Microbial α-L-arabinofuranosidases: diversity, properties, and biotechnological applications.

Arabinoxylans (AXs) are hemicellulosic polysaccharides consisting of a linear backbone of ß-1,4-linked xylose residues branched by high content of α-L-arabinofuranosyl (Araf) residues along with other side-chain substituents, and are abundantly found in various agricultural crops especially cereals. The efficient bioconversion of AXs into monosaccharides, oligosaccharides and/or other chemicals depends on the synergism of main-chain enzymes and de-branching enzymes. Exo-α-L-arabinofuranosidases (ABFs) catalyze the hydrolysis of terminal non-reducing α-1,2-, α-1,3- or α-1,5- linked α-L-Araf residues from arabinose-substituted polysaccharides or oligosaccharides. ABFs are critically de-branching enzymes in bioconversion of agricultural biomass, and have received special attention due to their application potentials in biotechnological industries. In recent years, the researches on microbial ABFs have developed quickly in the aspects of the gene mining, properties of novel members, catalytic mechanisms, methodologies, and application technologies. In this review, we systematically summarize the latest advances in microbial ABFs, and discuss the future perspectives of the enzyme research.

Functional characterization of a new 3-dehydroshikimate dehydratase from Eupenicillium parvum and its potential for protocatechuic acid production.

Protocatechuic acid (PCA) is an important phenolic compound with diverse industrial values. Conversion of 3-dehydroshikimate (DHS) to PCA by dehydroshikimate dehydratase (DSD) provides an efficient approach for the production of the molecule. Herein, a new DSD from fungus Eupenicillium parvum was functionally investigated after recombinant expression in Escherichia coli. The DSD displayed 30%-35% sequence identities with the known fungal DSDs. The recombinant protein showed catalysis activity against DHS, with the optimal temperature of 40 °C and pH of 7.5. The specific activity and Km of the protein were 910 mU per mg protein and 0.83 m m, respectively. Metal ion (Mg2+ or Mn2+) played a critical role in the enzymatic activity. Meanwhile, the thermal stability of the protein was improved by Mg2+ or Mn2+. Furthermore, the expression of the protein in E. coli resulted in de novo synthesis of 491 mg/L PCA in a modified M9 medium with glycerol as a carbon source.

Comparison of the Biochemical Properties and Roles in the Xyloglucan-Rich Biomass Degradation of a GH74 Xyloglucanase and Its CBM-Deleted Variant from Thielavia terrestris.

Xyloglucan is closely associated with cellulose and still retained with some modification in pretreated lignocellulose; however, its influence on lignocellulose biodegradation is less understood. TtGH74 from Thielavia terrestris displayed much higher catalytic activity than previously characterized fungal GH74 xyloglucanases. The carbohydrate-binding module 1 (CBM1) deleted variant (TtGH74ΔCBM) had the same optimum temperature and pH but an elevated thermostability. TtGH74 displayed a high binding affinity on xyloglucan and cellulose, while TtGH74ΔCBM completely lost the adsorption capability on cellulose. Their hydrolysis action alone or in combination with other glycoside hydrolases on the free xyloglucan, xyloglucan-coated phosphoric acid-swollen cellulose or pretreated corn bran and apple pomace was compared. CBM1 might not be essential for the hydrolysis of free xyloglucan but still effective for the associated xyloglucan to an extent. TtGH74 alone or synergistically acting with the CBH1/EG1 mixture was more effective in the hydrolysis of xyloglucan in corn bran, while TtGH74ΔCBM showed relatively higher catalytic activity on apple pomace, indicating that the role and significance of CBM1 are substrate-specific. The degrees of synergy for TtGH74 or TtGH74ΔCBM with the CBH1/EG1 mixture reached 1.22-2.02. The addition of GH10 xylanase in TtGH74 or the TtGH74ΔCBM/CBH1/EG1 mixture further improved the overall hydrolysis efficiency, and the degrees of synergy were up to 1.50-2.16.

Two C1-oxidizing AA9 lytic polysaccharide monooxygenases from Sordaria brevicollis differ in thermostability, activity, and synergy with cellulase.

