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1.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639118

RESUMO

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0-9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10-3 mM s-1), and Kcat/Km (763.92 mM-1 s-1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.


Assuntos
Anoxybacillus/enzimologia , Galactosídeos/biossíntese , Temperatura Alta , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Homologia de Sequência , Especificidade por Substrato , alfa-Galactosidase/química
2.
Int J Syst Evol Microbiol ; 71(10)2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34596507

RESUMO

Twelve thermophilic Anoxybacillus strains were isolated from sediment and water samples from a Karvachar hot spring located in the northern part of Nagorno-Karabakh. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, one of the isolates, designated strain K1T, was studied in detail. The cells are straight, motile rods that are 0.2-0.4×2.3-7.2 µm in size. The strain is a Gram-stain-positive, moderately thermophilic facultative anaerobe with an optimum growth temperature of 60-65 °C and a growth temperature range of 45-70 °C. Growth of strain K1T was observed at pH 6-11 (optimum, pH 8-9) and was inhibited in the presence of NaCl concentrations above 2.5 % (optimum, 1-1.5 %). The isolate could utilize a wide variety of carbon sources, including d-arabinose, d-ribose, d-galactose, d-fructose, d-mannitol, maltose, aesculin, melibiose, sucrose, trehalose, raffinose, amidone, glycogen, turanose, d-lyxose, d-tagatose, potassium gluconate and 2-keto-gluconate. The strain was able to hydrolyse starch, casein and gelatin, was positive for oxidase and catalase, and reduced nitrate to nitrite, but was negative for H2S production. Production of urease and indole was not observed. The major cellular fatty acids were C15 : 0 iso, C16 : 0 and C17 : 0 iso (52.5, 13.6 and 19.6 % of total fatty acids, respectively). Strain K1T shares >99 % 16S rRNA sequence similarity and a genomic average nucleotide identity value of 94.5 % with its closest relative, Anoxybacillus flavithermus DSM 2641T, suggesting that it represents a separate and novel species, for which the name Anoxybacillus karvacharensis sp. nov. is proposed. The type strain of Anoxybacillus karvacharensis is K1T (=DSM 106524T=KCTC 15807T).


Assuntos
Anoxybacillus , Fontes Termais , Anoxybacillus/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
3.
Biomed Res Int ; 2021: 1869748, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34258259

RESUMO

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.


Assuntos
Anoxybacillus/genética , Análise de Dados , Fontes Termais/microbiologia , Sequenciamento Completo do Genoma , Sequência de Aminoácidos , Anoxybacillus/enzimologia , Anoxybacillus/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Circular/genética , Genoma Bacteriano , Glicosídeo Hidrolases/metabolismo , Anotação de Sequência Molecular , Nepal , Fases de Leitura Aberta/genética , Filogenia , Xilose/metabolismo
4.
Ecotoxicol Environ Saf ; 214: 112084, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33640726

RESUMO

Direct Black G (DBG) is a highly toxic synthetic azo dye which is difficult to degrade. Biological treatment seems to be a promising option for the treatment of azo dye containing effluent. A thermophilic bacterial strain (Anoxybacillus sp. PDR2) previously isolated from the soil can effectively remove DBG. However, the molecular underpinnings of DBG degradation and the microbial detoxification ability remains unknown. In the present study, the genetic background of PDR2 for the efficient degradation of DBG and its adaptation to azo dye-contaminated environments was revealed by bioinformatics. Moreover, the possible biodegradation pathways were speculated based on the UV-vis spectral analysis, FTIR, and intermediates identified by LC-MS. Additionally, phytotoxicity and the comet experiment studies clearly indicated that PDR2 converts toxic azo dye (DBG) into low toxicity metabolites. The combination of biodegradation pathways and detoxification analysis were utilized to explore the molecular degradation mechanism and bioremediation of azo dye for future applications. These findings will provide a valuable theoretical basis for the practical treatment of azo dye wastewater.


