苏云金芽孢杆菌FZ62蛋白酶分离纯化及性质研究
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摘要
苏云金芽孢杆菌FZ62蛋白酶发酵液,分别经过硫酸铵沉淀、Sephadex G-25分子筛脱盐、DEAE Sepharose Fast Flow离子交换柱层析、Sephadex G-75分子筛柱层析对蛋白酶进行纯化,获得SDS-PAGE电泳单一条带的蛋白酶。该纯化过程中,酶的总回收率为9.28%,其比活力为3159.44 U/mg。将蛋白印迹到PVDF膜上后进行N-端测序,前10个残基为:1-GGNNKVGSGK-10。测量蛋白酶的理化性质,其分子量为38.92 kDa,催化酪蛋白水解的最适pH为7.2,在pH 7-12区域内较稳定,pH＞13时酶迅速失活,酶促最适温度为55℃,50℃以下酶的热稳定性好。以酪蛋白为底物,酶促反应符合米氏方程,K_m=0.71 g/L,V_m=2000.00μg/mL/min,活化能为50.22kJ/mol,55℃时的活化焓变为47.49 kJ/mol、活化自由能为84.48 kJ/mol、活化熵变为-0.113 kJ/mol/k。
研究金属离子对蛋白酶活力的影响。结果表明,Na~+、K~+、Li~+和Mg~(2+)等金属离子对酶活力没有任何影响。Ca~(2+)对酶活力有微弱抑制作用,Zn~(2+)、Cu~(2+)、Hg~(2+)和Al~(3+)对该酶有不同程度的抑制作用。研究Zn~(2+)、Cu~(2+)、Hg~(2+)和Al~(3+)的抑制动力学,结果表明:这些金属离子对蛋白酶表现为可逆抑制作用,Zn~(2+)的抑制类型为反竞争型,抑制常数K_I为1.35g/L。Cu~(2+)为混合型类型,抑制常数K_I为1.39g/L。Hg~(2+)为混合型,抑制常数K_I为1.2g/L。Al~(3+)为竞争性抑制,抑制常数K_I为0.6 g/L。
金属螯合剂(EDTA)处理蛋白酶,浓度为1.2 mmol/L时酶活力完全丧失,说明该酶是金属蛋白酶。探究Fe~(3+)对酶活力的影响,结果显示:加入Fe~(3+)后酶活力最可增大2.5倍,在经EDTA处理后的蛋白酶液中,加入Fe~(3+)其终浓度为2mmol/L时,酶活力可恢复59.77%,初步判定Fe~(3+)是该金属蛋白酶的活性中心辅基。
研究几种有机溶剂对蛋白酶活力的影响。结果表明:甲醇、乙醇、正戊醇三种伯醇对酶均有抑作用,抑制强度相近。甲醇对酶的抑制作用略比其他两种伯醇弱。异丙醇对酶效应为可逆抑制作用,IC_(50)为14%,其抑制机理表现为竞争型抑制,K_I为0.243 g/L。甲醛对酶的活性有很强抑制作用,其IC_(50)为88 mmol/L,是一种可逆反竞争型抑制作用,抑制常数K_I为0.036 g/L。
选择氯胺-T、顺丁烯二酸酐、DTT、β-巯基乙醇、N-溴代丁二酰亚胺(NBS)、溴乙酸(BrAc)、乙酰丙酮、苯甲基磺酸氟(PMSF)、N-乙酰咪唑为修饰剂,对蛋白酶进行修饰。结果表明:NBS、BrAc、PMSF对酶有相当的抑制作用。NBS修饰蛋白酶分子中的色氨酸残基,结合荧光发射光谱和紫外吸收光谱的变化分析,经NBS修饰后,酶活力丧失的同时,441 nm处的荧光发射峰峰高下降及277 nm处的紫外吸收峰消失,说明色氨酸是该蛋白酶的活性中心基团。BrAc、PMSF修饰酶后,酶活力也受到影响,从结果显示,组氨酸、丝氨酸是维持酶活性特定结构的重要基团。
The proteinase from Bacillus thuringiensis FZ62 was purified by using ammonium sulfate, column chromatography on Sephadex G-25, DEAE Sepharose Fast Flow and Sephadex G-75. Its fineness was checked to be about a single band by SDS-PAGE. Purification times and recovery rate of enzyme were 14.92 and 9.28 % respectively. The specific activity of the enzyme was 3159.44 U/mg. The series was mensurated after albumen transferred on PVDF. The N-terminal partial sequence was GGNNKVGSGK. The molecular weight of the purified enzyme was estimated to be 38.92 kDa by SDS-PAGE. The optimum pH was determined to be at pH 7.2 and showed to be stable in the range 7-12. The optimum temperature was 55℃, and stability of the enzyme was very well bellow 50℃. Casein as substrate was hydrolyzed to study the enzyme kinetics of proteinase. The enzyme followed typical Michaelis-Menten equation and the K_m and V_m values were determined to be 0.71g/L and 3159.44U/mg. Ea, enthalpy, free-energy and entropy of this enzyme were determined to be 50.22 kJ/mol, 47.49 kJ/mol, 84.48 kJ/mol and 0.11 kJ/mol/k respectively.
The effects of metal ions on the enzyme activity of proteinase were studied. The results showed that Na~+, K~+, Li~+ and Mg~(2+) ions had no any effects on the enzyme activity. Ca~(2+) ions had a few effects on the enzyme activity, while Zn~(2+), Cu~(2+), Hg~(2+)and Al~(3+) inhibited the enzyme. The inhibitory kinetics of Zn~(2+), Cu~(2+), Hg~(2+) and Al~(3+) on the enzyme was further studied. The results showed that Zn~(2+) was a reversible competitive inhibitor of the enzyme and was determined to be non-competitive inhibitor. The inhibition constants of free enzyme (K_I) was 1.35g/L. Cu~(2+)was a reversible mixed-type inhibitor of the enzyme, and the inhibition constants (K_I) was determined to be 1.39 g/L. The inhibition of Hg~(2+) on the enzyme was a reversible mixed-type inhibitor. The inhibition constants of free enzyme (K_I) with Hg~(2+) was 1.2 g/L. Al~(3+) was reversible competitive inhibitor. The inhibition constant of free enzyme (K_I) with Al~(3+) was 0.6 g/L.
The activity of enzyme treated with metal chelating agent-ethylenediaminetetraacetic acid disodium (EDTA) and the enzyme activity was completely lost with concentration at 1.2 mmol/L, which indicated that the enzyme was a metallic-proteinase. Effects of Fe~(3+)on enzyme activity, the results showed that the enzyme activity was accelerated to be 2.5 times. Lost activity of the enzyme would comeback 59.77%, when concentration of Fe~(3+) was 2mmol/L. So Fe~(3+)was judged to the agon of proteinase center primarily.
