Acinetobacter sp.胆固醇酯酶的分离纯化与酶学特性的研究
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摘要
胆固醇酯酶(CHE:EC 3.1.1.13)是一种重要的甾体酯酶,在食品、医药、造纸等方面有着广泛的用途。本研究的目的是为医药与工业提供合适的酶源,主要开展了以下几个方面工作。
首先,经过筛选从大型肉食动物肠道中获得9株胆固醇酯酶高产菌株,其中1株表现出高于其他菌株的产酶能力,命名为CHE4-1。经形态及生理生化鉴定、16S rDNA(GenBank登录ID号:FJ649602)分析,CHE4-1鉴定为Acinetobactersp。迄今为止,未见不动杆菌属(Acinetobacter)产胆固醇酯酶的报道。
其次,对该菌株的生长与产酶条件进行了研究,提出了分段控制的发酵策略。产酶条件优化研究表明,其最适培养基为:胆固醇油酸酯0.2%,用0.15%TritonX-100助溶,蛋白胨0.05%,NaNO_3 0.008%,硼酸0.0075%,CaCl_20.03%,MgSO_4.7H_2O 0.0125%,FeSO_4.7H_2O 0.0005%,KH_2PO_4 0.037%;其最适培养条件为:起始pH6.9,250ml发酵瓶最佳装液量为35ml,30℃,发酵时间48 h;酶活力达到950U/L。
然后,对胆固醇酯酶进行纯化。发酵上清液经超滤浓缩,DEAE-Sepharose离子交换层析、Phenyl-Sepharose CL-4B疏水层析和Superdex-50凝胶层析分离纯化获得胆固醇酯酶。纯化后的胆固醇酯酶比活力为7.15 U/mg,纯化收率为27.02%,纯化倍数为9.67倍。纯化后的胆固醇酯酶经HPLC纯度检测达98.22%,SDS-PAGE为单一条带。SDS-PAGE和Superdex-75凝胶层析分析显示为约6.5kDa的单链蛋白,是目前为止所发现的最小的胆固醇酯酶。
最后,对胆固醇酯酶的酶学特性进行了研究,结果表明,该胆固醇酯酶的pH稳定范围为4-10:最适pH和最适温度分别为pH7.0和45℃,该酶催化胆固醇亚油酸酯、胆固醇油酸酯的米氏常数Km分别为1.75×10~(-3)mol/L、5.36×10~(-4)mol/L。该酶对长链和短链胆固醇酯均具有较高的催化活力。大多数金属离子对胆固醇酯酶抑制作用不明显;Ca~(2+)、Zn~(2+)、硼酸对胆固醇酯酶有微弱激活作用;Cu~(2+)、TritonX-100对胆固醇酯酶不具明显作用,显示了良好的临床应用价值。
Cholesterol esterase(CHE;EC 3.1.1.13) is an important steroidal esterase, it is widely used in food processing and medical assay and paper industry.In present study,several cholesterol esterase-producing bacterial strains were isolated,and one of them with highiest cholesterol esterase acticity which was identified as Acinetobacter sp.,was further studied.
The objective of this work is to obtain an appropriate source of cholesterol esterases for industrial and medicinal needs.Nine bacterial strains that express high level of inducible extracellular cholesterol esterase(CHE) were isolated from carnivore feces.One of these strains,named CHE4-1,belonging to the genus Acinetobacter sp., was found to produce the highest level of cholesterol esterase.No reports about the cholesterol esterase(CHE) from Acinetobacter sp.and the bacteria secreting cholesterol esterase(CHE) from carnivore feces have been reported by far.
The growth and fermentation have been studied and the strategy of the control of sub-fermentation have been advanced,Fermentation process is optimized.The optimum culture medium is constituted as:cholesterol oleate 0.2%,TritonX-100 0.15%,peptone0.05%,NaNO_3 0.008%,boracic acid 0.0075%,CaCl_2 0.03%, MgSO_4·7H_2O 0.0125%,FeSO_4.7H_20 0.0005%,KH_2PO_4 0.037%.Fermentation condition for the strain was optimized as intitative pH 6.9,volume ofsubstrate 30ml, 30℃,cultivit for 48h.Emzyme activity reach at 950U/L.
