圆弧青霉碱性脂肪酶基因的克隆、定点突变及其表达的研究
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摘要
本论文以圆弧青霉BD26(Penicillium cyclopium BD26)为出发菌株,采用RT-PCR扩增出编码脂肪酶的774bp成熟肽基因片段。序列测定结果表明,该序列与圆弧青霉基因序列(GenBank登录号:AF274320)和扩展青霉脂肪酶基因序列(GenBank登录号:AAG22769)同源性高达99%。
脂肪酶基因Lip PD克隆入大肠杆菌表达载体pET-28a、pET-25b和pTrc99a,构建重组工程菌BL21/pET-28a-Lip PD、BL21/pTrc99a-Lip PD和BL21/pET-25b-Lip PD。三种重组菌体在固体琼脂显色平板上都形成了明显透明圈。SDS-PAGE电泳显示,在三种宿主菌中都含有分子量约为27 kDa的活性脂肪酶蛋白。对工程菌BL21/pTrc99a-Lip PD表达条件优化结果表明:该脂肪酶在pTrc99a原核表达系统中,培养至OD600为0.8时,加入IPTG至终浓度为0.5mmol/L,在37℃诱导培养2h后比酶活为11.74U/mg。对工程菌BL21/pET-28a-Lip PD表达条件优化结果表明:该脂肪酶在pET原核表达系统中,培养至OD600为0.5时,加入IPTG至终浓度为1.0mmol/L ,在32℃诱导培养1h比酶活达29.30U/mg。工程菌BL21/pET-25b-Lip PD周质溶液经NaOH滴定法测定,比酶活为24.19U/mg。
经定点突变证明,S132、D188和H241的确为Lip PD的三联体催化活性中心。LipG24C的最适反应温度仍为25℃,比酶活是20.73U/mg,为野生型的85.71%。LipA252C的最适反应温度为30℃,与原酶相比提高了5℃,比酶活为34.59U/mg,是野生型的1.43倍。
将该酶成熟肽基因片段克隆入酵母表达载体pPIC9K中,电转化毕赤酵母宿主GS115。经过抗性和表型筛选,从抗性为3.00mg/mL G418的平板上挑选15株重组工程菌GS115/pPIC9k-LipPD,在其发酵上清液中没有检测到酶活,而在其胞内检测到了脂肪酶酶活,仅为10.53U/mg。SDS-PAGE电泳显示,重组蛋白在宿主菌GS115中表达量不高。
Total RNA was isolated from Penicillium Cyclopium BD26 with TRIzol reagent by one step method.A 774 bp Lip PD gene was amplified by RT-PCR. Analysis of nucleotide sequence of the fragment suggested that Lip PD gene shared high similarity with the gene sequences of Penicillium Cyclopium(GenBank:AF274320)and Penicillium Expansum (GenBank:AAG22769)up to 99% identity.
The cDNA encoding the mature peptide was cloned into expression plasmid pET-28a(+)、pTrc-99a and pET-25b respectively, and expressed in Escherichia coli BL21(DE3) by IPTG induction respectively. A specific protein band of 27 kDa was detected by SDS–PAGE. With the halos appeared on solid agar plate,the activity of the recombinant lipase protein was detected. The further study on optimizing induction of BL21/pET-28a-Lip PD showed that the expression level of lipase was up to the highest specific activities of 29.30 U/mg with induction temperature 32℃, induction time 1h, the concentration of IPTG 1.0 mmol/L, strain density OD600 0.5. The further study on optimizing induction of BL21/ pTrc99a-Lip PD showed that the expression level of lipase exceeded the highest specific activity of 11.74U/mg with induction temperature 32℃, induction time 2h, the concentration of IPTG 0.5mmol/L, strain density OD600 0.8. The recombinant lipase protein of BL21/pET-25a-Lip PD was expressed in E. coli periplasm and the specific activity of the recombinant lipase was up to 24.19U/mg.
The active site triad residues of Lip PD included Ser132,Asp188 and His241and they were identified by site-directed mutagenesis LipS132A、LipD188A and LipH241 ,respectively.
The specific activity of LipG24C was 20.73U/mg at 25℃, which was 85.71% of the wild type at the same conditions. The optimum temperature of mutant was the same to the wild type . The optimum temperature for LipA252C was higher by 5℃than that of the wild type Lip PD. The specific activity of LipA252C was 34.59U/mg , which was 143% of the wild type at the same conditions.
The gene of mature peptide was inserted into Pichia pastoris expression vector pPIC9K, resulting in the recombinant plasmid pPIC9K-LipPD. The pPIC9K-Lip PD was linearized by Sal I ,and then transformed into P. pastoris GS115 by electroporation. The intracellular activity of the recombinant lipase was up to 10.53U/mg. A specific protein band of 27 kDa was only detected by SDS–PAGE and the protein level was very low.