Relation of different organic solvents and the enzyme activity was studied .The results showed that primary alcohols including methanol, ethanol and n-amyl alcohol inhibited the enzyme, and their extents of inhibition were approximate. Effect of methanol was more feebleness than the other primary alcohol. Isopropyl alcohol was a reversible competitive inhibitor of the enzyme; the inhibition constants (K_I) and half deadly concentration (IC_(50)) were determined to be 0.243g/L and 14%. The inhibition of formaldehyde on the enzyme underwent a reversible reaction, and was a non-competitive inhibitor. The inhibition constant of enzyme and IC_(50) with formaldehyde were 0.036g/L and 88mmol/L.
Effects of chloramines-T, cis-butenedioic anhydride, dithiothreitol,β-mercaptoe-thanol, N-bromo-succinimide, monobromo-acetic acid, acetylacetone, benzyl sulfonic acid fluoride and acet-imidazole on the enzyme were investigated. The results showed that NBS, BrAc, PMSF inhibited the enzyme.When Try residue of the enzyme was modified by NBS, the relation between Try residue and activity of enzyme could be studied according to the fluorescence emission spectra and the ultraviolet spectra. The result showed that the fluorescence emission peak at 441nm and the ultraviolet absorbed peak at 277 nm were disappeared with the loss of the activity of the enzyme after the enzyme was modified by NBS. Try was the activity center residue of this proteinase. The enzyme was modified by BrAc and PMSF, which influenced enzyme activity. From results, His and Ser were very importment to keep configuration of activity center.
研究金属离子对蛋白酶活力的影响。结果表明,Na~+、K~+、Li~+和Mg~(2+)等金属离子对酶活力没有任何影响。Ca~(2+)对酶活力有微弱抑制作用,Zn~(2+)、Cu~(2+)、Hg~(2+)和Al~(3+)对该酶有不同程度的抑制作用。研究Zn~(2+)、Cu~(2+)、Hg~(2+)和Al~(3+)的抑制动力学,结果表明:这些金属离子对蛋白酶表现为可逆抑制作用,Zn~(2+)的抑制类型为反竞争型,抑制常数K_I为1.35g/L。Cu~(2+)为混合型类型,抑制常数K_I为1.39g/L。Hg~(2+)为混合型,抑制常数K_I为1.2g/L。Al~(3+)为竞争性抑制,抑制常数K_I为0.6 g/L。
金属螯合剂(EDTA)处理蛋白酶,浓度为1.2 mmol/L时酶活力完全丧失,说明该酶是金属蛋白酶。探究Fe~(3+)对酶活力的影响,结果显示:加入Fe~(3+)后酶活力最可增大2.5倍,在经EDTA处理后的蛋白酶液中,加入Fe~(3+)其终浓度为2mmol/L时,酶活力可恢复59.77%,初步判定Fe~(3+)是该金属蛋白酶的活性中心辅基。
研究几种有机溶剂对蛋白酶活力的影响。结果表明:甲醇、乙醇、正戊醇三种伯醇对酶均有抑作用,抑制强度相近。甲醇对酶的抑制作用略比其他两种伯醇弱。异丙醇对酶效应为可逆抑制作用,IC_(50)为14%,其抑制机理表现为竞争型抑制,K_I为0.243 g/L。甲醛对酶的活性有很强抑制作用,其IC_(50)为88 mmol/L,是一种可逆反竞争型抑制作用,抑制常数K_I为0.036 g/L。
选择氯胺-T、顺丁烯二酸酐、DTT、β-巯基乙醇、N-溴代丁二酰亚胺(NBS)、溴乙酸(BrAc)、乙酰丙酮、苯甲基磺酸氟(PMSF)、N-乙酰咪唑为修饰剂,对蛋白酶进行修饰。结果表明:NBS、BrAc、PMSF对酶有相当的抑制作用。NBS修饰蛋白酶分子中的色氨酸残基,结合荧光发射光谱和紫外吸收光谱的变化分析,经NBS修饰后,酶活力丧失的同时,441 nm处的荧光发射峰峰高下降及277 nm处的紫外吸收峰消失,说明色氨酸是该蛋白酶的活性中心基团。BrAc、PMSF修饰酶后,酶活力也受到影响,从结果显示,组氨酸、丝氨酸是维持酶活性特定结构的重要基团。
The proteinase from Bacillus thuringiensis FZ62 was purified by using ammonium sulfate, column chromatography on Sephadex G-25, DEAE Sepharose Fast Flow and Sephadex G-75. Its fineness was checked to be about a single band by SDS-PAGE. Purification times and recovery rate of enzyme were 14.92 and 9.28 % respectively. The specific activity of the enzyme was 3159.44 U/mg. The series was mensurated after albumen transferred on PVDF. The N-terminal partial sequence was GGNNKVGSGK. The molecular weight of the purified enzyme was estimated to be 38.92 kDa by SDS-PAGE. The optimum pH was determined to be at pH 7.2 and showed to be stable in the range 7-12. The optimum temperature was 55℃, and stability of the enzyme was very well bellow 50℃. Casein as substrate was hydrolyzed to study the enzyme kinetics of proteinase. The enzyme followed typical Michaelis-Menten equation and the K_m and V_m values were determined to be 0.71g/L and 3159.44U/mg. Ea, enthalpy, free-energy and entropy of this enzyme were determined to be 50.22 kJ/mol, 47.49 kJ/mol, 84.48 kJ/mol and 0.11 kJ/mol/k respectively.
The effects of metal ions on the enzyme activity of proteinase were studied. The results showed that Na~+, K~+, Li~+ and Mg~(2+) ions had no any effects on the enzyme activity. Ca~(2+) ions had a few effects on the enzyme activity, while Zn~(2+), Cu~(2+), Hg~(2+)and Al~(3+) inhibited the enzyme. The inhibitory kinetics of Zn~(2+), Cu~(2+), Hg~(2+) and Al~(3+) on the enzyme was further studied. The results showed that Zn~(2+) was a reversible competitive inhibitor of the enzyme and was determined to be non-competitive inhibitor. The inhibition constants of free enzyme (K_I) was 1.35g/L. Cu~(2+)was a reversible mixed-type inhibitor of the enzyme, and the inhibition constants (K_I) was determined to be 1.39 g/L. The inhibition of Hg~(2+) on the enzyme was a reversible mixed-type inhibitor. The inhibition constants of free enzyme (K_I) with Hg~(2+) was 1.2 g/L. Al~(3+) was reversible competitive inhibitor. The inhibition constant of free enzyme (K_I) with Al~(3+) was 0.6 g/L.
The activity of enzyme treated with metal chelating agent-ethylenediaminetetraacetic acid disodium (EDTA) and the enzyme activity was completely lost with concentration at 1.2 mmol/L, which indicated that the enzyme was a metallic-proteinase. Effects of Fe~(3+)on enzyme activity, the results showed that the enzyme activity was accelerated to be 2.5 times. Lost activity of the enzyme would comeback 59.77%, when concentration of Fe~(3+) was 2mmol/L. So Fe~(3+)was judged to the agon of proteinase center primarily.
