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1.
Electron J Biotechnol ; 49: 64-71, Jan. 2021. ilus, tab, graf
Article in English | LILACS | ID: biblio-1291923

ABSTRACT

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.


Subject(s)
Oligosaccharides/metabolism , beta-Mannosidase/metabolism , Plant Gums/chemistry , Mannans , In Vitro Techniques , Enzyme Stability , Sphingomonas , Prebiotics , Fermentation
2.
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
3.
Rev. chil. nutr ; 47(3): 381-389, jun. 2020. tab, graf
Article in Spanish | LILACS | ID: biblio-1126135

ABSTRACT

El género Pseudomonas es una fuente importante de proteasas; sin embargo, su uso está restringido en la industria alimentaria. El clonaje permite aprovechar la capacidad catalítica de estas enzimas mediante su producción en microorganismos inocuos. Por otro lado, las leguminosas son fuentes ricas en proteínas, a partir de las cuales se pueden obtener compuestos con valor agregado mediante procesos de hidrólisis enzimática. En este estudio, se produjo y caracterizó una proteasa recombinante (PT4) alcalina y termoestable de Pseudomonas aeruginosa M211, para la obtención de hidrolizados proteicos de leguminosas. Para ello, el gen de la proteasa se clonó en el vector pJET1.2/blunt utilizando E. coli DHalfa como hospedero. El análisis de la secuencia nucleotídica parcial de la proteasa indicó un 99 % de similitud con Peptidasas de la Familia M4 de Pseudomonas aeruginosa. La enzima recombinante presentó un peso molecular de 80 kDa, demostró ser activa y estable en condiciones alcalinas y termófilas con un pH y temperatura óptimos de 8 y 60 °C, respectivamente, y fue inhibida por EDTA. Además, hidrolizó proteínas de semillas de Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis, obteniéndose fracciones peptídicas menores a 40 kDa. Esta proteasa recombinante se podría utilizar en la elaboración de hidrolizados proteicos funcionales a partir proteínas de distintas fuentes y residuos agroalimentarios.


The genus Pseudomonas is an important source of proteases; however, in the food industry the use of this bacterium is restricted. Cloning allows for the use of the proteolytic activity of Pseudomonas proteases through their production in innocuous microorganisms. Leguminous are protein-rich sources from which value-added compounds can be obtained through enzymatic hydrolysis. In this study, an alkaline and thermostable recombinant protease (PT4) from Pseudomonas aeruginosa M211 was cloned and characterized in order to obtain protein hydrolysates from leguminous. Therefore, protease gene was cloned into the pJET1.2 / blunt vector using E. coli DHalpha as a host. Analysis of protease partial nucleotide sequence showed 99% homology with Peptidases M4 Family from Pseudomonas aeruginosa. The molecular weight of the recombinant enzyme was 80 kDa, it was active and stable under alkaline and thermophilic conditions, presented an optimum pH and temperature of 8 and 60 °C, respectively, and was inhibited by EDTA. In addition, it hydrolysed Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis proteins, obtaining peptide fractions less than 40 kDa. This recombinant protease could be used in the elaboration of functional hydrolysates using protein from different sources and agricultural waste.


Subject(s)
Peptide Hydrolases/metabolism , Protein Hydrolysates/metabolism , Pseudomonas aeruginosa/enzymology , Recombinant Proteins/metabolism , Peptide Hydrolases/genetics , Temperature , Enzyme Stability , Cloning, Molecular , Hydrogen-Ion Concentration , Fabaceae
4.
Electron. j. biotechnol ; 43: 1-7, Jan. 2020. tab, graf, ilus
Article in English | LILACS | ID: biblio-1087520

