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1.
J Agric Food Chem ; 67(40): 11198-11209, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31532988

RESUMO

The importance of inhibition sensitivity for xylanase functionality in bread making was investigated using mutants of the wild-type Bacillus subtilis xylanase (XBSTAXI), sensitive to Triticum aestivum xylanase inhibitor (TAXI). XBSNI, a mutant with reduced sensitivity to TAXI, and XBSTI, a mutant sensitive to all wheat endogenous proteinaceous inhibitors (TAXI, Xylanase Inhibiting Protein and Thaumatin-like Xylanase Inhibitor) were used. The higher inhibition sensitivity of XBSTAXI and XBSTI compared to XBSNI was associated with a respective 7- and 53-fold increase in enzyme dosage required for a maximal increase in bread loaf volume. XBSTI and XBSTAXI were only active during the mixing phase and the beginning of fermentation, while XBSNI was able to hydrolyze arabinoxylan until the end of fermentation. In spite of this difference in activity profile, no differences in loaf volume were observed for the different xylanases at optimal concentrations. Dough extensional viscosity analysis suggests that increased water availability as a result of xylanase activity favors starch-starch and starch-gluten interactions and drives the improvement in bread loaf volume.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Pão/análise , Endo-1,4-beta-Xilanases/antagonistas & inibidores , Endo-1,4-beta-Xilanases/química , Inibidores Enzimáticos/química , Proteínas de Plantas/química , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Inibidores Enzimáticos/metabolismo , Farinha/análise , Manipulação de Alimentos , Hidrólise , Mutação , Proteínas de Plantas/metabolismo , Triticum/química , Triticum/metabolismo , Viscosidade
2.
J Microbiol Biotechnol ; 29(8): 1281-1287, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370114

RESUMO

Nattokinase (NK, E.C. 3.4.21.62) is a serine protease produced by Bacillus subtilis natto that shows promise for the treatment of thrombotic disease. In this study, we assessed the effects of NK on the development of hepatocellular carcinoma (HCC), a principal malignancy of the liver that causes morbidity and mortality worldwide. Crude extracts of NK (NCE) were isolated from fermentation medium by centrifugation and separated into three fractions (<10 K, 100~30 K and >30K). Orthotopic HCC mouse models were established and NCE was administered by oral gavage. H&E staining was performed to examine the pathology of HCC livers. Immunohistochemistry and immunofluorescence were used to evaluate FOXM1, CD31, CD44 and vimentin expression in the liver. Compared to PBS groups, NCE increased the survival rates of HCC-bearing mice to 31% and decreased ascites. Low-intensity ultrasound imaging showed that the hypoechoic mass area was lower in NCE-treated mice and that tumor growth significantly decreased. IHC staining showed that the expression of FOXM1 was inhibited by NCE treatment. Immunofluorescence results revealed lower levels of CD31, CD44 and vimentin in the NCE groups. Taken together, these data demonstrate that NCE from Bacillus subtilis natto improves survival and inhibits tumor growth in HCC mice.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Misturas Complexas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Subtilisinas/farmacologia , Animais , Bacillus subtilis/enzimologia , Carcinoma Hepatocelular/patologia , Misturas Complexas/isolamento & purificação , Modelos Animais de Doenças , Fermentação , Fibrinolíticos/farmacologia , Proteína Forkhead Box M1/análise , Receptores de Hialuronatos/análise , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Subtilisinas/isolamento & purificação , Vimentina/análise
3.
J Agric Food Chem ; 67(33): 9314-9324, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352776

RESUMO

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Trealose/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Fermentação , Maltose/metabolismo , Engenharia Metabólica
4.
Nat Commun ; 10(1): 3099, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308373

RESUMO

The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Engenharia Metabólica/métodos , Optogenética/métodos , Fitocromo/genética , Proteínas Quinases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Luz , Ficobilinas/biossíntese , Ficocianina/biossíntese , Fitocromo/metabolismo , Regiões Promotoras Genéticas/efeitos da radiação , Proteínas Quinases/metabolismo
5.
World J Microbiol Biotechnol ; 35(8): 122, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346836

RESUMO

To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn't destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn't inhibited with Na+, K+ and Ca2+ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn't further degrade collagen in the time range of 8 h to 24 h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Bacteriano/isolamento & purificação , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fragmentação do DNA , DNA Bacteriano/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Microb Cell Fact ; 18(1): 100, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159804

