新疆鲜蒜中蒜氨酸酶的分离纯化及性质
摘要
大蒜（Allium Sativum L.）百合科葱属植物，是历史悠久的药食两用植物。它具有杀菌、降血脂、抗血栓、预防中风和高血压、保护肝脏和提高免疫力、抗癌等功效。大蒜中主要的功效物质是由蒜氨酸酶催化裂解蒜氨酸产生的极不稳定的大蒜辣素等含硫化合物。因此，蒜氨酸酶是大蒜产生药用成分的重要生理活性物质。本论文主要工作包括蒜氨蒜酶的分离纯化及其动力学性质的研究。
将新疆吉木萨尔白皮蒜鳞茎破碎细胞，离心去蒜渣，上清液经硫酸铵分级沉淀，透析脱盐，过葡聚糖G-200凝胶柱等步骤分离纯化蒜氨酸酶，通过酶活性测定和聚丙烯酰胺凝胶电泳检测，确定蒜氨酸酶纯化倍数和分离效果。结果表明，经过上述提纯步骤，获得了纯化14倍的蒜氨酸酶，经SDS-PAGE鉴定，基本为单一组分，分子量为53kD，与文献报道基本一致。
将分离纯化后的蒜氨酸酶液作为测试液，以蒜氨酸为底物研究了蒜氨酸酶的反应动力学及最适反应条件，确定了该酶最适反应温度为30℃，50℃以下稳定，动力学常数Km=0.693mmol/L、Vm=0.353?mol/min，通过等电聚焦电泳测定其等电点为4.8。采用化学修饰法测定蒜氨酸酶活性中心氨基酸，可推断色氨酸在蒜氨酸酶催化反应中起重要作用。
本实验从新疆吉木萨尔白皮鲜蒜中分离纯化蒜氨酸酶，并对其性质进行了测定，为今后新疆大蒜的开发利用奠定了基础。
Garlic (Allium Sativum L.) is a number of species of the liliaceae. It was a widely distributed plant and used in all parts of the world not only as a spice and a food, but also as a popular remedy. In addition, it effected on blood coagulation, lipid- and cholesterol-lowering, blood pressure, antithrombic, and anticancer activities were under discussion. Allicin and other sulfur containing compounds were suggested as active compounds in garlic. The extremely unstable allicin itself is liberated from the more stable alliin by the enzyme alliinase if fresh garlic is crunched. Therefore , products of allicin is due to alliinase. This paper included two parts, one part research was to purify the alliinase from fresh garlic, another one was to research optimum reactive conditions and reaction kinetics of alliinase.
Garlic bulb was blended, the precipitate was removed by centrifugation. Solid ammonium sulphate was added to the supernatant to give 25~55% saturation, and the precipitate was collected by centrifugation, then suspended in the phosphate-glycerol solution. Further purification was achieved by passing samples of the enzyme through sephadex G-200 column. By enzyme assay and gel electrophoresis of alliinase, the results showed that a 14-fold purification of alliinase was obtained, and one most intensive band at Mr =53kD was detected which is in accordance with literature values.
Purified alliinase optimum reactive conditions and reaction kinetics by alliin as basic substance were studied. The Km and Vm values for enzyme activity on alliin at 35℃ were 0.693mmol/L, 0.353?mol/min, which accords with Michaclis-Menten equation. The optimum temperature for activity was 30℃, range of thermal stability was below 50℃. Isoelectric focusing of alliinase,
the major bands were visualized at pH 4.82. Identification of an essential residue in alliinase by chemical modification, the results indicated that Trp residue played an important role in enzyme catalysis.
We had purified alliinase from fresh garlic, and studied on its properties. This study provided theoretical foundation for development and application of Garlic in xinjiang.
