ABSTRACT
BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.
Subject(s)
Acetylesterase , Esterases , Thermobifida , Acetylesterase/metabolism , Acetylesterase/genetics , Acetylesterase/chemistry , Hydrogen-Ion Concentration , Kinetics , Substrate Specificity , Thermobifida/enzymology , Thermobifida/genetics , Esterases/metabolism , Esterases/genetics , Esterases/chemistry , Enzyme Stability , Temperature , Escherichia coli/genetics , Escherichia coli/metabolism , Cloning, Molecular/methods , Hydrolysis , Xylans/metabolism , Butyrates/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , NitrophenolsABSTRACT
Background Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fne industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. Methods and results In this study, a gene encoding an esterase from Thermobifda fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purifed using afnity chromatography. The TfEst kinetic assay revealed catalytic efciencies of 0.58 s −1 mM−1, 1.09 s−1 mM−1, and 0.062 s−1 mM−1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. Conclusions The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with signifcant implications for the advancement of biotechnology and biofuels industries.
ABSTRACT
A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.
ABSTRACT
As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.
Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.
Subject(s)
Alginates/pharmacokinetics , Neocallimastix , Xylans/analysisABSTRACT
Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.
Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.
ABSTRACT
As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.(AU)
Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.(AU)
Subject(s)
Neocallimastix , Alginates/pharmacokinetics , Xylans/analysisABSTRACT
Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.
Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.
Subject(s)
Neocallimastix , Temperature , Escherichia coli/geneticsABSTRACT
The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: ⢠A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. ⢠TkPLC was recombinantly produced in Pichia pastoris and successfully purified. ⢠TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.
Subject(s)
Soybean Oil , Type C Phospholipases , Lecithins , Phospholipases , Phospholipids , Soybean Oil/chemistry , Type C Phospholipases/geneticsABSTRACT
BACKGROUND The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of a-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. RESULTS Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. CONCLUSIONS The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme
Subject(s)
Geobacillus stearothermophilus/metabolism , Geobacillus stearothermophilus/chemistry , Bacillus subtilis/metabolism , Bacillus subtilis/chemistry , Geobacillus stearothermophilus/isolation & purification , Geobacillus stearothermophilus/genetics , Bacillus subtilis/isolation & purification , Bacillus subtilis/genetics , Lipase/chemistryABSTRACT
Geoffroea decorticans is a xerophilous deciduous tree present in most arid forests of southern South America, which is commonly used in traditional medicine. The seeds of this tree have been previously investigated for their singular chemical composition, but their protein content has been poorly investigated. Herein, we report the isolation, purification, and characterization of a set of thermostable peptides derived from Geoffroea decorticans seeds (GdAPs) with strong antioxidant and anticoagulant activities. The most potent antioxidant peptides showed a half maximal inhibitory concentration (IC50) of 35.5 ± 0.3 µg/mL determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH). They also caused a dose-dependent prolongation of the aPTT clotting time with an IC50 value of ~82 µg/mL. Interestingly, MALDI-TOF/MS analysis showed the presence of three major peptides with low molecular weights of 2257.199 Da, 2717.165 Da, and 5422.002 Da. The derived amino-acid sequence of GdAPs revealed their unique structural features, exhibiting homology with various proteins present in the genome of Arachis hypogaea. All in all, our data suggest a direct applicability of GdAPs for pharmaceutical purposes.
ABSTRACT
Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.
Subject(s)
Glucosides/chemistry , Phytosterols/chemistry , beta-Glucosidase/metabolism , Biofuels/analysis , Cloning, Molecular/methods , Enzymes/chemistry , Hydrolysis , Plant Oils , beta-Glucosidase/biosynthesisABSTRACT
rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.
Subject(s)
Arthropod Proteins/chemistry , Molecular Dynamics Simulation , Rhipicephalus/chemistry , Serine Proteinase Inhibitors/chemistry , Animals , Arthropod Proteins/genetics , Arthropod Proteins/pharmacology , Disease Models, Animal , Humans , Leukocyte Elastase/antagonists & inhibitors , Leukocyte Elastase/metabolism , Mice , Protein Stability , Pulmonary Emphysema/drug therapy , Pulmonary Emphysema/metabolism , Pulmonary Emphysema/pathology , Rhipicephalus/genetics , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/pharmacologyABSTRACT
Biocatalytic decolorization of azo dyes is hampered by their recalcitrance and the characteristics of textile effluents. Alkaline pH and heavy metals present in colored wastewaters generally limit the activity of enzymes such as laccases of fungal origin; this has led to an increasing interest in bacterial laccases. In this work, the dye decolorization ability of LAC_2.9, a laccase from the thermophilic bacterial strain Thermus sp. 2.9, was investigated. Its resistance towards different pHs and toxic heavy metals frequently present in wastewaters was also characterized. LAC_2.9 was active and highly stable in the pH range of 5.0 to 9.0. Even at 100 mM Cd+2, As+5 and Ni+2 LAC_2.9 retained 99%, 86% and 75% of its activity, respectively. LAC_2.9 was capable of decolorizing 98% of Xylidine, 54% of RBBR, 40% of Gentian Violet, and 33% of Methyl Orange after 24 h incubation at pH 9, at 60 °C, without the addition of redox mediators. At acidic pH, the presence of the mediator 1-hydroxybenzotriazole generally increased the catalytic effectiveness. We analyzed the degradation products of laccase-treated Xylidine and Methyl Orange by capillary electrophoresis and mass spectrometry, and propose a degradation pathway for these dyes. For its ability to decolorize recalcitrant dyes, at pH 9, and its stability under the tested conditions, LAC_2.9 could be effectively used to decolorize azo dyes in alkaline and heavy metal containing effluents.
