| 深海沉积物宏基因组文库中产蛋白酶克隆CAPRO2的筛选及酶学性质分析 |
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| 引用本文： | 徐辉,赵晶,曾润颖.深海沉积物宏基因组文库中产蛋白酶克隆CAPRO2的筛选及酶学性质分析[J].台湾海峡,2011,30(4):522-527. |
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| 作者姓名： | 徐辉 赵晶 曾润颖 |
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| 作者单位： | 1. 国家海洋局第三海洋研究所、海洋生物遗传资源重点实验室,福建厦门,361005
2. 厦门大学海洋与环境科学学院,福建厦门,361005 |
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| 基金项目： | 国家863计划资助项目，中国大洋矿产资源研究开发协会资助项目 |
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| 摘 要： | 利用选择性筛选培养基从所构建的深海沉积物宏基因组文库中筛选得到一株产蛋白酶的克隆（CAPR0002），对其进行了酶学性质分析．结果表明该酶的最适作用温度为65cc，最适作用pH值为9．0．该蛋白酶具有较好的热稳定性，在40cC以下的温度中可长期保持稳定，在50℃中处理6h后仍能保持60％的活力，在60℃下保温30rain后仍能保持约60％的活力．Ca^2＋、Mg^2＋、sr^2＋、co^2＋对该蛋白酶有明显的促进作用，而且ca^2＋的存在可明显提高该蛋白酶的热稳定性，ca^2＋、sr^2＋、c0^2＋这3种离子均在3．0mmol／dm^3。浓度时具有最高的促进作用，当浓度高于3．0mmol／dm’时，这3种离子对酶活力的促进作用减弱．Hg^2＋、Fe^2＋、cu¨对酶有明显的抑制作用．CAPR02蛋白酶在pH值为7。5～9．5时活力较高，pH值为7。5时可保持约80％的活力，pH值为9．5时保持60％的活力，而在pH值高于9．5的条件下酶活力下降较快，pH值为10．0时活力降为约15％，表明CAPR02属于碱性蛋白酶．丝氨酸蛋白酶抑制剂PMSF、E一64和AEBSF对CAPR02蛋白酶均无抑制作用，显示该酶不属于丝氨酸蛋白酶，而EDTA对酶有明显的抑制作用，表明该酶属于金属蛋白酶．
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| 关 键 词： | 海洋生物学 深海沉积物 宏基因组文库 蛋白酶 热稳定性 |
Screening and characterization of a protease-producing clone CAPRO2 from metagenomic library of deep sea sediments |
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| XU Hui,ZHAO Jing,ZENG Run-ying.Screening and characterization of a protease-producing clone CAPRO2 from metagenomic library of deep sea sediments[J].Journal of Oceanography In Taiwan Strait,2011,30(4):522-527. |
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| Authors: | XU Hui ZHAO Jing ZENG Run-ying |
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| Institution: | XU Hui1,ZHAO Jing2,ZENG Run-ying1(1.Third Institute of Oceanography/ Key lab of Marine Biogenetic Resources,SOA,Xiamen 361005,China,2.College of Oceanography and Environmental Science,Xiamen University,China) |
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| Abstract: | A protease-producing clone(CAPRO2) was screened from metagenomic library of deep sea sediments using selective medium,and the characteristics of protease was studied.The results showed that the optimal temperature and pH for CAPRO2 protease activity were 65℃ and 9.0,respectively.The enzyme had excellent thermal stability.It was stable at temperature below 40℃,and retained about 60% of residual activity after 6 hours incubation at 50℃ and 30 minutes incubation at 60℃.The presence of Ca2+,Mg2+,Sr2+ and Co2+ increased the activity obviously,and Ca2+ enhanced the thermal stability distinctly.Three ions,Ca2+,Sr2+ and Co2+,increased the activity of the enzyme significantly at concentration of 3.0 mmol/dm3,and the activity-increasing effect was weak when the concentrations of these 3 ions were higher than 3.0 mmol/dm3.The ions Hg2+,Fe3+ and Cu2+ inhibited the enzyme activity notably.The CAPRO2 protease showed higher activity at pH 7.5 ~ 9.5,retaining 80% and 60% of residual activity at pH 7.5 and 9.5,respectively.The residual activity decreased rapidly at pH higher than 9.5 and reduced to about 15% at pH 10.0.It indicates that the enzyme belongs to alkaline protease.The serine protease inhibitors,PMSF,E-64 and AEBSF,showed no inhibition to the CAPRO2 enzyme activity,which indicates that the enzyme was not serine protease.On the other hand,EDTA inhibited the enzyme activity strongly,indicating that the enzyme belongs to metalloproteinase. |
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| Keywords: | marine biology deep sea sediment metagenomic library protease thermal stability |
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