Cellulolytic fungi usually have multiple genes for C1-oxidizing auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) in their genomes, but their potential functional differences are less understood. In this study, two C1-oxidizing AA9 LPMOs, SbLPMO9A and SbLPMO9B, were identified from Sordaria brevicollis, and their differences, particularly in terms of thermostability, reducing agent specificity, and synergy with cellulase, were explored. The two enzymes exhibited weak binding to cellulose and intolerance to hydrogen peroxide. Their oxidative activity was influenced by cellulose crystallinity and surface morphology, and both enzymes tended to oxidize celluloses of lower crystallinity and high surface area. Comparably, SbLPMO9A had much better thermostability than SbLPMO9B, which may be attributed to the presence of a carbohydrate binding module 1 (CBM1)-like sequence at its C-terminus. In addition, the two enzymes exhibited different specificities and responsivities toward electron donors. SbLPMO9A and SbLPMO9B were able to boost the catalytic efficiency of endoglucanase I (EGI) on physically and chemically pretreated substrates but with different degrees of synergy. Substrate- and enzyme-specific synergism was observed by comparing the synergistic action of SbLPMO9A or SbLPMO9B with commercial Celluclast 1.5L on three kinds of cellulosic substrates. On regenerated amorphous cellulose and PFI (Papirindustriens Forskningsinstitut)-fibrillated bleached eucalyptus pulp, SbLPMO9B showed a higher synergistic effect than SbLPMO9A, while on delignified wheat straw, the synergistic effect of SbLPMO9A was higher than that of SbLPMO9B. On account of its excellent thermostability and boosting effect on the enzymatic hydrolysis of delignified wheat straw, SbLPMO9A may have high application potential in biorefineries for lignocellulosic biomass. KEY POINTS: • C1-oxidizing SbLPMO9A displayed higher thermostability than SbLPMO9B, probably due to the presence of a CBM1-like module. • The oxidative activity of the two SbLPMO9s on celluloses increased with decreasing cellulose crystallinity or increasing beating degree. • The two SbLPMO9s boosted the catalytic efficiency of cellulase, but the synergistic effect was substrate- and enzyme-specific.

Characterization and functional analysis of two novel thermotolerant α-L-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum.

Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-L-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-L-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. KEY POINTS: • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

Expression and characterization of two glucuronoyl esterases from Thielavia terrestris and their application in enzymatic hydrolysis of corn bran.

The thermophilic fungus Thielavia terrestris when cultured on cellulose produces a cocktail of thermal hydrolases with potential application in saccharification of lignocellulosic biomass and other biotechnological areas. Glucuronoyl esterases are considered to play a unique role as accessory enzymes in lignocellulosic material biodegradation by cleaving the covalent ester linkage between 4-O-methyl-D-glucuronic acid (MeGlcA) and lignin in lignin-carbohydrate complexes (LCCs). Two glucuronoyl esterases from T. terrestris named TtGE1 and TtGE2 were expressed in Pichia pastoris. Both esterases displayed features of thermophilic enzymes, with the optimal temperature at 45 °C and 55 °C. TtGE1 and TtGE2 exhibited activity towards methyl (4-nitrophenyl ß-D-glucopyranosid) uronate (Me-GlcA-pNP) but no catalytic activity to benzyl-D-glucuronate (BnzGlcA), indicating the difference in substrate specificity from previously studied fungal GEs. A substantial increase in the release of monomeric sugars and glucuronic acid from autohydrolysis of corn bran was observed by the supplementing TtGEs into commercial xylanase; the results clearly demonstrated that the TtGEs played a significant role in this degradation process. This research on TtGEs enriches our knowledge of this novel class of fungal GEs. These newly characterized TtGEs could be used as promising accessory enzymes to improve the hydrolysis efficiency of commercial enzymes in saccharification of lignocellulosic materials due to their thermophilic characteristics.

Heterologous expression of two Aspergillus niger feruloyl esterases in Trichoderma reesei for the production of ferulic acid from wheat bran.