CHE from strain CHE4-1 was purified from the culture supematant by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography,and then by Superdex-50 gel filtration.The specifiv activity of pure products reached 7.15U/mg,and the yield of enzyme activity is 27.02%,with a 9.67 purification factor.The purified cholesterol esterase migrated as a single protein band on reduced SDS-PAGE.The purity analyzed by HPLC is 98.22%. SDS-PAGE electrophoresis and gel filtration chromatography of CHE.showed that the purified enzyme was a monomer with a molecular weight of about 6.5kDa,and exhibited maximum absorption at 280 nm.It is the smallest cholesterol esterase among those reported by far.
The kinetic property of cholesterol esterase from Acinetobacter sp.had been investgated.The enzyme was found to be stable from 4.0-10.0,and be stable from 0℃-55℃,and with optimum activity at pH7.0 and 45℃;The Km value for hydrolyzation of cholesterol oleate by this enzyme was 5.36×10~(-4)mol/L,the Km value for hydrolyzation of cholesterol linoleate by this enzyme was 1.75×10~(-3)mol/L.The enzyme showed higher vitality to both long-chain cholesterol ester and short-chain cholesterol ester.The studies of the effects of some metal ion and organic compounds on the enzyme activity also indicated its potential as a clinical diagnostic reagent.
首先,经过筛选从大型肉食动物肠道中获得9株胆固醇酯酶高产菌株,其中1株表现出高于其他菌株的产酶能力,命名为CHE4-1。经形态及生理生化鉴定、16S rDNA(GenBank登录ID号:FJ649602)分析,CHE4-1鉴定为Acinetobactersp。迄今为止,未见不动杆菌属(Acinetobacter)产胆固醇酯酶的报道。
其次,对该菌株的生长与产酶条件进行了研究,提出了分段控制的发酵策略。产酶条件优化研究表明,其最适培养基为:胆固醇油酸酯0.2%,用0.15%TritonX-100助溶,蛋白胨0.05%,NaNO_3 0.008%,硼酸0.0075%,CaCl_20.03%,MgSO_4.7H_2O 0.0125%,FeSO_4.7H_2O 0.0005%,KH_2PO_4 0.037%;其最适培养条件为:起始pH6.9,250ml发酵瓶最佳装液量为35ml,30℃,发酵时间48 h;酶活力达到950U/L。
然后,对胆固醇酯酶进行纯化。发酵上清液经超滤浓缩,DEAE-Sepharose离子交换层析、Phenyl-Sepharose CL-4B疏水层析和Superdex-50凝胶层析分离纯化获得胆固醇酯酶。纯化后的胆固醇酯酶比活力为7.15 U/mg,纯化收率为27.02%,纯化倍数为9.67倍。纯化后的胆固醇酯酶经HPLC纯度检测达98.22%,SDS-PAGE为单一条带。SDS-PAGE和Superdex-75凝胶层析分析显示为约6.5kDa的单链蛋白,是目前为止所发现的最小的胆固醇酯酶。
最后,对胆固醇酯酶的酶学特性进行了研究,结果表明,该胆固醇酯酶的pH稳定范围为4-10:最适pH和最适温度分别为pH7.0和45℃,该酶催化胆固醇亚油酸酯、胆固醇油酸酯的米氏常数Km分别为1.75×10~(-3)mol/L、5.36×10~(-4)mol/L。该酶对长链和短链胆固醇酯均具有较高的催化活力。大多数金属离子对胆固醇酯酶抑制作用不明显;Ca~(2+)、Zn~(2+)、硼酸对胆固醇酯酶有微弱激活作用;Cu~(2+)、TritonX-100对胆固醇酯酶不具明显作用,显示了良好的临床应用价值。
Cholesterol esterase(CHE;EC 3.1.1.13) is an important steroidal esterase, it is widely used in food processing and medical assay and paper industry.In present study,several cholesterol esterase-producing bacterial strains were isolated,and one of them with highiest cholesterol esterase acticity which was identified as Acinetobacter sp.,was further studied.
