一种纤溶酶SPFE-Ⅲ的生物制备、分离纯化及酶学性质研究
|
摘要
血栓栓塞性疾病主要包括脑血栓、心肌梗死、静脉血栓等,是目前临床上死亡率最高的疾病之一,并且在世界范围内的发病率正逐年上升,因此人们一直都在研制开发新型的溶血栓药物。本文从亚洲传统发酵食品——虾酱中筛选到一株产纤溶酶能力较强的菌株,由中国典型培养物保藏中心定名为Bacillus sp.nov.SK006(CCTCC NO.:M 205071),已经证明该菌株能产生四种不同的纤溶酶,本文在优化该菌产酶条件的基础上,主要对芽孢杆菌Bacillus sp.nov.SK006产生的其中一种纤溶酶(SPFE-Ⅲ)作了深入的研究。
首先在摇瓶水平上考察了培养基组成及培养条件对Bacillus sp.nov.SK006发酵产纤溶酶能力的影响,结果表明该菌株产酶最佳的培养基组分为:葡萄糖20 g/L,胰蛋白胨30g/L,Na_2HPO_4·12H_2O 15g/L,NaH_2PO_4·2H_2O 1.3g/L,MgSO_4·7H_2O 0.5g/L,CaCl_20.1g/L;最佳培养条件:种龄18h,接种量3%,发酵周期24h,初始pH为7.0,摇床转速180r/min,发酵温度37℃,在此条件下发酵液纤溶活性(以plasmin为标准)高达2.63 U/mL,是优化前0.70 U/mL的3.76倍。
芽孢杆菌Bacillus sp.nov.SK006的发酵液经乙醇沉淀、离子交换层析(DEAE-Sepharose CL-6B)、凝胶过滤层析(Superdex 75)后分离纯化出一种电泳纯的纤溶酶SPFE-Ⅲ,分子量约为42.8 kDa,酶活为7.1 U/mg(以plasmin为标准)。SPFE-Ⅲ对纤维蛋白原的降解过程为最先降解α链,其次是γ链,而对β链的降解最缓慢;利用加热纤维蛋白平板法研究纤溶酶SPFE-Ⅲ对纤维蛋白的作用方式,结果发现该酶对纤维蛋白有直接降解作用,而对纤溶酶原的敏感性较低。
其次研究了纯化后纤溶酶SPFE-Ⅲ的酶学性质:最适反应温度为30-40℃,最适反应pH范围为7.0-8.0,在40℃下保温1h剩余酶活力约为60%,而在50℃保温1h剩余酶活力只有30%左右,纤溶酶SPFE-Ⅲ对高温较为敏感。金属离子实验表明:在实验选择的范围内,除Ba~(2+)外其它金属离子对纤溶酶SPFE-Ⅲ纤溶活性均有一定程度的抑制作用,其中Ca~(2+)、Mn~(2+)、Fe~(3+)和Zn~(2+)的抑制作用较强,抑制率为50%左右。抑制剂实验表明:蛋白酶抑制剂对纤溶酶SPFE-Ⅲ有不同程度的抑制作用,EDTA和β-巯基乙醇对纤溶酶SPFE-Ⅲ都有较强的抑制作用。
最后分别选择了三种人工合成的发光底物D-Val-Leu-Arg-pNA、D-Val-Leu-Lys-pNA和N-Succ-Ala-Ala-Pro-Phe-pNA与纤溶酶SPFE-Ⅲ反应。SPFE-Ⅲ对底物N-Succ-Ala-Ala-Pro-Phe-pNA具有最强的专一性,其酰胺水解活性是75.77AU,该底物是胰凝乳蛋白酶和枯草杆菌蛋白酶的最适底物。对N-Succ-Ala-Ala-Pro-Phe-pNA的米氏常数K_m为0.239 mmol/L,酶反应转换数k_(cat)为475.5s~(-1),表明纤溶酶SPFE-Ⅲ对该底物的亲和性较好,反应速度较高。
The incidence of thrombotic disorder including cerebral stroke, myocardial infarction, and venous thromboembolism are rapidly increasing throughout the world. A tremendous amount of research has been done in the area of prevention and the treatment of the diseases. An extracellar fibrinolytic strain, isolated from fermented shrimp paste, a popular seasoning in Asian countries, was shown to have a strong fibrinolytic activity. It was named as Bacillus sp. nov. SK006 by CCTCC. The strain was proved to produce four fibrinolytic isoenzymes. In this study, one fibrinolytic enzyme (SPFE-Ⅲ) from Bacillus sp. nov. SK006 was intensively investigated.
The medium composition and fermentation conditions for fibrinolytic enzyme production were optimized in the research. The maximal yield of fibrinolytic enzyme (2.63 U/mL) could be obtained in the medium with concentrations of 20 g/L glucose, 30g/L tryptone, 15 g/L Na_2HPO_4·12H_2O, 1.3 g/L NaH_2PO_4·2H_2O, 0.5 g/L MgSO_4·7H_2O, 0.1 g/L CaCl_2, respectively, under the optimized cultivation conditions of pH 7.0, rotation speed 180 r/min and inoculation size 3% in 50 mL of culture medium in 250 mL shake flasks at 37℃for 24 h. The fibrinolytic enzyme production of Bacillus sp. nov. SK006 was 3.76 times of the control.
A fibrinolytic enzyme (SPFE-Ⅲ) which produced from Bacillus sp. nov. SK006, was isolated using a combination of ethanol precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and Superdex 75 gel filtration chromatography. The purified SPFE-Ⅲthat has a molecular weight of 42.8 kDa was assessed homogeneous by SDS-PAGE. The specific activity was determined to be 7.1 U/mg using plasmin as a standard. SPFE-Ⅲeffectively degraded fibrin clots by direct fibrinolysis. During the degradation process of fibrinogen,α-subunits of fibrinogen were cleaved first, followed by slower release of theγ-chains.β-chains were resistant to the enzyme digestion.
