离子诱变选育纤溶酶（Plasmin）高产菌以及部分酶学性质研究
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摘要
以实验室保存菌株BL.PY为出发菌株，该菌为地衣芽孢杆菌(Bacillus licheniformis)，可以产生血纤维蛋白溶解酶(Plasmin)。该菌株经过复壮后，酶活大约为800U。用低能N离子注入纤溶酶产生菌，以20kev,剂量为1×10~(14)至4×10~(15)ions／cm~2·s的N离子为诱变剂，产生可遗传的变异，一定范围内纤溶酶产生菌的正向突变率，致死率与离子注入剂量成正相关，离子注入后纤溶酶产生菌的菌体形态发生一定程度上的改变。从大约120株突变株中筛选到一株编号为BL.Z高产菌，摇瓶发酵表明该突变菌株酶产量可提高51.13％，达到1210U。通过单因素实验以及正交分析法探索该菌株的最佳发酵条件为(％)：蔗糖 3，黄豆粉 0.2，酵母膏0.1， MgSO_4·7H_2O 0.1， CaCl_2 0.03，K_2HPO_4 0.3， KH_2PO_4 0.04，通气量为1：0.5—1：1，转速为480rpm，37℃发酵4天，其纤溶酶活力达到1320U，较出发菌株提高了65％。
发酵液通过硫酸铵分级盐析沉淀，CM-Sephadex C-25阳离子交换层析，DEAE-Sephadex A-50阴离子交换层析和Sephadex G-75凝胶过滤，获得一种具有纤溶酶活性的蛋白酶BK，其分子量大约为23KD。以苯胺-硫酸法测定中性糖含量为7.55％，以3，5-二硝基水杨酸法(DNS法)测总还原糖含量分别为7.45％。以硅胶G薄层层析法鉴定糖的种类，该酶所含糖蛋白主要为中性戊糖，同时研究了一些金属离子的添加对纤溶酶的活性的影响，EDTA对酶活影响不大，而PMSF对酶活有较强的抑制作用，说明该酶的活性中心有丝氨酸参与，属丝氨酸酶系，而其作用中心无Ca~(2+)，Mg~(2+)等金属离子参与。脲和SDS变性剂对酶有一定
的抑制作用，但较弱，可以看出酶的抗变性剂能力较强。
A novel plasmin-producing bacterial strain PY,which had been identified as Bacillus Licheniformao
Using a dosage of about 1 X 1014 to 4X 1015 N ions/cm2 -s and 20kev, the strain B L.PY. was treated by N ion implantation
and a stable mutant strain with high yield of plasmin is obtained .In general, the rate of mutation and death grows as the
increasing of ion dosage. The shape of strains changed. Shaking fermentation indicates that compared with that of normal
strain about 120 strians, the enzymetic production of the mutant one increases 51.13%, reaching 1210U. Fermentation conditions
for enzyme production have been established. Composition of the medium (%) is edible sucrose 3, soybean poder 1.2, yeast extract 0.2, MgSO4 7H2O 0.1, CaCl2 0.03, K2HPO4 0.3, KH2PO40.04, pH 7.2, After fermentation in 1.5 liter of fermentorat 37"C with an aeration rate of 1: 0.5 - 1:1 for 4 days, the average fibrinolytic activity was about 1320U/ml culture broth,increase by 65% over the mutant.
The purification of enzyme BK include saline extraction,, ion-exchange chromatography on CM-Sephadex C-25 and DEAE-Sephadex A-50, and gel-filtration on Sephadex G-75 ,with a molecular weight of 23 KD. The protocols used were phenols.sulfuric acid method,3,5-dinitrosalicylic acid method and thin layer chromatography of salic gel G. Reducing sugar ang the tatol sugar were
measured with 3,5-2nitro-salcylic acid.We found that their contents were 7.45% and 7.49% respectnely.The categories of sugar were obtained using silica gel sheet chromatography .The glyco proteins that this enzyme contains mortly were hetrosamine .In the same time,a research was performed to find out how the additionnal metal hydronium would affect the activity of the fibrinolytic enzyme. We could draw an inclusion that EDTA did not have an obvipns inhibitoryaction.These showed that serine was one of the conponents of the active center while metal hydromium wasn't Urea and denaturant SDS inhibited the activity of the enzyme to a degree,but we could still find that the enzyme's ability of anti-denaturant was strong.