RESUMO

BACKGROUND: Bacillus subtilis spores have been commonly used for the surface display of various food-related or human antigens or enzymes. For successful display, the target protein needs to be fused with an anchor protein. The preferred anchored proteins are the outer-coat proteins of spores; outer-coat proteins G (CotG) and C (CotC) are commonly used. In this study, mutant trehalose synthase (V407M/K490L/R680E TreS) was displayed on the surface of B. subtilis WB800n spores using CotG and CotC individually or in combination as an anchoring protein. RESULTS: Western blotting, immunofluorescence, dot blot, and enzymatic-activity assays detected TreS on the spore surface. The TreS activity with CotC and CotG together as the anchor protein was greater than the sum of the enzymatic activities with CotC or CotG alone. The TreS displayed on the spore surface with CotC and CotG together as the anchoring protein showed elevated and stable specific activity. To ensure spore stability and prevent spore germination in the trehalose preparation system, two germination-specific lytic genes, sleB and cwlJ, were deleted from the B. subtilis WB800n genome. It was demonstrated that this deletion did not affect the growth and spore formation of B. subtilis WB800n but strongly inhibited germination of the spores during transformation. The conversion rate of trehalose from 300 g/L maltose by B. subtilis strain WB800n(ΔsleB, ΔcwlJ)/cotC-treS-cotG-treS was 74.1% at 12 h (350 U/[g maltose]), and its enzymatic activity was largely retained, with a conversion rate of 73% after four cycles. CONCLUSIONS: The spore surface display system based on food-grade B. subtilis with CotC and CotG as a combined carrier appears to be a powerful technology for TreS expression, which may be used for the biotransformation of D-maltose into D-trehalose.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Glucosiltransferases/genética , Esporos Bacterianos/enzimologia , Trealose/biossíntese , Bacillus subtilis/genética , Técnicas de Inativação de Genes , Esporos Bacterianos/genética
7.
J Agric Food Chem ; 67(24): 6837-6846, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31180217

RESUMO

Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , beta-Manosidase/química , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclização , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Engenharia de Proteínas , beta-Manosidase/genética , beta-Manosidase/metabolismo
8.
Protein Pept Lett ; 26(9): 702-716, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31215367

RESUMO

OBJECTIVE: Dynamic communication caused by mutation affects protein stability. The main objective of this study is to explore how mutations affect communication and to provide further insight into the relationship between heat resistance and signal propagation of Bacillus subtilis lipase (Lip A). METHODS: The relationship between dynamic communication and Lip A thermostability is studied by long-time MD simulation and residue interaction network. The Dijkstra algorithm is used to get the shortest path of each residue pair. Subsequently, time-series frequent paths and spatio-temporal frequent paths are mined through an Apriori-like algorithm. RESULTS: Time-series frequent paths show that the communication between residue pairs, both in wild-type lipase (WTL) and mutant 6B, becomes chaotic with an increase in temperature; however, more residues in 6B can maintain stable communication at high temperature, which may be associated with the structural rigidity. Furthermore, spatio-temporal frequent paths reflect the interactions among secondary structures. For WTL at 300K, ß7, αC, αB, the longest loop, αA and αF contact frequently. The 310-helix between ß3 and αA is penetrated by spatio-temporal frequent paths. At 400K, only αC can be frequently transmitted. For 6B, when at 300K, αA and αF are in more tight contact by spatio-temporal frequent paths though I157M and N166Y. Moreover, the rigidity of the active site His156 and the C-terminal of Lip A are increased, as reflected by the spatio-temporal frequent paths. At 400K, αA and αF, 310-helix between ß3 and αA, the longest loop, and the loop where the active site Asp133 is located can still maintain stable communication. CONCLUSION: From the perspective of residue dynamic communication, it is obviously found that mutations cause changes in interactions between secondary structures and enhance the rigidity of the structure, contributing to the thermal stability and functional activity of 6B.


Assuntos
Bacillus subtilis/enzimologia , Lipase/química , Simulação de Dinâmica Molecular , Domínio Catalítico , Estabilidade Enzimática , Temperatura Alta , Cinética , Lipase/genética , Mutação , Estrutura Secundária de Proteína , Temperatura Ambiente , Termodinâmica
9.
Eur J Med Chem ; 174: 252-264, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31048140

RESUMO

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Šcrystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.


