Exploring Bacillus species xylanases for industrial applications: screening via thermostability and reaction modelling.

CONTEXT: Xylanases derived from Bacillus species hold significant importance in various large-scale production sectors, with increasing demand driven by biofuel production. However, despite their potential, the extreme environmental conditions often encountered in production settings have led to their underutilisation. To address this issue and enhance their efficacy under adverse conditions, we conducted a theoretical investigation on a group of five Bacillus species xylanases belonging to the glycoside hydrolase GH11 family. Bacillus sp. NCL 87-6-10 (sp_NCL 87-6-10) emerged as a potent candidate among the selected biocatalysts; this Bacillus strain exhibited high thermal stability and achieved a transition state with minimal energy requirements, thereby accelerating the biocatalytic reaction process. Our approach aims to provide support for experimentalists in the industrial sector, encouraging them to employ structural-based reaction modelling scrutinisation to predict the ability of targeted xylanases. METHODS: Utilising crystal structure data available in the Carbohydrate-Active enzymes database, we aimed to analyse their structural capabilities in terms of thermal-stability and activity. Our investigation into identifying the most prominent Bacillus species xylanases unfolds with the help of the semi-empirical quantum mechanics MOPAC method integrated with the DRIVER program is used in calculations of reaction pathways to understand the activation energy. Additionally, we scrutinised the selected xylanases using various analyses, including constrained network analyses, intermolecular interactions of the enzyme-substrate complex and molecular orbital assessments calculated using the AM1 method with the MO-G model (MO-G AM1) to validate their reactivity.

Purification and Characterization of a Novel Fibrinolytic Enzyme from Marine Bacterium Bacillus sp. S-3685 Isolated from the South China Sea.

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 µM and 49.0 µmol min-1 mg-1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment.

Marine Bacillus haynesii chitinase: Purification, characterization and antifungal potential for sustainable chitin bioconversion.

The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0-9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL-1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL-1 and Vmax of 5.75 mmol min-1. Kcat and catalytic efficiency were measured at 1.91 s-1 and 191 mL mg-1 s-1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent.

Synergistic biocementation: harnessing Comamonas and Bacillus ureolytic bacteria for enhanced sand stabilization.

Biocementation, driven by ureolytic bacteria and their biochemical activities, has evolved as a powerful technology for soil stabilization, crack repair, and bioremediation. Ureolytic bacteria play a crucial role in calcium carbonate precipitation through their enzymatic activity, hydrolyzing urea to produce carbonate ions and elevate pH, thus creating favorable conditions for the precipitation of calcium carbonate. While extensive research has explored the ability of ureolytic bacteria isolated from natural environments or culture conditions, bacterial synergy is often unexplored or under-reported. In this study, we isolated bacterial strains from the local eutrophic river canal and evaluated their suitability for precipitating calcium carbonate polymorphs. We identified two distinct bacterial isolates with superior urea degradation ability (conductivity method) using partial 16 S rRNA gene sequencing. Molecular identification revealed that they belong to the Comamonas and Bacillus genera. Urea degradation analysis was performed under diverse pH (6,7 and 8) and temperature (15 °C,20 °C,25 °C and 30 °C) ranges, indicating that their ideal pH is 7 and temperature is 30 °C since 95% of the urea was degraded within 96 h. In addition, we investigated these strains individually and in combination, assessing their microbially induced carbonate precipitation (MICP) in silicate fine sand under low (14 ± 0.6 °C) and ideal temperature 30 °C conditions, aiming to optimize bio-mediated soil enhancement. Results indicated that 30 °C was the ideal temperature, and combining bacteria resulted in significant (p ≤ 0.001) superior carbonate precipitation (14-16%) and permeability (> 10- 6 m/s) in comparison to the average range of individual strains. These findings provide valuable insights into the potential of combining ureolytic bacteria for future MICP research on field applications including soil erosion mitigation, soil stabilization, ground improvement, and heavy metal remediation.

Improvement of combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase by functionalization of cross-linker for maltooligosaccharides synthesis.