发酵液通过硫酸铵分级盐析沉淀，CM-Sephadex C-25阳离子交换层析，DEAE-Sephadex A-50阴离子交换层析和Sephadex G-75凝胶过滤，获得一种具有纤溶酶活性的蛋白酶BK，其分子量大约为23KD。以苯胺-硫酸法测定中性糖含量为7.55％，以3，5-二硝基水杨酸法(DNS法)测总还原糖含量分别为7.45％。以硅胶G薄层层析法鉴定糖的种类，该酶所含糖蛋白主要为中性戊糖，同时研究了一些金属离子的添加对纤溶酶的活性的影响，EDTA对酶活影响不大，而PMSF对酶活有较强的抑制作用，说明该酶的活性中心有丝氨酸参与，属丝氨酸酶系，而其作用中心无Ca~(2+)，Mg~(2+)等金属离子参与。脲和SDS变性剂对酶有一定
的抑制作用，但较弱，可以看出酶的抗变性剂能力较强。
A novel plasmin-producing bacterial strain PY,which had been identified as Bacillus Licheniformao
Using a dosage of about 1 X 1014 to 4X 1015 N ions/cm2 -s and 20kev, the strain B L.PY. was treated by N ion implantation
and a stable mutant strain with high yield of plasmin is obtained .In general, the rate of mutation and death grows as the
increasing of ion dosage. The shape of strains changed. Shaking fermentation indicates that compared with that of normal
strain about 120 strians, the enzymetic production of the mutant one increases 51.13%, reaching 1210U. Fermentation conditions
for enzyme production have been established. Composition of the medium (%) is edible sucrose 3, soybean poder 1.2, yeast extract 0.2, MgSO4 7H2O 0.1, CaCl2 0.03, K2HPO4 0.3, KH2PO40.04, pH 7.2, After fermentation in 1.5 liter of fermentorat 37"C with an aeration rate of 1: 0.5 - 1:1 for 4 days, the average fibrinolytic activity was about 1320U/ml culture broth,increase by 65% over the mutant.
The purification of enzyme BK include saline extraction,, ion-exchange chromatography on CM-Sephadex C-25 and DEAE-Sephadex A-50, and gel-filtration on Sephadex G-75 ,with a molecular weight of 23 KD. The protocols used were phenols.sulfuric acid method,3,5-dinitrosalicylic acid method and thin layer chromatography of salic gel G. Reducing sugar ang the tatol sugar were
measured with 3,5-2nitro-salcylic acid.We found that their contents were 7.45% and 7.49% respectnely.The categories of sugar were obtained using silica gel sheet chromatography .The glyco proteins that this enzyme contains mortly were hetrosamine .In the same time,a research was performed to find out how the additionnal metal hydronium would affect the activity of the fibrinolytic enzyme. We could draw an inclusion that EDTA did not have an obvipns inhibitoryaction.These showed that serine was one of the conponents of the active center while metal hydromium wasn't Urea and denaturant SDS inhibited the activity of the enzyme to a degree,but we could still find that the enzyme's ability of anti-denaturant was strong.
