一株耐盐芽孢杆菌的筛选及其蛋白酶的酶学特性研究
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摘要
从浙江舟山的盐场中分离出了一株耐盐菌,结合生理生化实验及16S rDNA基因序列鉴定结果,表明菌株CK-1为Bacillus的未定种,其系统分类学关系与花津浦芽孢杆菌(Bacillushwajinpoensis)种最近,命名为Bacillussp.CK-1。经研究,该菌最适生长NaCl浓度为4%,且能在20%NaCl浓度下生长,表明其为耐盐菌。对其分泌的蛋白酶进行了表征和培养条件的优化。经研究发现,该蛋白酶在pH 8.0和45℃具有最高的蛋白酶活性,并在8%NaCl浓度下仍能保持44.9%的活性。对发酵培养基的优化结果表明,蔗糖、淀粉和麦芽糖均对蛋白酶的生产有明显的促进作用。随着发酵培养基中NaCl浓度的测试表明,该菌分泌的蛋白酶活性随着NaCl浓度的变化呈现先增大后减小的趋势,并在12%NaCl浓度时该菌分泌的蛋白酶酶活达到最高。Ca~(2+)对发酵液中的蛋白酶活性具有明显的促进作用,表明其为Ca~(2+)依赖蛋白酶。经优化,在含有KCl 2.0 g·L~(-1)、Na HCO3 0.06 g·L~(-1)、FeCl3 0.001 g·L~(-1)、NaCl 120.0 g·L~(-1)、CaCl2·2H2O 1 mmol·L~(-1)、麦芽糖10 g·L~(-1)、tryptone 10 g·L~(-1)和初始pH 7.5的培养基中,于40℃发酵培养5 h后,Bacillus sp. CK-1所产蛋白酶具有最大的活性。
A halotolerant bacterium CK-1 was isolated form a saltworks of ZheJiang Zhoushan. The strain CK-1 was an undetermined species of Bacillus based on its 16 S rDNA sequence as well as physiological and biochemical analysis, and its systematic taxonomic relationship was closest to the species of Bacillus hwajinpoensis,and The Bacillus sp. CK-1 could grow at the concentration of 20% NaCl, indicating it as a halotolerant bacteria. The Na Cl concentration for the optimal growth of Bacillus sp. CK-1 was 4%. The Bacillus sp. CK-1 secreted proteases during growth. The proteases had the highest activity at pH 8.0 and 45℃, and remained 44.9% activity in the presence of 8% NaCl. Sucrose, starch and maltose significantly improved the production of proteases. The activity of proteases increased firstly and then decreased with the increase of NaCl concentration in the fermentation medium.The activity of proteases reached the maximum in the presence of 12% NaCl. The activity of proteases is significantly promoted by Ca~(2+), suggesting that they were calcium-dependent proteases.The maximal activity was obtained in the optimized medium(KCl 2.0 g·L~(-1),NaHCO_3 0.06 g·L~(-1),Fe Cl3 0.001 g·L~(-1), NaCl 120.0 g·L~(-1),CaCl_2·2 H_2O 1 mmol·L~(-1), maltose 10 g·L~(-1), tryptone 10 g·L~(-1), initial ph7.5) after fermentation at 40℃ for 5 h.
A halotolerant bacterium CK-1 was isolated form a saltworks of ZheJiang Zhoushan. The strain CK-1 was an undetermined species of Bacillus based on its 16 S rDNA sequence as well as physiological and biochemical analysis, and its systematic taxonomic relationship was closest to the species of Bacillus hwajinpoensis,and The Bacillus sp. CK-1 could grow at the concentration of 20% NaCl, indicating it as a halotolerant bacteria. The Na Cl concentration for the optimal growth of Bacillus sp. CK-1 was 4%. The Bacillus sp. CK-1 secreted proteases during growth. The proteases had the highest activity at pH 8.0 and 45℃, and remained 44.9% activity in the presence of 8% NaCl. Sucrose, starch and maltose significantly improved the production of proteases. The activity of proteases increased firstly and then decreased with the increase of NaCl concentration in the fermentation medium.The activity of proteases reached the maximum in the presence of 12% NaCl. The activity of proteases is significantly promoted by Ca~(2+), suggesting that they were calcium-dependent proteases.The maximal activity was obtained in the optimized medium(KCl 2.0 g·L~(-1),NaHCO_3 0.06 g·L~(-1),Fe Cl3 0.001 g·L~(-1), NaCl 120.0 g·L~(-1),CaCl_2·2 H_2O 1 mmol·L~(-1), maltose 10 g·L~(-1), tryptone 10 g·L~(-1), initial ph7.5) after fermentation at 40℃ for 5 h.
引文
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[2]ELLEUCHE S,SCHR?DER C,SAHM K,et al.Extremozymes--biocatalysts with unique properties from extremophilic microorganisms.[J].Curr Opin Biotech,2014,29(1):116-123.
[3]中华人民共和国国家技术监管局.GB/T 23527-2009ICS67.220.22X69-2009.蛋白酶制剂[S].北京标准出版社,2009.
[4]布洛克.微生物生物学[M].四川大学等《微生物生物学》翻译组,译.北京:人民教育出版社,1980.
[5]斯塔尼尔.微生物世界[M].《微生物世界》翻译组,译.北京:科学出版社,1983.
[6]郇惠杰,钟泓波,雷芬芬,等.产蛋白酶海洋细菌的筛选、鉴定及发酵培养基的研究[J].食品工业科技,2013,34(24):181-185.
[7]CHI Z,MA C,WANG P,et al.Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans[J].Bioresource Technol,2007,98(3):534-538.
[8]DAOUD L,JLIDI M,HMANI H,et al.Characterization of thermo-solvent stable protease from Halobacillus sp.CJ4 isolated from Chott Eldjerid hypersaline lake in Tunisia[J].J Basic Microb,2017,57(2):104.
[9]SANTOS A F,VALLE R S,PACHECO C A,et al.Extracellular proteases of Halobacillus blutaparonensis strain M9,a new moderately halophilic bacterium[J].Braz JMicrobiol,2013,44(4):1299-1304.
[10]NAMWONG S,HIRAGA K,TAKADA K,et al.A halophilic serine proteinase from Halobacillus sp.SR5-3 isolated from fish sauce:purification and characterization[J].Biosci Biotech Bioch,2006,70(6):1395-1401.
[11]KARBALAEI-HEIDARI H R,AMOOZEGAR M A,HAJIGHASEMI M,et al.Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium Halobacillus karajensis[J].J Ind Microbiol Biot,2009,36(1):21-27.
[12]VALASAKI K,STAIKOU A,THEODOROU L G,et al.Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus,from kefir with possible biotechnological interest[J].Bioresource Technol,2008,99(13):5804-5813.
[13]LI X,YU H Y,LIU X X,et al.Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile,Halobacillus sp.LY6[J].Folia Microbiol,2011,56(4):329-334.