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1 (Combi-CLEAs-CM) were successfully developed to synthesis maltooligosaccharides (MOS). Yet, the poor cross-linking performance between chitosan (cross-linker) and enzymes resulting low activity recovery and catalytic efficiency. In this study, we proposed the functionalization of cross-linkers with the integration of computational analysis to study the influences of different functional group on cross-linkers in combi-CLEAs development. From in-silico analysis, O-carboxymethyl chitosan (OCMCS) with the highest binding affinity toward both enzymes was chosen and showed alignment with the experimental result, in which OCMCS was synthesized as cross-linker to develop improved activity recovery of Combi-CLEAs-CM-ocmcs (74 %). The thermal stability and deactivation energy (205.86 kJ/mol) of Combi-CLEAs-CM-ocmcs were found to be higher than Combi-CLEAs-CM (192.59 kJ/mol). The introduction of longer side chain of carboxymethyl group led to a more flexible structure of Combi-CLEAs-CM-ocmcs. This alteration significantly reduced the Km value of Combi-CLEAs-CM-ocmcs by about 3.64-fold and resulted in a greater Kcat/Km (3.63-fold higher) as compared to Combi-CLEAs-CM. Moreover, Combi-CLEAs-CM-ocmcs improved the reusability with retained >50 % of activity while Combi-CLEAs-CM only 36.18 % after five cycles. Finally, maximum MOS production (777.46 mg/g) was obtained by Combi-CLEAs-CM-ocmcs after optimization using response surface methodology.

Surface-associated residues in subtilisins contribute to poly-L-lactic acid depolymerization via enzyme adsorption.

Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a biodegradable commodity. Enzymatic recycling is one strategy for ensuring circularity of PLLA, but this approach requires a thorough understanding of enzymatic mechanisms and protein engineering strategies to enhance activity. In this study, we engineer PLLA depolymerizing subtilisin enzymes originating from Bacillus species to elucidate the molecular mechanisms dictating their PLLA depolymerization activity and to improve their function. The surface-associated amino acids of two closely related subtilisin homologues originating from Bacillus subtilis (BsAprE) and Bacillus pumilus (BpAprE) were compared, as they were previously engineered to have nearly identical active sites, but still varied greatly in PLLA depolymerizing activity. Further analysis identified several surface-associated amino acids in BpAprE that lead to enhanced PLLA depolymerization activity when engineered into BsAprE. In silico protein modelling demonstrated increased enzyme surface hydrophobicity in engineered BsAprE variants and revealed a structural motif favoured for PLLA depolymerization. Experimental evidence suggests that increases in activity are associated with enhanced polymer binding as opposed to substrate specificity. These data highlight enzyme adsorption as a key factor in PLLA depolymerization by subtilisins.

Thermo-Alkali-Tolerant Recombinant Laccase from Bacillus swezeyi and Its Degradation Potential against Zearalenone and Aflatoxin B1.

The enzymatic biodegradation of mycotoxins in food and feed has attracted the most interest in recent years. In this paper, the laccase gene from Bacillus swezeyi was cloned and expressed in Escherichia coli BL 21(D3). The sequence analysis indicated that the gene consisted of 1533 bp. The purified B. swezeyi laccase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis -12% with an estimated molecular weight of 56.7 kDa. The enzyme is thermo-alkali-tolerant, displaying the optimal degradation of zearalenone (ZEN) and aflatoxin B1 (AFB1) at pH 8 and 9, with incubation temperatures of 55 and 50 °C, respectively, within 24 h. The degradation potentials of the 50 µg of the enzyme against ZEN (5.0 µg/mL) and AFB1 (2.5 µg/mL) were 99.60 and 96.73%, respectively, within 24 h. To the best of our knowledge, this is the first study revealing the recombinant production of laccase from B. swezeyi, its biochemical properties, and potential use in ZEN and AFB1 degradation in vitro and in vivo.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Ultrarobust stable ABTS radical cation prepared using Spore@Cu-TMA biocomposites for antioxidant capacity assay.

Herein, spore@Cu-trimesic acid (TMA) biocomposites were prepared by self-assembling Cu-based metal-organic framework on the surface of Bacillus velezensis spores. The laccase-like activity of spore@Cu-TMA biocomposites was enhanced by 14.9 times compared with that of pure spores due to the reaction of Cu2+ ions with laccase on the spore surface and the microporous structure of Cu-TMA shell promoting material transport and increasing substrate accessibility. Spore@Cu-TMA rapidly oxidized and transformed 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into ABTS●+ without using H2O2. Under optimum conditions, the ABTS●+ could be stored for 21 days at 4 °C and 7 days at 37 °C without the addition of any stabilizers, allowing for the large-scale preparation and long-term storage of ABTS●+. The ultrarobust stable ABTS●+ obtained with the use of Cu-TMA could effectively reduce the "back reaction" by preventing the leaching of the metabolites released by the spores. On the basis of these findings, a rapid, low-cost, and eco-friendly colorimetric platform was successfully developed for the detection of antioxidant capacity. Determination of antioxidant capacity for several antioxidants such as caffeic acid, glutathione, and Trolox revealed their corresponding limits of detection at 4.83, 8.89, and 7.39 nM, respectively, with linear ranges of 0.01-130, 0.01-140, and 0.01-180 µM, respectively. This study provides a facile way to prepare ultrarobust stable ABTS●+ and presents a potential application of spore@Cu-TMA biocomposites in food detection and bioanalysis.

