Bispora Sp Research Articles

More efficient barley malting under catalyst: Thermostability improvement of a β-1,3-1,4-glucanase through surface charge engineering with higher activity

β-1,3–1,4-Glucanase is an indispensable biocatalyst in barley brewing industry for its crucial effect in reducing the viscosity of mash. However, the unsatisfactory thermostability greatly limited its application performance. In this study, structure-based surface charge engineering was conducted aiming at thermostability improvement of BisGlu16B, a highly active β-1,3–1,4-glucanase from Bispora sp. MEY-1. By applying the enzyme thermal stability system (ETSS), residues D47, D213, and D253 were inferred to be critical sites for thermal properties. Single (D47A, D213A, and D253A) and combination (D47A/D213A/D253A) mutants were generated and compared with BisGlu16B. Among all improved mutants, D47A/D213A/D253A outstanded in thermostability. In comparison with BisGlu16B, its T50 and Tm were respectively increased by 7.0 °C and 4.3 °C, while the t1/2 at 70 °C was 8.1 times that of the wild type. Furthermore, the catalytic activity of D47A/D213A/D253A also increased by 42.5%, compared with BisGlu16B (42,900 ± 300 U/mg vs. 30,100 ± 800 U/mg). Comparing with BisGlu16B and commercial enzyme treatment groups, under simulated malting conditions, efficiency improvement was observed in decreasement of viscosity (35.5% and 90.7%) and filtration time (30.9% and 34.6%) for D47A/D213A/D253A treatment group. Molecular dynamics simulation showed that mutation sites A47, A213, and A253 increased the protein rigidity by lowering the overall root mean square deviation (RMSD). This study may bring optimization of technology and improvement of producing efficiency to the present brewing industry.

Improvement of XYL10C_\u2206N catalytic performance through loop engineering for lignocellulosic biomass utilization in feed and fuel industries

BackgroundXylanase, an important accessory enzyme that acts in synergy with cellulase, is widely used to degrade lignocellulosic biomass. Thermostable enzymes with good catalytic activity at lower temperatures have great potential for future applications in the feed and fuel industries, which have distinct demands; however, the potential of the enzymes is yet to be researched.ResultsIn this study, a structure-based semi-rational design strategy was applied to enhance the low-temperature catalytic performance of Bispora sp. MEY-1 XYL10C_∆N wild-type (WT). Screening and comparisons were performed for the WT and mutant strains. Compared to the WT, the mutant M53S/F54L/N207G exhibited higher specific activity (2.9-fold; 2090 vs. 710 U/mg) and catalytic efficiency (2.8-fold; 1530 vs. 550 mL/s mg) at 40 °C, and also showed higher thermostability (the melting temperature and temperature of 50% activity loss after 30 min treatment increased by 7.7 °C and 3.5 °C, respectively). Compared with the cellulase-only treatment, combined treatment with M53S/F54L/N207G and cellulase increased the reducing sugar contents from corn stalk, wheat bran, and corn cob by 1.6-, 1.2-, and 1.4-folds, with 1.9, 1.2, and 1.6 as the highest degrees of synergy, respectively.ConclusionsThis study provides useful insights into the underlying mechanism and methods of xylanase modification for industrial utilization. We identified loop2 as a key functional area affecting the low-temperature catalytic efficiency of GH10 xylanase. The thermostable mutant M53S/F54L/N207G was selected for the highest low-temperature catalytic efficiency and reducing sugar yield in synergy with cellulase in the degradation of different types of lignocellulosic biomass.Graphic

Loop engineering of a thermostable GH10 xylanase to improve low-temperature catalytic performance for better synergistic biomass-degrading abilities