The enzyme had an optimal pH of 7.0-8.0. It had an optimal reaction temperature of 30℃-40℃, and kept 60% and 30% of the initial activity after heating at 40℃and 50℃for 1 hour. In addition, barium ion stimulated the enzyme activity whereas Zn~(2+), Ca~(2+), Fe~(3+), Mn~(2+) and Cu~(2+) caused deactivation. SPFE-Ⅲwas completely inhibited either by 10 mmol/L EDTA or 1 mmol/L 2-mercaptoethanol. The fibrinolytic activity was partially inhibited by other protease inhibitor.
SPFE-Ⅲhad the highest affinity for N-Succ-Ala-Ala-Pro-Phe-pNA, which is a well-known substrate for subtilisin or chymotrypsin. SPFE-Ⅲalso degraded D-Val-Leu-Lys-pNA (for plasmin). However, it showed a lower activity for D-Val-Leu-Arg-pNA (for kallikrein) compared with other investigated fibrinolytic enzymes. K_(m) and k_(cat) for N-Succ-Ala-Ala-Pro-Phe-pNA were 0.239 mmol/L and 475.5 s~(-1), respectively.
In conclusion, SPFE-Ⅲ, a new type of fibrinolytic enzyme, has some potential for practical application as a source of functional foods and new therapeutic agents to treat thrombosis.
首先在摇瓶水平上考察了培养基组成及培养条件对Bacillus sp.nov.SK006发酵产纤溶酶能力的影响,结果表明该菌株产酶最佳的培养基组分为:葡萄糖20 g/L,胰蛋白胨30g/L,Na_2HPO_4·12H_2O 15g/L,NaH_2PO_4·2H_2O 1.3g/L,MgSO_4·7H_2O 0.5g/L,CaCl_20.1g/L;最佳培养条件:种龄18h,接种量3%,发酵周期24h,初始pH为7.0,摇床转速180r/min,发酵温度37℃,在此条件下发酵液纤溶活性(以plasmin为标准)高达2.63 U/mL,是优化前0.70 U/mL的3.76倍。
芽孢杆菌Bacillus sp.nov.SK006的发酵液经乙醇沉淀、离子交换层析(DEAE-Sepharose CL-6B)、凝胶过滤层析(Superdex 75)后分离纯化出一种电泳纯的纤溶酶SPFE-Ⅲ,分子量约为42.8 kDa,酶活为7.1 U/mg(以plasmin为标准)。SPFE-Ⅲ对纤维蛋白原的降解过程为最先降解α链,其次是γ链,而对β链的降解最缓慢;利用加热纤维蛋白平板法研究纤溶酶SPFE-Ⅲ对纤维蛋白的作用方式,结果发现该酶对纤维蛋白有直接降解作用,而对纤溶酶原的敏感性较低。
其次研究了纯化后纤溶酶SPFE-Ⅲ的酶学性质:最适反应温度为30-40℃,最适反应pH范围为7.0-8.0,在40℃下保温1h剩余酶活力约为60%,而在50℃保温1h剩余酶活力只有30%左右,纤溶酶SPFE-Ⅲ对高温较为敏感。金属离子实验表明:在实验选择的范围内,除Ba~(2+)外其它金属离子对纤溶酶SPFE-Ⅲ纤溶活性均有一定程度的抑制作用,其中Ca~(2+)、Mn~(2+)、Fe~(3+)和Zn~(2+)的抑制作用较强,抑制率为50%左右。抑制剂实验表明:蛋白酶抑制剂对纤溶酶SPFE-Ⅲ有不同程度的抑制作用,EDTA和β-巯基乙醇对纤溶酶SPFE-Ⅲ都有较强的抑制作用。
最后分别选择了三种人工合成的发光底物D-Val-Leu-Arg-pNA、D-Val-Leu-Lys-pNA和N-Succ-Ala-Ala-Pro-Phe-pNA与纤溶酶SPFE-Ⅲ反应。SPFE-Ⅲ对底物N-Succ-Ala-Ala-Pro-Phe-pNA具有最强的专一性,其酰胺水解活性是75.77AU,该底物是胰凝乳蛋白酶和枯草杆菌蛋白酶的最适底物。对N-Succ-Ala-Ala-Pro-Phe-pNA的米氏常数K_m为0.239 mmol/L,酶反应转换数k_(cat)为475.5s~(-1),表明纤溶酶SPFE-Ⅲ对该底物的亲和性较好,反应速度较高。
The incidence of thrombotic disorder including cerebral stroke, myocardial infarction, and venous thromboembolism are rapidly increasing throughout the world. A tremendous amount of research has been done in the area of prevention and the treatment of the diseases. An extracellar fibrinolytic strain, isolated from fermented shrimp paste, a popular seasoning in Asian countries, was shown to have a strong fibrinolytic activity. It was named as Bacillus sp. nov. SK006 by CCTCC. The strain was proved to produce four fibrinolytic isoenzymes. In this study, one fibrinolytic enzyme (SPFE-Ⅲ) from Bacillus sp. nov. SK006 was intensively investigated.
The medium composition and fermentation conditions for fibrinolytic enzyme production were optimized in the research. The maximal yield of fibrinolytic enzyme (2.63 U/mL) could be obtained in the medium with concentrations of 20 g/L glucose, 30g/L tryptone, 15 g/L Na_2HPO_4·12H_2O, 1.3 g/L NaH_2PO_4·2H_2O, 0.5 g/L MgSO_4·7H_2O, 0.1 g/L CaCl_2, respectively, under the optimized cultivation conditions of pH 7.0, rotation speed 180 r/min and inoculation size 3% in 50 mL of culture medium in 250 mL shake flasks at 37℃for 24 h. The fibrinolytic enzyme production of Bacillus sp. nov. SK006 was 3.76 times of the control.
