Xylanase is a hemicellulose-degrading enzyme that breaks down the xylan glucose polymer into xylooligosaccharides and D-xylose. Xylanase can be produced by microorganisms through fermentation processes involving bacteria or fungi. Bacillus halodurans CM1 has been identified as a potential xylanase-producing bacteria for applications in pulp and paper and other industries. This study aimed to optimise the production of xylanase enzyme by B. halodurans CM1 using an economical mineral salt medium (MSM) and xylan as an inducer, through Box–Behnken design response surface methodology (BBD-RSM). The study focused on identifying the optimal fermentation conditions (xylan concentration, temperature, pH, and agitation) and to evaluate the effects of metal ions, solvents, and surfactants on enzyme activity and stability. The optimum conditions for achieving the highest specific xylanase enzyme activity of 33.8 U/mg were xylan inducer concentration of 2%, temperature of 37 °C, pH 9, and agitation at 200 rpm. The optimum activity of B. halodurans CM1 xylanase was at temperature 70 °C, pH 9. Enhanced by metal ion activators Mg2+, Cu2+, K+, Na+, Co2+, and Ca2+. Inhibited by metal ions Zn2+, Ni2+, Fe³+, as well as organic solvents including ethanol, methanol, DMSO, formaldehyde, and chloroform. Non-ionic surfactants such as Triton X, Tween-80, EDTA, and Chem 9201 surfactant did not significantly affect enzyme activity. Ionic surfactants SDS, Tween-20, and Teepol exhibited inhibitory effects. B. halodurans CM1 xylanase properties with such unique characteristics of thermo alkali tolerant has a diverse range of application in paper and pulp, deinking, biomass utilisation and food feed industries.

Exenatide is used for the treatment of type 2 diabetes. Exenatide is normally produced using solid or liquid phase chemical synthesis which requires protecting side chain groups. The C-terminal is then amidated while the protecting groups are still in place. The novel production of exenatide, using a microorganism with the correct coding sequence, does not require protecting groups. The exenatide is then amidated using a PAM enzyme system. An UPLC-QTOF-MS method was developed to separate and analyze the non-amidated exenatide molecule from the final active amidated exenatide. The non-amidated exenatide was produced using an expression construct comprising a carrier protein open reading frame (ORF) Yarrowia lipolytica lipase or truncated Bacillus halodurans flagellin cloned in frame with the coding sequence for exenatide. The non-amidated and amidated exenatide differ by 1 Dalton in mass and typically co-elute during analysis. The method developed was able to separate the two compounds and could be used to measure the amidated exenatide produced during the bioconversion. The aim of the study was to investigate the feasibility of producing exenatide and monitoring the amidation during the bioconversion for possible scale-up and commercialization. The results showed complete amidation of the glycine-extended heterologous exenatide. The reproducibility of the analytical method was evaluated and found that the retention times and peaks areas of the detected exenatide were stable, making this analytical method suitable for reaction monitoring.

The aim of the study was to investigate potent alkaline protease producing indigenous bacterial strains from alkaline soils of Vindhya region of Madhya Pradesh, India. Accordingly, isolation and screening of alkaline protease producing alkaliphilic bacteria was performed from different habitats of Vindhya region of Madhya Pradesh, India. Selection and identification of promising alkaline protease producing isolates, enzyme activity assay and partial characterization of crude alkaline protease filtrate were done. Many alkaline proteases producing bacterial isolates were isolated from poultry farm soils of Vindhya region, Madhya Pradesh, India. Out of several proteolytic isolates, most potent new bacterial strain was identified as Bacillus halodurans RSCVS-PF21 (Genbank Accession No. MT279908) based on phenotypic, biochemical and molecular characterizations. Crude alkaline protease was stable and effective at high temperature. It was also highly stable in different organic solvents, so it can be used in many industrial processes.