Assuntos
Anoxybacillus/metabolismo , Compostos Azo/metabolismo , Biodegradação Ambiental , Anoxybacillus/genética , Bactérias/metabolismo , Cor , Corantes/metabolismo , Humanos , Solo , Águas Residuárias
5.
Arch Microbiol ; 203(5): 2101-2118, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33604750

RESUMO

Exopolysaccharides (EPS/EPSs) possess several various applications in the food and pharmaceutical industries. This study was performed to investigate the biological (antibiofilm and antitumor), rheological (temperature, shear rate, and density) and chemical (solubility, carbohydrate and protein content, composition, molecular weight, functional group analysis, thermal analysis, X-ray diffraction pattern and scanning electron microscopy) properties of the EPS, which was purified from the locally isolated thermophilic bacterium Anoxybacillus pushchinoensis G11 (MN720646). EPS was found to have antibiofilm and antitumor [lung (A-549) and colon (Caco-2 and HT-29) cancer] activities. The viscosity of EPS showing Newtonian flow was temperature dependent. As chemical properties, the EPS was found to be a heteropolysaccharide containing arabinose (57%), fructose (26%), glucose (12%), and galactose (5%). EPS contained 93% carbohydrates and 1.08% protein. The molecular weight of EPS was determined as 75.5 kDa. The FTIR analysis confirmed the presence of sulfate ester (band at 1217 cm-1), an indication of the antitumor effect. The EPS was semi-crystalline. It could maintain 36% of its weight at 800 °C and crystallization and melting temperatures were 221 and 255.6 °C. This is the first report on the EPS production potential and the biological activity of A. pushchinoensis.


Assuntos
Anoxybacillus/química , Biofilmes/efeitos dos fármacos , Polissacarídeos Bacterianos/farmacologia , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HT29 , Humanos , Peso Molecular , Polissacarídeos Bacterianos/isolamento & purificação , Temperatura , Viscosidade
6.
Anal Bioanal Chem ; 413(4): 1107-1116, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33388846

RESUMO

This paper proposes the use of Anoxybacillus flavithermus SO-15 immobilized on iron oxide nanoparticles (NPs) as a novel magnetized biosorbent for the preconcentrations of uranium (U) and thorium (Th). The SPE procedure was based on biosorption of U(VI) and Th(IV) on a column of iron oxide NPs loaded with dead and dried thermophilic bacterial biomass prior to U(VI) and Th(IV) measurements by ICP-OES. The biosorbent characteristicswere explored using FT-IR, SEM, and EDX. Significant operational factors such as solution pH, volume and flow rate of the sample solution, amounts of dead bacteria and iron oxide nanoparticles, matrix interference effect, eluent type, and repeating use of the biosorbent on process yield were studied. The biosorption capacities were found as 62.7 and 56.4 mg g-1 for U(VI) and Th(IV), respectively. The novel extraction process has been successfullyapplied to the tap, river, and lake water samples for preconcentrations of U(VI) and Th(IV).


Assuntos
Anoxybacillus/química , Nanopartículas Magnéticas de Óxido de Ferro/química , Extração em Fase Sólida/métodos , Tório/isolamento & purificação , Urânio/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Adsorção , Células Imobilizadas/química
7.
Ecotoxicol Environ Saf ; 203: 111047, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888598

RESUMO

Understanding azo dye degrading enzymes and the encoding of their functional genes is crucial for the elucidation of their molecular mechanisms. In this study, a thermophilic strain capable of degrading azo dye was isolated from the soil near a textile dye manufacturing factory. Based on its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis, the strain was identified as Anoxybacillus sp. PDR2. The decolorization ratios of 100-600 mg/L Direct Black G (DBG) by strain PDR2 reached 82.12-98.39% within 48 h of dyes. Genome analysis revealed that strain PDR2 contains a circular chromosome of 3791144 bp with a G + C content of 42.48%. The genetic basis of azo dye degradation by strain PDR2 and its capacity to adapt to harsh environments, were further elucidated through bioinformatics analysis. RNA-Seq and qRT-PCR technology confirmed that NAD(P)H-flavin reductase, 2Fe-2S ferredoxin and NAD(P)-dependent ethanol dehydrogenase genes expressed by strain PDR2, were the key genes involved in DBG degradation. The combination of genome and transcriptome analysis was utilized to explore the key genes of strain PDR2 involved in azo dye biodegradation, with these findings providing a valuable theoretical basis for the practical treatment of azo dye wastewater.


