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首页> 外文期刊>BMC Biotechnology >A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillus sp. SK37
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A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillus sp. SK37

机译：一种新型的枯草杆菌酶，具有来自Virgibacillus sp。的NaCl激活和氧化稳定活性。 SK37

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Background Microbial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications. Results A gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX). Conclusions The stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.
机译：背景技术微生物蛋白酶是最商业上有价值的酶之一，其中芽孢杆菌属的枯草蛋白酶或碱性蛋白酶已占据了最大的市场份额。尽管有关微生物蛋白酶的信息很多，但对于这种高度复杂的酶类别的基本和应用方面，仍然仍需要寻找具有独特性质的新型蛋白酶。业已证明，氧化剂稳定的蛋白酶（OSP）在洗涤剂和漂白行业中具有广泛的应用，最近已成为各种生物技术应用中最有吸引力的酶之一。结果从Virgibacillus sp中分离到一个编码枯草蛋白酶超家族新成员的基因。 SK37，从泰国鱼露发酵中分离出的一种产生蛋白酶的细菌。通过在包含1 mM IPTG和3％脱脂乳的Luria-Bertani（LB）琼脂板上基于基因组DNA表达文库的基于活性的筛选来克隆该基因。在筛选的100,000个克隆中，所有六个分离的阳性克隆均包含一个重叠的开放阅读框，与来自芽孢杆菌属的aprX基因有45％的同一性。将该基因命名为aprX-sk37克隆到pET21d（+）中，并在大肠杆菌BL21（DE3）中过表达。通过固定的金属离子亲和色谱法纯化命名为AprX-SK37的酶产物，使其具有明显的均一性并进行表征。根据含有5 mM CaCl 2 的偶氮酪蛋白分析，AprX-SK37酶在pH 9.5和55°C时显示最佳的催化条件。发现在1 M NaCl时具有最大催化活性，而在3 M NaCl时残留活性为30％。 1 M NaCl还增强了酶的热稳定性。该酶绝对是钙依赖性的，在15 mM时具有最佳浓度的CaCl 2 。苯甲磺酰氟和乙二胺四乙酸的抑制作用表明该酶是金属依赖性丝氨酸蛋白酶。酶的活性对还原剂，尿素和SDS敏感，但在H 2 O 2 。系统发育分析表明，AprX-SK37属于枯草杆菌蛋白酶超家族的一个新家族。我们提议将此新家族命名为碱性丝氨酸蛋白酶X（AprX）。结论对H 2 O 2 的稳定性和中等耐光度和耐热性AprX-SK37酶对各种生物技术应用具有吸引力。

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