Identification and characterization of a promiscuous metallohydrolase in metallo-ß-lactamase superfamily from a locally isolated organophosphate-degrading Bacillus sp. strain S3wahi.

In this present study, characteristics and structure-function relationship of an organophosphate-degrading enzyme from Bacillus sp. S3wahi were described. S3wahi metallohydrolase, designated as S3wahi-MH (probable metallohydrolase YqjP), featured the conserved αß/ßα metallo-ß-lactamase-fold (MBL-fold) domain and a zinc bimetal at its catalytic site. The metal binding site of S3wahi-MH also preserves the H-X-H-X-D-H motif, consisting of specific amino acids at Zn1 (Asp69, His70, Asp182, and His230) and Zn2 (His65, His67, and His137). The multifunctionality of S3wahi-MH was demonstrated through a steady-state kinetic study, revealing its highest binding affinity (KM) and catalytic efficiency (kcat/KM) for OP compound, paraoxon, with values of 8.09 × 10-6 M and 4.94 × 105 M-1 s-1, respectively. Using OP compound, paraoxon, as S3wahi-MH native substrate, S3wahi-MH exhibited remarkable stability over a broad temperature range, 20 °C - 60 °C and a broad pH tolerance, pH 6-10. Corresponded to S3wahi-MH thermal stability characterization, the estimated melting temperature (Tm) was found to be 72.12 °C. S3wahi-MH was also characterized with optimum catalytic activity at 30 °C and pH 8. Additionally, the activity of purified S3wahi-MH was greatly enhanced in the presence of 1 mM and 5 mM of manganese (Mn2+), showing relative activities of 1323.68 % and 2073.68 %, respectively. The activity of S3wahi-MH was also enhanced in the presence of DMSO and DMF, showing relative activities of 270.37 % and 307.41 %, respectively. The purified S3wahi-MH retained >60 % residual activity after exposure to non-ionic Tween series surfactants. Nevertheless, the catalytic activity of S3wahi-MH was severely impacted by the treatment of SDS, even at low concentrations. Considering its enzymatic properties and promiscuity, S3wahi-MH emerges as a promising candidate as a bioremediation tool in wide industrial applications, including agriculture industry.

Unlocking the potential of Algerian lignocellulosic biomass: exploring indigenous microbial diversity for enhanced enzyme and sugar production.

Nowadays, natural resources like lignocellulosic biomass are gaining more and more attention. This study was conducted to analyse chemical composition of dried and ground samples (500 µm) of various Algerian bioresources including alfa stems (AS), dry palms (DP), olive pomace (OP), pinecones (PC), and tomato waste (TW). AS exhibited the lowest lignin content (3.60 ± 0.60%), but the highest cellulose (58.30 ± 2.06%), and hemicellulose (20.00 ± 3.07%) levels. DP, OP, and PC had around 30% cellulose, and 10% hemicellulose. OP had the highest lignin content (29.00 ± 6.40%), while TW contained (15.70 ± 2.67% cellulose, 13.70 ± 0.002% hemicellulose, and 17.90 ± 4.00% lignin). Among 91 isolated microorganisms, nine were selected for cellulase, xylanase, and/or laccase production. The ability of Bacillus mojavensis to produce laccase and cellulase, as well as B. safensis to produce cellulase and xylanase, is being reported for the first time. In submerged conditions, TW was the most suitable substrate for enzyme production. In this conditions, T. versicolor K1 was the only strain able to produce laccase (4,170 ± 556 U/L). Additionally, Coniocheata hoffmannii P4 exhibited the highest cellulase activity (907.62 ± 26.22 U/L), and B. mojavensis Y3 the highest xylanase activity (612.73 ± 12.73 U/L). T. versicolor K1 culture showed reducing sugars accumulation of 18.87% compared to initial concentrations. Sucrose was the predominant sugar detected by HPLC analysis (13.44 ± 0.02 g/L). Our findings suggest that T. versicolor K1 holds promise for laccase production, while TW represents a suitable substrate for sucrose production.

A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Antagonistic and enzymatic activities of Bacillus species isolated from the fish gastrointestinal tract as potential probiotics use in Artemia culture.

