Automated optimization of the solubility of a hyper-stable α-amylase.

Most successes in computational protein engineering to date have focused on enhancing one biophysical trait, while multi-trait optimization remains a challenge. Different biophysical properties are often conflicting, as mutations that improve one tend to worsen the others. In this study, we explored the potential of an automated computational design strategy, called CamSol Combination, to optimize solubility and stability of enzymes without affecting their activity. Specifically, we focus on Bacillus licheniformis α-amylase (BLA), a hyper-stable enzyme that finds diverse application in industry and biotechnology. We validate the computational predictions by producing 10 BLA variants, including the wild-type (WT) and three designed models harbouring between 6 and 8 mutations each. Our results show that all three models have substantially improved relative solubility over the WT, unaffected catalytic rate and retained hyper-stability, supporting the algorithm's capacity to optimize enzymes. High stability and solubility embody enzymes with superior resilience to chemical and physical stresses, enhance manufacturability and allow for high-concentration formulations characterized by extended shelf lives. This ability to readily optimize solubility and stability of enzymes will enable the rapid and reliable generation of highly robust and versatile reagents, poised to contribute to advancements in diverse scientific and industrial domains.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Regulator DegU can remarkably influence alkaline protease AprE biosynthesis in Bacillus licheniformis 2709.

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5'-TAAAT……AAAAT…….AACAT…TAAAA-3') located upstream -91 to -87 bp, -101 to -97 bp, -195 to -191 bp, -215 to -211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism.

Functional characterization of glycine oxidase from Bacillus licheniformis in Escherichia coli and transgenic plants.

OBJECTIVES: To find glycine oxidase genes that can be applied to the breeding of glyphosate resistant crops. RESULTS: The glycine oxidase (GO, EC 1.4.3.19) gene (GenBank No: KC831746) from Bacillus licheniformis (B. licheniformis) was chemically synthesized and transformed into glyphosate-sensitive Escherichia coli (E. coli). The GO gene was transformed into Arabidopsis and rice through Agrobacterium-mediated transformation. The test results confirmed that transgenic plants containing GO genes are more resistant to glyphosate than wild-type plants. On solid Murashige and Skoog (MS) (Murashige and Skoog1962 ) medium containing 200 µM glyphosate, transgenic Arabidopsis thaliana grew normally, while wild-type plants were stunted and root growth was restricted. In a solution containing 500 µM glyphosate, wild-type rice showed severe yellowing, while transgenic rice grew normally. In addition, when sprayed with 10 mM glyphosate solution, wild-type rice withered and died, while transgenic rice grew well. The function of GO gene in glyphosate resistance and the application value of GO gene in the cultivation of glyphosate-resistant crops is proved. CONCLUSIONS: The glycine oxidase gene from B. licheniformis enhances the resistance of E. coli, Arabidopsis and rice to glyphosate.

Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis.

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.

Characterization of a salt-resistant fibrinolytic protease of Bacillus licheniformis HJ4 isolated from Hwangseokae jeotgal, a traditional Korean fermented seafood.

Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27 kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84 h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27 kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively.

Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes.

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP.

Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.

Non-classical secretion of a type I L-asparaginase in Bacillus subtilis.

L-asparaginase (EC 3.5.1.1) showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia (ALL), lymphoid system malignancies and Hodgkin disease, and also to its use in the prevention of acrylamide formation in fried and baked foods. In this study, a type I L-asparaginase gene from Bacillus licheniformis Z-1 (BlAase) was cloned and expressed in Bacillus subtilis RIK 1285. Results showed that even without the mediation of any N-terminal signal peptides, BlAase can efficiently secrete into the medium. Further investigation indicated that the secretion of the BlAase was via neither Sec- nor Tat-dependent secretion pathway, and both the N- and C-terminal regions of the BlAase were essential for its expression and secretion, implying that BlAase might be secreted via a non-classical secretion pathway. To explore its secretion ability, BlAase was used as a signal peptide to direct the secretion of various heterologous proteins, where two of five proteins were successfully secreted with the mediation of BlAase. To the best of our knowledge, this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B. subtilis, and provide a potential tool for secretion of recombinant proteins expressed in B. subtilis using BlAase as a signal peptide.

