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


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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    • "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(1):410. DOI:10.1186/2193-1801-2-410
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    • "Adding these enzymes to cleaning solutions allows the use of fewer toxic chemicals such as solvents and corrosive substances, decreasing their environmental impact [6]. Currently, the majority of proteases used in detergents are serine proteases [7] and their catalytic activity depends on the interplay of a nucleophile, a general base and an acid. In the two largest groups of serine-proteases, the (chymo)trypsin and subtilisin families, the catalytic triad is composed of serine, histidine and aspartate residues which exhibits similar spatial arrangements, but the order of the residues in the amino acid sequence and tertiary structure is different [8] [9]. "
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    ABSTRACT: This work reports the purification of a novel alkaline protease enzyme from a putative new thermophilic fungus Myceliophthora sp. The molecular weight of the enzyme was determined as 28.2kDa by using MALDI-TOF MS and it was inhibited by PMSF indicating it is a serine-protease. The optimum pH and temperature were 9.0 and 40–45°C, respectively. Mg was the only tested cation able to promote an increase of the protease activity. The N-terminal sequence of the purified protease (GVVGVC) presented identity and homology when compared to other proteases from fungi. This study also provides biochemical information about substrate specificity using fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp. The results showed that Abz-KLISSKQ-EDDnp is the best substrate among those tested for the purified protease (kcat/Km=1275.3mM−1s−1). Also, the specificity data suggest that subsites S1, S2, S3 and S1′, S2′, S3′, in general, present a preference for hydrophobic residues with the exception of Glu in P3, His in P2′ and Arg in P3′. The highest values for the specificity constant kcat/Km were obtained for P1, P2 and P2′.
    PROCESS BIOCHEMISTRY 11/2011; 46(11):2137-2143. DOI:10.1016/j.procbio.2011.08.014 · 2.52 Impact Factor
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