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1.
Braz. j. biol ; 82: e244735, 2022. tab, graf
Article in English | LILACS | ID: biblio-1249280

ABSTRACT

Abstract L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G - 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.


Resumo A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G - 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.


Subject(s)
Humans , Asparaginase/biosynthesis , Asparaginase/pharmacology , Pyrococcus abyssi/enzymology , Antineoplastic Agents/pharmacology , Substrate Specificity , Enzyme Stability , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , Caco-2 Cells , Escherichia coli/genetics , Molecular Docking Simulation , Hydrogen-Ion Concentration
2.
Chinese Journal of Biotechnology ; (12): 2623-2632, 2021.
Article in Chinese | WPRIM | ID: wpr-887828

ABSTRACT

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.


Subject(s)
Glycoside Hydrolases/metabolism , Hydrogen-Ion Concentration , Rhamnose , Substrate Specificity , Temperature
3.
Chinese Journal of Biotechnology ; (12): 2513-2521, 2021.
Article in Chinese | WPRIM | ID: wpr-887817

ABSTRACT

Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.


Subject(s)
Humans , Kinetics , Mutation , Phospholipases A2, Secretory , Phospholipids , Saccharomycetales , Substrate Specificity
4.
Chinese Journal of Biotechnology ; (12): 4215-4230, 2021.
Article in Chinese | WPRIM | ID: wpr-921500

ABSTRACT

Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish β-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure β-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.


Subject(s)
Amino Acids , Catalysis , Glycine , Glycine Hydroxymethyltransferase/metabolism , Kinetics , Substrate Specificity , Threonine
5.
Chinese Journal of Biotechnology ; (12): 3439-3458, 2021.
Article in Chinese | WPRIM | ID: wpr-921440

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants, which have received widespread attentions due to their carcinogenic and mutagenic toxicity. The microbial degradation of PAHs are usually started from the hydroxylation, followed by dehydrogenation, ring cleavage and step-by-step removal of branched chains, and finally mineralized by the tricarboxylic acid cycle. Rieske type non-heme iron aromatic ring-hydroxylating dioxygenases (RHOs) or cytochrome P450 oxidases are responsible for the conversion of hydrophobic PAHs into hydrophilic derivatives by the ring hydroxylation. The ring hydroxylation is the first step of PAHs degradation and also one of the rate-limiting steps. Here, we review the distribution, substrate specificity, and substrate recognition mechanisms of RHOs, along with some techniques and methods used for the research of RHOs and PAHs.


Subject(s)
Bacteria/metabolism , Biodegradation, Environmental , Dioxygenases/metabolism , Iron , Polycyclic Aromatic Hydrocarbons , Substrate Specificity
6.
Chinese Journal of Biotechnology ; (12): 1298-1311, 2021.
Article in Chinese | WPRIM | ID: wpr-878632

ABSTRACT

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.


Subject(s)
Escherichia coli/metabolism , Hydrolases/metabolism , Rhodospirillaceae , Substrate Specificity
7.
Chinese Journal of Biotechnology ; (12): 580-592, 2021.
Article in Chinese | WPRIM | ID: wpr-878583

ABSTRACT

A novel β-glucosidase BglD2 with glucose and ethanol tolerant properties was screened and cloned from the deep-sea bacterium Bacillus sp. D1. The application potential of BglD2 toward polydatin-hydrolyzing was also evaluated. BglD2 exhibited the maximal β-glucosidase activity at 45 °C and pH 6.5. BglD2 maintained approximately 50% of its origin activity after incubation at 30 °C and pH 6.5 for 20 h. BglD2 could hydrolyze a variety of substrates containing β (1→3), β (1→4), and β (1→6) bonds. The activity of β-glucosidase was enhanced to 2.0 fold and 2.3 fold by 100 mmol/L glucose and 150 mmol/L xylose, respectively. BglD2 possessed ethanol-stimulated and -tolerant properties. At 30 °C, the activity of BglD2 enhanced to 1.2 fold in the presence of 10% ethanol and even remained 60% in 25% ethanol. BglD2 could hydrolyze polydatin to produce resveratrol. At 35 °C, BglD2 hydrolyzed 86% polydatin after incubation for 2 h. Thus, BglD2 possessed glucose and ethanol tolerant properties and can be used as the potential candidate of catalyst for the production of resveratrol from polydatin.