The objective of this work is to obtain an appropriate source of cholesterol esterases for industrial and medicinal needs.Nine bacterial strains that express high level of inducible extracellular cholesterol esterase(CHE) were isolated from carnivore feces.One of these strains,named CHE4-1,belonging to the genus Acinetobacter sp., was found to produce the highest level of cholesterol esterase.No reports about the cholesterol esterase(CHE) from Acinetobacter sp.and the bacteria secreting cholesterol esterase(CHE) from carnivore feces have been reported by far.
The growth and fermentation have been studied and the strategy of the control of sub-fermentation have been advanced,Fermentation process is optimized.The optimum culture medium is constituted as:cholesterol oleate 0.2%,TritonX-100 0.15%,peptone0.05%,NaNO_3 0.008%,boracic acid 0.0075%,CaCl_2 0.03%, MgSO_4·7H_2O 0.0125%,FeSO_4.7H_20 0.0005%,KH_2PO_4 0.037%.Fermentation condition for the strain was optimized as intitative pH 6.9,volume ofsubstrate 30ml, 30℃,cultivit for 48h.Emzyme activity reach at 950U/L.
CHE from strain CHE4-1 was purified from the culture supematant by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography,and then by Superdex-50 gel filtration.The specifiv activity of pure products reached 7.15U/mg,and the yield of enzyme activity is 27.02%,with a 9.67 purification factor.The purified cholesterol esterase migrated as a single protein band on reduced SDS-PAGE.The purity analyzed by HPLC is 98.22%. SDS-PAGE electrophoresis and gel filtration chromatography of CHE.showed that the purified enzyme was a monomer with a molecular weight of about 6.5kDa,and exhibited maximum absorption at 280 nm.It is the smallest cholesterol esterase among those reported by far.
The kinetic property of cholesterol esterase from Acinetobacter sp.had been investgated.The enzyme was found to be stable from 4.0-10.0,and be stable from 0℃-55℃,and with optimum activity at pH7.0 and 45℃;The Km value for hydrolyzation of cholesterol oleate by this enzyme was 5.36×10~(-4)mol/L,the Km value for hydrolyzation of cholesterol linoleate by this enzyme was 1.75×10~(-3)mol/L.The enzyme showed higher vitality to both long-chain cholesterol ester and short-chain cholesterol ester.The studies of the effects of some metal ion and organic compounds on the enzyme activity also indicated its potential as a clinical diagnostic reagent.
引文
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[1]季文明,等。比色法测定胆固醇氧化酶酶活[J]。无锡轻工大学学报,19(3):251-254.
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[1]谭晓晶。Rhodococcus sp.胆固醇酯酶分离纯化与基本性质研究[M]。四川大学,2006.
[2]Hongyu Xiang,Shunsuke Masuo,Takayuki Hoshino,Naoki Takaya 。 Novel family of cholesterol esterases produced by actinomycetes bacteria[J]。
[3]Hongyu Xiang,Naoki Takaya,Takayuki Hoshino.Novel cholesterol esterase secreted by Streptomyces persists during aqueous long-term storage.The Society for Biotechnology.2006.101:1.19-25.
[4]SU,966115,1983.
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[7]OLGA CALERO-RUEDA,et al.Production,isolation and characterization of a sterol esterase from Ophiostoma piceae.Biochim.Biophys.Acta 2002,1599:28.
[8]杨光礼,等。假单胞菌胆固醇酯酶的生产,Ⅰ.菌种筛选和发酵条件的研究。Ⅱ.提取、纯化和酶学性质的研究。医药工业,1987,18:112:18:486.