ABSTRACT

Background: Textile industry not only plays a vital role in our daily life but also a prominent factor in improving global economy. One of the environmental concern is it releases huge quantities of toxic dyes in the water leading to severe environmental pollution. Bacterial laccase and azoreductase successfully oxidize complex chemical structure of nitrogen group-containing azo dyes. Additionally, the presence of textile dye infuriates bacterial peroxidase to act as a dye degrading enzyme. Our present study deals with three textile dye degrading enzymes laccase, azoreductase, and peroxidase through analyzing their structural and functional properties using standard computational tools. Result: According to the comparative analysis of physicochemical characteristics, it was clear that laccase was mostly made up of basic amino acids whereas azoreductase and peroxidase both comprised of acidic amino acids. Higher aliphatic index ascertained the thermostability of all these three enzymes. Negative GRAVY value of the enzymes confirmed better water interaction of the enzymes. Instability index depicted that compared to laccase and preoxidase, azoreductase was more stable in nature. It was also observed that the three model proteins had more than 90% of total amino acids in the favored region of Ramachandran plot. Functional analysis revealed laccase as multicopper oxidase type enzyme and azoreductase as FMN dependent enzyme, while peroxidase consisted of α-ß barrel with additional haem group. Conclusion: Present study aims to provide knowledge on industrial dye degrading enzymes, choosing the suitable enzyme for industrial set up and to help in understanding the experimental laboratory requirements as well.


Subject(s)
Azo Compounds/metabolism , Peroxidase/chemistry , Laccase/chemistry , NADH, NADPH Oxidoreductases/chemistry , Temperature , Azo Compounds/chemistry , Textile Industry , Biodegradation, Environmental , Computer Simulation , Enzyme Stability , Peroxidase/metabolism , Lactase/metabolism , Coloring Agents/metabolism , NADH, NADPH Oxidoreductases/metabolism
5.
Chinese Journal of Biotechnology ; (12): 920-931, 2020.
Article in Chinese | WPRIM | ID: wpr-826884

ABSTRACT

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.


Subject(s)
Animals , Cyclization , Endo-1,4-beta Xylanases , Chemistry , Metabolism , Enzyme Stability , Industrial Microbiology , Methods , Microbiota , Protein Engineering , Rumen , Microbiology , Temperature
6.
Chinese Journal of Biotechnology ; (12): 932-941, 2020.
Article in Chinese | WPRIM | ID: wpr-826883

ABSTRACT

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.


Subject(s)
Cloning, Molecular , Enzyme Stability , Escherichia coli , Genetics , Gene Expression , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase , Genetics , Metabolism , Recombinant Proteins , Genetics , Metabolism , Streptomyces , Genetics
7.
Electron. j. biotechnol ; 41: 60-71, sept. 2019. graf, tab, ilus
Article in English | LILACS | ID: biblio-1087169

ABSTRACT

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.


Subject(s)
Sordariales/enzymology , Glucan 1,3-beta-Glucosidase/chemistry , Temperature , Enzyme Stability , Cellulases , Glucan 1,3-beta-Glucosidase/isolation & purification , Electrophoresis, Polyacrylamide Gel , Tandem Mass Spectrometry , Enzyme Assays , Hydrogen-Ion Concentration
8.
Electron. j. biotechnol ; 39: 91-97, may. 2019. ilus, graf, tab
Article in English | LILACS | ID: biblio-1052260

ABSTRACT

BACKGROUND: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. RESULTS: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and ß-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.


Subject(s)
Penicillium/enzymology , Enzyme Stability , Detergent Industry , Lipase/metabolism , Penicillium/isolation & purification , Penicillium/genetics , Polymerase Chain Reaction/methods , Cold Temperature , Alkalies , Biocatalysis , Hydrophobic and Hydrophilic Interactions , Hydrogen-Ion Concentration , Lipase/isolation & purification , Lipase/genetics , Mutation
9.
Chinese Journal of Biotechnology ; (12): 159-168, 2019.
Article in Chinese | WPRIM | ID: wpr-771390

ABSTRACT

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.


Subject(s)
Angelica sinensis , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protein Isoforms , Temperature
10.
Chinese Journal of Biotechnology ; (12): 513-521, 2019.
Article in Chinese | WPRIM | ID: wpr-771356

