Yuling Hao, Yu Jin, Aodi Zhang, Xinran Jiang, Ming Gong, Cunlong Lu, Ruru Pan, Shaoxing Chen
{"title":"Identification and biochemical characterization of a novel halolysin from Halorubellus sp. PRR65 with a relatively high temperature activity.","authors":"Yuling Hao, Yu Jin, Aodi Zhang, Xinran Jiang, Ming Gong, Cunlong Lu, Ruru Pan, Shaoxing Chen","doi":"10.1007/s11274-024-04149-x","DOIUrl":null,"url":null,"abstract":"<p><p>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 Asp<sup>154</sup>-His<sup>193</sup>-Ser<sup>348</sup>. 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 K<sub>m</sub>, V<sub>max</sub> and K<sub>cat</sub> for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg<sup>-1</sup> and 1538.09 S<sup>-1</sup>, 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.</p>","PeriodicalId":23703,"journal":{"name":"World journal of microbiology & biotechnology","volume":"40 11","pages":"340"},"PeriodicalIF":4.0000,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"World journal of microbiology & biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s11274-024-04149-x","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
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.