Lignocellulosic biorefining for producing biofuels poses technical challenges. It is usually conducted over a long time using heat, making it energy intensive. In this study, we lowered the energy consumption of this process through an optimized enzyme and pretreatment strategy. First, the dominant mutant M137E/N269G of Bispora sp. MEY-1XYL10C_ΔN was obtained by directed evolution with highcatalytic efficiency (970 mL/s∙mg)and specific activity (2090 U/mg)at 37 °C, and thermostability was improved (T50 increased by5 °C). After pretreatment with seawater immersionfollowing steam explosion,bagasse was co-treated with cellulase and M137E/N269G under mild conditions (37 °C), the resulting highest yield of fermentable sugars reached 219 µmol/g of bagasse,46% higher than that of the non-seawater treatment group, with the highest degree of synergy of 2.0. Pretreatment with seawater following steam explosion and synergistic hydrolysis through high activity xylanase and cellulase helped to achieve low energy degradation of lignocellulosic biomass.

Development and characterization of a thermostable GH11/GH10 xylan degrading chimeric enzyme

Xylanases are categorized into different family groups, two of which are glycoside hydrolases 10 (GH10) and 11 (GH11) families. These well-characterized xylanases demonstrate different modes of action in hydrolysis of xylans. Imitating certain types of microorganisms to produce bifunctional enzymes such as engineered xylanases has gained considerable attention among researchers. In this study, a recombinant chimeric enzyme (X11-10) was designed by fusing two thermostable xylanases through a peptide linker. The recombinant parental enzymes, xylanase 10 from fungus Bispora sp. MEY-1 (X10) and xylanase 11 from bacterium Thermobacillus xylanilyticus (X11), and their chimera were successfully expressed in Pichia pastoris (P. pastoris), purified, and characterized. Being active over a wide pH range, X11-10 chimera showed higher thermal stability, possessed a lower Km, and a higher catalytic efficiency (kcat/Km) in comparison to the parental enzymes. Also, molecular dynamics simulation (MDS) of X11-10 revealed that its active site residues were free to interact with substrate. This novel chimeric xylanase may have potential applications in different industrial processes since it can substitute two separate enzymes and therefore minimize the production costs.

Isolation of Endophytic Fungi from Vitex trifolia L and Antagonism Test against Sclerotium rolfsii and pathogenic bacteria

Plant damage by pathogenic fungi is often found in plants, one of which is caused by Sclerotium rolfsii. Biological control strategy offers a promising alternative for managing disease in plants because they are environmental friendly compared to pesticides application. One of the biological control offered is by using endophytic fungi isolated from Vitex trivolia L. The aim of the study was to isolate, to identify macroscopic and microscopic endophytic fungi from Vitex trifolia L and to test their antagonism potency against the pathogenic fungus Sclerotium rolfsii in vitro. The isolation obtained 7 endophytic fungi isolates identified based on their genus characteristics, nsmely Periconia sp, Aspergillussp, Dendrophoma sp, Geotrichum sp, Ampulliferina sp, Chalara sp, dan Bispora sp and 2 isolates have not been identified. The Antibacterial test of the fungi isolate on the 4 tested bacteria showed that of all the fungi isolate have low activity. The antagonism test using the direct opposition method with the PIRG formula, showed that the 3 isolates had high percentage of growth inhibition, in which ALJ1, BLJ5, and ALJ3 isolate has  85%, 90%, and 100% respectively. This potency could be used as biological agents on the pathogenic fungus Sclerotium rolfsii.

Improvement of BsAPA Aspartic Protease Thermostability via Autocatalysis-Resistant Mutation.

An aspartic protease gene (Bsapa) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. The recombinant BsAPA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of BsAPA. However, heat inactivation still limits the application of BsAPA. To further improve its thermostability, an autocatalysis site (L205-F206) in BsAPA was identified and three mutants (F193W, K204P, and A371V) were generated based on the analysis of the structure neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2-, and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold increased half-life at 80 and a 10.7 °C increased Tm, compared with those of the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Furthermore, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.

