Increased Malbranchea pulchella β-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs.
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ABSTRACT:
SUBMITTER: Oliveira Monteiro LM
PROVIDER: S-EPMC8081928 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 β-glucosidases.
| S-EPMC7181827 | biostudies-literature
Project description:BACKGROUND:The search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis. RESULTS:Xylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (?/?)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses. CONCLUSIONS:MpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.
| S-EPMC4364333 | biostudies-literature
Project description:An extract from a PDB static culture of Malbranchea dendritica exhibited α-glucosidase and PTP-1B inhibitory activities. Fractionation of the active extract led to the isolation of gymnoascolide A (1), a γ-butenolide, and xanthones sydowinin A (2), sydowinin B (3), and AGI-B4 (4), as well as orcinol (5). Compound 1 exhibited important inhibitory activity against yeast α-glucosidase (IC50 = 0.556 ± 0.009 mM) in comparison to acarbose (IC50 = 0.403 ± 0.010 mM). Kinetic analysis revealed that 1 is a mixed-type inhibitor. Furthermore, compound 1 significantly reduced the postprandial peak in mice during a sucrose tolerance test at the doses of 5.16 and 10 mg/kg. Compound 1 was reduced with Pd/C to yield a mixture of enantiomers 1a and 1b; the mixture showed similar activity against α-glucosidase (IC50 = 0.396 ± 0.003 mM) and kinetic behavior as the parent compound but might possess better drug-likeness properties according to SwissADME and Osiris Property Explorer tools. Docking analysis with yeast α-glucosidase (pdb: 3A4A) and the C-terminal subunit of human maltase-glucoamylase (pdb: 3TOP) predicted that 1, 1a, and 1b bind to an allosteric site of the enzymes. Compounds 1-5 were evaluated against PTP-1B, but only xanthone 3 moderately inhibited in a noncompetitive fashion the enzyme with an IC50 of 0.081 ± 0.004 mM. This result was consistent with that of docking analysis, which revealed that 3 might bind to an allosteric site of the enzyme. From the inactive barley-based semisolid culture of M. dendritica, the natural pigment erythroglaucin (6) and the nucleosides deoxyadenosine (7), adenosine (8), thymidine (9), and uridine (10) were also isolated and identified.
| S-EPMC8427789 | biostudies-literature
Project description:The specificity of thermomycolase toward glucagon and the oxidized A and B chains of insulin was investigated. Extensive digestion of glucagon occurred when conducted at pH 7.0 and 45 degrees C for 40 min, whereas hydrolysis of only three peptide bonds occurred at pH 7.0 and 28 degrees C for 5 min. A similar situation was observed for the oxidized B chain of insulin, which exhibited only a single major cleavage after 5 min at 25 degrees C. No well-defined specificity for particular amino acid residues was evident, but ready hydrolysis of peptide bonds occurred within sequences containing non-polar residues. This endoproteinase must therefore possess an extended hydrophobic binding site for polypeptides. Thermomycolase hydrolysed acetylalanylalanylalanine methyl ester and elastin-Congo Red at 22 and 8.5 times the rate of porcine elastase respectively. A limited degradation of native collagen and significant hydrolysis of benzyloxycarbonyl-Gly-Pro-Leu-Gly-Pro were suggestive of some collagenase-like activity. No keratinase activity was apparent.
| S-EPMC1172400 | biostudies-other
Project description:BACKGROUND: The thermostable ?-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-?-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. RESULTS: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on ?-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as Tm by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (?T of 5-10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in KM and turnover. The KM-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for pNPGlc, while the KM decreased a corresponding extent for Q3.Turnover was only significantly changed using pNPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in KM for this substrate. CONCLUSION: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both KM and turnover. An affinity change, leading to a decreased KM, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site.
| S-EPMC3056771 | biostudies-literature
Project description:?-Glucosidase is claimed as a key enzyme in cellulose hydrolysis. The cellulosic fibers are usually entrapped with hemicelluloses containing xylose. So there is ongoing interest in searching for glucose- and xylose-stimulated ?-glucosidases to increase the efficiency of hydrolysis of cellulosic biomass.A thermostable ?-glucosidase gene (Bglp) was cloned from Anoxybacillus flavithermus subsp. yunnanensis E13T and characterized. Optimal enzyme activity was observed at 60 °C and pH 7.0. Bglp was relatively stable at 60 °C with a 10-h half-life. The kinetic parameters V max and K m for p-nitrophenyl-?-D-glucopyranoside (pNPG) were 771?±?39 ?mol/min/mg and 0.29?±?0.01 mM, respectively. The activity of Bglp is dramatically stimulated by glucose or xylose at concentrations up to 1.4 M. After Bglp was added to Celluclast® 1.5 L, the conversion of sugarcane bagasse was 48.4?±?0.8%, which was much higher than of Celluclast® 1.5 L alone. Furthermore, Bglp showed obvious advantages in the hydrolysis when initial concentrations of glucose and xylose are high.The supplementation of BglP significantly enhanced the glucose yield from sugarcane bagasse, especially in the presence of high concentrations of glucose or xylose. Bglp should be a promising candidate for industrial applications.
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Project description:The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C-C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF2· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF2· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC(O)-Trp (C-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong "red-shifting" of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV-visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical "HC(O)·" equivalents enables first examples of C-C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally "edited" into biology.
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