Subject(s)
Enzyme Stability , Glucose , Glucosides/pharmacology , Hydrogen-Ion Concentration , Stilbenes/pharmacology , Substrate Specificity , Temperature , Xylose , beta-Glucosidase/genetics
8.
Article in Chinese | WPRIM | ID: wpr-878545

ABSTRACT

The formation of most proteins consists of two steps: the synthesis of precursor proteins and the synthesis of functional proteins. In these processes, propeptides play important roles in assisting protein folding or inhibiting its activity. As an important polypeptide chain coded by a gene sequence in lipase gene, propeptide usually functions as an intramolecular chaperone, assisting enzyme molecule folding. Meanwhile, some specific sites on propeptide such as glycosylated sites, have important effect on the activity, stability in extreme environment, methanol resistance and the substrate specificity of the lipase. Studying the mechanism of propeptide-mediated protein folding, as well as the influence of propeptide on lipases, will allow to regulate lipase by alternating the propeptide folding behavior and in turn pave new ways for protein engineering research.


Subject(s)
Lipase/metabolism , Molecular Chaperones/metabolism , Protein Folding , Protein Precursors , Substrate Specificity
9.
Chinese Journal of Biotechnology ; (12): 868-878, 2020.
Article in Chinese | WPRIM | ID: wpr-826889

ABSTRACT

2-Haloacid dehalogenases (EC 3.8.1.X) catalyze the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. The enzymes not only degrade xenobiotic halogenated pollutants, but also show wide substrate profile and astonishing efficiency for enantiomer resolution, making them valuable in environmental protection and the green synthesis of optically pure chiral compounds. A variety of 2-haloacid dehalogenases have been biochemically characterized so far. Further studies have been made in protein crystal structures and catalytic mechanisms. Here, we review the recent progresses of 2-haloacid dehalogenases in their source, protein structures, reaction mechanisms, catalytic properties and application. We also suggest further research directions for 2-haloacid dehalogenase.


Subject(s)
Catalysis , Halogenation , Hydrolases , Chemistry , Metabolism , Hydrolysis , Research , Substrate Specificity
10.
Chinese Journal of Biotechnology ; (12): 1021-1030, 2020.
Article in Chinese | WPRIM | ID: wpr-826874

ABSTRACT

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.


Subject(s)
Carboxylic Ester Hydrolases , Chemistry , Metabolism , Catalytic Domain , Crystallography , Pectins , Metabolism , Protein Structure, Tertiary , Substrate Specificity
11.
Biomédica (Bogotá) ; 39(supl.1): 96-106, mayo 2019. tab, graf
Article in English | LILACS | ID: biblio-1011458

ABSTRACT

Abstract Introduction: The treatment of urinary tract infections has become more challenging due to the increasing frequency of multidrug-resistant Escherichia coli in human populations. Objective: To characterize multidrug-resistant E. coli isolates causing community-acquired urinary tract infections in Cumaná, Venezuela, and associate possible risk factors for infection by extended-spectrum beta-lactamases (ESBL)-producing isolates. Materials and methods: We included all the patients with urinary tract infections attending the urology outpatient consultation and emergency unit in the Hospital de Cumaná, Estado Sucre, Venezuela, from January through June, 2014. blaTEM, blaSHV and blaCTX-M genes detection was carried out by PCR. Results: We found a high prevalence of multidrug-resistant E. coli (25.2%) with 20.4% of the isolates producing ESBL. The ESBL-producing isolates showed a high frequency (66.7%) of simultaneous resistance to trimethoprim-sulphamethoxazole, fluoroquinolones and aminoglycosides compared to non-producing isolates (2.4%). Of the resistant isolates, 65.4% carried the blaTEM gene, 34.6% the blaCTX-M and 23.1% the blaSHV. The blaCTX-M genes detected belonged to the CTX-M-1 and CTX-M-2 groups. Plasmid transfer was demonstrated by in vitro conjugation in 17 of the 26 ESBL-producing isolates. All three genes detected were transferred to the transconjugants. Age over 60 years, complicated urinary tract infections and previous use of a catheter predisposed patients to infection by ESBL-producing E. coli. Conclusions: The high frequency of multidrug-resistant ESBL-producing isolates should alert the regional health authorities to take measures to reduce the risk of outbreaks caused by these types of bacteria in the community.


Resumen Introducción. El tratamiento de las infecciones urinarias constituye un reto creciente por el aumento de Escherichia coli proveniente de la comunidad multirresistente a los medicamentos. Objetivo. Caracterizar aislamientos de E. coli multirresistente causantes de infecciones urinarias adquiridas en la comunidad en Cumaná, Venezuela, y detectar los posibles riesgos de infección por aislamientos productores de betalactamasas de espectro extendido (BLEE). Materiales y métodos. Se incluyeron todos los pacientes atendidos en la consulta externa de urología y en urgencias del Hospital de Cumaná entre enero y junio de 2014 y que evidenciaban infecciones urinarias. La detección de los genes blaTEM, blaSHV y blaCTX-M se hizo mediante la reacción en cadena de la polimerasa (PCR). Resultados. Se encontró una alta prevalencia de E. coli multirresistente a los medicamentos (25,2 %), con 20,4 % de aislamientos productores de BLEE y una gran frecuencia de resistencia simultánea a trimetoprim-sulfametoxazol, fluoroquinolonas y aminoglucósidos (66,7 %) comparados con los no productores (2,4 %). En el 65,4 % de los aislamientos resistentes, se encontró el gen blaTEM; en 34,6 %, el blaCTX-M, y en 23,1 %, el blaSHV. Los genes blaCTX-M detectados pertenecían a los grupos CTX-M-1 y CTX-M-2. Se demostró la transferencia in vitro de plásmidos por conjugación en 17 de los 26 aislamientos productores de BLEE. Los tres tipos de genes detectados se transfirieron a los transconjugantes. La edad mayor de 60 años, las infecciones urinarias con complicaciones y el uso previo de catéter, predispusieron a la infección por cepas de E. coli productoras de BLEE. Conclusiones. La gran frecuencia de aislamientos multirresistentes productores de BLEE debería alertar a las autoridades sanitarias para tomar medidas que reduzcan el riesgo de epidemias causadas por este tipo de bacterias en la comunidad.


