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1.
Biotechnol Lett ; 46(3): 443-458, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38523202

RESUMO

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.


Assuntos
Estabilidade Enzimática , Esterases , Geobacillus , Geobacillus/enzimologia , Geobacillus/genética , Esterases/genética , Esterases/química , Esterases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Desenho Assistido por Computador , Clonagem Molecular
2.
Int J Biol Macromol ; 263(Pt 2): 130438, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38408579

RESUMO

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.


Assuntos
Asparaginase , Geobacillus , Asparaginase/química , Geobacillus/genética , Geobacillus/metabolismo , Mercaptoetanol , Proteínas Recombinantes/genética , Estabilidade Enzimática
3.
Extremophiles ; 28(1): 18, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38353731

RESUMO

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.


Assuntos
Geobacillus , Ribonucleotídeo Redutases , Ribonucleotídeo Redutases/genética , Oxigenases de Função Mista/genética , Geobacillus/genética , Alcanos
4.
J Environ Manage ; 354: 120416, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38408391

RESUMO

Hydrogen sulfide (H2S) is a toxic gas massively released during chicken manure composting. Diminishing its release requires efficient and low cost methods. In recent years, heterotrophic bacteria capable of rapid H2S oxidation have been discovered but their applications in environmental improvement are rarely reported. Herein, we investigated H2S oxidation activity of a heterotrophic thermophilic bacterium Geobacillus thermodenitrificans DSM465, which contains a H2S oxidation pathway composed by sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). This strain rapidly oxidized H2S to sulfane sulfur and thiosulfate. The oxidation rate reached 5.73 µmol min-1·g-1 of cell dry weight. We used G. thermodenitrificans DSM465 to restrict H2S release during chicken manure composting. The H2S emission during composting process reduced by 27.5% and sulfate content in the final compost increased by 34.4%. In addition, this strain prolonged the high temperature phase by 7 days. Thus, using G. thermodenitrificans DSM465 to control H2S release was an efficient and economic method. This study provided a new strategy for making waste composting environmental friendly and shed light on perspective applications of heterotrophic H2S oxidation bacteria in environmental improvements.


Assuntos
Compostagem , Geobacillus , Sulfeto de Hidrogênio , Animais , Galinhas , Esterco , Proteínas de Bactérias/metabolismo , Sulfetos/metabolismo , Geobacillus/metabolismo , Oxirredução
5.
J Basic Microbiol ; 64(4): e2300653, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38212247

RESUMO

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.


Assuntos
Detergentes , Geobacillus , alfa-Amilases , alfa-Amilases/genética , alfa-Amilases/química , Amido/metabolismo , Farinha , Filogenia , Triticum/metabolismo , Hidrólise , Concentração de Íons de Hidrogênio
6.
Int J Biol Macromol ; 257(Pt 2): 128679, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38072346

RESUMO

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable ß-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.


Assuntos
Geobacillus , Xilosidases , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Xilosidases/química , Xilanos/metabolismo , Especificidade por Substrato
7.
Rev Argent Microbiol ; 56(1): 102-111, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37704517

RESUMO

The genus Geobacillus is composed of thermophilic bacteria that exhibit diverse biotechnological potentialities. Specifically, Geobacillus stearothermophilus is included as a test bacterium in commercial microbiological inhibition methods, although it exhibits limited sensitivity to aminoglycosides, macrolides, and quinolones. Therefore, this article evaluates the antibiotic susceptibility profiles of five test bacteria (G. stearothermophilus subsp. calidolactis C953, Geobacillus thermocatenulatus LMG 19007, Geobacillus thermoleovorans LMG 9823, Geobacillus kaustophilus DSM 7263 and Geobacillus vulcani 13174). For that purpose, the minimum inhibitory concentrations (MICs) of 21 antibiotics were determined in milk samples for five test bacteria using the radial diffusion microbiological inhibition method. Subsequently, the similarities between bacteria and antibiotics were analyzed using cluster analysis. The dendrogram of this multivariate analysis shows an association between a group formed by G. thermocatenulatus and G. stearothermophilus and another by G. thermoleovorans, G. kaustophilus and G. vulcani. Finally, future microbiological methods could be developed in microtiter plates using G. thermocatenulatus as test bacterium, as it exhibits similar sensitivities to G. stearothermophilus. Conversely, G. vulcani, G. thermoleovorans and G. kaustophilus show higher MICs than G. thermocatenulatus.