Alkali-stable amylases offer potential for integrating textile desizing and scouring processes. To meet industrial requirements, molecular engineering strategies were employed to enhance oxidative stability and catalytic efficiency of alkaline amylase. In this study, alkali-stable amylase Amy I from Bacillus halodurans was engineered by modifying multiple highly flexible regions. The sequential truncation of HFR I's N-terminal 40 residues yielded mutant T-40 with 77% higher residual activity than Amy I after incubation at 70 °C for 1 h. Saturation mutagenesis of HFR II/III generated mutant M4, showing 38.9% activity enhancement. CBM-25 substitution in HFR IV further increased activity by 7%. Finally, the integrated mutant Amy I-ML demonstrated exceptional performance with 14.5-fold higher specific activity and 29.6-fold extended half-life (29.4 min at 100 °C) compared to wild-type Amy I. These engineered features-combining thermostability reinforcement with catalytic optimization-establish Amy I-ML as a promising biocatalyst for industrial textile processing. The multiregion engineering approach provided a strategic framework for developing robust industrial enzymes through rational flexibility modulation.

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes. Therefore, we designed hybrid cellulase complexes from 49 biotinylated catalytic domain (CD) and 30 biotinylated cellulose-binding domain (CBD) libraries on streptavidin-conjugated nanoparticles to enhance cellulose hydrolysis by mimicking the cellulosome structure. The hybrid cellulase complex, incorporating both native CD and CBD, significantly improved reducing sugar production from cellulose compared to free native modular enzymes. The optimal CBD for each hybrid cellulase complex differed from that of the native enzyme. The most effective hybrid cellulase complex was observed with the combination of CD6-4 from Thermobifida fusca YX and CBD46 from the Bacillus halodurans C-125. The hybrid cellulase complex/CD6-4-CBD46 and -CD6-4-CBD2-5 combinations showed increased reducing sugar production. Similar results were also observed in microcrystalline cellulose degradation. Furthermore, clustering on nanoparticles enhanced enzyme thermostability. Our results demonstrate that hybrid cellulase complex structures improve enzyme function through synergistic effects and extend the lifespan of the enzyme.

YqeK is a bacterial HD-domain metalloprotein that hydrolyzes the putative second messenger diadenosine tetraphosphate (Ap4A). Elevated Ap4A levels are primarily observed upon exposure of bacteria to factors such as heat or oxidative stress and cause pleiotropic effects, including antibiotic sensitivity and disrupted biofilm formation. Ap4A thus plays a central role in bacterial physiology and metabolism, and its hydrolysis by YqeK is intimately linked to the ability of these microbes to cope with stress. Although YqeK is reported to hydrolyze Ap4A under aerobic conditions, all four existing crystal structures reveal an active site that consists of a diiron center, portraying a cryptic chemical nature for the active metallocofactor. This study examines two YqeK proteins from two ecologically diverse parent organisms: the obligate anaerobe Clostridium acetobutylicum and the facultative aerobe Bacillus halodurans. Both enzymes utilize Fe-based cofactors for catalysis, while under ambient or oxidative conditions, Bh YqeK hydrolyzes Ap4A more efficiently compared to Ca YqeK. This redox-dependent activity difference stems from the following two molecular mechanisms: the incorporation of mixed-metal, Fe-based bimetallic cofactors, in which the second metal is redox inert (i.e., Fe–Zn) and the upshift of the Fe–Fe cofactor reduction potentials. In addition, three strictly conserved, positively charged residues vicinal to the active site are critical for tuning Ap4A hydrolysis. In conclusion, YqeK is an Fe-dependent phosphohydrolase that appears to have evolved to permit Ap4A hydrolysis under different environmental niches (aerobic vs. anaerobic) by expanding its cofactor configuration and O2 tolerance.

An improved approach was developed for the genetic manipulation of the Gram-positive extremophile Halalkalibacterium halodurans (formerly called Bacillus halodurans). We describe an allelic replacement method originally developed for Staphylococcus aureus that allows the deletion, mutation, or insertion of genes without leaving markers or other genetic scars. In addition, a protocol for rapid in vitro plasmid methylation and transformation is presented. The combined methods allow the routine genetic manipulation of H. halodurans from initial transformation to the desired strain in 8 days. These methods improve H. halodurans as a model organism for the study of extremophiles.

