地衣芽孢杆菌胞外蛋白酶的纯化及特性分析
PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR
-
摘要: 研究不同条件对地衣芽孢杆菌De株产生胞外蛋白酶的量及其酶活性的影响,结果表明在pH为7.4-8.2范围内,温度为30℃时,培养8-12h的菌株所分泌胞外产物中的蛋白酶活性最高。实验先以半透膜法收集芽孢杆菌的胞外产物,然后再经过硫酸铵沉淀过夜S、ephadex G-100凝胶层析和DEAE-Cellulose离子交换层析及聚丙烯酰胺凝胶电泳等四个步骤的分离纯化后,可以得到含有3种主要蛋白质(BLP1、BLP2、BLP3)成分的胞外蛋白酶,其分子量分别为66.2KD、31.0KD及约20.1KD,所得纯化蛋白酶的蛋白浓度为0.773μg/mL,蛋白回收率为11.66%。实验还发现,纯化的胞外蛋白酶在100℃下作用30min,仍可保持其活力,可见具有相当的热稳定性,而其酶活最佳的pH和温度条件分别为7.8和45-65℃。酶活抑制实验显示EDTA、铜、钴、镁离子等均可成为其酶活抑制因子;而丝氨酸蛋白酶抑制剂甲基磺酰氟(PMSF)、铁、锰、钡、钙离子等对酶活性没有明显影响;锌则会令之酶活性其部分丧失。Abstract: The research of Bacillus as a kind of probiotic for aquatic animals is increasing with the demand for environment friendly aquaculture, like biocontrol when the treatment is antagonistic to pathogens or bioremediation when water quality is improved.The Bacillus lichenif ormis srain De isolated from white shrimp( Litopanaeus vannamie)cultural ponds can produce many kinds of extracelluar enzyme, moreover, the activity of extracelluar protease is very high. Therefor, the study was carried out to collect itps extracellular protease and the physicochemical characters of the enzyme were investigated. The extracellular protease of the strain De was collected followed the semiperable membrane method and was purified followed a four-step purification procedure, including ammounium sulfate precipitation, Sephadex G- 100 gel filtration chromatography, anion- exchange chromatography on DEAE- cellulose and polyacrylamide gel electrophoresis. The activities of extracelluar protease was tested by theAzocasein digestion method.As results showed that extracellular protease secreted by strain De obtained a higher enzymatic activity when it was cultivated 12-18h at 30℃and pH 7.4- 8.2. Three predominant protein of the extracellular products BLP1、BLP2、BLP3 were obtained,whose molecular mass measured by polyacrylamide gel electrophoresis respectively were 66.2KD 31.0KD and rough 20.1KD. The protein content of purified extracellular protease was 0.773Lg/mL, comparatively, its＇ ppurified protein recovery rate was 11. 66%.Moreover, the optimum pH and temperature of the extracellular protease was found to be 7.8 and 45-65℃. The extracellular protease is thermophilic, since full biological activity is retained after heating at 100 e for 30min. Enzyme testing for inhibit ion of the extracellular protease indicated that only EDTA、Cu2+、Co2+and Mg2+ can inhibite enzymatic activity, but PMSF、Fe3+、Mn2+、Ba2+、Ca2+ can not, and Zn2+ can inhibite enzymatic activity to some extent.