引文
(1) D.ColIen .D.C.Stump ,H.K.Gold,Annu,Rev,Med 1998,39:405-423
(2) D.J Collen cell Biochem 1987 33 :77-86．
(3) Jeon Ok-Hee Moon Woong-Joon. and Kion Doo-Sik, 1995． An auticoagulant/flbrinolytic protease from Lumbricus rubellus. J. Biochenl. mol. Biol.28(2) :138-142
(4) .Fujata M, K.Hong Y.Ltom et al Biol phasm Ball.,1995,18:1386-1391．
(5) G.Darta, A.Dong .J.Witl et al. Arch. Bil chem .. biophys., 1995,317:36-373．
(6) W.Kim,K.chio,Y.kim etal Appl Envion,Microbiol., 1996,62:2482-2485
(7) S.A.Maksoud,N.H,Tharnat.Acta Pharm,Ture., 1995,37: 11 4-129
(8) S.A,el.-Aassar,Biotechnol,lett., 1995,7:943-948
(9) Yu zengliang,Yang jianbo,Wu yuelin et at Nucl instr and Meth, 1993,880/81 : 1328-1331
(10)Yu zengliang,Yang jianbo,He jianjun et at Mutation breeding by ion implantation, Nucl instr and Meth, 1991,B59/60:705-708
(11)Huang weidong,Yu zengliang,Wang xiangqin,et,aI.Redial Phys Chem: 1996,48(3):319-323
(12)Chen Y R, Mo S W ,Qiu S H. Effects of N~+ ion implantation on wheat growth. Nucl instr and Meth,1987;23B:344
(13)谢立清，张荫芬，陈如意等。离子注入抗生素产生菌诱变效应的研究核技术，1995；18(6)：324
(14)朱凤绥，卫俊智，孙元枢，N~+离子引起的黑卖染色体易位，作物学报，1993；19(4)：299
(15)陈宇等 离子注入红霉素产生菌诱变高产菌株及其机理初步研究 中国抗生素杂志 1997：22(6) 410
(16)宋道军 姚建铭 吴丽芳 离子注入对微生物细胞的刻蚀与对DNA的损伤及修复 遗传 221(4)37-40 1999
(17)罗大珍 王宇钢 朱文 离子注入右旋糖酐生产菌的诱变效应研究 微生物学报 37(4)：312-315，1997
(18)Mihara H,Sumi H.,Akazawa K.,Yoneta T. and Mizumota H, 1983. Fibinolytic enzyme extracted form the earthworm,Throms,Haemmosts,50:258
(19)路英华，金汝成，1986。蚯蚓纤溶酶的提取，性质鉴定和溶解血栓的研究。兰州大学学报(自然科学版)。22(1)：95-100
(20)张伟，刘秀丽，赵晓渝，1997。蚯蚓纤溶酶的糖成分分析。生物技术，7(4)：19-22
(21)周远聪，朱洪，陈远聪，陶宗晋。赤子爱蚯蚓(Eiseniafoelide)纤溶酶的分离纯化。1988，生物化学与生物物理学报。20(1)：35-41
(22)Mihara H.,Sumi H.,Yoneta.,Mizumoton H.,Ikedo R.,Seiki M and Marayama M. 1991, A novel fibrinolytic enzyme extracted form the earthworm, Lumbricus rubellus. Jpn.J. Physial, 41(3)461-472
(23)Nobuyoshi N.,Hisashi M.,and Hiroyuki S. 1993,Chracterization of popent fibrinolytic enzyme in earthworm, Lumbricus rubellus.Biosei.Biotech. Biochem.57(10): 1726-1736
(24)Sumi H.,Nakajima N.and Mihara H. 1993.Avery stable and potent ibrinolytic enzyme found in earthworm, Lumbricus rubellus autolysate .Comparative Biochemistry and physiology B
Comparative Biochemistry.106(3):763-766
(25) Jeon Ok-Hee Moon Woong -Joon. and Kion Doo-Sik, 1995. An auticoagulant/fibrinolytic protease from Lumbricus rubellus. J. Biochenl. mol. Biol.28(2): 138-142
(26) Mao, J. P and Y.L, Xiong A New fibrinogense from the Venom of Trimeresums mucrosguamatas Chinese habu sake, Asiatic Hepertology Reseach 1994 5(4): 408-415.
(27) 范秀容，李文武，沈萍，1982，微生物学实验(第二版)，北京，高等教育出版社，260
(28) Kondo I, and Fujise K,1997,Serotype B staphylococcal bacteriophage singly convening staphylokinase. Infect. Immum. 18:266-272
(29) 李荣萍，李晶，越晓祥，张淑海，张云湖，杨志兴。1996。一种具有纤溶活性的枯草芽孢杆菌蛋白激酶的研究。Ⅰ。高产酶菌株的筛选与鉴定。生物技术。6(3)：21-23
(30) Astrup T. and Mullertz S. 1952. The fibrin plate metted for estmating fibrinotytic activity.Arch .Biochem. Biopheys .40;346-351.