Assuntos
Anoxybacillus/isolamento & purificação , Compostos Azo/análise , Corantes/análise , Genes Bacterianos , Microbiologia do Solo , Anoxybacillus/genética , Anoxybacillus/metabolismo , Compostos Azo/metabolismo , Biodegradação Ambiental , China , Corantes/metabolismo , Perfilação da Expressão Gênica , Genômica , RNA Ribossômico 16S/genética , Solo/química , Indústria Têxtil
8.
Elife ; 92020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32735215

RESUMO

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.


Assuntos
Anoxybacillus/metabolismo , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Antiporters/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Transporte Biológico Ativo , Cátions/metabolismo , Microscopia Crioeletrônica , Escherichia coli , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Prótons , Sódio/metabolismo
9.
J Basic Microbiol ; 60(9): 809-815, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32602226

RESUMO

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.


Assuntos
Anoxybacillus/metabolismo , Extremófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Anoxybacillus/classificação , Anoxybacillus/isolamento & purificação , Anoxybacillus/fisiologia , Brasil , Estabilidade Enzimática , Extremófilos/classificação , Extremófilos/isolamento & purificação , Extremófilos/fisiologia , Concentração de Íons de Hidrogênio , Queratinas/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Temperatura , Termolisina/química , Termolisina/metabolismo , Termotolerância
10.
J Food Prot ; 83(12): 2074-2079, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32663274

RESUMO

ABSTRACT: Thermophilic spore-forming bacteria are found ubiquitously in natural environments and, therefore, are present in a number of agricultural food products. Spores produced by these bacteria can survive harsh environmental conditions encountered during food processing and have been implicated in food spoilage. During research efforts to develop a standardized method for enumerating spores in dairy powders, the dairy powder-associated thermophilic sporeformer Anoxybacillus flavithermus was discovered growing in uninoculated control plates of tryptic soy agar (TSA) supplemented with 1% (w/v) starch, after incubation at thermophilic (55°C) growth temperatures. This article reports the investigation into the source of this thermophilic sporeformer in TSA medium components and characterization of the bacterial isolates collected. Aqueous solutions of tryptic soy broth powder from four suppliers and four agar-agar powders (two manufacturing lots from one supplier [agar A_1 and agar A_2] and two from separate suppliers [agar B and agar C]) were subjected to two different autoclave cycle times (121°C for 15 min or 121°C for 30 min) and then prepared as TSA. After incubation at 55°C for 48 h, bacterial growth was observed only in media prepared from both lots of agar A agar-agar powder, and only when they were subjected to a 15-min autoclave cycle, implicating these powders as a source of the sporeformer contamination. Genetic characterization of 49 isolates obtained indicated the presence of five unique rpoB allelic types of the thermophilic sporeformer Geobacillus spp. in agar-agar powder from agar A. These results not only highlight the importance of microbiological controls but also alert researchers to the potential for survival of thermophilic sporeformers such as Anoxybacillus and Geobacillus in microbiological media used for detection and enumeration of these same thermophilic sporeformers in products such as dairy powders.


Assuntos
Leite , Esporos Bacterianos , Ágar , Animais , Anoxybacillus , Bactérias , Pós , Esporos
11.
Artigo em Inglês | MEDLINE | ID: mdl-32416322

RESUMO

In this study, it was hypothesis that A. mongoliensis could be used as bioindicator for Ni (II) and Co (II). Thus, Ni (II) and Co (II) resistance, removal, bioaccumulation, and the impacts of them on antioxidant enzyme systems of thermophilic Anoxybacillus mongoliensis were investigated in details. The bioaccumulation of Ni (II) and Co (II) on the cell membrane of thermophilic A. mongoliensis, variations on surface macrostructure and functionality by FT-IR and SEM, and determination of antioxidant enzyme activities were also tested. The highest bioaccumulation values of Co (II) and Ni (II) were detected as 102.0 mg metal/g of dry bacteria at 10 mg/L for the 12th h and 90.4 mg metal/g of dry bacteria for the 24th h, respectively, and the highest Ni (II) and Co (II) cell membrane bioaccumulation capacities of A. mongoliensis were determined as 268.5 and 274.9 mg metal/g wet membrane, respectively at the 24th h. In addition, increasing on SOD and CAT activities were observed on depend of concentration of Ni (II) and Co (II) with respect to control. The antioxidant enzyme activity results also indicated that A. mongoliensis might be used as a bioindicator for Ni (II) and Co (II) pollution in environmental water specimens.


Assuntos
Anoxybacillus/crescimento & desenvolvimento , Antioxidantes/metabolismo , Catalase/metabolismo , Cobre/metabolismo , Poluentes Ambientais/metabolismo , Níquel/metabolismo , Superóxido Dismutase/metabolismo , Anoxybacillus/efeitos dos fármacos , Anoxybacillus/enzimologia , Anoxybacillus/metabolismo , Bioacumulação , Cobre/isolamento & purificação , Cobre/toxicidade , Poluentes Ambientais/isolamento & purificação , Poluentes Ambientais/toxicidade , Níquel/isolamento & purificação , Níquel/toxicidade
12.
Prep Biochem Biotechnol ; 50(6): 578-584, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32011972

RESUMO

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R2 = 0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4 IU/g at an optimum temperature of 57.5 °C, pH 8.31, moisture 50% and 1.2 mg of bio-surfactant. Experimental production of 30.3 IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.


Assuntos
Anoxybacillus/enzimologia , Proteínas de Bactérias/biossíntese , Detergentes/química , Lipase/biossíntese , Modelos Estatísticos , Anoxybacillus/genética , DNA Bacteriano/genética , Detergentes/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Fermentação , Concentração de Íons de Hidrogênio , Índia , Mostardeira/química , Filogenia , Óleos Vegetais/química , RNA Ribossômico 16S/genética , Temperatura
13.
Int J Biol Macromol ; 152: 584-592, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097739

RESUMO

L-asparaginase is an important enzyme with diverse applications in food industry and therapeutics. However, the enzyme currently employed in the treatment of leukaemias, comes with undesired L-glutaminase activity. A gene encoding 38 kDa L-asparaginase form Anoxybacillus flavithermus was cloned and expressed in Escherichia coli as a soluble and active enzyme. Heat treatment and Ni-affinity column chromatography provided highly purified enzyme possessing a specific activity of 165 units mg-1. The enzyme exhibited allosteric behaviour with a Hill coefficient of 1.60 and K0.5 of 25 mM with L-asparagine as specific substrate. No detectable activity was observed in the presence of D-asparagine, l-glutamine and d-glutamine. Purified AfASNase showed optimum activity at 60 °C and pH 7.0. The enzyme had ability to withstand up to 6 M urea and showed complete inactivation when treated with 1 M guanidine hydrochloride. Protein-Ligand docking and molecular dynamic simulations indicated that the regulatory site is formed by T262-T263-C265-G269-Thr294 and is located on a domain different from the one carrying the well-established active site. AfASNase is reported as first thermostable L-asparaginase with allosteric regulation. Hitherto, AfASNase presents the first characterization of recombinant L-asparaginase from the genus Anoxybacillus.


Assuntos
Anoxybacillus/enzimologia , Asparaginase/química , Sítio Alostérico , Asparagina/genética , Proteínas de Bactérias/química , Domínio Catalítico , Cromatografia de Afinidade , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/metabolismo , Guanidina/química , Cinética , Ligantes , Conformação Molecular , Proteínas/química , Especificidade por Substrato
14.
Environ Sci Pollut Res Int ; 27(13): 15842-15855, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32095964

RESUMO

The efficiency of the proteolytic strain Anoxybacillus kamchatkensis M1V in the fermentation of speckled shrimp by-product was investigated for the recovery of a deproteinized bioactive hydrolysate. The biological activities of the resulting hydrolysate were also examined by applying several antioxidant and enzyme inhibitory assays. The strain M1V was found to produce high level of protease activity (2000 U/mL) when grown in media containing only shrimp powder at 25 g/L. The crude protease displayed a significant deproteinization capabiliy, with the best efficiency (48%) being recorded for an enzyme to substrate (E/S) ratio of 30 U/mg. Following the deproteinization, chitin was recovered and the authenticity was confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis. On the other hand, the obtained hydrolysate showed a significant enzymatic inhibitory potential against acetylcholinesterase, tyrosinase, amylase, and angiotensin I convertase, and a strong antioxidant activity. Graphical Abstract.