Assuntos
Anti-Infecciosos , Geobacillus , Animais , DNA Ribossômico/análise , Leite/química , RNA Ribossômico 16S , Geobacillus/genética , Antibacterianos/farmacologia , Antibacterianos/análise
8.
Biotechnol Appl Biochem ; 71(1): 162-175, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37908087

RESUMO

Microbial lipases are utilized in various biotechnological areas, including pharmaceuticals, food, biodiesel, and detergents. In this study, we cloned and sequenced Lip21 and Lip33 genes from Geobacillus sp. GS21 and Geobacillus sp. GS33, then we in silico and experimentally analyzed the encoded lipases. For this purpose, Lip21 and Lip33 were cloned, sequenced, and their amino acid sequences were investigated for determination of biophysicochemical characteristics, evolutionary relationships, and sequence similarities. 3D models were built and computationally affirmed by various bioinformatics tools, and enzyme-ligand interactions were investigated by docking analysis using six ligands. Biophysicochemical property of Lip21 and Lip33 was also determined experimentally and the results demonstrated that they had similar isoelectric point (pI) (6.21) and Tm (75.5°C) values as Tm was revealed by denatured protein analysis of the circular dichroism spectrum and pI was obtained by isoelectric focusing. Phylogeny analysis indicated that Lip21 and Lip33 were the closest to lipases from Geobacillus sp. SBS-4S and Geobacillus thermoleovorans, respectively. Alignment analysis demonstrated that S144-D348-H389 was catalytic triad residues in Lip21 and Lip33, and enzymes possessed a conserved Gly-X-Ser-X-Gly motif containing catalytic serine. 3D structure analysis indicated that Lip21 and Lip33 highly resembled each other and they were α/ß hydrolase-fold enzymes with large lid domains. BANΔIT analysis results showed that Lip21 and Lip33 had higher thermal stability, compared to other thermostable Geobacillus lipases. Docking results revealed that Lip21- and Lip33-docked complexes possessed common residues (H112, K115, Q162, E163, and S141) that interacted with the substrates, except paranitrophenyl (pNP)-C10 and pNP-C12, indicating that these residues might have a significant action on medium and short-chain fatty acid esters. Thus, Lip21 and Lip33 can be potential candidates for different industrial applications.


Assuntos
Geobacillus , Geobacillus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Estabilidade Enzimática
9.
Int J Biol Macromol ; 256(Pt 1): 128331, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38013084

RESUMO

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotype-phenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst-95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.


Assuntos
Carboxilesterase , Geobacillus , Carboxilesterase/química , Mutagênese , Hidrolases de Éster Carboxílico/química , Mutagênese Sítio-Dirigida
10.
Extremophiles ; 28(1): 6, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38036917

RESUMO

This study investigated the metabolism of Geobacillus sp. LC300, a promising biorefinery host organism with high substrate utilization rates. A new defined medium was designed and tested that allows for exponential growth to elevated cell densities suitable for quantitative physiological studies. Screening of the metabolic requirements of G. sp. LC300 revealed prototrophy for all essential amino acids and most vitamins and only showed auxotrophy for vitamin B12 and biotin. The effect of temperature and pH on growth rate was investigated, adjusting the optimal growth temperature to several degrees lower than previously reported. Lastly, studies on carbon source utilization revealed a capability for fast growth on several common carbon sources, including monosaccharides, oligosaccharides, and polysaccharides, and the highest ever reported growth rate in defined medium on glucose (2.20 h-1) or glycerol (1.95 h-1). These findings provide a foundation for further exploration of G. sp. LC300's physiology and metabolic regulation, and its potential use in bioproduction processes.


