Article

Identification and characterization of alkaline serine protease from goat skin surface metagenome

Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, India 625021. .
AMB Express 03/2011; 1(1):3. DOI: 10.1186/2191-0855-1-3
Source: PubMed

ABSTRACT Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.

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    • " There have been several novel proteases discovered through functional metagenomic methods . For instance , Biver et al . ( 2013a ) identified an oxidant - stable alkaline serine protease from a forest - soil metagenomic library . An alkaline serine protease was also identified in a metagenomic library constructed from goat skin surface samples by Pushpam et al . ( 2011 ) . These alkaline proteases are examples of microbial enzymes with potential industrial applications , mainly in the detergent industry ."
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    ABSTRACT: Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i) the identification of enzymes with desirable technological properties, capable of catalyzing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii) the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii) the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.
    Frontiers in Microbiology 06/2015; 6:672. DOI:10.3389/fmicb.2015.00672 · 3.94 Impact Factor
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    • "A novel protease belonging to chymotrypsin-like S1 serine proteases was isolated by Niehaus et al. [37]. Neveu et al. also isolated two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts [38], while Pushpam et al. identified and characterized metagenomic alkaline serine protease from the metagenome of goat skin surface [39]. This class of enzymes had not been described earlier for use in laundry and cleaning applications. "
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    ABSTRACT: Metagenomics deals with the isolation of genetic material directly recovered from environmental samples. Metagenomics as an approach has emerged over the past two decades to elucidate a host of microbial communities inhabiting a specific niche with the goal of understanding their genetic diversity, population structure, and ecological role played by them. A number of new and novel molecules with significant functionalities and applications have been identified through this approach. In fact, many investigators are engaged in this field to unlock the untapped genetic resources with funding from governments sector. The sustainable economic future of modern industrialized societies requires the development of novel molecules, enzymes, processes, products, and applications. Metagenomics can also be applied to solve practical challenges in the field of medicine, agriculture, sustainability, and ecology. Metagenomics promises to provide new molecules and novel enzymes with diverse functions and enhanced features compared to the enzymes from the culturable microorganisms. Besides the application of metagenomics for unlocking novel biocatalysts from nature, it also has found applications in fields as diverse as bioremediation, personalized medicine, xenobiotic metabolism, and so forth.
    08/2014; 2014:1-7. DOI:10.1155/2014/146030
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    • "This strategy has been applied to several ecosystems, allowing detection of diverse enzymes such as cellulases, xylanases, glucosidases, amylases, and esterases (Duan and Feng 2010; Kennedy et al. 2011; Nimchua et al. 2012; Rondon et al. 2000). To date, only a few proteases have been isolated by functional metagenomics (Lee et al. 2007; Neveu et al. 2011; Pushpam et al. 2011). Here, a forest-soil library previously screened with success for lipolytic and antimicrobial activities was used to search for new proteases . "
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    ABSTRACT: A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-2-410) contains supplementary material, which is available to authorized users.
    SpringerPlus 08/2013; 2:410. DOI:10.1186/2193-1801-2-410
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