Assuntos
Penaeidae , Animais , Anoxybacillus , Quitina , Endopeptidases , Fermentação
15.
Appl Biochem Biotechnol ; 191(3): 942-954, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31939086

RESUMO

Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (PulAC) derived from the pullulanase (PulA) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type PulA was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and kcat/Km value of the PulAC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type PulA. The optimum temperature of the mutant PulAC was 65 °C. The PulAC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type PulA. Due to its high optimum temperature, thermostability, and specific activity, the mutant PulAC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.


Assuntos
Anoxybacillus/enzimologia , Glicosídeo Hidrolases/metabolismo , Arginina/química , Dicroísmo Circular , Dissulfetos/química , Glicina/química , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial , Cinética , Lisina/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Amido , Especificidade por Substrato , Propriedades de Superfície , Temperatura
16.
Bioelectrochemistry ; 133: 107450, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31978857

RESUMO

Common alloys used for the manufacture of aircrafts are subject to different forms of environmental deterioration. A major one is corrosion, and there is a strong body of evidence suggesting that environmental microorganisms initiate and accelerate it. The development of an appropriate strategy to reduce this process depends on the knowledge concerning the factors involved in corrosion. In this work, a biofilm forming bacterial consortium was extracted in situ from the corrosion products formed in an aircraft exposed to Antarctic media. Two thermophilic bacteria, an Anoxybacillus and a Staphylococcus strain, were successfully isolated from this consortium. Two extracellular enzymes previously speculated to participate in corrosion, catalase and peroxidase, were detected in the extracellular fraction of the consortium. Additionally, we assessed the individual contribution of those thermophilic microorganisms on the corrosion process of 7075-T6 aluminum alloy, which is widely used in aeronautical industry, through electrochemical methods and surface analysis techniques.


Assuntos
Ligas/química , Alumínio/química , Anoxybacillus/fisiologia , Biofilmes , Anoxybacillus/enzimologia , Anoxybacillus/isolamento & purificação , Regiões Antárticas , Corrosão , Oxirredução , Staphylococcus/enzimologia , Staphylococcus/isolamento & purificação , Staphylococcus/fisiologia , Propriedades de Superfície
17.
Spectrochim Acta A Mol Biomol Spectrosc ; 230: 118055, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31955121

RESUMO

Cyclomaltodextrinase (CDase) is a member of the alpha-amylase family GH13, the subfamily GH13_20. In addition to CDase and neopullulanase, this subfamily also contains maltogenic amylase. They have common structural features, but different substrate specificity. In current work, a combination of bioinformatics and experimental tools were used for designing and constructions of single and double mutants of a new variant of CDase from Anoxybacillus flavithermus. Considering the evolutionary variable positions 123 and 127 at the dimer interface of subunits in the alpha-amylase family, these positions in CDase were modified and three mutants, including A123V, C127Q and A123V/C127Q were constructed. The tertiary structure of WT and mutants were made with the MODELLER program, and the phylogenetic tree of homologous protein sequences was built with selected programs in Phylip package. Enzyme kinetic studies revealed that the catalytic efficiency of mutants, especially double one, is lower than the WT enzyme. Heat-induced denaturation experiments were monitored by measuring the UV/Vis signal at 280 nm, and it was found that WT protein is structurally more stable at 25 °C. However, it is more susceptible to changes in temperature compared to the double mutant. It was concluded that the positions 123 and 127 at the dimeric interface of CDase, not only could affect the conformational stability; but also; the catalytic properties of the enzyme by setting up the active site configuration in the dimeric state.


