胆固醇氧化酶产生菌Enterobacter sp.的筛选及其酶分离纯化的研究
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摘要
胆固醇是机体内重要的固醇类物质,它具有多种多样的生理功能,但是体内过多的胆固醇却是引起冠心病、动脉硬化和心肌梗塞的危险因素之一。胆固醇氧化酶(COD,EC 1.1.3.6)是一类黄素蛋白,属于GMC氧化还原酶体系,它能催化胆固醇分解成胆甾-4-烯-3-酮和过氧化氢,是胆固醇代谢过程中的第一个酶,也是一个关键酶,它能够快速准确地检测出血清中胆固醇的浓度,用来诊断动脉硬化和其他脂质紊乱疾病;能够降低食品中的胆固醇含量,有益于人类健康;能够有效地杀灭鞘翅目、鳞翅目、双翅目、直翅目和同翅目的害虫,是一种安全有效的生物杀虫剂;此外,胆固醇氧化酶氧化胆固醇生成的胆甾-4-烯-3-酮在治疗肥胖等方面具有应用前景。
本研究的目的是获得大量来源的胆固醇氧化酶,以满足工业和医疗领域的需要,从不同的环境中筛选得到了14株高产胞外胆固醇氧化酶(COD)的菌株,其中胆固醇氧化酶活性最高的一株菌,命名为COX8-9,通过菌体形态,菌落特征,生理生化反应和16SrDNA基因全序列分析,初步鉴定该菌属于肠杆菌属(Enterobacter)。
利用单因子实验和正交实验对菌株COX8-9产胆固醇氧化酶摇瓶发酵的培养基及培养条件进行优化。结果表明,发酵培养基的最佳成份为:胆固醇0.2%,酵母粉0.5%,可溶性淀粉0.5%,CuSO4·5H2O 0.001%,吐温-80 0.3%, NH4NO30.1%,KH2PO40.025%,MgSO4·7H2O 0.025%, FeSO40.0001%,pH 7.0 ,最适培养条件为接种量5%,种龄15h~18h,摇瓶装量为30mL培养基/250mL三角瓶,30℃,200r/min,发酵48 h,胆固醇氧化酶活力可达到748U/L。
菌株COX8-9的发酵上清液经过超滤后,连续两次通过不同pH值的Q-sepharose柱,再过superdex-75凝胶过滤色谱,得到了纯化的胆固醇氧化酶。纯化的胆固醇氧化酶是分子量为58 kDa的单体酶,在280nm处有最大吸收峰。该酶氧化胆固醇的Km值为1.19×10-4 mol/L,最适温度为25℃,在40℃以下都比较稳定;最适pH为7.0,而在pH9.0时最稳定;金属离子Cu2+可以提高胆固醇氧化酶的活力,可将其提高三倍;在30℃温度下,该酶在长期的水溶液储存条件下都比较稳定,表明了该酶作为临床诊断试剂的潜力。其他筛选菌株的胆固醇氧化酶的鉴定及制备工作正在进行中。
Cholesterol is an important sterol in organism and it exhibited many physiologic function,but too much cholesterol in vivo is one of the dangerous factors that cause coronary artery disease, arteriosclerosis and heart infarction. Cholesterol oxidase(COD,EC1.1.3.6) is a kind of flavoprotein and belongs to GMC oxidation reduction enzyme,which catalyzes the oxidation of cholesterol (5-cholesten-3β-ol) to cholest-4-en-3-one and hydrogen peroxide, is the first and the key enzyme in the pathway of cholesterol metabolism.It may determine the cholesterol concentration in serum quickly and exactly and may be used to diagnose angiosclerosis and other ester derangement disease; it may decrease cholesterol content in food and benefit for health ;it may kill pest of Coleoptera, Lepidoptera, Diptera, Orthoptera and Homoptera and is a kind of safe and useful bioinsecticide; besides, cholest-4-en-3-one which formed by oxidation of cholesterol has the application perspective to heal obesity.
The objective of this work is to obtain an abundant source of cholesterol oxidases to meet the needs of industry and medicinal field. Fourteen bacteria strains that produce high level of extracellular cholesterol oxidase (COD) were isolated from different environment. One of these strains,named COX8-9, was found to produce the highest level of cholesterol oxidase,according to the strain morphology,colony character and physiological and biochemistric character , 16SrDNA gene of COX8-9 sequencing results,it can be identified as Enterobacter.
The shake flask fermentation condition and medium of strain COX8-9 producing cholesterol oxidase was optimized by single factor and orthogonal experiment.The results indicated that ,the optimal composition of fermentation medium were: cholesterol 0.2%,yeast 0.5%,amidulin 0.5%,CuSO4·5H2O 0.001%,Tween-80 0.3%, NH4NO30.1%,KH2PO40.025%,MgSO4·7H2O 0.025%, FeSO40.0001%,pH 7.0,the optimal cultivation conditions were:5% inoculum size ,15h~18h bacterium age ,volume of substrate was 30ml medium/250ml erlenmeyer flask, 30℃,200r/min,48h fermentation time, cholesterol oxidase activity reached at 748U/L.