ABSTRACT

To explore the immobilization of target proteins for screening libraries of ligand mixtures, magnetic submicron particles (MSP) functionalized with Ni²⁺-NTA and carboxyl were compared for the immobilization of Mycobacterium tuberculosis dihydrofolate reductase (MtDHFR). MtDHFR fused with 6×His was expressed, purified and characterized for kinetics. MtDHFR was immobilized on Ni²⁺-NTA-functionalized MSP directly and carboxyl-functionalized MSP upon activation. The immobilization capacity, residual activity, thermostability and affinities for putative inhibitors were characterized. MtDHFR immobilized on Ni²⁺-NTA-functionalized MSP retained about 32% activity of the free one with the immobilization capacity of (93±12) mg/g of MSP (n=3). Ni²⁺ and EDTA synergistically inhibited MtDHFR activity, while Fe³⁺ had no obvious interference. MtDHFR immobilized on carboxyl-functionalized MSP retained (87±4)% activity of the free one with the immobilization capacity of (8.6±0.6) mg/g MSP (n=3). In 100 mmol/L HEPES (pH 7.0) containing 50 mmol/L KCl, there was no significant loss of the activities of the free and immobilized MtDHFR after storage at 0 °C for 16 h, but nearly 60% and 35% loss of their activities after storage at 25 °C for 16 h, respectively. The inhibition effects of methotrexate on the immobilized and free MtDHFR were consistent (P>0.05). The immobilization of MtDHFR on carboxyl-functionalized MSP was thus favorable for higher retained activity and better thermostability, with promise for rapid screening of its ligand mixtures.


Subject(s)
Enzyme Stability , Enzymes, Immobilized , Hydrogen-Ion Concentration , Kinetics , Ligands , Magnetite Nanoparticles , Mycobacterium tuberculosis , Temperature , Tetrahydrofolate Dehydrogenase
11.
Chinese Journal of Biotechnology ; (12): 616-625, 2019.
Article in Chinese | WPRIM | ID: wpr-771347

ABSTRACT

Glucoamylase is a critical ingredient for saccharification in the starch decomposition, and widely used in food, pharmaceutical and fermentation industries. Glucoamylases are usually thermostable and have peak activities at high temperature, as required for the industrial process of glucose production. In this study, a glucoamylase gene belonging to the glycoside hydrolase (GH) family 15, Tlga15A, was cloned from Talaromyces leycettanus JCM12802, and successfully expressed in Pichia pastoris GS115. Recombinant glucoamylase TlGA showed optimal activities at pH 4.5 and 75 °C. The result of thermostability analysis showed that TlGA retained above 70% activity after incubating for 1 h at 65 °C, and 43% residual activity after 30 min at 70 °C. Moreover, TlGA had high resistance to most metal ions and chemical reagents tested. Various starch substrates could be hydrolyzed by TlGA, including soluble starch (255.6±15.3) U/mg, amylopectin (342.3±24.7) U/mg, glycogen (185.4±12.5) U/mg, dextrin (423.3±29.3) U/mg and pullulan (65.7±8.1) U/mg. The primary, secondary and tertiary structures of glucoamylase were further analyzed. The low ratio of Gly in the primary structure and low exposed nonpolarity solvent accessible surface in the tertiary structure may be the main reasons for TlGA's thermostability. These results show that TlGA is great promising for potential use in the commercial production of glucose syrups. Moreover, this research will provide knowledge and innovating ideas for the improvement of glucoamylase thermostability.


Subject(s)
Cloning, Molecular , Enzyme Stability , Glucan 1,4-alpha-Glucosidase , Hydrogen-Ion Concentration , Pichia , Talaromyces , Temperature
12.
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
13.
Electron. j. biotechnol ; 35: 18-24, sept. 2018. ilus, tab, graf
Article in English | LILACS | ID: biblio-1047727

ABSTRACT

Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies


Subject(s)
Geobacillus/enzymology , L-Lactate Dehydrogenase/metabolism , Computer Simulation , Enzyme Stability , Polymerase Chain Reaction , Cloning, Molecular , Escherichia coli/metabolism , Molecular Docking Simulation , Glycolysis , L-Lactate Dehydrogenase/genetics
14.
Electron. j. biotechnol ; 34: 29-36, july. 2018. ilus, tab, graf
Article in English | LILACS | ID: biblio-1045993

ABSTRACT

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.


Subject(s)
Bacillus/enzymology , Cellulases/biosynthesis , Temperature , Enzyme Stability , Gene Expression , Cell Wall/enzymology , Polymerase Chain Reaction , Cloning, Molecular , Cellulases/isolation & purification , Cellulases/metabolism , Escherichia coli/metabolism , Plant Cells/enzymology , Hydrogen-Ion Concentration , Hydrolysis
15.
Electron. j. biotechnol ; 33: 39-45, May. 2018. tab, graf, ilus
Article in English | LILACS | ID: biblio-1022849

ABSTRACT

Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. T he molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and ßmercaptoethanol, but it was reduced by Cu+2 and Pb+2 . The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.