Assuntos
Anoxybacillus/genética , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Anoxybacillus/química , Anoxybacillus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Mutagênese , Mutação , Filogenia , Conformação Proteica , Multimerização Proteica , Alinhamento de Sequência , Homologia Estrutural de Proteína
18.
Enzyme Microb Technol ; 131: 109421, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615670

RESUMO

The hydrolytic activity of a thermophilic cyclomaltodextrinase (CMD) from Anoxybacillus flavithermus ZNU-NGA and a representative single mutant were investigated against soluble substrates including α-, ß- and γ-cyclomaltodestrines (CDs). Based on the occurrence of arginine (Arg) at position 403 in some homologue proteins, His403 in Wild-type (WT) CMD was replaced with Arg (H403R variant) with site-directed mutagenesis procedures. According to bioinformatics data, Arg403 in mutant protein is located near Glu357 as one of the catalytic residues in a manner that they are able to create a medium-range attractive electrostatistic interaction. Structural studies by Far UV-CD showed that this mutation is accompanied by conversion of a small fraction of α-helix to ß-form structure. Fluorescence data reveals that, the hydrophobic regions at the surface of protein, as the binding sites for ANS (8-Anilinonaphthalene-1-sulfonic acid) increase in mutant protein, demonstrating relative inflation of H403R variant compared with WT protein. However, the polarity of microenvironment around chromophores did not change upon mutation. Activity measurement in different ranges of pH and temperatures showed that the optimum values of pH and temperature in mutant enzyme is the same as WT enzyme, however; the activity at optimum points increased in H403R variant. It was also revealed that the H403R variant had slightly improved catalytic efficiency for γ-CD. The same value of activation parameters for both protein variants indicates that mutation does not alter the mechanism of catalysis during enzyme-substrate formation.


Assuntos
Substituição de Aminoácidos , Anoxybacillus/enzimologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínio Catalítico , Dicroísmo Circular , Biologia Computacional , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Conformação Proteica , Temperatura
19.
Enzyme Microb Technol ; 131: 109385, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615674

RESUMO

From ecological and industrial perspectives, Anoxybacillus flavithermus species that lives in a thermophilic environment, are extremely important bacteria due to their potential in producing highly interesting compounds and enzymes. In order to understand the genetic makeup of these thermophiles, we have performed a comparative genomics study of 12 genome-sequenced strains of Anoxybacillus flavithermus bacteria. The genome size of Anoxybacillus flavithermus strains is from 2.5Mbp to 3.7Mbp and on average containing a low percentage of G + C genomic content (˜41.9%). We show that, on the basis of the total gene-content, Anoxybacillus flavithermus strains are grouped in three different subgroups. In the future, it would be interesting to explore these strain subgroups to further understand the lifestyle of thermophilic bacteria. Focussing on the Anoxybacillus flavithermus AK1 strain, which was isolated from a Hot Spring in Saudi Arabia and closely related to A. flavithermus NBRC strain, we identified a unique list of 75 genes specific to AK1 strain, of which 63 of them have homologs in other taxonomically related species. We speculate that these AK1-specific genes might be resulted due to horizontal gene transfer from other bacteria in order to adapt to the extreme environmental conditions. Moreover, we predicted three potential secondary metabolite gene clusters in the AK1 strain that further need to be experimentally characterised. Genomic annotation, secondary metabolite gene clusters and outcomes of the strain genomic comparisons from this study would be the basis for the strain-specific mathematical model for exploiting the metabolism for the industrial and ecological applications.


Assuntos
Anoxybacillus/genética , Genoma Bacteriano , Genômica , Anoxybacillus/isolamento & purificação , Composição de Bases , Genótipo , Fontes Termais , Arábia Saudita
20.
Extremophiles ; 23(6): 687-706, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31407121

RESUMO

A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.


Assuntos
Anoxybacillus/enzimologia , Proteínas de Bactérias/química , Detergentes/química , Temperatura Alta , Peptídeo Hidrolases/química , Anoxybacillus/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática , Peptídeo Hidrolases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
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