Feruloyl esterase (FAE)-encoding genes AnfaeA and AnfaeB were isolated from Aspergillus niger 0913. For overexpression of the two genes in Trichoderma reesei, constitutive and inductive expression plasmids were constructed based on parental plasmid pAg1-H3. The constructed plasmids contained AnfaeA or AnfaeB gene under the control of glyceraldehyde-3-phosphate dehydrogenase A gene (gpdA) promoter (from A. nidulans) or cellobiohydrolases I (cbh I) gene promoter (from T. reesei), and cbh I terminator from T. reesei. The target plasmids were transferred into T. reesei D-86271 (Rut-C30) by Agrobacterium tumefaciens-mediated transformation (ATMT), respectively. A high level of feruloyl esterase was produced by the recombinant fungal strains under solid-state fermentation, and the cbh I promoter was more efficient than the gpdA promoter in the expression of AnfaeA. The optimum temperatures and pH values were 50 °C and 5.0 for AnFAEA, and 35 °C and 6.0 for AnFAEB. The maximum production levels were 20.69 U/gsd for AnFAEA and 15.08 U/gsd for AnFAEB. The recombinant fungal enzyme systems could release 62.9% (for AnFAEA) and 52.2% (for AnFAEB) of total ferulic acids from de-starched wheat bran, which was higher than the 46.3% releasing efficiency of A. niger 0913. The supplement of xylanase from T. longibrachiatum in the enzymatic hydrolysis led to a small increment of the ferulic acids release.

Characterization of a thermo-alkali-stable laccase from Bacillus subtilis cjp3 and its application in dyes decolorization.

In this work, a novel bacterial strain exhibiting laccase activity was isolated from black liquor and identified as Bacillus subtilis cjp3. The CotA-laccase gene was cloned from strain cjp3 and expressed in Escherichia coli. The purified recombinant laccase has a maximum activity of 7320 U/L, maintaining high stabilities under a wide pH range and high temperature conditions. Nearly no loss of laccase activity was observed even at pH 9.0 after 10 h of incubation. Reactive blue 19, reactive black 5 and indigo carmine could be efficiently decolorized by the purified laccase in the presence of a mediator ABTS. More than 86% of tested dyes were removed in 4 h at pH = 9.0. The recombinant laccase can work well in a broad range of temperatures of 20-80°C(>80% relative activity). These special properties indicated the potential use of the CotA-laccase in treating wastewater containing synthetic dyes.

Replacement of carbohydrate binding modules improves acetyl xylan esterase activity and its synergistic hydrolysis of different substrates with xylanase.

BACKGROUND: Acetylation of the xylan backbone was a major obstacle to enzymatic decomposition. Removal of acetyl groups by acetyl xylan esterases (AXEs) is essential for completely enzymatic hydrolysis of xylan. Appended carbohydrate binding modules (CBMs) can promote the enzymatic deconstruction of plant cell walls by targeting and proximity effects. Fungal acetyl xylan esterases are strictly appended to cellulose-specific CBM1. It is still unclear whether xylan-specific CBMs have a greater advantage than CBM1 in potentiating the activity of fungal deacetylating enzymes and its synergistic hydrolysis of different substrates with xylanase. RESULTS: Three recombinant AXE1s fused with different xylan-specific CBMs, together with wild-type AXE1 with CBM1 and CBM1-deleted mutant AXE1dC, were constructed in this study. The optimal temperature and pH of recombinant AXE1s was 50 °C and 8.0 (except AXE1dC-CBM6), respectively. Cellulose-specific CBM1 in AXE1 obviously contributed to its catalytic action against substrates compared with AXE1dC. However, replacement of CBM1 with xylan-specific CBM4-2 significantly enhanced AXE1 thermostability and catalytic activity against soluble substrate 4-methylumbelliferyl acetate. Whereas replacements with xylan-specific CBM6 and CBM22-2 were more effective in enzymatic release of acetic acid from destarched wheat bran, NaClO2-treated wheat straw, and water-insoluble wheat arabinoxylan compared to AXE1. Moreover, replacement with CBM6 and CBM22-2 also resulted in higher degree releases of reducing sugar and acetic acid from different substrates when simultaneous hydrolysis with xylanase. A good linear relationship exists between the acetic acid and reducing sugar release. CONCLUSIONS: Our findings suggested that the replacement with CBM6 and CBM22-2 not only significantly improved the catalysis efficiency of AXE1, but also increased its synergistic hydrolysis of different substrates with xylanase, indicating the significance of targeting effect in AXE1 catalysis mediated by xylan-specific CBMs.

