Purification and properties of endo-1,4-β-xylanase from Trichosporon cutaneum

Summary The endoxylanase (1,4-D-xylan xylanohydrolase, EC 3.2.1.8) was purified 3,7 fold from the culture filtrate of the yeast Trichosporon cutaneum grown on oathusk xylan. The final enzyme preparation gave a single protein band on disc gel electrophoresis and has a molecular weight of approx. 45000. The enzyme has a pH optimum of 5.0 and a temperatur optimum of 50°C. Patterns of hydrolysis demonstrate that this xylanase is an endo-splitting enzyme able to break down xylans at random giving xylobiose, xylotriose and xylose as the main end-products. Since the enzyme seems not to be capable of liberating L-arabinose from arabino-xylan branched arabinose-containing xylooligosaccharides are formed, too. This enzyme contains carbohydrates in a noncovalent manner, indicating that this extracellular xylanase, is not a glycoprotein.     

A novel strain of Streptomyces malaysiensis isolated from Brazilian soil produces high endo-β-1,4-xylanase titres

One hundred and sixty two actinomycete strains isolated from Brazilian soils were screened for xylanase activity, according to the size of the hydrolysis zones observed in oat spelts xylan agar plates. The strain AMT-3, later identified as Streptomyces malaysiensis, was selected as the best producer. In subsequent shake flasks fermentations using growth media contanning 1% (w/v) of either birchwood, or oat spelts xylan, plus organic nitrogen and salts, high endo--1,4-xylanase titres (EC 3.2.1.8) (116 U ml^–1) were observed in the larchwood medium within 6 days. This is the first report concerning xylanase production by streptomyces malaysiensis, which has been recently described as a new species.     

Biocatalytic properties and substrate-binding ability of a modular GH10 β-1,4-xylanase from an insect-symbiotic bacterium,Streptomyces mexicanus HY-14

The gene (1350-bp) encoding a modular β-1,4-xylanase (XylU), which consists of an N-terminal catalytic GH10 domain and a C-terminal carbohydrate-binding module 2 (CBM 2), from Streptomyces mexicanus HY-14 was cloned and functionally characterized. The purified His-tagged recombinant enzyme (rXylU, 44.0 kDa) was capable of efficiently hydrolyze diverse xylosidic compounds, p-nitrophenyl-cellobioside, and p-nitrophenyl-xylopyranoside when incubated at pH 5.5 and 65°C. Especially, the specific activities (649.8 U/mg and 587.0 U/mg, respectively) of rXylU toward oat spelts xylan and beechwood xylan were relatively higher than those (<500.0 U/mg) of many other GH10 homologs toward the same substrates. The results of enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) revealed that rXylU preferentially hydrolyzed the substrates to xylobiose (>75%) as the primary degradation product. Moreover, a small amount (4%<) of xylose was detected as the degradation product of the evaluated xylosidic substrates, indicating that rXylU was a peculiar GH10 β-1,4-xylanase with substrate specificity, which was different from its retaining homologs. A significant reduction of the binding ability of rXylU caused by deletion of the C-terminal CBM 2 to various insoluble substrates strongly suggested that the additional domain might considerably contribute to the enzyme-substrate interaction.     

A highly active endo-β-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, β-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-β-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.     

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.     

Biochemical characterization of a GH53 endo-β-1,4-galactanase and a GH35 exo-β-1,4-galactanase from Penicillium chrysogenum