(31) Deogny L., Weidenbach A and Hampton W. 1975. Improved fibrin plate method for fibrinolytie activty measuremevts:use of bentonite precipitation and agar solidifiachion Clinica.Chimica. Acta. 160:85-89.
(32) 谢立清等，1995，离子束注入抗生素产生菌诱变效应的衙门就，核技术1995(6)
(33) 李春善 王文林，1997，《生物统计学》科学出版社
(34) 张龙翔，张庭芳，李令媛。1997，生化实验方法和技术(第一版)，北京：高等教育出版社
(35) D.R.马歇克，J.T.门永，R.R.布格斯，M.W.克努特，W.A.布伦南，S-H林(朱厚础等译)，1999，蛋白质纯化与实验指南，北京，科学出版社，11-56
(36) F．奥斯伯，R。布伦特，R。E。金斯顿，D。D穆尔，J。G。塞得曼，J。A。史密斯，K。斯特拉尔(颜子颖，王海林译)，1988，精编分子生物学实验指南，北京；科学出版社，333-338
(37) 张伟，刘秀丽，赵晓谕，1997，蚯蚓纤溶酶的糖成分分析。生物技术，7(4)：19-22，1997
(38) (日)微生物研究法讨论会；(程光胜，李玲阁，张启先，樊洪业译)1983，微生物学实验法 北京，科学出版社 198-200
(39)王骏，王敏，王以光，生物工程学报，1999(15)：147—152。
(40)工业发酵分析，无锡轻工业学院编。轻工业出版社，1991：224-241
(41)彭先风，陶建宁，杨继虞，彭余田，鲍锦库，赤子爱胜蚓五种纯化纤溶酶的理化性质研究。华西药学杂志，1999，14(2)：98—101 ’
(2) D.J Collen cell Biochem 1987 33 :77-86．
(3) Jeon Ok-Hee Moon Woong-Joon. and Kion Doo-Sik, 1995． An auticoagulant/flbrinolytic protease from Lumbricus rubellus. J. Biochenl. mol. Biol.28(2) :138-142
(4) .Fujata M, K.Hong Y.Ltom et al Biol phasm Ball.,1995,18:1386-1391．
(5) G.Darta, A.Dong .J.Witl et al. Arch. Bil chem .. biophys., 1995,317:36-373．
(6) W.Kim,K.chio,Y.kim etal Appl Envion,Microbiol., 1996,62:2482-2485
(7) S.A.Maksoud,N.H,Tharnat.Acta Pharm,Ture., 1995,37: 11 4-129
(8) S.A,el.-Aassar,Biotechnol,lett., 1995,7:943-948
(9) Yu zengliang,Yang jianbo,Wu yuelin et at Nucl instr and Meth, 1993,880/81 : 1328-1331
(10)Yu zengliang,Yang jianbo,He jianjun et at Mutation breeding by ion implantation, Nucl instr and Meth, 1991,B59/60:705-708
(11)Huang weidong,Yu zengliang,Wang xiangqin,et,aI.Redial Phys Chem: 1996,48(3):319-323
(12)Chen Y R, Mo S W ,Qiu S H. Effects of N~+ ion implantation on wheat growth. Nucl instr and Meth,1987;23B:344
(13)谢立清，张荫芬，陈如意等。离子注入抗生素产生菌诱变效应的研究核技术，1995；18(6)：324
(14)朱凤绥，卫俊智，孙元枢，N~+离子引起的黑卖染色体易位，作物学报，1993；19(4)：299
(15)陈宇等 离子注入红霉素产生菌诱变高产菌株及其机理初步研究 中国抗生素杂志 1997：22(6) 410
(16)宋道军 姚建铭 吴丽芳 离子注入对微生物细胞的刻蚀与对DNA的损伤及修复 遗传 221(4)37-40 1999
(17)罗大珍 王宇钢 朱文 离子注入右旋糖酐生产菌的诱变效应研究 微生物学报 37(4)：312-315，1997
(18)Mihara H,Sumi H.,Akazawa K.,Yoneta T. and Mizumota H, 1983. Fibinolytic enzyme extracted form the earthworm,Throms,Haemmosts,50:258
(19)路英华，金汝成，1986。蚯蚓纤溶酶的提取，性质鉴定和溶解血栓的研究。兰州大学学报(自然科学版)。22(1)：95-100
