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1.
Biosci Biotechnol Biochem ; 85(8): 1830-1838, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34021568

RESUMO

Information about the inulosucrase of nonlactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the Gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0-9.0 and 50-55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multiangle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3-27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.


Assuntos
Bacillaceae/enzimologia , Hexosiltransferases/metabolismo , Bacillaceae/genética , Cromatografia Líquida de Alta Pressão/métodos , Clonagem Molecular , Meios de Cultura , Genes Bacterianos , Hexosiltransferases/genética , Hexosiltransferases/isolamento & purificação , Inulina/biossíntese , Espectroscopia de Ressonância Magnética/métodos , Peso Molecular , Filogenia , Temperatura
2.
Carbohydr Polym ; 262: 117968, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838833

RESUMO

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Glicosídeos/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilose/química , Amilose/metabolismo , Bacillaceae/enzimologia , Digestão , Glicosídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Rhodothermus/enzimologia , Amido/metabolismo
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540582

RESUMO

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.


Assuntos
Oxirredutases do Álcool/genética , Bacillaceae/enzimologia , Mutação , Oxirredutases do Álcool/metabolismo , Bacillaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Cinética , Metanol/metabolismo
4.
N Biotechnol ; 62: 49-56, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33486119

RESUMO

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.


Assuntos
Bacillaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares
5.
Int J Biol Macromol ; 166: 557-566, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33186653

RESUMO

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH2 nanospheres showed high loading capacity of 103.8 µg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications.


Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Nanosferas/química , Biocatálise/efeitos dos fármacos , Detergentes/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Nanosferas/ultraestrutura , Oxidantes/farmacologia , Porosidade , Salinidade , Dióxido de Silício/química , Solventes/química , Tensoativos/farmacologia , Temperatura
6.
FEBS Lett ; 595(3): 351-359, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33277689

RESUMO

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin-carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α-glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α-glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α-glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate.


Assuntos
Proteínas de Bactérias/metabolismo , Esterases/metabolismo , Glicosídeo Hidrolases/metabolismo , Lignina/metabolismo , Polissacarídeos/metabolismo , Xilanos/metabolismo , Bacillaceae/química , Bacillaceae/enzimologia , Proteínas de Bactérias/genética , Betula/química , Biomassa , Ensaios Enzimáticos , Esterases/genética , Gammaproteobacteria/química , Gammaproteobacteria/enzimologia , Expressão Gênica , Ácido Glucurônico/metabolismo , Glicosídeo Hidrolases/genética , Hidrólise , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Madeira/química
7.
J Sci Food Agric ; 101(8): 3308-3318, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33222223

RESUMO

BACKGROUND: Gracilibacillus alcaliphilus SK51.001, a strain that produces ß-CGTase (ß-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the ß-CGTase gene from G. alcaliphilus SK51.001. RESULTS: According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The ß-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 µg mL-1 and 0.13 µmol mL-1  min-1 , respectively. CONCLUSION: Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, ß-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of ß-CD could allow the enzyme to be used in ß-CD production. © 2020 Society of Chemical Industry.


Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Bacillaceae/química , Bacillaceae/genética , Bacillaceae/isolamento & purificação , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/metabolismo , Temperatura Alta , Cinética , Peso Molecular , Microbiologia do Solo , Amido/metabolismo , Especificidade por Substrato , gama-Ciclodextrinas/metabolismo
8.
Subcell Biochem ; 96: 355-372, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252736

RESUMO

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has already been applied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.


Assuntos
Leucina Desidrogenase/química , Leucina Desidrogenase/metabolismo , Bacillaceae/enzimologia , Microscopia Crioeletrônica , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Leucina Desidrogenase/ultraestrutura , Sporosarcina/enzimologia
9.
Bioprocess Biosyst Eng ; 44(2): 225-234, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32888092

RESUMO

Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05T proteases which have potential for the valorization of organic wool as well as for other industrial applications.


Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias , Peptídeo Hidrolases , Proteômica , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação
10.
Prep Biochem Biotechnol ; 51(1): 28-34, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32633612

RESUMO

Organic solvent-tolerant proteases have many applications in the synthesis of peptides. In this study, we have developed a low-cost and convenient method to produce highly concentrated organic solvent-tolerant protease. Organic solvent tolerant protease (OSP) gene from Bacillus sphaericus DS11 was cloned and expressed in Bacillus subtilis WB800. The optimum pH of the recombinant protease was 9.0. The optimum temperature of the recombinant protease was 40 °C. The recombinant protease was purified by ethanol with the yield of (87.33%). The yield of OSP enriched by ethanol was higher than that of by Ni-chelating affinity chromatography, which indicated that precipitation of the recombinant OSP with ethanol is a relatively low-cost and fast method for organic solvent -tolerant protease preparation. These results showed that this enzyme could be very useful in different industrial applications.


Assuntos
Bacillaceae/enzimologia , Bacillaceae/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/química , Solventes/química , Proteínas de Bactérias/genética , Precipitação Química , Detergentes/química , Estabilidade Enzimática , Etanol/química , Genes Bacterianos , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/genética , Proteínas Recombinantes/isolamento & purificação , Temperatura
11.
Biosci Biotechnol Biochem ; 84(11): 2293-2302, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32741269

RESUMO

High collagenolytic activity has been detected in pathogenic bacteria. Collagenase plays an essential role in the invasion step in animals and humans. In this study, we characterized collagenase found in the nonpathogenic bacterium Lysinibacillus sphaericus VN3, which was isolated from soil in Vietnam. The collagenase activity of the purified enzyme was strongly inhibited by Cu2+, but it was significantly increased by Zn2+. The purified enzyme with a molecular mass of approximately 110 kDa exhibited collagenolytic, gelatinolytic, and caseinolytic activity. The kinetic studies showed that this enzyme had greater hydrolyzing activity toward collagen and gelatin compared with casein. Based on the ratio V max/K m, collagen is likely to be the best substrate among three proteins. We found that this collagenase could digest small pieces of bovine skin and tendon into a collagen solution. Interestingly, at pH 6.0-8.0, the soluble collagen could form a collagen membrane, which is useful as a wound-healing biomaterial.


Assuntos
Bacillaceae/enzimologia , Colagenases/metabolismo , Hidrólise , Temperatura
12.
Int J Biol Macromol ; 161: 1456-1469, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777411

RESUMO

A new serine alkaline protease (designated as SAPGB) from Gracilibacillus boraciitolerans strain LO15, was produced (9000 U/mL), purified, and characterized. SAPGB has a monomer structure with a precise molecular weight of 30,285.03 kDa as learnt from matrix-assisted laser desorption/ionization-time of flight/mass spectroscopy (MALDI-TOF/MS) exploration. The NH2-terminal amino-acid succession revealed significant identity with Bacillus proteases. The SAPGB was irreversibly inhibited by diiodopropyl fluorophosphates (DFP) and phenylmethylsulfonyl fluoride (PMSF). The enzyme displayed optimum activity at 65 °C and pH 10. The maximal activity was achieved in the range 0.5-5 M NaCl and about 52% of the activity was preserved across the broad salinity range of 0-30%. SAPGB exhibited a considerable catalytic efficiency (ratio kcat/Km) and degree of hydrolysis (DH). In addition, SAPGB showed a high tolerance to several organic solvents and an excellent detergent compatibility than SAPV, SAPA, Thermolysin type X, and Esperase 8.0 L. These properties make SAPGB a potential candidate for detergent formulations. On the other hand, sapGB gene was cloned and expressed in E. coli BL21(DE3)pLysS and the biochemical properties of the purified extracellular recombinant protease (rSAPGB) were similar to those of SAPGB. Finally, a 3D structural model of SAPGB was constructed by homology modeling.


Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/química , Endopeptidases/química , Modelos Moleculares , Conformação Proteica , Serina Proteases/química , Sequência de Aminoácidos , Bacillaceae/genética , Proteínas de Bactérias/genética , Sequência de Bases , Fenômenos Químicos , Endopeptidases/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Serina Proteases/genética , Solventes , Especificidade por Substrato , Temperatura
13.
Microbiology (Reading) ; 166(9): 800-816, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32744496

RESUMO

The genus Geobacillus, belonging to the phylum Firmicutes, is one of the most important genera and comprises thermophilic bacteria. The genus Geobacillus was erected with the taxonomic reclassification of various Bacillus species. Taxonomic studies of Geobacillus remain in progress. However, there is no comprehensive review of the characteristic features, taxonomic status and study of various applications of this interesting genus. The main aim of this review is to give a comprehensive account of the genus Geobacillus. At present the genus acomprises 25 taxa, 14 validly published (with correct name), nine validly published (with synonyms) and two not validly published species. We describe only validly published species of the genera Geobacillus and Parageobacillus. Vegetative cells of Geobacillus species are Gram-strain-positive or -variable, rod-shaped, motile, endospore-forming, aerobic or facultatively anaerobic, obligately thermophilic and chemo-organotrophic. Growth occurs in the pH range 6.08.5 and a temperature of 37-75 °C. The major cellular fatty acids are iso-C15:o, iso-C16:0 and iso-C17:o. The main menaquinone type is MK-7. The G-+C content of the DNA ranges between 48.2 and 58 mol%. The genus Geobacillus is widely distributed in nature, being mostly found in many extreme locations such as hot springs, hydrothermal vents, marine trenches, hay composts, etc. Geobacillus species have been widely exploited in various industrial and biotechnological applications, and thus are promising candidates for further studies in the future.


Assuntos
Bacillaceae/classificação , Bacillaceae/fisiologia , Geobacillus/classificação , Geobacillus/fisiologia , Bacillaceae/enzimologia , Bacillaceae/genética , Biodegradação Ambiental , Biocombustíveis , Evolução Biológica , Biotecnologia , Sistemas CRISPR-Cas , Ambientes Extremos , Geobacillus/enzimologia , Geobacillus/genética , Microbiologia Industrial , Filogenia , Temperatura
14.
Molecules ; 25(11)2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32512695

RESUMO

Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.


Assuntos
Bacillaceae/enzimologia , Bacillaceae/crescimento & desenvolvimento , Fontes Termais/microbiologia , Temperatura Alta , Nitrogênio/metabolismo , Peptídeo Hidrolases/metabolismo , Bacillaceae/isolamento & purificação , Técnicas de Cultura de Células , Concentração de Íons de Hidrogênio , Filogenia
15.
Microbiologyopen ; 9(8): e1059, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32485072

RESUMO

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP+ -dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. Moreover, d-amino acid dehydrogenase (d-AADHs) derived from protein-engineered meso-DAPDH is useful for one-step synthesis of d-amino acids with high optical purity. Here, we report the identification and functional characterization of a novel NAD(P)+ -dependent meso-DAPDH from Numidum massiliense (NmDAPDH). After the gene encoding the putative NmDAPDH was expressed in recombinant Escherichia coli cells, the enzyme was purified 4.0-fold to homogeneity from the crude extract through five purification steps. Although the previously known meso-DAPDHs use only NADP+ as a coenzyme, NmDAPDH was able to use both NADP+ and NAD+ as coenzymes. When NADP+ was used as a coenzyme, NmDAPDH exhibited an approximately 2 times higher kcat /Km value toward meso-DAP than that of meso-DAPDH from Symbiobacterium thermophilum (StDAPDH). NmDAPDH also catalyzed the reductive amination of corresponding 2-oxo acids to produce acidic d-amino acids such as d-aspartate and d-glutamate. The optimum pH and temperature for the oxidative deamination of meso-DAP were about 10.5 and 75°C, respectively. Like StDAPDH, NmDAPDH exhibited high stability: it retained more than 75% of its activity after 30 min at 60°C (pH 7.2) or at pHs ranging from 5.5 to 13.0 (50°C). Alignment of the amino acid sequences of NmDAPDH and the known meso-DAPDHs suggested NmDAPDH has a hexameric structure. Given its specificity for both NADP+ and NAD+ , high stability, and a broad range of reductive amination activity toward 2-oxo acids, NmDAPDH appears to offer advantages for engineering a more effective d-AADH.


