嗜热脂肪芽孢杆菌氨基酰化酶基因在大肠杆菌中的克隆，表达及细胞固定化研究
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摘要
甲硫氨酸是一个含硫的必需氨基酸,在饲料、医药、保健和食品工业领域都有着广泛的用途。甲硫氨酸是一个高附加值产品,也是我国为数不多需从国外大量进口的氨基酸之一。寻找成本低,产量高的甲硫氨酸生产工艺和方法成了近年来国内氨基酸生产领域的一个研究热点。
本研究以嗜热脂肪芽孢杆菌为原始菌,从中克隆出氨基酰化酶基因,并转化到大肠杆菌中进行诱导表达,同时对菌体细胞进行了直接固定化研究,以N-乙酰-DL-甲硫氨酸为底物,探讨适合工业化拆分DL-甲硫氨酸的应用可能性。
方法是以嗜热脂肪芽孢杆菌基因组DNA为模板,通过PCR扩增出编码氨基酰化酶amaA基因,与pMD-18T vector重组,对阳性克隆菌的序列分析显示,与Genebank中amaA(Y08753)序列完全一致。用NdeⅠ和EcoRⅠ双酶切pET-28a(+)和pMD-18T vector阳性克隆,T4 DNA连接酶连接,构建了amaA-pET28a(+)的重组表达载体,转化到E.coli BL21中进行诱导表达。SDS-PAGE分析和氨基酰化酶活性测定都表明,amaA基因在大肠杆菌中获得了高效表达。研究了IPTG的浓度,IPTG加入的时间和诱导表达的时间等。结果显示的最佳条件为:37℃,200rpm培养,菌浓度OD_(600)在0.8～1.0之间时加入IPTG诱导,IPTG终浓度0.4mmol/L,诱导表达6h后,获得了氨基酰化酶活性为1,043U/g湿菌体。
以N-乙酰-DL-甲硫氨酸为底物的拆分研究显示:底物浓度＜0.08mol/L时,反应速度随底物浓度增加呈线性关系;55℃酶反应,游离菌的K_m为4.5mmol/L,固定化后的K_m为14.7mmol/L。酶学性质研究同时表明,65℃酶的催化效率最高,最适pH为7.0;固定化后酶pH稳定范围更广,热稳定性也更高;连续10批次拆分N-乙酰-DL-甲硫氨酸,活性仅损失20%左右;4℃条件下保存23天仍保留有固定化时73.6%的酶活性。
菌体直接固定化研究显示,最佳条件为:3%卡拉胶包埋30%菌体细胞,以1.25%多乙烯多胺渗透交联固定化菌体细胞10min和0.1%戊二醛硬化处理20min,与未固定菌体比较,菌体固定化后仍保留有83%的酶活性。
本研究显示,重组大肠杆菌表达的氨基酰化酶有着很好的应用前景。
Methionine is a sulfur-bearing essential amino acid,which is widely used in forage,pharmaceuticals,health and food industry.Methionine is a high value-added product and one of the imported-dependent amino acids in China.So recent years, looking for a producing method and process of methionine with lower cost and higher output has become a research hot spot in domestic amino acid industry areas.
In this study,Bacillus Stearothermophilus was used as an original strain,the amaA gene encoding aminoacylase was cloned and expressed in E.coli.Using N-acetyl-DL-methionine as a substrate,the recombinant E.coli cell immobilization was investigated to explore the application prospect on L-methionine industry.
The genome DNA from Bacillus Stearothermophilus was used as a template for PCR.The PCR product was cloned into pMD-18T vector for sequence analysis which indicated the same identity of amaA(Y08753) on Genebank.The plasmid pET-28a(+) and pMD-18T with amaA gene was digested with NdeⅠand EcoRⅠand ligated by T4 DNA ligase to construct amaA-pET28a(+).The recombinant plasmid was transformed into E.coli BL22(DE3) to express the amaA gene.Both SDS-PAGE and aminoacylase assay showed that the cloned strain was effective expressed.The expression condition study included concentration of IPTG,inducing period and induced time etc. The optimized condition was:37℃200rpm shaking;added 0.4mmol/L IPTG when OD_(600) reached 0.8～1.0,culturing 6 hours with IPTG inducement.The enzyme activity was 1,043U/g cells under the optimized condition.
Cell immobilization research showed that the optimized condition was 30%of E.coli cell were embed by 3%carrageenan,treated for 20 minutes with 2.25% polyethylene polyamines to permeate and crosslink,then hardened these cells for 20 minutes with 0.15%glutaraldehyde.After immobilization,the cell preserved 83%of enzyme activity.
