Abstract
BackgroundMannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-β-mannosidase or 1,4-β-D-mannanase (EC 3.2.1.78), commonly named β-mannanase, is an enzyme that can catalyze random hydrolysis of β-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-β-mannosidase gene (manB) from B. licheniformis.ResultsThe mannan endo-1,4-β-mannosidase gene (manB), commonly known as β-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 × 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°C. The recombinant β-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50°C, and within pH 6 - 9 after incubation at 50°C for 24 h. The enzyme was stable at temperatures up to 50°C with a half-life time of activity (τ1/2) of approximately 80 h at 50°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 β-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.
Highlights
Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose
Cloning and expression of mannan endo-1,4-βmannosidase from Bacillus licheniformis The oligonucleotide primers for cloning of the manB gene encoding mannan endo-1,4-β-mannosidase from B. licheniformis DSM13 were designed from its complete genomic database, according to the DNA sequence of gene ydhT (NCBI accession number NC006322), encoding a hypothetical protein similar to mannan endo-1,4beta-mannosidase
The manB gene was under control of the tac promoter and could be induced for high expression using Isopropyl β-D-1-thiogalactopyranoside (IPTG)
Summary
Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. 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; there has been no previous report on the cloning and expression of mannan endo-1,4-βmannosidase gene (manB) from B. licheniformis. The enzyme has found a number of applications in different sectors [4], including food, feed, pharmaceutical, and pulp/paper industries, gas well stimulation [1], as well as pre-treatment of lignocellulosic biomass for the production of second generation biofuel [2]. Bacillus licheniformis is a Gram-positive endosporeforming microorganism that belongs to the B. subtilis group of the genus Bacillus It is generally non-pathogenic and has been used extensively for large-scale industrial production of exoenzymes such as subtilisins or amylase, and the antibiotic bacitracin [9]. The properties of the recombinant enzyme are reported, and our results demonstrate that recombinant mannan endo-1,4-β-mannosidase from B. licheniformis is thermo- and alkali-stable, and suitable for various industrial applications
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