橡胶树内生沙雷氏菌脂肪酶基因SmLipase1的克隆和表达分析
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摘要
脂肪酶在洗涤剂、食品、纸浆以及生物柴油等多个工业领域被广泛应用。不同的应用领域对脂肪酶的酶学特性有不同的要求,因此挖掘脂肪酶基因资源具有重要意义。本研究报道从橡胶树内生菌Serratiamarcescens ITBB5-1中克隆得到一个脂肪酶基因,命名为Sm Lipase1,编码662个氨基酸,pI 4.82,能分泌脂肪酶到培养基中。构建重组表达载体pET22b-Sm Lipase-1,转化大肠杆菌菌株BL21(DE3)。本研究表明该转基因菌株能高效表达脂肪酶,并在平板培养基上形成较大的透明圈,转基因菌株发酵培养所获得的培养液酶活性达到26 U/mL。此外,该酶还具有催化油脂与甲醇反应生产脂肪酸甲酯的活性,在生物柴油领域具有一定的开发价值。
Lipases are widely used in multiple industries such as detergent, food, paper pulp and biodiesel industry.Different application fields require different enzymatic properties of lipase, therefore, study on lipase gene resources is beneficiary to the industries. We cloned a lipase gene named as SmLipase1 from an endophytic bacterial strain ITBB5-1 from rubber tree. This gene encoded a total of 662 amino acids with pI of 4.82, which can secret lipase into the medium. Recombinant plasmid pET22 b-SmLipase-1 was constructed to transform Escherichia coli strain BL21(DE3). The study result indicated that the gene strain could high-effectively express lipase, and form a large transparent circle on the plate medium. The enzyme activity of the medium obtained by fermentation and culture of transgenic strains reached 26 U/mL. Besides, this enzyme could catalyze oil to react with methanol so as to produce fatty acid methyl ester, which has certain development value in the field of biodiesel.
Lipases are widely used in multiple industries such as detergent, food, paper pulp and biodiesel industry.Different application fields require different enzymatic properties of lipase, therefore, study on lipase gene resources is beneficiary to the industries. We cloned a lipase gene named as SmLipase1 from an endophytic bacterial strain ITBB5-1 from rubber tree. This gene encoded a total of 662 amino acids with pI of 4.82, which can secret lipase into the medium. Recombinant plasmid pET22 b-SmLipase-1 was constructed to transform Escherichia coli strain BL21(DE3). The study result indicated that the gene strain could high-effectively express lipase, and form a large transparent circle on the plate medium. The enzyme activity of the medium obtained by fermentation and culture of transgenic strains reached 26 U/mL. Besides, this enzyme could catalyze oil to react with methanol so as to produce fatty acid methyl ester, which has certain development value in the field of biodiesel.
引文
Akoh C.C.,Chang S.W.,Lee G.C.,and Shaw J.F.,2007,Enzymatic approach to biodiesel production,J.Agric.Food Chem.,55(22):8995-9005
Arpigny J.L.,and Jaeger K.E.,1999,Bacterial lipolytic enzymes:classification and properties,Biochem.J.,343(1):177-183
Contesini F.J.,Lopes D.B.,Macedo G.A.,Nascimento M.D.G.,and Carvalho P.D.O.,2010,Aspergillus,sp.lipase:Potential biocatalyst for industrial use,Journal of Molecular Catalysis B Enzymatic,67(3-4):163-171
Fernandez-Lafuente R.,2010,Lipase from Thermomyces lanuginosus:uses and prospects as an industrial biocatalyst,Journal of Molecular Catalysis B Enzymatic,62(3-4):197-212
Gupta R.,Rathi P.,Gupta N.,and Bradoo S.,2006,Lipase assays for conventional and molecular screening:an overview,Biotechnol.Appl.Biochem.,37(1):63-71
Holmquist M.,1998,Insights into the molecular basis for fatty acyl specificities of lipase s from geotrichum candidum and candida rugosa,Chem.Phys.Lipids,93(1-2):57-66
