海洋贝类肠道弧菌21Z1碱性蛋白酶的分离纯化及酶学性质
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摘要
由威海海域贝类肠道中分离出一株产胞外碱性蛋白酶的细菌,经16S rDNA分析鉴定为弧菌21Z1。该菌发酵液上清经60%饱和度的硫酸铵溶液沉淀、透析、Sephadex-G100分子筛层析等步骤,获得电泳纯的21Z1碱性蛋白酶,聚丙烯酰胺凝胶电泳(SDS-PAGE)显示其分子量约为31 kDa。酶学性质测定结果显示:该酶最适反应温度为40℃,在20~40℃稳定;最适反应pH为8.5,在pH=8.5~11.0间稳定;Na~+、K~+、Ca~(2+)、Mn~(2+)对该酶具有激活作用,Fe~(2+)、Fe~(2+)、Zn~(2+)及Cu~(2+)则不同程度地抑制其活性;该酶的耐盐性检测结果显示,在Na~+浓度为4 mol/L时,残余酶活在62%以上,表明21Z1碱性蛋白酶具有较强的耐盐性。
A strain was isolated from intestinal of shellfish in Weihai waters which could produce extracellular alkaine protease,and was identified as Vibrio sp. 21Z1 by 16S rDNA analysis. The purified alkaine enzyme 21Z1 was obtained through ammonium sulfate precipitation,dialysed and Sephadex-G100 column chromatography. Its molecular weight was determined as about 31 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) analysis. The results show that the optimal temperature of this enzyme is 40 ℃,and it is stable at 20 ~ 40 ℃. The optimal pH of this enzyme is 8.5,and it is stable at pH ranging from 8.5 ~ 11.0. Na~+,K~+,Ca~(2+),and Mn~(2+)have effect on its enzyme activity while Fe~(2+),Fe~(2+),Zn~(2+)and Cu~(2+)have various degrees of inhibition effect upon this enzyme. The salt tolerance test of enzyme 21Z1 exhibits the residual protease activity remains more than 62% at 4 mol/L concentration of Na~+,so alkaine protease 21Z1 has good salt resistance.
A strain was isolated from intestinal of shellfish in Weihai waters which could produce extracellular alkaine protease,and was identified as Vibrio sp. 21Z1 by 16S rDNA analysis. The purified alkaine enzyme 21Z1 was obtained through ammonium sulfate precipitation,dialysed and Sephadex-G100 column chromatography. Its molecular weight was determined as about 31 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) analysis. The results show that the optimal temperature of this enzyme is 40 ℃,and it is stable at 20 ~ 40 ℃. The optimal pH of this enzyme is 8.5,and it is stable at pH ranging from 8.5 ~ 11.0. Na~+,K~+,Ca~(2+),and Mn~(2+)have effect on its enzyme activity while Fe~(2+),Fe~(2+),Zn~(2+)and Cu~(2+)have various degrees of inhibition effect upon this enzyme. The salt tolerance test of enzyme 21Z1 exhibits the residual protease activity remains more than 62% at 4 mol/L concentration of Na~+,so alkaine protease 21Z1 has good salt resistance.
引文
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[20]JOO H S,CHANG C S.Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52:enhanced production and simple purification[J].J Appl Microbiol,2005,98(2):491-497.
[2]JSALEEM M,REHMAN A,YASMIN R,et al.Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain[J].Molecular Biology Reports,2012,39(6):6399-6408.
[3]RAJKUMAR R,KOTHILMOZIAN J,RAMASAMY R.Production and characterization of a novel protease from Bacillus sp.RRM1under solid state fermentation[J].Journal of Microbiology and Biotechnology,2011,21(6):627-636.
[4]DENG A,WU J,ZHANG Y,et al.Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp.B001[J].Bioresource Technology,2010,101(18):7111-7117.
[5]路炳声,黄志强,林白雪,等.海洋氧化短杆菌15E产碱性蛋白酶的发酵条件[J].福建农林大学学报(自然科学版),2007,26(6):591-595.
[6]杨冠东,杜少平,杨础华,等.产碱性蛋白酶的嗜热菌株筛选及发酵条件研究[J].兰州大学学报(自然科学版),2001,27(6):80-84.
[7]孙谧,王跃军,张云波,等.一株产低温碱性蛋白酶嗜冷海洋细菌YS-9412-130的分离和培养条件研究[J].海洋水产研究,2000,21(4):1-5.
[8]梅承芳,江晓路,牟海津,等.碱湖高产碱性蛋白酶菌的选育和产酶条件研究[J].中国海洋大学学报,2005,35(4):613-617.
[9]冯静,荆谷,谢慧君,等.海洋细菌Pseudomnas sp.7-11产碱性蛋白酶的研究[J].山东大学学报(理学版),2002,37(3):264-267.
[10]肖峰,张浩,王斌等.一株产中性蛋白酶海洋细菌的筛选与初步鉴定[J].食品与药品,2011,13(3):89-93.
[11]SALEEM M,REHMAN A,YASMIN R,et al.Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain[J].Molecular Biology Reports,2012,39(4):6399-6408.
[12]SUNDARARAJAN S,KANNAN C N,CHITTIBABU S.Alkaline protease from Bacillus cereus VITSN04:potential application as a dehairing agent[J].Journal of Bioscience and Bioengineering,2011,111(2):128-133.
[13]ELIBOL M,MOREIRA A R.Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation[J].Process Biochemstry,2005,40(5):1951-1956.
[14]SCHILDKRAUT C,LIFSON S.Dependence of the melting temperature of DNA on salt concentration[J].Biopolymers,1965,3(2):195-208.
[15]GUEST S,NGO V,HEFFORD M.Circular dichroism reveals sensitivity of glucagon solution structure to fluoroalcohols,p H and ionic strength[J].Protein Peptide Lett,2008,15(8):811-817.
[16]LEE S K,HWANG J Y,CHOI S H,et al.Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste[J].Food Sci Biotech,2010,19(2):327-334.
[17]FENG Y Z,CAI Y,SU G W,et al.Evaluation of aroma differences between high-salt liquid-state fermentation and low-salt solid-state fermentation soy sauces from China[J].Food Chemistry,2014,145(7):126-134.
[18]冯滢滢,段杉,李远志.食盐浓度对虾油风味成分形成的影响研究[J].现代食品科技,2013,2(3):269-273.
[19]KIM Sang-moo.Accelerating effect of squid viscera on the fermention of Alaska Pollack scraps sauce[J].Food Science and Nutrition,2000,4(2):103-106.
[20]JOO H S,CHANG C S.Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52:enhanced production and simple purification[J].J Appl Microbiol,2005,98(2):491-497.