Assuntos
Geobacillus , Geobacillus/metabolismo , Carbono/metabolismo , Temperatura , Glucose/metabolismo
11.
Int J Biol Macromol ; 253(Pt 8): 127656, 2023 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-37884253

RESUMO

Plastic pollution is one of the biggest environmental problems plaguing the modern world. Polyester-based plastics contribute significantly to this ecological safety concern. In this study, lipolytic biocatalysts GD-95RM and GDEst-lip developed based on lipase/esterase produced by Geobacillus sp. 95 strain were applied for the degradation of polycaprolactone films (Mn 45.000 (PCL45000) and Mn 80.000 (PCL80000)). The degradation efficiency was significantly enhanced by the addition of short chain alcohols. Lipase GD-95RM (1 mg) can depolymerize 264.0 mg and 280.7 mg of PCL45000 and PCL80000, films respectively, in a 24 h period at 30 °C, while the fused enzyme GDEst-lip (1 mg) is capable of degrading 145.5 mg PCL45000 and 134.0 mg of PCL80000 films in 24 h. The addition of ethanol (25 %) improves the degradation efficiency ~2.5 fold in the case of GD-95RM. In the case of GDEst-lip, 50 % methanol was found to be the optimal alcohol solution and the degradation efficiency was increased by ~3.25 times. The addition of alcohols not only increased degradation speeds but also allowed for simultaneous synthesis of industrially valuable 6-hydroxyhexonic acid esters. The suggested system is an attractive approach for removing of plastic waste and supports the principles of bioeconomics.


Assuntos
Ésteres , Geobacillus , Lipase/metabolismo , Esterases/metabolismo , Álcoois
12.
Viruses ; 15(8)2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37632033

RESUMO

We report a detailed characterization of five thermophilic bacteriophages (phages) that were isolated from compost heaps in Vilnius, Lithuania using Geobacillus thermodenitrificans strains as the hosts for phage propagation. The efficiency of plating experiments revealed that phages formed plaques from 45 to 80 °C. Furthermore, most of the phages formed plaques surrounded by halo zones, indicating the presence of phage-encoded bacterial exopolysaccharide (EPS)-degrading depolymerases. Transmission Electron Microscopy (TEM) analysis revealed that all phages were siphoviruses characterized by an isometric head (from ~63 nm to ~67 nm in diameter) and a non-contractile flexible tail (from ~137 nm to ~150 nm in length). The genome sequencing resulted in genomes ranging from 38,161 to 39,016 bp. Comparative genomic and phylogenetic analysis revealed that all the isolated phages had no close relatives to date, and potentially represent three new genera within siphoviruses. The results of this study not only improve our knowledge about poorly explored thermophilic bacteriophages but also give new insights for further investigation of thermophilic and/or thermostable enzymes of bacterial viruses.


Assuntos
Bacteriófagos , Compostagem , Geobacillus , Filogenia , Técnicas de Tipagem Bacteriana , Bacteriófagos/genética , Geobacillus/genética
13.
J Agric Food Chem ; 71(31): 12015-12028, 2023 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-37495598

RESUMO

Bacterial 1,4-α-glucan branching enzymes (GBEs) provide a viable strategy for glycosidic bond rearrangement in starch and regulation of its digestion rate. However, the exponential increase in paste viscosity during starch gelatinization has a detrimental effect on the catalytic action of GBEs, thereby limiting productivity and product performance. Here, we designed an enzymatic treatment on corn starch granules by the GBE from Rhodothermus obamensis STB05 (Ro-GBE) prior to the glycosidic bond rearrangement of gelatinized starch catalyzed using the GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE). Specifically, a moderate amount of Ro-GBE was required for the pretreatment stage. The dual GBE modification process enabled the treatment of more concentrated starch slurry (up to 20%, w/w) and effectively reduced starch digestibility. The resulting product contained a rapidly digestible starch fraction of 66.0%, which was 11.4% lower than that observed in the single Gt-GBE-modified product. The mechanistic investigation showed that the Ro-GBE treatment promoted swelling and gelatinization of starch granules, reduced starch paste viscosity, and increased the mobility of water molecules in the starch paste. It also created a preferable substrate for Gt-GBE. These changes improved the transglycosylation efficiency of Gt-GBE. These findings provide useful guidance for designing an efficient process to regulate starch digestibility.


Assuntos
Zea mays , Zea mays/química , Zea mays/metabolismo , Amido/química , Amido/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Geobacillus/enzimologia , Amilose/química , Viscosidade , Especificidade por Substrato
14.
Extremophiles ; 27(2): 18, 2023 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-37428266

RESUMO

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.


Assuntos
Geobacillus , Lacase , Lacase/química , Regiões Antárticas , Cobre/metabolismo , Geobacillus/genética , Geobacillus/metabolismo , Vermelho Congo/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Temperatura
15.
CRISPR J ; 6(3): 278-288, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37134217

RESUMO

Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.