A fibrinolytic enzyme (SPFE-Ⅲ) which produced from Bacillus sp. nov. SK006, was isolated using a combination of ethanol precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and Superdex 75 gel filtration chromatography. The purified SPFE-Ⅲthat has a molecular weight of 42.8 kDa was assessed homogeneous by SDS-PAGE. The specific activity was determined to be 7.1 U/mg using plasmin as a standard. SPFE-Ⅲeffectively degraded fibrin clots by direct fibrinolysis. During the degradation process of fibrinogen,α-subunits of fibrinogen were cleaved first, followed by slower release of theγ-chains.β-chains were resistant to the enzyme digestion.
The enzyme had an optimal pH of 7.0-8.0. It had an optimal reaction temperature of 30℃-40℃, and kept 60% and 30% of the initial activity after heating at 40℃and 50℃for 1 hour. In addition, barium ion stimulated the enzyme activity whereas Zn~(2+), Ca~(2+), Fe~(3+), Mn~(2+) and Cu~(2+) caused deactivation. SPFE-Ⅲwas completely inhibited either by 10 mmol/L EDTA or 1 mmol/L 2-mercaptoethanol. The fibrinolytic activity was partially inhibited by other protease inhibitor.
SPFE-Ⅲhad the highest affinity for N-Succ-Ala-Ala-Pro-Phe-pNA, which is a well-known substrate for subtilisin or chymotrypsin. SPFE-Ⅲalso degraded D-Val-Leu-Lys-pNA (for plasmin). However, it showed a lower activity for D-Val-Leu-Arg-pNA (for kallikrein) compared with other investigated fibrinolytic enzymes. K_(m) and k_(cat) for N-Succ-Ala-Ala-Pro-Phe-pNA were 0.239 mmol/L and 475.5 s~(-1), respectively.
In conclusion, SPFE-Ⅲ, a new type of fibrinolytic enzyme, has some potential for practical application as a source of functional foods and new therapeutic agents to treat thrombosis.
引文
1.Mine Y, Wong A H, Jiang B. Fibrinolytic enzymes in Asian traditional fermented foods[J]. Food Rev.In., 2005, 38: 243-250
2.Gorman P R, Bundy G L, Peterson D C. et al. Inhibition of human Platelet thromboxane synthetase by 9,11-azoprosta-5,13-dienotic acid[J]. Proc Natl Acac Sci USA, 1977, 74(9): 4007-4011
3.张丽萍.血栓形成及抗血栓药物研究的进展[J].心功能杂志,1998,10(1):29-34
4.冯周琴.实用血栓病学[M].郑州:河南科学技术出版社,1998
5.王振义,立家增,阮长耿主编.血栓与止血.基础理论与临床(第二版)[M].上海:上海科学技术出版社.1996.21
6.王鸿利,王学锋.血栓病临床新技术[M].北京:人民军医出版社,2003
7.王中枢.纤维蛋白溶解的生物化学[M].北京:科学出版社,1991
8.陈撷珠.血栓溶解治疗的进展[M].国内医学内科学分册,1991,18(2):50-54
9.O' reilly M S, Hol M L, Shing Y, et al. Angiostatin: A novel angiogenesis inhibitor that mediateds the suppression of metastases by a lewis lung carcinoma[J]. 1994, 79: 315-328
10.Can Y, Chen A, Anss G, et al. Kringle 5 of plasmingogenisa novel inhibitor of endothelial cell growth[J]. J Biol chem, 1997, 272(360): 22924-22928
11.范彬,张英起,颜真等.人纤溶酶原饼环区5基因的原核表达及活性测定[J].中国生物化学与分子生物学学报,2001,17(60):710-714
12.上海第一医学院.医用生物化学(上册)[M].北京:人民卫生出版社,1979:59-62
13.Chavakis T, Robin A, Pixley I, et al. A novel antithronbotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor function[J]. J Biol Chem, 2002, 277(36): 32677-32682
14.Urona T, Hayato I, Kazuo U, et al. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor typel[J]. J Biol Chem, 2001, 276(27):24690-24696
15.路福平,杜连祥,杜冰等.微生物发酵产生纤溶酶的研究[J].天津轻工业学院学报,1999,1:5-9
16.韩玉珉,申同健.水蛭素的分子生物学研究[J].生物化学与生物物理进展,1991,18(2):88-90,117
17.陆核,阮长耿主编.血栓与止血(第一版)[M].南京:江苏科技出版社,1994:335-368
18.Nirav J M, Khan M D, Facc M D. Cardiology's 10 Greatest Discoverise of the 20th century[J]. Texas Heart Institude Journal, 2002,29(3): 164-171
19.孙天霄,徐长法.溶栓剂的蛋白质工程[J].生物工程进展,1996,16(2):43-49
20.Mullertz S. Fibrinolysis: An overview, Simin[J]. Thromb. Hemost, 1984,1:1-15
21.Marder V J, Sherry S. Thrombolytic therapy: current status[J]. N Engl J Med., 1998, 318: 1512-1520
22.黄柄辉,陈春麟.临床溶栓药UK与SK生化,药理和临床应用比较[J].生化药物杂志,1990,52(2):19-20
23.Rijken D C. Measurement of human tissue type plasminogen activator by a two-sites immunoradiometric assay[J]. J. Lab.Clin. Med., 1983(101): 274-284
24.马大龙.生物技术药物[M].北京:科学出版社,2001
25.刘征辉,阮长耿.第三代溶栓药物研究进展[J].国外医学输血及血液学分册,1997,20(4):228-231