将新疆吉木萨尔白皮蒜鳞茎破碎细胞，离心去蒜渣，上清液经硫酸铵分级沉淀，透析脱盐，过葡聚糖G-200凝胶柱等步骤分离纯化蒜氨酸酶，通过酶活性测定和聚丙烯酰胺凝胶电泳检测，确定蒜氨酸酶纯化倍数和分离效果。结果表明，经过上述提纯步骤，获得了纯化14倍的蒜氨酸酶，经SDS-PAGE鉴定，基本为单一组分，分子量为53kD，与文献报道基本一致。
将分离纯化后的蒜氨酸酶液作为测试液，以蒜氨酸为底物研究了蒜氨酸酶的反应动力学及最适反应条件，确定了该酶最适反应温度为30℃，50℃以下稳定，动力学常数Km=0.693mmol/L、Vm=0.353?mol/min，通过等电聚焦电泳测定其等电点为4.8。采用化学修饰法测定蒜氨酸酶活性中心氨基酸，可推断色氨酸在蒜氨酸酶催化反应中起重要作用。
本实验从新疆吉木萨尔白皮鲜蒜中分离纯化蒜氨酸酶，并对其性质进行了测定，为今后新疆大蒜的开发利用奠定了基础。
Garlic (Allium Sativum L.) is a number of species of the liliaceae. It was a widely distributed plant and used in all parts of the world not only as a spice and a food, but also as a popular remedy. In addition, it effected on blood coagulation, lipid- and cholesterol-lowering, blood pressure, antithrombic, and anticancer activities were under discussion. Allicin and other sulfur containing compounds were suggested as active compounds in garlic. The extremely unstable allicin itself is liberated from the more stable alliin by the enzyme alliinase if fresh garlic is crunched. Therefore , products of allicin is due to alliinase. This paper included two parts, one part research was to purify the alliinase from fresh garlic, another one was to research optimum reactive conditions and reaction kinetics of alliinase.
Garlic bulb was blended, the precipitate was removed by centrifugation. Solid ammonium sulphate was added to the supernatant to give 25~55% saturation, and the precipitate was collected by centrifugation, then suspended in the phosphate-glycerol solution. Further purification was achieved by passing samples of the enzyme through sephadex G-200 column. By enzyme assay and gel electrophoresis of alliinase, the results showed that a 14-fold purification of alliinase was obtained, and one most intensive band at Mr =53kD was detected which is in accordance with literature values.
Purified alliinase optimum reactive conditions and reaction kinetics by alliin as basic substance were studied. The Km and Vm values for enzyme activity on alliin at 35℃ were 0.693mmol/L, 0.353?mol/min, which accords with Michaclis-Menten equation. The optimum temperature for activity was 30℃, range of thermal stability was below 50℃. Isoelectric focusing of alliinase,
the major bands were visualized at pH 4.82. Identification of an essential residue in alliinase by chemical modification, the results indicated that Trp residue played an important role in enzyme catalysis.
We had purified alliinase from fresh garlic, and studied on its properties. This study provided theoretical foundation for development and application of Garlic in xinjiang.
引文
孙君社,高孔荣.大蒜的化学与药效及利用.广州食品工业科技.1994, S1:8-11
顾银娜,黄蕴慧.大蒜有效成份的提取及药理作用.南京军医学院学报. 2000,(22):249-250
吉木萨尔白皮蒜.葱姜蒜专家咨询系统.
周静,徐以玲.新疆大蒜营养成分分析.石河子医学院学报.1990,(12):22~23
曾凡骏,张月天,陈松波.大蒜油软胶囊保健食品的研究.食品与发酵工业. 2001,（27）：24-27
Ferrwick G R,Hanley A B.CRC Crit Rev.Food Sci Nutr.1985,22(3):273.
李萍.大蒜提取技术.科技交流.
吴传茂,陈士英.蒜氨酸酶和无臭大蒜.医药原理.2000,(13):268
李素民,杨文昌,田彩锁等.大蒜的现代研究进展.中医药学报.1997, (6):30-32
Stoll A,seeback E.The decomposition of alliinase and the properties of alliinase. Helv Chim Acta,1949,32:197-205
Mazelis,M(1963) Phytochemistry 2,15
Mazelis,Mcreveling,R.K.(1975)Biochem.J.147,485-491
Nomura,J.,Nishizuka,Y.,Hayaishi,O.(1963)J.Boil.Chem.238,1141-1446
Mazelis ,M.Crews,L.Purification of the alliin lyase of garlic ,allium sativum L.Biochem.J.(1968)108,725-730
Kazaryan,R.A.,Goryachenkova,E.V.(1978)Biochemistry 43,1502-1508
Nock LP,Mazelis M.The C-S lyases of higher plants:preparation and properties of homogeneouds alliin lyase from garlic (allium sativum).Arch Biochem Biophys 1986 Aug 249(1):27-33
Herwig J,Bernd M,Karl K.Characterzation of an alliin lyase preparation from garlic (allium sativum).Planta Medica 55(1989).434-439
Rabinkov A,Zhu XZ,grafi G,Mirelman D .Alliin lyase (alliinase)from garlic (allium sativum).biochemical characterization and cDNA cloning.Appl Biotechnol 1994 sep;149-171
Krest and M.keusgen. Stabilization and pharmaceutical use of alliinase . Pharmazie 1999 Apr;54(4):289-293
Ingo krest and Michael keusgen.quality of herbal remedies from allium satium:differences between alliinase from garlic power and fresh garlic . Planta Medica 65(1999)139-143
Kuettner BE. Hilgenfeld R. Weiss SM. Purification, characterization, and crystallization of alliinase from garlic. Arch Biochem Biophys, 2002, 402: 192-200.
