Identification and Characterization of Bacterial Alliinase: Resource and Substrate Stereospecificity.

Limited alliinase resources cause difficulties in the biosynthesis of thiosulfinates (e.g., allicin), restricting their applications in the agricultural and food industries. To effectively biosynthesize thiosulfinates, this study aimed to excavate bacterial alliinase resources and elucidate their catalytic properties. Two bacterial cystathionine ß-lyases (MetCs) possessing high alliinase activity (>60 U mg -1) toward L-(-)-alliin were identified from Allium sativum rhizosphere isolates. Metagenomic exploration revealed that cystathionine ß-lyase from Bacillus cereus (BcPatB) possessed high activity toward both L-(±)-alliin and L-(+)-alliin (208.6 and 225.1 U mg -1), respectively. Although these enzymes all preferred l-cysteine S-conjugate sulfoxides as substrates, BcPatB had a closer phylogenetic relationship with Allium alliinases and shared several similar features with A. sativum alliinase. Interestingly, the Trp30Ile31Ala32Asp33 Met34 motif in a cuspate loop of BcPatB, especially sites 31 and 32 at the top of the motif, was modeled to locate near the sulfoxide of L-(+)-alliin and is important for substrate stereospecificity. Moreover, the stereoselectivity and activity of mutants I31V and A32G were higher toward L-(+)-alliin than those of mutant I31L/D33E toward L-(-)-alliin. Using bacterial alliinases and chemically synthesized substrates, we obtained thiosulfinates with high antimicrobial and antinematode activities that could provide insights into the protection of crops and food.

Reversible conjugation of a CBASS nucleotide cyclase regulates bacterial immune response to phage infection.

Prokaryotic antiviral defence systems are frequently toxic for host cells and stringent regulation is required to ensure survival and fitness. These systems must be readily available in case of infection but tightly controlled to prevent activation of an unnecessary cellular response. Here we investigate how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a type II CBASS system from Bacillus cereus into the model organism Bacillus subtilis, we show that the protein-conjugating Cap2 (CBASS associated protein 2) enzyme links the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. The cyclase-PspA conjugation is reversed by the deconjugating isopeptidase Cap3 (CBASS associated protein 3). We propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage, with conjugation released upon infection, priming the cyclase for activation.

Effect of calcium on Pseudomonas aeruginasa and Bacillus cereus metabolites / Efeito do cálcio nos metabólitos de Pseudomonas aeruginasa e Bacillus cereus

The effects of Calcium (Ca+²) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+² on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+² on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.

Os efeitos do cálcio (Ca+²) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+² sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+² na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.

Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids.

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system. The thermodynamically unfavorable oxidation reaction was driven by the reduction reaction. The efficiency of the biocatalytic reaction was evaluated using 12 different substrate combinations, and a significant variation was observed in substrate conversion, which was subsequently explained by the differences in enzyme kinetics parameters. The reaction with the selected model substrates 2-oxobutanoic acid and L-leucine reached 90.3% conversion with a high total turnover number of 9.0 × 106 under the optimal reaction conditions. Furthermore, complete conversion was achieved by adjusting the ratio of addition of the two substrates. The constructed reaction mode can be applied to other amino acid dehydrogenases in future studies to synthesize a wider range of valuable products.

Isocitrate dehydrogenase of Bacillus cereus is involved in biofilm formation.

Isocitrate dehydrogenase (IDH), a key enzyme in the TCA cycle, participates in the formation of biofilms in Staphylococcus aureus, but it remains to be clarified whether it is involved in the formation of Bacillus cereus biofilms. In this study, we scanned the genome of B. cereus 0-9 and found a gene encoding isocitrate dehydrogenase (FRY47_22620) named icdH. The IcdH protein was expressed and purified. The enzyme activity assay showed that the protein had IDH activity dependent on NADP+, indicating that this gene encoded an IDH. The ΔicdH mutant and its complemented strains were obtained by a homologous recombination strategy, and crystal violet data and CLSM were measured. The results showed that the biofilm yield of the mutant ΔicdH decreased, and the biofilm morphology also changed, while the growth of ΔicdH was not affected. The extracellular pH and citric acid content results showed that the ΔicdH mutant exhibited citric acid accumulation and acidification of the extracellular matrix. In addition, the addition of excess Fe3+ restored the biofilm formation of the ΔicdH mutant. It is speculated that IDH in B. cereus may regulate biofilm formation by modulating intracellular redox homeostasis. In addition, we found that the icdH deletion of B. cereus 0-9 could result in a reduced sporulation rate, which was significantly different from sporulation in B. subtilis caused by interruption of the stage I sporulation process due to icdH loss. All the above results provide us with new insights for further research on IDH.

Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties.

Low-molecular-weight (low Mr ) thioredoxin reductases (TrxRs) are homodimeric NADPH-dependent dithiol flavoenzymes that reduce thioredoxins (Trxs) or Trx-like proteins involved in the activation networks of enzymes, such as the bacterial class Ib ribonucleotide reductase (RNR). During the last few decades, TrxR-like ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs) have been discovered and characterized in several types of bacteria, including those not encoding the canonical plant-type FNR. In Bacillus cereus, a TrxR-like FNR has been shown to reduce the flavodoxin-like protein NrdI in the activation of class Ib RNR. However, some species only encode TrxR and lack the homologous TrxR-like FNR. Due to the structural similarity between TrxRs and TrxR-like FNRs, as well as variations in their occurrence in different microorganisms, we hypothesized that low Mr TrxR may be able to replace TrxR-like FNR in, for example, the reduction of NrdI. In this study, characterization of TrxR from B. cereus has revealed a weak FNR activity toward NrdI reduction. Additionally, the crystal structure shows that only one out of two binding sites of the B. cereus TrxR homodimer is occupied with NADPH, indicating a possible asymmetric co-substrate binding in TrxR.

Sphingomyelinase-Mediated Multitimescale Clustering of Ganglioside GM1 in Heterogeneous Lipid Membranes.

Several signaling processes in the plasma membrane are intensified by ceramides that are formed by sphingomyelinase-mediated hydrolysis of sphingomyelin. These ceramides trigger clustering of signaling-related biomolecules, but how they concentrate such biomolecules remains unclear. Here, the spatiotemporal localization of ganglioside GM1, a glycolipid receptor involved in signaling, during sphingomyelinase-mediated hydrolysis is described. Real-time visualization of the dynamic remodeling of the heterogeneous lipid membrane that occurs due to sphingomyelinase action is used to examine GM1 clustering, and unexpectedly, it is found that it is more complex than previously thought. Specifically, lipid membranes generate two distinct types of condensed GM1: 1) rapidly formed but short-lived GM1 clusters that are formed in ceramide-rich domains nucleated from the liquid-disordered phase; and 2) late-onset yet long-lasting, high-density GM1 clusters that are formed in the liquid-ordered phase. These findings suggest that multiple pathways exist in a plasma membrane to synergistically facilitate the rapid amplification and persistence of signals.

Improved production of 2'-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus.

BACKGROUND: 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides in human milk. It constitutes an authorized functional additive to improve infant nutrition and health in manufactured infant formulations. As a result, a cost-effective method for mass production of 2'-FL is highly desirable. RESULTS: A microbial cell factory for 2'-FL production was constructed in Saccharomyces cerevisiae by expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus (FutBc) and enhancing the de novo GDP-L-fucose biosynthesis. When enabled lactose uptake, this system produced 2.54 g/L of 2'-FL with a batch flask cultivation using galactose as inducer and carbon source, representing a 1.8-fold increase compared with the commonly used α-1, 2-fucosyltransferase from Helicobacter pylori (FutC). The production of 2'-FL was further increased to 3.45 g/L by fortifying GDP-mannose synthesis. Further deleting gal80 enabled the engineered strain to produce 26.63 g/L of 2'-FL with a yield of 0.85 mol/mol from lactose with sucrose as a carbon source in a fed-batch fermentation. CONCLUSION: FutBc combined with the other reported engineering strategies holds great potential for developing commercial scale processes for economic 2'-FL production using a food-grade microbial cell factory.