An endo-β-1,4-galactanase (PcGAL1) and an exo-β-1,4-galactanase (PcGALX35C) were purified from the culture filtrate of Penicillium chrysogenum 31B. Pcgal1 and Pcgalx35C cDNAs encoding PcGAL1 and PcGALX35C were isolated by in vitro cloning. The deduced amino acid sequences of PcGAL1 and PcGALX35C are highly similar to a putative endo-β-1,4-galactanase of Aspergillus terreus (70 % amino acid identity) and a putative β-galactosidase of Neosartorya fischeri (72 %), respectively. Pfam analysis revealed a “Glyco_hydro_53” domain in PcGAL1. PcGALX35C is composed of five distinct domains including “Glyco_hydro_35,” “BetaGal_dom2,” “BetaGal_dom3,” and two “BetaGal_dom4_5” domains. Recombinant enzymes (rPcGAL1 and rPcGALX35C) expressed in Escherichia coli and Pichia pastoris, respectively, were active against lupin galactan. The reaction products of lupin galactan revealed that rPcGAL1 cleaved the substrate in an endo manner. The enzyme accumulated galactose and galactobiose as the main products. The smallest substrate for rPcGAL1 was β-1,4-galactotriose. On the other hand, rPcGALX35C released only galactose from lupin galactan throughout the reaction, indicating that it is an exo-β-1,4-galactanase. rPcGALX35C was active on both β-1,4-galactobiose and triose, but not on lactose, β-1,3- or β-1,6-galactooligosaccharides even after 24 h of incubation. To our knowledge, this is the first report of a gene encoding a microbial exo-β-1,4-galactanase. rPcGAL1 and rPcGALX35C acted synergistically in the degradation of lupin galactan and soybean arabinogalactan. Lupin galactan was almost completely degraded to galactose by the combined actions of rPcGAL1 and rPcGALX35C. Surprisingly, neither rPcGAL1 nor rPcGALX35C released any galactose from sugar beet pectin.     

Purification and properties of the endo-1,4-β-glucanase III from Ruminococcus albus

Endoglucanase III (EGIII) was purified from Ruminococcus albus culture supernatant. An enzyme having a molecular weight of 53,000 was stabilized by mercaptoethanol and inhibited by sulfhydryl group-blocking reagents, and exhibited its highest CMC-degrading activity of pH 5.7 and 55°C. The enzyme hydrolyzed cellobiose (G2) and cellotriose (G3) only negligibly, but significantly hydrolyzed cellotetraose (G4), cellopentaose (G5) and cellohexaose (G6). The major hydrolysis reactions conducted by the enzyme were G4→2G2, G5→G2+G3, G6→G2+G4 and G6→2G3. The V_max values of these reactions increased remarkably while the K_m values decreased significantly with an increase in degree of polymerization of the substrate.     

Catalytic properties of endo-1,3-β-D-glucanase from the Vietnamese edible mussel Perna viridis

A β-1,3-glucanase with a molecular mass of 33 kDa was isolated in the homogeneous state from a crystalline stalk of the commercially available Vietnamese edible mussel Perna viridis. It hydrolyzes β-1,3-bonds in glucans and is capable of catalyzing the transglycosylation reaction. The β-1,3-glucanase has a K _m value of 0.3 mg/ml for the hydrolysis of laminaran and shows a maximum activity in the pH range from 4 to 6.5 and at 45°C. Its half-inactivation time is 180 min at 45°C and 20 min at 50°C. The enzyme was ascribed to glucan-endo-(1 → 3)-β-D-glucosidases (EC 3.2.1.39). The enzyme could be used in the structure determination of β-1,3-glucans and enzymatic synthesis of new carbohydrate-containing compounds.     

Structural insights into the acidophilic pH adaptation of a novel endo-1,4-β-xylanase from Scytalidium acidophilum

In this study, the crystal structure of a novel endo-1,4-β-xylanase from Scytalidium acidophilum, XYL1, was solved at 1.9 Å resolution. This is one of the few solved crystal structures of acidophilic proteins. The enzyme has the overall fold typical to family 11 xylanases. Comparison of this structure with other homologous acidophilic, neutrophilic and alkalophilic xylanases provides additional insights into the general features involved in low pH adaptation (stability and activity). Several sequence and structure modifications appeared to be responsible for the acidophilic characteristic: (a) the presence of an aspartic acid H bonded to the acid/base catalyst (b) the nature of specifically conserved residues in the active site (c) the negative potential at the surface (d) the decreased number of salt bridges and H bonds in comparison with highly alkaline enzymes.     