Assuntos
Aminoácido Oxirredutases/metabolismo , Bacillaceae/enzimologia , Ácido D-Aspártico/metabolismo , Ácido Glutâmico/metabolismo , NADP/metabolismo , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Bacillaceae/genética , Clostridiales/enzimologia , Escherichia coli/genética , Cinética , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato
16.
Curr Microbiol ; 77(8): 1558-1568, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32248284

RESUMO

The current study was designed to isolate, identify and characterize a Bacillus sp. capable of producing protease and exhibiting antifungal activity. A highly potent bacterium capable of producing protease abundantly was isolated from the soil collected from the waste pit near Microbiology Laboratory of Birendra Multiple Campus, Bharatpur and later on identified as Lysinibacillus fusiformis strain SK on the basis of morphological, physiological, biochemical and 16S rDNA gene sequencing techniques. The strain SK showed 98.36% similarity with L. fusiformis strain NBRC 15717. Using R-programming statistical analysis tool, the optimum incubation time for the highest average protease production (APP) (47.2 U/mL) was found to be 22 h at 50 °C and both incubation time and temperature showed a significant impact on the production of protease (P < 0.01). The maximum average relative protease activity (ARPA) was observed at pH 7.8 and 48 °C, whereas the least ARPA was observed in the presence of 80 g/L NaCl and 10 g/L HgCl2 (P < 0.01). The newly isolated bacterial strain also exhibited strong antifungal activity against aflatoxigenic Aspergillus flavus and Aspergillus parasiticus suggesting that it can be a potential candidate for protease production and activity over a wider range of temperature and pH as well as for synthesizing effective antifungal compounds.


Assuntos
Antibiose , Bacillaceae/enzimologia , Peptídeo Hidrolases/metabolismo , Microbiologia do Solo , Aspergillus , Aspergillus flavus , Bacillaceae/classificação , Bacillaceae/isolamento & purificação , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Nepal , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
17.
Antonie Van Leeuwenhoek ; 113(7): 1049-1059, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32318981

RESUMO

Gracilibacillus dipsosauri is a moderately-halophilic Gram-positive bacterium which forms an extracellular α-amylase that is induced by starch, repressed by D-glucose, and active in 2.0 M KCl. Previous studies showed that while enzyme activity could be measured with the synthetic substrate 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3), other assays were inconsistent and the protein showed aberrant mobility during nondenaturing gel electrophoresis. To clarify the properties of this enzyme, the genome of G. dipsosauri was sequenced and was found to be 4.19 Mb in size with an overall G+C content of 36.9%. A gene encoding an α-amylase composed of 691 amino acids was identified. The protein was a member of the glycosyl hydrolase 13 family, which had a molecular mass of 77,396 daltons and a pI of 4.39 due to an unusually large number of aspartate and glutamate residues (95/691 or 13.7%). BLAST analysis of the amino acid sequence revealed significant matches to other proteins with cyclodextrin glycosyltransferase activity. Partial purification of the protein from G. dipsosauri showed that fractions catalyzing the hydrolysis of CNPG3 and p-nitrophenyl-D-maltoheptoside also catalyzed the formation of ß-cyclodextrin but not α-cyclodextrin or γ-cyclodextrin. Formation of ß-cyclodextrin was not stimulated by high salt concentrations but did occur with rice, potato, wheat, and corn starches and amylopectin. These studies explain the unusual features of the α-amylase from G. dipsosauri and indicate it should be classified as EC 2.4.1.19. The availability of the complete genomic sequence of G. dipsosauri will provide the basis for studies on other enzymes from this halophile which may be useful for biotechnology.


