Bacillus Subtilis DB104 Research Articles

Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822T.

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822T (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H2 O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2 O2 , suggest it has potential for biotechnological applications.

New robust subtilisins from halotolerant and halophilic Bacillaceae

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.Key points• Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins.• Four new subtilisins were biochemically characterised in detail.• The four proteases show potential for future biotechnological applications.

Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T.

Halophilic and halotolerant microorganisms represent a promising source of salt‐tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high‐alkaline subtilisin from Alkalihalobacillus okhensis Kh10‐101T. The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high‐alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0–12.0 and temperature 20–80 °C, optimally at pH 9.0–9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.

Expression and Purification ofPseudomonas aeruginosaKeratinase inBacillus subtilisDB104 Expression System

The DNA encoding Pseudomonas aeruginosa keratinase was ligated into pRPA expression vector and transformed into Bacillus subtilis DB104. Recombinant keratinase (rK), secreted by B. subtilis after 72 h of incubation, was purified to electrophoretical homogeneity by nickel affinity chromatography and found to have a molecular mass of 33 kDa. The rK had an optimal pH and temperature at 8.0 and 60 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-50 degrees C. It was strongly inhibited by Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, and ethylene diamine tetraacetic acid but activated by Ca(2+), Mg(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity, the rK was considered to be a metalloprotease.

Expression, Purification, and Characterization of Phosphatidylserine Synthase from Escherichia coli K12 in Bacillus subtilis

Although phosphatidylserine synthase (PSS) from Escherichia coli is an ideal enzyme for phospholipid production, its application in the food industry has been limited because of the low PSS yield. In this study, the pss gene was cloned from E. coli K(12) and expressed in Bacillus subtilis DB104, and the recombinant PSS was characterized subsequently. PSS was purified to 39.59-fold, and the highest activity was detected as 13.62 U/mg. The enzyme was found to be stable in a pH range of 6.5-9.5, with optimal pH values of 8.0 for hydrolysis and 7.0 for transphosphatidylation, respectively. The optimal temperature for PSS activity was 35 degrees C. The enzyme activity could be detected after 1 h of heating at 65 degrees C. Among the detected detergents and metal ions, Triton X-100, Ca(2+), Mn(2+), and Co(2+) could improve PSS activity. The transformation of phosphatidylcholine to phosphatidylserine under PSS catalyzation was carried out in a biphasic system, which confirmed the actual catalyzing ability of the recombinant protein.

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg(-1). As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65 degrees C and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 degrees C.

Bacillus subtilis에서 β-agarase의 분비형 과발현 및 효소분해산물의 항균활성

The gene for of an Agarivorans sp. JA-1 was expressed in Bacillus subtilis DB104, 168 and ISW1214 strains for mass-production. Among 3 host strains, B. subtilis ISW1214 secreted the highest amount of recombinant with a specific activity of 201 U/mg and 360 mg of protein into culture broth. This was approximately 130-fold higher than the production in E. coli as an expression host. Recombinant enzyme produced neoagarooligosaccharides such as neoagarohexaose, neoagarotetraose, and neoagarobiose from agar. Produced neoagarooligosaccharides showed antibacterial activities against gram-negative E. coli and gram-positive B. subtilis at a concentration of 1.5%. These data suggest that neoagarooligosaccharides could be an useful preservative for food industry.

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala.

The 3-D structure of subtilisin YaB was computer modelled using the structures of subtilisin BPN', subtilisin Carlsberg and thermitase as templates. Gly124 and Gly151 located on both sides of the waist of the S1 pocket were selected for site-directed mutagenesis based on the modelled structure. The mutated ale genes coding for the mutant subtilisin YaB were expressed in Bacillus subtilis DB104. All of the G124 and G151 series of mutants exhibited an increase of relative catalytic activity for elastin-orcein against casein and myofibrillar proteins. The S1 substrate specificity of G124A, G124V and G151A mutants were assessed using various carbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro or Val)-(Ala, Phe or Leu)-p-nitroanilide [AA(P/V) (A/F/L)]. While G124A and G124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzed Ala, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPF and AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Km values approximately 3-10-fold higher than those of the wild-type enzyme. The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVA with kcat/Km values approximately 1-4-fold higher than those of the wild-type enzyme. These results clearly indicate that the S1 substrate specificity of G124A and G124V mutants was restricted to Ala and Gly, and G151A mutant to Ala, Gly and Leu.

Simultaneous overproduction of extracellular endoglucanase and intracellular ?-glucosidase by genetically engineered Bacillus subtilis

Simultaneous overproduction of intracellular β-glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the β-glucosidase-endoglucanase gene(βE) or the endoglucanase-β-glucosidase gene(Eβ) under the control of a strong engineered promoter, BJ27UΔ88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the β-glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.

Nucleotide sequence and possible functional mechanism of the transcriptional activator gene ( preL) for neutral protease from Lactobacillus sp.

A positive regulatory gene ( preL) from Lactobacillus sp. no. 1, which enhanced production of neutral protease (NprL), was cloned in Bacillus subtilis DB104 together with the nprL structural gene. When nprL was expressed with the regulatory gene ( preL) on the same plasmid, extracellular protease production in a 12 h culture was 28-fold higher (14.2 U/ml) than protease production without preL expression (0.5 U/ml). The nucleotide sequence of the preL was determined and the deduced amino acid sequence revealed that PreL was relatively homologous (33%) to that of a positive regulatory protein (NprA) for protease production from Bacillus stearothermophilus, whose function is similar to that of preL. Northern hybridization analysis revealed that preL, in multicopy, enhanced transcription of the nprL gene. A consensus DNA binding motif (helix-turn-helix) was found within the amino acid sequence of the PreL. These results suggest that preL is a transcriptional activator of nprL. Indeed, a possible target sequence of PreL with a stable palindromic structure containing putative — 10 region was found in the promoter region of nprL. It is speculated that PreL melts this palindromic structure and assists RNA polymerase in binding effectively to the nprL promoter. Furthermore, a similar palindromic structure was also found in the promoter region of preL itself. Enhanced production of NprL in the stationary phase may possibly be due to this auto-activation by preL.

Cloning and nucleotide sequence of the regulatory gene, degM, for minor serine protease in Bacillus subtilis

Abstract The regulatory gene (degM) from Bacillus sp. B21-2 which enhanced the production of minor serine protease was cloned in Bacillus subtilis DB104 (Npr− Apr−). Nucleotide sequence analysis showed that the degM gene was composed of 1119 bases (373 amino acid residues). The deduced amino acid sequence was fairly homologous with those of sensor proteins of a two-component regulatory system in Bacillus sp.

Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus

A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18. In B. subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B. sphaericus.