Assuntos
Adenosina/análogos & derivados , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/química , Sulfonamidas/química , Adenosina/síntese química , Aminoacil-tRNA Sintetases/química , Aminoacilação , Bacillus subtilis/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/síntese química , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Pectobacterium chrysanthemi/enzimologia , Sulfolobus/enzimologia , Sulfonamidas/síntese química , Thermus thermophilus/enzimologia
10.
Pol J Microbiol ; 68(1): 105-114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050258

RESUMO

Bacteria from the genus Bacillus are a rich source of commercial enzymes, including amylases, proteases, cellulases, glucose isomerase, and pullulanase. Cellulases account for 15% of the global market of industrial enzymes; thus, new microorganisms producing cellulases in a higher concentration and new ingredients, which can enhance the level of enzyme synthesis, are still needed. Many of cellulose-degrading microorganisms have been isolated so far and characterized in various regions of the world. In this study, we were looking for the bacteria isolated from the natural environment with the high cellulolytic potential, which could be used as components of a biopreparation to accelerate decomposition of postharvest leftovers in agriculture. The 214 bacterial strains were isolated from environmental samples rich in cellulose and their ability to synthesize cellulases were examined using the diffusion method. Six strains, which have the highest diameter of clearing zone both for biomass and supernatant, were selected for identification. Optimization of biosynthesis of the cellulose-degrading enzymes indicated that optimal temperature of this process fluctuated in the range of 21-42°C (depending on the strain and carbon source). The highest cellulolytic activity was observed for the isolates designed as 4/7 (identified as Bacillus subtilis) and 4/18 (identified as Bacillus licheniformis) in a temperature of 32°C. With the use of a desirability function methodology, the optimal medium composition to achieve a simple, cost-efficient process of cellulases production was developed for both strains. These experiments show that microorganisms isolated from natural environmental samples have unique properties and potential for commercial applications (e.g. for biopreparations production).Bacteria from the genus Bacillus are a rich source of commercial enzymes, including amylases, proteases, cellulases, glucose isomerase, and pullulanase. Cellulases account for 15% of the global market of industrial enzymes; thus, new microorganisms producing cellulases in a higher concentration and new ingredients, which can enhance the level of enzyme synthesis, are still needed. Many of cellulose-degrading microorganisms have been isolated so far and characterized in various regions of the world. In this study, we were looking for the bacteria isolated from the natural environment with the high cellulolytic potential, which could be used as components of a biopreparation to accelerate decomposition of postharvest leftovers in agriculture. The 214 bacterial strains were isolated from environmental samples rich in cellulose and their ability to synthesize cellulases were examined using the diffusion method. Six strains, which have the highest diameter of clearing zone both for biomass and supernatant, were selected for identification. Optimization of biosynthesis of the cellulose-degrading enzymes indicated that optimal temperature of this process fluctuated in the range of 21­42°C (depending on the strain and carbon source). The highest cellulolytic activity was observed for the isolates designed as 4/7 (identified as Bacillus subtilis) and 4/18 (identified as Bacillus licheniformis) in a temperature of 32°C. With the use of a desirability function methodology, the optimal medium composition to achieve a simple, cost-efficient process of cellulases production was developed for both strains. These experiments show that microorganisms isolated from natural environmental samples have unique properties and potential for commercial applications (e.g. for biopreparations production).


Assuntos
Bacillus licheniformis/enzimologia , Bacillus licheniformis/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Celulases/metabolismo , Celulose/metabolismo , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus licheniformis/isolamento & purificação , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Biomassa , Nitrogênio/metabolismo , Microbiologia do Solo , Temperatura Ambiente
11.
Prep Biochem Biotechnol ; 49(6): 578-583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30957714

RESUMO

(S)-1-(2, 6-dichloro-3-fluorophenyl) ethanol, the key chiral intermediate of crizotinib, was prepared from 1-(2, 6-dichloro-3-fluorophenyl) ethanone using the alcohol dehydrogenases from Lactobacillus kefir (ADH-LK) with a tetrad mutant (ADH-LKM, F147L/Y190P/V196L/A202W), coupled with glucose dehydrogenase (GDH). In the present study, ADH-LKM and GDH were successfully heterologous expressed in recombinant Escherichia coli. During the regeneration of NADPH with GDH, 150 g/L substrate was totally transformed into target chiral alcohol with an enantiomeric excess value of 99.9% after 12 h at 30 °C (pH 7.0). Our study demonstrates the potential for industrial green production of the key chiral intermediate of crizotinib.