Relation of different organic solvents and the enzyme activity was studied .The results showed that primary alcohols including methanol, ethanol and n-amyl alcohol inhibited the enzyme, and their extents of inhibition were approximate. Effect of methanol was more feebleness than the other primary alcohol. Isopropyl alcohol was a reversible competitive inhibitor of the enzyme; the inhibition constants (K_I) and half deadly concentration (IC_(50)) were determined to be 0.243g/L and 14%. The inhibition of formaldehyde on the enzyme underwent a reversible reaction, and was a non-competitive inhibitor. The inhibition constant of enzyme and IC_(50) with formaldehyde were 0.036g/L and 88mmol/L.
Effects of chloramines-T, cis-butenedioic anhydride, dithiothreitol,β-mercaptoe-thanol, N-bromo-succinimide, monobromo-acetic acid, acetylacetone, benzyl sulfonic acid fluoride and acet-imidazole on the enzyme were investigated. The results showed that NBS, BrAc, PMSF inhibited the enzyme.When Try residue of the enzyme was modified by NBS, the relation between Try residue and activity of enzyme could be studied according to the fluorescence emission spectra and the ultraviolet spectra. The result showed that the fluorescence emission peak at 441nm and the ultraviolet absorbed peak at 277 nm were disappeared with the loss of the activity of the enzyme after the enzyme was modified by NBS. Try was the activity center residue of this proteinase. The enzyme was modified by BrAc and PMSF, which influenced enzyme activity. From results, His and Ser were very importment to keep configuration of activity center.
引文
[1]相沢孝亮,黄文涛,胡学智译.酶应用手册[M]1版.上海科学技术出版社,1989
[2]胡学智.蛋白酶的生产和应用[M].江苏调味副食品,2006,23(6):1-5
[3]郑毅.苏云金芽孢杆菌FS140菌株蛋白酶的研究[M].福建农林大学,2008
[4]刘欣,徐红华,李铁晶.微生物蛋白酶改性大豆分离蛋白的研究进展[J].大豆通报,2005,4:27-28
[5]MALAB, APARNAM T, MOHINI S.ChATYGE et al. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology eview[J]. 1998, 62 (3): 597-635
[6]GUPTA R, BEG Q K, LORENZ P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 2002; 59:15-32.
[7]周虓.苏云金芽孢杆菌耐热蛋白酶的菌株筛选、发酵优化及酶学特性的研究[M].福建师范大学,2007
[8]周德庆.微生物学教程[M].高等教育出版社,1998
[9] PAVLOVA I N, ZhOLNER L G. The Serine proteinase of thermophilic Bacillus [J].Microbiol, 1994, 56 (5) : 8-16
[10] PEEK K, VEITCH D P. Some characterization of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli comparison with the native enzyme and its processing in E. Coli and in vitro [J]. Appl Environ Microbiol, 1993, 59(4) :68-75
[11] FUJIWARAN, MASUIA, MANAKAT. Purification and propertes of the highly thermostable alkaline protease from and alkaliphilican thermophilic Bacillus sp [J].J Biotechnol, 1993, 30 (2) : 245-246
[12] MILONOA, MANACHINIPL, PARINIC, et al. Sequence of the gene encoding an alkaline serine protease of thermophilic Bacillus smithii [J]. Gene, 1994, 145(1) : 149-150
[13] VECEREK B, KYSLIK P. Cloning and seuencing of the neutral proteinase 2 encoding gene from a thermophilic strain of Bacillus sp [J]. Gene, 1995, 158(1):147-148
[14] PRESCOTT M, PEEK K, DANIEL R M. Characterization of a thermostable pepstatin insensitive acid protenase from a Bacillus sp [J] . Int Biochem Cell Biol,1995, 27 (7) : 729-739
[15] KUMAR C G. Purification and characterization of a thermostable alkaline protease from alkealiphilic Bacillus pumilus[J].Lett Appl Micoobiol, 2002, 34(1) : 13-17
[16] BURLINI N, MAGNANI P. A heatstable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus [J] . Biochem Biophys Acta,1992, 1122 (3) : 283-292
[17] LEE Y C, MIYATA Y, TERADA I, et al. Involvemet of NH2-teminal pro-sequence in the produsts of active aqualysin I in Escherichia coli [ J]. Agric Biochem, 1991,55 (12) : 3027-3032
[18] 堀越毅弘.日本特许[M].1972:2794
[19] ONG, P.S. Ferment Technol Today[M]. 1972: 217
[20] LAUWERS, A.M.et al. Arch.Microbiol[J]. 1973: 91, 241
[21] MIZUSAWA, K.etal. Appl.Microbiol[J]. 1969, 17: 366.
[22] 横塚保.特许公报[M].1975:9870
[23] 水沢清.化学と生物[M].1974,12(5):311
[24] 张随榜,张兴.生物农药的研究与应用[J].西北农业学报,2003,12(2):75-79
[25] 朱玮,赵兵,王晓东,王玉春.生物农药苏云金芽孢杆菌的研究进展[J].过程工程学报,2004,4(3):282-288
[26] 丁学知,邹先琼,孙运军,付祖姣,高必达,夏立秋.苏云金芽孢杆菌4.0718菌株晶体毒素性质的研究[J].农业生物技术学报,2005,13(3):365-371
[27] 喻子牛.苏云金杆菌[M].北京:科学出版社,1990
[28] 戴莲韵,王学聘.苏云金芽孢杆菌研究进展[J].北京:科学出版社,1997
[29] KUMAR N S, VENKATEWERLU G. Endogenous protease activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis [J].FEMS Microbiol Lett, 1998, 159:113-120.
[30] REDDY Y C , VENKATESWERLU G. Intracellular proteases of Bacillus thuringiensis subsp. kurstaki and a protease-deficient mutant Btk-q [J]. Curr Microbiol, 2002, 45:405-409
[31] BRAR S K., VERMA M., TYAGI R D, SURAMPALLI R Y, BARMABE S,VALERO J R. Bacillus thuringiensis proteases: production and role in growth,spomlation and synergism[M]. Process Biochemstry, 2007, 42:773-790
[32] KUNITATE A, 0KAM0T0M, OHMORI I. Purification and characterization of a thermostable serine protease from Bacillus thuringiensis [J]. Agric Biol Chem,1989, 53:3251-3260.