Ramie (Boehmeria nivea L. Gaud) is a versatile plant with potential as a cotton alternative in textiles. Its cellulose content, second only to cotton, requires degumming to remove fiber gum before industrial use. This study scaled up enzymatic degumming to 1 kg of ramie fiber, employing a dual enzyme approach: local Bacillus halodurans CM1 xylanase and commercial pectinase. This was optimized under varying conditions, including temperatures (30, 50, and 70 °C), durations (1, 2, and 3 h), and solid-liquid ratios (SLRs; 1:15, 1:22.5, 1:30), utilizing response surface methodology. Optimal outcomes were achieved at 50 °C and a 1:15 ratio, with a 3-h treatment duration, resulting in the highest reducing sugar yield (5.25 mg/mL) and a 102.03% enhancement in ramie fiber brightness compared to the enzyme-free control. This enzymatic process effectively separated gum from fibers while improving the quality, fineness, and tensile strength of cellulose fibers.

Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture

Coproduction of alkaline protease and xylanase from genetically modified Indonesian local Bacillus halodurans CM1 using corncob as an inducing substrate

Ornate, large, extremophilic (OLE) RNAs comprise a class of large noncoding RNAs in bacteria whose members form a membrane-associated ribonucleoprotein (RNP) complex. This complex facilitates cellular adaptation to diverse stresses such as exposure to cold, short-chain alcohols, and elevated Mg2+ concentrations. Here, we report additional phenotypes exhibited by Halalkalibacterium halodurans (formerly called Bacillus halodurans) strains lacking functional OLE RNP complexes. Genetic disruption of the complex causes restricted growth compared to wild-type cells when cultured in minimal media (MM) wherein glucose is replaced with alternative carbon/energy sources. Genetic suppressor selections conducted in glutamate MM yielded isolates that carry mutations in or near genes relevant to Mn2+ homeostasis (ykoY and mntB), phosphate homeostasis (phoR), and putative multidrug resistance (bmrCD). These functional links between OLE RNA, carbon/energy management, and other fundamental processes including protein secretion are consistent with the hypothesis that the OLE RNP complex is a major contributor to cellular adaptation to unfavorable growth conditions.

The demand for amylases with improved storage and operational properties for the economical production of textiles has been on the rise in recent years. An alkaline amylase (Amy LBW 5117) from alkaliphilic Bacillus halodurans LBW 5117 was characterized and used to desize woven cotton. The enzyme had a shelf life of 6 weeks when stored at 4-30°C. It exhibited pH and temperature optima of 10 and 60 o C, respectively. Its activity was stimulated or insignificantly affected by up to 10 mM K + , Ca 2+ , Mg 2+ , Fe 3+ , Na + , and Zn 2+ , as well as Cu 2+ (≤1.0 mM), but was partially inhibited by Mn 2+ (0.5 mM). The enzyme lost all of its activity after 3 h of incubation at 60°C, but this improved to 96% in the presence of 1.0 mM Ca 2+ and 0.05 mM Tween 20. The enzyme was cellulase-free and hydrolyzed starch in an endo-fashion. Furthermore, it degraded and eliminated 8.2% starch size from woven cotton and yielded a fabric that exhibited a TEGEWA rating of 7-8 (residual starch content = 0.0725%) under its optimum operating conditions. This shows that Amy LBW 5117 has good storage and operational properties that potentially make it an effective desizing agent. Amy LBW 5117 is an alkaline endo-α-1-4-amylase that can desize woven cotton. • At high pH, implying less contamination from neutrophils. • At low temperatures, implying low-energy costs. • In the presence of metal ion impurities, implying savings on the purchase of chelators.

