欧文氏杆菌甘露聚糖酶基因同源高效表达与缺失表达研究
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摘要
草本纤维生物提取法是一种利用微生物分泌复合酶系降解草本纤维原料中的非纤维素物质而获得纯净天然纤维素纤维的方法,本室筛选和改造的欧文氏杆菌变异菌株CXJZ95-198和CXJZU120是迄今报道草本纤维提取效率最高的菌株,为了研究CXJZ95-198甘露聚糖酶基因的特点,提高CXJZU-120甘露聚糖酶表达水平,笔者进行了以下研究：
利用生物信息学方法对欧文氏杆菌变异菌CXJZ95-198的甘露聚糖酶基因manA及其翻译产物进行了相似度比对,信号肽,跨膜区,系统进化树,二级结构和同源三维建模的分析、预测,结果表明manA核酸序列与其他来源的甘露聚糖酶基因序列相似度很低,翻译产物是胞外酶,N端前27AA可能为信号肽序列,根据三维结构预测结果设计引物,用PCR的方法分别扩增出manA 5'端截去0,81,111,141bp的四个甘露聚糖酶基因部分缺失片段,连接pET28a在E.coli BL21(DE3)中表达,结果证明,manA的翻译产物N端27个AA为信号肽序列,跨膜分泌时会被切割,N端27-37个AA缺失后对酶活无明显影响,N端37-47个AA缺失会引起酶活性丧失。
将manA基因连接pEASY E1 express载体转入E.coli BL21(DE3)中,经透明圈,抗生素筛选后再将重组质粒从BL21(DE3)重组菌中提取出来,导入欧文氏杆菌变异菌株CXJZU-120中进行同源表达,用透明圈法发现CXJZU-120重组菌较原始菌在甘露聚糖酶筛选培养平板上产生的水解圈大2/3,培养25h后酶活测定结果表明,重组菌的甘露聚糖酶酶活较原始菌提高2.1倍。
Bio-extraction of fiber from crops is a method using bacteria which producing series of enzymes to decompose hemicellulose of the crops.Two Erwinia carotovora CXJZ95-198 and CXJZU-120 have been isolated in this laboratory, which are reported as the most efficient deguming bacteria. In this study, the property of mannanase from CXJZ95-198,and the mannanase expression level on CXJZU-120 were investigated to promote their production, the results are as follows.
manA, the mannanase gene of CXJZ95-198 has been analysed by bioinformatics method in homology alignment, signal peptide prediction, transmembrane region prediction, phylogenic tree foundation, second structure and crystal structure foundation. The results show that manA has low similarity in the alignment with other source of mannanase genes, and its translation product is extracellular. Signal peptides of 27AA from the N-terminal were found at the manA's translation product. Four truncated mannanase genes were designed according to the homologous model, in which 0,81,111,141bp were deleted from the N-terminal respectively. N-truncated gene expression proved the 27aa aside the N terminal were signal peptide, and loss of the 37aa from the N terminal show little effect on mannanase activity. However, the enzyme will be inactivatied while the number of amino acid deltion was more than 47.
The manA was cloned into plasmid pEASY El express and transferred into E.coli BL21 (DE3).The recombinant plasmids were extracted from the positive transformant, and transferred into Erwinia carotovor variant CXJZU-120. Measured by circle decomposing method, the mannanase expression level of the homologous recombinant was 2/3 higher than the original host CXJZU-120, and the promotion of mannanase activity is 2.1 times.
利用生物信息学方法对欧文氏杆菌变异菌CXJZ95-198的甘露聚糖酶基因manA及其翻译产物进行了相似度比对,信号肽,跨膜区,系统进化树,二级结构和同源三维建模的分析、预测,结果表明manA核酸序列与其他来源的甘露聚糖酶基因序列相似度很低,翻译产物是胞外酶,N端前27AA可能为信号肽序列,根据三维结构预测结果设计引物,用PCR的方法分别扩增出manA 5'端截去0,81,111,141bp的四个甘露聚糖酶基因部分缺失片段,连接pET28a在E.coli BL21(DE3)中表达,结果证明,manA的翻译产物N端27个AA为信号肽序列,跨膜分泌时会被切割,N端27-37个AA缺失后对酶活无明显影响,N端37-47个AA缺失会引起酶活性丧失。
将manA基因连接pEASY E1 express载体转入E.coli BL21(DE3)中,经透明圈,抗生素筛选后再将重组质粒从BL21(DE3)重组菌中提取出来,导入欧文氏杆菌变异菌株CXJZU-120中进行同源表达,用透明圈法发现CXJZU-120重组菌较原始菌在甘露聚糖酶筛选培养平板上产生的水解圈大2/3,培养25h后酶活测定结果表明,重组菌的甘露聚糖酶酶活较原始菌提高2.1倍。
Bio-extraction of fiber from crops is a method using bacteria which producing series of enzymes to decompose hemicellulose of the crops.Two Erwinia carotovora CXJZ95-198 and CXJZU-120 have been isolated in this laboratory, which are reported as the most efficient deguming bacteria. In this study, the property of mannanase from CXJZ95-198,and the mannanase expression level on CXJZU-120 were investigated to promote their production, the results are as follows.
