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1.
Molecules ; 26(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34577096

RESUMO

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.


Assuntos
Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Bacillus/enzimologia , Bacillus/genética , Celulases/genética , Celulases/metabolismo , Clostridium/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulose/metabolismo , Clonagem Molecular , Hidrólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Folia Microbiol (Praha) ; 66(5): 787-795, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34128186

RESUMO

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.


Assuntos
Bacillus licheniformis , Alimentos e Bebidas Fermentados , Alimentos Marinhos , Bacillus licheniformis/enzimologia , Ativação Enzimática/efeitos dos fármacos , Alimentos e Bebidas Fermentados/microbiologia , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , República da Coreia , Alimentos Marinhos/microbiologia , Cloreto de Sódio/farmacologia
3.
Chem Commun (Camb) ; 57(36): 4460-4463, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33949502

RESUMO

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.


Assuntos
Oxirredutases do Álcool/metabolismo , Escherichia coli/metabolismo , Compostos Férricos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lacase/metabolismo , Lipase/metabolismo , Oxirredutases do Álcool/química , Bacillus licheniformis/enzimologia , Bacillus subtilis/enzimologia , Candida tropicalis/enzimologia , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Compostos Férricos/química , Proteínas de Fluorescência Verde/química , Histidina/química , Histidina/metabolismo , Lacase/química , Lipase/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo
4.
Carbohydr Polym ; 264: 118059, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33910709

RESUMO

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.


Assuntos
Bacillus licheniformis/enzimologia , Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Bacillus licheniformis/química , Domínio Catalítico , Celulases/química , Celulases/metabolismo , Celulose/química , Cristalografia por Raios X/métodos , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Especificidade por Substrato
5.
Vet Res ; 52(1): 59, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863379

RESUMO

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.


Assuntos
Bacillus licheniformis/química , Encefalopatia Espongiforme Bovina/etiologia , Temperatura Alta , Peptídeo Hidrolases/metabolismo , Proteínas Priônicas/química , Proteólise , Animais , Bacillus licheniformis/enzimologia , Bovinos , Camundongos Transgênicos
6.
Int J Biol Macromol ; 180: 677-683, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33757855

RESUMO

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.


Assuntos
Asparaginase/metabolismo , Bacillus licheniformis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/metabolismo , Asparaginase/genética , Bacillus licheniformis/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Western Blotting , Biologia Computacional/métodos , Sinais Direcionadores de Proteínas/genética
7.
Food Chem ; 354: 129475, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-33744660

RESUMO

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.


Assuntos
Sais/química , alfa-Amilases/metabolismo , Bacillus licheniformis/enzimologia , Sítios de Ligação , Estabilidade Enzimática , Metais/química , Simulação de Dinâmica Molecular , Temperatura , alfa-Amilases/química
8.
J Food Sci ; 86(4): 1475-1487, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33655547

RESUMO

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.


Assuntos
Bacillus licheniformis/enzimologia , Esterases/isolamento & purificação , Esterases/metabolismo , Piretrinas/metabolismo , Cromatografia em Gel , Estabilidade Enzimática , Esterases/química , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Temperatura
9.
Int J Biol Macromol ; 176: 126-136, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548313

RESUMO

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.


Assuntos
Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lipase/química , Lipase/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Bacillus licheniformis/genética , Bacillus licheniformis/isolamento & purificação , Proteínas de Bactérias/genética , Biocatálise , Biodegradação Ambiental , Cinnamomum camphora/microbiologia , Detergentes , Estabilidade Enzimática , Herbicidas/metabolismo , Microbiologia Industrial , Lipase/genética , Peso Molecular , Mapeamento de Peptídeos , Filogenia , Solventes , Especificidade por Substrato , Tensoativos
10.
Molecules ; 26(2)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33451050

RESUMO

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.


