印尼热泉中产嗜热碱性蛋白酶菌株筛选及酶学性质研究  

Screening and Characterization of a Crude Thermophilic Alkaline Protease From an Isolated Strain of Brevibacillus PBI in Indonesia's Hot Spring

作　　者：王帅[1] 林学政[1] 黄晓航[1] 郑立[1] ZILDA Dewi Seswita 

机构地区：[1]国家海洋局第一海洋研究所海洋生物活性物质重点实验室,山东青岛266061 [2]印度尼西亚海洋与渔业研究局海洋生物技术与水产品加工研究中心,雅加达999006

出　　处：《海洋科学进展》2012年第2期244-251,共8页Advances in Marine Science

基　　金：中央级公益性科研院所基本科研业务项目--印尼热泉生态系统微生物多样性及其产嗜热酶研究(2010G13);海洋专性解烃菌Cycloclasticus spp.的代谢特征及协同降解高分子量多环芳烃的研究(2010G23)

摘　　要：采用MSM寡营养培养基从印度尼西亚Pantai cermin,Kalianda和Banyuwedang三个地区的热泉水样、泥样以及沉积物样品中分离获得细菌菌株,通过检测蛋白酶产生透明圈和福林酚蛋白酶活性测定相结合的方法,从中筛选出1株产嗜热蛋白酶的菌株PBI,该菌株经初步鉴定为短杆菌属(Brevibacillus),并对其酶学性质进行了研究。结果表明,菌株PBI产蛋白酶的最适酶活温度为60℃,最适pH值为8.0～9.0,具有较好的热稳定性和pH稳定性,60℃时酶的半衰期为30min,70℃条件下20min仍保持46.1%的酶活,该酶在pH值为5.0～9.0范围的缓冲液中酶活相对稳定。其产嗜热蛋白酶的酶活力最高可达到60.53U/mL,在100℃条件下仍能保留26.37%的相对酶活。Fe2+,Fe3+,Cu2+和Zn2+对嗜热蛋白酶活性具有明显的抑制作用。该嗜热蛋白酶对EDTA敏感,苯甲基磺酰氟(PMSF)、亮抑酶肽(Leupeptin)、苄眯(Benzamidine)和胃蛋白酶抑制剂(Pepstain A)对该嗜热蛋白酶都有一定的抑制作用,说明其酶活性受到丝氨酸、半胱氨酸残基的影响。结果表明,该菌株是1株具有进一步改造利用价值的产蛋白酶菌株。Thermophilic bacteria strains were isolated from water samples, soil samples and sediment samples of three Indonesia＇s hot spring area （Pantai cermin, Kalianda and Banyu wedang） by using MSM oligomedium. A strain of bacterium, by detecting hydrolysis halos of protease and its activity. The protease belonged to thermophilic alkaline protease, which showed high-temperature stability and pH stability. In addition, the results revealed that the optimal temperature and pH were 60 ℃ and 8.0-9.0 for protease activity, respectively. The enzyme activity decreased faintly at 60 ℃ for 10 min, the enzyme stability declined with increasing temperature, the enzyme half-life is 30rain at 60 ℃, enzyme activity remained 46.1 % at 70 ℃ for 20 rain, the enzyme activity was relatively stable in the range of pH5.0-9.0. And the highest protease activity was 60.53 U/mL. At 100 ℃, the protease still remained 26.37% relative enzyme activity. The effects of different metal ions （Ca^2＋ , Mg^2＋ , Mn^2＋ , Fe^2＋ , Fe^3＋ , Cu^2＋ , Zn^2＋ , Ba^2＋ and Sr^2＋） and protease inhibitor （EDTA, PMSF, Leupeptin, Pepstain A and Benzamidine） on thermophilic protease activity were investigated. It was found that low concentration of Fe^3＋ could promote the production of thermophilic protease. In contrast, the protease activity was inhibited markedly by Fe^3＋ , Cu^2＋ and Zn^2＋. The thermophilic protease was sensitive to EDTA, its activity was decreased with the increased of EDTA concentration. And the protease activity was also inhibited in a certain extent by PMSF, Leupeptin, Benzamidine and Pepstain A, it showed that the enzyme activity declined was due to the effects of serine, cysteine residues. Based on the above results, the strain PBI had shown interesting properties in producing thermophilic protease.

关 键 词：热泉 嗜热细菌 嗜热蛋白酶 鉴定 

分 类 号：Q93[生物学—微生物学]