26.Datar R V, Cartwright T, Rosen C G. Process economic of animal cell and bacterial fermentations: A case study analysis of tissue plasminoen activator[J]. Biol Technol, 1993,11:349-355
27.赵海霞.组织型纤溶酶原激活物研究进展[J].国外医学输血及血液学分册,1993,18(1):19-23
28.Nakajima N, Ishihara K, Sugimoto M, et al. Chemical modification of earthworm fibrinolytic enzyme with human serum albumin fragment and characterization of the protease as a therapeutic enzyme[J].Biosci Biotechnol Biochem, 1996,60(2): 293-300
29.Egen NB, Russell FE, Sammons DW, et al. Isolation by preparative isoelectric focussing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contorix (Southern copperhead )[J]. Toxicon, 1987,25(11): 1189-1198
30.Baker BJ, Tu AT. Atroxase-a fibrinolytic enzyme isolated from the venom of western diamondback rattlesnake. Isolation, characterization and cloning[J]. Adv Exp Med Biol, 1996, 391: 203-211
31.Siigur, Samel M, Tonismagi K, Subbi J,et al. Biochemical characterization of lebetase, a direct-acting fibrinolytic enzyme from vipera lebetina snake venom[J]. Thomb Res, 1998,90(1): 39-49
32.Lee JW, Park W. cDNA cloning of brevinase, a heterogeneous two-chain fibrinolytic enzyme from Agkistrsdon blomhoffii brevicaudus snake venom, by serial hybridization-polymerase chain reaction[J].Arch Biochem Biophys, 2000, 377(2):234-240
33.Witt W, Baldus B, Bringmann P, et al. Thomolytic properties of desmodus rofundus salivary plasminogen activator in experimental pulmonary embolism in rats[J]. Blood, 1992,79: 1213-1218
34.裴光源,王中枢.蒲黄“纤溶酶”的分离纯化及部分性质的研究[J].生物化学与生物物理学报,1991,23(1):14-19
35.Egorov NS, Kochetov GA and Khaidarova NV. Isolation and properties of the fibrinolytic ernzyme from the Actinomyces thermovulgaris cultural broth[J]. Mikrobiologiia, 1976,45: 455459
36. Matsubara K, Hori K, Matsuura Y and Miyazawa K. Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum[J]. Comp. Biochem. Physiol. B-Biochem. Mol. Biol, 2000, 125(1): 137-143
37. Matsubara K, Hori K, Matsuura Y and Miyazawa K. A fibrinolytic enzyme from a marine green alga,Codium latum[J]. Phytochemistry, 1999, 52(6): 993-999
38. Bono F, Savi P, Tuong A, etal. Purification and characterization of a novel protease from culture filtrates of a Strptomyces sp. FEMS Microbiol Lett, 1996, 141: 213-222
39. Chitte R R, Dey S. Potent fibrinolytic enzy W a from a thermophilicStreptomyces megasporus strain SDS[J]. Lett Appl Microbiol, 2000, 31: 405-410
40. 王骏,王敏,王以光.链霉菌产生的新型纤溶酶的纯化和性质研究[J].生物工程学报,1999,15(2):147-153
41. 武临专,王以光.链霉菌C-3662产生的纤溶活性蛋白酶的纯化和理化性质[J].中国生物化学与分子生物学报,2001,17(1):85-90
42. Toshiaki N. Yoshiyuki O, et al. Isolation and characterization of fibrinogenase from Candida albicans NH-1[J]. Int J Biochem, 1993, 25(12): 815-822
43. Su Tao, Liu Beihui, Li Penh, et al. New solid-state fermentation process for repeated batch prodution of fibrinolytic enzyme by Fusarium Oxysporum[J]. Proc Biochem, 1998, 33(4): 419-422
44. Sumi H, Hamada H,Tsushima H, et al.A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a typical and popular soybean food in the Japanese diet[J]. Experientia, 1987, 43(10):1110-1111
45. Fujita M, Nomura K, Hong K, et al. Prufication and charaterization of a strong fibrinolytic enzyme(nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan[J]. Biochem. Biophy. Res. comman., 1993, 197(3): 1340-1347
46. Kim W, Choi K, Kim Y, et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Strain CK 11-4 screened from Chung kook-Jang[J]. Appl. Environ. Microbial, 1996,62(7): 2482-2488
47. Kim SH and Choi NS. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp.strain DJ-4 screened from Doen-Jang[J]. Biosci. Biotech. Biochem, 2000, 64: 1722-1725
48. Sumi H, Nakajima N, Yatagai C. A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack "Shiokara", a Japanese traditional fermented food. Comp. Biochem. Physiol. 1995, 112, 543-547
49.Noh K A, Kim D H, Choi N S, et al. Isolation of fibrinolytic Enzyme producing strains from kimchi[J].Korean Journal of Food Science and Technology, 1999, 31: 219-223