张学武等.蛋白质水解的动力学模型.中国食品学报.1998,6(6):1-6
Michaelis L.,Menten M.L.;Biochem .Zeitschr.,1913,49,333
易蕴玉等.酸性脲酶反应动力学研究.无锡轻工大学学报,1998.17(2):25-29
Linweaver H.,Burk D;J.Am.Chem..soc.,1949,56,658
Vance K.et al.Starch hydroiysis kinetics of bacillus licheniformis α-amylase.J.Chem..Tech.Biotech.,1991,51:209
万梦嘉编著.应用数理统计.北京:中国农业大学,1996
Ingvorsen K.,Zehnder A.J.B.et al Kinetics of sulphate and acetake by desulfobactor postgatei.Appl.Envir.Microbial,1984,46:403-408
Schwimmol/Ler,s.(1964)Biochem.Biophys.Acta 81,377-385
Schwimmol/Ler,s.,kjaer,,A.(1960)Biochem .Biophys.Acta42,316
Keusgen M.A high-throughput method for the quantitative determination of alliin.Planta Medica64(1998)736-740
Tomofumi m,Asako H,Mitsuyo S:Aliinase from allium tuberosum(Chinese chive)purification,localization,cDNA cloning and heterologous functional
expression .Eur.J.Biochem.257,21-30(1998)
Burger,et al,Garlic supplementation and lipoportein oxidation susceotibility , Lipids,1993;28(5)475~477
Landshuter J.,Lohmüller Eva-Maria ,and Knobloch K.Purification and characterization of a c-s lyase from ramson,the wild garlic ,allium ursinum. Planta Medica 60(1994)343-347
Randle WM,Bussard ML.Streamlining onion pungency analysis . Hortsci, 1993,28:60
Tobkin HE,Mazelis M.Alliinase lyase :preparation and characterization of homogeneous enzyme from onion bulbs.Arch Biochem Biophys 1978, 193 :150-157
Lancaster JE,Shaw M L,Joyce MD,McCallum JA,Mc ManusMT.A novel alliinase from onion roots.biochemical characterization and cDNA cloning . Plant Physiol 2000 Apr;122(4):1267-1279
Willy J.Peumans,Koen Smeets,Karel Van Nerum et al.Lectin and alliinase are the predominant proteins in nectar from leek(allium porrum L) flowers. Planta(1997)201:298-302
Manabe T,Hasumi A,Sugiyama M,Yamazaki M,Saito K,Alliinase from allium tuberosum (chinese chive)-purification localization cDNA cloning and heterologous functional expression .eur J biochem 1998 Oct 1:257 (1): 21-30
Van Dammol/Le EJM,Smeets K,Towekens S et al.Isolation and characterization of alliinase cDNA clones from garlic and related species .EUR J biochem 1992,209:751-757
Nock PL,Mazelis M. Lack of homology between the alliin lyases of garlic and onion .Phytochem,1989,28:729-731
Herwig Jansen, Bernd Müller ,and Knobloch. Alliin lyase from garlic,allium sativum: investigation on enzyme/substrate, enzyme /inhibitor interactions, and on a new
coenzyme .planta medica 55(1989)440-445
Weik R, Francky A,Striedner G,raspor P,bayer K,mattanovich D.recombinant expression of alliin lyase from garlic (Allium sativum)in bacteria and yeast. Planta Medica 1998 May ;64(4):440-445
W?fler U,Shaw ML,Lancaster JE.Effect of freezing upon alliinase activity in onion extracts and pure enzyme preparations .J Sci Food Agric 1994, 64: 315-318
乔旭光,张振华,张雅珊等.蒜氨酸酶动力学特性的研究.山东农业大学学报,1999,30(1):42-46
Young Nam Jin,Yong-Hoon Choi,and Chul-Hak Yang:Identification of an essential tryptophan residue in alliinase from garlic(allium sativum) by chemical modification .Bull.Korean Chem.Soc.2001(22):68-78.
