Deduced Amino Acid Sequence and Possible Catalytic Residues of a Thermostable, Alkaline Cellulase from an Alkaliphilic <i>Bacillus</i> Strain

Dipicolinic acid (DPA) is a multi-functional agent for cosmetics, antimicrobial products, detergents, and functional polymers. The aim of this study was to design a new method for producing DPA from renewable material. The Bacillus subtilis spoVF operon encodes enzymes for DPA synthase and the part of lysine biosynthetic pathway. However, DPA is only synthesized in the sporulation phase, so the productivity of DPA is low level. Here, we report that DPA synthase was expressed in vegetative cells, and DPA was produced in the culture medium by replacement of the spoVFA promoter with other highly expressed promoter in B. subtilis vegetative cells, such as spoVG promoter. DPA levels were increased in the culture medium of genetically modified strains. DPA productivity was significantly improved up to 29.14 g/L in 72 h culture by improving the medium composition using a two-step optimization technique with the Taguchi methodology.

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“…KSM-237. 14) pC194 contains the chloramphenicol-resistant cassette. 20) Media and cultivation conditions.…”

Section: Methodsmentioning

confidence: 99%

“…So we tried to replace the promoter of spoVFA with other promoter that expressed in B. subtilis vegetative cells. We used the promoters of a cellulase gene, egl-237 14) or early stage sporulation-associated gene, spoVG, 15) which are regulated by σ A and σ H , respectively.…”

mentioning

confidence: 99%

Increased dipicolinic acid production with an enhanced spoVF operon in Bacillus subtilis and medium optimization

Takahashi

Sumitomo

Hagihara

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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“…pHYS237 and pHP237-K16, expression vectors for the alkaline cellulase Egl-237 from Bacillus sp. strain KSM-S237 (24) and the subtilisin-like alkaline protease M protease from Bacillus clausii strain KSM-K16 (31,34), respectively, were used for evaluation of exogenous enzyme production (39). pHYS237 was previously constructed by introducing a 3.1-kb DNA fragment containing the promoter, Shine-Dalgarno (SD) sequence, and coding region for the signal sequence, mature protein, and terminator of egl-237, which was PCR amplified from the Bacillus sp.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we constructed a multiple deletion mutant, MGB874, by the sequential deletion of 865 genes (874 kb; 20.7%) from the total genomic DNA of B. subtilis strain 168, including all prophage and prophage-like sequences, the pks and pps operons, and 11 nonessential gene clusters (3,39). Notably, compared to strain 168, strain MGB874 showed enhanced production of the exogenous extracellular alkaline cellulase Egl-237 (24) and the subtilisin-like alkaline protease M protease (31,34) from plasmid-borne genes in modified 2xL-Mal medium, a model medium for industrial protein production. Although enzyme production in wild-type 168 cells was arrested after the transition state, the production levels continued to increase in strain MGB874 throughout the culture period (39).…”

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confidence: 99%

Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874

Manabe

Kageyama

Morimoto

et al. 2011

Appl Environ Microbiol

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Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.Due to recent advances in genetic engineering technology, a variety of useful substances, including enzymes, have been produced industrially by use of microorganisms. To further improve production efficiencies on an industrial scale, several approaches, such as mutational breeding, have been used to generate hyperproducing microbial cells. In addition, strategies for genome reduction (18), which represents a relatively new field in synthetic genomics, have been used with Escherichia coli (25,44) and Bacillus subtilis (3,39,54) to investigate microbial genomic architecture and to improve characteristics relevant to protein production.B. subtilis, a Gram-positive sporiferous bacillus, is an attractive organism for industrial use for a variety of reasons, including its high growth rate, protein secretion ability, and GRAS (generally regarded as safe) status (46, 49). B. subtilis is also one of the best-characterized model microorganisms, by biochemical, genetic, and molecular biological studies. Furthermore, the complete genomic sequence of B. subtilis strain 168 has been determined, facilitating genetic engineering of this industrially useful strain (4, 35).In the 4.2-Mb genome of B. subtilis strain 168, only 271 of the 4,106 identified genes are indispensable for the growth of this organism in rich medium (33). In addition, B. subtilis has numerous genes that are activated only under specific conditions or in response to environmental stresses. Therefore, under controlled cond...

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“…Because of the stability of the cellulolytic thermozymes and advantage that they can be synthesized in the laboratory at large scale, the cellulases from thermophiles are realized to be the promising options for industrial applications. Attempts have also been made to engineer organisms/enzymes to confer thermostability and enhanced catalytic rates [11,12].…”

Section: Introductionmentioning

confidence: 99%

Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

Krishnaraj¹,

Samanta²,

Kumar³

et al. 2017

Can J Biotech

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Cellulolytic enzymes are promising candidates for the use of cellulose in any bioprocess operations and for the disposal of the cellulosic wastes in an environmentally benign manner. Cellulases from thermophiles have the advantage of hydrolyzing cellulose at wider range of operating conditions unlike the normal enzymes. Herein we report the modeled structures of cellulolytic enzymes (endoglucanase, cellobiohydrolase and β-glucosidase) from a thermophilic bacterium, Clostridium thermocellum and their validation using Root Mean Square Deviation (RMSD) and Ramachandran plot analyses. Further, the molecular interactions of the modeled enzyme with cellulose were analyzed using molecular docking technique. The results of molecular docking showed that the endoglucanase, cellobiohydrolase and β-glucosidase had the binding affinities of -10.7, -9.0 and -10.8 kcal/mol, respectively. A correlation between the binding affinity of the endoglucanase with cellulose and the enzyme activity was also demonstrated. The results showed that the binding affinities of cellulases with cellulose could be used as a tool to assess the hydrolytic activity of cellulases. The results obtained could be used in virtual screening of cellulolytic enzymes based on the molecular interactions with the substrate, and aid in developing systems biology models of thermophiles for industrial biotechnology applications.

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Deduced Amino Acid Sequence and Possible Catalytic Residues of a Thermostable, Alkaline Cellulase from an Alkaliphilic Bacillus Strain

Cited by 41 publications

References 9 publications

Increased dipicolinic acid production with an enhanced spoVF operon in Bacillus subtilis and medium optimization

Increased dipicolinic acid production with an enhanced spoVF operon in Bacillus subtilis and medium optimization

Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874

Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

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