The desirable salt bridges in amylases: Distribution, configuration and location.

The α-amylases are the most widely used industrial enzymes, and are particularly useful as liquifying enzymes in industrial processes based upon starch. Since starch liquefication is carried out at evaluated temperatures, typically above 60 °C, there is substantial demand for thermostable α -amylases. Most naturally occurring α -amylases exhibit moderate thermostability, so substantial effort has been invested in attempts to increase their thermostability. One structural feature that has the potential to increase protein thermostability is the introduction of salt bridges. However, not every salt bridge contributes to protein thermostability. The salt bridges in amylases have their characteristics in terms of distribution, configuration and location. The summary of these features helps to introduce new salt bridges based on the characteristics. This review focuses on salt bridges of α-amylases, both naturally present and introduced using mutagenesis. Its aim is to provide a bird's eye view of distribution, configuration, location of desirable salt bridges.

Purification and characterization of a novel cypermethrin-hydrolyzing esterase from Bacillus licheniformis B-1.

Cypermethrin (CY) is a synthetic pyrethroid widely used to control insect pests and it elicits a toxic effect on the human body. In this study, Bacillus licheniformis B-1 isolated from tea garden soil was used to degrade CY effectively. A specific enzyme was mainly localized in the extracellular compartments of B-1. This enzyme was identified as an esterase that could be produced without CY. The enzyme was purified 23.03-fold to apparent homogeneity with 8.38% overall recovery by ammonium sulfate precipitation, anion exchange chromatography, and gel filtration chromatography. The molecular mass of the CY-degrading enzyme was 66.4 kDa, and its optimal pH and temperature were 8.5 and 40 °C, respectively. Appropriate Zn2+ , Mn2+ , Mg2+ , Tween 80, SDS, Triton X-100, and BSA concentrations could greatly increase the activity of this enzyme. By contrast, EDTA, 1,10-phenanthroline, NaF, and PMSF strongly inhibited its activity. The purified enzyme showed Km and Vmax values were 5.532 nmol/mL and 33.445 nmol/min. The CY residue in lettuce and cherry tomatoes could be removed more than 50% under the conditions of the treatment concentration for 500 mg/L and the enzyme preparation dilution of 100 times. These results suggested that the CY-degrading enzyme, a constitutive enzyme that mainly exists in the extracellular space, was a novel esterase that might be used to detoxify CY, and could remove CY in vegetables effectively. PRACTICAL APPLICATION: Our research found a novel cypermethrin-hydrolyzing esterase from Bacillus licheniformis B-1 and proved that the enzyme could remove cypermethrin in vegetables effectively.

Engineered pro-peptide enhances the catalytic activity of keratinase to improve the conversion ability of feather waste.

Keratinase is an attractive industrial enzyme that can specifically catalyze keratin waste to obtain value-added products. A challenge to the application of keratinase is improving catalytic capacity to achieve efficient hydrolysis. In this study, we effectively expressed the keratinase gene from Bacillus licheniformis BBE11-1 in Bacillus subtilis WB600 based on pro-peptide engineering. Partial deletion of the pro-peptide sequence and the substitution of amino acid at the pro-peptide cleavage site (P1) suggested that the "chaperone effect" and "cleavage efficiency" of the pro-peptide determine the activity of the mature enzyme. Subsequently, seven target sites that can increase the activity of the mature enzyme by 16%-66% were obtained through the multiple sequence alignment of pro-peptides and site-directed mutation. We further performed combinatorial mutations at six sites based on the design principle of three-codon saturation mutations and obtained mutant 2-D12 (236.8 KU/mg) with a mature enzyme activity of 186% of the original (127.6 KU/mg). Finally, continuous fermentation was carried out in a 5-L bioreactor for 22 h, and the activity of the 2-D12 mature enzyme was increased to 391.6 KU/mg. Most importantly, 2-D12 could degrade more than 90% of feather waste into amino acids and peptides within 12 h with the aid of sulfite.

Characterization of a novel lipase from Bacillus licheniformis NCU CS-5 for applications in detergent industry and biodegradation of 2,4-D butyl ester.

