Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis

Bo-liang, Gong; Mao, Runqian; Xiao, Yumei; Jia, Mei-Lu; Zhong, Xiaolin; Liang, Yan; Xu, Peng; Li, Gang

doi:10.1016/j.enzmictec.2017.06.015

Cited by 26 publications

(11 citation statements)

References 27 publications

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“…In another work (Ashraf et al, 2019), a serine protease from Pseudomonas aeruginosa was modified at two positions (A29G and V336I); as a consequence, they achieved a 5 °C increase in the temperature of observed residual activity and 1.4-fold enhancement of the catalytic activity (Ashraf et al, 2019). In yet another study (Gong et al, 2017), statistical mutagenesis of an alkaline-protease gene discovered during a metagenomic analysis increased the enzymatic activity by 6.6-fold.…”

Section: The Latest Research On the Modification Of Alkaline Serine Pmentioning

confidence: 99%

Production of subtilisin proteases in bacteria and yeast

et al. 2021

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In this review, we discuss the progress in the study and modif ication of subtilisin proteases. Despite longstanding applications of microbial proteases and a large number of research papers, the search for new protease genes, the construction of producer strains, and the development of methods for their practical application are still relevant and important, judging by the number of citations of the research articles on proteases and their microbial producers. This enzyme class represents the largest share of the industrial production of proteins worldwide. This situation can explain the high level of interest in these enzymes and points to the high importance of designing domestic technologies for their manufacture. The review covers subtilisin classif ication, the history of their discovery, and subsequent research on the optimization of their properties. An overview of the classes of subtilisin proteases and related enzymes is provided too. There is a discussion about the problems with the search for (and selection of) subtilases from natural strains of various microorganisms, approaches to (and specif ics of) their modif ication, as well as the relevant genetic engineering techniques. Details are provided on the methods for expression optimization of industrial subtilases of various strains: the details of the most important parameters of cultivation, i. e., composition of the media, culture duration, and the inf luence of temperature and pH. Also presented are the results of the latest studies on cultivation techniques: submerged and solid-state fermentation. From the literature data reviewed, we can conclude that native enzymes (i. e., those obtained from natural sources) currently hardly have any practical applications because of the decisive advantages of the enzymes modif ied by genetic engineering and having better properties: e. g., thermal stability, general resistance to detergents and specif ic resistance to various oxidants, high activity in various temperature ranges, independence from metal ions, and stability in the absence of calcium. The vast majority of subtilisin proteases are expressed in producer strains belonging to different species of the genus Bacillus. Meanwhile, there is an effort to adapt the expression of these enzymes to other microbes, in particular species of the yeast Pichia pastoris.

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Section: The Latest Research On the Modification Of Alkaline Serine Pmentioning

confidence: 99%

Production of subtilisin proteases in bacteria and yeast

et al. 2021

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“…Protease enzymes play a significant role in the cell's metabolism because these enzymes can digest long protein chains in shorter fragments through the hydrolysis of peptide bonds (Gong et al, 2017). Alcalase ® is a bacterial alkaline protease produced by Bacillus licheniformis and has been considered by many researchers to be one of the best enzymes for protein hydrolysis (Memon et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Assessment of obtaining sunflower oil from enzymatic aqueous extraction using protease enzymes

et al. 2022

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The aim of this work was to maximize the enzymatic aqueous extraction (EAE) of sunflower seed oil using protease enzymes from the evaluation of various temperatures, pH and enzyme concentrations, using a Box-Behnken experimental design. The effect of a thermal pre-treatment of sunflower seeds on free oil yield (FOY) and oil quality was also determined. In the experimental range adopted, a lower temperature (40 °C) provided higher FOY values, as well as the intermediate pH (8.00) and maximum enzyme concentration (9% v/v). Thermal pre-treatment provided an increase in FOY in the initial extraction times (60 to 180 min) and decreased of the extraction time of 4 to 3 h to obtain the highest FOY value (~16%). The fatty acid composition of the oils obtained showed a predominance of oleic (~47.5%) and linoleic acids (~39.5%). The total phytosterol content in the samples was hardly affected by the heat pre-treatment of the seeds, while the fatty acid profile, tocopherol content and oxidative stability were not altered.

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“…Metagenomic strategy provides an access to genomes of the uncultured microorganisms skipping culturing microorganisms and have been applied to screen novel biocatalysts for industrial applications in the past decades (Handelsman 2004). Various novel biocatalysts have been successfully identi ed from soil metagenomic library, such as lipolytic enzymes (Li et al 2018;Park et al 2020), proteases (Gong et al 2017), cellulases (Garg et al 2016;Yang et al 2016), and novel natural products (Katz et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

et al. 2021

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A novel lipolytic gene, estq7, was identi ed from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910 S -1 , respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Homology modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking reveled the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property, and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

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Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis

Cited by 26 publications

References 27 publications

Production of subtilisin proteases in bacteria and yeast

Production of subtilisin proteases in bacteria and yeast

Assessment of obtaining sunflower oil from enzymatic aqueous extraction using protease enzymes

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

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