[33] KUMAR N S , VENKATESWERLU G. Involvement of an endogenous metalloprotease in the activation of protoxin in Bacillus thuringiensis subsp.kurstaki[J].Biochem Mol Biol Intl, 1997, 42:901-908
[34] KUMAR N S, VENKATESWERLU G. Intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki and their role in protoxin activation[J]. FEMS Microbiol Lett, 1998, 166:377-382
[35] 刘凯于,阎江洪,康薇,郑进,洪华珠.一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响[J].昆虫知识,2005,42(5):540-543
[36] 徐艳聆,王振营,何康来,白树雄,张杰.对Bt抗性和敏感亚洲玉米螟解毒酶和中肠蛋白酶的比较[J].农业生物技术学报,2006,14(6):889-893
[37] YOSHIHISA N, YASUHIRO S, SHOZABURO K. Purification and Some Properties of Extracelluar Protease of Euglena gracilisz [J]. Agric.Biol.Chem., 1979,43 (2) :222-223
[38] TAKAO T, KOHEI O, MATASHI K, SAWAO M. Purification and some properties of carboxyl proteinase in Mycelium of Lentinus edodes[J]. Agric. Biol. Chem.,1981, 45 (9) :1929-1930
[39] KARL E E and BERT P.Purification and partial characterizeation of two acidic proteases from the whiterot fungus Sporotrichum pulverulentum [J]. Eur. J.Biochem, 1982, 124:635-64
[40] YASUHIRO S, TOYOKAZU N, SAWAO M. Purifcauan and characterization of membrane bound serine protease of Bacillus subtilis IFO 3027[J]. Agric.Biol.Chem.,1983, 47 (8) :1775-1780
[41] KOICHI K, TOSHIHIRO Y, KZAUO M. Purification and characterization of chymotrypsin-like proteinase from Euphausias uperba. Agric [J].Biol. Chem., 1985, 49 (6) :1599-1600
[42]TETSUYA K, YOUHEI Y, TERUYOSHI A. Purification of a new eatracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action[J]. Biosci. Biotech. Biochem, 1992,56 (7) :1166-1170
[43]KATSUMI T, YASUO N, HRIOTSUGU S, TETSU K. Purification and characterization of a thermostable alkaline protease from Alkalophilic Thermoactinomyces sp.HS682. Biosci [J]. Biotech.Biochem, 1992, 56 (2):246-250
[44]曹志云.黑曲霉产酸性蛋白酶催化机制和稳定剂研究[J].2005:29-34
[45]王全富,缪锦来,李光友,侯艳华,王国栋.南极嗜冷菌Colwellia sp.NJ341产适冷蛋白酶及低温适应性的研究[J].中国水产科学,2005,12(4):437-444
[46]黄光荣,应铁进,戴德慧,活泼,将家新.嗜热芽孢杆菌蛋白酶HS08的分离纯化研究[J].食品科学,2007,128(4):179-182
[47]曹煜成,李卓佳,吴灶和,冯娟.地衣芽孢杆菌胞外蛋白酶的纯化及特性分析[J].水生生物学报,2006,30(3):262-268
[48]谢晶,葛绍荣,陶勇,高平,刘昆,刘世贵.多粘类芽孢杆菌BS04拮抗成分分离纯化及其特性.化学研究与应用[J],2004,16(6):775-777
[49]贺稚非,周泽扬,李洪军,高兆建,宫春波.假单胞菌胞外弹性蛋白酶的纯化研究[J].中国食品学报,2005, (2):22-27
[50]沈锦玉,尹文林,曹铮,潘晓艺,吴颖蕾.枯草芽孢杆菌B115抗菌蛋白的分离纯化及部分性质[J].水生生物学报,2005,29(6):689-693
[51]刘永锋,张杰,陈志谊,周明国,宋福平,刘邮洲,聂亚锋,罗楚平.枯草芽孢杆菌Bs2916胞外抗菌蛋白质的生物功能分析[J].江苏农业学报,2008,24(3):269-273
[52]邢介帅,李然,赵蕾,梁元存,竺晓平.生防芽孢杆菌T2胞外蛋白酶的纯化及其抗真菌作用[J].植物病理学报,2008,38(4):377-381
[53]杨星勇,王中康,夏玉先,卢晓风,裴炎.球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性[J].菌物系统,2000,19(2):254-260
[54]BLACKSHEAR P J. System for Polyacrylamide Gel Electrophoresis [J].Meth Enzymol, 1984, 104: 237-255
[55]LAEMMI U K. Cleavage of Structural Proteins during the Assembly of the Head of BacteriophageT4[J]. Nature, 1970, 227: 680-685
[56]汪家政,范明.蛋白质技术手册[M]1版.北京:科学出版社,2002
[57]郭尧君.SDS电泳技术的实验考虑及最新进展[J].生物化学与生物物理进展,1991,18(1):32-37
[58]邓军卫,游学科,汤虹,余鸥.滤纸条方法SDS电泳[J].湖南医科大学学报,1996,21(3):259-261
[59]郭尧君,闻弢.SDS电泳半干新技术[J].生物化学与生物物理进展,1994,21(3):265-267
[60]武建国.实用临床免疫学检验[M].江苏科学技术出版社,1990:67-70
[61]李晓军,秦浚川,武建国.蛋白印迹技术研究进展[J].临床检验杂志,2004,22(3):227-229
[62]KURIEN B T, SCOFIELD R H. Multiple immunoblots after non 2 electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens[J].J Immunol Methods , 1997, 205: 91
[63]LASNE F. Double 2 blotting: a solution to the problemof nonspecific binding of secondary antibodies in immunoblotting procedures [J]. J Immunol Methods, 2003,276:223
[64]LOWE G. Slice blotting: a method for detecting the release of immunoreactive substances from living brain tissue [J]. J Neurosci Methods, 1990, 90: 117
[65]REESE G, SCHMECHELD, AYUSOR, et al. Grid2immunoblotting: a fast and simple technique to test multiple allergens with small amounts of antibody for cross 2 reactivity [J]. J Chromatogr.B Biomed Sci, 2001, 756:151
[66]NOYA O, ALARCONDE N B. The multiple antigen blot assay (MABA) :a simple immunoenzymatic technique for simultaneous screening of multiple antigens[J]. Immunol Lett, 1998, 63:53
[67]SZAKAI D, SCHNEIDER G, PAI T.A colony blot immunoassay to identify enteroinvasive Escherichia coli and Shigella in stool sample[J]. Diagn Microbiol Infect Dis, 2003, 45: 1651
[68]王镜岩.生物化学[M].高等教育出版社,2002
[69]李勃,党永,马瑜,陈颖恰.蛋壳内膜分解菌所产蛋白酶的纯化及其N-末端氨基酸序列测定[J].微生物学通报,2008,35(8):1182-1185
[70]母智深,白英,赵广华,胡小松.荧光假单胞杆菌胞外蛋白酶的纯化及热稳定性[J].高等学校化学学报,2008,29(4):762-766
[71]谢必峰,曹治云,郑腾,王水顺.黑曲霉Aspergillus niger SL2-111所产酸性蛋白酶的分离纯化及酶学特性[J].应用与环境生物学报,2005,11(5):618-622