N- and C-terminal truncations of a GH10 xylanase significantly increase its activity and thermostability but decrease its SDS resistance.

XynII from Volvariella volvacea has high sodium dodecyl sulfate (SDS) resistance, with the potential for industrial applications under harsh conditions. It consists of a single glycoside hydrolase family 10 (GH10) catalytic domain but contains an additional unique 10 and 4 amino acid residues at the N- and C-terminus, respectively. In this study, five XynII derivatives with N- and/or C-terminus deletions were constructed to determine the effects of these regions on enzyme activity, substrate specificity, thermostability, and SDS resistance. Our results revealed that N- and/or C-terminal truncations significantly increased enzyme activity and thermostability, but reduced SDS resistance. Specifically, the XynIIΔNC4 mutant had 2.53-fold more catalytic efficiency (k cat/K m) towards beechwood xylan than wild-type and 3.0-fold more thermostability (t 1/2 [55°C]). XynIIΔNC4 displayed 3.33-, 4.38-, 1.37-, and 1.98-fold more activity against xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively, than XynII did. However, its half-life (t 1/2) in 4 % SDS was only 1.72 h, while that of XynII was 4.65 h. Circular dichroism analysis revealed that deletion of N- and C-terminal segments caused minor changes in secondary structure. Our observations suggest that the extra N- and C-terminal segments in wild-type XynII evolved to strengthen the interaction between these regions of the protein, making the local structure more rigid and reducing structural flexibility. In this way, N- and C-terminal truncations increased the thermostability and activity of XynII on different xylans and linear xylooligosaccharides, but reduced its resistance to SDS.

An organic solvent-tolerant phenolic acid decarboxylase from Bacillus licheniformis for the efficient bioconversion of hydroxycinnamic acids to vinyl phenol derivatives.

A new phenolic acid decarboxylase gene (blpad) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The full-length blpad encodes a 166-amino acid polypeptide with a predicted molecular mass and pI of 19,521 Da and 5.02, respectively. The recombinant BLPAD displayed maximum activity at 37 °C and pH 6.0. This enzyme possesses a broad substrate specificity and is able to decarboxylate p-coumaric, ferulic, caffeic, and sinapic acids at the relative ratios of specific activities 100:74.59:34.41:0.29. Kinetic constant K m values toward p-coumaric, ferulic, caffeic, and sinapic acids were 1.64, 1.55, 1.93, and 2.45 mM, and V max values were 268.43, 216.80, 119.07, and 0.78 U mg(-1), respectively. In comparison with other phenolic acid decarboxylases, BLPAD exhibited remarkable organic solvent tolerance and good thermal stability. BLPAD showed excellent catalytic performance in biphasic organic/aqueous systems and efficiently converted p-coumaric and ferulic acids into 4-vinylphenol and 4-vinylguaiacol. At 500 mM of p-coumaric and ferulic acids, the recombinant BLPAD produced a total 60.63 g l(-1) 4-vinylphenol and 58.30 g l(-1) 4-vinylguaiacol with the conversion yields 97.02 and 70.96 %, respectively. The low yield and product concentration are the crucial drawbacks to the practical bioproduction of vinyl phenol derivatives using phenolic acid decarboxylases. These unusual properties make BLPAD a desirable biocatalyst for commercial use in the bioconversion of hydroxycinnamic acids to vinyl phenol derivatives via enzymatic decarboxylation in a biphasic organic/aqueous reaction system.

Biochemical characteristics of an alkaline pectate lyase PelA from Volvariella volvacea: roles of the highly conserved N-glycosylation site in its secretion and activity.

Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60 °C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca(2+) was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96 IU mg(-1) by the addition of 0.1 mM Ca(2+), whereas the K m values were decreased from 0.681 to 0.514 mg ml(-1). Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16 %) to modest (27.37 %) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site.

The chitinase C gene PsChiC from Pseudomonas sp. and its synergistic effects on larvicidal activity.