-
Keywords:
- Bacillus lichenif ormis /
- Extracellular proteases /
- Purification /
- Characterization
-
-
[1] Zhang Q,Li Z J, Chen K D. The effects of microbiological compound on ecological factors in culture water[J]. J Shanghai Fisheries university, 1999, 8: 43-47[张庆, 李卓佳, 陈康德. 复合微生物对水体生态因子的影响. 上海水产大学学报,1999, 8: 43- 47]
[2] Gat esoupe F J.The use of probiot ics in aquaculture [ J]. Aquaculture,1999, 160: 177-203
[3] Pinar calik, Esra Bilir,G?zide calik,et al. Influence of pH conditions on metabolic regulations in serine alkaline prot ease production byBacillus lichenif ormis[J]. Enzyme and Microbial Technology. 2002,31: 685-697
[4] Maitin V, Kavitha R, Umesh-Kumar S. Properties of an extracellular amylase purified from a Bacil lus species[J].World Journal of Microbiology and Biotechnol ogy. 2001, 17: 823-826
[5] Mulalo B N, Hugh-George P, Andr?v an T,et al. Over- expression and properties of a purified recombinantBacillus lichenif ormis lipase: a comparative report on Bacillus lipase[J].Enzyme and MicrobialTechnology. 2001, 28: 705-712
[6] Sogarrd. H,Demark T S.Microbials f or feed beyond lactic acid bact eria[J]. Feed International, 1990, 11(4): 32- 38
[7] Guo X H.The basis and applicat ion of probiot ics[M]. Beijing: Beijing Science and Technology Press. 2002, 32-34[郭兴华. 益生菌基础与应用. 北京: 北京科学技术出版社. 2002, 32- 34]
[8] Chen XG. Study on vaccines against Vibrio harveyi inestuary cod( Epinephelus coides)[D].Mast er papers of Shanghai Fisheries university, 2003, 31- 33 [ 陈献稿. 斜带石斑鱼哈氏弧菌疫苗研究[D]. 上海水产大学硕士学位论文, 2003, 31-33]
[9] Li H R,ChenH Q,Lu C P. Purificat ion and charact erization of ext racellular prot ease ECPase54 from Aeromonas hydrophil[J]. J Najing Agriculture University. 1996, 19( 3): 88-94[ 李焕荣, 陈怀青, 陆承平. 嗜水气单胞菌胞外蛋白酶ECPase54 的纯化及特性分析.南京农业大学学报, 1996, 19(3): 88-94]
[10] Allan B J, St evenson R M W. Extracellular virulence factors of a in fish inf ections [J]. Canadian Journal of Microbiology, 1981, 27:1114-1122
[11] Li H R. Selection, inhibitory activities and applicat ion in culture ofPenaeus japoni cus of marine probiotics[D].Doctor papers of Ocean University of Qingdao, 2001, 48-66 [ 李会荣. 海洋有益菌的筛选、抑菌作用及其在日本对虾养殖中的应用. 青岛海洋大学博士学位论文, 2001, 48-66]
[12] Wellingt a C A N,Meire L L M. Production and propert ies of an ext racellular prot ease from thermophilic Bacillussp.[ J].Brazilian journalof mi crobiology, 2004, 35: 91- 96
[13] Mi Z Q, Xie J,Pan B D, et al.Relationship among the phytoplankton,physicochemical fact ors and shrimp disease in intensive shrimp farming pond[ J]. J Shanghai Fisheries university, 1999, 8(4):304-308[米振琴, 谢骏, 潘德博, 等. 精养虾池浮游植物、理化因子与虾病的关系. 上海水产大学学报, 1999, 8(4): 304-308]
[14] Ding X, Li Z J, Chen Y Q, et al. Effects of probiotics on growth and activit ies of digestive enzymes ofPennaus vannamei[J].J Fishery Sciences of China, 2004, 11(6):581-585[丁贤, 李卓佳, 陈永青等. 芽孢杆菌对凡纳对虾生长和消化酶活性的影响. 中国水产科学, 2004, 11( 6) : 581-585]
[15] Huang X H, Li C L, Zheng L, et al. Effects of the immobilized microalgae on the Quantity dynamicsof Vibrio In the Shrimp ponds[J].ActaHydrobiologica Sinica, 2005, 29(5): 684-688[ 黄翔鹄, 李长玲, 郑莲, 等. 固定化微藻对虾池弧菌数量动态的影响. 水生生物学报, 2005, 29(5) : 684-688]
[16] Huang Xianghu, Li Changling, Liu Chuwu, et al. Studies on two microalgae improving environment of shrimp pond and strengthening anti-disease ability of Penaeus vannamei[J]. Acta Hydrobi ologica Sinica,2002, 26( 4):342- 347[ 黄翔鹄, 李长玲, 刘楚吾, 等. 两种微藻改善虾池环境增强凡纳对虾抗病力的研究. 水生生物学报,2002, 26( 4):342-347]
[17] Qi Z, Yang J P. Applicat ion of microbiological preparat ion and microalgae in aquaculture[J].Acta Hydrobiol ogica Sinica
计量
- 文章访问数: 982
- HTML全文浏览量: 5
- PDF下载量: 568
下载: