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1.
Biochemistry ; 60(25): 2011-2021, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34105957

RESUMO

We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent Km values for α-R and ATP were 358 and 297 µM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and ß-ribazole (ß-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate ß-N-linked glycosides like ß-adenosine or ß-R. Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (S. enterica) strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated α-Ado in vitro. When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica α-Ado transport may be inefficient. We conclude that GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.


Assuntos
Proteínas de Bactérias/química , Geobacillus/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ribonucleosídeos/química , Proteínas de Bactérias/isolamento & purificação , Ensaios Enzimáticos , Cinética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Purina-Núcleosídeo Fosforilase/química , Ribonucleosídeos/síntese química , Salmonella/enzimologia , Especificidade por Substrato
2.
PLoS One ; 16(6): e0251751, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061877

RESUMO

5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters.


Assuntos
Geobacillus/enzimologia , Lipase/química , Lipase/genética , Simulação de Acoplamento Molecular , Mutação , Geobacillus/genética , Lipase/metabolismo , Conformação Proteica
3.
Biochim Biophys Acta Bioenerg ; 1862(8): 148436, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33940039

RESUMO

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.


Assuntos
Corynebacterium glutamicum/enzimologia , Grupo dos Citocromos b/metabolismo , Eletrodos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Geobacillus/enzimologia , Oxirredutases/metabolismo , Oxigênio/metabolismo , Catálise , Oxigênio/química
4.
Int J Biol Macromol ; 179: 576-585, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33676984

RESUMO

Superoxide dismutases are the enzymes involved in dismutation of superoxide radicals into oxygen and hydrogen peroxide. The present work reports a thermostable Fe/Mn SOD of Geobacillus sp. strain PCH100 (GsSOD) isolated from glacial soil. Purified recombinant GsSOD is a dimeric protein of ~57 kDa that exhibited highest activity at a temperature of 10 °C and pH of 7.8. Maximum enzyme velocity and Michaelis constant of the GsSOD were 1098.90 units/mg and 0.62 µM, respectively. At 80 °C, thermal inactivation rate constant and half-life of GsSOD were 3.33 × 10-3 min-1 and 208 min, respectively. Interestingly, GsSOD tolerated a temperature of 100 °C and 130 °C up to 15 min and 5 min, respectively. Circular dichroism and differential scanning calorimetry confirmed thermostable nature of GsSOD. Apoenzyme of GsSOD regained enzymatic activity in the presence of Fe2+ and Mn2+ as metal ion cofactors. GsSOD was stable under varying concentrations of chemicals, namely ethylenediaminetetraacetic acid, potassium cyanide, hydrogen peroxide, chloroform-ethanol, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, Tween-20, Triton X-100, urea, and guanidine hydrochloride. The enzyme exhibited >70% activity in presence of 10 mM metal ions. Owing to its thermostable nature and resistance to chemical inhibitors, GsSOD is a potential enzyme for industrial applications.


Assuntos
Proteínas de Bactérias/química , Geobacillus/enzimologia , Superóxido Dismutase/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Índia , Cinética , Microbiologia do Solo , Superóxido Dismutase/isolamento & purificação , Temperatura
5.
Org Biomol Chem ; 19(12): 2773-2783, 2021 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-33690764

RESUMO

Different Pd-complexes containing orthometallated push-pull oxazolones were inserted by supramolecular Pd-amino acid coordination on two genetically engineered modified variants of the thermoalkalophilic Geobacillus thermocatenolatus lipase (GTL). Pd-lipase conjugation was performed on the solid phase in the previously immobilized form of GTL under mild conditions, and soluble conjugated Pd-GTL complexes were obtained by simply desorbing by washing with an acetonitrile aqueous solution. Three different Pd complexes were incorporated into two different genetically modified enzyme variants, one containing all the natural cysteine residues changed to serine residues, and another variant including an additional Cys mutation directly in the catalytic serine (Ser114Cys). The new Pd-enzyme conjugates were fluorescent even at ppm concentrations, while under the same conditions free Pd complexes did not show fluorescence at all. The Pd conjugation with the enzyme extremely increases the catalytic profile of the corresponding Pd complex from 200 to almost 1000-fold in the hydrogenation of arenes in aqueous media, achieving in the case of GTL conjugated with orthopalladated 4a an outstanding TOF value of 27 428 min-1. Also the applicability of GTL-C114 conjugated with orthopalladated 4b in a site-selective C-H activation reaction under mild conditions has been demonstrated. Therefore, the Pd incorporation into the enzyme produces a highly stable conjugate, and improves remarkably the catalytic activity and selectivity, as well as the fluorescence intensity, of the Pd complexes.


Assuntos
Complexos de Coordenação/química , Fluorescência , Lipase/química , Oxazolona/química , Paládio/química , Engenharia de Proteínas , Adsorção , Catálise , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , Geobacillus/enzimologia , Lipase/genética , Lipase/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxazolona/metabolismo , Paládio/metabolismo
6.
Biochem Biophys Res Commun ; 547: 96-101, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33610046

RESUMO

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA's have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA's the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3- when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Geobacillus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Magnésio/química , Prótons , Alinhamento de Sequência , Relação Estrutura-Atividade , Zinco/química
7.
J Microbiol Biotechnol ; 31(3): 483-491, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33622993

RESUMO

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDSPAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50°C and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and in silico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.