(20)张伟，刘秀丽，赵晓渝，1997。蚯蚓纤溶酶的糖成分分析。生物技术，7(4)：19-22
(21)周远聪，朱洪，陈远聪，陶宗晋。赤子爱蚯蚓(Eiseniafoelide)纤溶酶的分离纯化。1988，生物化学与生物物理学报。20(1)：35-41
(22)Mihara H.,Sumi H.,Yoneta.,Mizumoton H.,Ikedo R.,Seiki M and Marayama M. 1991, A novel fibrinolytic enzyme extracted form the earthworm, Lumbricus rubellus. Jpn.J. Physial, 41(3)461-472
(23)Nobuyoshi N.,Hisashi M.,and Hiroyuki S. 1993,Chracterization of popent fibrinolytic enzyme in earthworm, Lumbricus rubellus.Biosei.Biotech. Biochem.57(10): 1726-1736
(24)Sumi H.,Nakajima N.and Mihara H. 1993.Avery stable and potent ibrinolytic enzyme found in earthworm, Lumbricus rubellus autolysate .Comparative Biochemistry and physiology B
Comparative Biochemistry.106(3):763-766
(25) Jeon Ok-Hee Moon Woong -Joon. and Kion Doo-Sik, 1995. An auticoagulant/fibrinolytic protease from Lumbricus rubellus. J. Biochenl. mol. Biol.28(2): 138-142
(26) Mao, J. P and Y.L, Xiong A New fibrinogense from the Venom of Trimeresums mucrosguamatas Chinese habu sake, Asiatic Hepertology Reseach 1994 5(4): 408-415.
(27) 范秀容，李文武，沈萍，1982，微生物学实验(第二版)，北京，高等教育出版社，260
(28) Kondo I, and Fujise K,1997,Serotype B staphylococcal bacteriophage singly convening staphylokinase. Infect. Immum. 18:266-272
(29) 李荣萍，李晶，越晓祥，张淑海，张云湖，杨志兴。1996。一种具有纤溶活性的枯草芽孢杆菌蛋白激酶的研究。Ⅰ。高产酶菌株的筛选与鉴定。生物技术。6(3)：21-23
(30) Astrup T. and Mullertz S. 1952. The fibrin plate metted for estmating fibrinotytic activity.Arch .Biochem. Biopheys .40;346-351.
(31) Deogny L., Weidenbach A and Hampton W. 1975. Improved fibrin plate method for fibrinolytie activty measuremevts:use of bentonite precipitation and agar solidifiachion Clinica.Chimica. Acta. 160:85-89.
(32) 谢立清等，1995，离子束注入抗生素产生菌诱变效应的衙门就，核技术1995(6)
(33) 李春善 王文林，1997，《生物统计学》科学出版社
(34) 张龙翔，张庭芳，李令媛。1997，生化实验方法和技术(第一版)，北京：高等教育出版社
(35) D.R.马歇克，J.T.门永，R.R.布格斯，M.W.克努特，W.A.布伦南，S-H林(朱厚础等译)，1999，蛋白质纯化与实验指南，北京，科学出版社，11-56
(36) F．奥斯伯，R。布伦特，R。E。金斯顿，D。D穆尔，J。G。塞得曼，J。A。史密斯，K。斯特拉尔(颜子颖，王海林译)，1988，精编分子生物学实验指南，北京；科学出版社，333-338
(37) 张伟，刘秀丽，赵晓谕，1997，蚯蚓纤溶酶的糖成分分析。生物技术，7(4)：19-22，1997
(38) (日)微生物研究法讨论会；(程光胜，李玲阁，张启先，樊洪业译)1983，微生物学实验法 北京，科学出版社 198-200
(39)王骏，王敏，王以光，生物工程学报，1999(15)：147—152。
(40)工业发酵分析，无锡轻工业学院编。轻工业出版社，1991：224-241
(41)彭先风，陶建宁，杨继虞，彭余田，鲍锦库，赤子爱胜蚓五种纯化纤溶酶的理化性质研究。华西药学杂志，1999，14(2)：98—101 ’