50.Kim J H, Kim Y S. A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom,Armillariella mellea[J]. Biosci. Biotechnol. Biochem. 1999, 63,2130-2136
51.彭勇,张义正.豆豉溶栓酶产生菌的筛选及其酶学性质的初步研究[J].高技术通讯,2002,12(2):30-34
52.Wong A H, Mine Y. A novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning[J]. Agric. Food. Chem. 2004, 52, 980-986
53.李荣萍,李晶,赵晓祥等.一种具有纤溶活性的枯草杆菌蛋白激酶的研究[J].生物技术,1996,6(3):21-23
54.傅俐,李荣萍,李晶等.一株具有纤溶活性的枯草杆菌(Bacillus subtilis)的研究——液体发酵条件的选择[J].物工程进展,199717(3):31-33
55.钟青萍,谢俊杰,余世望.纳豆菌的分离鉴定及其杭菌活性[J].食品科学,2002,23(10):109-112
56.董明胜,江晓,江汉湖等.溶栓纳豆菌的筛选鉴定及其发酵特性研究[J].南京农业大学学报,2001,24(1):95-98
57.杨艳燕,李顺意,高尚等.豆豉中纳豆杆菌的筛选和纳豆激酶的初步分离[J].沈阳药科大学学报,2001,18(6):436-438,442
58.彭勇,张义正.解淀粉芽孢杆菌(Bacillus amyloliquefaciens)DC-4产豆豉溶栓酶发酵条件的优化[J].食品与发酵工业,2002,28(4):19-22
59.陈波,贺新生,张玲.折多山纳豆生产菌株的分离与筛选[J].食品与发酵工业,2002,28(5):24-25
60.谢秋玲,郭勇.纳豆激酶液体发酵条件的优化[J].华南理工大学学报(自然科学版),1999,27(5):127-130
61.姚劲挺.生物微胶囊固定化溶栓酶产生菌及酿酒酵母微囊化培养模型化的初步研究[D].浙江大学硕士学位论文(2000):39-51
62.刘北域,宋后燕.纳豆激酶基因的克隆及其在枯草杆菌中的表达[J].生物化学与生物物理学报,2002,34(3):338-340
63.彭勇,黄庆,张义正.枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E.coli中的表达[J].生物化学与分子生物学报,2002,18(5):559-563
64.罗立新,黄志立,杨汝德等.纳豆激酶基因在E.coli HB101中的初步表达研究[J].微生物学通报,2002,29(3):62-66
65.余榕捷,汪炬,谢秋玲等.纳豆激酶酶原基因和纳豆激酶基因的克隆及在大肠杆菌中表达[J].生 物技术,2002,12(3):24
66.阎家麟,童岩,藏莹安.豆豉纤溶酶的纯化及其性质研究[J].药物生物技术,2000 7(3):49-152
67.董明盛,江晓,刘诚等.胞外纤溶酶产生菌的筛选及其产酶条件研究[J].食品与发酵工业,2002,27(1):23-27
68.韩润林,张小勇,张建安.枯草杆菌溶栓酶的分离与纯化研究[J].中国生化药物杂志,2000 21(5):219-222
69.吴思方,向梅,陶琳等.豆豉纤溶酶产生菌发酵条件研究[J].中国医药工业杂志,2004,35(6):332-335
70.齐海萍,钱和,王璋等.高活性豆豉纤溶酶产生菌株的筛选[J].中国调味品,2004,2:12-15
71.谢秋玲,郭勇,林剑.纳豆激酶活性测定方法[J].广东药学,2000,10(6):8-9
72.Astrup T. Mullerfz S. The fibrinplate method for estimating fibrinolytic activity[J]. Arch, Biochem.Biopys, 1995,40: 346-351
73.Loewy AG, Santer UV. A simple economic in vitro fibrinolysis rate assay[J]. Tlwombo Res. 1992. 68:201-204
74.#12
75.HaraT, Tadokoro Y, Satoyma T. A simple, easy and routine assay of fibrinolytic enzyme activity[J].Jpn. Soc. Food Sci. Tech, 1996,43 (2): 172-175
76.#12
77.Yukiv Y, Nakagawa T. Fujita M, et al. A sandwich enzyme-linked immunosorbent assay for nattokinase[J]. Biosci Biotech Biochem, 1994,58(2): 366-370
78.黄忠立,罗立新,杨汝德等,纳豆激酶生命的化学[M].2000,20(2):82-83
79.王贤舜,路阳.枯草杆菌碱性蛋白酶E的纯化和性质[J].生物化学与生物物理进展,1992,19(5):398-400
80.Ying Hua, Bo Jiang, Yoshinori Mine, et al. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste[J]. J.Agric. Food Chem. 2008,56,1451-1457
81.北京大学生物系化学教研室.生物化学实验指导[M].北京:高等教育出版社,1984.2
82.Reese E T and Maguire A. Surfactants as stimulants of enzyme production by microorganisms.Applied Microbiology [J]. Fed. 1969, 242-245
83.Sutton D L and Foy C L. Effect of diquat and several surfactants on membrane permeability in red beet root tissue. Bot. Gaz[J]. 1971,132: 299-304
84.Asther M, Corrieu G and Drapron R. Effect of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microbiol. Technol[J]. 1987,9: 245-249
85.董明盛,江晓.溶栓纳豆菌的筛选鉴定及其发酵特性研究[J].南京农业大学学报,2001,24(1)95-98
86.阎家麒,童岩,藏莹安.豆豉纤溶酶的纯化及其性质研究[J].药物生物技术,2000,7(3):149-152
87.沈同,王镜岩,生物化学.北京;高等教育出版社,1989.209-210
88.竹内商,三迟悟.纤溶活性蛋白酶制照法[P].JP公开特许6-153977,1994.06-03
89.付利,杨志兴.纳豆激酶的研究与应用[J].生物工程进展,1995.15(5):46-49
90.Wong A H, Mine Y. A novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning[J]. Agric. Food. Chem. 2004,52,980-986
91.Jeong Y K, Park J U, Baek H, et al. Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17[J]. World J. Microbiol. Biotechnol. 2001,17, 89-92
92.Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature[J]. 1970,227, 680-685
93.Lowry O H, Rosenbrough N J, Farr A L, et al. Protein measurement with the Folin phenol reagent[J].Biol Chem.. 1951,193, 265-275
94.EI-Aassar S. Production and properties enzyme in solid state cultures of Fusarium pallidoroseum[J].Biotechnol. Lett., 1990,17(9): 943-948
95.Wang C T, Ji B P, Li B, et al. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi[J]. Ind. Microbiol. Biotechnol. 2006, 33,750-758
96.Peng Y, Huang Q, Zhang R H, et al. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food[J]. Comp. Biochem. Physiol. B 2003, 134,45-52
