RESUMO
Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 µg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.
Assuntos
Aflatoxina B1 , Bacillus amyloliquefaciens , Proteínas de Bactérias , Biodegradação Ambiental , Aflatoxina B1/metabolismo , Aflatoxina B1/química , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/química , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Células Hep G2 , Alimentos Fermentados/microbiologia , MultiômicaRESUMO
BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.
Assuntos
Bacillus amyloliquefaciens , Celulase , Clonagem Molecular , Simulação de Acoplamento Molecular , Oryza , Celulase/genética , Celulase/biossíntese , Celulase/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Oryza/microbiologia , Fermentação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.
Assuntos
Bacillus amyloliquefaciens , Proteínas de Bactérias , Engenharia Metabólica , Monoéster Fosfórico Hidrolases , Plasmídeos , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Bacillus amyloliquefaciens/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Técnicas de Inativação de GenesRESUMO
Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.
Assuntos
Bacillus amyloliquefaciens , Proteínas de Bactérias , Endopeptidases , Proteínas de Fluorescência Verde , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Endopeptidases/metabolismo , Endopeptidases/genética , Endopeptidases/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Simulação de Acoplamento Molecular , Sinais Direcionadores de Proteínas , Proteínas de Membrana , Serina Endopeptidases , Proteínas de Membrana TransportadorasRESUMO
BACKGROUND: Nattokinase (NK) is a naturally occurring fibrinolytic protease enzyme obtained from the traditional Japanese food called Natto and has several uses in the pharmaceutical and medical industries. Nowadays, the most often used thrombolytic agent in the clinical field is NK, in part because it is less expensive than other thrombolytic medicines. OBJECTIVES: The objective of this study is to investigate the screening, isolation and characterization of the NK enzyme-producing Bacillus strain from fermented Soya beans. METHODS: The sample of fermented soya beans were tested for the presence of fibrinolytic protease- producing bacteria, followed by the screening, extraction, characterization and clot lysis assays. RESULTS: A total of three isolates were screened for caseinolytic activities by casein hydrolysis assay. Out of those isolates, MRS18 was found to be potent in producing the enzyme proteinase. To determine the taxonomy and phylogeny of these isolates, biochemical and molecular characterization has been carried out. Bacillus amyloliquefaciens MRS18 has been found with the highest caseinolytic activity. The clot lysing ability of the potent strain Bacillus amyloliquefaciens was found to be 61.7% after 120 min, and on further purification, by ammonium sulphate precipitation method, the lysis percentage was found to be 656% after 120 min. CONCLUSION: From the results of the present study, we concluded that Bacillus amyloliquefaciens isolated from the fermented soya beans produced an NK enzyme that exhibits immense potential to lyse blood clots.
Assuntos
Bacillus amyloliquefaciens , Fermentação , Subtilisinas , Subtilisinas/química , Subtilisinas/metabolismo , Bacillus amyloliquefaciens/enzimologia , Glycine max/microbiologia , FilogeniaRESUMO
Aflatoxins, widely found in feed and foodstuffs, are potentially harmful to human and animal health because of their high toxicity. In this study, a strain of Bacillus amyloliquefaciens B10 with a strong ability to degrade aflatoxin B1 (AFB1) was screened; it could degrade 2.5 µg/mL of AFB1 within 96 h. The active substances of Bacillus amyloliquefaciens B10 for the degradation of AFB1 mainly existed in the culture supernatant. A new laccase with AFB1-degrading activity was separated by ammonium sulfate precipitation, diethylaminoethyl (DEAE) and gel filtration chromatography. The results of molecular docking showed that B10 laccase and aflatoxin had a high docking score. The coding sequence of the laccase was successfully amplified from cDNA by PCR and cloned into E. coli. The purified laccase could degrade 79.3% of AFB1 within 36 h. The optimum temperature for AFB1 degradation was 40 °C, and the optimum pH was 6.0-8.0. Notably, Mg2+ and dimethyl sulfoxide (DMSO) could enhance the AFB1-degrading activity of B10 laccase. Mutation of the three key metal combined sites of B10 laccase resulted in the loss of AFB1-degrading activity, indicating that these three metal combined sites of B10 laccase play an essential role in the catalytic degradation of AFB1.