Probiotics have been used successfully in aquaculture to enhance disease resistance, nutrition, and/or growth of cultured organisms. Six strains of Bacillus were isolated from the intestinal tracts of fish and recognised by conventional biochemical traits. The six isolated strains were Bacillus cereus and Bacillus subtilis using MALDI-TOF-MS technique. The probiotic properties of these Bacillus strains were studied. The tested bacillus strains exhibit antibacterial activity against the different pathogens. The strain S5 gave the important inhibition zones against most pathogens (20.5, 20.33, 23, and 21 mm against Vibrio alginolyticus, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella typhimurium, respectively). According to our results, all Bacillus strains have extracellular components that can stop pathogenic bacteria from growing. The enzymatic characterization showed that the tested strains can produce several biotechnological enzymes such as α-glucosidase, naphtol-AS-BI-Phosphohydrolase, esterase lipase, acid phosphatase, alkaline phosphatase, amylase, lipase, caseinase, and lecithinase. All Bacillus strains were adhesive to polystyrene. The adding Bacillus strains to the Artemia culture exerted significantly greater effects on the survival of Artemia. The challenge test on Artemia culture showed that the protection against pathogenic Vibrio was improved. These findings allow us to recommend the examined strains as prospective probiotic options for the Artemia culture, which will be used as food additives to improve the culture conditions of crustacean larvae and marine fish.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.

Biodegradation of Keratin Waste by Bacillus velezensis HFS_F2 through Optimized Keratinase Production Medium.

Enormous aggregates of keratinous wastes are produced annually by the poultry and leather industries which cause environmental degradation globally. To combat this issue, microbially synthesized extracellular proteases known as keratinase are used widely which is effective in degrading keratin found in hair and feathers. In the present work, keratinolytic bacteria were isolated from poultry farm soil and feather waste, and various cultural conditions were optimized to provide the highest enzyme production for efficient keratin waste degradation. Based on the primary and secondary screening methods, the potent keratinolytic strain (HFS_F2T) with the highest enzyme activity 32.65 ± 0.16 U/mL was genotypically characterized by 16S rRNA sequencing and was confirmed as Bacillus velezensis HFS_F2T ON556508. Through one-variable-at-a-time approach (OVAT), the keratinase production medium was optimized with sucrose (carbon source), beef extract (nitrogen source) pH-7, inoculum size (5%), and incubation at 37 °C). The degree of degradation (%DD) of keratin wastes was evaluated after 35 days of degradation in the optimized keratinase production medium devoid of feather meal under submerged fermentation conditions. Further, the deteriorated keratin wastes were visually examined and the hydrolysed bovine hair with 77.32 ± 0.32% degradation was morphologically analysed through Field Emission Scanning Electron Microscopy (FESEM) to confirm the structural disintegration of the cuticle. Therefore, the current study would be a convincing strategy for reducing the detrimental impact of pollutants from the poultry and leather industries by efficient keratin waste degradation through the production of microbial keratinase.

Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production.

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Chitosanase-immobilized magnetite-agar gel particles as a highly stable and reusable biocatalyst for enhanced production of physiologically active chitosan oligosaccharides.

A novel immobilized chitosanase was developed and utilized to produce chitosan oligosaccharides (COSs) via chitosan hydrolysis. Magnetite-agar gel particles (average particle diameter: 338 µm) were prepared by emulsifying an aqueous agar solution dispersing 200-nm magnetite particles with isooctane containing an emulsifier at 80 °C, followed by cooling the emulsified mixture. The chitosanase from Bacillus pumilus was immobilized on the magnetite-agar gel particles chemically activated by introducing glyoxyl groups with high immobilization yields (>80%), and the observed specific activity of the immobilized chitosanase was 16% of that of the free enzyme. This immobilized chitosanase could be rapidly recovered from aqueous solutions by applying magnetic force. The thermal stability of the immobilized chitosanase improved remarkably compared with that of free chitosanase: the deactivation rate constants at 35 °C of the free and immobilized enzymes were 8.1 × 10-5 and 3.9 × 10-8 s-1, respectively. This immobilized chitosanase could be reused for chitosan hydrolysis at 75 °C and pH 5.6, and 80% of its initial activity was maintained even after 10 cycles of use. COSs with a degree of polymerization (DP) of 2-7 were obtained using this immobilized chitosanase, and the product content of physiologically active COSs (DP ≥ 5) reached approximately 50%.

A polylactic acid degrading lipase from Bacillus safensis: Characterization and structural analysis.

A polylactic acid degrading triacylglycerol lipase (TGL) was identified from Bacillus safensis based on genome annotation and validated by real-time quantitative PCR. TGL displayed optimal activity at pH 9.0 and 55 °C. It maintained stability at pH 9.0 and temperatures 45 °C. The activity of TGL was found to benefit from the presence of potassium sodium ions, and low concentrations of Triton X-100. The TGL could erode the surface of polylactic acid films and increase its hydrophilicity. The hydrolysis products of polylactic acid by TGL were lactate monomer and dimer. TGL contains a classical catalytic triad structure of lipase (Ser77, Asp133, and His156) and an Ala-X-Ser-X-Gly sequence. Compared with some lipases produced by the same genus Bacillus, TGL is highly conserved in its amino acid sequence, mainly reflected in the amino acid residues that exercise the enzyme activity, including the catalytic activity center and the substrate binding sites.