97.Chang C T, Fan M H, Kuo F C, et al. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1[J]. Agric. Food Chem. 2000,48, 3210-3216
98.Fujita M, Nomura K, Hong K, et al. Purification and characterization of a strong fibrinolytic enzyme(Nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan[J].Biochem Biophy Res Commun 1993, 197(3): 1340-1346
99.杨志兴,张淑梅,张云湖等.一株具有纤溶活性的枯草杆菌蛋白激酶的初步研究[J].药物生物技术,1996 3(3):133-136
100.王卫东,孙月娥.豆豉纤溶酶产生菌发酵液营养和酶活成分分析[J].现代食品科技,2006,22(2):58-60
101.李江伟,冉国侠,陈新梅.豆豉溶栓酶的分离纯化及其体外溶栓作用[J].中国生化医药杂志,1999,20(3):148-150
102.Nakamura T, Youher Y, Eiji I. Nucleotide sequence of the Subtilisin NAT, aprN, of Bacillus subtilis(natto)[J]. Biosci Biotech Biochem. 1992. 56(11): 1869-1871
103.袁勤生.现代酶学[M].上海:华东理工大学出版社,2001
104.陈石根,周润琦.酶学[M].上海:复旦大学出版社,2001
105.Hase C C, Finkelstein R A. Bacterial extracellular zinc-containing metalloproteases[J]. Microbiol Rev,1993, 57(4):823-837
106.张树政.酶制剂工业[M].北京:科学出版社,1984
2.Gorman P R, Bundy G L, Peterson D C. et al. Inhibition of human Platelet thromboxane synthetase by 9,11-azoprosta-5,13-dienotic acid[J]. Proc Natl Acac Sci USA, 1977, 74(9): 4007-4011
3.张丽萍.血栓形成及抗血栓药物研究的进展[J].心功能杂志,1998,10(1):29-34
4.冯周琴.实用血栓病学[M].郑州:河南科学技术出版社,1998
5.王振义,立家增,阮长耿主编.血栓与止血.基础理论与临床(第二版)[M].上海:上海科学技术出版社.1996.21
6.王鸿利,王学锋.血栓病临床新技术[M].北京:人民军医出版社,2003
7.王中枢.纤维蛋白溶解的生物化学[M].北京:科学出版社,1991
8.陈撷珠.血栓溶解治疗的进展[M].国内医学内科学分册,1991,18(2):50-54
9.O' reilly M S, Hol M L, Shing Y, et al. Angiostatin: A novel angiogenesis inhibitor that mediateds the suppression of metastases by a lewis lung carcinoma[J]. 1994, 79: 315-328
10.Can Y, Chen A, Anss G, et al. Kringle 5 of plasmingogenisa novel inhibitor of endothelial cell growth[J]. J Biol chem, 1997, 272(360): 22924-22928
11.范彬,张英起,颜真等.人纤溶酶原饼环区5基因的原核表达及活性测定[J].中国生物化学与分子生物学学报,2001,17(60):710-714
12.上海第一医学院.医用生物化学(上册)[M].北京:人民卫生出版社,1979:59-62
13.Chavakis T, Robin A, Pixley I, et al. A novel antithronbotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor function[J]. J Biol Chem, 2002, 277(36): 32677-32682
14.Urona T, Hayato I, Kazuo U, et al. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor typel[J]. J Biol Chem, 2001, 276(27):24690-24696
15.路福平,杜连祥,杜冰等.微生物发酵产生纤溶酶的研究[J].天津轻工业学院学报,1999,1:5-9
16.韩玉珉,申同健.水蛭素的分子生物学研究[J].生物化学与生物物理进展,1991,18(2):88-90,117
17.陆核,阮长耿主编.血栓与止血(第一版)[M].南京:江苏科技出版社,1994:335-368
18.Nirav J M, Khan M D, Facc M D. Cardiology's 10 Greatest Discoverise of the 20th century[J]. Texas Heart Institude Journal, 2002,29(3): 164-171
19.孙天霄,徐长法.溶栓剂的蛋白质工程[J].生物工程进展,1996,16(2):43-49
20.Mullertz S. Fibrinolysis: An overview, Simin[J]. Thromb. Hemost, 1984,1:1-15
21.Marder V J, Sherry S. Thrombolytic therapy: current status[J]. N Engl J Med., 1998, 318: 1512-1520
22.黄柄辉,陈春麟.临床溶栓药UK与SK生化,药理和临床应用比较[J].生化药物杂志,1990,52(2):19-20
23.Rijken D C. Measurement of human tissue type plasminogen activator by a two-sites immunoradiometric assay[J]. J. Lab.Clin. Med., 1983(101): 274-284
24.马大龙.生物技术药物[M].北京:科学出版社,2001
25.刘征辉,阮长耿.第三代溶栓药物研究进展[J].国外医学输血及血液学分册,1997,20(4):228-231
26.Datar R V, Cartwright T, Rosen C G. Process economic of animal cell and bacterial fermentations: A case study analysis of tissue plasminoen activator[J]. Biol Technol, 1993,11:349-355
27.赵海霞.组织型纤溶酶原激活物研究进展[J].国外医学输血及血液学分册,1993,18(1):19-23
28.Nakajima N, Ishihara K, Sugimoto M, et al. Chemical modification of earthworm fibrinolytic enzyme with human serum albumin fragment and characterization of the protease as a therapeutic enzyme[J].Biosci Biotechnol Biochem, 1996,60(2): 293-300
29.Egen NB, Russell FE, Sammons DW, et al. Isolation by preparative isoelectric focussing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contorix (Southern copperhead )[J]. Toxicon, 1987,25(11): 1189-1198
30.Baker BJ, Tu AT. Atroxase-a fibrinolytic enzyme isolated from the venom of western diamondback rattlesnake. Isolation, characterization and cloning[J]. Adv Exp Med Biol, 1996, 391: 203-211
31.Siigur, Samel M, Tonismagi K, Subbi J,et al. Biochemical characterization of lebetase, a direct-acting fibrinolytic enzyme from vipera lebetina snake venom[J]. Thomb Res, 1998,90(1): 39-49