Assuntos
Aflatoxina B1 , Bacillus amyloliquefaciens , Lacase , Aflatoxina B1/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Escherichia coli/metabolismo , Lacase/metabolismo , Simulação de Acoplamento MolecularRESUMO
Rhizobacteria from pearl millet were screened to produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase and to evaluate its role in alleviating drought stress. Amongst 96 isolates, 28 were positive for ACC deaminase production, with MMR04 offering maximum activity of 2196.23 nmol of α-ketobutyrate produced mg-1 of protein h-1. The ACC deaminase producing rhizobacteria with multiple beneficial properties along with root colonization and non-pathogenic were selected [Bacillus amyloliquefaciens (MMR04), Bacillus subtilis (MMR18) and Stenotrophomonas maltophilia (MMR36)] to confirm the presence of ACC deaminase gene. A significant enhancement in seed germination (91.75%) and seedling vigor (1213.73) was noted upon seed treatment with MMR04 and hence further evaluated for its ability to induce drought stress. The seed treatment with MMR04 improved plant growth parameters and total chlorophyll and RWC in plants grown under severe drought stress (G5) conditions compared to control plants. In addition, MMR04 seed treatment enhanced proline, APX and SOD activity while decreased the MDA content up to 2.3 fold compared to untreated plants (G5). Gene expression studies revealed a significant decrease of 3.3 and 1.8 fold in the relative expression of drought-responsive (DREB-1E) and ethylene-responsive factor (ERF-1B) marker genes, respectively and an increase of 2.2 and 2.9 fold in the relative expression of APX1 and SOD1, respectively in MMR04 treated plants grown under G5 conditions over control. The results confirmed that ACC deaminase producing B. amyloliquefaciens MMR04 could defend the pearl millet plants against drought stress through an antioxidative system, thereby warranting its application in drought stress management.
Assuntos
Bacillus amyloliquefaciens , Secas , Interações entre Hospedeiro e Microrganismos , Pennisetum , Antioxidantes/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Carbono-Carbono Liases/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Pennisetum/microbiologiaRESUMO
DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.
Assuntos
5-Metilcitosina/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Escherichia coli K12/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Acinetobacter calcoaceticus/enzimologia , Acinetobacter calcoaceticus/genética , Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/genética , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Sequência de Bases , Clostridium acetobutylicum/enzimologia , Clostridium acetobutylicum/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas de Restrição do DNA/genética , Escherichia coli K12/enzimologia , Regulação Bacteriana da Expressão Gênica , Haemophilus/enzimologia , Haemophilus/genética , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Humanos , Microbiota/genética , Análise de Sequência de DNA , Pele/microbiologiaRESUMO
To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.
Assuntos
Fermentação , Glycine max/microbiologia , Peptídeo Hidrolases/genética , Alimentos de Soja/microbiologia , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/metabolismo , Biossíntese Peptídica/genética , Peptídeo Hidrolases/química , Peptídeos/química , Peptídeos/genética , Álcool Feniletílico/química , Álcool Feniletílico/metabolismo , Glycine max/metabolismoRESUMO
BACKGROUND: 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. RESULTS: A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. CONCLUSIONS: The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable.
Assuntos
Bacillus amyloliquefaciens/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carboxiliases/química , Carboxiliases/genética , Engenharia de Proteínas , Bacillus amyloliquefaciens/química , Bacillus amyloliquefaciens/genética , Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Catálise , Ácidos Cumáricos/metabolismo , Mutagênese Sítio-Dirigida , Domínios ProteicosRESUMO
We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.
Assuntos
Escherichia coli/enzimologia , Transglutaminases/biossíntese , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Reatores Biológicos , Meios de Cultura , Escherichia coli/genética , Glucose , Plasmídeos/genética , Transglutaminases/genéticaRESUMO
Bacillus amyloliquefaciens strain PPL shows a potential for the control of phytopathogenic fungi. In the present study, upon growing the strain PPL on various forms of chitosan (0.5 % powder, 0.1 % soluble, and 0.15 % colloidal) as the carbon source, the antifungal activity on tomato Fusarium wilt correlated with the activity of chitosanase and ß-1,3-glucanase. The colloidal substrate-based strain PPL fermentation displayed the highest degree of spore germination inhibition (79.5 %) and biocontrol efficiency (76.0 %) in tomato by increased biofilm formation. The colloidal culture upregulated the expression of chitosanase gene (5.9-fold), and the powder attributed to the expression of cyclic lipopeptides-genes (2.5-5.7 fold). Moreover, the three chitosan cultures induced the morphological changes of Fusarium oxysporum. These results suggest that the choice of growth substrate synergistically affects the production of secondary metabolites by PPL strain, and consequently its antifungal activity.