Assuntos
Catequina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Palmitatos/farmacologia , Polifenóis/química , Chá/química , Bacillus licheniformis/enzimologia , Catequina/síntese química , Catequina/química , Catequina/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Ligantes , Simulação de Acoplamento Molecular , Palmitatos/síntese química , Palmitatos/química , Saccharomyces cerevisiae/enzimologia , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo
11.
Int J Biol Macromol ; 166: 1491-1498, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33166558

RESUMO

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.


Assuntos
Bacillus licheniformis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Fatores de Transcrição/metabolismo , Transcriptoma , Bacillus licheniformis/enzimologia , Bacillus licheniformis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Transcrição/genética
12.
Food Chem ; 344: 128599, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33223297

RESUMO

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.


Assuntos
Bacillus licheniformis/enzimologia , Pão/análise , Glicosídeo Hidrolases/metabolismo , Bacillus licheniformis/classificação , Bacillus licheniformis/genética , Elasticidade , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Dureza , Concentração de Íons de Hidrogênio , Estabilidade Proteica , RNA Ribossômico 16S/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Reologia , Temperatura
13.
Int J Biol Macromol ; 167: 1393-1405, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33202275

RESUMO

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.


Assuntos
Bacillus licheniformis/genética , Corantes/química , Escherichia coli/metabolismo , Lacase/química , Bacillus licheniformis/enzimologia , Clonagem Molecular , Inibidores Enzimáticos/química , Expressão Gênica , Temperatura Alta , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Íons/química , Lacase/antagonistas & inibidores , Lacase/isolamento & purificação , Metais/química , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes , Especificidade por Substrato
14.
J Agric Food Chem ; 69(1): 223-231, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33371681

RESUMO

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.


Assuntos
Asparaginase/química , Asparaginase/metabolismo , Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Asparaginase/genética , Asparagina/química , Asparagina/metabolismo , Bacillus licheniformis/química , Bacillus licheniformis/genética , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Especificidade por Substrato
15.
Protein Expr Purif ; 177: 105748, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32911063

RESUMO

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.


Assuntos
Antioxidantes/farmacologia , Bacillus licheniformis/química , Plumas/química , Queratinas/farmacologia , Peptídeos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Bacillus licheniformis/enzimologia , Compostos de Bifenilo/antagonistas & inibidores , Compostos de Bifenilo/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Temperatura Alta , Humanos , Hidrólise , Queratinas/química , Queratinas/isolamento & purificação , Peso Molecular , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Peptídeos/química , Peptídeos/isolamento & purificação , Picratos/antagonistas & inibidores , Picratos/metabolismo
16.
Mol Biol (Mosk) ; 54(5): 858-871, 2020.
Artigo em Russo | MEDLINE | ID: mdl-33009795

RESUMO

At the International Space Station (ISS), artificial living conditions are created and maintained to satisfy human needs, these conditions are also favorable for the growth of numerous microorganisms, molds and bacteria. Among the microorganisms detected on the ISS are those from the automicroflora of crew members, and a significant number of spore-forming bacteria. In most cases, this group of microorganisms gives rise to strains that are able to colonize, grow and reproduce on interior materials and equipment of stations, and may be involved in biodestructive processes. These bacteria show increased resistance to various stress factors, for example, DNA-damaging and oxidizing agents. The molecular mechanisms of this resistance to stress are poorly understood. As part of the sanitary-microbiological monitoring of the ISS habitat, the Bacillus licheniformis 24 strain was isolated. Here, we demonstrated that this strain has increased resistance to hydrogen peroxide and Paraquat when compared to the "terrestrial" B. licheniformis B-10956 strain. B. licheniformis 24 overexpressed genes encoding enzymes that neutralize reactive oxygen species, such as KatX catalase and the superoxide dismutases SodA and SodF. Apart from this, in comparison with B. licheniformis B-10956, of B. licheniformis 24 cells had lower hydrogen sulfide production that was associated with sharply reduced expression of the cysIJ operon that encodes sulfite reductase. The results indicate that enzymatic antioxidant protective systems make a more significant contribution to the hyper-resistance of Bacillus strains to oxidizing agents than components of non-enzymatic systems, such as hydrogen sulfide.