Assuntos
Proteína 9 Associada à CRISPR , Edição de Genes , Geobacillus , Edição de Genes/métodos , Humanos , Genoma , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR/metabolismo , Geobacillus/metabolismo , Engenharia Genética/métodos , Escherichia coli , Células HEK293
16.
World J Microbiol Biotechnol ; 39(6): 139, 2023 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-36995480

RESUMO

The Bacillaceae family members are considered to be a good source of microbial factories for biotechnological processes. In contrast to Bacillus and Geobacillus, Anoxybacillus, which would be thermophilic and spore-forming group of bacteria, is a relatively new genus firstly proposed in the year of 2000. The development of thermostable microbial enzymes, waste management and bioremediation processes would be a crucial parameter in the industrial sectors. There has been increasing interest in Anoxybacillus strains for biotechnological applications. Therefore, various Anoxybacillus strains isolated from different habitats have been explored and identified for biotechnological and industrial purposes such as enzyme production, bioremediation and biodegradation of toxic compounds. Certain strains have ability to produce exopolysaccharides possessing biological activities including antimicrobial, antioxidant and anticancer. This current review provides past and recent discoveries regarding Anoxybacillus strains and their potential biotechnological applications in enzyme industry, environmental processes and medicine.


Assuntos
Anoxybacillus , Bacillaceae , Bacillus , Geobacillus , Biotecnologia , Bacillus/genética , Geobacillus/genética
17.
J Biosci Bioeng ; 135(4): 282-290, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36806411

RESUMO

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst-Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit ß (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst-Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use.


Assuntos
Geobacillus , Recombinases , Recombinases/genética , Recombinases/metabolismo , DNA Polimerase Dirigida por DNA/genética , Geobacillus/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade
18.
Food Chem ; 406: 134506, 2023 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-36463594

RESUMO

Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01 mg/kg and the yield of oil reached to 98.80 % after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.


Assuntos
Geobacillus , Óleo de Soja , Óleo de Soja/química , Lisofosfolipase/metabolismo , Monoacilglicerol Lipases , Lisofosfatidilcolinas , Glycine max/metabolismo
19.
Environ Sci Pollut Res Int ; 30(15): 42596-42607, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35670947

RESUMO

The microbial interactions with plant hosts were known to establish plant growth and beneficial productivity. Some bacteria, fungi, actinomycetes, yeast, and algae have proven as potential effective microbes in agricultural field. In this study, the insecticidal effect of Geobacillus thermodenitrificans PS41 secondary metabolites was tested against third instar larvae of Spodoptera litura, with mortality rate 60.26 ± 1.5% which might influence the agropest management. The test bacterial metabolites were subjected to GC-MS analysis. Totally, 17 different compounds were identified from the ethyl acetate extract metabolites of PS41 strain. The highest peak was obtained with behenic alcohol compound followed by 1-octadecene and penta erythrityl tetrachloride. The Geobacillus thermodenitrificans PS41 secondary metabolites showed potential antifungal activity against plant pathogenic fungi. The highest inhibit was attained against Cladosporium sp., (25 mm) followed by Rhizoctonia solani and Alternaria brassicola (23 mm). However, no toxic effect was exerted upon earthworm (Perionyx excavatus) when treated with PS41 bacterial metabolites. The potential PS41 strain was also found supporting the plant growth. The potential bacterial strain PS41 did not show antagonistic activity against soil bacteria such as Rhizobium sp., Azotobacter sp., Azospirullum brasilense, Pseudomonas fluorescens and Bacillus megaterium. The potential test organism, Geobacillus thermodenitrificans PS41, possessing biopesticide and biofertilizer properties can be a suitable ecofriendly organic applicant in agricultural field for enhancing crop production.


Assuntos
Fungicidas Industriais , Geobacillus , Fungicidas Industriais/farmacologia , Antifúngicos/farmacologia , Desenvolvimento Vegetal , Fungos
20.
Biotechnol Appl Biochem ; 70(3): 1100-1108, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36455188

RESUMO

Alpha-L-arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most-active and the least-active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid-base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C-C bond between C171 and C213 by becoming closer to each other.


Assuntos
Geobacillus , Geobacillus/genética , Glicosídeo Hidrolases/química , Estabilidade Enzimática
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