Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes

Yamabhai, Montarop; Buranabanyat, Bancha; Jaruseranee, Nanthnit; Songsiriritthigul, Chomphunuch

doi:10.4161/bbug.2.1.13419

Cited by 18 publications

(12 citation statements)

References 14 publications

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“…pET vector was used to express the recombinant aprX-sk37 gene in this study. This system has been shown to be efficient for the expression of various Bacillus hydrolytic enzymes [37,38]. The canonical ribosomal binding site in this vector is identical to that of Virgibacillus aprX-sk37 .…”

Section: Discussionmentioning

confidence: 99%

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

et al. 2011

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BackgroundMicrobial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.ResultsA gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).ConclusionsThe stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.

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Section: Discussionmentioning

confidence: 99%

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

et al. 2011

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“…It has been verified that both E. coli OmpA and native signal peptide could be used to direct the secretion of Bacillus extracellular enzymes in E. coli expression system [9,10]. It is the first report that the signal peptide of chitosanase from Microbacterium sp.…”

Section: Discussionmentioning

confidence: 96%

“…EF159153) [7,8]. It has been verified both the E. coli OmpA and native signal peptide could be used to direct the secretion of Bacillus extracellular enzymes in E. coli expression systems [9,10]. A signal peptide was predicted by online software (SignalP 4.1), in the deduced amino acid sequence of mschito gene at the position 1-25.…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

2014

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A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 9 His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria-Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap TM FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS-PAGE analysis. Detected by 4700 MALDI-TOF-TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.

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“…The expression plasmid was transformed into E. coli Top10 (Invitrogen, Carlsbad, California, USA) and we found that this strain of E. coli is suitable not only for the cloning but also for the expression of various hydrolytic enzymes (Yamabhai et al, 2011). The cells harbouring the mannanase gene were grown at 310 K in Luria-Bertani medium containing 100 mg l À1 ampicillin.…”

Section: Expression and Purification Of Recombinant B Licheniformis mentioning

confidence: 99%

Crystallization and preliminary crystallographic analysis of β-mannanase fromBacillus licheniformis

et al. 2011

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The mannan endo-1,4-β-mannosidase (ManB) from Bacillus licheniformis strain DSM13 was overexpressed in Escherichia coli. Purification of the thermostable and alkali-stable recombinant mannanase yielded approximately 50 mg enzyme per litre of culture. Crystals were grown by hanging-drop vapour diffusion using a precipitant solution consisting of 12%(w/v) PEG 8000, 0.2 M magnesium acetate tetrahydrate and 0.1 M MES pH 6.5. The protein crystallized in the monoclinic space group P2(1), with two molecules per asymmetric unit and unit-cell parameters a = 48.58, b = 91.75, c = 89.55 Å, β = 98.29°, and showed diffraction to 2.3 Å resolution.

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Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes

Cited by 18 publications

References 14 publications

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

Crystallization and preliminary crystallographic analysis of β-mannanase fromBacillus licheniformis

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