32.Lee JW, Park W. cDNA cloning of brevinase, a heterogeneous two-chain fibrinolytic enzyme from Agkistrsdon blomhoffii brevicaudus snake venom, by serial hybridization-polymerase chain reaction[J].Arch Biochem Biophys, 2000, 377(2):234-240
33.Witt W, Baldus B, Bringmann P, et al. Thomolytic properties of desmodus rofundus salivary plasminogen activator in experimental pulmonary embolism in rats[J]. Blood, 1992,79: 1213-1218
34.裴光源,王中枢.蒲黄“纤溶酶”的分离纯化及部分性质的研究[J].生物化学与生物物理学报,1991,23(1):14-19
35.Egorov NS, Kochetov GA and Khaidarova NV. Isolation and properties of the fibrinolytic ernzyme from the Actinomyces thermovulgaris cultural broth[J]. Mikrobiologiia, 1976,45: 455459
36. Matsubara K, Hori K, Matsuura Y and Miyazawa K. Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum[J]. Comp. Biochem. Physiol. B-Biochem. Mol. Biol, 2000, 125(1): 137-143
37. Matsubara K, Hori K, Matsuura Y and Miyazawa K. A fibrinolytic enzyme from a marine green alga,Codium latum[J]. Phytochemistry, 1999, 52(6): 993-999
38. Bono F, Savi P, Tuong A, etal. Purification and characterization of a novel protease from culture filtrates of a Strptomyces sp. FEMS Microbiol Lett, 1996, 141: 213-222
39. Chitte R R, Dey S. Potent fibrinolytic enzy W a from a thermophilicStreptomyces megasporus strain SDS[J]. Lett Appl Microbiol, 2000, 31: 405-410
40. 王骏,王敏,王以光.链霉菌产生的新型纤溶酶的纯化和性质研究[J].生物工程学报,1999,15(2):147-153
41. 武临专,王以光.链霉菌C-3662产生的纤溶活性蛋白酶的纯化和理化性质[J].中国生物化学与分子生物学报,2001,17(1):85-90
42. Toshiaki N. Yoshiyuki O, et al. Isolation and characterization of fibrinogenase from Candida albicans NH-1[J]. Int J Biochem, 1993, 25(12): 815-822
43. Su Tao, Liu Beihui, Li Penh, et al. New solid-state fermentation process for repeated batch prodution of fibrinolytic enzyme by Fusarium Oxysporum[J]. Proc Biochem, 1998, 33(4): 419-422
44. Sumi H, Hamada H,Tsushima H, et al.A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a typical and popular soybean food in the Japanese diet[J]. Experientia, 1987, 43(10):1110-1111
45. Fujita M, Nomura K, Hong K, et al. Prufication and charaterization of a strong fibrinolytic enzyme(nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan[J]. Biochem. Biophy. Res. comman., 1993, 197(3): 1340-1347
46. Kim W, Choi K, Kim Y, et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Strain CK 11-4 screened from Chung kook-Jang[J]. Appl. Environ. Microbial, 1996,62(7): 2482-2488
47. Kim SH and Choi NS. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp.strain DJ-4 screened from Doen-Jang[J]. Biosci. Biotech. Biochem, 2000, 64: 1722-1725
48. Sumi H, Nakajima N, Yatagai C. A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack "Shiokara", a Japanese traditional fermented food. Comp. Biochem. Physiol. 1995, 112, 543-547
49.Noh K A, Kim D H, Choi N S, et al. Isolation of fibrinolytic Enzyme producing strains from kimchi[J].Korean Journal of Food Science and Technology, 1999, 31: 219-223
50.Kim J H, Kim Y S. A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom,Armillariella mellea[J]. Biosci. Biotechnol. Biochem. 1999, 63,2130-2136
51.彭勇,张义正.豆豉溶栓酶产生菌的筛选及其酶学性质的初步研究[J].高技术通讯,2002,12(2):30-34
52.Wong A H, Mine Y. A novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning[J]. Agric. Food. Chem. 2004, 52, 980-986
53.李荣萍,李晶,赵晓祥等.一种具有纤溶活性的枯草杆菌蛋白激酶的研究[J].生物技术,1996,6(3):21-23
54.傅俐,李荣萍,李晶等.一株具有纤溶活性的枯草杆菌(Bacillus subtilis)的研究——液体发酵条件的选择[J].物工程进展,199717(3):31-33
55.钟青萍,谢俊杰,余世望.纳豆菌的分离鉴定及其杭菌活性[J].食品科学,2002,23(10):109-112
56.董明胜,江晓,江汉湖等.溶栓纳豆菌的筛选鉴定及其发酵特性研究[J].南京农业大学学报,2001,24(1):95-98
57.杨艳燕,李顺意,高尚等.豆豉中纳豆杆菌的筛选和纳豆激酶的初步分离[J].沈阳药科大学学报,2001,18(6):436-438,442
58.彭勇,张义正.解淀粉芽孢杆菌(Bacillus amyloliquefaciens)DC-4产豆豉溶栓酶发酵条件的优化[J].食品与发酵工业,2002,28(4):19-22
59.陈波,贺新生,张玲.折多山纳豆生产菌株的分离与筛选[J].食品与发酵工业,2002,28(5):24-25
60.谢秋玲,郭勇.纳豆激酶液体发酵条件的优化[J].华南理工大学学报(自然科学版),1999,27(5):127-130
61.姚劲挺.生物微胶囊固定化溶栓酶产生菌及酿酒酵母微囊化培养模型化的初步研究[D].浙江大学硕士学位论文(2000):39-51
62.刘北域,宋后燕.纳豆激酶基因的克隆及其在枯草杆菌中的表达[J].生物化学与生物物理学报,2002,34(3):338-340