COD from strain COX8-9 was purified from the culture supernatant by ultra filtration followed with two consecutive Q-sepharose chromatographies in different pH values, and then by superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The Km value for oxidation of cholesterol by this enzyme was 1.19×10-4 mol/L, with optimum activity at 25℃and pH7.0,it was stable under 40℃and at pH9.0; enzymatic activity was enhanced 3-fold in the presence of metal ion Cu2+, and the enzyme was stable during long-term aqueous storage under 30℃, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way.
本研究的目的是获得大量来源的胆固醇氧化酶,以满足工业和医疗领域的需要,从不同的环境中筛选得到了14株高产胞外胆固醇氧化酶(COD)的菌株,其中胆固醇氧化酶活性最高的一株菌,命名为COX8-9,通过菌体形态,菌落特征,生理生化反应和16SrDNA基因全序列分析,初步鉴定该菌属于肠杆菌属(Enterobacter)。
利用单因子实验和正交实验对菌株COX8-9产胆固醇氧化酶摇瓶发酵的培养基及培养条件进行优化。结果表明,发酵培养基的最佳成份为:胆固醇0.2%,酵母粉0.5%,可溶性淀粉0.5%,CuSO4·5H2O 0.001%,吐温-80 0.3%, NH4NO30.1%,KH2PO40.025%,MgSO4·7H2O 0.025%, FeSO40.0001%,pH 7.0 ,最适培养条件为接种量5%,种龄15h~18h,摇瓶装量为30mL培养基/250mL三角瓶,30℃,200r/min,发酵48 h,胆固醇氧化酶活力可达到748U/L。
菌株COX8-9的发酵上清液经过超滤后,连续两次通过不同pH值的Q-sepharose柱,再过superdex-75凝胶过滤色谱,得到了纯化的胆固醇氧化酶。纯化的胆固醇氧化酶是分子量为58 kDa的单体酶,在280nm处有最大吸收峰。该酶氧化胆固醇的Km值为1.19×10-4 mol/L,最适温度为25℃,在40℃以下都比较稳定;最适pH为7.0,而在pH9.0时最稳定;金属离子Cu2+可以提高胆固醇氧化酶的活力,可将其提高三倍;在30℃温度下,该酶在长期的水溶液储存条件下都比较稳定,表明了该酶作为临床诊断试剂的潜力。其他筛选菌株的胆固醇氧化酶的鉴定及制备工作正在进行中。
Cholesterol is an important sterol in organism and it exhibited many physiologic function,but too much cholesterol in vivo is one of the dangerous factors that cause coronary artery disease, arteriosclerosis and heart infarction. Cholesterol oxidase(COD,EC1.1.3.6) is a kind of flavoprotein and belongs to GMC oxidation reduction enzyme,which catalyzes the oxidation of cholesterol (5-cholesten-3β-ol) to cholest-4-en-3-one and hydrogen peroxide, is the first and the key enzyme in the pathway of cholesterol metabolism.It may determine the cholesterol concentration in serum quickly and exactly and may be used to diagnose angiosclerosis and other ester derangement disease; it may decrease cholesterol content in food and benefit for health ;it may kill pest of Coleoptera, Lepidoptera, Diptera, Orthoptera and Homoptera and is a kind of safe and useful bioinsecticide; besides, cholest-4-en-3-one which formed by oxidation of cholesterol has the application perspective to heal obesity.
The objective of this work is to obtain an abundant source of cholesterol oxidases to meet the needs of industry and medicinal field. Fourteen bacteria strains that produce high level of extracellular cholesterol oxidase (COD) were isolated from different environment. One of these strains,named COX8-9, was found to produce the highest level of cholesterol oxidase,according to the strain morphology,colony character and physiological and biochemistric character , 16SrDNA gene of COX8-9 sequencing results,it can be identified as Enterobacter.
The shake flask fermentation condition and medium of strain COX8-9 producing cholesterol oxidase was optimized by single factor and orthogonal experiment.The results indicated that ,the optimal composition of fermentation medium were: cholesterol 0.2%,yeast 0.5%,amidulin 0.5%,CuSO4·5H2O 0.001%,Tween-80 0.3%, NH4NO30.1%,KH2PO40.025%,MgSO4·7H2O 0.025%, FeSO40.0001%,pH 7.0,the optimal cultivation conditions were:5% inoculum size ,15h~18h bacterium age ,volume of substrate was 30ml medium/250ml erlenmeyer flask, 30℃,200r/min,48h fermentation time, cholesterol oxidase activity reached at 748U/L.
COD from strain COX8-9 was purified from the culture supernatant by ultra filtration followed with two consecutive Q-sepharose chromatographies in different pH values, and then by superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The Km value for oxidation of cholesterol by this enzyme was 1.19×10-4 mol/L, with optimum activity at 25℃and pH7.0,it was stable under 40℃and at pH9.0; enzymatic activity was enhanced 3-fold in the presence of metal ion Cu2+, and the enzyme was stable during long-term aqueous storage under 30℃, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way.
引文
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