Subject(s)
Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Young Adult , Urinary Tract Infections/microbiology , Drug Resistance, Multiple, Bacterial , Escherichia coli/drug effects , Escherichia coli Infections/epidemiology , Outpatients , Substrate Specificity , Urinary Tract Infections/epidemiology , Venezuela/epidemiology , beta-Lactamases/analysis , beta-Lactamases/genetics , Risk , Prevalence , Retrospective Studies , Risk Factors , Community-Acquired Infections/microbiology , Community-Acquired Infections/epidemiology , beta-Lactam Resistance , Escherichia coli/isolation & purification , Escherichia coli/genetics
12.
Biomédica (Bogotá) ; 39(supl.1): 199-220, mayo 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-1011464

ABSTRACT

Resumen Las betalactamasas, enzimas con capacidad hidrolítica frente a los antibióticos betalactámicos, son responsables del principal mecanismo de resistencia en bacterias Gram negativas; las de mayor impacto clínico y epidemiológico en los hospitales, son las betalactamasas de espectro extendido (BLEE), las de tipo AmpC y las carbapenemasas. El incremento en su frecuencia y su diseminación a nivel mundial ha limitado cada vez más las opciones terapéuticas tanto en infecciones adquiridas en los hospitales como las que se generan en la comunidad. En Colombia, las redes de vigilancia y los grupos de investigación iniciaron su estudio desde finales de los años 90 y, así, se logró la caracterización molecular de las diferentes variantes; además, se reportó una gran prevalencia y diseminación en los hospitales de mediana y alta complejidad, y se describió el impacto clínico de las infecciones que causan. Dichos estudios han evidenciado el alto grado de endemia de algunas de estas betalactamasas y, en consecuencia, la necesidad de una inmediata implementación de programas para inducir el uso prudente de los antibióticos y de medidas de vigilancia, que permitan controlar y prevenir su diseminación, con el fin de disminuir la morbimortalidad en los pacientes y preservar las opciones terapéuticas disponibles en la actualidad. En esta revisión, se recopiló la información sobre las variantes, la distribución geográfica y la caracterización molecular de las betalactamasas en Colombia, así como los estudios llevados a cabo desde finales de la década de 90 hasta el 2016, lo cual permitió tener un panorama de las betalactamasas que circulan en diferentes regiones, su incremento en el tiempo y sus implicaciones clínicas.


Abstract Beta-lactamases are enzymes with hydrolytic activity over beta-lactam antibiotics and they are the main resistance mechanism in Gram-negative bacteria. Extended-spectrum beta-lactamases (ESBL), AmpC, and carbapenemases have the greatest clinical and epidemiological impact in hospital settings. The increasing frequency and worldwide spread of these enzymes have limited the therapeutic options in hospital-acquired infections and those originating in the community. In Colombia, surveillance networks and research groups began studying them in the late 90s. Different variants of these enzymes have been molecularly characterized and their high prevalence and dissemination in medium and high complexity hospitals, along with a high clinical impact, have been reported. Furthermore, many studies in Colombia have evidenced high endemicity for some of these beta-lactamases, which requires an urgent implementation of antimicrobial stewardship programs in order to preserve the few therapeutic options and infection control strategies to prevent and limit their dissemination. In this publication, we carried out a review of the different enzyme variants, geographic distribution, and molecular characterization of these beta-lactamases in Colombia. Additionally, we describe the available information in the literature regarding studies conducted between the late 1990s and 2016, which provide an overview of the beta-lactamases circulating in different regions of Colombia, their increase over time, and their clinical implications.