Identification, heterologous expression and characterization of a novel glycoside hydrolase family 30 xylanase from the fungus Penicillium purpurogenum

Penicillium purpurogenum grows on a variety of natural carbon sources and secretes to the medium a large number of enzymes that degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel xylanase (XynC) belonging to family 30 of the glycoside hydrolases (GH), has been identified in the genome of the fungus. The enzyme has been expressed in Pichia pastoris and characterized. The mature XynC has 454 amino acid residues and a calculated molecular weight of 49 240. The purified protein shows a molecular weight of 67 000, and it is partially deglycosylated using EndoH. Its pH optimum is in the range of 3–5, and the optimal temperature is 45 °C. It is active on both arabinoxylan and glucuronoxylan, similarly to other fungal GH 30 xylanases. It liberates a set of oligosaccharides, which have been detected by thin-layer chromatography, thus indicating that it is an endo-acting xylanase. It hydrolyzes xylooligosaccharides, releasing mainly xylobiose, in contrast to other fungal GH family 30 enzymes which generate chiefly xylose. Highest sequence identity to a characterized family 30 xylanase is found with the enzyme from the fungus Bispora sp (53%). This is the first GH 30 xylanase described from a Penicillium.

Functional Analysis of a Highly Active β-Glucanase from Bispora sp. MEY-1 Using Its C-terminally Truncated Mutant.

A β-1,3-1,4-glucanase-encoding gene, Bisglu16B, was identified in Bispora sp. MEY-1. The deduced BisGlu16B consists of an N-terminal signal peptide, a catalytic module of glycoside hydrolase family 16 (GH16), and a C-terminal serine/proline-rich module. After expression in Pichia pastoris GS115, the purified recombinant BisGlu16B showed maximal activity at pH 4.0 and 55 °C and had broad substrate specificity (β-1,3-/β-1,4-mixed, β-1,3-, β-1,4-, and β-1,6-linked glucan, and β-1,4-mannan). The enzyme possessed high specific activities toward barley β-glucan (34 700 U·mg-1), lichenan (23 900 U·mg-1), and laminarin (9 000 U·mg-1). After removing the C-terminal module, the truncated mutant, BisGlu16B-ΔC, retained similar enzymatic properties to the wild type but displayed significantly enhanced activities (up to 2.5-fold). Functional and structural analyses indicated that the C-terminal module plays a key role in the substrate binding of BisGlu16B. This study provided an excellent candidate glucanase for industrial purposes and revealed the functions of a C-terminal serine/proline-rich region.

A key residue for the substrate affinity enhancement of a thermophilic endo-polygalacturonase revealed by computational design.

Protein engineering has been a research hotspot to improve the catalytic efficiency of industrially important enzymes. In the present study, a novel computational strategy was developed to in silico screen mutants with enhanced binding interaction between enzyme and substrate as well as catalytic efficiency. Through homology modeling and molecular dynamics (MD) simulation, four key residues related to substrate binding were identified in the endo-polygalacturonase BiPG28A from Bispora sp. MEY-1. Further analyses of the conformation, hydrogen bond interactions, and binding free energy revealed that lysine at position 129 (subsite - 2) has the strongest affinity to substrate. Biochemical and calorimetry experiments confirmed the functional role of Lys129 in substrate binding through non-covalent interactions. The common role of Lys129 was also verified in another GH28 endo-polygalacturonase. Distinguished from other protein engineering strategies involving structure resolution and construction of certain enzymes, this computational strategy represents an insightful and efficient approach to develop a "designed" enzyme with significantly enhanced binding affinity and catalytic efficiency.

Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1.

Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency.

