Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Recombinant cellobiohydrolase of Myceliophthora thermophila: characterization and applicability in cellulose saccharification

ABSTRACT: Graphical Abstract A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4–12) and temperatures (40–100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL−1, 222.2 μmol mg−1 min−1, 2492 s−1 and 778.7 s−1 mg−1 mL−1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13568-021-01311-8.

SUBMITTER: Dadwal A

PROVIDER: S-EPMC8568750 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Enhanced endoxylanase production by Myceliophthora thermophila with applicability in saccharification of agricultural substrates.

Project description:The production of enzymes by solid-state fermentation is an interesting process and currently used worldwide as it can be carried out in solid matrix in absence of free water. In present study, Myceliophthora thermophila BJTLRMDU3 produced high titres of endoxylanase (890.55 U/g DR, dry residue) using 5 g rice straw at pH 7.0 and at 45 °C with 1:7 (w/v) solid-to-moisture ratio with inoculum rate of 12 × 106 spores/ml after 4 days in solid-state fermentation. High enzyme titre was produced after moistening the rice straw with solution containing ammonium sulphate (0.4%), K2HPO4 (1.0%), MgSO4·7H2O (0.3%), FeSO4·7H2O (0.03%) and CaCl2 (0.03%). Addition of sucrose (2% w/v) and ammonium nitrate (2% w/v) further enhanced the endoxylanase production. A high endoxylanase production was achieved at water activity (a W) of 0.95 (1639.80 U/g DR) that declined drastically below this value. Among different surfactants, Tween 20 (3% v/v) enhanced the secretion of endoxylanase (2047.91 U/g DR). Furthermore, on optimization of K2HPO4 concentration, it was found that 0.5% K2HPO4 improved (2191.28 U/g DR) endoxylanase production and overall 4.35-folds increase in production of endoxylanase was achieved after optimization of culture conditions. The enzyme has potential to liberate monomeric (xylose) as well as oligomeric (xylotiose, xylotetrose, and xylopantose) sugars from xylan. On saccharification of rice straw and corncob with endoxylanase, maximum yield of reducing sugars was 135.61 and 132.61 mg/g of substrate recorded after 48, and 36 h, respectively.

| S-EPMC6525679 | biostudies-literature

Characterization of Two Endo-?-1, 4-Xylanases from Myceliophthora thermophila and Their Saccharification Efficiencies, Synergistic with Commercial Cellulase.

Project description:The xylanases with high specific activity and resistance to harsh conditions are of high practical value for biomass utilization. In the present study, two new GH11 xylanase genes, MYCTH_56237 and MYCTH_49824, have been cloned from thermophilic fungus Myceliophthora thermophila and expressed in Pichia pastoris. The specific activities of purified xylanases reach approximately 1,533.7 and 1,412.5 U/mg, respectively. Based on multiple template-based homology modeling, the structures of their catalytic domains are predicted. Enzyme activity was more effective in 7.5 L fermentor, yielding 2,010.4 and 2,004.2 U/mL, respectively. Both enzymes exhibit optimal activity at 60°C with pH of 6.0 and 7.0, respectively. Their activities are not affected by EDTA and an array of metal ions. The kinetic constants have been determined for MYCTH_56237 (Km = 8.80 mg/mL, Vmax = 2,380 U/mg) and MYCTH_49824 (Km = 5.67 mg/mL, Vmax = 1,750 U/mg). More importantly, both xylanases significantly cooperate with the commercial cellulase Celluclast 1.5 L in terms of the saccharification efficiency. All these biochemical properties of the xylanases offer practical potential for future applications.

| S-EPMC5817056 | biostudies-literature

Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from <i>Myceliophthora thermophila</i>.