Subject(s)
Humans , beta-Lactamases/analysis , Gram-Negative Bacterial Infections/microbiology , beta-Lactam Resistance/genetics , Gram-Negative Bacteria/enzymology , Substrate Specificity , beta-Lactamases/classification , beta-Lactamases/genetics , Gram-Negative Bacterial Infections/epidemiology , Colombia/epidemiology , Geography, Medical , Antimicrobial Stewardship , Genes, Bacterial , Gram-Negative Bacteria/drug effects
13.
Chinese Journal of Biotechnology ; (12): 1234-1246, 2019.
Article in Chinese | WPRIM | ID: wpr-771805

ABSTRACT

1,3-1,4-β-glucanase (E.C.3.2.1.73) is an important industrial enzyme which cleave β-glucans into oligosaccharides through strictly cutting the β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units. Microbial 1,3-1,4-β-glucanase belongs to retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold structure. The present paper reviews the industrial application and protein engineering of microbial β-glucanases in the last decades and forecasts the research prospects of microbial β-glucanases.


Subject(s)
Amino Acid Sequence , Glycoside Hydrolases , Models, Molecular , Protein Engineering , Substrate Specificity
14.
Chinese Journal of Biotechnology ; (12): 1843-1856, 2019.
Article in Chinese | WPRIM | ID: wpr-771748

ABSTRACT

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.


Subject(s)
Directed Molecular Evolution , High-Throughput Screening Assays , Protein Engineering , Proteins , Chemistry , Genetics , Metabolism , Substrate Specificity
15.
Chinese Journal of Biotechnology ; (12): 816-826, 2019.
Article in Chinese | WPRIM | ID: wpr-771328

ABSTRACT

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.


Subject(s)
Carboxylic Ester Hydrolases , Metabolism , Catalysis , Molecular Weight , Pichia , Genetics , Metabolism , Polymerization , Protein Engineering , Substrate Specificity
16.
Braz. j. microbiol ; 49(3): 647-655, July-Sept. 2018. graf
Article in English | LILACS | ID: biblio-951810

ABSTRACT

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.


Subject(s)
Aspergillus fumigatus/enzymology , Fungal Proteins/genetics , Fungal Proteins/chemistry , Gene Expression , Cellulase/genetics , Cellulase/chemistry , Cloning, Molecular , Aspergillus fumigatus/genetics , Substrate Specificity , Enzyme Stability , Kluyveromyces/genetics , Kluyveromyces/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Fungal Proteins/metabolism , Cellulase/metabolism , Hot Temperature , Hydrogen-Ion Concentration
17.
Braz. j. microbiol ; 49(2): 429-442, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889226

ABSTRACT

Abstract Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and β-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times β-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.


Subject(s)
Animals , Cattle , Bacillus/enzymology , Cellulase/genetics , Cellulase/metabolism , Evolution, Molecular , Bacillus/growth & development , Bacillus/isolation & purification , Cellulose/metabolism , Computational Biology , Feces/microbiology , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , INDEL Mutation , Sequence Analysis, DNA , Sequence Homology , Substrate Specificity , Zea mays/metabolism
18.
An. acad. bras. ciênc ; 90(1,supl.1): 943-992, 2018. tab, graf
Article in English | LILACS | ID: biblio-886937

ABSTRACT

ABSTRACT Several enzymatic reactions of heteroatom-containing compounds have been explored as unnatural substrates. Considerable advances related to the search for efficient enzymatic systems able to support a broader substrate scope with high catalytic performance are described in the literature. These reports include mainly native and mutated enzymes and whole cells biocatalysis. Herein, we describe the historical background along with the progress of biocatalyzed reactions involving the heteroatom(S, Se, B, P and Si) from hetero-organic substrates.


Subject(s)
Bacteria/metabolism , Biotransformation , Enzymes/metabolism , Biocatalysis , Fungi/metabolism , Substrate Specificity , Biosensing Techniques , Enzymes/chemistry
19.
Chinese Journal of Biotechnology ; (12): 1996-2006, 2018.
Article in Chinese | WPRIM | ID: wpr-771409

ABSTRACT

Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene, Tcxyn10a, was cloned from Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in Pichia pastoris GS115. The recombinant protein was then purified and characterized. The pH and temperature optima of TcXyn10A were determined to be 5.0 and 65-70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0-11.0) or after 1-h treatment at 60 °C. The specific activities of TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose and lichenan were (1 480±26) U/mg, (2 055±28) U/mg, (7.4±0.2) U/mg and (10.9±0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional TcXyn10A has a single catalytic domain, in which the substrate xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.


Subject(s)
Cellulase , Endo-1,4-beta Xylanases , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Docking Simulation , Pichia , Substrate Specificity , Thermoascus
20.
Electron. j. biotechnol ; 27: 37-43, May. 2017. tab, ilus, graf
Article in English | LILACS | ID: biblio-1010283

ABSTRACT

Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.


Subject(s)
Aspergillus niger/enzymology , beta-Galactosidase/chemistry , Substrate Specificity , Kinetics , Amino Acid Sequence , Cloning, Molecular , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
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