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Cloning and characterization of mersacidin like bacteriocin from Bacillus licheniformis MKU3 in Escherichia coli.

ABSTRACT: A putative gene encoding mersacidin like lantibiotic bacteriocin (lanA) was identified in Bacillus licheniformis genome. The lanA open reading frame codes for 74 amino acids with calculated isoelectric point of 6.7 and molecular mass of 8.2 kDa. The lanA gene was amplified from B. licheniformis MKU3, cloned in pQE30 vector and overexpressed in Escherichia coli M15. The recombinant peptide was purified to homogeneity using Ni-NTA chromatography and the SDS-PAGE analysis of the purified peptide revealed it to be a monomer with molecular mass of ~8.5 kDa. The purified bacteriocin showed wide spectrum activity against gram-positive pathogens. The peptide was found to be stable under in wide range of pH, temperature tolerant and resistant to the proteolytic enzymes. The stable nature of the bacteriocin to high temperature and resistant to various chemicals it also exhibited antimicrobial activity against food-borne pathogens make this bacteriocin as potent attractive antimicrobial agent in food products.

SUBMITTER: Kayalvizhi N

PROVIDER: S-EPMC4921080 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Cloning and characterization of mersacidin like bacteriocin from Bacillus licheniformis MKU3 in Escherichia coli.

Kayalvizhi Nagarajan N Rameshkumar Neelamegam N Gunasekaran Paramasamy P

Journal of food science and technology 20160527 5

A putative gene encoding mersacidin like lantibiotic bacteriocin (lanA) was identified in Bacillus licheniformis genome. The lanA open reading frame codes for 74 amino acids with calculated isoelectric point of 6.7 and molecular mass of 8.2 kDa. The lanA gene was amplified from B. licheniformis MKU3, cloned in pQE30 vector and overexpressed in Escherichia coli M15. The recombinant peptide was purified to homogeneity using Ni-NTA chromatography and the SDS-PAGE analysis of the purified peptide re ...[more]

PMID: 27407196

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Project description:BackgroundMannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis.ResultsThe mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose.ConclusionOur study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

Dataset Information

Cloning and characterization of mersacidin like bacteriocin from Bacillus licheniformis MKU3 in Escherichia coli.

Publications

Cloning and characterization of mersacidin like bacteriocin from Bacillus licheniformis MKU3 in Escherichia coli.

Similar Datasets

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