Rhodococcus sp.胆固醇酯酶分离纯化与基本性质研究
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摘要
胆固醇酯酶(COE，EC3.1.1.13)在食品、医药等方面的功能日益受到重视，为此本课题从以下四个方面研究胆固醇酯酶，从而为它的应用提供依据：胆固醇酯酶高产菌株的筛选、菌株鉴定；胆固醇酯酶高产菌株培养条件的优化；胆固醇酯酶的分离纯化及纯度鉴定；胆固醇酯酶的酶学特性研究。
经过筛选，从21株菌株中筛选出一株胆固醇酯酶的高产菌株作为胆固醇酯酶的生产菌种。经鉴定，该菌株为马红球菌，其菌体呈橙红、橙黄色，为多形态的革兰阳性杆菌，生化反应不活泼，不能发酵任何糖、醇类，触酶呈阳性反应。药敏试验结果表明：马红球菌对环丙沙星、万古霉素等敏感率达100％；对青霉素等呈耐药反应。
以马红球菌作为研究对象，对此菌株胆固醇酯酶形成条件进行研究表明：马红球菌产生的胆固醇酯酶为诱导酶，其胞外活性高于胞内活性。当油酸和卵磷脂作为诱导物且是培养基中唯一碳源时产酶量最高，蛋白胨和牛肉膏是产酶的适合氮源。马红球菌经过诱导及发酵培养条件的优化，确定其最适培养基组成为(％)：蛋白胨1.3，KCl0.5，牛肉膏0.1，Na_2HPO_40.632，MgSO_40.06，油酸0.4，卵磷脂0.25；最适培养条件为：pH7.4，250mL三角瓶中装量40mL，接种量6.0％，摇床旋转速度180r／min，温度30℃，培养时间24h。
The significant functions of cholesterol esterase(COE, EC3.1.1.13) in food processing and medical assay were recognized gradually and showed more and more potential. In this paper, four topics were investigated: screening and identification the strain produces cholesterol esterase, optimization of cholesterol esterase-forming conditions from Rhodococcus sp., purification and identification of cholesterol esterase from Rhodococcus sp., characterization of cholesterol esterase.
Strain one clone that could produce cholesterol esterase was screened from 21clones. After been identificated, could know it was Rhodococcus sp. Rhodococcus sp. is a clone of polymorphous gram positive bacteria, orange thalli, inertial biochemical reaction, cannot ferment any glucide or alcohols. The result of drug sensitive test shows that: Rhodococcus sp. is sensitively with penitricin、 vancomycin, and non-sensitive with penicillin.
Cholesterol esterase-forming conditions of Rhodococcus sp. were investigated. The results were obtained as follow: cholesterol esterase from Rhodococcus sp. was inducible enzyme, and extra-cellular enzyme activity was higher than intra-cellular enzyme. Oleic acid and lecithin, the substrates of the cholesterol esterase, were the best carbon sources, and they are also the best inducer. Peptone and Beef-extract were the best nitrogen sources for enzyme formation. The optimization of cultivation conditions was selected through
经过筛选，从21株菌株中筛选出一株胆固醇酯酶的高产菌株作为胆固醇酯酶的生产菌种。经鉴定，该菌株为马红球菌，其菌体呈橙红、橙黄色，为多形态的革兰阳性杆菌，生化反应不活泼，不能发酵任何糖、醇类，触酶呈阳性反应。药敏试验结果表明：马红球菌对环丙沙星、万古霉素等敏感率达100％；对青霉素等呈耐药反应。
以马红球菌作为研究对象，对此菌株胆固醇酯酶形成条件进行研究表明：马红球菌产生的胆固醇酯酶为诱导酶，其胞外活性高于胞内活性。当油酸和卵磷脂作为诱导物且是培养基中唯一碳源时产酶量最高，蛋白胨和牛肉膏是产酶的适合氮源。马红球菌经过诱导及发酵培养条件的优化，确定其最适培养基组成为(％)：蛋白胨1.3，KCl0.5，牛肉膏0.1，Na_2HPO_40.632，MgSO_40.06，油酸0.4，卵磷脂0.25；最适培养条件为：pH7.4，250mL三角瓶中装量40mL，接种量6.0％，摇床旋转速度180r／min，温度30℃，培养时间24h。
The significant functions of cholesterol esterase(COE, EC3.1.1.13) in food processing and medical assay were recognized gradually and showed more and more potential. In this paper, four topics were investigated: screening and identification the strain produces cholesterol esterase, optimization of cholesterol esterase-forming conditions from Rhodococcus sp., purification and identification of cholesterol esterase from Rhodococcus sp., characterization of cholesterol esterase.
Strain one clone that could produce cholesterol esterase was screened from 21clones. After been identificated, could know it was Rhodococcus sp. Rhodococcus sp. is a clone of polymorphous gram positive bacteria, orange thalli, inertial biochemical reaction, cannot ferment any glucide or alcohols. The result of drug sensitive test shows that: Rhodococcus sp. is sensitively with penitricin、 vancomycin, and non-sensitive with penicillin.
