Mitsraria sp.1412产壳聚糖酶的分离纯化及性质研究
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摘要
以实验室选育的Mitsuaria sp.1412菌株发酵产壳聚糖酶,采用硫酸铵分级沉淀,表明沉淀饱和度为60%～90%效果较好,壳聚糖酶纯化倍数达1.85倍。然后依次采用离心超滤、离子交换层析和Sephadex G-200凝胶柱层析进行纯化。SDS-PAGE凝胶电泳检测表明,产物仅有一条清晰的条带,显示该壳聚糖酶的分子量为33.6 kD.酶学性质结果表明,该酶的最适反应pH和温度分别为5.5、50℃,并且该酶在pH5.0～8.0和35～45℃范围内,稳定性良好。酶最大反应速度V_(max)为2.8μmol/min,常数K_m为168.4 mmol/L.对该酶的酶解产物进行分析发现其酶解产物为壳二糖和壳三糖。
Firstly, the chitosanase from fermented broth of Mitsuaria sp. 1412 was purified by ammonium sulfate fractional precipitation. The result showed the optimum saturation of ammonium sulfate was 60%～90%, and the purification fold of was 1.85. Then centrifugal ultrafiltration, Ion-exchangchromatography, and Sephadex G-200 column chromatography were conducted to purify chitosanase. The result showed that there was only one clear band in the SDS-PAGE gel electrophoretogram, and the molecular weight of the chitosanase was 33.6 kD. Finally, the characterizations of a chitosanase were studied, and the results showed that the optimum pH and temperature of chitosanase were 5.5 and 50 °C, respectively; the chitosanase activity were relatively stable in the range of pH 5.0～8.0 and 35～45 °C; the kinetic parameter V_(max) was 2.8 μmol/min, and K_m was 168.4 mmol/L; the hydrolysates of chitosanase were chitobiose and chitotriose.
Firstly, the chitosanase from fermented broth of Mitsuaria sp. 1412 was purified by ammonium sulfate fractional precipitation. The result showed the optimum saturation of ammonium sulfate was 60%～90%, and the purification fold of was 1.85. Then centrifugal ultrafiltration, Ion-exchangchromatography, and Sephadex G-200 column chromatography were conducted to purify chitosanase. The result showed that there was only one clear band in the SDS-PAGE gel electrophoretogram, and the molecular weight of the chitosanase was 33.6 kD. Finally, the characterizations of a chitosanase were studied, and the results showed that the optimum pH and temperature of chitosanase were 5.5 and 50 °C, respectively; the chitosanase activity were relatively stable in the range of pH 5.0～8.0 and 35～45 °C; the kinetic parameter V_(max) was 2.8 μmol/min, and K_m was 168.4 mmol/L; the hydrolysates of chitosanase were chitobiose and chitotriose.
引文
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[11]韩宝芹, 杨菊林, 刘万顺, 等. 壳聚糖酶的分离纯化及性质研究[J]. 中国海洋大学学报(自然科学版), 2006,(02): 255～260.
[12]Yoon H G, Kim H Y, Kim H K, et al. Thermostable Chitosanase from Bacillus sp. Strain CK4: Its Purification,Characterization,and Reaction Patterns[J]. Biosci Biotechnol Biochem, 2001,65(4): 802～809.
[13]张翔, 张彦昊, 刘孝永, 等. 产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质[J]. 化工进展, 2018,37(06): 2354～2363.
[14]袁建平, 侯晓强. 壳聚糖酶的分离纯化及性质研究[J]. 食品科技, 2013,38(09): 165～167.
[15]李佳茵, 王慧敏, 祖国仁. 一株产壳聚糖酶海洋真菌MF-08的分离鉴定及酶学性质研究[J]. 食品工业科技, 2015,36(22): 193～197.
[16]Zhu X F, Tan H Q, Zhu C, et al. Cloning and overexpression of a new chitosanase gene from Penicillium sp. D-1[J]. AMB Express, 2012,2(1): 13～13.
[2]Wang, S L, Chen S J, Wang C L. Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate[J]. Carbohydr Res, 2008,343(7): 1171～1179.
[3]Yun, C S , Matsuda H, Kawamukai M. Directed Evolution to Enhance Secretion Efficiency and Thermostability of Chitosanase from Mitsuaria chitosanitabida 3001[J]. Bioscience Biotechnology & Biochemistry, 2006,70(2): 559～563.
[4]Gamal, R F, El-Tayeb T S , Raffat E I, et al. Optimization of chitin yield from shrimp shell waste by Bacillus subtilis and impact of gamma irradiation on production of low molecular weight chitosan[J]. International Journal of Biological Macromolecules, 2016,91: 598～608.
[5]方祥年, 杜昱光, 黄秀梨, 等. 球孢白僵菌胞外壳聚糖酶的纯化和性质[J]. 菌物系统, 2002,(01): 77～83.
[6]胡远亮, 洪文, 谭光迅. 产壳聚糖酶菌株的筛选与鉴定[J]. 中南民族大学学报(自然科学版), 2016,35(02): 42～45.
[7]Zhang X, Dai A, Zhang X, et al. Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold, Aspergillus oryzae IAM2660[J]. Bioscience Biotechnology & Biochemistry, 2000,64(9): 1896～1902.
[8]Araújo N K D , Pagnoncelli M G B , Pimentel V C, et al. Single-step purification of chitosanases from Bacillus cereus using expanded bed chromatography[J]. International Journal of Biological Macromolecules, 2016,82: 291.
[9]谭光迅. Mitsuaria sp.141-2产壳聚糖酶发酵条件研究及低分子量壳聚糖的制备[D].武汉:华中农业大学, 2009.
[10]Chen X E , Xia W, Yu X. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326[J]. Food Research International, 2005,38(3): 315～322.
[11]韩宝芹, 杨菊林, 刘万顺, 等. 壳聚糖酶的分离纯化及性质研究[J]. 中国海洋大学学报(自然科学版), 2006,(02): 255～260.
[12]Yoon H G, Kim H Y, Kim H K, et al. Thermostable Chitosanase from Bacillus sp. Strain CK4: Its Purification,Characterization,and Reaction Patterns[J]. Biosci Biotechnol Biochem, 2001,65(4): 802～809.
[13]张翔, 张彦昊, 刘孝永, 等. 产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质[J]. 化工进展, 2018,37(06): 2354～2363.
[14]袁建平, 侯晓强. 壳聚糖酶的分离纯化及性质研究[J]. 食品科技, 2013,38(09): 165～167.
[15]李佳茵, 王慧敏, 祖国仁. 一株产壳聚糖酶海洋真菌MF-08的分离鉴定及酶学性质研究[J]. 食品工业科技, 2015,36(22): 193～197.
[16]Zhu X F, Tan H Q, Zhu C, et al. Cloning and overexpression of a new chitosanase gene from Penicillium sp. D-1[J]. AMB Express, 2012,2(1): 13～13.