Influence of lignin-derived phenolic compounds on the Clostridium thermocellum endo-β-1,4-xylanase XynA

Phenolic compounds released during pretreatment of lignocellulosic biomass influence its enzymatic hydrolysis. To understand the effects of these compounds on the kinetic properties of xylan-degrading enzymes, the present study employed the recombinant cellulosomal endo-β-1,4-xylanase, thermostable GH11 XynA protein from Clostridium thermocellum, as an enzyme model to evaluate the effects of 4-hydroxybenzoic acid, gallic acid, vanillin, tannic acid, p-coumaric acid, ferulic acid, syringaldehyde, and cinnamic acid. XynA was deactivated by the assayed phenols at 60 °C, presenting the strongest deactivation in the presence of tannic acid, with an activity reduction of about 80 %. Thermal stability of XynA was influenced by ferulic acid, syringaldehyde, cinnamic acid, 4-hydroxybenzoic acid, and p-coumaric acid. The hydrolysis rate of oat-spelt xylan by XynA was influenced by temperature, being unable to hydrolyze at 40 °C in the presence of tannic acid. On hydrolysis at 60 °C, the presence of gallic and tannic acid caused a major reduction in reducing sugar production, generating 3.74 and 2.15 g.L^-1 of reducing sugar, respectively, whereas the reaction in the absence of phenols generated 4.41 g.L^-1. When XynA was pre-deactivated by phenols it could recover most of its activity at 40 °C, however, at 60 °C activity could not be reestablished.     

Characterization of production and enzyme properties of an endo-β-1,4-glucanase fromBacillus subtilis CK-2 isolated from compost soil

Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo--1,4-glucanase. Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium. Enzyme production did not require CMC or other cellulose containing materials. The endo--1,4-glucanase activity was optimal at pH 5.6–5.8 and at 65 MoC, and achieved thermal stability up to 55 MoC. The activity was inhibited by Hg²⁺. The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme. The enzyme activity was irreversibly inhibited by SDS. Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence.Abbreviations CMC carboxymethylcellulose - DNS dinitrosalicylic - SDS sodium dodecyl sulfate     

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(?)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.     

Purification, Characterization of GH11 Endo-β-1,4-xylanase from Thermotolerant Streptomyces sp. SWU10 and Overexpression in Pichia pastoris KM71H

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1–11), more than 80 % of initial activity remained at pH 2–11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.     

Characterization of endo-1,3–1,4-β-glucanases in GH family 12 from Magnaporthe oryzae

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze β-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3–1,4-β-glucans, such as barley β-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3–1,4-β-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3–1,4-β-glucan during infection and spore formation.     

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those of Thermomonospora fusca. Genes encoding two different endo-β-1,4-xylanases were cloned from T.␣alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48 456 Da. The protein contains a 38-amino-acid leader peptide with six Arg⁺ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/xylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 °C, and had broad pH stability in the range from pH 4.0 to pH 10.0. Received: 9 January 1997 / Received revision: 27 March 1997 / Accepted: 13 April 1997     

Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli

• 1.1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile “Caldocellum saccharolyticum strain Tp8T6.3.3.1. It was found to have an M_r of 42,000 and an isoelectric point of approx. 5.0.
• 2.2. The enzyme showed optimum activity at pH 5.0–7.7 and had an activation energy of 44 kJ mol⁻¹. It was stable at room temperature at pH 4.5–11.5 in the presence of 0.5 mg ml⁻¹ bovine serum albumin. The half-life of the enzyme at 75°C was 20 min at pH 6.0 in the presence of 0.5 mg ml⁻¹ bovine serum albumin.
• 3.3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The K_m for oat spelts xylan was 0.021% (w/v) at pH6.0.
• 4.4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside, but could not hydrolyse xylobiose.
• 5.5. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. Xylose did not inhibit the enzyme.

     

Characterization of endo-β-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein⁻¹, one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28°C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28°C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene (eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601.     

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.     

Purification and characterization of a thermostable endo-β-1,4-glucanase from a novel strain of Penicillium purpurogenum

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. P. purpurogenum produced one of the highest levels of EG (5.6 U mg-protein^?1) with rice straw and corn steep powder as carbon and nitrogen sources, respectively. The extracellular EG was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The purified EG was a monomeric protein with a molecular weight of 37 kDa and showed broad substrate specificity with maximum activity towards lichenan. P. purpurogenum EG showed t_1/2 value of 2 h at 70 °C and catalytic efficiency of 118 ml mg^?1 s^?1, one of the highest levels seen for EG-producing microorganisms. Although EGs have been reported elsewhere, the high catalytic efficiency and thermostability distinguish P. purpurogenum EG.