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We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.

Pullulanase (EC 3.2.1.41) is an important debranching enzyme that plays a critical role in maximizing the abundant energy present in branched polysaccharides. Its unique ability to efficiently degrade branched polysaccharides makes it crucial in industries like biofuels, food, and pharmaceuticals. Therefore, discovering microbes that produce pullulanase and thrive in harsh industrial conditions holds significant potential for optimizing large-scale bioprocessing. This unique property has made pullulanase an important enzyme in the industry. Thus, the search for microbes that have the pullulanase production properties and capacity to withstand harsh industrial conditions will be of high industrial relevance. Therefore, this study aimed to amplify, sequence, and molecularly characterize the pullulanase gene encoding extracellular pullulanase in Bacillus licheniformis strain FAO.CP7 (Accession No: MN150530.1.) which was obtained from cocoa pods using several bioinformatics tools. The amplified PulA gene had a nucleotide sequence of 2247 base pairs encoding a full-length open reading frame (ORF) pullulanase protein of 748 amino-acids residues with molecular weight 82.39 kDa and theoretical isoelectric point of 6.47, respectively. The deduced pullulanase protein had an aliphatic index of 77.66. Using BLASTp, the deduced amino acid sequence of the pullulanase gene showed 85% homologies with those from B. licheniformis strains. Multiple sequence alignment of PulA protein sequence showed that it contains YNWGYNP motif which is also found in all type I pullulanase protein sequences analysed. The restriction mapping of the gene showed that it can be digested with several restriction enzymes. Further analysis revealed that the deduced protein had a hydrophobicity score of − 0.37 without a transmembrane helix. Overall, this study revealed the PulA gene of B. licheniformis strain FAO.CP7 obtained from cocoa pods and its deduced protein show significant potential for enhancing starch bioprocessing. With further optimization, it could offer substantial benefits to starch-based biotechnological industries.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Poly-γ-glutamic acid (γ-PGA) has diverse applications from cosmetic to drug delivery. The production of γ-PGA primarily relies on microbial fermentation using Bacillus spp. supplemented with l -glutamate supplementation. However, the high cost of l -glutamate supplementation limits industrial production. This study aimed to achieve direct γ-PGA production from glucose using a Corynebacterium glutamicum-Bacillus licheniformis coculture system. To create such a coculture system, we utilized B. licheniformis ATCC 9945a, a natural l -glutamate-dependent γ-PGA producing strain, and C. glutamicum F343, which exhibited an excellent capacity to produce l -glutamate from glucose. B. licheniformis ATCC 9945a grew well and produced small amounts of γ-PGA in the medium of C. glutamicum F343. Subsequently, B. licheniformis ATCC 9945a was cultured using the supernatant collected from the C. glutamicum F343 fermentation broth to investigate its effect on the fermentation profile. It was found that B. licheniformis ATCC 9945a produced more γ-PGA in the supernatant compared to when exogenously supplemented with l -glutamate. Moreover , nine intracellular metabolites were discovered to be strongly connected to γ-PGA synthesis by UPLC-MS. Finally, the coculture of C. glutamicum F343 and B. licheniformis ATCC 9945a to produce γ-PGA was conducted. We successfully achieved direct γ-PGA production from glucose under optimal conditions, including an inoculation time of 4 h for B. licheniformis after C. glutamicum inoculation, a 75% inoculum ratio of C. glutamicum , and a total inoculum size of 10% culture volume. The coculture system produced 12.49 g/L of γ-PGA in a shake flask and 22.7 g/L in a 5-L fermentor. Key points • C. glutamicum F343 could produce L-glutamate from glucose as a precursor for PGA synthesis by B. licheniformis ATCC 9945a . • The C. glutamicum-B. licheniformis coculture system could produce γ-PGA up to 22.7 g/L . • Nine intracellular metabolites demonstrated a remarkable influence on γ-PGA synesis by UPLC-MS and metabolite profiling .