Regulation of ß-amylase synthesis: a brief overview.

BACKGROUND: The major activity of ß-amylase (BMY) is the production of maltose by the hydrolytic degradation of starch. BMY is found to be produced by some plants and few microorganisms only. The industrial importance of the enzyme warrants its application in a larger scale with the help of genetic engineering, for which the regulatory mechanism is to be clearly understood. RESULTS AND CONCLUSION: In plants, the activities of BMY are regulated by various environmental stimuli including stress of drought, cold and heat. In vascular plant, Arabidopsis sp. the enzyme is coded by nine BAM genes, whereas in most bacteria, BMY enzymes are coded by the spoII gene family. The activities of these genes are in turn controlled by various compounds. Production and inhibition of the microbial BMY is regulated by the activation and inactivation of various BAM genes. Various types of transcriptional regulators associated with the plant- BMYs regulate the production of BMY enzyme. The enhancement in the expression of such genes reflects evolutionary significance. Bacterial genes, on the other hand, as exemplified by Bacillus sp and Clostridium sp, clearly depict the importance of a single regulatory gene, the absence or mutation of which totally abolishes the BMY activity.

Statistical optimization of thermostable alkaline protease from Bacillus cereus KM 05 using response surface methodology.

OBJECTIVES: Proteases have gained great attention due to their enormous applications in food, tannery, detergent, photography and many other industries. Proteases rank third position in the production of enzymes. This paper targets to isolate a bacterium with high alkaline protease activity and optimization of its production conditions using Response Surface Methodology (RSM). RESULTS: A bacterium isolated from soil contaminated with detergent exhibited clearance zone on skim milk agar medium with a protease activity of 22 U/ml. The bacterial strain was identified as Bacillus cereus KM05 and optimization of its production conditions were performed using statistical methods. Further optimization with Box Behnken design resulted in an increase in protease activity by 1.5-fold (28.6 U/ml). The protease enzyme was thermotolerant up to 70 °C with stability towards alkaline pH (pH 9). The enzyme was not affected by most of the metal ions and solvents. Moreover, the protease was also compatible with six commercial detergents tested. Densitometric analysis of the destained fabric materials following the detergent-enzyme treatment, revealed a stain removal efficiency of 97%. CONCLUSION: The alkaline protease enzyme obtained was stable at different conditions with stain removal efficacy. Hence, the present alkaline protease could be used for detergent formulations.

Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs.

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Characterization of recombinant E. coli expressing a novel fucosidase from Bacillus cereus 2-8 belonging to GH95 family.

Fucoidan oligosaccharides possesses diverse physicochemical and biological activities. Specific glycoside hydrolases are valuable tools for degrading polysaccharides to produce oligosaccharides. In this study, BcFucA, a novel fucosidase belonging to GH95 family from Bacillus cereus 2-8, was cloned into pET21a vector, expressed in E. coli BL21 (DE3) and characterized. The protein consists of 1136 amino acid residues encoded by 3411 bases and has a molecular weight of 125.35 kDa. The optimal temperature and pH of this enzyme are 50 °C and pH 4.0. In addition, this study showed that the unknown function domain (encoding Lys261-Thr681) defined as a linker is quite important for its activity. The obtained novel enzyme BcFucA will contribute to the effective degradation of fucoidan and future industrial applications.

A nucleotide-sensing endonuclease from the Gabija bacterial defense system.

The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.

Coexistence of endonuclease and exonuclease activities in a novel RecJ from Bacillus cereus.