Project description:Endoglucanase (EG) is a key enzyme during enzymatic preparation of cellulose nanocrystals (CNCs). Myceliophthora thermophila is a thermophilic fungus that has thermal properties and a high secretion of endoglucanases (EGs), and could serve as potential sources of EGs for the preparation of CNCs. In this work, four different GH families (GH5, GH7, GH12, and GH45) of EGs from M. thermophila were expressed and purified, and their enzymatic characteristics and feasibility of application in CNC preparation were investigated. It was shown that the MtEG5A from M. thermophila has good potential in the enzymatic preparation of CNCs using eucalyptus dissolving pulp as feedstock. It was also observed that there was a synergistic effect between the MtEG5A and other MtEGs in the preparation of CNCs, which improved the yield and properties of CNCs obtained by enzymatic hydrolysis. This study provides a reference for understanding the enzymatic characteristics of different families of EGs from M. thermophile and their potential application in nanocellulose production.

| S-EPMC10341860 | biostudies-literature

Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila.

Project description:The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

| S-EPMC6017366 | biostudies-literature

Extracellular aldonolactonase from Myceliophthora thermophila.

Project description:Fungi secrete many different enzymes to deconstruct lignocellulosic biomass, including several families of hydrolases, oxidative enzymes, and many uncharacterized proteins. Here we describe the isolation, characterization, and primary sequence analysis of an extracellular aldonolactonase from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile). The lactonase is a 48-kDa glycoprotein with a broad pH optimum. The enzyme catalyzes the hydrolysis of glucono-δ-lactone and cellobiono-δ-lactone with an apparent second-order rate constant, k(cat)/K(m), of ~1 × 10(6) M(-1) s(-1) at pH 5.0 and 25°C but is unable to hydrolyze xylono-γ-lactone or arabino-γ-lactone. Sequence analyses of the lactonase show that it has distant homology to cis-carboxy-muconate lactonizing enzymes (CMLE) as well as 6-phosphogluconolactonases present in some bacteria. The M. thermophila genome contains two predicted extracellular lactonase genes, and expression of both genes is induced by the presence of pure cellulose. Homologues of the M. thermophila lactonase, which are also predicted to be extracellular, are present in nearly all known cellulolytic ascomycetes.

| S-EPMC3020561 | biostudies-literature

Decolorization and detoxification of textile wastewaters by recombinant Myceliophthora thermophila and Trametes trogii laccases.

Project description:Laccases are multi-copper oxidoreductases with broad biotechnological applications. Here, we report detailed biochemical characterization of purified recombinant laccases originating from Myceliophthora thermophila (MtL) and Trametes trogii (TtL). We identified optimal conditions for decolorization of commercial dyes and textile wastewater samples. We also tested the toxicity of decolorized wastewater samples using human peripheral blood mononuclear cells. MtL and TtL were expressed in Saccharomyces cerevisiae, and secreted enzymes were purified by consecutive hydrophobic and gel chromatography. The molecular masses of TtL (~?65 kDa) and MtL (>?100 kDa) suggested glycosylation of the recombinant enzymes. Deglycosylation of MtL and TtL led to 25% and 10% decreases in activity, respectively. In a thermal stability assay, TtL retained 61% and MtL 86% of the initial activity at 40 °C. While TtL retained roughly 50% activity at 60 °C, MtL lost stability at temperatures higher than 40 °C. MtL and TtL preferred syringaldazine as a substrate, and the catalytic efficiencies for ABTS oxidation were 7.5 times lower than for syringaldazine oxidation. In the presence of the mediator HBT, purified TtL almost completely decolorized dyes within 30 min and substantially decolorized wastewater samples from a textile factory (up to 74%) within 20 h. However, products of TtL-catalyzed decolorization were more toxic than MtL-decolorized products, which were almost completely detoxified.

| S-EPMC6269326 | biostudies-literature

Saccharification of cellulose by recombinant Rhodococcus opacus PD630 strains.

Project description:The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml(-1), but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.

| S-EPMC3753941 | biostudies-literature

Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila.

Project description:Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism. Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulans. MtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.

| S-EPMC6101365 | biostudies-literature

Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from <i>Myceliophthora thermophila</i>.

Project description:The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (KD = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E0' ∼ -118 mV), and a large increase in the CYP505A30 heme domain FeIII/FeII redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30's ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.

| S-EPMC6044835 | biostudies-literature

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1.

Project description:By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.

| S-EPMC5095424 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data