[72]张东方,周美环,单玉,杨松松.参环毛蚓中纤溶活性蛋白酶研究[J].药物生物技术,2006,13(1):49-50
[73]王骏,王敏,王以光.链霉菌产生的新型纤溶酶的纯化和性质的研究[J].生物工程学报,1999,15(2):147-153
[74]于声,段斯亮,苏晓庆.贵阳腐霉类枯草杆菌蛋白酶(Pr1)的纯化与部分特性研究[J].贵阳医学院学报,2007,32(2):126-130
[75]张树政.酶制剂工业[M].北京:科学出版社,1998
[76]李奇,王昭,张纪周,洪敏.一种新的具有纤溶活性的N-V蛋白酶酶学性质分析[J].吉林大学学报(医学版),2008,34(3):377-381
[77]王荣新,雷大卫,苏晓庆.贵阳腐霉Pr1蛋白酶的初步纯化及其酶学特性研究[J].安徽农业大学学报,2007,34(4):505-508
[78]袁康培,郑春丽,冯明光.黑曲霉HU53菌株产酸性蛋白酶的条件和酶学性质[J].食品科学,2003,24(8):46-49
[79]袁铸,王忠彦,胡承,胡永松.地衣芽孢杆菌JF-UN122碱性蛋白酶的分离纯化与性质[J].工业微生物,2003,33(3):25-29
[80]孙谧,修朝阳,王跃军,陈丽芳,张云波,洪义国,阎晓玲,韩艳波,李晶,陈良.黄海黄杆菌YS-9412-130低温碱性蛋白酶性质鉴定[J].海洋水产研究,2001,22(2):1-10
[81]张淑娟,田利强,王辂,王美芳,李利军.米曲霉菌今野菌株所产蛋白酶性质的研究[J].中国调味品,2001,11:15-17
[82]齐崴,何志敏,史德青.酪蛋白酶解制备酪蛋白磷酸肽研究[J].食品科学,2001,22(8),37-40
[83]YANS, QINGXC, QIN W, KANG K S, LING Q. Inhibitory effects of cinnamic acid its derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase[J]. Food chemistry, 2005, 92:707-712
[84]XIAO LX, MIN G, QING X C. Inhibition kinetics of hydrogen peroxide on β-N-acetyl-D-glucosaminidase from prawn (peneaus vannamei) [J]. Journal of enzyme inhibiton and medicinal chemistry, 2006, 21 (1) :55-60
[85]邱全胜,张楠.胰蛋白酶处理对质膜H~+-ATPase钒酸钠抑制效应的影响[J].植物学报,2001,43(1):24-28
[86]黄小丽,汪开毓,苏秀梅,耿毅,邓永强.嗜麦芽寡养单胞菌胞外蛋白酶的化学修饰[J].化学学报,2007,65(7):651-654
[87]黄光荣,戴德慧,活泼,王龙军,蒋家新.嗜热芽孢杆菌高温蛋白酶HS08活性中心研究[J].食品工业科技,2007,1:204-206
[88]TOGNI G, SANGLARD D, FALCHETO R et al. Isolation and nucleotide sequence of the extracellularac idp roteaseg ene ( ACP ) fromt hey eastC andidatr opicalis[J].FEBSL et, Ju ly29, 1991;286 (1-2) : 181-185.
[89]SHINTANI, ICHISHIMA E. Primary structure of aspergillopepsin I deduced from nucleotide sequence of the gene and aspartic acid-76 is an essential active site of the enzyme for trypsinogen activation[J]. Biochim Biophys Acta.1994 Feb 16;1204 (2) :257-264
[90]储卫华,陆承平.嗜水气单胞菌胞外蛋白酶的化学修饰[J].中国生物化学与分子生物学报,2001,17(3):372-375
[91]池政豪,陈吉祥,杨慧,刘斌,李筠.鳗弧菌胞外金属蛋白酶的化学修饰研究[J].生物技术通报(增刊),2006:34-437
[92]舒薇,房师松,崔堂兵,郭勇.木瓜凝乳蛋白酶的单甲氧基聚乙二醇化学修饰及其对酶活力和抗原性的影响[J].华南农业大学学报,2006,27(1):65-68
[93]J.萨姆布鲁克,E.F弗里奇,T.曼尼阿蒂斯著,金冬雁等译,侯云德校.分子克隆试验指南[M].2版.北京:科学出版社,1998
[94]叶海梅.米曲霉FZ58 β-葡萄糖苷酶固体发酵优化及酶学特性的研究[M].福建师范大学,2008
[95]张寒俊,刘大川,杨国燕.紫外光谱法定量测定不同种蛋白酶活力的研究[J].粮食与饲料工业,2004,(9):44-45
[96]B.施特尔马赫,钱嘉渊.酶的测定方法[J].中国轻工业出版社,1992
[97]SB汀10317-199.蛋白酶活力测定法中华人民共和国专业标准[M].1987
[98]李建武.生物化学试验原理和方法[M].北京大学出版社,1994
[99]LOWRY O H, ROSEBROUGH N J, FARR A L et al. Protein measurement with the Folinphenol reagent [J]. J. Biol. Chem., 1951, 193:2650-95
[100]COOPER T G著,徐晓利主译.《生物化学工具》[M].人民卫生出版社,1980
[101]陶慰孙.蛋白质分子基础[M].人民教育出版社,1982
[102]杨学敏.效应物对锯缘青蟹N-乙酰-β-D-氨基葡萄糖苷酶的抑制作用研究[M].厦门大学,2006
[103]龚敏.南美白对虾N-乙酰-β-D氨基葡萄糖苷酶的活力调控及动力学研究[M].厦门大学,2006
[104]北京大学生物系生物化学教研室.生物化学实验指导[M].高等教育出版社,1988:277-280
[105]OTHMER G. Analytical diso gel electrophoresis [M]. Methods in Enzymology,1971, 22:565-573
[106]陈宏惠.效应物对中华绒螯蟹(Eriocheir sinensis)N-乙酰-β-D-氨基葡萄糖苷酶的影响[M].福建农林大学,2007
[107]罗秀针.枯草芽孢杆菌FB123产生的枯草菌素的研究[M].福建师范大学,2008:62-66
[108]孔令泉,蒲莹晖,马仕坤,涂刚,任国胜.快慢转法及不同滤膜和显色检测法在Western blotting中的应用分析[J].南方医科大学学报,2008:28(1)
[109]黄小红,王寿昆,黄一帆,陈宏惠.日本沼虾(Macrobrachium,nipponense)N-乙酰-β-D-氨基葡萄糖苷酶初步纯化及部分性质[J].应用与环境生物学报2006,12(6):804-808
[110]王长康,黄一帆,黄小红,陈宏惠.番鸭(Cairina moschata)睾丸N-乙酰-β-D-氨基葡萄糖苷酶的提取及性质研究[J].畜牧兽医学报,2007,38(6):552-557
[111]黄小红.棉铃虫N-乙酰氨基葡萄糖苷酶的分离纯化及性质研究[M].福建农林大学,2005:46-105
[112]王君,黄小红,陈清西.金属离子对棉铃虫N-乙酰-β-D-氨基葡萄糖苷酶活力的影响[J].厦门大学学报(自然科学版),2005,(44)6:843-846
[113]CHEN Q X,, ZHENG W Z, LIN J Y et al. Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase[J]. Int J Biochem Cell Biol, 2000,32 (8): 879-885
[114]肖炎农,王明祖,王道本.淡紫拟青霉几丁质酶的纯化和活性影响因子[J].植物病理学报,1998,28(3):75-280
[115]KOGAD, HOSHIKAH, MATSUSHITAM, TANAKAA, IDEA, KONOM.Purification and characterization of beta-N-acetylhexosaminidase from the liver of a prawn, Penaeus japonicus[J]. Biosci. Biotechnol. Biochem., 1996, 60 (2):194-199
[116]刘鹏虎,周刚,陈大福,梁勤.金属离子对蜜蜂球囊菌几丁质酶活力的影响[J].福建农林大学学报,2008,37(1):96-98
[117]汤鸣强,郑挺,曾小芳,朱晓清.酿造酱油米曲霉产中性蛋白酶提取与酶学性质研究[J].中国调味品,2008,2:38-40
[118]彭立凤.有机溶剂对酶催化活性和选择性的影响[J].化学进展,2000,12(3):296-304
[119]FREISHEIM J H, HUENNEKENS F M. Effect of N-Bromosuccinimide on Dinydrofolate Reductase [J]. Biochemistry, 1969, 8:2271-2276
[2]胡学智.蛋白酶的生产和应用[M].江苏调味副食品,2006,23(6):1-5
[3]郑毅.苏云金芽孢杆菌FS140菌株蛋白酶的研究[M].福建农林大学,2008
[4]刘欣,徐红华,李铁晶.微生物蛋白酶改性大豆分离蛋白的研究进展[J].大豆通报,2005,4:27-28
[5]MALAB, APARNAM T, MOHINI S.ChATYGE et al. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology eview[J]. 1998, 62 (3): 597-635
[6]GUPTA R, BEG Q K, LORENZ P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 2002; 59:15-32.
