). The results showed that the Cel9 C gene(1943 bp with 647 amino acids) was effectively expressed in E.coli. The optimum condition of the enzyme was 45℃ and p H 6.0. The enzyme showed a strong activity towards Barley-β-glucan(114 U/mg) and Konjac glucomannan(57 U/mg), while it showed an extremely low activity towards brichwood xylan and galacto mannan, suggesting that this enzyme should be a kind of glucanase.
引文
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[2]孟雷,陈冠军,王怡,等.纤维素酶的多型性[J].纤维素科学与技术,2002,10(2):47-55.
[3]Sakka M,Goto M,Fujii T,et al.Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A Bioscience[J].Biotechnology,and Biochemistry,2010,74(10):2077-2082.
[4]康传红,韩晓云,关卫夫,等.甜菜基因组DNA三种提取方法的对比研究[J].中国糖料,2002,2(1):1-4.
[5]李海玲,彭书明,李凛,等.4种常用蛋白浓度测定方法的比较[J].中国生化药物杂志,2008,29(4):277-282.
[6]赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(8):534-536.
[7]Nelson N.A photometric adaptation of the Somogyi method for the determination of glucose[J].J Biol Chem,1944,153(2):375-380.
[8]Bischoff K M,Rooney A P,LI X L,et al.Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis[J].Biotechnology Letters,2006,28(21):1761-1765.
[9]颜军,郭晓强,李晓光,等.TLC快速分析多糖的单糖组成[J].食品科学,2006,27(12):603-607.
[10]Sakka M,Higashi Y,Kimura T,et al.Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D,a xylanase that contains a family 3 carbohydrate-binding module[J].Applied and Environmental Microbiology,2011,77(12):4260-4263.
[11]吴琪,谢红云,段垒,等.β-葡聚糖酶的酶学性质研究[J].饲料研究,2010(2):34-37.
[12]张秀艳,何国庆.微生物源β-葡聚糖酶的稳定性研究[J].浙江大学学报:农业与生命科学版,2007,33(3):387-391.
[13]齐军茹,廖劲松.β-甘露聚糖酶的制备及其应用研究进展[J].中国食品添加剂,2002(6):12-15.
[14]营红磊,张卫明,蒋建新,等.半乳甘露聚糖胶酶法改性研究进展[J].中国野生植物资源,2009,28(2):5-10.
[15]Fujii T,Fang X,Inoue H,et al.Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials[J].Biotechnology for Biofuels,2009,2(1):24.
[16]李剑芳,邬敏辰,程科,等.β-甘露聚糖酶制备魔芋葡甘露低聚糖的研究[J].食品与发酵工业,2007,33(1):21-24.
[17]宫春波,谢丽源.β-葡聚糖酶及在啤酒工业中的应用[J].广州食品工业科技,2002,18(1):58-59.
[18]邹东恢,江洁.β-葡聚糖酶的开发与应用研究[J].农产品加工学刊,2005(8):7-9.
[19]常巧玲.热纤梭菌β-1,4-内切葡聚糖酶基因cel D在大肠杆菌和毕赤酵母中的表达[D]:杭州:浙江大学,2006.