Assuntos
Álcool Desidrogenase/metabolismo , Álcoois Benzílicos/metabolismo , Crizotinibe/química , Glucose 1-Desidrogenase/metabolismo , Kefir/microbiologia , Lactobacillus/enzimologia , Acetofenonas/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Álcoois Benzílicos/química , Biotransformação/efeitos dos fármacos , Escherichia coli/genética , Química Verde/métodos , Concentração de Íons de Hidrogênio , Lactobacillus/genética , NADP/metabolismo , Estereoisomerismo , Temperatura Ambiente
12.
J Biosci Bioeng ; 128(2): 156-161, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30862433

RESUMO

Food processing technology such as protein hydrolysis using proteases has been receiving a lot of attention, and it is important to accurately understand the cleavage specificity of each protease for selecting a protease suited to aims. Although numerous methods have been reported to reveal the substrate specificity of proteases, there is no method to evaluate simply, quickly, reasonably, and accurately. This study set out to devise Pep-MS assay, a novel assay system that can be used to comprehensively clarify positions at which proteases cleave, by combining a mass spectrometer and a photo-cleavable peptide array. First, we evaluated peptide array corresponding to the primary sequences of αS1-casein, αS2-casein and ß-casein with trypsin to verify the accuracy of the Pep-MS assay. The evaluation of cleavage positions by the trypsin protease reagent using the Pep-MS assay resulted in a matching rate of about 96.8% to rational cleavage positions. Next, we confirmed the cleavage positions in αS2-casein or ß-lactoglobulin by an industrial bacterial protease from Bacillus subtilis at some protease reaction temperatures or reaction times. The Pep-MS assay clarified the differences in the cleavage patterns due to the reaction temperature, and the change in the cleavage strength with the reaction time. Pep-MS assay is a promising method for evaluating the substrate specificity of proteases, which will be useful to find effective production conditions for functional peptide from foods and effective hydrolysis conditions for decreasing allergen of food proteins.


Assuntos
Caseínas/metabolismo , Lactoglobulinas/metabolismo , Espectrometria de Massas , Processos Fotoquímicos , Análise Serial de Proteínas , Tripsina/metabolismo , Bacillus subtilis/enzimologia , Manipulação de Alimentos , Hidrólise , Cinética , Proteólise , Especificidade por Substrato , Temperatura Ambiente
13.
Bioengineered ; 10(1): 43-51, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30876377

RESUMO

α-keto acids are organic compounds that contain an acid group and a ketone group. L-amino acid deaminases are enzymes that catalyze the oxidative deamination of amino acids for the formation of their corresponding α-keto acids and ammonia. α-keto acids are synthesized industrially via chemical processes that are costly and use harsh chemicals. The use of the directed evolution technique, followed by the screening and selection of desirable variants, to evolve enzymes has proven to be an effective way to engineer enzymes with improved performance. This review presents recent studies in which the directed evolution technique was used to evolve enzymes, with an emphasis on L-amino acid deaminases for the whole-cell biocatalysts production of α-keto acids from their corresponding L-amino acids. We discuss and highlight recent cases where the engineered L-amino acid deaminases resulted in an improved production yield of phenylpyruvic acid, α-ketoisocaproate, α-ketoisovaleric acid, α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and pyruvate.