Improvement of the catalytic performance of a hyperthermostable GH10 xylanase from Talaromyces leycettanus JCM12802

A xylanase gene of GH 10, Tlxyn10A, was cloned from Talaromyces leycettanus JCM12802 and expressed in Pichia pastoris. Purified recombinant TlXyn10A was acidic and hyperthermophilic, and retained stable over the pH range of 2.0–6.0 and at 90°C. Sequence analysis of TlXyn10A identified seven residues probably involved in substrate contacting. Three mutants (TlXyn10A_P, _N and _C) were then constructed by substituting some or all of the residues with corresponding ones of hyperthermal Xyl10C from Bispora sp. MEY-1. TlXyn10A_P with mutations at subsites +2 to +4 exhibited improved specific activity (by 0.44-fold) and pH stability (2.0–10.0). Molecular dynamics simulation analysis indicated that mutations E229I and F232E probably weaken the substrate affinity at subsites +3 to +4, and G149D may introduce a new hydrogen bond. These modifications altogether account for the improved performance of TlXyn10A_P. Moreover, TlXyn10A_P was able to hydrolyze wheat straw persistently, and has the application potentials in various industries.

A thermostable glucoamylase from Bispora sp. MEY-1 with stability over a broad pH range and significant starch hydrolysis capacity.

BackgroundGlucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.Methodology/Principal FindingsA glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.Conclusion/SignificanceGLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

BackgroundIncorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.Methodology/Principal FindingsHere we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract.Conclusion/SignificanceOur study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

Identifikasi Jamur pada rizofir Tanaman Nenas (Ananas comosus L.) dan Uji Indikasi Antagonisnya terhadap patogen Thielaviopsis Paradoxa di Desa Rimbo Panjang Kecamatan Tambang Kabupaten Kampar

The objective of this research was to identify the fungi at pineapple rizoshere and indication of its antagonism to Thielaviopsis paradoxa. It was conducted at laboratory from April to Juli 2006 and sampling method was purposive sampling. The parameters of this research were identification of the rizophere fungi and the ability of rizophere fungi ti inhibit the growth of Thielaviopsis paradoxa. The reasult of this research can be summarized that the pineapple rizosphere fungi identified were: 1). Bispora Sp, 2). Trichocladium Sp, 3). Trichoderma harzianicum, 4). Trichoderma longibrachiatum. Bispora Sp. and Trichocladium Sp. Do not have the ability to inhibit the growth of Thielaviopsis paradoxa, while Trichoderma harzianicum and Trichoderma longibrachiatum have the ability y to inhibit the growth of Thielaviopsis paradoxa.

Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization.

BackgroundMannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.Methodology/Principal FindingsThe man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.Conclusion/SignificanceThis study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

Dense fouling in acid transfer pipelines by an acidophilic rubber degrading fungus

An unique case of dense fouling by an acidophilic, hard rubber (polymerized rubber) degrading fungus in the acid transfer pipelines of a boron enrichment plant located at Kalpakkam, India is reported. In spite of a highly adverse environment for survival (pH 1.5, no dissolved nutrients), the fungus thrived and clogged the pipeline used for transferring 0.1N hydrochloric acid (HCl). Detailed investigations were carried out to isolate and identify the fungus and examine the nutrient source for such profuse growth inside the system. Microscopic observation showed the presence of a thick filamentous fungal biomass. Molecular characterization by 18S rRNA gene sequencing showed 98% similarity of the isolate with the acidophilic fungus Bispora sp. In laboratory studies the fungus showed luxuriant growth (specific growth rate of 13 mg day−1) when scrapings of the hard rubber were used as the sole source of carbon. Scanning electron microscopy revealed extensive incursion of the fungus into the hard rubber matrix. In the laboratory, fungal growth was completely inhibited by the antifungal agent sodium omadine. The study illustrates an interesting example of biofouling under extreme conditions and demonstrates that organisms canphysiologically adapt to grow under unfavourable conditions, provided that a nutrient source is available and competition is low. The use of this fungal strain in biodegradation and in development of environmentally compatible processes for disposal of rubber wastes is envisaged.