[7]周虓.苏云金芽孢杆菌耐热蛋白酶的菌株筛选、发酵优化及酶学特性的研究[M].福建师范大学,2007
[8]周德庆.微生物学教程[M].高等教育出版社,1998
[9] PAVLOVA I N, ZhOLNER L G. The Serine proteinase of thermophilic Bacillus [J].Microbiol, 1994, 56 (5) : 8-16
[10] PEEK K, VEITCH D P. Some characterization of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli comparison with the native enzyme and its processing in E. Coli and in vitro [J]. Appl Environ Microbiol, 1993, 59(4) :68-75
[11] FUJIWARAN, MASUIA, MANAKAT. Purification and propertes of the highly thermostable alkaline protease from and alkaliphilican thermophilic Bacillus sp [J].J Biotechnol, 1993, 30 (2) : 245-246
[12] MILONOA, MANACHINIPL, PARINIC, et al. Sequence of the gene encoding an alkaline serine protease of thermophilic Bacillus smithii [J]. Gene, 1994, 145(1) : 149-150
[13] VECEREK B, KYSLIK P. Cloning and seuencing of the neutral proteinase 2 encoding gene from a thermophilic strain of Bacillus sp [J]. Gene, 1995, 158(1):147-148
[14] PRESCOTT M, PEEK K, DANIEL R M. Characterization of a thermostable pepstatin insensitive acid protenase from a Bacillus sp [J] . Int Biochem Cell Biol,1995, 27 (7) : 729-739
[15] KUMAR C G. Purification and characterization of a thermostable alkaline protease from alkealiphilic Bacillus pumilus[J].Lett Appl Micoobiol, 2002, 34(1) : 13-17
[16] BURLINI N, MAGNANI P. A heatstable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus [J] . Biochem Biophys Acta,1992, 1122 (3) : 283-292
[17] LEE Y C, MIYATA Y, TERADA I, et al. Involvemet of NH2-teminal pro-sequence in the produsts of active aqualysin I in Escherichia coli [ J]. Agric Biochem, 1991,55 (12) : 3027-3032
[18] 堀越毅弘.日本特许[M].1972:2794
[19] ONG, P.S. Ferment Technol Today[M]. 1972: 217
[20] LAUWERS, A.M.et al. Arch.Microbiol[J]. 1973: 91, 241
[21] MIZUSAWA, K.etal. Appl.Microbiol[J]. 1969, 17: 366.
[22] 横塚保.特许公报[M].1975:9870
[23] 水沢清.化学と生物[M].1974,12(5):311
[24] 张随榜,张兴.生物农药的研究与应用[J].西北农业学报,2003,12(2):75-79
[25] 朱玮,赵兵,王晓东,王玉春.生物农药苏云金芽孢杆菌的研究进展[J].过程工程学报,2004,4(3):282-288
[26] 丁学知,邹先琼,孙运军,付祖姣,高必达,夏立秋.苏云金芽孢杆菌4.0718菌株晶体毒素性质的研究[J].农业生物技术学报,2005,13(3):365-371
[27] 喻子牛.苏云金杆菌[M].北京:科学出版社,1990
[28] 戴莲韵,王学聘.苏云金芽孢杆菌研究进展[J].北京:科学出版社,1997
[29] KUMAR N S, VENKATEWERLU G. Endogenous protease activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis [J].FEMS Microbiol Lett, 1998, 159:113-120.
[30] REDDY Y C , VENKATESWERLU G. Intracellular proteases of Bacillus thuringiensis subsp. kurstaki and a protease-deficient mutant Btk-q [J]. Curr Microbiol, 2002, 45:405-409
[31] BRAR S K., VERMA M., TYAGI R D, SURAMPALLI R Y, BARMABE S,VALERO J R. Bacillus thuringiensis proteases: production and role in growth,spomlation and synergism[M]. Process Biochemstry, 2007, 42:773-790
[32] KUNITATE A, 0KAM0T0M, OHMORI I. Purification and characterization of a thermostable serine protease from Bacillus thuringiensis [J]. Agric Biol Chem,1989, 53:3251-3260.