Cholesterol esterase-forming conditions of Rhodococcus sp. were investigated. The results were obtained as follow: cholesterol esterase from Rhodococcus sp. was inducible enzyme, and extra-cellular enzyme activity was higher than intra-cellular enzyme. Oleic acid and lecithin, the substrates of the cholesterol esterase, were the best carbon sources, and they are also the best inducer. Peptone and Beef-extract were the best nitrogen sources for enzyme formation. The optimization of cultivation conditions was selected through
引文
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[5] SITUMORANG, M., ALEXANDER, P. W., HIBBERT. D. B. Flow injection potentiometry for enzymatic assay of cholesterol with a tungsten electrode sensor. Talanta. 1999.49: 639.
[6] SHUMYANTSEVA, V., DELUCA, G., BULKO, T., et al. Cholesterol amperometric biosensor based on cytochrome P540 scc. Biosensors and Bioelectonics. 2004. 19: 971.
[7] 王深琪，李世普，闫玉华．测量胆固醇含量的生物传感器研究进展．传感器技术．1998．17(5)．
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[9] 刘泽民．酶法直接测定血清总胆固醇方法探讨．中华医学检验杂志．1984．2：69．
[10] 陈文祥，李培英，王抒，等．中华医学会检验学会血脂测定推荐方法(二)，中华医学检验杂志．1995．18(3)：185．
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[1] 王抒等．假单胞菌胆固醇酯酶的性能及其在血清总胆固醇测定中的应用．中华医学检验杂志，1990，13(2)：65．
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[14] 钟彦伟，夏光明，张维国，等．肝硬化患者血培养分离出L型马红球菌1例．临床检验杂志，1999，17(3)：139．
[1] HERANADEX, H.H., et al. J. Biol. Chem., 1957, 228: 447.
[2] UWAJIMA T, et al. Purification and properties of cholesterol esterase from pseudomonas fluorescens.Agr. Biol. Chem., 1975, 39: 1511.
[3] SU, 966115, 1983.
[4] UR, 1571877, 1980.
[5] JE公开特许，昭53-109992，1987．
[6] OLGA CALERO-RUEDA, et al. Production, isolation and characterization of a sterol esterase from Ophiostoma piceae. Biochim. Biophys. Acta. 2002, 1599: 28.
[7] 黄秀梨．微生物学实验指导．高等教育出版社，施普林格出版社．1999：114．
[8] ALLAIN, C. C., LUCY S. POON, CICELY S. G. CHAN., et al. Enzymatic determination of total serum cholesterol. 1974. Clin. Chem. 20(4): 470.
[9] 汪家政，范明。蛋白质技术手册．科学出版社，2002，4．
[10] 杨德．实验设计与分析．中国农业出版社，2002，1．
[11] 何少华，文竹青，娄涛．实验设计与数据处理．国防科技大学出版社，2002，1．
[12] 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．
[13] 黄武华，方定一，崔玉兰，方一澄．假单胞菌1299产胆固醇酯酶的研究，Ⅰ．适宜的产酶条件．
[1] DEYKINA, D., GOODMAN, D. S. The hydrolysis of long chain fatty acid esters of cholesterol with rat liver enzymes. J. Biol. Chem. 1962. 237: 3649.
[2] CALAME, K.B., GALLO, L., CHERIATHUNDAM, E., et al. Purification and properties of subunits of sterol ester hydrolase from rat pancreas. Arch. Biochem. Biophys. 1975. 168: 57.
[3] TRZECIAK, W. H., BOYD, G.S. Activation of cholesteryl esterase in bovine adrenal cortex. Eur. J. Biochem. 1974.46: 201.
[4] BEHRMAN, H.R., GREEP, R.O. Hormonal dependence of cholesterol ester hydrolase in the corpus luteum and adrenals. Horm. Metab. Res. 1972. 4: 206.
[5] DURHAM, L. A., GROGAN, W. M. Temperature sensitivity of cholesteryl ester hydrolases in the rat testis. Lipids. 1982. 17: 970.
[6] MARTINEZ, M. J., BOTHAM, K. M. Cholesteryl ester hydrolase: three activities in the lactating rat mammary gland. Biochem. Soc. Trans. 1990.18: 619.
[7] ETO, Y., SUZUKI, K. Cholesterol esters in developing rat brain: enzymes in choleaterol ester metabolism. J. Neuroche. 1972.19: 109-115.
[8] COOK, K. G., COLBRAN, R.J., SNEE, J., et al. Cytosolic cholesterol ester hydrolase rom bovine corpus luteum: its purification, identification and relationship to hormone sensitive lipase. Biochim. Biophys. Acta. 1983.752: 46-53.