Assuntos
Proteínas de Bactérias/metabolismo , Geobacillus/enzimologia , Lipase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , DNA Bacteriano , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Geobacillus/genética , Concentração de Íons de Hidrogênio , Lipase/genética , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Temperatura
8.
Int J Biol Macromol ; 173: 421-434, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33493559

RESUMO

In this study lipolytic biocatalysts GD-95RM, GDEst-95 and GDEst-lip were immobilized by encapsulation in calcium alginate beads. All three immobilized biocatalysts demonstrated significantly increased thermal stability at 60-70 °C temperatures and the activity of GD-95RM lipase increased by 50% at 70-80 °C following the immobilization. Moreover, encapsulated GDEst-95 esterase retained higher than 50% lipolytic activity after 3 months of incubation with butanol (25%) and ethanol (50%); GDEst-lip enzyme possessed 50% activity after 2 months of treatment with ethanol (25%) and methanol (25%); and GD-95RM lipase displayed higher that 50% activity after two-week incubation with methanol (50%). All three immobilized enzymes displayed long-term storage capability (>50% activity) at least until 3 months at 4 °C. It was also detected that immobilized GD-95RM and GDEst-lip can perform flow hydrolysis of both avocado oil and p-NP dodecanoate in prototype packed-bed column reactor. The analysis of continuous transesterification of avocado or sunflower oil with ethanol or methanol as substrates confirmed that encapsulated GD-95RM and GDEst-lip enzymes is a useful approach to produce fatty acid alkyl esters.


Assuntos
Geobacillus/enzimologia , Lipase/química , Lipase/metabolismo , Óleos Vegetais/química , Alginatos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Butanóis/farmacologia , Cápsulas , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Esterificação , Etanol/farmacologia , Meia-Vida , Temperatura Alta , Hidrólise , Ácidos Láuricos/química , Metanol/farmacologia , Persea/química , Óleo de Girassol/química
9.
Appl Biochem Biotechnol ; 193(5): 1574-1584, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33507494

RESUMO

Proteases account for approximately 60% of the enzyme market in the world, and they are used in various industrial applications including the detergent industry. In this study, production and characterization of a novel serine protease of thermophilic Geobacillus sp. GS53 from Balçova geothermal region, Izmir, Turkey, were performed. The thermostable protease was purified through ammonium sulfate precipitation and anion-exchange chromatography. The results showed that the protease had 137.8 U mg-1 of specific activity and optimally worked at 55 oC and pH 8. It was also active in a broad pH (4-10) and temperature (25-75 °C) ranges. The protease was highly stable at 85 °C and demonstrated relative stability at pH 4, 7, and 10. Also, the enzyme had high stability against organic solvents and surfactants; enzyme relative activity did not decrease below 81% upon preincubation for 10 min. Ca2+, Cu2+, and Zn2+ ions slightly induced protease activity. The protease was highly specific to casein, skim milk, Hammerstein casein, and BSA substrates. These results revealed that the protease might have a potential effect in a variety of industrial fields, especially the detergent industry, because of its high thermostability and stability to surfactants.


Assuntos
Geobacillus/enzimologia , Cálcio/metabolismo , Cromatografia por Troca Iônica , Cobre/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Peso Molecular , Serina Proteases/metabolismo , Especificidade por Substrato , Tensoativos/química , Zinco/metabolismo
10.
J Agric Food Chem ; 69(3): 1011-1019, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33428404

RESUMO

Luo Han Guo fruit extract (Siraitia grosvenorii), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources (Paenibacillus macerans, Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.


Assuntos
Proteínas de Bactérias/química , Cucurbitaceae/química , Glucosídeos/biossíntese , Glucosiltransferases/química , Extratos Vegetais/química , Edulcorantes/síntese química , Biocatálise , Cromatografia Líquida de Alta Pressão , Frutas/química , Geobacillus/enzimologia , Glucosídeos/química , Paenibacillus/enzimologia , Extratos Vegetais/síntese química , Espectrometria de Massas por Ionização por Electrospray , Edulcorantes/química , Thermoanaerobacter/enzimologia
11.
Prep Biochem Biotechnol ; 51(4): 350-360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32940138

RESUMO

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.


Assuntos
Proteínas de Bactérias/biossíntese , Geobacillus/enzimologia , Lipase/biossíntese , Modelos Estatísticos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Temperatura , Biomassa , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Metanol/metabolismo
12.
Int J Biol Macromol ; 168: 261-271, 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33301847

RESUMO

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.


Assuntos
Esterases/química , Geobacillus/enzimologia , Lipase/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Catálise , Estabilidade Enzimática , Esterases/metabolismo , Geobacillus/metabolismo , Cinética , Lipase/metabolismo , Lipólise , Especificidade por Substrato
13.
Int J Biol Macromol ; 165(Pt B): 2338-2348, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33132126

RESUMO

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.