[33] KUMAR N S , VENKATESWERLU G. Involvement of an endogenous metalloprotease in the activation of protoxin in Bacillus thuringiensis subsp.kurstaki[J].Biochem Mol Biol Intl, 1997, 42:901-908
[34] KUMAR N S, VENKATESWERLU G. Intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki and their role in protoxin activation[J]. FEMS Microbiol Lett, 1998, 166:377-382
[35] 刘凯于,阎江洪,康薇,郑进,洪华珠.一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响[J].昆虫知识,2005,42(5):540-543
[36] 徐艳聆,王振营,何康来,白树雄,张杰.对Bt抗性和敏感亚洲玉米螟解毒酶和中肠蛋白酶的比较[J].农业生物技术学报,2006,14(6):889-893
[37] YOSHIHISA N, YASUHIRO S, SHOZABURO K. Purification and Some Properties of Extracelluar Protease of Euglena gracilisz [J]. Agric.Biol.Chem., 1979,43 (2) :222-223
[38] TAKAO T, KOHEI O, MATASHI K, SAWAO M. Purification and some properties of carboxyl proteinase in Mycelium of Lentinus edodes[J]. Agric. Biol. Chem.,1981, 45 (9) :1929-1930
[39] KARL E E and BERT P.Purification and partial characterizeation of two acidic proteases from the whiterot fungus Sporotrichum pulverulentum [J]. Eur. J.Biochem, 1982, 124:635-64
[40] YASUHIRO S, TOYOKAZU N, SAWAO M. Purifcauan and characterization of membrane bound serine protease of Bacillus subtilis IFO 3027[J]. Agric.Biol.Chem.,1983, 47 (8) :1775-1780
[41] KOICHI K, TOSHIHIRO Y, KZAUO M. Purification and characterization of chymotrypsin-like proteinase from Euphausias uperba. Agric [J].Biol. Chem., 1985, 49 (6) :1599-1600
[42]TETSUYA K, YOUHEI Y, TERUYOSHI A. Purification of a new eatracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action[J]. Biosci. Biotech. Biochem, 1992,56 (7) :1166-1170
[43]KATSUMI T, YASUO N, HRIOTSUGU S, TETSU K. Purification and characterization of a thermostable alkaline protease from Alkalophilic Thermoactinomyces sp.HS682. Biosci [J]. Biotech.Biochem, 1992, 56 (2):246-250
[44]曹志云.黑曲霉产酸性蛋白酶催化机制和稳定剂研究[J].2005:29-34
[45]王全富,缪锦来,李光友,侯艳华,王国栋.南极嗜冷菌Colwellia sp.NJ341产适冷蛋白酶及低温适应性的研究[J].中国水产科学,2005,12(4):437-444
[46]黄光荣,应铁进,戴德慧,活泼,将家新.嗜热芽孢杆菌蛋白酶HS08的分离纯化研究[J].食品科学,2007,128(4):179-182
[47]曹煜成,李卓佳,吴灶和,冯娟.地衣芽孢杆菌胞外蛋白酶的纯化及特性分析[J].水生生物学报,2006,30(3):262-268
[48]谢晶,葛绍荣,陶勇,高平,刘昆,刘世贵.多粘类芽孢杆菌BS04拮抗成分分离纯化及其特性.化学研究与应用[J],2004,16(6):775-777
[49]贺稚非,周泽扬,李洪军,高兆建,宫春波.假单胞菌胞外弹性蛋白酶的纯化研究[J].中国食品学报,2005, (2):22-27
[50]沈锦玉,尹文林,曹铮,潘晓艺,吴颖蕾.枯草芽孢杆菌B115抗菌蛋白的分离纯化及部分性质[J].水生生物学报,2005,29(6):689-693
[51]刘永锋,张杰,陈志谊,周明国,宋福平,刘邮洲,聂亚锋,罗楚平.枯草芽孢杆菌Bs2916胞外抗菌蛋白质的生物功能分析[J].江苏农业学报,2008,24(3):269-273
[52]邢介帅,李然,赵蕾,梁元存,竺晓平.生防芽孢杆菌T2胞外蛋白酶的纯化及其抗真菌作用[J].植物病理学报,2008,38(4):377-381
[53]杨星勇,王中康,夏玉先,卢晓风,裴炎.球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性[J].菌物系统,2000,19(2):254-260
[54]BLACKSHEAR P J. System for Polyacrylamide Gel Electrophoresis [J].Meth Enzymol, 1984, 104: 237-255
[55]LAEMMI U K. Cleavage of Structural Proteins during the Assembly of the Head of BacteriophageT4[J]. Nature, 1970, 227: 680-685
[56]汪家政,范明.蛋白质技术手册[M]1版.北京:科学出版社,2002
[57]郭尧君.SDS电泳技术的实验考虑及最新进展[J].生物化学与生物物理进展,1991,18(1):32-37
[58]邓军卫,游学科,汤虹,余鸥.滤纸条方法SDS电泳[J].湖南医科大学学报,1996,21(3):259-261
[59]郭尧君,闻弢.SDS电泳半干新技术[J].生物化学与生物物理进展,1994,21(3):265-267
[60]武建国.实用临床免疫学检验[M].江苏科学技术出版社,1990:67-70
[61]李晓军,秦浚川,武建国.蛋白印迹技术研究进展[J].临床检验杂志,2004,22(3):227-229
[62]KURIEN B T, SCOFIELD R H. Multiple immunoblots after non 2 electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens[J].J Immunol Methods , 1997, 205: 91
[63]LASNE F. Double 2 blotting: a solution to the problemof nonspecific binding of secondary antibodies in immunoblotting procedures [J]. J Immunol Methods, 2003,276:223
[64]LOWE G. Slice blotting: a method for detecting the release of immunoreactive substances from living brain tissue [J]. J Neurosci Methods, 1990, 90: 117
[65]REESE G, SCHMECHELD, AYUSOR, et al. Grid2immunoblotting: a fast and simple technique to test multiple allergens with small amounts of antibody for cross 2 reactivity [J]. J Chromatogr.B Biomed Sci, 2001, 756:151
[66]NOYA O, ALARCONDE N B. The multiple antigen blot assay (MABA) :a simple immunoenzymatic technique for simultaneous screening of multiple antigens[J]. Immunol Lett, 1998, 63:53
[67]SZAKAI D, SCHNEIDER G, PAI T.A colony blot immunoassay to identify enteroinvasive Escherichia coli and Shigella in stool sample[J]. Diagn Microbiol Infect Dis, 2003, 45: 1651
[68]王镜岩.生物化学[M].高等教育出版社,2002
[69]李勃,党永,马瑜,陈颖恰.蛋壳内膜分解菌所产蛋白酶的纯化及其N-末端氨基酸序列测定[J].微生物学通报,2008,35(8):1182-1185
[70]母智深,白英,赵广华,胡小松.荧光假单胞杆菌胞外蛋白酶的纯化及热稳定性[J].高等学校化学学报,2008,29(4):762-766
[71]谢必峰,曹治云,郑腾,王水顺.黑曲霉Aspergillus niger SL2-111所产酸性蛋白酶的分离纯化及酶学特性[J].应用与环境生物学报,2005,11(5):618-622
[72]张东方,周美环,单玉,杨松松.参环毛蚓中纤溶活性蛋白酶研究[J].药物生物技术,2006,13(1):49-50
[73]王骏,王敏,王以光.链霉菌产生的新型纤溶酶的纯化和性质的研究[J].生物工程学报,1999,15(2):147-153
[74]于声,段斯亮,苏晓庆.贵阳腐霉类枯草杆菌蛋白酶(Pr1)的纯化与部分特性研究[J].贵阳医学院学报,2007,32(2):126-130
[75]张树政.酶制剂工业[M].北京:科学出版社,1998
[76]李奇,王昭,张纪周,洪敏.一种新的具有纤溶活性的N-V蛋白酶酶学性质分析[J].吉林大学学报(医学版),2008,34(3):377-381
[77]王荣新,雷大卫,苏晓庆.贵阳腐霉Pr1蛋白酶的初步纯化及其酶学特性研究[J].安徽农业大学学报,2007,34(4):505-508
[78]袁康培,郑春丽,冯明光.黑曲霉HU53菌株产酸性蛋白酶的条件和酶学性质[J].食品科学,2003,24(8):46-49
[79]袁铸,王忠彦,胡承,胡永松.地衣芽孢杆菌JF-UN122碱性蛋白酶的分离纯化与性质[J].工业微生物,2003,33(3):25-29
[80]孙谧,修朝阳,王跃军,陈丽芳,张云波,洪义国,阎晓玲,韩艳波,李晶,陈良.黄海黄杆菌YS-9412-130低温碱性蛋白酶性质鉴定[J].海洋水产研究,2001,22(2):1-10
[81]张淑娟,田利强,王辂,王美芳,李利军.米曲霉菌今野菌株所产蛋白酶性质的研究[J].中国调味品,2001,11:15-17
[82]齐崴,何志敏,史德青.酪蛋白酶解制备酪蛋白磷酸肽研究[J].食品科学,2001,22(8),37-40
[83]YANS, QINGXC, QIN W, KANG K S, LING Q. Inhibitory effects of cinnamic acid its derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase[J]. Food chemistry, 2005, 92:707-712
[84]XIAO LX, MIN G, QING X C. Inhibition kinetics of hydrogen peroxide on β-N-acetyl-D-glucosaminidase from prawn (peneaus vannamei) [J]. Journal of enzyme inhibiton and medicinal chemistry, 2006, 21 (1) :55-60
[85]邱全胜,张楠.胰蛋白酶处理对质膜H~+-ATPase钒酸钠抑制效应的影响[J].植物学报,2001,43(1):24-28
[86]黄小丽,汪开毓,苏秀梅,耿毅,邓永强.嗜麦芽寡养单胞菌胞外蛋白酶的化学修饰[J].化学学报,2007,65(7):651-654
[87]黄光荣,戴德慧,活泼,王龙军,蒋家新.嗜热芽孢杆菌高温蛋白酶HS08活性中心研究[J].食品工业科技,2007,1:204-206
[88]TOGNI G, SANGLARD D, FALCHETO R et al. Isolation and nucleotide sequence of the extracellularac idp roteaseg ene ( ACP ) fromt hey eastC andidatr opicalis[J].FEBSL et, Ju ly29, 1991;286 (1-2) : 181-185.