[9] 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．
[10] 中国科学院微生物研究所．胆固醇酯酶的研制及应用．鉴定会资料1988．
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[13] ALLAIN, C. C., LUCY S. POON, CICELY S. G. CHAN., et al. Enzymatic determination of total serum cholesterol. 1974. Clin. Chem. 20(4): 470-475.
[14] 张金红，柳振宇．疏水层析法分离纯化脂肪酶的研究．南开大学学报(自然科学版)．2000，33(2)：95．
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[2] UWAZIMA T et al. Agr Biol Chem, 1976, 40: 1957.
[3] 日本公开特许 79-138188 (CA 1980，92：71903g．
[2] CHARPENTIER, L., MURR, I. E. Amperometric determination of cholesterol in serum with use of a renewable surface peroxidase electrode. Analytica Chimaca Acta. 1995. 318:89.
[3] MARTIN, S.P., LAMB, D.J., LYNCH, J.M., et al. Enzyme-based determination of cholesterol using the quartz crystal acoustic wave sensor. Analytica Chimaca Acta. 2003. 487: 91.
[4] TATSUMA, T., WATANABE, T. Oxidase/peroxidase bilayer-modified electrodes as sensors for lactate, cholesterol and uric acid. Analytica Chimaca Acta. 1991. 242: 85.
[5] SITUMORANG, M., ALEXANDER, P. W., HIBBERT. D. B. Flow injection potentiometry for enzymatic assay of cholesterol with a tungsten electrode sensor. Talanta. 1999.49: 639.
[6] SHUMYANTSEVA, V., DELUCA, G., BULKO, T., et al. Cholesterol amperometric biosensor based on cytochrome P540 scc. Biosensors and Bioelectonics. 2004. 19: 971.
[7] 王深琪，李世普，闫玉华．测量胆固醇含量的生物传感器研究进展．传感器技术．1998．17(5)．
[8] CHARLES C. ALLAIN., LUCY S. POON., C1CELY S. G. CHAN., et al. Enzymatic determination of total serum cholesterol. Clin. Chem. 1974. 20(4): 470.
[9] 刘泽民．酶法直接测定血清总胆固醇方法探讨．中华医学检验杂志．1984．2：69．
[10] 陈文祥，李培英，王抒，等．中华医学会检验学会血脂测定推荐方法(二)，中华医学检验杂志．1995．18(3)：185．
[11] YUZO KAYAMORI, HIROYUKI HAISUYAMA, et al. Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol hydrogenase. Clin. Chem. 1999.45: 2158.
[12] AMUNDOSN, D.M., ZHOU. Flurometric method for the enzymatic determination of cholesterol.J. Biochem. Biophys. Methods. 1999. 38: 43.
[13] ZHANG S, WRIGHT, G, YANG, Y. Materials and techniques for electrochemical biosensor design and construction. Biosensors &Bioelectronics. 2001.15: 273.
[14] KUMAR, A., MALHOTRA, R., MALHOTTRA, B.D., et al. Co-immobilization of cholesterol oxidase and horseradish peroxidase in a sol-tel film. Anal. Chim. Acta. 2000. 414: 43.
[15] XIE B, RAMANATHAN, K, et al. Mini/micro thermal biosensor and other related device for biochemical/clinical analysis and monitoring. Trends in Analytical Chemistry. 2000. 19: 340.
[16] RAGHAVAN, V, RAMANATHAN, K., et al. An enzyme thermistor based assay for total and free cholesterol. Clin. Chin. Acta. 1999. 289: 145.
[17] DEYKINA, D., GOODMAN, D. S. The hydrolysis of long chain fatty acid esters of cholesterol with rat liver enzymes. J. Biol. Chem. 1962. 237: 3649.
[18] CALAME, K.B., GALLO, L., CHERIATHUNDAM, E., et al. Purification and properties of subunits of sterol ester hydrolase from rat pancreas. Arch. Biochem. Biophys. 1975. 168:57.
[19] TRZECIAK, W. H., BOYD, G.S. Activation of cholesteryl esterase in bovine adrenal cortex. Eur. J. Biochem. 1974. 46: 201.
[20] BEHRMAN, H.R., GREEP, R.O. Hormonal dependence of cholesterol ester hydrolase in the corpus luteum and adrenals. Horm. Metab. Res. 1972. 4: 206.
[21] DURHAM, L. A., GROGAN, W. M. Temperature sensitivity of cholesteryl ester hydrolases in the rat testis. Lipids. 1982.17: 970.
[22] MARTINEZ, M. J., BOTHAM, K. M. Cholesteryl ester hydrolase: three activities in the lactating rat mammary gland. Biochem. Soc. Trans. 1990. 18: 619.
[23] ETO, Y, SUZUKI, K. Cholesterol esters in developing rat brain: enzymes in cholesterol ester metabolism. J. Neuroche. 1972. 19: 109.
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