Subject(s)
Penicillium chrysogenum/metabolism , Polysaccharides/metabolism , Endo-1,4-beta Xylanases/biosynthesis , Temperature , Enzyme Stability , Biomass , Endo-1,4-beta Xylanases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Hydrolysis
16.
Braz. j. biol ; 78(2): 281-288, May-Aug. 2018. tab, graf
Article in English | LILACS | ID: biblio-888879

ABSTRACT

Abstract Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.


Resumo O conhecimento da atividade enzimática é essencial para formular uma correta dieta específica para espécie, além de estarem correlacionadas com o hábito da alimentação e capacidade digestive. Neste estudo determinamos e caracterizamos a atividade enzimática presente no intestino, estômago e fígado do linguado Paralichthys orbignyanus. Os efeitos da temperatura e pH sobre a atividade enzimática também foram avaliados utilizando substratos específicos. O uso de substratos e inibidores específicos mostrou uma forte evidência da presença da tripsina (BApNA = 0,51 ± 0,2 mU mg-1), quimotripsina (SAPNA = 2,62 ± 1,8 mU mg-1), e as aminopeptidases (Leu-p-Nan = 0,97 ± 0,83 mU mg-1) no intestino. O pH ótimo observado para a atividade de tripsina, quimotripsina, leucino aminopeptidase, amilase e pepsina foi 9,5, 9,0, 8,0, 7,5 e 3,5, respectivamente. A temperatura ótima observada foi 50, 50, 50, 40 e 45 °C, respectivamente. Estes resultados fornecem informações adicionais sobre a biologia do linguado brasileiro e pode ser usado como base para novos estudos sobre fisiologia alimentar.


Subject(s)
Animals , Flounder/physiology , Fish Proteins/metabolism , Fish Proteins/chemistry , Gastrointestinal Tract/enzymology , Aminopeptidases/metabolism , Aminopeptidases/chemistry , Temperature , Enzyme Stability , Brazil , Serine Endopeptidases/metabolism , Serine Endopeptidases/chemistry , Hydrogen-Ion Concentration , Liver/enzymology
17.
Electron. j. biotechnol ; 31: 61-66, Jan. 2018. graf, ilus, tab
Article in English | LILACS | ID: biblio-1022044

ABSTRACT

Background: Study of correlation between pretreatment of yeast with ultraviolet radiation and efficiency of further fermentation of wort made of ultrafine grain particles to ethanol. Results: We investigated three races of industrial yeast Saccharomyces cerevisiae (native and irradiated by ultraviolet). Physiological properties during fermentation of starchy wort were tested in all variants. It was shown that activation of the yeast by ultraviolet radiation allows to further increase the ethanol yield by 25% on average compared with the native yeast races when using thin (up to micro- and nano-sized particles) or standard grain grinding. Conclusions: Using mechanical two-stage grinding of starchy raw materials and ultraviolet pretreatment of yeast, the efficiency of saccharification of starch and fermentation of wort to ethanol was increased.


Subject(s)
Saccharomyces cerevisiae/radiation effects , Ultraviolet Rays , Yeasts/radiation effects , Ethanol/radiation effects , Saccharomyces/metabolism , Starch , Temperature , Yeasts/metabolism , Enzyme Stability , Ethanol/metabolism , Fermentation , Glucose , Amylases
18.
Electron. j. biotechnol ; 31: 84-92, Jan. 2018. graf, tab, ilus
Article in English | LILACS | ID: biblio-1022139

ABSTRACT

Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and ß-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of ß-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the ß-glucosidase activity was optimal at pH 6.0. Both CMCase and ß-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and ß-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.