[89]SHINTANI, ICHISHIMA E. Primary structure of aspergillopepsin I deduced from nucleotide sequence of the gene and aspartic acid-76 is an essential active site of the enzyme for trypsinogen activation[J]. Biochim Biophys Acta.1994 Feb 16;1204 (2) :257-264
[90]储卫华,陆承平.嗜水气单胞菌胞外蛋白酶的化学修饰[J].中国生物化学与分子生物学报,2001,17(3):372-375
[91]池政豪,陈吉祥,杨慧,刘斌,李筠.鳗弧菌胞外金属蛋白酶的化学修饰研究[J].生物技术通报(增刊),2006:34-437
[92]舒薇,房师松,崔堂兵,郭勇.木瓜凝乳蛋白酶的单甲氧基聚乙二醇化学修饰及其对酶活力和抗原性的影响[J].华南农业大学学报,2006,27(1):65-68
[93]J.萨姆布鲁克,E.F弗里奇,T.曼尼阿蒂斯著,金冬雁等译,侯云德校.分子克隆试验指南[M].2版.北京:科学出版社,1998
[94]叶海梅.米曲霉FZ58 β-葡萄糖苷酶固体发酵优化及酶学特性的研究[M].福建师范大学,2008
[95]张寒俊,刘大川,杨国燕.紫外光谱法定量测定不同种蛋白酶活力的研究[J].粮食与饲料工业,2004,(9):44-45
[96]B.施特尔马赫,钱嘉渊.酶的测定方法[J].中国轻工业出版社,1992
[97]SB汀10317-199.蛋白酶活力测定法中华人民共和国专业标准[M].1987
[98]李建武.生物化学试验原理和方法[M].北京大学出版社,1994
[99]LOWRY O H, ROSEBROUGH N J, FARR A L et al. Protein measurement with the Folinphenol reagent [J]. J. Biol. Chem., 1951, 193:2650-95
[100]COOPER T G著,徐晓利主译.《生物化学工具》[M].人民卫生出版社,1980
[101]陶慰孙.蛋白质分子基础[M].人民教育出版社,1982
[102]杨学敏.效应物对锯缘青蟹N-乙酰-β-D-氨基葡萄糖苷酶的抑制作用研究[M].厦门大学,2006
[103]龚敏.南美白对虾N-乙酰-β-D氨基葡萄糖苷酶的活力调控及动力学研究[M].厦门大学,2006
[104]北京大学生物系生物化学教研室.生物化学实验指导[M].高等教育出版社,1988:277-280
[105]OTHMER G. Analytical diso gel electrophoresis [M]. Methods in Enzymology,1971, 22:565-573
[106]陈宏惠.效应物对中华绒螯蟹(Eriocheir sinensis)N-乙酰-β-D-氨基葡萄糖苷酶的影响[M].福建农林大学,2007
[107]罗秀针.枯草芽孢杆菌FB123产生的枯草菌素的研究[M].福建师范大学,2008:62-66
[108]孔令泉,蒲莹晖,马仕坤,涂刚,任国胜.快慢转法及不同滤膜和显色检测法在Western blotting中的应用分析[J].南方医科大学学报,2008:28(1)
[109]黄小红,王寿昆,黄一帆,陈宏惠.日本沼虾(Macrobrachium,nipponense)N-乙酰-β-D-氨基葡萄糖苷酶初步纯化及部分性质[J].应用与环境生物学报2006,12(6):804-808
[110]王长康,黄一帆,黄小红,陈宏惠.番鸭(Cairina moschata)睾丸N-乙酰-β-D-氨基葡萄糖苷酶的提取及性质研究[J].畜牧兽医学报,2007,38(6):552-557
[111]黄小红.棉铃虫N-乙酰氨基葡萄糖苷酶的分离纯化及性质研究[M].福建农林大学,2005:46-105
[112]王君,黄小红,陈清西.金属离子对棉铃虫N-乙酰-β-D-氨基葡萄糖苷酶活力的影响[J].厦门大学学报(自然科学版),2005,(44)6:843-846
[113]CHEN Q X,, ZHENG W Z, LIN J Y et al. Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase[J]. Int J Biochem Cell Biol, 2000,32 (8): 879-885
[114]肖炎农,王明祖,王道本.淡紫拟青霉几丁质酶的纯化和活性影响因子[J].植物病理学报,1998,28(3):75-280
[115]KOGAD, HOSHIKAH, MATSUSHITAM, TANAKAA, IDEA, KONOM.Purification and characterization of beta-N-acetylhexosaminidase from the liver of a prawn, Penaeus japonicus[J]. Biosci. Biotechnol. Biochem., 1996, 60 (2):194-199
[116]刘鹏虎,周刚,陈大福,梁勤.金属离子对蜜蜂球囊菌几丁质酶活力的影响[J].福建农林大学学报,2008,37(1):96-98
[117]汤鸣强,郑挺,曾小芳,朱晓清.酿造酱油米曲霉产中性蛋白酶提取与酶学性质研究[J].中国调味品,2008,2:38-40
[118]彭立凤.有机溶剂对酶催化活性和选择性的影响[J].化学进展,2000,12(3):296-304
[119]FREISHEIM J H, HUENNEKENS F M. Effect of N-Bromosuccinimide on Dinydrofolate Reductase [J]. Biochemistry, 1969, 8:2271-2276