BACKGROUND: All RecJ proteins are known to date only perform exonuclease activity. The present study reports that a novel RecJ protein obtained from Bacillus cereus isolated from marine sediments has both endonuclease and exonuclease activities. METHODS: Analysis of the BcRecJ expression induction in E. coli BL21 revealed that the BcRecJ protein cleaved plasmids and genomic DNA in the host cell, and led to cell death and decreased the DNA content. Further, the BcRecJ protein had the ability to degrade supercoiled plasmid DNA into circular or linear forms in vitro. Meanwhile, the BcRecJ protein loaded with an S-modified guide facilitated plasmid linearization and reduced smear formation. RESULTS: The results suggested that this novel BcRecJ protein was different from any reported RecJs and had a longer C-terminus. Testing the BcRecJ mutants indicated that the endonuclease activity was affected by two residues of BcRecJ (D561, E637) after testing the BcRecJ mutants. CONCLUSION: The discovery of the type of protein is a new breakthrough for the RecJ proteins, which has both endonuclease and exonuclease activities.

Identification and antioxidant activity of bovine bone collagen-derived novel peptides prepared by recombinant collagenase from Bacillus cereus.

Millions of tons of collagen-rich bovine bone are produced as byproducts of the consumption of beef. Hydrolyzing bovine bone collagen (BBC) is an effective measure for both increasing its added value and protecting the environment. In this study, a kind of recombinant bacterial collagenase mining from Bacillus cereus was successfully performed and applied to hydrolyze BBC to collagen-soluble peptides (CPP). Response surface methodology (RSM) was applied to optimize the processing conditions of antioxidant CPP, attaining a distinguished ABTS free radical scavenging activity of 99.21 ± 0.35% while keeping DPPH free radical scavenging activity and reducing power at high levels under the optimal condition. Furthermore, we identified five new antioxidant peptides by LC-MS/MS with typical collagen repeated Gly-Xaa-Yaa sequence units within the CPP. These results suggest that our recombinant collagenase is a powerful tool for degrading collagen and the CPP are promising candidates for antioxidant and related functional food applications.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Phosphonate as a Stable Zinc-Binding Group for "Pathoblocker" Inhibitors of Clostridial Collagenase H (ColH).

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.

Insights into substrate specificity of proteases for screening efficient dehairing enzymes.

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Biochemical characterization of two chitinases from Bacillus cereus sensu lato B25 with antifungal activity against Fusarium verticillioides P03.

Bacterial chitinases are a subject of intense scientific research due to their biotechnological applications, particularly their use as biological pesticides against phytopathogenic fungi as a green alternative to avoid the use of synthetic pesticides. Bacillus cereus sensu lato B25 is a rhizospheric bacterium that is a proven antagonist of Fusarium verticillioides, a major fungal pathogen of maize. This bacterium produces two chitinases that degrade the fungal cell wall and inhibit its growth. In this work, we used a heterologous expression system to purify both enzymes to investigate their biochemical traits in terms of Km, Vmax, optimal pH and temperature. ChiA and ChiB work as exochitinases, but ChiB exhibited a dual substrate activity and it is also an endochitinase. In this work, the direct addition of these chitinases inhibited fungal conidial germination and therefore they may play a major role in the antagonism against F. verticillioides.

Increasing chitosanase production in Bacillus cereus by a novel mutagenesis and screen method.

Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides. One of the prerequisites of enzyme fermentation production is to select and breed enzyme-producing cells with good performance. So in the process of fermentation production, the low yield of chitosanase cannot meet the current requirement. In this paper, a strain producing chitosanase was screened and identified, and a novel mutagenesis system (Atmospheric and Room Temperature Plasma (ARTP)) was selected to increase the yield of chitosanase. Then, the fermentation medium was optimized to further improve the enzyme activity of the strain. A strain of Bacillus cereus capable of producing chitosanase was screened and identified from soil samples. A mutant strain of B.cereus was obtained by Atmospheric and Room Temperature Plasma mutagenesis and bioscreening method, and chitosanase activity was 2.49 folds that of the original bacterium. After an optimized fermentation medium, the enzyme activity of the mutant strain was 1.47 folds that of the original bacterium. Combined with all the above optimization experiments, the enzyme activity of mutant strain increased by 3.66 times. The results showed that the Atmospheric and Room Temperature Plasma mutagenesis and bioscreening method could significantly increase the yield of chitosanase in B.cereus, and had little effect on the properties of the enzyme. These findings have potential applications in the mutagenesis of other enzyme-producing microorganisms.