Subject(s)
Azo Compounds , Laccase , Wastewater , Biodegradation, Environmental , Color , Coloring AgentsABSTRACT
Microorganisms present in Antarctica have to deal not only with cold temperatures but also with other environmental conditions, such as high UV radiation, that trigger the generation of reactive oxygen species. Therefore, Antarctic microorganisms must have an important antioxidant defense system to prevent oxidative damage. One of these defenses are antioxidant enzymes, such as catalase, which is involved in the detoxification of hydrogen peroxide produced under oxidative conditions. Here, we reported the isolation and partial characterization of an Antarctic bacterium belonging to the Serratia genus that was resistant to UV-C radiation and well-adapted to cold temperatures. This microorganism, denominated strain I1P, was efficient at decreasing reactive oxygen species levels produced after UV-C irradiation. Genomic and activity assays suggested that the enzymatic antioxidant defense mechanisms of strain I1P, especially its catalase enzyme, may confer UV resistance. This catalase was active in a wide range of temperatures (20-70 °C), showing optimal activity at 50 °C (at pH 7.0), a remarkable finding considering its psychrotolerant origin. In addition, this enzyme was thermostable, retaining around 60% of its activity after 6 h of incubation at 50 °C. The antioxidant defense systems of strain I1P, including its surprisingly thermoactive and thermostable catalase enzyme, make this microorganism a good source of biocompounds with potential biotechnological applications.
ABSTRACT
BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.
Subject(s)
Antigens/immunology , Immunoglobulins/metabolism , Occlusion Body Matrix Proteins/chemistry , Peptides/chemistry , Vaccines/immunology , Animals , Antigens/chemistry , Drug Stability , Female , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/immunology , Immunization , Mice , Nanoparticles , Particle Size , Protein Aggregates , Recombinant Fusion Proteins/immunology , Thermodynamics , Vaccines/chemistryABSTRACT
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/metabolismABSTRACT
Orange juice is a major agricultural product, and oranges are among the most widely sold fresh fruits in several countries. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, affects almost every species of citrus, causing a esthetic depreciation of the fruit and fruit drop, with consequent financial loss for its in natura commercialization. Fungicides are the major control measure for CBS, but have limited efficiency and high cost, and give rise to fungal strains resistant to these products. This work assayed the production of antifungal compounds by Bacillus spp. isolates and tested the potential of two isolates from B. subtilis (ACB-AP3 and ACB-83) for controlling CBS under field conditions with two previously untested orange varieties. A total of 15 isolates produced cell-free, thermostable or volatile compounds effective in suppressing P. citricarpa growth in vitro. It was possible to detect the production of two antibiotics (iturin and surfactin) by B. subtilis ACB-83. Additionally, B. subtilis isolates ACB-AP3 and ACB-83 displayed significant effects in controlling the disease under field conditions.
Subject(s)
Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Ascomycota/drug effects , Bacillus/isolation & purification , Bacillus/metabolism , Citrus/microbiology , Ascomycota/growth & development , Ascomycota/pathogenicity , Fruit/microbiology , Fungicides, Industrial/metabolism , Fungicides, Industrial/pharmacology , Plant Diseases/microbiology , Spores, FungalABSTRACT
Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60⯰C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.
Subject(s)
Cellulase/chemistry , Cellulase/metabolism , Cellulose/metabolism , Enterobacter/enzymology , beta-Glucans/metabolism , Argentina , Carboxymethylcellulose Sodium/metabolism , Cellulase/genetics , Enterobacter/genetics , Enterobacter/isolation & purification , Enzyme Stability , Glucans/metabolism , Glucose/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Oligosaccharides/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Soil Microbiology , Substrate Specificity , Temperature , beta-Glucans/chemistryABSTRACT
Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purified enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specificity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharification. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignification of lignocellulosic biomass.
ABSTRACT
In this study, two hundred fifty-seven bacterial isolates from a suppressive soil library were screened to study their secretion of alkali-thermostable xylanases for potential use in cellulose pulp biobleaching. Xylanase activity was evaluated in solid and liquid media using xylan as the carbon source. Isolates were initially evaluated for the degradation of xylan in solid media by the congo red test. Selected strains were evaluated in liquid media for enzymatic activity and determination of total protein concentration using a crude protein extract (CPE). An isolate identified as Bacillus species TC-DT13 produced the highest amount of xylanase (1808 U mL-1). The isolate was active and stable at 70°C and pH 9.0, conditions which are necessary for the paper industry. This isolate can grow and produce xylanase in medium containing wheat fiber as a substrate. The CPE of this isolate was used in preliminary testing on cellulose pulp bleaching; enzyme treatment of the pulp resulted in a 5% increase of whiteness.