Subject(s)
Penicillium/enzymology , Cellulase/biosynthesis , beta-Glucosidase/biosynthesis , Oligosaccharides , Temperature , Trichoderma/enzymology , Enzyme Stability , Cellulase/metabolism , beta-Glucosidase/metabolism , Amazonian Ecosystem , Biocatalysis , Fermentation , Hydrogen-Ion Concentration , Hydrolysis , Lignin/metabolism
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.
São Paulo; s.n; s.n; 2018. 91 p. graf, tab, ilus.
Thesis in Portuguese | LILACS | ID: biblio-998299

ABSTRACT

A doença de Chagas representa um problema de saúde pública em muitos países e regiões. O tratamento consiste em fármacos tóxicos, com eficácia discutível, principalmente, na fase crônica da doença. Assim, faz-se necessário o planejamento de novos quimioterápicos, mais seguros e eficazes. Os dendrímeros são novas arquiteturas moleculares formadas por um foco central e ramificações partindo desse foco. Apresentam diversas aplicações biológicas como, por exemplo, atuar como transportadores de fármacos. Face ao exposto, o objetivo deste trabalho foi o estudo de condições para ligar o ácido anacárdico (AA) em derivado dendrimérico com potencial ação na doença de Chagas, o qual tem como foco central o ácido succínico (AS) e ramificações compostas por arginina (Arg) e lisina (Lys). Sabe-se que a cruzaína, uma cisteíno-protease do T. cruzi, catalisa a hidrólise de ligação peptídica entre lisina e arginina. A síntese dos compostos em fase sólida forneceu os derivados brutos: (1) pró-fármaco AA-K-R-NH2 e (2) G.05 AA-K(AS)-R-NH2, que foram purificados e caracterizados por Cromatografia Líquida de Alta Eficiência e espectrometria de massas. Os compostos purificados AA-K-R-NH2 e AA-K(AS)-R-NH2 apresentaram rendimentos de 34% e 47%, com pureza de 88% e 98%, respectivamente. Os resultados dos experimentos enzimáticos utilizando o AA-K-R-NH2 não foram conclusivos. Acredita-se que a baixa solubilidade e/ou baixa concentração podem ter contribuído para tal. Já na estabilidade química em pH 7,4 (que simula pH sanguíneo), pH 1,2 (que simula pH estomacal) e pH 8,5 (que simula pH intestinal), observou-se que o AA-K(AS)-R-NH2 foi estável durante as 24 h de ensaio. Estes últimos resultados são interessantes, pois espera-se que o pró-fármaco dendrimérico alcance o T. cruzi estruturalmente integro, sofrendo hidrólise e liberação do composto ativo no interior do parasita


Chagas disease is a public health problem in many countries and regions. The treatment consists of toxic drugs, with debatable efficacy, mainly, in the chronic phase of the disease. Thus, it is necessary to plan new chemotherapeutics, safer and more effective than those drugs. Dendrimers are new molecular architectures composed by a central focus and branching from that focus. They present several biological applications, such as acting as drug carriers. Thereby, the goal of this work was the study of conditions to bind anacardic acid (AA) in a dendrimeric derivative with potential action in Chagas disease, which was composed by a central focus of succinic acid (AS) and branches of arginine (Arg) and lysine (Lys). Cruzain, a T. cruzi cysteine protease, is known to catalyze the peptide-binding hydrolysis between lysine and arginine. Synthesis of the solid phase compounds provided the crude derivatives: (1) prodrug AA-KR-NH2 and (2) G.05 AA-K(AS)-R-NH2, which were purified and characterized by High Performance Liquid Chromatography (HPLC) and mass spectrometry. The purified AA-K-R-NH2 and AA-K(AS)-R-NH2 compounds showed yields of 34% and 47%, with purity of 88% and 98% respectively. The results of the enzymatic experiments using AA-K-R-NH2 were not conclusive. It is believed that the low solubility and/or low concentration may have contributed for this. On the chemical stability at pH 7.4 (which simulates blood pH), pH 1.2 (which simulates stomach pH) and pH 8.5 (which simulates intestinal pH), it was observed that AA-K(AS)R-NH2 was stable for 24 hours. These latter results are interesting because the dendrimeric prodrug is expected to reach structurally integral T. cruzi, undergoing hydrolysis and release of the active compound within the parasite


Subject(s)
Chagas Disease/classification , Dendrimers/analysis , Enzyme Stability , Pharmaceutical Preparations/analysis , Anacardic Acids
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