Hsu A.J.K.,Jones K.,Foglia T.A.,and Marmer W.N.,2002,Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel,Biotechnol.Appl.Biochem.,36(3):181-186
Jaeger K.E.,Ransac S.,Dijkstra B.W.,Colson C.,Van H.M.,and Misset O.,1994,Bacterial lipases,FEMS Microbiology Reviews,15(1):29-63
Jaeger K.E.,and Reetz M.T.,1998,Microbial lipases form versatile tools for biotechnology,Trends in Biotechnology,16(9):396-403
Jaeger K.E.,Dijkstra B.W.,and Reetz M.T.,1999,Bacterial biocatalysts:molecular biology,three-dimensional structures,and biotechnological applications of lipases,Annu.Rev.Microbiol.,53(1):315-351
Li C.L.,Cui J.Z.,Wang Y.,and Cui C.X.,2005,The host system of medical molecular farming,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(1):140-146(李翠玲,崔继哲,王洋,崔彩霞,2005,医药分子农业的受体系统评述,分子植物育种,3(1):140-146)
Liu H.,Liu J.,Zhang Y.,Xiao Q.,and Wang Z.,2009,The latest exploratory development trends on microbial lipase,Shipin Yanjiu Yu Kaifa(Food Research and Development),30(1):141-143(刘海洲,刘均洪,张媛媛,肖强,王志萍,2009,微生物脂肪酶的最新应用研究进展,食品研究与开发,30(1):141-143)
Ma Q.L.,Sun X.P.,Gong S.F.,and Zhang J.M.,2010,Screening and identification of a highly lipolytic bacterial strain from barbecue sites in Hainan and characterization of its lipase,Annals of Microbiology,60(3):429-437
Ma Q.L.,and Zhang J.M.,2010,Screening and classfication for lipase-producing strain C7828-5 and optimization of its fer mentation conditions,Gongye Weishengwu(Industrial Microbiology),40(6):39-44(麻琼丽,张家明,2010,产脂肪酶菌株C7828-5的筛选、鉴定以及产酶条件的优化,工业微生物,40(6):39-44)
Noureddini H.,Gao X.,and Philkana R.S.,2005,Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil,Bioresour.Technol.,96(7):769-777
Qian Z.,and Lutz S.,2005,Improving the catalytic activity of Candida antarctica lipase B by circular permutation,Journal of the American Chemical Society,127(39):13466-13467
Tan D.G.,Fu L.L.,Han B.Y.,Sun X.P.,Zheng P.,and Zhang J.M.,2015,Identification of an endophytic antifungal bacterial strain isolated from the rubber tree and its application in the biological control of banana fusarium wilt,PLoS One,10(7):e0131974
Ying M.,and Chen G.,2007,Study on the production of biodiesel by magnetic cell biocatalyst based on lipase-producing Bacillus subtilis,Appl.Biochem.Biotechnol.,137(1-12):793-803
Zhu Q.,Chen Y.,Zhang B.,and Huang R.,2010,Cloning,expression and characterization of the lipase gene from Serratia marcescens,Shengwu Jishu(Biotechnology),20(5):12-16(朱绮霞,陈英,张搏,黄日波,2010,粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究,生物技术,20(5):12-16)
Zhu H.Y.,Wu C.X.,Hu B.Z.,Li W.B.,and Han T.F.,2004,Progess in plant-derived oral vaccine production,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),2(3):443-448(朱会英,吴存祥,胡宝忠,李文彬,韩天富,2004,利用植物生物反应器生产口服疫苗的进展,分子植物育种,2(3):443-448)
Zhou W.,Yang J.K.,Huang Y.,and Yan Y.J.,2007,Study on production of biodiesel with compound lipases,Shengwu Jiagong Guocheng(Chinese Journal of Bioprocess Engineering),5(3):20-26(周位,杨江科,黄瑛,闫云君,2007,复合脂肪酶催化生物柴油的初步研究,生物加工过程,5(3):20-26)
Arpigny J.L.,and Jaeger K.E.,1999,Bacterial lipolytic enzymes:classification and properties,Biochem.J.,343(1):177-183
Contesini F.J.,Lopes D.B.,Macedo G.A.,Nascimento M.D.G.,and Carvalho P.D.O.,2010,Aspergillus,sp.lipase:Potential biocatalyst for industrial use,Journal of Molecular Catalysis B Enzymatic,67(3-4):163-171
Fernandez-Lafuente R.,2010,Lipase from Thermomyces lanuginosus:uses and prospects as an industrial biocatalyst,Journal of Molecular Catalysis B Enzymatic,62(3-4):197-212