脂肪酶基因Lip PD克隆入大肠杆菌表达载体pET-28a、pET-25b和pTrc99a,构建重组工程菌BL21/pET-28a-Lip PD、BL21/pTrc99a-Lip PD和BL21/pET-25b-Lip PD。三种重组菌体在固体琼脂显色平板上都形成了明显透明圈。SDS-PAGE电泳显示,在三种宿主菌中都含有分子量约为27 kDa的活性脂肪酶蛋白。对工程菌BL21/pTrc99a-Lip PD表达条件优化结果表明:该脂肪酶在pTrc99a原核表达系统中,培养至OD600为0.8时,加入IPTG至终浓度为0.5mmol/L,在37℃诱导培养2h后比酶活为11.74U/mg。对工程菌BL21/pET-28a-Lip PD表达条件优化结果表明:该脂肪酶在pET原核表达系统中,培养至OD600为0.5时,加入IPTG至终浓度为1.0mmol/L ,在32℃诱导培养1h比酶活达29.30U/mg。工程菌BL21/pET-25b-Lip PD周质溶液经NaOH滴定法测定,比酶活为24.19U/mg。
经定点突变证明,S132、D188和H241的确为Lip PD的三联体催化活性中心。LipG24C的最适反应温度仍为25℃,比酶活是20.73U/mg,为野生型的85.71%。LipA252C的最适反应温度为30℃,与原酶相比提高了5℃,比酶活为34.59U/mg,是野生型的1.43倍。
将该酶成熟肽基因片段克隆入酵母表达载体pPIC9K中,电转化毕赤酵母宿主GS115。经过抗性和表型筛选,从抗性为3.00mg/mL G418的平板上挑选15株重组工程菌GS115/pPIC9k-LipPD,在其发酵上清液中没有检测到酶活,而在其胞内检测到了脂肪酶酶活,仅为10.53U/mg。SDS-PAGE电泳显示,重组蛋白在宿主菌GS115中表达量不高。
Total RNA was isolated from Penicillium Cyclopium BD26 with TRIzol reagent by one step method.A 774 bp Lip PD gene was amplified by RT-PCR. Analysis of nucleotide sequence of the fragment suggested that Lip PD gene shared high similarity with the gene sequences of Penicillium Cyclopium(GenBank:AF274320)and Penicillium Expansum (GenBank:AAG22769)up to 99% identity.
The cDNA encoding the mature peptide was cloned into expression plasmid pET-28a(+)、pTrc-99a and pET-25b respectively, and expressed in Escherichia coli BL21(DE3) by IPTG induction respectively. A specific protein band of 27 kDa was detected by SDS–PAGE. With the halos appeared on solid agar plate,the activity of the recombinant lipase protein was detected. The further study on optimizing induction of BL21/pET-28a-Lip PD showed that the expression level of lipase was up to the highest specific activities of 29.30 U/mg with induction temperature 32℃, induction time 1h, the concentration of IPTG 1.0 mmol/L, strain density OD600 0.5. The further study on optimizing induction of BL21/ pTrc99a-Lip PD showed that the expression level of lipase exceeded the highest specific activity of 11.74U/mg with induction temperature 32℃, induction time 2h, the concentration of IPTG 0.5mmol/L, strain density OD600 0.8. The recombinant lipase protein of BL21/pET-25a-Lip PD was expressed in E. coli periplasm and the specific activity of the recombinant lipase was up to 24.19U/mg.
The active site triad residues of Lip PD included Ser132,Asp188 and His241and they were identified by site-directed mutagenesis LipS132A、LipD188A and LipH241 ,respectively.
The specific activity of LipG24C was 20.73U/mg at 25℃, which was 85.71% of the wild type at the same conditions. The optimum temperature of mutant was the same to the wild type . The optimum temperature for LipA252C was higher by 5℃than that of the wild type Lip PD. The specific activity of LipA252C was 34.59U/mg , which was 143% of the wild type at the same conditions.
The gene of mature peptide was inserted into Pichia pastoris expression vector pPIC9K, resulting in the recombinant plasmid pPIC9K-LipPD. The pPIC9K-Lip PD was linearized by Sal I ,and then transformed into P. pastoris GS115 by electroporation. The intracellular activity of the recombinant lipase was up to 10.53U/mg. A specific protein band of 27 kDa was only detected by SDS–PAGE and the protein level was very low.
引文
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2. G.D.Haki,S.K.Rakshit.Developments in industrially important thermostable enzy- mes: a review[J]. Bioresource Technology,2003,89(1):17—34
3.邬显章,邬敏辰.脂肪酶分子生物学的研究进展[J].食品与生物技术,2002,21(1): 94—98
4.张树政主编.酶制剂工业[M]:下册.北京:科学出版社,1984.655—670
5. Benjamin S,Pandey A.Candida rugosa lipases:molecular biology and versatility in biotechnology[J]. Yeast,1998,14(12):1069—1087
6. Schloss P D,Handelsman J.Biotechnological prospects from metagenomics[J].Curr. Opin.Biotechnol, 2003,14(3):303—310
7. Streit W R,Daniel R,Jaeger K E.Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms[J].Curr Opin Biotechno1,2004,15(4):285—290
8. Daniel R.The soil metagenome-a rich resource for the discovery of novel natural products[J].Curr.Opin.Biotechnol,2004,15(3):199—204
9. Yun J,Kang S,Park S,et al.Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library[J].Appl.Environ.Microbiol,2004, 70 (12): 7229—7235
10. Lee S W,Won K,Lim H K et al.Screening for novel lipolytic enzymes from uncultured soil microorganisms[J].Appl.Microbiol.Biotechnol,2004,65(6):720—726.
11.宋欣,魏留梅,刘瑞田,等.脂肪酶高产菌株选育和育种库的建立[J].微生物学通报,2002,29(1):6—10.
12. Yeoh H H,Wong F M,Lim G..Screening for Fungallipase using chromogenic lipid substrates[J].Mycologia,1986,78(2):298—300.
13.谢舜珍,吴琼发,徐家立.脂肪酶产生菌Geotrichum sp.AS 2.1135产酶条件一般性质的研究[J].微生物学报,1986,26(2):262—264.
14.王美英,徐家立.白地酶一新变种的鉴定及其脂肪酶的研究[J].微生物学报,1989,29(1):1—6.
15.陶文沂,邬显章,向瑞春,等.脂肪酶产生菌地霉7203菌株的分离和诱变[J].微生物学报,1990,30(3):216—222
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