Assuntos
Quitosana/química , Polímeros/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/crescimento & desenvolvimento , Bacillus amyloliquefaciens/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Biofilmes , Fusarium/efeitos dos fármacos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Lipopeptídeos/metabolismo , Solanum lycopersicum/microbiologiaRESUMO
High lead (Pb) concentration in soils is becoming a severe threat to human health. It also deteriorates plants, growth, yield and quality of food. Although the use of plant growth-promoting rhizobacteria (PGPR), biochar and compost can be effective environment-friendly amendments for decreasing Pb stress in crop plants, the impacts of their simultaneous co-application has not been well documented. Thus current study was carried, was conducted to investigate the role of rhizobacteria and compost mixed biochar (CB) under Pb stress on selected soil properties and agronomic parameters in mint (Mentha piperita L.) plants. To this end, six treatments were studied: Alcaligenes faecalis, Bacillus amyloliquefaciens, CB, PGPR1 + CB, PGPR2 + CB and control. Results showed that the application A. faecalis + CB significantly decreased soil pH and EC over control. However, OM, nitrogen, phosphorus and potassium concentration were significantly improved in the soil where A. faecalis + CB was applied over control. The A. faecalis + CB treatment significantly improved mint plant root dry weight (58%), leaves dry weight (32%), chlorophyll (37%), and N (46%), P (39%) and K (63%) leave concentration, while also decreasing the leaves Pb uptake by 13.5% when compared to the unamended control. In conclusion, A. faecalis + CB has a greater potential to improve overall soil quality, fertility and mint plant productivity under high Pb soil concentration compared to the sole application of CB and A. faecalis.
Assuntos
Carvão Vegetal/metabolismo , Compostagem/métodos , Chumbo/toxicidade , Mentha/efeitos dos fármacos , Rizosfera , Poluentes do Solo/toxicidade , Alcaligenes faecalis/enzimologia , Alcaligenes faecalis/metabolismo , Aminoidrolases/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Frutas/química , Chumbo/metabolismo , Mentha/microbiologia , Poluentes do Solo/metabolismo , Estresse Fisiológico , Verduras/químicaRESUMO
A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram's staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS-PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10-90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with KM and Vmax of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H2O2), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens. The structure of this protease and its highest-priority substrate ß-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein-protein (protease from HM48 and ß-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.
Assuntos
Bacillus amyloliquefaciens/enzimologia , Temperatura Alta , Peptídeo Hidrolases/metabolismo , Microbiologia do Solo , Animais , Bacillus amyloliquefaciens/citologia , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/isolamento & purificação , Caseínas/metabolismo , Galinhas , Ácido Edético/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Plumas , Geografia , Concentração de Íons de Hidrogênio , Índia , Íons , Cinética , Metais/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Peso Molecular , Oxidantes/farmacologia , Peptídeo Hidrolases/genética , Proteólise/efeitos dos fármacos , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Solventes , Especificidade por Substrato/efeitos dos fármacos , Tensoativos/farmacologiaRESUMO
Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.
Assuntos
Bacillus amyloliquefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/biossíntese , Bacillus amyloliquefaciens/química , Bacillus amyloliquefaciens/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Quitina/metabolismo , Quitosana/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Especificidade por SubstratoRESUMO
The spore laccase enzyme production by B. amyloliquefaciens was optimized. It was characterized and tested for its textile dye decolorization potential. LB medium was found to be the most promising growth medium with addition of glucose (1-2%), yeast extract (0.1%), FeCl3 (0.01 mM) and MnCl2 (0.001 mM). The optimum spore laccase production was at pH 8, 30 °C, 1:5 medium to air ratio, 2% inoculum size and 7 days incubation. The characterization study of the enzyme showed the maximum activity at 60 °C and pH 6-7.5. It was induced by Ca+2, Mg+2, Fe+3, Zn+2, Cu+2 and Na+ at 1 mM concentration. Also, it was stable in the presence of methanol, ethanol, acetone and chloroform. In addition, it enhanced about 34% by 5 mM H2O2 and it was nearly stable at 10-20 mM H2O2. Furthermore, mediators such as ABTS, syrengaldazine and 2, 6 dimethyl phenol enhanced the spore laccase activity. The spore laccase enzyme efficiently decolorized direct red 81 and acid black 24 after 24 h. Phytotoxicity of the direct red 81 solution after decolorization by tested spore laccase was lower than that of the untreated dye solution. Finally, this study added a promising spore laccase candidate for ecofriendly and cost-effective dye wastewater bio-decolorization.
Assuntos
Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/isolamento & purificação , Corantes/metabolismo , Lacase/metabolismo , Esporos Bacterianos/enzimologia , Têxteis , Águas Residuárias/microbiologia , Descoloração da Água/métodos , Poluentes Químicos da Água/metabolismo , Compostos Azo/metabolismo , Compostos Azo/farmacologia , Biodegradação Ambiental , Corantes/farmacologia , Meios de Cultura , Temperatura Alta , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Lens (Planta)/efeitos dos fármacos , Sementes/efeitos dos fármacos , Poluentes Químicos da Água/farmacologiaRESUMO
Laccases are versatile oxidases that are capable of decolorizing various synthetic dyes. Recombinant Bacillus amyloliquefaciens laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) for application in dye decolorization. Several parameters influencing the activity recovery were evaluated during the synthesis of M-CLEAs. With ammonium sulfate as precipitant, maximum activity was recovered by cross-linking with 0.16% glutaraldehyde for 1 h. The prepared M-CLEAs exhibited improved activity under alkaline conditions. It remained 74% activity after incubation at 60 °C for 5 h. Enhanced tolerance towards NaCl was also observed for the M-CLEAs, with 68% activity remaining in the presence of 1 M NaCl. The immobilized laccase could rapidly decolorize more than 93% of reactive black 5 and indigo carmine in 1 h, while its catalytic efficiency towards reactive blue 19 was relatively low. After four cycles of consecutive reuse, the M-CLEAs could decolorize 92% of indigo carmine. The easy recovery and reusability of M-CLEAs facilitate the potential application of bacterial laccase in dye decolorization.