Pseudomonas sp. strain TXG6-1, a chitinolytic gram-negative bacterium, was isolated from a vegetable field in Taixing city, Jiangsu Province, China. In this study, a Pseudomonas chitinase C gene (PsChiC) was isolated from the chromosomal DNA of this bacterium using a pair of specific primers. The PsChiC gene consisted of an open reading frame of 1443 nucleotides and encoded 480 amino acid residues with a calculated molecular mass of 51.66 kDa. The deduced PsChiC amino acid sequence lacked a signal sequence and consisted of a glycoside hydrolase family 18 catalytic domain responsible for chitinase activity, a fibronectin type III-like domain (FLD) and a C-terminal chitin-binding domain (ChBD). The amino acid sequence of PsChiCshowed high sequence homology (> 95%) with chitinase C from Serratia marcescens. SDS-PAGE showed that the molecular mass of chitinase PsChiC was 52 kDa. Chitinase assays revealed that the chitobiosidase and endochitinase activities of PsChiCwere 51.6- and 84.1-fold higher than those of pET30a, respectively. Although PsChiC showed little insecticidal activity towards Spodoptera litura larvae, an insecticidal assay indicated that PsChiC increased the insecticidal toxicity of SpltNPV by 1.78-fold at 192 h and hastened death. These results suggest that PsChiC from Pseudomonas sp. could be useful in improving the pathogenicity of baculoviruses.

A novel cellulolytic/xylanolytic SbAA14 from Sordaria brevicollis with a branched chain preference and its synergistic effects with glycoside hydrolases on lignocellulose.

In this study, the novel auxiliary activity (AA) family 14 lytic polysaccharide monooxygenase (LPMO) SbAA14 from Sordaria brevicollis was successfully characterized. It was active against heteroxylan, xyloglucan and cellulose in ß-cellulose and released native oligosaccharides and corresponding C1- and/or C4-oxidized products. SbAA14 showed a branched chain preference, because partial removal of arabinosyl substituents from heteroxylan led to a decrease in activity. SbAA14 had synergistic effects with the debranching enzyme EpABF62C in an enzyme- and ascorbic acid-dependent manner. SbAA14 had synergistic effects with the GH10 endoxylanase EpXYN1, and the degree of synergy was greater with step-by-step addition than with simultaneous addition. SbAA14 could also synergize with Celluclast® 1.5 L on NaOH-pretreated wheat straw and on NaOH-pretreated and hydrogen peroxide-acetic acid (HPAC)-H2SO4-pretreated bamboo substrates. The greatest synergistic effect between SbAA14 and Celluclast® 1.5 L was observed for HPAC-H2SO4-200 mM pretreated bamboo, in which the degree of synergy reached approximately 1.61. The distinctive substrate preference of SbAA14 indicated that it is a novel AA14 LPMO that may act mainly on heteroxylan with numerous arabinosyl substituents between cellulose fibers rather than on recalcitrant xylan tightly associated with cellulose. These findings broaden the understanding of enigmatic AA14 LPMOs and provide new insights into the substrate specificities and biological functionalities of AA14 LPMOs in fungi.

A novel AA14 LPMO from Talaromyces rugulosus with bifunctional cellulolytic/hemicellulolytic activity boosted cellulose hydrolysis.

BACKGROUND: The recently discovered PcAA14A and B from white-rot basidiomycete Pycnoporus coccineus enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation. RESULTS: In this study, a novel AA14 LPMO was characterized from the ascomycete Talaromyces rugulosus. TrAA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables TrAA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of TrAA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from PcAA14B, despite TrAA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, PcAA14B and NcLPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of TrAA14A. CONCLUSIONS: The extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that TrAA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.

Processivity and enzymatic mode of a glycoside hydrolase family 5 endoglucanase from Volvariella volvacea.

EG1 is a modular glycoside hydrolase family 5 endoglucanase from Volvariella volvacea consisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses.

Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus.

