Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY.
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ABSTRACT:
SUBMITTER: Genov N
PROVIDER: S-EPMC1153848 | biostudies-other | 1982 Nov
REPOSITORIES: biostudies-other
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Project description:BACKGROUND:?-Mannanase catalyzes the cleavage of ?-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable ?-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline ?-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high ?-mannanase activity at alkaline condition. In this study, this ?-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied. RESULTS:A thermo-alkaline ?-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0-11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing ?-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg-1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l-1 and 1238.9 s-1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml-1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2?×?SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml-1 after 72 h cultivation with 6% inoculum size by shake flask fermentation. CONCLUSIONS:The thermo-alkaline ?-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported ?-mannanases and was a promising thermo-alkaline ?-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml-1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline ?-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other ?-mannanases in B. subtilis.
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Project description:We report a case of bacteremia caused by Bacillus subtilis variant natto after a gastrointestinal perforation in a patient in Japan. Genotypic and phenotypic studies of biotin identified B. subtilis var. natto. This case and 3 others in Japan may have been caused by consuming natto (fermented soybeans).
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Project description:Sco-like proteins contain copper bound by two cysteines and a histidine residue. Although their function is still incompletely understood, there is a clear involvement with the assembly of cytochrome oxidases that contain the Cu(A) center in subunit 2, possibly mediating the transfer of copper into the Cu(A) binuclear site. We are investigating the reaction chemistry of BSco, the homologue from Bacillus subtilis. Our studies have revealed that BSco behaves more like a redox protein than a metallochaperone. The essential H135 residue that coordinates copper plays a role in stabilizing the Cu(II) rather than the Cu(I) form. When H135 is mutated to alanine, the oxidation rate of both hydrogen peroxide and one-electron outer-sphere reductants increases by 3 orders of magnitude, suggestive of a redox switch mechanism between the His-on and His-off conformational states of the protein. Imidazole binds to the H135A protein, restoring the N superhyperfine coupling in the EPR, but is unable to rescue the redox properties of wild-type Sco. These findings reveal a unique role for H135 in Sco function. We propose a hypothesis that electron transfer from Sco to the maturing oxidase may be essential for proper maturation and/or protection from oxidative damage during the assembly process. The findings also suggest that interaction of Sco with its protein partner(s) may perturb the Cu(II)-H135 interaction and thus induce a sensitive redox activity to the protein.
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Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes.
2015-01-26 | GSE65272 | GEO
Project description:Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape maintenance, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Apparently-redundant proteins can be important for enabling an organism to cope with environmental stressors. In this study, we evaluated the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of PBPs in B. subtilis change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are favored for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly-redundant periplasmic enzymes.IMPORTANCEMicrobes adapt to ever-changing environments and thrive over a vast range of conditions. While bacterial genomes are relatively small, significant portions encode for "redundant" functions. Apparent redundancy is especially pervasive in bacterial proteins that reside outside of the inner membrane. While conditions within the cytoplasm are carefully controlled, those of the periplasmic space are largely determined by the cell's exterior environment. As a result, proteins within this environmentally exposed region must be capable of functioning under a vast array of conditions, and/or there must be several similar proteins that have evolved to function under a variety of conditions. This study examines the activity of a class of enzymes that is essential in cell wall construction to determine if individual proteins might be adapted for activity under particular growth conditions. Our results indicate that a subset of these proteins are preferred for growth under alkaline conditions, while others are readily dispensable.
| S-EPMC10807460 | biostudies-literature
Project description:The filament of a bacterial flagellum is a tube-like organelle made of a single protein-flagellin-and assembled into multiple polymorphic forms. The filament can be further discretized into four subunit domains (D0, D1, D2, and D3) along the radial direction. However, it remains unclear which subunit domain plays an important role in regulating the rigidity of the filament. In this article, we address how the absence of two outer subunit domains (D2 and D3) affects the bending stiffness of the bacterium B. subtilis' flagellar filament. We first shear off flagellar filaments from the cell body, anchor one of its ends to the wall of a microfluidic channel, and correlate the elongation of the filament with the driving background flow. A numerical model is then applied to determine the bending stiffness of the filament. We find that the bending stiffness does not change drastically when the filament transforms from normal to hyperextended forms, which is estimated to be 2-3 pN⋅μm2. Furthermore, B. subtilis' flagellar filament has similar bending stiffness to Salmonella's, although the radius of the former is almost half of that of the latter, suggesting that the rigidity comes from the inner D0 and D1 subunit domains.