Using N-acetyl-DL-methionine as a substrate,the properties of immobilized bacilli were studied.The results revealed a linear relationship between reaction velocity and substrate concentration when N-acetyl-DL-methionine was under 0.08mol/L. The K_m of free cell was 4.5mmol/L while immobilized cell was 14.7mmol/L at 55℃. Enzyme properties showed that the optimum temperature and pH are 65℃and 7.0 respectively.Compare to free cell,the immobilized cell had a higher thermal and pH stability.After reacting continuously for 10 times,the enzyme activity lost just only 20%.When placed at 4℃for 23 days,73.6%of enzyme activity was preserved compared to original immobilized cell.
These results indicated that there is an excellent industrial application prospect of immobilized recombinant E.coli with amaA gene.
本研究以嗜热脂肪芽孢杆菌为原始菌,从中克隆出氨基酰化酶基因,并转化到大肠杆菌中进行诱导表达,同时对菌体细胞进行了直接固定化研究,以N-乙酰-DL-甲硫氨酸为底物,探讨适合工业化拆分DL-甲硫氨酸的应用可能性。
方法是以嗜热脂肪芽孢杆菌基因组DNA为模板,通过PCR扩增出编码氨基酰化酶amaA基因,与pMD-18T vector重组,对阳性克隆菌的序列分析显示,与Genebank中amaA(Y08753)序列完全一致。用NdeⅠ和EcoRⅠ双酶切pET-28a(+)和pMD-18T vector阳性克隆,T4 DNA连接酶连接,构建了amaA-pET28a(+)的重组表达载体,转化到E.coli BL21中进行诱导表达。SDS-PAGE分析和氨基酰化酶活性测定都表明,amaA基因在大肠杆菌中获得了高效表达。研究了IPTG的浓度,IPTG加入的时间和诱导表达的时间等。结果显示的最佳条件为:37℃,200rpm培养,菌浓度OD_(600)在0.8～1.0之间时加入IPTG诱导,IPTG终浓度0.4mmol/L,诱导表达6h后,获得了氨基酰化酶活性为1,043U/g湿菌体。
以N-乙酰-DL-甲硫氨酸为底物的拆分研究显示:底物浓度＜0.08mol/L时,反应速度随底物浓度增加呈线性关系;55℃酶反应,游离菌的K_m为4.5mmol/L,固定化后的K_m为14.7mmol/L。酶学性质研究同时表明,65℃酶的催化效率最高,最适pH为7.0;固定化后酶pH稳定范围更广,热稳定性也更高;连续10批次拆分N-乙酰-DL-甲硫氨酸,活性仅损失20%左右;4℃条件下保存23天仍保留有固定化时73.6%的酶活性。
菌体直接固定化研究显示,最佳条件为:3%卡拉胶包埋30%菌体细胞,以1.25%多乙烯多胺渗透交联固定化菌体细胞10min和0.1%戊二醛硬化处理20min,与未固定菌体比较,菌体固定化后仍保留有83%的酶活性。
本研究显示,重组大肠杆菌表达的氨基酰化酶有着很好的应用前景。
Methionine is a sulfur-bearing essential amino acid,which is widely used in forage,pharmaceuticals,health and food industry.Methionine is a high value-added product and one of the imported-dependent amino acids in China.So recent years, looking for a producing method and process of methionine with lower cost and higher output has become a research hot spot in domestic amino acid industry areas.
In this study,Bacillus Stearothermophilus was used as an original strain,the amaA gene encoding aminoacylase was cloned and expressed in E.coli.Using N-acetyl-DL-methionine as a substrate,the recombinant E.coli cell immobilization was investigated to explore the application prospect on L-methionine industry.
The genome DNA from Bacillus Stearothermophilus was used as a template for PCR.The PCR product was cloned into pMD-18T vector for sequence analysis which indicated the same identity of amaA(Y08753) on Genebank.The plasmid pET-28a(+) and pMD-18T with amaA gene was digested with NdeⅠand EcoRⅠand ligated by T4 DNA ligase to construct amaA-pET28a(+).The recombinant plasmid was transformed into E.coli BL22(DE3) to express the amaA gene.Both SDS-PAGE and aminoacylase assay showed that the cloned strain was effective expressed.The expression condition study included concentration of IPTG,inducing period and induced time etc. The optimized condition was:37℃200rpm shaking;added 0.4mmol/L IPTG when OD_(600) reached 0.8～1.0,culturing 6 hours with IPTG inducement.The enzyme activity was 1,043U/g cells under the optimized condition.