Assuntos
Amidoidrolases/metabolismo , Aminoácidos/metabolismo , Amônia-Liases/metabolismo , Evolução Molecular Direcionada/métodos , Microbiologia Industrial/métodos , Engenharia de Proteínas/métodos , Amidoidrolases/química , Amidoidrolases/genética , Aminoácidos/química , Amônia-Liases/química , Amônia-Liases/genética , Bacillus subtilis/química , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Cetoácidos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metionina/análogos & derivados , Metionina/biossíntese , Proteus/química , Proteus/enzimologia , Proteus/genética , Ácido Pirúvico/metabolismo
14.
Eur J Med Chem ; 171: 209-220, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925337

RESUMO

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis that is essential for bacterial survival. Therefore, FabH has been identified as an attractive target for the development of new antibacterial agents. We present here the discovery of a promising new series of Pyrazol-Benzimidazole amides with low toxicity and potent FabH inhibitory. Twenty-seven novel compounds have been synthesized, and all the compounds were characterized by 1H NMR, 13C NMR and MS. Afterwards they were evaluated for in-vitro antibacterial activities against E. coli, P. aeruginosa, B. subtilis and S. aureus, along with E. coli FabH inhibition and cytotoxicity test. Some compounds proved to be of low toxicity and potent, especially compound 31 exhibited the most potential to be a new drug with MIC of 0.49-0.98 µg/mL against the tested bacterial strains and IC50 of 1.22 µM against E. coli FabH. Eight analogues 16, 28, 30, 31, 33, 34, 35 and 36 with low range MIC against wild type Xanthomonas Campestris exhibited no inhibition against FabH-deficient mutant strain, which firmly proved the class of compounds arrived at antibacterial activity via interacting with FabH. In silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) evaluation also pointed out that these compounds are potential for druggability. Further, effective overall docking scores of all the compounds have been recorded, and docking simulation of compound 31 into E. coli FabH binding pocket has been conducted, where solid binding interactions has been identified.


Assuntos
Bacillus subtilis/enzimologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
15.
Nat Chem ; 11(4): 335-341, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804500

RESUMO

Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.


Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Aminoácidos/química , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Ensaios Enzimáticos/métodos , Cinética , Streptomyces/enzimologia , Streptomyces/metabolismo
16.
Int J Biol Macromol ; 128: 237-243, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30703417

RESUMO

Levan is a fructan whose backbone is composed of ß-(2-6) linkages. It is synthetized by the enzyme levansucrase (EC 2.4.1.10). The effect of Fe3+, K+, Mg2+, Mn2+, and Zn2+ in the range of 0-1 mM on parameters of levan was tested using design of response surface experiment. The bimodal distribution of levan was observed, however, the concentration of high molecular mass fraction (weight average molar mass, Mw = 1.9 ·â€¯107 ±â€¯3.4 ·â€¯104 g/mol and intrinsic viscosity, [η] = 0.206 ±â€¯0.016 dL/g,) was not >4% of the total yield of levan. The metal ions in the reaction medium had an effect on parameters of low molecular mass levan and the efficiency of synthesis. Molar mass distribution of abundant, low molecular mass fraction was in the range of 4.33 ·â€¯104 g/mol to 9.77 ·â€¯104 g/mol; [η] was 0.040-0.075 dL/g while the efficiency of transfructosylation was within the range of 61.4to 69.1%. It was observed that the molar mass of levan depends on Fe3+ concentration while intrinsic viscosity is affected by the concentration of Mn2+ in the reaction medium. The GPC (with triple detection) data was analyzed using response surface methodology (RSM), k-means and principal component analysis (PCA).


Assuntos
Bacillus subtilis/enzimologia , Hexosiltransferases/química , Íons/química , Metais/química , Hexosiltransferases/síntese química , Microscopia de Força Atômica , Viscosidade
17.
Food Chem ; 282: 101-108, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711093

RESUMO

A one-step method to immobilize xylanase onto cellulosic material by fusion of expansin from Bacillus subtilis to xylanase LC9 without the requirement of prior purification of enzyme has been developed. Fusion enzyme EXLX-R2-XYN was specifically adsorbed onto corncob residue with high loading capacity due to bio-affinity adsorption of expansin onto cellulose. The immobilization yield was close to 100%, with a recovered activity of 82.4%. The immobilized EXLX-R2-XYN retained 45.3% of its activity after incubation at 70 °C for 3 h, whereas only 16.3% of the activity was left in free form under the same conditions. The conversion yield of XOS by using immobilized EXLX-R2-XYN reached up to 515 mg/g xylan from 2% corncob extracted xylan, which was higher than that of the free enzyme. The hydrolysis products were mainly xylobiose (57.5%) and xylotriose (38.4%), without undesirable xylose production. After five cycles of hydrolysis, more than 70% of conversion was obtained.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Bactérias/genética , Cromatografia de Afinidade , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/genética , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucuronatos/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/isolamento & purificação , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Reciclagem , Temperatura Ambiente , Trissacarídeos/metabolismo
18.
J Biotechnol ; 293: 66-71, 2019 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-30703469