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[13]韩文君,路新枝,肖琳,等。一株产褐藻酸多糖的海洋假单胞细菌Pseudomonas sp.QDA 的筛选和鉴定[J]。中国海洋大学学报(自然科学版),2004,31(4):060-064。
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[15]季文明,等。比色法测定胆固醇氧化酶酶活[J]。无锡轻工大学学报,19(3):251-254
[16]Atsushi Maeda,Takayuki Mizuno,Masanori Bunya,et al Characterization of Novel Cholesterol Esterase from Trichoderma sp.AS59 with High Ability to Synthesize Steryl Esters [J]. Bioscience and Bioengineering. 2008,105(4): 341-349.
[1]季文明,等。比色法测定胆固醇氧化酶酶活[J]。无锡轻工大学学报,19(3):251-254.
[2]叶盛。腺苷甲硫氨酸高产菌株的筛选、诱变选育及其培养条件优化研究[M]。四川师范大学,2007.
[3]李海军,等。45株不动杆菌细菌营养结果的分析[J]。四川省卫生管理干部学院学报。2004,23(1):9-10.
[4]成方,等。啤酒中乳酸细菌分离用培养基的优化[J]。中国酿造。2007,7:28-31.
[5]董如何,等。正交试验设计的理论分析方法及应用[J]。安徽建筑工业学院学报(自然科学版)。2004,12(6):103-106.
[6]杨铭。用SPSS的Conjoint模块功能进行正交设计与数据处理[J]。药学服务与研究。2004,4(4):380-381.
[7]李春喜,等。生物统计学[M]。北京:科学出版社,2005.
[8]曲音波,等。微生物技术开发原理[M]。北京:化学工业出版社,2005.
[1]汪家政,等。蛋白质技术手册[M]。北京:科学出版社,2000.
[2]郭尧君。蛋白质电泳实验技术[M]。北京:科学出版社,2005.
[3]陆建,等。蛋白质纯化技术及应用[M]。北京:化学工业出版社,2005.
[4]Hongyu Xiang,Naoki Takaya,Takayuki Hoshino.Novel cholesterol esterase secreted by Streptomyces persists during aqueous long-term storage[J].The Society for Biotechnology.2006.101:1.19-25.
[5]谭晓晶。Rhodococcus sp.胆固醇酯酶分离纯化与基本性质研究[M]。四川大学,2006.
[6]杨光礼,等。假单胞菌胆固醇酯酶的生产,Ⅰ。菌种筛选和发酵条件的研究。Ⅱ。提取、纯化和酶学性质的研究。医药工业,1987,18:112;18:486.
[7]Hanna Kontkanen. Novel steryl esterases as biotechnological tools[J]. VTT PUBLICATIONS 600 , Text preparing properties Vahala, VTT, Otamedia Oy, Espoo 2006
[8]Hongyu Xiang, Shunsuke Masuo, Takayuki Hoshino, Naoki Takaya 。 Novel family of cholesterol esterases produced by actinomycetes bacteria[J]。 The Society for Biotechnology. 2006. 101:32-28.
[11]Hongyu Xiang, Naoki Takaya, Takayuki Hoshino. Novel cholesterol esterase secreted by Streptomyces persists during aqueous long-term storage. The Society for Biotechnology. 2006. 101:1. 19-25.
[1]谭晓晶。Rhodococcus sp.胆固醇酯酶分离纯化与基本性质研究[M]。四川大学,2006.
[2]杨光礼,等。假单胞菌胆固醇酯酶的生产,Ⅰ。菌种筛选和发酵条件的研究。Ⅱ。提取、纯化和酶学性质的研究。医药工业,1987,18:112;18.486.
[3]Hongyu Xiang,Naoki Takaya,Takayuki Hoshino.Novel cholesterol esterase secreted by Streptomyces persists during aqueous long-term storage.The Society for Biotechnology.2006.101:1.19-25.
[4]US 1977,4011138(Ger Offer 1976,2527068,CA 1976,84:149233V).
[5]UWAZIMA T et al.Agr Biol Chem,1976,40:1957.
[6]日本公开特许 79-138188 CA 1980,92:71903g.