Cloning, expression and characterization of endo-β-1,4-mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris (original) (raw)
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Microbial Cell Factories, 2009
Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic mannooligosaccharides (MOS).
Enzyme and Microbial Technology, 2010
Bacillus stearothermophilus secretes ,-mannanase and oa-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of aL-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified P-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The P-D-mannanase activity exhibited thermostabiity, retaining nearly full activity after incubation for 24 h at 70°C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for V.. and Km of 455 60 U/mg and 1.5 0.3 mg/ml, respectively, at 70°C and pH 6.5.
2016
A cDNA encoding β-mannanase was cloned from <i>Aspergillus niger</i> BCC4525 and expressed in <i>Pichia pastoris</i> KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high <i>k</i><sub>cat</sub>/<i>K</i><sub><i>m</i></sub> (461 mg<sup>−1</sup> s<sup>−1</sup> mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-β-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An <i>in vitro</i> digestibility test simulating the gastrointestinal tract system ...
A mutant MS301 of Aspergillus niger MS82 showed 1.5 to 2.5-fold improved endoglucanase and β-glucosidase activity when grown on crude lignocellulosic substrates under solid-state and submerged conditions. Indicators of thermal stability of enzymes (T m and T 1/2 ) showed that the wild type and mutant endoglucanase was more heat-resistant compared to βglucosidase. However, mutant and parent enzymes shared almost the same values for melting temperatures and half-lives. Endoglucanase and β-glucosidase from both the strains showed optimum activity under acidic pH. Energy of activation (Ea) of mutant β-glucosidase was substantially lower than the parent enzyme while E a of mutant endoglucanase was slightly less than the parent. The lowered E a values can be attributed to the improved β-glucosidase activity of the mutant strain. Moreover, the MS301 enzymes were better in hydrolyzing purified and crude cellulosic materials than the parent MS82.
The wide variety of bacteria in the environment permits screening for more efficient cellulases to help overcome current challenges in biofuel production. A β-1-4 - endoglucanase produced by a newly two Bacillus strains, RL1 and RL2, isolated from soil (Jin Yun mountain, 800 m), Chong Qing province, China. Were sequnced of the 16S rRNA genes of cellulase positive isolates were amplified and sequenced, then BLASTed to determine likely genera. Phylogenetic analysis revealed genera belonging to major Phyla of Gram positive bacteria. The sequence had 1513bp of newly two strains Bacillus sp. RL1 and Bacillus sp. RL2 and G+C content of the sequence was found at 53.46% and 53.6% respectively. Both EGBRL1 and EGBRL2 purified to homogeneity from culture supernatants, enzymes had a molecular mass of 30 kDa and 27 kDa, respectively, while isoelectric point of 6.5 and 8.15, respectively. The sequence analysis using NCBI tool BLAST revealed an open reading frame (ORF) of 822 bp in Bacillus sp. RL1 -1, 4-endoglucanase and 723 bp in Bacillus sp. RL2 -1, 4-endoglucanase, encoding 273 amino acid and 240 amino acid respectively. EgI-RL1 showed 100% homology to that of the sequences Bacillus thuringiensis serovar berliner ATCC10792, Bacillus cereus m1550, Bacillus thuringiensis serovar pakistani str especially the sequence from amino acid 1 to 260 of EgI-RL1. While the translated amino acid sequence of EgI-RL2 showed 99% homology with published Bacillus cereus m1550, Bacillus cereus ATCC4342 and 97% homology with Bacillus cereus MM3 and Bacillus pseudomycoides DSM 12442. A novel EgI-RL1 and EgI-RL2 were found in the full genome sequence of Bacillus sp. RL1 and Bacillus sp. RL2 these genes were successfully amplified from the genomic DNA by polymerase chain reaction (PCR) The PCR product of 1400 bp and 1500bp respectively were purified and ligated into pMD19T vector. Combination E. coli strain DHa 50 was transformed with this recombinant plasmids separately for propagation. For expression of EgI-RL1 and EgI-RL2 genes separately were ligated into pPIC9K