Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.     

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila

Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K _m and k _cat values are 0.4 mM and 15 sec^?1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K _m and k _cat values are 1.3 mg/ml and 67 sec^?1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K _m and k _cat values are 0.08 mM and 35 sec^?1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.     

Induction and regulation of alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14 (ATCC 48104).

The alpha-amylase enzyme synthesis was higher when M. thermophila D-14 (ATCC 48104) was grown in culture medium incorporated with starch or other carbohydrates containing maltose units. Maximum enzyme production was attained with 1% starch followed by a gradual decrease with increasing concentration. Marked decrease in alpha-amylase synthesis occurred with the addition of glucose to the culture medium and this decreasing activity was proportional to the concentration of glucose. The enzyme synthesis was resumed as soon as the glucose concentration fell below a critical level. The addition of cAMP did not eliminate the repressive activity of glucose. The findings suggest that extracellular alpha-amylase synthesis in M. thermophila D-14 was inducible and subject to catabolite repression.     

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC₅₀ 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC₅₀ 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

     

Genome-wide identification,annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila

Cytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXR and CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off (>90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of >65 °C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the well-characterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea).     

Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential

Myceliophthora thermophila syn. Sporotrichum thermophile is a ubiquitous thermophilic mould with a strong ability to degrade organic matter during optimal growth at 45?°C. Both genome analysis and experimental data have suggested that the mould is capable of hydrolyzing all major polysaccharides found in biomass. The mould is able to secrete a large number of hydrolytic enzymes (cellulases, laccases, xylanases, pectinases, lipases, phytases and some other miscellaneous enzymes) employed in various biotechnological applications. Characterization of the biomass-hydrolyzing activity of wild and recombinant enzymes suggests that this mould is highly efficient in biomass decomposition at both moderate and high temperatures. The native enzymes produced by the mould are more efficient in activity than their mesophilic counterparts beside their low enzyme titers. The mould is able to synthesize various biomolecules, which are used in multifarious applications. Genome sequence data of M. thermophila also supported the physiological data. This review describes the biotechnological potential of thermophilic mould, M. thermophila supported by genomic and experimental evidences.     

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively). The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.     

Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library

The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.     

Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions

Applied Microbiology and Biotechnology - After publication of the original article, authors found that there has been a minor mistake in the units of kcat and kcat/Km in Table 2. The units should...     

High-level expression of a xylanase gene from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5?l fermentor culture, the total extracellular protein was 8.1?g?l^?1 with an activity of 52,940?U?ml^?1. The enzyme was purified to homogeneity with a recovery of 48?%. The recombinant xynA was optimally active at 75?°C, as measured over 10?min, and at pH 7. The enzyme was stable up to 80?°C for 30?min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.     

Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction

An endoglucanase gene from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 7, was functionally expressed in methylotrophic yeast Pichia pastoris. The putative endoglucanase from the genomic DNA was successfully cloned in P. pastoris X-33 and the recombinant enzyme was purified to its homogeneity (65 kDa) and subsequently characterized. Substrate specificity analysis revealed that the enzyme exhibits high activity on substrates containing β-1,4-glycosidic bonds such as carboxymethyl cellulose, barley β-glucan, and cello-oligosaccharides, as well as activity on xylan-containing substrates, including arabinoxylan and oat spelt xylan. MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating its key role to the initial step of hydrolysis of high-solids lignocellulose substrates. High thermostability of the endoglucanase reflects potential commercial significance of the enzyme.     

Purification and characterization of alpha-galactosidase from a thermophilic fungus Thermomyces lanuginosus

An extracellular alpha-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl alpha-D-galactosides but efficiently hydrolyzed alpha-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl alpha-galactopyranoside and galactosyl(1)mannotriose, followed by (1)H NMR spectroscopy, pointed out the alpha-anomer of D-galactose was the primary product of hydrolysis from which the beta-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.     

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification.     

Two endoxylanases active and stable at alkaline pH from the newly isolated thermophilic fungus, Myceliophthora sp. IMI 387099

Two extra-cellular endoxylanases (Xyl Ia and Ib) were purified to homogeneity from the newly isolated thermophilic fungus, Myceliophthora sp. IMI 387099. Xyl Ia and Ib, having a molecular mass of approximately 53 kDa and pI of 5.2 and 4.8, respectively, were optimally active at 75 degrees C and at pH 6.0. They were stable at pH 9.2 at 60 degrees C for 2 h, but less stable at pH 6.0 and above 50 degrees C. Mg+2, Zn+2, Ca+2, Co+2 and DTT increased their activity by 1.5-3.0-folds, while SDS and NBS completely inhibited their activity. Both xylanases were active on pNPX and pNPC, but their activity on pNPC was three times higher than that on pNPX. Xyl Ia was more active than Xyl Ib on pNP-alpha-L-Arap, while the latter preferred pNP-alpha-L-Araf. Both xylanases showed two to four times higher activity on rye and wheat arabinoxylans than on birchwood xylan, but Xyl Ib was more active than Xyl Ia on oat spelt xylan. Wheat insoluble pentosan was a good substrate for Xyl Ia, while Xyl Ib preferred wheat soluble arabinoxylan. Xyl Ia had lower Km and higher kcat/Km ratios than Xyl Ib towards all three xylans tested. Both xylanases degraded X4-X6 in an endo-fashion and catalysed hydrolysis and trans-xylosylation reactions. HPLC and LC/MS analysis showed that Xyl Ia and Ib released the unsubstituted X2-X6 as well as mono and di-methyl glucuronic acid substituted X3 and X2 from arabinoxylans.