Assuntos
Bacillaceae/enzimologia , Bacillaceae/genética , Genômica , Tolerância ao Sal/fisiologia , Sais/química , alfa-Amilases/genética , alfa-Amilases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Composição de Bases , Sequência de Bases , DNA Bacteriano/genética , Genes Bacterianos/genética , Genoma Bacteriano , Glucosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Conformação Proteica , alfa-Amilases/química
18.
Biointerphases ; 15(1): 011002, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31948239

RESUMO

The implementation of self-assembled biomolecules on solid materials, in particular, sensor and electrode surfaces, gains increasing importance for the design of stable functional platforms, bioinspired materials, and biosensors. The present study reports on the formation of a planar hybrid lipid/polymer membrane on a crystalline surface layer protein (SLP) lattice. The latter acts as a connecting layer linking the biomolecules to the inorganic base plate. In this approach, chemically bound lipids provided hydrophobic anchoring moieties for the hybrid lipid/polymer membrane on the recrystallized SLP lattice. The rapid solvent exchange technique was the method of choice to generate the planar hybrid lipid/polymer membrane on the SLP lattice. The formation process and completeness of the latter were investigated by quartz crystal microbalance with dissipation monitoring and by an enzymatic assay using the protease subtilisin A, respectively. The present data provide evidence for the formation of a hybrid lipid/polymer membrane on an S-layer lattice with a diblock copolymer content of 30%. The hybrid lipid/polymer showed a higher stiffness compared to the pure lipid bilayer. Most interestingly, both the pure and hybrid membrane prevented the proteolytic degradation of the underlying S-layer protein by the action of subtilisin A. Hence, these results provide evidence for the formation of defect-free membranes anchored to the S-layer lattice.


Assuntos
Proteínas de Bactérias/química , Lipídeos/química , Polímeros/química , Subtilisinas/química , Bacillaceae/enzimologia , Proteínas de Bactérias/metabolismo , Cristalização , Ensaios Enzimáticos , Interações Hidrofóbicas e Hidrofílicas , Técnicas de Microbalança de Cristal de Quartzo , Subtilisinas/metabolismo , Propriedades de Superfície
19.
J Microbiol Biotechnol ; 30(4): 604-614, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31893610

RESUMO

The application of steroids has steadily increased thanks to their therapeutic effects. However, alternatives are required due their severe side effects; thus, studies on the activities of steroid derivatives are underway. Sugar derivatives of nandrolone, which is used to treat breast cancer, as well as cortisone and prednisone, which reduce inflammation, pain, and edema, are unknown. We linked O-glucose to nandrolone and testosterone using UDP-glucosyltransferase (UGT-1) and, then, tested their bioactivities in vitro. Analysis by NMR showed that the derivatives were 17ß-nandrolone ß-D-glucose and 17ß-testosterone ß-D-glucose, respectively. The viability was higher and cytotoxicity was evident in PC12 cells incubated with rotenone and, testosterone derivatives, compared to the controls. SH-SY5Y cells incubated with H2O2 and nandrolone derivatives remained viable and cytotoxicity was attenuated. Both derivatives enhanced neuronal protective effects and increased the amounts of cellular ATP.


Assuntos
Bacillaceae/enzimologia , Glucosiltransferases/metabolismo , Glicosídeos/metabolismo , Congêneres da Testosterona/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biotransformação , Linhagem Celular Tumoral , Metabolismo Energético/efeitos dos fármacos , Glucose/química , Glucose/metabolismo , Glicosídeos/química , Glicosídeos/farmacologia , Humanos , Nandrolona/química , Nandrolona/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Células PC12 , Ratos , Testosterona/química , Testosterona/metabolismo , Congêneres da Testosterona/química , Congêneres da Testosterona/farmacologia
20.
J Biochem ; 167(1): 89-99, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31599938

RESUMO

Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.


Assuntos
Bacillaceae/enzimologia , Lipase/biossíntese , Óleos/metabolismo , Temperatura , Águas Residuárias/química , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/genética , Lipase/isolamento & purificação , Óleos/química
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