vector in the EcoRl and Notl sites. The resultant plasmid pPIC9K-EgI-RL1 and pPIC9K-EgI-RL2 were linearized with SaIl and transformed to P. pastoris GS115. The recombinant endo-β-1,4-glucanase of Bacillus sp.RL1activity reached 130.9% within 72 h after 1% methanol induction in shaking flasks comparative with parent endo-β-1,4-glucanase . These results indicate that the genes for EGBRL1 and EGBRL2 in Bacillus sp.RL1 and Bacillus sp.RL2 belong to the glycoside hydrolase family8 (GHF). The endoglucanases were optimally active in the pH 7.0. The optimum temperature was 35°C for both enzymes. The pH stability of both EGBRL1 and EGBRL2 was carried out between pH 4.0 and 10 was determined by incubating the enzyme in 0.05M citrate buffer of different pH values at 45°C for 30 min and then assaying the residual enzyme activity under standard assay conditions at pH 7.0. Both EG were clearly stable between pH 4.0 and 10. The stability over a broad pH range seems to be characteristic of many Bacillus endoglucanases. This pH stability range is good for this enzyme to be utilized in alkaline conditions such as paper pulp processing. In order to assess the thermal stability of EGBRL1 was determined at various temperature from 30 -90°C at pH 7.0 the enzyme retain 85.5% of the original EGBRL1 activity was maintained at temperature 90°C after 30min pre-incubation. More than 70% of the original EGBRL1 activity was maintained at abroad temperatures throughout the experiments. The results show that EGBRL2 was stable at different temperature carried out and 90.3% at 90°C. Due to its high temperature stability (up to 90o The enzymes showed highest activity with xylose as a carbon source substrate followed by starch and pineapple shell. Beef extract served as a suitable nitrogen source for best production for both endoglucanases from two bacteria, followed by yeast extract and tryptone. Significant activity was also observed with crystalline forms of cellulose such as filter paper for both the enzymes. For carboxy methycellulose, EGBRL1 and EGBRL2 had a Km of 0.2797 and 0.0947 mg mlC), our EGBRL1 and EGBRL2 might be useful for some industrial applications which will be investigated in further study. -1, respectively, and Vmax of 0.2124 and 0.1646 mmol glucose min-1 mg protein-1, respectively. FeSO4.7H2O and CO2+ ions caused a significant increase on EGBRL1 activity 275 and 157%, respectively. While FeSO4.7H2O, Na2SO3, K+, Li+, Ca2+ and Co2+ stimulated EGBRL2 activity 393.49, 114.92, 111.30, 103.53, 128.01 and 211.08%, respectively. Overall, both our study and others indicate that Ca2+ Keywords: β-1-4 – endoglucanase; identification; gene clone; expression is very important for the endoglucanase activity.
Protein Expression and Purification, 2008
The present study demonstrates cloning, expression, and characterization of hyperthermostable L-asparaginase from Thermococcus kodakarensis KOD1 in Escherichia coli BLR(DE3). The recombinant 6Â His-tagged protein L-asparaginase from T. kodakarensis (TkAsn), was purified to homogeneity by heat treatment followed by affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The molecular mass of the purified enzyme was found to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties, such as optimum temperature and pH, were 90°C and 8.0, respectively. Its appearent K m , V max , and K cat values were 2.6 mM, 1121 mmol min À1 mg À1 , and 694 S À1 , respectively. The enzyme displayed high thermal stability at optimum temperature with an insignificant loss in enzymatic activity, retaining almost 90% of its activity over a time period of 32 h. The relative activity of the enzyme was significantly inhibited by the supplementation of Cu 2þ and Ni 2þ ions, while moderately inhibited by other ions. In contrast, Mg 2þ ions enhanced the relative activity compared to the control. The acrylamide contents in baked dough were reduced to sixty percent after treatment with recombinant TkAsn as compared to the untreated control. Results of the present study revealed that the enzyme was highly active at broader range of temperatures and pH, which reflect the potential of recombinant TkAsn in the food processing industry. In addition, the high thermal stability of the enzyme may facilitates its handling, storage, and transportation.