Biotechnology Division, CSIR-North East Institute of Science and Technology, Jorhat, 785006, Assam, India.
Folia Microbiol (Praha). 2012 Mar;57(2):129-37. doi: 10.1007/s12223-012-0097-6. Epub 2012 Feb 29.
Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099(T)), Pseudomonas aureofaciens (NCIM2026(T)), and Pseudomonas aeruginosa (MTCC2582(T)). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH(4))(2)SO(4) precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn(2+), Cu(2+), and Ni(2+) ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.
从印度阿萨姆邦卡齐兰加附近地区绿豆的根际土壤中分离出 50 株荧光假单胞菌，并对其胞外蛋白酶生产进行了测定。在这些分离物中，有 20 株在蛋白酶生产方面表现突出。通过 BOX-PCR 指纹图谱和 16S rDNA-RFLP 以及 3 个参考菌株（荧光假单胞菌（NCIM2099（T））、金黄色葡萄球菌（NCIM2026（T））和铜绿假单胞菌（MTCC2582（T）））分析了 20 个分离物的遗传多样性。BOX-PCR 在 56%相似系数下产生了两个不同的聚类，并有 7 个不同的 BOX 图谱。16S rDNA-RFLP 用三种四切割酶（HaeIII、AluI 和 MspI）显示了两个主要聚类 A 和 B；聚类 A 仅包含单个分离株 FPS9，而其余 22 个分离株属于聚类 B。根据表型特征和 16S rDNA 序列相似性，所有 8 株高蛋白酶产生菌株均与铜绿假单胞菌有关。蛋白酶从两种最突出的菌株（KFP1 和 KFP2）中提取，通过（NH4）2SO4 沉淀、凝胶过滤和离子交换层析三步过程进行纯化。该酶的最适 pH 为 8.0，在 60°C 和 37°C 时，KFP1 和 KFP2 的酶活性最高。比活分别为 75050（KFP1）和 81320 U/mg（KFP2）。纯化后的酶在天然和 SDS-PAGE 上均为单带，分子量为 32 kDa。Zn2+、Cu2+和 Ni2+离子抑制酶活性。EDTA 的存在也抑制了酶的活性，这表明它们是金属蛋白酶。