Enzymatic degradation has become the most promising approach to degrading organic ester compounds. In this study, Bacillus licheniformis NCU CS-5 was isolated from the spoilage of Cinnamomum camphora seed kernel, and its extracellular lipase was purified, with a specific activity of 192.98 U/mg. The lipase was found to be a trimeric protein as it showed a single band of 27 kDa in SDS-PAGE and 81 kDa in Native-PAGE. It was active in a wide range of temperatures (5-55 °C) and pH values (6.0-9.0), and the optimal temperature and pH value were 40 °C and 8.0, respectively. The enzyme was active in the presence of various organic solvents, metal ions, inhibitors and surfactants. Both crude and purified lipase retained more than 80% activity after 5 h in the presence of commercial detergents, suggesting its great application potential in detergent industry. The highest activity was found to be towards medium- and long-chain fatty acids (C6-C18). Peptide mass spectrometric analysis of the purified lipase showed similarity to the lipase family of B. licheniformis. Furthermore, it degraded more than 90% 2,4-D butyl ester to its hydrolysate 2,4-D within 24 h, indicating that the novel lipase may be applied to degrade organic ester pesticides.

Synthesis, Stability, and Antidiabetic Activity Evaluation of (-)-Epigallocatechin Gallate (EGCG) Palmitate Derived from Natural Tea Polyphenols.

This work describes a novel approach for the synthesis of (-)-epigallocatechin gallate (EGCG) palmitate by a chemical-synthesis method, where the elevated stability of the EGCG derivative is achieved. Various parameters affecting the acylation process, such as the base, solvent, as well as the molar ratio of palmitoyl chloride, have been studied to optimize the acylation procedure. The optimized reaction condition was set as follows: EGCG/palmitoyl chloride/sodium acetate was under a molar ratio of 1:2:2, with acetone as the solvent, and the reaction temperature was 40 °C. Under the optimized condition, the yield reached 90.6%. The EGCG palmitate (PEGCG) was isolated and identified as 4'-O-palmitoyl EGCG. Moreover, the stability of PEGCG under different conditions was proved significantly superior to EGCG. Finally, PEGCG showed better inhibition towards α-amylase and α-glucosidase, which was 4.5 and 52 times of EGCG, respectively. Molecular docking simulations confirmed the in vitro assay results. This study set a novel and practical synthetic approach for the derivatization of EGCG, and suggest that PEGCG may act as an antidiabetic agent.

Stability and cytotoxicity of DPPH inhibitory peptides derived from biodegradation of chicken feather.

The antioxidant activity and cell viability of feather hydrolysates obtained with the Bacillus licheniformis were evaluated using an in-vitro model. The results indicate that feathers-derived peptides under 3 kDa have antioxidant activity with IC50 values of 5.03 ± 0.215 mg/mL by using DPPH antioxidant assay. Although the antioxidant activity of the peptides under 3 kDa preserved after applying diverse heating (from 20 to 100 °C), they lost their activity under strongly acidic or alkaline conditions. Antioxidant activity of the mixed feather bioactive peptides (MFBPs) obtained with partial purification of peptides under 3 kDa was with IC50 amount of 0.169 mg/mL ± 0.004 using DPPH radical scavenging assay. Also, MFBPs within an amount range of from 0.0048 to 5.0 mg/mL, illustrated no cytotoxicity to gingival fibroblast blood cell lines. In light of our results, the obtained value-added peptides could be useful in different food products as a future functional ingredient with antioxidant potency.

Transcriptome based functional identification and application of regulator AbrB on alkaline protease synthesis in Bacillus licheniformis 2709.