Currently, we report the preparation of transition metal complexes Co(II), Ni(II), and Cu(II) of hydrazone Schiff base ligands, which are obtained by the condensation reaction of substituted salicylaldehyde and hydrazines. The synthesized hydrazone ligands and their metal complexes were characterized by spectroscopic methods such as Fourier transform infrared (FT-IR), UV-vis, nuclear magnetic resonance (1H NMR and C13 NMR), and mass spectrometry analyses. All of the quantum chemistry calculations were performed using DFT executed in the Gaussian 09 software package. The geometry was optimized by using the density functional theory (DFT) approximation at the B3LYP level with a basis set of 6-31G (d, p). There was excellent agreement between the FT-IR values obtained experimentally and those obtained theoretically for the test compounds. It is worth noting that none of the optimized geometries for any of the Schiff base and metal complexes had any eigenvalues that were negative, indicating that these geometries represent the true minimum feasible energy surfaces. We also analyzed the electrostatic potential of the molecule and NBO calculation at the same level of theory. Gauss View 6 was utilized for the file organization of the input data. Gauss View 6.0, Avogadro, and Chemcraft were used to determine the data. Additionally, synthesized compounds were screened for antimicrobial activity against Gram-negative bacteria (Salmonella typhi, Escherichia coli) and Gram-positive bacteria (Bacillus halodurans, Micrococcus luteus) and two fungal strains (Aspergillus flavus, Aspergillus niger). These research findings have established the potential of ligands and their metal complexes as antimicrobial agents. Additionally, the compounds demonstrated promising nonlinear optical (NLO) properties, with potential applications across a wide range of contemporary technologies.

X-ray diffraction study on microbial calcite precipitation in concrete with Bacillus subtilis and Bacillus halodurans

Green synthesis of iron oxide nanoparticles using Bombax malabaricum for antioxidant, antimicrobial and photocatalytic applications

The crude oil reserves in Oman mainly consist of heavy oil. Microbial enhanced heavy oil recovery (MEOR) has been proved to be an efficient technique in the tertiary heavy oil recovery. Five Bacillus species potential for enhanced heavy oil recovery (EHOR) were isolated and the biodegradation ability of these isolates was studied. As heavy crude oil comprises of aromatic hydrocarbons rather than aliphatic ones, the aromatic catabolism gene, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) were the genes of interest in this study along with the reference gene, 16S rDNA. The copy number variation of these genes was determined using droplet digital PCR (ddPCR). The primers and probes for ddPCR assay were designed targeting these genes. It was observed that the heavy crude oil biodegradation potential of the isolates correlated with the copy number of C23O gene in the microbial genomes. The isolate, Paenibacillus ehimensis BS1 had the highest C23O gene copy number (1.057) followed by Bacillus firmus BG4 (0.895) and Bacillus halodurans BG5 (0.031) as demonstrated by their biodegradation potential. This is one of the few studies deploying ddPCR in the field of heavy crude oil biodegradation by spore forming bacteria.

The study was carried out with the influence of four Bacillus species, namely Bacillus subtilis (BS), Bacillus licheniformis (BL), Bacillus halodurans (BH), and Bacillus cereus (BC), in order to improve the impermeable nature of concrete. The selected bacterial agents were cultured according to the nutrient broth medium method and prepared with two variable cell concentrations of 108 and 109. In this study, bacteria were used in two different modes, i.e., as an additive to the concrete and as a curing agent. However, all the concrete specimens were cracked with a 65% stress level concentration and then cured in calcium lactate (only for bacteria-induced concrete specimens) and bacterial solution (only for normal concrete specimens) for crack healing. The durability behavior of these concrete specimens was monitored before crack, after crack, and after healing. In this regard, the following durability tests were performed: Rapid Chloride Permeability Test (RCPT), Water absorption test, Open porosity test, and Acid attack test. From the experimental observations, a decline in the passage of coulombs by 1352 C, water absorption by 3.47%, open porosity by 4.61%, and increased resistance against acid attack was found in normal concrete specimens cured under bacterial solution (especially in Bacillus halodurans with cell concentrations of 109) compared to other ones. Based on the analysis of the results, bacterial cultures have enriched the durability of the concrete by filling the voids and cracks with calcite crystals. However, Bacillus halodurans has shown better durability performance in both types of concrete, i.e., bacterial concrete cured in calcium lactate and normal concrete cured in bacterial solutions.

