Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 507
Filtrar
1.
Nat Commun ; 12(1): 5707, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588455

RESUMO

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Geobacillus/fisiologia , Fatores de Transcrição/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/ultraestrutura
2.
J Agric Food Chem ; 69(34): 9859-9868, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34427087

RESUMO

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.


Assuntos
Geobacillus , Sistema da Enzima Desramificadora do Glicogênio , Pão , Geobacillus/genética , Glucanos , Sistema da Enzima Desramificadora do Glicogênio/genética
3.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299026

RESUMO

Pseudomonas aeruginosa and Sphingobacterium sp. are well known for their ability to decontaminate many environmental pollutants while Geobacillus sp. have been exploited for their thermostable enzymes. This study reports the annotation of genomes of P. aeruginosa S3, Sphingobacterium S2 and Geobacillus EC-3 that were isolated from compost, based on their ability to degrade poly(lactic acid), PLA. Draft genomes of the strains were assembled from Illumina reads, annotated and viewed with the aim of gaining insight into the genetic elements involved in degradation of PLA. The draft genome of Sphinogobacterium strain S2 (435 contigs) was estimated at 5,604,691 bp and the draft genome of P. aeruginosa strain S3 (303 contigs) was estimated at 6,631,638 bp. The draft genome of the thermophile Geobacillus strain EC-3 (111 contigs) was estimated at 3,397,712 bp. A total of 5385 (60% with annotation), 6437 (80% with annotation) and 3790 (74% with annotation) protein-coding genes were predicted for strains S2, S3 and EC-3, respectively. Catabolic genes for the biodegradation of xenobiotics, aromatic compounds and lactic acid as well as the genes attributable to the establishment and regulation of biofilm were identified in all three draft genomes. Our results reveal essential genetic elements that facilitate PLA metabolism at mesophilic and thermophilic temperatures in these three isolates.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Genoma Bacteriano , Geobacillus/genética , Poliésteres/metabolismo , Pseudomonas aeruginosa/genética , Sphingobacterium/genética , Biodegradação Ambiental , DNA Bacteriano/análise , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Filogenia
4.
J Biotechnol ; 337: 24-34, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34197821

RESUMO

A thermostable T1 lipase from Geobacillus zalihae exhibits broad substrate specificity and good potential application in fats and oils. However, structural insight into the enzyme against substrates is poorly understood at the molecular level. Herein, the study aimed to examine interactions between a mutant T1 lipase (Mut-T1 lipase) and selected fatty acids (caprylic, myristic, stearic, oleic, linoleic and linolenic acids) by performing molecular docking and molecular dynamics (MD) simulation. The structure of Mut-T1 lipase obtained by homology modeling was reliable for molecular docking and MD simulation. Molecular docking revealed that Mut-T1 lipase showed low binding affinity for caprylic acid (-4.97 kcal/mol) compared to the other fatty acids (-5.65 to -6.88 kcal/mol). However, the conformation of Mut-T1 lipase-caprylic acid complex was comparably stable during the simulation, in terms of less root-mean square fluctuation. Besides, solvent accessible surface area value of Mut-T1 lipase-fatty acid complexes decreased with increasing chain length of fatty acid. van der Waals interactions were requisite in maintaining complex stability during the binding process. This work provides structural insight into interactions between the lipase and the fatty acids, which will facilitate design and applications of new mutants of T1 lipase in modifying fats and oils.


Assuntos
Ácidos Graxos , Simulação de Dinâmica Molecular , Geobacillus , Lipase/genética , Lipase/metabolismo , Simulação de Acoplamento Molecular
5.
Extremophiles ; 25(4): 403-412, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34191121

RESUMO

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.


Assuntos
Geobacillus , Transcriptoma , Escherichia coli/genética , Geobacillus/genética , Proteínas Recombinantes/genética
6.
Braz J Biol ; 82: e239449, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34105678

RESUMO

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.


Assuntos
Escherichia coli , alfa-Amilases , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Geobacillus , Concentração de Íons de Hidrogênio , Temperatura , alfa-Amilases/genética , alfa-Amilases/metabolismo
7.
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
8.
Biomolecules ; 11(5)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069858

RESUMO

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1200 MHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.


Assuntos
Geobacillus/metabolismo , Vírus da Influenza A/metabolismo , Proteínas de Membrana/química , Neisseria gonorrhoeae/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/instrumentação , Proteínas da Membrana Bacteriana Externa/química , Humanos , Campos Magnéticos , Modelos Moleculares , Proteínas Quinases/química , Estrutura Secundária de Proteína , Proteínas da Matriz Viral/química , Canais de Ânion Dependentes de Voltagem/química
9.
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
10.
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
11.
Sci Rep ; 11(1): 8006, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850195

RESUMO

Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent KM value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo3.


Assuntos
Grupo dos Citocromos b , Geobacillus , Consumo de Oxigênio
12.
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
13.
J Food Biochem ; 45(5): e13716, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33788288

RESUMO

Xylanases have great attention to act as a potential role in agro-industrial processes. In this study, production, characterization, and fruit juice application of novel xylanase from thermophilic Geobacillus vulcani GS90 (GvXyl) were performed. GvXyl was purified via acetone precipitation and gel-filtration chromatography. The results showed that GvXyl had 1,671.4 U/mg of specific activity and optimally worked at pH 8 and 55°C. It was also active in a wide pH (3-9) and temperature (30-90ºC) ranges. GvXyl was highly stable at 90ºC and relatively stable at pH 3-9. The kinetic parameters of GvXyl were obtained as Km , Vmax , and kcat ; 10.2 mg/ml, 4,104 µmol min-1  mg-1 , and 3,542.6 s-1 , respectively. GvXyl had higher action than commercial xylanase in fruit juice enrichment. These results revealed that GvXyl might possess a potential influence in fruit juice processing because of its high specific activity and great thermal stability. PRACTICAL APPLICATIONS: Polysaccharides include starch, pectin, and hemicellulose create problems by lowering fruit juice quality in beverages. To overcome this problem, various clarification processes might be applied to natural fruit juices. Even though chemicals are widely used for this purpose, recently enzymes including xylanases are preferred for obtaining high-quality products. In this study, we reported the production and biochemical characterization of novel thermostable xylanase from thermophilic G. vulcani GS90 (GvXyl). Also, apple and orange juice enrichment were performed with the novel xylanase to increase the quality in terms of yield, clarity, and reducing sugar substance. The improved quality features of apple and orange juices with GvXyl was then compared to commercially available ß-1,4-xylanase. The results revealed that GvXyl might possess a potential influence in fruit juice processing because of its high specific activity and great thermal stability.


Assuntos
Sucos de Frutas e Vegetais , Geobacillus , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
14.
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
15.
J Mol Graph Model ; 105: 107897, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33770705

RESUMO

Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The km and kcat values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.


Assuntos
Geobacillus , Lipase , Estabilidade Enzimática , Geobacillus/genética , Geobacillus/metabolismo , Lipase/genética , Lipase/metabolismo , Metanol , Solventes
16.
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
17.
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
18.
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
19.
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
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...