Bacillus licheniformis 2709 is the major alkaline protease producer, which has great potential value of industrial application, but how the high-producer can be regulated rationally is still not completely understood. It's meaningful to understand the metabolic processes during alkaline protease production in industrial fermentation medium. Here, we collected the transcription database at various enzyme-producing stages (preliminary stage, stable phase and decline phase) to specifically research the synthesized and regulatory mechanism of alkaline protease in B. licheniformis. The RNA-sequencing analysis showed differential expression of numerous genes related to several processes, among which genes correlated with regulators were concerned, especially the major differential gene abrB on enzyme (AprE) synthesis was investigated. It was further verified that AbrB is a repressor of AprE by plasmid-mediated over-expression due to the severely descending enzyme activity (11,300 U/mL to 2695 U/mL), but interestingly it is indispensable for alkaline protease production because the enzyme activity of the null abrB mutant was just about 2279 U/mL. Thus, we investigated the aprE transcription by eliminating the theoretical binding site (TGGAA) of AbrB protein predicated by computational strategy, which significantly improved the enzyme activity by 1.21-fold and gene transcription level by 1.77-fold in the mid-log phase at a cultivation time of 18 h. Taken together, it is of great significance to improve the production strategy, control the metabolic process and oriented engineering by rational molecular modification of regulatory network based on the high throughput sequencing and computational prediction.

A new maltogenic amylase from Bacillus licheniformis R-53 significantly improves bread quality and extends shelf life.

Maltogenic amylase suppressed starch retrogradation in baked products. Here, a maltogenic amylase-producing strain of bacteria was screened and identified as Bacillus licheniformis R-53. Its coding gene was cloned and over-expressed in Bacillus subtilis WB600. Recombinant maltogenic amylase BLMA exhibited activity of 3235 U/mg under optimal conditions (60 °C and pH 6.5), with a good thermostability and pH stability. Mixolab experiment showed that a concentration of 60 ppm BLMA significantly improved the operating characteristics of dough. Baking test indicated the recombinant BLMA reduced bread hardness by 2.12 times compared with the control. Compared with maltogenic amylase from Novozymes (Novamyl 3D BG) and Angel Yeast Co. Ltd. (MAM100), BLMA has better effect on improving the bread volume, and almost the same effect on reducing hardness, improving elasticity and maintaining sensory as Novamyl 3D BG. Adding BLMA improved bread quality, increased bread volume and decreased hardness during storage, thus extending its shelf life.

Structures of l-asparaginase from Bacillus licheniformis Reveal an Essential Residue for its Substrate Stereoselectivity.

l-Asparaginase, which catalyzes the hydrolysis of l-asparagine, is an important enzyme in both the clinical and food industry. Exploration of efficient l-asparaginase with high substrate specificity, especially high chiral selectivity, is essential for extending its use. Herein, various crystal structures of type I l-asparaginase from Bacillus licheniformis (BlAsnase) have been resolved, and we found that there are two additional tyrosines in BlAsnase, contributing to the binding and catalysis of d-asparagine. Strikingly, the substitution of Tyr278 with methionine impaired the interaction with d-asparagine via water molecules due to the small hydrophobic side chain of methionine, which forced the ligand to the deep side of the active site toward the catalytic residues and thus resulted in the loss of hydrolyzing function. Our investigation of the substrate recognition mechanism of BlAsnase is significant for both a better understanding of l-asparaginase and its rational design to achieve high specificity for clinical and industrial applications.

Enzymatic characterization, molecular dynamics simulation, and application of a novel Bacillus licheniformis laccase.

A new laccase gene from newly isolated Bacillus licheniformis TCCC 111219 was actively expressed in Escherichia coli. This recombinant laccase (rLAC) exhibited a high stability towards a wide pH range and high temperatures. 170% of the initial activity was detected at pH 10.0 after 10-d incubation, and 60% of the initial activity was even kept after 2-h incubation at 70 °C. It indicated that only single type of extreme environment, such as strong alkaline environment (300 K, pH 12) or high temperature (370 K, pH 7), did not show obvious impact on the structural stability of rLAC during molecular dynamics simulation process. But the four loop regions of rLAC where the active site is situated were seriously destroyed when strong alkaline and high temperature environment existed simultaneously (370 K, pH 12) because of the damage of hydrogen bonds and salt bridges. Moreover, this thermo- and alkaline-stable enzyme could efficiently decolorize the structurally differing azo, triphenylmethane, and anthraquinone dyes with appropriate mediator at pH 3.0, 7.0, and 9.0 at 60 °C. These rare characteristics suggested its high potential in industrial applications to decolorize textile dyeing effluent.