Assuntos
Bacillus amyloliquefaciens/enzimologia , Biotecnologia/métodos , Corantes/química , Microbiologia Industrial/métodos , Lacase/química , Magnetismo , Sulfato de Amônio/química , Carmim/química , Catálise , Domínio Catalítico , Reagentes de Ligações Cruzadas , Enzimas Imobilizadas , Glutaral/química , Concentração de Íons de Hidrogênio , Índigo Carmim/química , Proteínas Recombinantes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Fatores de TempoRESUMO
A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo- was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo- achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.
Assuntos
Bacillus amyloliquefaciens , Proteínas de Bactérias , Edição de Genes , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , alfa-Amilases , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , alfa-Amilases/biossíntese , alfa-Amilases/genéticaRESUMO
Ethyl carbamate (EC) is a carcinogen detected in fermented foods and alcohol beverages. Excessive intake of EC is possibly harmful to health. Enzymatic degradation is one of the most effective approaches for reducing EC in fermented foods. Urease catalyzes the hydrolysis of both EC and urea. This confers urease a good application prospect in reducing EC and its precursor urea in fermented foods. Currently, degradation of EC in alcohol beverages by urease is inefficient due to its low urethanase activity and poor affinity to EC. Urease from Bacillus amyloliquefaciens JP-21 was successfully expressed in Escherichia coli at the level of 3 292 U/L urease and 227.3 U/L urethanase. Two key residues M326 and M374 were characterized that might block the binding of enzyme to EC, through simulating docking the structure of catalytic subunit UreC of urease with EC. Three mutants (M374A, M374T and M326V) of urease with improved urethanase activity were obtained by performing point saturated mutagenesis approach. Using EC as the substrate, Km values of M374A, M374T and M326V were detected to be 101.8 mmol/L, 129.5 mmol/L and 121.7 mmol/L, respectively, which were decreased by 37.47%-50.82% compared with that of the wild type urease. These mutants can degrade more than 97% of urea in rice wine and mutant M374T shows the highest degradation of EC in rice wine. EC content in rice wine was reduced from 525 µg/L to 393 µg/L by using M374T, and the EC degradation rate of it is 0.97 folds higher than that of the wild type urease. The results are of great significance for engineering the catalytic properties of urease and improving its industrial properties, and lays a good foundation for developing strategies to reducing microbial metabolic ammonia (amine) hazards in fermented foods.
Assuntos
Bacillus amyloliquefaciens , Microbiologia de Alimentos , Oryza , Urease , Uretana , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Carcinógenos/metabolismo , Mutagênese Sítio-Dirigida , Urease/genética , Urease/metabolismo , Uretana/metabolismo , Vinho/microbiologiaRESUMO
L-theanine, a unique amino acid in green tea with health benefits, can be enzymatically synthesized by γ-glutamyltranspeptidase (γ-GT; EC 2.3.2.2). Here, a salt-tolerant γ-glutamyltranspeptidase from a marine bacterium Bacillus amyloliquefaciens was expressed in Escherichia. coli BL21 (DE3) and was shown to be optimally active at 55 °C, pH 8.5 and alkali stable. A mutant, with higher transpeptidation activity, was obtained following two rounds of directed evolution using error-prone PCR and site-saturation mutagenesis. The mutation increased the ratio of transpeptidation to hydrolysis from 1.6 to 35.6. Additionally, Kinetic analysis exhibited 17.5% decrease of Km, 13.0-fold increase of Kcat, and 16.3-fold increase of Kcat/Km in mutant V319A/S437 G versus the wild-type. The 3-D modelling analysis revealed a tighter binding pocket in mutant V319A/S437 G. The frequency of hydrogen bond between donor substrate and two residues in the catalytic pocket (Gly437 and Thr375) was enhanced, which stabilized the ligand binding and thus improved the catalytic efficiency. The optimal conditions for the biocatalytic synthesis were determined as pH 10.0, 20 µg mL-1BaGT, 200 mM L-glutamine, 2 M ethylamine, and a reaction time of 5 h. The V319A/S437 G mutant was shown to increase the percentage yield of L-theanine from 58% to 83%. These results indicate the great potential of V319A/S437 G in L-theanine production after further study.