RESUMO

Enzymatic degradation of urea, the precursor of carcinogenic compound ethylcarbamate in rice wine, is always attractive. In the present study, we achieved high efficient production of Bacillus paralicheniformis iron-containing urease (Bp_Urease) in B. subtilis with the food-grade expression system. After reassembly of the urease gene cluster with inserting ribosome binding site (RBS), the production was increased from 38 U/L to 187 U/L. After altering the position of ureC and co-expressing the iron transporter encoding gene ureH, the activity was further increased to 1307 U/L. Eventually, the urease production was improved to 21,964 U/L in 3-L fermentor, which is the highest reported value to date. Food-grade production of the iron-containing urease would be favorable to the practical applications in food industries.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Urease/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Alimentos , Ferro , Urease/genética
19.
Enzyme Microb Technol ; 124: 32-40, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30797477

RESUMO

Endosulfan is one of the most widely used organochlorine cyclodiene insecticides. Microbial oxidation of endosulfan forms endosulfan sulfate, which is more or less toxic and persistent as endosulfan. Due to lack of specificity and efficiency of microbial bioremediation technique in the field conditions, enzymatic bioremediation is receiving huge attention to clean-up the environment. In the present study, X-ray crystal structures of enzymes from Brookhaven Protein Data Bank were screened for their potential to degrade endosulfan and endosulfan sulfate using molecular docking and molecular dynamics simulation techniques. A phenol hydroxylase, 1PN0 from Trichosporon cutaneum was found to have the potential to degrade both α-endosulfan and endosulfan sulfate while a bacterial CotA laccase, 3ZDW from Bacillus subtilis has the potential to degrade α-endosulfan. The in silico result correlate with in vitro degradation study using two different strains of Trichosporon cutaneum. In vitro degradation study found that the fungal strain was capable of degrading 60.36% α-endosulfan, 70.73% ß-endosulfan, and 52.08% endosulfan sulfate. The presence of phenol hydroxylase inhibitor in the sulfur-free medium with endosulfan and endosulfan sulfate as sole sulfur source inhibits the growth of both the fungal strains. Such in silico techniques can provide an easy and reliable way to speed up the development of bioremediation processes through rapid identification of potential enzymes and microbes to counter the ever-increasing number of toxic compounds in the environment.


Assuntos
Basidiomycota/metabolismo , Endossulfano/análogos & derivados , Endossulfano/metabolismo , Inseticidas/metabolismo , Oxigenases de Função Mista/metabolismo , Simulação de Acoplamento Molecular , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Basidiomycota/enzimologia , Basidiomycota/crescimento & desenvolvimento , Biodegradação Ambiental , Bases de Dados de Proteínas , Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores
20.
Enzyme Microb Technol ; 124: 54-62, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30797479

RESUMO

Enzymatic production of chitooligosaccharides has high value in medicine and other fields. However, low chitinase activity and yield of chitooligosaccharides limit the production and application. Herein, we used a series of molecular biology strategies to increase the expression of chitinase in Bacillus subtilis WB600. Upon addition of the signal peptide NprB, Chisb was successfully secreted to the outside of the cell and extracellular expression level reached 35.54 U/mL. Furthermore, optimizing Ribosome Binding Sites (RBSs) with spacer sequences, and combining molecular docking technology with site-directed mutagenesis, the expression level and the specific activity of Chisb was further increased to 51.67 U/mL and 249.62 U/mg, respectively. When colloidal chitin was used as the substrate, the chitooligosaccharides detected by ion chromatography were (GlcNAc)1-5, and the total yield of chitooligosaccharides was 14.4%. Our results indicate that strategies for increasing Chisb expression contribute to the study and application of chitinase and the production of chitooligosaccharides.


Assuntos
Bacillus subtilis/enzimologia , Quitina/análogos & derivados , Quitinases/genética , Quitinases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sítios de Ligação , Quitina/metabolismo , Quitinases/isolamento & purificação , Clonagem Molecular , DNA Espaçador Ribossômico/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura Ambiente
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