Background Proteases are essential in various industries due to their unique substrate specificities and robustness in different operational conditions. Bacillus strains consist of a genotype favorable for rapid growth whilst secreting enzymes extracellularly, thereby simplifying recombinant protease production. Despite the widespread use of batch and fed-batch fermentations for their ease and robustness, these cultivation types are often marred by significant energy requirements and prolonged downtimes. The switch towards continuous cultivation methods promises reduced carbon footprints and improved equipment efficiency. Yet, research focusing on Bacillus strains is limited, therefore we aimed to establish a continuous cultivation as a competitive alternative to fed-batch. Results Therefore, this study aimed to explore the potential of chemostat cultivations for producing a protease from Bacillus licheniformis utilizing a derepressed induction system, and comparing specific productivities and space-time yields to fed-batch cultivations. The continuous cultivations were described in a hybrid model, considering the effect of productivity as function of the applied dilution rate as well as the generation time. The workflow of this study demonstrates that screenings in a fed-batch mode and chemostat cultivations conducted at the same growth rate, result in different specific productivities for derepressible systems. Conclusion The results of this study highlight that the feeding rate’s impact on specific productivity varies significantly between fed-batch and chemostat cultivations. These differences suggest that fed-batch screenings may not be adequate for developing a continuous process using a derepressed promoter system in B. licheniformis . Although the space-time yield of fed-batch cultivations has not been surpassed by stable continuous operations—achieving only a third of the highest space-time yield observed in fed-batch—valuable mechanistic insights have been gained. This knowledge could facilitate the transition towards a more sustainable mode of cultivation for industrial protease production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12 g/l and extracellular protein concentration containing xylanase 400 mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50 °C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60 °C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2 mg/ml, Vmax 2.7 ± 0.2 U/ml and and Kcat 1.8 ± 0.2 s−1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48 h and 24 h at 100 µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn’t observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.

Biosurfactants are environmentally friendly alternatives for chemical surfactants and have a broad spectrum of applications in different industries such as cosmetics, oleochemistry, pharmaceuticals, and detergents. It has been established that Bacillus licheniformis produces several lipopeptide-type biosurfactants, including lichenysin and iturin. However, in order to enhance the biosurfactant production by Bacillus licheniformis, it is necessary to either extend the already performed media optimization to circumvent the current limitations or defeat the product inhibition. Cyclic oligosaccharides made of glucose monomers called cyclodextrins (CD) have been shown to improve the biomass synthesis of other microorganisms, which may also increase the output of biosurfactants. The efficient fermentative production of biosurfactants is often limited by the inhibitory/toxic effect of the product on the producer cells itself. Therefore, in this work, we demonstrated that CDs may entrap biosurfactants from the broth, decreasing product inhibition. Thus, we also tested the media supplementation with three different types of cyclodextrins including alpha-, beta-, and gamma-CD and a derivative (dimethyl-beta-cyclodextrin, DIMEB); notably, DIMEB at 2.0 g/L enhanced biosurfactant production by up to 41.43% and specific product formation (g product/g cells) by 79,6% compared to the control, while mitigating the growth inhibition observed at lower concentrations. This study demonstrates, for the first time, the distinct advantage of DIMEB over native CDs in reducing product toxicity and boosting biosurfactant yields, highlighting its potential as a simple additive strategy for improving sustainable bioprocesses.

Background Cellulose is the major part of lignocellulosic biomass. It can be hydrolyzed into glucose units via specific enzymes called cellulases that have been applied in many commercial fields. There are several studies illustrate the influence of enzymes on apple juice clarification. However, to the best of our knowledge, the effect of microbial cellulase on volatile compounds of apple juice is not well known. The present study aimed to assess the effect of cellulase from a new bacterial isolate on the physicochemical properties of apple juice as well as volatile compounds. The hydrolysis of some polysaccharides (cellulose, hemicellulose, pectin) and polyphenols during apple juice production is necessary to reduce cloud sedimentation or color deterioration and increase the yield of juice. So, enzymes from new microbial isolates serve as processing aids to obtain clear juice with a high yield. Results Cellulase-producing bacterium was isolated, characterized and molecularly identified as Bacillus licheniformis strain-MA1 with an accession number of ON840115. Optimization of medium parameters was implemented using Plackett–Burman design (PBd) followed by Box-Behnken design (BBd) of response surface methodology (RSM). The PBd revealed the three most important (significant) variables including carboxymethyl cellulose (CMC), corn cob, and peptone that had positive impact on cellulase production. Additionally, using the agricultural residue (corn cob) by the bacterial strain as a carbon source helps in reducing the costs of enzyme production, recycling the by-products, and preserving the environment. The optimized medium using PBd and BBd enhanced cellulase production from B. licheniformis strain-MA1 by 6.8-fold. A remarkable increase was observed in juice yield in enzyme treated-juice sample (88.2 ± 0.15%) in comparison with control juice (75.4 ± 0.09%). The total phenolic contents in cloudy and clarified apple juices were 0.957 ± 0.09 and 0.412 ± 0.03 mg/mL, respectively. Also, DPPH and FRAP assays showed a remarkable increase in antioxidant activity (Low IC 50 ) in the control sample compared to enzyme treatment. Twenty-seven volatile compounds were extracted using headspace solid-phase microextraction-gas and analysis was performed by GC–MS. The identified volatile constituents belonged to several chemical classes: 15 esters; 6 alcohols; 4 aldehydes and 2 acids. The predominant class in apple juice volatile fraction was esters with a sweet and fruity odor. Conclusion The crude cellulase obtained from the novel bacterial isolate B. licheniformis strain-MA1 was successfully applied as a clarifying agent in apple juice.