Gupta R.,Rathi P.,Gupta N.,and Bradoo S.,2006,Lipase assays for conventional and molecular screening:an overview,Biotechnol.Appl.Biochem.,37(1):63-71
Holmquist M.,1998,Insights into the molecular basis for fatty acyl specificities of lipase s from geotrichum candidum and candida rugosa,Chem.Phys.Lipids,93(1-2):57-66
Hsu A.J.K.,Jones K.,Foglia T.A.,and Marmer W.N.,2002,Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel,Biotechnol.Appl.Biochem.,36(3):181-186
Jaeger K.E.,Ransac S.,Dijkstra B.W.,Colson C.,Van H.M.,and Misset O.,1994,Bacterial lipases,FEMS Microbiology Reviews,15(1):29-63
Jaeger K.E.,and Reetz M.T.,1998,Microbial lipases form versatile tools for biotechnology,Trends in Biotechnology,16(9):396-403
Jaeger K.E.,Dijkstra B.W.,and Reetz M.T.,1999,Bacterial biocatalysts:molecular biology,three-dimensional structures,and biotechnological applications of lipases,Annu.Rev.Microbiol.,53(1):315-351
Li C.L.,Cui J.Z.,Wang Y.,and Cui C.X.,2005,The host system of medical molecular farming,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(1):140-146(李翠玲,崔继哲,王洋,崔彩霞,2005,医药分子农业的受体系统评述,分子植物育种,3(1):140-146)
Liu H.,Liu J.,Zhang Y.,Xiao Q.,and Wang Z.,2009,The latest exploratory development trends on microbial lipase,Shipin Yanjiu Yu Kaifa(Food Research and Development),30(1):141-143(刘海洲,刘均洪,张媛媛,肖强,王志萍,2009,微生物脂肪酶的最新应用研究进展,食品研究与开发,30(1):141-143)
Ma Q.L.,Sun X.P.,Gong S.F.,and Zhang J.M.,2010,Screening and identification of a highly lipolytic bacterial strain from barbecue sites in Hainan and characterization of its lipase,Annals of Microbiology,60(3):429-437
Ma Q.L.,and Zhang J.M.,2010,Screening and classfication for lipase-producing strain C7828-5 and optimization of its fer mentation conditions,Gongye Weishengwu(Industrial Microbiology),40(6):39-44(麻琼丽,张家明,2010,产脂肪酶菌株C7828-5的筛选、鉴定以及产酶条件的优化,工业微生物,40(6):39-44)
Noureddini H.,Gao X.,and Philkana R.S.,2005,Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil,Bioresour.Technol.,96(7):769-777
Qian Z.,and Lutz S.,2005,Improving the catalytic activity of Candida antarctica lipase B by circular permutation,Journal of the American Chemical Society,127(39):13466-13467
Tan D.G.,Fu L.L.,Han B.Y.,Sun X.P.,Zheng P.,and Zhang J.M.,2015,Identification of an endophytic antifungal bacterial strain isolated from the rubber tree and its application in the biological control of banana fusarium wilt,PLoS One,10(7):e0131974
Ying M.,and Chen G.,2007,Study on the production of biodiesel by magnetic cell biocatalyst based on lipase-producing Bacillus subtilis,Appl.Biochem.Biotechnol.,137(1-12):793-803
Zhu Q.,Chen Y.,Zhang B.,and Huang R.,2010,Cloning,expression and characterization of the lipase gene from Serratia marcescens,Shengwu Jishu(Biotechnology),20(5):12-16(朱绮霞,陈英,张搏,黄日波,2010,粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究,生物技术,20(5):12-16)
Zhu H.Y.,Wu C.X.,Hu B.Z.,Li W.B.,and Han T.F.,2004,Progess in plant-derived oral vaccine production,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),2(3):443-448(朱会英,吴存祥,胡宝忠,李文彬,韩天富,2004,利用植物生物反应器生产口服疫苗的进展,分子植物育种,2(3):443-448)
Zhou W.,Yang J.K.,Huang Y.,and Yan Y.J.,2007,Study on production of biodiesel with compound lipases,Shengwu Jiagong Guocheng(Chinese Journal of Bioprocess Engineering),5(3):20-26(周位,杨江科,黄瑛,闫云君,2007,复合脂肪酶催化生物柴油的初步研究,生物加工过程,5(3):20-26)