Kuettner BE. Hilgenfeld R. Weiss SM. The active principle of garlic at atomic resolution. JBC Papers. 2002.9 ,1-18.
Schwimmol/Ler,S;Mazelis ,M: Charaterization of allium cepa (onion).arch. biochem. biophys.100,66(1963)
Miron T,Rabinkov A,Mirelman D,Weiner L,wilchek M. A spectrophotometric assay for allicin and alliinase activity :reaction of 2-nitro-5-thiobenzoate with thiosulfinates. Anal Biochem 1998 Dec 15;265(2):317-325
孙君社,高孔荣.蒜氨酸酶及其动力学研究.食品科学,1995 Vol.16 No.9:13-15
Bernhard L Berl,Georg Winkler ,Bernd Miiller and Karlknobloch. Quantitative determination of allicin and alliin from garlic by HPLC.Planta Medica 1990 (56):320-326
张龙翔等.生化实验方法和技术.人民教育出版社.1982:128-132
Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein –dye binding [J]. Anal
Biochem,1976,72:248~254
王秀奇等.基础生物化学实验. 高等教育出版社.2003:219-222
何忠孝.电泳.科学出版社.2000:129-138
张树政,孟广震,何忠效.酶学研究技术.科学出版社.1987.
顾银娜,黄蕴慧.大蒜有效成份的提取及药理作用.南京军医学院学报. 2000,(22):249-250
吉木萨尔白皮蒜.葱姜蒜专家咨询系统.
周静,徐以玲.新疆大蒜营养成分分析.石河子医学院学报.1990,(12):22~23
曾凡骏,张月天,陈松波.大蒜油软胶囊保健食品的研究.食品与发酵工业. 2001,（27）：24-27
Ferrwick G R,Hanley A B.CRC Crit Rev.Food Sci Nutr.1985,22(3):273.
李萍.大蒜提取技术.科技交流.
吴传茂,陈士英.蒜氨酸酶和无臭大蒜.医药原理.2000,(13):268
李素民,杨文昌,田彩锁等.大蒜的现代研究进展.中医药学报.1997, (6):30-32
Stoll A,seeback E.The decomposition of alliinase and the properties of alliinase. Helv Chim Acta,1949,32:197-205
Mazelis,M(1963) Phytochemistry 2,15
Mazelis,Mcreveling,R.K.(1975)Biochem.J.147,485-491
Nomura,J.,Nishizuka,Y.,Hayaishi,O.(1963)J.Boil.Chem.238,1141-1446
Mazelis ,M.Crews,L.Purification of the alliin lyase of garlic ,allium sativum L.Biochem.J.(1968)108,725-730
Kazaryan,R.A.,Goryachenkova,E.V.(1978)Biochemistry 43,1502-1508
Nock LP,Mazelis M.The C-S lyases of higher plants:preparation and properties of homogeneouds alliin lyase from garlic (allium sativum).Arch Biochem Biophys 1986 Aug 249(1):27-33
Herwig J,Bernd M,Karl K.Characterzation of an alliin lyase preparation from garlic (allium sativum).Planta Medica 55(1989).434-439
Rabinkov A,Zhu XZ,grafi G,Mirelman D .Alliin lyase (alliinase)from garlic (allium sativum).biochemical characterization and cDNA cloning.Appl Biotechnol 1994 sep;149-171
Krest and M.keusgen. Stabilization and pharmaceutical use of alliinase . Pharmazie 1999 Apr;54(4):289-293
Ingo krest and Michael keusgen.quality of herbal remedies from allium satium:differences between alliinase from garlic power and fresh garlic . Planta Medica 65(1999)139-143
Kuettner BE. Hilgenfeld R. Weiss SM. Purification, characterization, and crystallization of alliinase from garlic. Arch Biochem Biophys, 2002, 402: 192-200.
