Metagenomics of un-culturable bacteria in cow rumen: Construction of cel9E–xyn10A fusion gene by site-directed mutagenesis

Kang, Young Min; Kim, Min Keun; An, Jin Mi; Haque, Md. Azizul; Cho, Kye Man

doi:10.1016/j.molcatb.2014.11.010

Cited by 8 publications

(4 citation statements)

References 35 publications

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“…To increase coupled catalytic action of single enzymes, several strategies were tested including enzyme cocktails, artificial cellulosomes and fusion enzymes (Bülow et al 1985 ; Elleuche 2015 ; Morais et al 2012 ; Resch et al 2013 ; Rizk et al 2012 ). In this context, end-to-end gene fusion has been proven to be a competent method for the construction of lignocellulose degrading bi- and multifunctional enzymes (Adlakha et al 2012 ; Fan et al 2009a ; Hong et al 2007 ; Kang et al 2015 ; Lee et al 2011 ). Using this method, a polypeptide is capable of catalyzing two or more distinct reactions.…”

Section: Introductionmentioning

confidence: 99%

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

Neddersen

Elleuche

2015

AMB Expr

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Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0122-7) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

Neddersen

Elleuche

2015

AMB Expr

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“…Several recent studies already suggest that new organisms such as uncultured bacteria are likely to harbor new antimicrobials (Degen et al, 2014 ; Doroghazi et al, 2014 ; Gavrish et al, 2014 ; Wilson et al, 2014 ) and underexplored complex microbial communities, including the rumen, very likely represent rich sources of novel antimicrobials. These microbiomes have the potential to revive the platform of natural product discovery in a new culture-independent perspective, unbiased by the culturing aptitude of microbial species (Lewis, 2012 ; McCann et al, 2014 ; Kang et al, 2015 ). The potential for application of metagenomics to biotechnology seems endless as functional screens can be used to identify new enzymes, antibiotics and other biological agents in libraries from diverse environments (Gillespie et al, 2002 ; Lorenz and Schleper, 2002 ; Piel, 2002 ; Voget et al, 2003 ; Berdy, 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis

et al. 2017

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Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

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“…It is estimated that unculturable microbes reach 99% of the population of microbes in the nature [2]. Metagenomics is the applications method of genomics technology to unculturable microbiota from its environment ( [6]; [7]) and reservoir genes (gut, gastrointestinal [8], rumen [9], etc) with making genomic libraries, followed by analyzing the genetic information contained therein. The metagenomics approach incorporates several fields of science, such as genetics, microbiology, and bioinformatics [10].…”

Section: Genes Exploration Using the Metagenomics Cdna Approachmentioning

confidence: 99%

Metagenomic DNA Library: Exploration of Novel Genes Encoding Glycoside Hydrolases

Kurniawati¹,

Sudiyono²,

Purkan³

et al. 2018

IJET

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Metagenomic is a potential approach to explore novel genes from a reservoir of genes. Metagenomic cDNA library as part of metagenomic approach be able to explore abundant quantity of microbiota genes. Metagenomic library collection was screened by functional and sequence analysis. Functional analysis can be done through induction genes expression substrat. Positive clones can be screened by expression of interest genes. Data of sequence analysis is very important in the process of product identification.

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Metagenomics of un-culturable bacteria in cow rumen: Construction of cel9E–xyn10A fusion gene by site-directed mutagenesis

Cited by 8 publications

References 35 publications

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis

Metagenomic DNA Library: Exploration of Novel Genes Encoding Glycoside Hydrolases

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