| S-EPMC9247555 | biostudies-literature
Project description:In this study, we have identified a bacterium that can inhibit the growth of Staphylococcus aureus, and further analyzed its antibacterial activity and other biological characteristics and laid the foundation for its future application. Through isolation and culture of the unknown bacteria, the culture characteristics, morphology observation, biochemical test, preliminary antibacterial test, 16S rRNA PCR amplification, sequence analysis, and homology analysis were performed. It was found that the bacteria are Gram positive spore chain Bacillus. The bacteria could only ferment glucose for acid production, but could not utilize lactose and maltose. The VP test for this bacteria was positive, while indole and methyl red tests were negative. Further analysis showed that these bacteria shared a homology up to 99.4% with Bacillus subtilis DQ198162.1. Thus, this newly identified bacterium was classified as Bacillus subtilis. Importantly, the crude bacteriocin of this Bacillus subtilis could inhibit the growth of Staphylococcus aureus, Escherichia coli, Enterococcus and Salmonella, which implies its potential usage in the future.
| S-EPMC5880821 | biostudies-literature
Project description:Alkaline proteases have applications in numerous industries. In this study, we have isolated and screened proteolytic bacteria from poultry wastes mixed soil and identified two bacterial isolates as Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 based on 16S rDNA sequencing. Maximum level of protease production was achieved after 24 h of fermentation in a basal medium. The optimal temperature, initial pH of the media and agitation for alkaline protease production by these two isolates were 30 °C, pH 9.0 and 120 rpm, respectively. The both bacterial isolates produced maximum level of protease with 3.0% organic municipal solid wastes (OMSW) as the sole source of carbon and nitrogen under previously optimized fermentation conditions. In comparison with the shake flask, protease production increased about 2.5-fold in the bioreactor with reduction in fermentation period. The partial purification of protease resulted in a final 45.67 and 34.86-fold purified protease with a specific activity of 8335.34 and 9918.91 U/mg protein and a typical yield of 9.75 and 9.41% from B. subtilis and E. indicum, respectively. The optimum temperature and pH of the partially purified protease from the both sources was 40 °C and pH 9.0, respectively. Protease from the both isolates was stable at pH 7.0-12.0 and at temperatures up to 50 °C. The effects of protease inhibitors indicated that the protease from B. subtilis might be serine and cysteine type and from E. indicum might be cysteine type. Mg2+, K+ and Ca2+ stimulated but Zn2+, Hg2+, Co2+ and Fe3+ strongly inhibited the protease activity. The partially purified protease from B. subtilis substantially dehaired cow skin and decomposed gelatinous compound from X-ray film. Our study revealed that OMSW can be used as raw material for production of bacterial extracellular protease and alkaline protease from B. subtilis might be potential for industrial and biotechnological applications.
| S-EPMC6042311 | biostudies-literature
Project description:BackgroundBacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.ResultsCompared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l(-1)) of AmyK38 was obtained using strain MGB874.ConclusionsWe demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.
| S-EPMC3424145 | biostudies-literature
Project description:A recently cloned Bacillus subtilis open reading frame (hemN) upstream of the dnaK operon was identified as encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation. B. subtilis hemN functionally complemented two Salmonella typhimurium hemF hemN double mutants under aerobic and anaerobic conditions. A B. subtilis hemN mutant accumulated coproporphyrinogen III only under anaerobic conditions. Interestingly, growth experiments using the B. subtilis hemN mutant revealed normal aerobic and anaerobic growth, indicating the presence of an alternative oxygen-independent enzymatic system. Northern blot experiments identified hemN mRNA as part of an approximately 7-kb pentacistronic transcript consisting of lepA, hemN, hrcA, grpE, and dnaK. One potential start site for aerobic and anaerobic transcription was located 37 bp upstream of the translational start codon of lepA. Comparable amounts of hemN transcript were observed under aerobic and anaerobic growth conditions. No experimental evidence for the presence of hemF in B. subtilis was obtained. Moreover, B. subtilis hemY did not substitute for hemF hemN deficiency in S. typhimurium. These results indicate the absence of hemF and suggest the presence of a second hemN-like gene in B. subtilis.
| S-EPMC179663 | biostudies-other