张学武等.蛋白质水解的动力学模型.中国食品学报.1998,6(6):1-6
Michaelis L.,Menten M.L.;Biochem .Zeitschr.,1913,49,333
易蕴玉等.酸性脲酶反应动力学研究.无锡轻工大学学报,1998.17(2):25-29
Linweaver H.,Burk D;J.Am.Chem..soc.,1949,56,658
Vance K.et al.Starch hydroiysis kinetics of bacillus licheniformis α-amylase.J.Chem..Tech.Biotech.,1991,51:209
万梦嘉编著.应用数理统计.北京:中国农业大学,1996
Ingvorsen K.,Zehnder A.J.B.et al Kinetics of sulphate and acetake by desulfobactor postgatei.Appl.Envir.Microbial,1984,46:403-408
Schwimmol/Ler,s.(1964)Biochem.Biophys.Acta 81,377-385
Schwimmol/Ler,s.,kjaer,,A.(1960)Biochem .Biophys.Acta42,316
Keusgen M.A high-throughput method for the quantitative determination of alliin.Planta Medica64(1998)736-740
Tomofumi m,Asako H,Mitsuyo S:Aliinase from allium tuberosum(Chinese chive)purification,localization,cDNA cloning and heterologous functional
expression .Eur.J.Biochem.257,21-30(1998)
Burger,et al,Garlic supplementation and lipoportein oxidation susceotibility , Lipids,1993;28(5)475~477
Landshuter J.,Lohmüller Eva-Maria ,and Knobloch K.Purification and characterization of a c-s lyase from ramson,the wild garlic ,allium ursinum. Planta Medica 60(1994)343-347
Randle WM,Bussard ML.Streamlining onion pungency analysis . Hortsci, 1993,28:60
Tobkin HE,Mazelis M.Alliinase lyase :preparation and characterization of homogeneous enzyme from onion bulbs.Arch Biochem Biophys 1978, 193 :150-157
Lancaster JE,Shaw M L,Joyce MD,McCallum JA,Mc ManusMT.A novel alliinase from onion roots.biochemical characterization and cDNA cloning . Plant Physiol 2000 Apr;122(4):1267-1279
Willy J.Peumans,Koen Smeets,Karel Van Nerum et al.Lectin and alliinase are the predominant proteins in nectar from leek(allium porrum L) flowers. Planta(1997)201:298-302
Manabe T,Hasumi A,Sugiyama M,Yamazaki M,Saito K,Alliinase from allium tuberosum (chinese chive)-purification localization cDNA cloning and heterologous functional expression .eur J biochem 1998 Oct 1:257 (1): 21-30
Van Dammol/Le EJM,Smeets K,Towekens S et al.Isolation and characterization of alliinase cDNA clones from garlic and related species .EUR J biochem 1992,209:751-757
Nock PL,Mazelis M. Lack of homology between the alliin lyases of garlic and onion .Phytochem,1989,28:729-731
Herwig Jansen, Bernd Müller ,and Knobloch. Alliin lyase from garlic,allium sativum: investigation on enzyme/substrate, enzyme /inhibitor interactions, and on a new
coenzyme .planta medica 55(1989)440-445
Weik R, Francky A,Striedner G,raspor P,bayer K,mattanovich D.recombinant expression of alliin lyase from garlic (Allium sativum)in bacteria and yeast. Planta Medica 1998 May ;64(4):440-445
W?fler U,Shaw ML,Lancaster JE.Effect of freezing upon alliinase activity in onion extracts and pure enzyme preparations .J Sci Food Agric 1994, 64: 315-318
乔旭光,张振华,张雅珊等.蒜氨酸酶动力学特性的研究.山东农业大学学报,1999,30(1):42-46
Young Nam Jin,Yong-Hoon Choi,and Chul-Hak Yang:Identification of an essential tryptophan residue in alliinase from garlic(allium sativum) by chemical modification .Bull.Korean Chem.Soc.2001(22):68-78.
Kuettner BE. Hilgenfeld R. Weiss SM. The active principle of garlic at atomic resolution. JBC Papers. 2002.9 ,1-18.
Schwimmol/Ler,S;Mazelis ,M: Charaterization of allium cepa (onion).arch. biochem. biophys.100,66(1963)
Miron T,Rabinkov A,Mirelman D,Weiner L,wilchek M. A spectrophotometric assay for allicin and alliinase activity :reaction of 2-nitro-5-thiobenzoate with thiosulfinates. Anal Biochem 1998 Dec 15;265(2):317-325
孙君社,高孔荣.蒜氨酸酶及其动力学研究.食品科学,1995 Vol.16 No.9:13-15
Bernhard L Berl,Georg Winkler ,Bernd Miiller and Karlknobloch. Quantitative determination of allicin and alliin from garlic by HPLC.Planta Medica 1990 (56):320-326
张龙翔等.生化实验方法和技术.人民教育出版社.1982:128-132
Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein –dye binding [J]. Anal
Biochem,1976,72:248~254
王秀奇等.基础生物化学实验. 高等教育出版社.2003:219-222
何忠孝.电泳.科学出版社.2000:129-138
张树政,孟广震,何忠效.酶学研究技术.科学出版社.1987.
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