Cell immobilization research showed that the optimized condition was 30%of E.coli cell were embed by 3%carrageenan,treated for 20 minutes with 2.25% polyethylene polyamines to permeate and crosslink,then hardened these cells for 20 minutes with 0.15%glutaraldehyde.After immobilization,the cell preserved 83%of enzyme activity.
Using N-acetyl-DL-methionine as a substrate,the properties of immobilized bacilli were studied.The results revealed a linear relationship between reaction velocity and substrate concentration when N-acetyl-DL-methionine was under 0.08mol/L. The K_m of free cell was 4.5mmol/L while immobilized cell was 14.7mmol/L at 55℃. Enzyme properties showed that the optimum temperature and pH are 65℃and 7.0 respectively.Compare to free cell,the immobilized cell had a higher thermal and pH stability.After reacting continuously for 10 times,the enzyme activity lost just only 20%.When placed at 4℃for 23 days,73.6%of enzyme activity was preserved compared to original immobilized cell.
These results indicated that there is an excellent industrial application prospect of immobilized recombinant E.coli with amaA gene.
引文
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5.徐铮奎.我国氨基酸工业现状与前景展望.中国制药信息2000,16(1)
6.毛忠贵.国际氨基酸产业发展动态.中国食品添加剂2001,1
7.陈燕珠.氨基酸的应用与发展趋势.化工时刊2001,7
8.余敦寿.氨基酸在医药及食品产业的发展趋势.中国食品卫生杂志.2004,9
9.Alessandra B.Production of D-phenylglycine from racemic DL-phenylglycine via isoelectrically trapped Penicillin G acylase.Biochem Bioeng,1998,60(4):454-461
10.侯仲轲.医药中间体市场动态与发展趋势.精细化工中间体.2006,36(6)
11.朱汪群,孙复华.赖氨酸市场前景及发展论述.发酵科技通讯2006,435(2)
12.王敏.甲硫氨酸的生产与应用.化工之友2001,2
13.温占玺.甲硫氨酸的制备与应用.化工之友1998,2
14.崔小明.甲硫氨酸的生产应用与市场前景.化工生产与技术2000,7(4)
15.王佩琳,徐泽辉.国内外DL-甲硫氨酸生产现状及市场分析.石油化工技术经济.2003(6)
16.中华人民共和国海关总署统计数据
17.James Gomes,Dharmendra Kumar.Production of L-methionine by submerged fermentation:A review.Enzyme and Microbial Technology,2005,37:3-18
18.张清玉,谭欣,赵林等.乙酰-D(L)-甲硫氨酸消旋工艺的研究.化学工业与工程2004,21(3)
19.Andreas S,Bommarius.Biocatalysis to amino acid-based chiral pharmaceuticals examples and perspectives.Journal of Molecular Catalysis B:Enzymatic 1998(5):1-11
20.Masanori Mitta,Ikunoshin Kato.The Primary Structure of Porcine Aminoacylase 1 Deduced from cDNA Sequence.J.Biochem,1992,Vol.112,No.6 737-742
21.王洪睿,周海梦.氨基酰化酶分了中并不存在二硫键.中国科学B辑1995,1
22.Schneider F.Chemical investigations on pig kidney aminoacylase.Biochim Biophys Acta,1976,445(2):446-45723.张彤,周海梦.Zn~(2+)对氨基酰化酶构象及其稳定性的影响.生物物理学报1994,6
24.Gottfried J,Palm I.Aminoacylase I from porcine kidney:Identification and characterization of two major protein domains.Journal of Protein Chemistry,1995,5 14(4)25.Gilles I.New isolation procedure for swine kidneyacylase Kinetics of Co~(2+),Mn~(2+),Mi~(2+) and enzymes.Z Naturforsch,1984,39(9-10):1017-1020
26.Holger Lindnera,Sabine H(o|¨)pfnera.The distribution of aminoacylase I among mammalian species and localization of the enzyme in porcine kidney.Biochim 2000(82):129-137
27.施华芳,黄伟达.水稻氨基酰化酶的分类、纯化与性质分析.生物化学杂志,1997,13(1)
28.Peter Curley,Douwe van Sinderen.Identification and characterisation of a gene encoding aminoacylase activity from Lactococcus lactis MG1363.FEMS Microbiology Letters,2000(183):177-182
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