Identification and biochemical characterization of a novel halolysin from Halorubellus sp. PRR65 with a relatively high temperature activity.

IF 4 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY World journal of microbiology & biotechnology Pub Date : 2024-10-03 DOI:10.1007/s11274-024-04149-x
Yuling Hao, Yu Jin, Aodi Zhang, Xinran Jiang, Ming Gong, Cunlong Lu, Ruru Pan, Shaoxing Chen
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Abstract

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp154-His193-Ser348. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The Km, Vmax and Kcat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg-1 and 1538.09 S-1, respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.

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从具有较高温度活性的 Halorubellus sp.
来自卤虫的胞外蛋白酶(又称卤素蛋白)的需求量越来越大,人们正在研究它们在调味品和皮革工业中的各种应用。本研究克隆了卤虫 PRR65 的胞外蛋白酶编码基因 hly65,并在大肠杆菌中进行了异源表达。通过研究苯甲磺酰氟(PMSF)对其酶活性的完全抑制作用,揭示了卤虫属新型卤素蛋白 Hly65 的特征。实验测定发现了一个由 Asp154-His193-Ser348 组成的三元催化活性中心。删除 C 端延伸部分(CTE)会导致酶活性丧失,而二硫苏糖醇(DTT)不会抑制酶活性,这表明 Hly65 可能以单体形式发挥作用。以氮杂霉素为底物,在 60 °C、pH 8.0 和 4.0 M NaCl 条件下,测定了 Hly65 的 Km、Vmax 和 Kcat 分别为 2.91 mM、1230.47 U-mg-1 和 1538.09 S-1。此外,本研究还获得了基于功能域的三维结构预测,这将有助于卤素蛋白的改造和重组,从而产生具有新生理活性的突变体。
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来源期刊
World journal of microbiology & biotechnology
World journal of microbiology & biotechnology 工程技术-生物工程与应用微生物
CiteScore
6.30
自引率
2.40%
发文量
257
审稿时长
2.5 months
期刊介绍: World Journal of Microbiology and Biotechnology publishes research papers and review articles on all aspects of Microbiology and Microbial Biotechnology. Since its foundation, the Journal has provided a forum for research work directed toward finding microbiological and biotechnological solutions to global problems. As many of these problems, including crop productivity, public health and waste management, have major impacts in the developing world, the Journal especially reports on advances for and from developing regions. Some topics are not within the scope of the Journal. Please do not submit your manuscript if it falls into one of the following categories: · Virology · Simple isolation of microbes from local sources · Simple descriptions of an environment or reports on a procedure · Veterinary, agricultural and clinical topics in which the main focus is not on a microorganism · Data reporting on host response to microbes · Optimization of a procedure · Description of the biological effects of not fully identified compounds or undefined extracts of natural origin · Data on not fully purified enzymes or procedures in which they are applied All articles published in the Journal are independently refereed.
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