63.彭勇,黄庆,张义正.枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E.coli中的表达[J].生物化学与分子生物学报,2002,18(5):559-563
64.罗立新,黄志立,杨汝德等.纳豆激酶基因在E.coli HB101中的初步表达研究[J].微生物学通报,2002,29(3):62-66
65.余榕捷,汪炬,谢秋玲等.纳豆激酶酶原基因和纳豆激酶基因的克隆及在大肠杆菌中表达[J].生 物技术,2002,12(3):24
66.阎家麟,童岩,藏莹安.豆豉纤溶酶的纯化及其性质研究[J].药物生物技术,2000 7(3):49-152
67.董明盛,江晓,刘诚等.胞外纤溶酶产生菌的筛选及其产酶条件研究[J].食品与发酵工业,2002,27(1):23-27
68.韩润林,张小勇,张建安.枯草杆菌溶栓酶的分离与纯化研究[J].中国生化药物杂志,2000 21(5):219-222
69.吴思方,向梅,陶琳等.豆豉纤溶酶产生菌发酵条件研究[J].中国医药工业杂志,2004,35(6):332-335
70.齐海萍,钱和,王璋等.高活性豆豉纤溶酶产生菌株的筛选[J].中国调味品,2004,2:12-15
71.谢秋玲,郭勇,林剑.纳豆激酶活性测定方法[J].广东药学,2000,10(6):8-9
72.Astrup T. Mullerfz S. The fibrinplate method for estimating fibrinolytic activity[J]. Arch, Biochem.Biopys, 1995,40: 346-351
73.Loewy AG, Santer UV. A simple economic in vitro fibrinolysis rate assay[J]. Tlwombo Res. 1992. 68:201-204
74.#12
75.HaraT, Tadokoro Y, Satoyma T. A simple, easy and routine assay of fibrinolytic enzyme activity[J].Jpn. Soc. Food Sci. Tech, 1996,43 (2): 172-175
76.#12
77.Yukiv Y, Nakagawa T. Fujita M, et al. A sandwich enzyme-linked immunosorbent assay for nattokinase[J]. Biosci Biotech Biochem, 1994,58(2): 366-370
78.黄忠立,罗立新,杨汝德等,纳豆激酶生命的化学[M].2000,20(2):82-83
79.王贤舜,路阳.枯草杆菌碱性蛋白酶E的纯化和性质[J].生物化学与生物物理进展,1992,19(5):398-400
80.Ying Hua, Bo Jiang, Yoshinori Mine, et al. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste[J]. J.Agric. Food Chem. 2008,56,1451-1457
81.北京大学生物系化学教研室.生物化学实验指导[M].北京:高等教育出版社,1984.2
82.Reese E T and Maguire A. Surfactants as stimulants of enzyme production by microorganisms.Applied Microbiology [J]. Fed. 1969, 242-245
83.Sutton D L and Foy C L. Effect of diquat and several surfactants on membrane permeability in red beet root tissue. Bot. Gaz[J]. 1971,132: 299-304
84.Asther M, Corrieu G and Drapron R. Effect of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microbiol. Technol[J]. 1987,9: 245-249
85.董明盛,江晓.溶栓纳豆菌的筛选鉴定及其发酵特性研究[J].南京农业大学学报,2001,24(1)95-98
86.阎家麒,童岩,藏莹安.豆豉纤溶酶的纯化及其性质研究[J].药物生物技术,2000,7(3):149-152
87.沈同,王镜岩,生物化学.北京;高等教育出版社,1989.209-210
88.竹内商,三迟悟.纤溶活性蛋白酶制照法[P].JP公开特许6-153977,1994.06-03
89.付利,杨志兴.纳豆激酶的研究与应用[J].生物工程进展,1995.15(5):46-49
90.Wong A H, Mine Y. A novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning[J]. Agric. Food. Chem. 2004,52,980-986
91.Jeong Y K, Park J U, Baek H, et al. Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17[J]. World J. Microbiol. Biotechnol. 2001,17, 89-92
92.Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature[J]. 1970,227, 680-685
93.Lowry O H, Rosenbrough N J, Farr A L, et al. Protein measurement with the Folin phenol reagent[J].Biol Chem.. 1951,193, 265-275
94.EI-Aassar S. Production and properties enzyme in solid state cultures of Fusarium pallidoroseum[J].Biotechnol. Lett., 1990,17(9): 943-948
95.Wang C T, Ji B P, Li B, et al. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi[J]. Ind. Microbiol. Biotechnol. 2006, 33,750-758
96.Peng Y, Huang Q, Zhang R H, et al. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food[J]. Comp. Biochem. Physiol. B 2003, 134,45-52
97.Chang C T, Fan M H, Kuo F C, et al. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1[J]. Agric. Food Chem. 2000,48, 3210-3216
98.Fujita M, Nomura K, Hong K, et al. Purification and characterization of a strong fibrinolytic enzyme(Nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan[J].Biochem Biophy Res Commun 1993, 197(3): 1340-1346
99.杨志兴,张淑梅,张云湖等.一株具有纤溶活性的枯草杆菌蛋白激酶的初步研究[J].药物生物技术,1996 3(3):133-136
100.王卫东,孙月娥.豆豉纤溶酶产生菌发酵液营养和酶活成分分析[J].现代食品科技,2006,22(2):58-60
101.李江伟,冉国侠,陈新梅.豆豉溶栓酶的分离纯化及其体外溶栓作用[J].中国生化医药杂志,1999,20(3):148-150
102.Nakamura T, Youher Y, Eiji I. Nucleotide sequence of the Subtilisin NAT, aprN, of Bacillus subtilis(natto)[J]. Biosci Biotech Biochem. 1992. 56(11): 1869-1871
103.袁勤生.现代酶学[M].上海:华东理工大学出版社,2001
104.陈石根,周润琦.酶学[M].上海:复旦大学出版社,2001
105.Hase C C, Finkelstein R A. Bacterial extracellular zinc-containing metalloproteases[J]. Microbiol Rev,1993, 57(4):823-837
106.张树政.酶制剂工业[M].北京:科学出版社,1984