Deoxynivalenol (DON) is one of the most prevalent food- and feed-associated mycotoxins. It frequently contaminates agricultural commodities and poses serious threats to human and animal health and leads to tremendous economic losses globally. Much attention has been paid to using microorganisms to detoxify DON. In this study, a Bacillus licheniformis strain named YB9 with a strong ability to detoxify DON was isolated and characterized from a moldy soil sample. YB9 could degrade more than 82.67% of 1 mg/L DON within 48 h at 37 °C and showed strong survival and DON degradation rate at simulated gastric fluid. The effects of YB9 on mice with DON intragastrical administration were further investigated by biochemical and histopathological examination and the gut microbiota was analyzed by 16S rRNA Illumina sequencing technology. The results showed that DON increased the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (Cr), decreased those of immunoglobulin G (IgG) and IgM in serum, and resulted in severe pathological damage of the liver, kidney, and spleen. By contrast, YB9 supplementation obviously inhibited or attenuated the damages caused by DON in mice. In addition, YB9 addition repaired the DON-induced dysbiosis of intestinal flora, characterized by recovering the balance of Firmicutes and Bacteroidetes to the normal level and decreasing the abundance of the potentially harmful bacterium Turicibacter and the excessive Lactobacillus caused by DON. Taken together, DON-degrading strain YB9 might be used as potential probiotic additive for improving food and feed safety and modulating the intestinal microbial flora of humans and animals.

Ectoine is a high-value protective agent with extensive applications in the fields of fine chemicals and biopharmaceuticals, and it is naturally synthesized by Halomonas in extreme environment, however, the current production level cannot meet the growing market demand. In this study, we aimed to develop an efficient and environmentally friendly ectoine production process using Bacillus licheniformis as the host organism. Through introducing ectoine synthetase gene cluster ectABC from Halomonas elongate , as well as optimizing ectABC Hs expression by promoter and 5’-UTR optimization, ectoine titer was increased to 0.55 g/L. Furthermore, subsequent introduction of exogenous phosphoenolpyruvate carboxylase PPC EC and down-regulated expression of phosphoenolpyruvate carboxykinase PCK optimized the carbon flux through C4 anaplerotic pathway, and further benefited ectoine synthesis. Furthermore, the carbon flux towards aspartic acid accumulation was increased through optimization of glyoxylate and TCA cycles, accompanied with introducing lysC T311I Cg and asd Cg , and blocking by-products pathways, ectoine titer produced by B. licheniformis ECT12 was 2.00 g/L. Moreover, NADPH supply was enhanced by overexpression of exogenous NADH kinase Pos5 Sc , and ectoine transportation was improved by introducing compatible solute transporter ProP from Escherichia coli , and the resulting B. licheniformis ECT14 was able to produce 2.60 g/L ectoine. Last but not the least, the ectoine yield of 3.29 g/L was attained in a 5-L fermenter. Taken together, this study not only established B. licheniformis as a framework for sustainable production of ectoine, but also paved the way for achieving the industrial production of ectoine and aspartic acid derivatives in the future.