Endo-1,4-β-xylanase catalyzes the random hydrolysis of β-1,4-D-xylosidic bonds in xylan, resulting in the formation of oligomers of xylose. This study aims to demonstrate the promise of endoxylanases from alkaliphilic Bacillus halodurans for the production of xylooligosaccharides (XOS) from oil palm empty fruit bunch (EFB) at high pH. Two enzyme preparations were employed: recombinant endoxylanase Xyn45 (GH10 xylanase) and nonrecombinant endoxylanases, a mixture of two extracellular endo-1,4-β-xylanases Xyn45 and Xyn23 (GH11 xylanase) produced by B. halodurans. EFB was first treated with an alkaline solution. Then, the dissolved xylan-containing fraction was retained, and a prepared enzyme was added to react at pH 8 to convert xylan into XOS. Compared with the use of only Xyn45, the combined use of Xyn45 and Xyn23 resulted in a higher yield of XOS, suggesting the synergistic effect of the two endoxylanases. The yield of XOS obtained from EFB was as high as 46.77% ± 1.64% (w/w), with the xylobiose-to-xylotriose ratio being 6:5. However, when the enzyme activity dose was low, the product contained more xylotriose than xylobiose. Four probiotic lactobacilli and bifidobacteria grew well on a medium containing XOS from EFB. The presence of XOS increased cell mass and reduced pH, suggesting that XOS promoted the growth of probiotics.

Bacillus halodurans C-125 is an alkaliphilic microorganism that grows best at pH 10 to 10.5. B. halodurans C-125 harbors the erm (erythromycin resistance methylase) gene as well as the mphB (macrolide phosphotransferase) and putative mef (macrolide efflux) genes, which confer resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. The Erm protein expressed in B. halodurans C-125 could be classified as ErmK because it shares 66.2% and 61.2% amino acid sequence identity with the closest ErmD and Erm(34), respectively. ErmK can be regarded as a dimethylase, as evidenced by reverse transcriptase analysis and the antibiotic resistance profile exhibited by E. coli expressing ermK. Although ErmK showed one-third or less in vitro methylating activity compared to ErmC', E. coli cells expressing ErmK exhibited comparable resistance to erythromycin and tylosin, and a similar dimethylation proportion of 23S rRNA due to the higher expression rate in a T7 promoter-mediated expression system. The less efficient methylation activity of ErmK might reflect an adaption to mitigate the fitness cost caused by dimethylation through the Erm protein presumably because B. halodurans C-125 has less probability to encounter the antibiotics in its favorable growth conditions and grows retardedly in neutral environments. IMPORTANCE Erm proteins confer MLSB antibiotic resistance (minimal inhibitory concentration [MIC] value up to 4,096 μg/mL) on microorganisms ranging from antibiotic producers to pathogens, imposing one of the most pressing threats to clinics. Therefore, Erm proteins have long been speculated to be plausible targets for developing inhibitor(s). In our laboratory, it has been noticed that there are variations in enzymatic activity among the Erm proteins, Erm in antibiotic producers being better than that in pathogens. In this study, it has been observed that Erm protein in B. halodurans C-125 extremophile is a novel member of Erm protein and acts more laggardly, compared to that in pathogen. While this sluggishness of Erm protein in extremophile might be evolved to reduce the fitness cost incurred by Erm activity adapting to its environments, this feature could be exploited to develop the more potent and/or efficacious drug to combat formidably problematic antibiotic-resistant pathogens.

Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA. Disruption of the genes for either OLE RNA, OapA, or OapB result in the inability of cells to overcome cold, alcohol, or Mg2+ stresses. In the current study, we used invivo crosslinking followed by OLE RNA isolation to identify the protein YbxF as a potential additional partner in the OLE RNP complex. Notably, a mutation in the gene for this same protein was also reported to be present in a strain wherein the complex is nonfunctional. The B.halodurans YbxF (herein renamed OapC) is homologous to a bacterial protein earlier demonstrated to bind kink turn (k-turn) RNA structural motifs. Invitro RNA-protein binding assays reveal that OLE RNA forms a previously unrecognized k-turn that serves as the natural binding site for YbxF/OapC. Moreover, B.halodurans cells carrying OLE RNAs with disruptive mutations in the k-turn exhibit phenotypes identical to cells lacking functional OLE RNP complexes. These findings reveal that the YbxF/OapC protein of B.halodurans is important for the formation of a functional OLE RNP complex.