Cloning, expression and characterization of an acidic endo-polygalacturonase from Bispora sp. MEY-1 and its potential application in juice clarification

An endo-polygalacturonase gene, pga1, was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. The 1455-bp full-length complementary DNA of pga1 encoded a 485-amino acid polypeptide (endo-PGA1) including a putative 21-residue signal peptide and a catalytic domain belonging to glycoside hydrolase family 28. Purified recombinant endo-PGA1 exhibited activity towards polygalacturonic acid and pectin was optimally active at pH 3.5 and 50 °C, and showed good stability at pH 2.0–7.0. When tested against pectin, endo-PGA1 exhibited 40% of maximum activity at pH 2.0 and over 50% maximum activity at pH 2.5–4.5. The K m,app and V max,app values for polygalacturonic acid were 1.25 mg/ml and 2526 μmol/min/mg, respectively. When treated apple juice at the concentration of 10 U/ml, endo-PGA1 reduced the intrinsic viscosity (7.7% vs. 8.0%) and increased the light transmittance (84% vs. 86%) almost on the same level as the commercial compound pectinase did. These properties make endo-PGA1 an interesting biocatalyst for acidic industrial processes, especially in the juice clarification.

An α-galactosidase from an acidophilic Bispora sp. MEY-1 strain acts synergistically with β-mannanase

An α-galactosidase gene ( AgalB) was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. The deduced amino acid sequence showed highest identity (35%) to the α-galactosidase from Penicillium simplicissimum, belonging to the glycosyl hydrolase family 27. The purified recombinant α-galactosidase (r-AgalB) exhibited optimal activity at pH 3.5 and 55 °C, was stable at pH 2.2–8.0, and showed higher hydrolytic activity towards galactomannan polysaccharides (guar gum and locust bean gum) than toward small galacto-oligosaccharides (melibiose, raffinose and stachyose). A synergistic (3-fold) increase in guar gum hydrolysis was observed when β-mannanase Man5A from Bispora sp. MEY-1 and r-AgalB were combined. Further, an increase in the reaction time from 5 h to 12 h or increase of the temperature from 37 °C to 55 °C enhanced guar gum degradation by the enzyme combination. These properties make r-AgalB a good candidate for extensive application in the pulp/paper, food, and feed industries.

An Acidophilic and Acid-Stable β-Mannanase from Phialophora sp. P13 with High Mannan Hydrolysis Activity under Simulated Gastric Conditions

A beta-mannanase gene, man5AP13, was cloned from Phialophora sp. P13 and expressed in Pichia pastoris. The deduced amino acid sequence of the mature enzyme, MAN5AP13, had highest identity (53%) with the glycoside hydrolase family 5 beta-mannanase from Bispora sp. MEY-1. The purified recombinant beta-mannanase was acidophilic and acid stable, exhibiting maximal activity at pH 1.5 and retaining >60% of the initial activity over the pH range 1.5-7.0. The optimum temperature was 60 degrees C. The specific activity, K(m) and V(max) for locust bean gum substrate were 851 U/mg, 2.5 mg/mL, and 1667.7 U/min.mg, respectively. The enzyme had excellent activity and stability under simulated gastric conditions, and the released reducing sugar of locust bean gum was significantly enhanced by one-fold in simulated gastric fluid containing pepsin in contrast to that without pepsin. All these properties make MAN5AP13 a potential additive for use in the food and feed industries.

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-beta-D-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 degrees C, maintained more than 60% of maximal activity when assayed at pH 1.5-4.0, and had good thermal stability at 60 degrees C and remained stable at pH 1.0-6.0. The enzyme activity was enhanced in the presence of Ni(2+) and beta-mercaptoethanol and inhibited by some metal irons (Hg(2+), Cu(2+), Pb(2+), Mn(2+), Li(+), and Fe(3+)) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-D-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg(-1), respectively. The apparent K (m) and V (max) values for beechwood xylan were 5.6 mg ml(-1) and 3,622 micromol min(-1) mg(-1), respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.