manA, the mannanase gene of CXJZ95-198 has been analysed by bioinformatics method in homology alignment, signal peptide prediction, transmembrane region prediction, phylogenic tree foundation, second structure and crystal structure foundation. The results show that manA has low similarity in the alignment with other source of mannanase genes, and its translation product is extracellular. Signal peptides of 27AA from the N-terminal were found at the manA's translation product. Four truncated mannanase genes were designed according to the homologous model, in which 0,81,111,141bp were deleted from the N-terminal respectively. N-truncated gene expression proved the 27aa aside the N terminal were signal peptide, and loss of the 37aa from the N terminal show little effect on mannanase activity. However, the enzyme will be inactivatied while the number of amino acid deltion was more than 47.
The manA was cloned into plasmid pEASY El express and transferred into E.coli BL21 (DE3).The recombinant plasmids were extracted from the positive transformant, and transferred into Erwinia carotovor variant CXJZU-120. Measured by circle decomposing method, the mannanase expression level of the homologous recombinant was 2/3 higher than the original host CXJZU-120, and the promotion of mannanase activity is 2.1 times.
引文
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2.张运雄,麻类生物脱胶与生物制浆酶系[J].中国麻作.2003,24(2)：14-16
3.刘正初,彭源德.苎麻生物脱胶新技术工业化生产应用研究[J].纺织学报,2001,
4.贾云鹤,张俊华,崔崇士.软腐欧文氏杆菌致病性的研究进展[J].东北农业大学学报.2007,38(1)：113-118
5.刘正初,彭德源,孙庆祥,冯湘沉,吕江南.兰麻生物脱胶工艺技术与设备.中国,发明专利,CNⅡ30216,1996.9.4
6.刘正初、彭源德、冯湘沉、邓硕苹、段盛文、郑科.一种草本纤维工厂化脱胶或制浆用高效菌剂制备方法.中国,发明专利,ZL02108820.9,20(3).10.29
7. Paul, J. Chemistry and biochemistry of semicellulose-relationship between semicellulose structure and enzymes required for hydrolysis[J].Macromol Symp.1997(120):183-196
8.Homrichhausen, Tanja M.,Hewitt, et al. Endo-beta-mannanase activity and completion of embryogenesis in imbibed carrot seed.http://abstracts.aspb.org/pb2003/public/p45/0629. html,2003,July 25-july 30
9.Wang A.,Li J.,Bewley D..Molecular cloning and characterization of an endo-beta-mannanase gene expressed in the lettuce endosperm following radical emergence[J].Seed Sci. Res.2004(14):267-276
10.Bourgault R.,Bewley J.D..Variation in C-terminal amino acids determines whether endo-p-mannanase is active or inactive in ripening tomato fruits of different cultivars[J]. plant physiol.2002(3):1254-1262
11.Goncalves P.,Cavalho B.D, Borhetti F. et al. Temperture-dependent germination and endo-β-mannanase activity in sesame seeds[J].R Bras Fisiol Veg.2001(2):139-148
12.史益敏,沈曾佑,张志良,颜季琼.魔芋球茎中的β-甘露聚糖酶[J].植物生理学报.1990(3)：306-310
13.Ootsuka S,Saga N, Suzuki KI, et al.isolation and cloning of an endo-beta-mannanase from pacific abalone aliotis discus hannai[J].J biotechol.2006,
14. Yamaura I,Nozaki Y, Matsumoto T, et al. Purification and some properties of an endo-β-mannanase from a marine molluse,littorina brevicula[J].Biosci Biotechnol Biochem.1996(4):674-676
15.Larsson A. M. Anderson L.,XU B.,et al. Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel mytilus edulis[J].J. Mol. Biol. 2006(5):1500-1510
16.李文玉,董志扬,崔福绵.枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质[J].微生物学报，2002,40(4)：420-424
17.余红英,孙远明,王慧军,杨跃生,枯草芽孢杆菌SA-22 β甘露聚糖酶及其特性[J].生物工程学报，2003,19(3)：327-331
18.Li W, Dong Z, Cui F. Purification and characterization of an endo-beta-1,4-mannanase from Bacillus subtilis BM9602[J].Acta microbiologica sinica.2000(4):420-424
19.张峻,何志敏,·胡鲲,冯耀宇,张志刚,地衣芽孢杆菌β-甘露聚糖酶的制备[J].食品发酵工业.2001,27(2)：5-7
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