Assuntos
Dendrímeros/química , Enzimas Imobilizadas/metabolismo , Geobacillus/enzimologia , Proteínas/metabolismo , Dióxido de Silício/química , Termolisina/metabolismo , Aminoácidos/análise , Animais , Bovinos , Estabilidade Enzimática , Estudos de Viabilidade , Hidrólise , Íons , Cinética , Metais/farmacologia , Peptídeos/análise , Soroalbumina Bovina/metabolismo
14.
Int J Mol Sci ; 21(20)2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33050217

RESUMO

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arginase/farmacologia , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Geobacillus/enzimologia , Proteínas Recombinantes/farmacologia , Arginase/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Geobacillus/genética , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Proteínas Mutantes , Proteínas Recombinantes/genética , Ureia/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
15.
J Am Chem Soc ; 142(43): 18652-18660, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-32966073

RESUMO

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.


Assuntos
Radicais Livres/química , Proteínas/química , S-Adenosilmetionina/química , Domínio Catalítico , Teoria da Densidade Funcional , Desoxiadenosinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Geobacillus/enzimologia , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Proteínas/genética , Proteínas/metabolismo , S-Adenosilmetionina/metabolismo
16.
Molecules ; 25(15)2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731607

RESUMO

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 °C and 76.0 °C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in ß-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.


Assuntos
Proteínas de Bactérias , Geobacillus , Lipase , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Geobacillus/enzimologia , Geobacillus/genética , Ligação de Hidrogênio , Lipase/química , Lipase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
17.
Protein Expr Purif ; 175: 105709, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32738436

RESUMO

Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 µmol min-1 mg-1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.


Assuntos
Aspartato Aminotransferases , Proteínas de Bactérias , Expressão Gênica , Geobacillus/genética , Aspartato Aminotransferases/biossíntese , Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Geobacillus/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
18.
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
19.
Int J Biol Macromol ; 164: 578-585, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32693140

RESUMO

In this study, the heterologous expression and biochemical characterization of a thermostable α-amylase from Geobacillus sp. GS33 was investigated. The recombinant α-amylase was overexpressed in Escherichia coli BL21 (λDE) and purified via anion exchange and size-exclusion chromatography. The purified α-amylase had a molecular weight of about 60 kDa, and was active in a broad range of pH 3-10 and temperature (40-90 °C) with maximum activity at pH 7-8 and 60 °C. The enzyme retained 50% residual activity at 65 °C, but only 20% at 85 °C after 16 h. At pH 9 and pH 7, the residual activity at 65 °C was 50% and 30%, respectively. The enzyme was remarkably activated by Co2+, Ca2+, Mg2+, PMSF, DTT, and Triton X-100, but partially inhibited by Cu2+, methanol, hexane, ethanol, acetone, SDS, and Tween 20. A molecular phylogeny analysis showed that the enzyme's amino acid sequence had the closest connection with an α-amylase from Geobacillus thermoleovorans subsp. stromboliensis nov. 3D-structure-based amino acid sequence alignments revealed that the three catalytic residues (D217, E246, D314) and the four Ca2+ ion coordination residues (N143, E177, D186, H221) were conserved in α-amylase from Geobacillus sp. GS33. The temperature stability and neutral pH optimum suggest that the enzyme may be useful for industrial applications.


Assuntos
Clonagem Molecular/métodos , Geobacillus/enzimologia , alfa-Amilases/química , alfa-Amilases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Estabilidade Enzimática , Evolução Molecular , Geobacillus/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peso Molecular , Filogenia , Conformação Proteica , Termodinâmica , alfa-Amilases/metabolismo
20.
J Agric Food Chem ; 68(25): 6892-6899, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32486647

RESUMO

A mannose-6-phosphate isomerase (MPI) from Geobacillus thermodenitrificans was expressed and successfully encapsulated into the Saccharomyces cerevisiae spores. Our results demonstrated that compared to the free enzyme, the MPI triple mutant encapsulated in osw2Δ spores exhibited much preferred enzymatic properties, such as enhanced catalytic activity, excellent reusability, thermostability, and tolerance to various harsh conditions. In combination with an l-arabinose isomerase (AI) also from G. thermodenitrificans, this technique of spore encapsulation was applied for producing a high-value rare sugar l-ribose from biomass-derived l-arabinose. Using a 10 mL reaction system, 350 mg of l-ribose was produced from 1 g of l-arabinose with a conversion yield of 35% by repeatedly reacting with 200 mg of AI-encapsulated spores and 300 mg of MPI-encapsulated spores. This study provides a very useful and concise approach for the synthesis of rare sugars and other useful compounds.


Assuntos
Proteínas de Bactérias/genética , Geobacillus/enzimologia , Manose-6-Fosfato Isomerase/genética , Ribose/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Expressão Gênica , Manose-6-Fosfato Isomerase/química , Manose-6-Fosfato Isomerase/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo
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