Assuntos
Antioxidantes/metabolismo , Bacillus licheniformis/enzimologia , Estresse Oxidativo , Bacillus licheniformis/genética , Catalase/genética , Catalase/metabolismo , Ambiente Controlado , Genes Bacterianos , Astronave , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
17.
Microb Cell Fact ; 19(1): 181, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32933546

RESUMO

Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N'-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.


Assuntos
Acetilglucosamina/biossíntese , Acetilglucosaminidase/metabolismo , Bacillus licheniformis/enzimologia , Quitina/metabolismo , Quitinases/biossíntese , Reguladores de Crescimento de Plantas/biossíntese , Saccharomycetales/metabolismo , Acetilglucosamina/farmacologia , Bacillus licheniformis/genética , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Biotecnologia , Quitinases/genética , Quitosana/farmacologia , Fermentação , Germinação/efeitos dos fármacos , Hidrólise , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Oligossacarídeos/farmacologia , Oryza/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Saccharomycetales/genética , Plântula/crescimento & desenvolvimento , Triticum/efeitos dos fármacos
18.
Int J Biol Macromol ; 163: 1458-1470, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32771518

RESUMO

Keratinases are valuable enzymes, given their application in keratin-rich waste recycling. Considering that keratinases usually require reducing agents to efficiently degrade keratin, improving the stability of keratinases under reducing conditions is highly desirable for practical applications. Here, we show that the introduction of several tyrosine derivatives containing para-substituted long-chain haloalkanes into the keratinase KerBL, which enabled proximity-triggered covalent crosslinking by rational design, could improve both the thermostability and autolytic resistance of the enzyme. After screening a series of noncanonical amino acid (ncAA)-based variants generated by rational design, two variants, N159C/Y260BprY and N159C/Y260BbtY, with enhanced keratinolytic activity were obtained. Both variants increased the Tm of the enzyme by approximately 10 °C. The potential mechanism underlying these improvements was investigated by molecular dynamics (MD) analysis. The results indicated that BprY-Cys and BbtY-Cys covalent bonds in the N159C/Y260TAG variant could significantly decrease the flexibility and fluctuations of the long loop (residues 151-162).


Assuntos
Bacillus licheniformis/enzimologia , Queratinas/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Bacillus licheniformis/genética , Sequência de Bases , Clonagem Molecular , Ativação Enzimática , Peptídeo Hidrolases/genética , Plasmídeos/genética , Conformação Proteica , Proteólise , Análise de Sequência , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 117(32): 19178-19189, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723819

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.


Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Quitina/metabolismo , Oxigenases de Função Mista/química , Oxigênio/metabolismo , Bacillus licheniformis/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Quitina/química , Cobre/química , Cobre/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Imageamento por Ressonância Magnética , Oxigenases de Função Mista/metabolismo , Oxigênio/química , Especificidade por Substrato
20.
Eur Biophys J ; 49(6): 435-447, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32683479

RESUMO

Bacterial esterases are highly versatile enzymes, currently widely used in detergents, biosurfactants, bioemulsifiers and as biocatalysts in paper and food industries. Present work describes heterologous expression, purification, and biophysical and biochemical characterization of a halotolerant esterase from Bacillus licheniformis (BlEstA). BlEstA preferentially cleaves pNP-octanoate and both activity and stability of the enzyme increased in the presence of 2 M NaCl, and also with several organic solvents (ethanol, methanol and DMSO). Furthermore, BlEstA has considerable emulsifying properties, particularly with olive oil as substrate. Our studies also show that the enzyme is monomeric in solution and its small-angle X-ray scattering low-resolution molecular envelope fits well its high-resolution homology model.


Assuntos
Bacillus licheniformis/enzimologia , Emulsificantes/química , Emulsificantes/metabolismo , Esterases/química , Esterases/metabolismo , Biocatálise , Concentração de Íons de Hidrogênio , Modelos Moleculares , Filogenia , Conformação Proteica , Cloreto de Sódio/farmacologia , Especificidade por Substrato , Temperatura
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