Two new laccase genes, named lac1 and lac2, were cloned from the edible basidiomycete Coprinus comatus. Comparison of the deduced amino acid sequences revealed two laccases showed 66.12 % identity and clustered with lac2 and lac3 from Coprinopsis cinerea in same phylogenetic group. Lac1 and lac2 encode proteins of 517 and 523 amino acids preceded by 18 and 21-residue signal peptides, respectively. Lac1 was functionally expressed in Pichia pastoris. The optimum pHs of recombinant Lac1 were 3.0, 6.0, 5.5 and 6.0 and the optimum temperatures were 65, 55, 70 and 50 °C for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The Km values of Lac1 were 34, 4,317, 7,611 and 14 µM, and the corresponding kcat values were 465.79, 7.67, 1.15 and 0.60 (s(-1) mM), for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The enzyme activity was completely inhibited by sodium azide (NaN(3)) and 1,4-dithiothreitol (DTT) at the concentration of 5 mM. Laccase activity was also inhibited by several metal ions, especially Fe(2+), while K(+) and NH(4) (+) slightly enhanced laccase activity. Twelve synthetic dyes belonging to anthraquinone, azo and triphenylmethane dyes were decolorized by the recombinant Lac1 at different extents. The recombinant Lac1 decolorized azo dye Reactive Dark Blue KR up to 90 % without any mediator and increasing to 96 % with mediator, indicating its potential in the treatment of industrial effluent containing some recalcitrant synthetic dyes.

A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase.

A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0-10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K(m) value towards beechwood xylan was 0.548 mg ml⁻¹, and the k(cat)/K(m) ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg⁻¹ s⁻¹ at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries.

Insights into the capability of the lignocellulolytic enzymes of Penicillium parvum 4-14 to saccharify corn bran after alkaline hydrogen peroxide pretreatment.

BACKGROUND: Corn bran is a major agro-industrial byproduct from corn starch processing. It contains abundant arabinoxylan that can be converted into value-added chemicals via biotechnology. Corn bran arabinoxylan (CBAX) is one of the most recalcitrant xylans for enzymatic degradation due to its particular heterogeneous nature. The present study aimed to investigate the capability of the filamentous fungus Penicillium parvum 4-14 to enzymatically saccharify CBAX and reveal the fungal carbohydrate-active enzyme (CAZyme) repertoire by genome sequencing and secretome analysis. RESULTS: CBAX1 and CBAX2 with different branching degrees, together with corn bran residue (CBR) were generated from corn bran after alkaline hydrogen peroxide (AHP) pretreatment and graded ethanol precipitation. The protein blends E_CBAX1, E_CBAX2, and E_CBR were produced by the fungus grown on CBAX1, CBAX2, or CBR, respectively. Under the optimal conditions, E_CBAX1 released more than 80% xylose and arabinose from CBAX1 and CBAX2. Almost complete saccharification of the arabinoxylans was achieved by combining E_CBAX1 and a commercial enzyme cocktail Cellic®CTec3. Approximately 89% glucose, 64% xylose, and 64% arabinose were liberated from CBR by E_CBR. The combination of E_CBR with Cellic®CTec3 enhanced the saccharification of CBR, with conversion ratios of 97% for glucose, 81% for xylose, and 76% for arabinose. A total of 376 CAZymes including plentiful lignocellulolytic enzymes were predicted in P. parvum based on the fungal genomic sequence (25.8 Mb). Proteomic analysis indicated that the expression of CAZymes in P. parvum varied between CBAX1 and CBR, and the fungus produced complete cellulases, numerous hemicellulases, as well as high levels of glycosidases under the culture conditions. CONCLUSIONS: This investigation disclosed the CAZyme repertoire of P. parvum at the genomic and proteomic levels, and elaborated on the promising potential of fungal lignocellulolytic enzymes upon saccharification of corn bran biomass after AHP pretreatment.

High-level production of cordycepin by the xylose-utilising Cordyceps militaris strain 147 in an optimised medium.

Cordycepin is an important active metabolite of Cordyceps militaris. Xylose, an attractive feedstock for producing chemicals through microbial fermentation, cannot be effectively utilised by many reported C. militaris strains. Herein, a xylose-utilising C. militaris strain 147 produced the highest level of cordycepin (3.03 g/L) in xylose culture. Xylose, alanine, and ammonium citrate were determined as the main affecting factors on the cordycepin production using a Plackett-Burman design. The combination of these factors was optimised using response surface methodology, and the maximal 6.54 g/L of cordycepin was produced by the fungus in the optimal medium. Transcriptome analysis revealed that xylose utilisation upregulated the transcriptional levels of genes participating in purine and energy metabolisms in the fungus, which may facilitate the formation of precursors for cordycepin biosynthesis. This investigation provides new insights into the efficient production of cordycepin and is conducive to the valorisation of biomass rich in xylose.