Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains

The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed. The xylanase produced by T. lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C. In comparison seven ATCC strains and the DSM 5826 strain of T. lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C. Culture filtrates of T. lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C. The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C. The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP. The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C. At 50 degrees C, the enzyme of T. lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0. Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h. The xylanase of T. lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.     

Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp

A xylanase purified from the thermophilic fungus Thermomyces lanuginosus CBS 288.54 was characterized and its potential application in wheat straw pulp biobleaching was evaluated. Xylanase was purified 33.6-fold to homogeneity with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa. The purified xylanase had a neutral optimum pH ranging from pH 7.0 to pH 7.5, and it was also stable over pH 6.5-10.0. The optimal temperature of the xylanase was 70-75 degrees C and it was stable up to 65 degrees C. The purified xylanase was found to be not glycosylated. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0, 4.7, 2.0 and 23.4 mg ml-1, respectively. The potential application of the xylanase was further evaluated in biobleaching of wheat straw pulp. The brightness of bleached pulps from the xylanase pretreated wheat straw pulp was 1.8-7.79% ISO higher than that of the control, and showed slightly lower tensile index and breaking length than the control. Although chlorine consumption was reduced by 28.3% during bleaching, the xylanase pretreated pulp (15 U g-1 pulp) still maintained its brightness at the control level. Besides, pretreatment of pulp with the xylanase was also effective at an alkaline pH as high as pH 10.0.     

Relatedness of Thermomyces lanuginosus strains producing a thermostable xylanase

Properties of an endo-beta-xylanase produced by a locally isolated Thermomyces lanuginosus strain SSBP was compared to seven other T. lanuginosus strains isolated from different geographical regions. Strain SSBP produced the highest xylanase activity of 59600 nkat ml(-1) when cultivated on corn cobs (maize) medium, whereas the seven other strains produced xylanase activities ranging from 6000 to 32000 nkat ml(-1). No cellulase activity was produced by the strains. Despite the variability in the production of xylanase, little difference in the other characteristics of the strains could be found. The optimal temperature and pH for xylanase production by the strains was either 40 or 50 degrees C and between pH 6 and 7, respectively. Optimal xylanase activity of the strains was observed at 70 degrees C and at pH 6 or 6.5. Culture supernatant analysis by SDS-PAGE and isoelectric focusing PAGE of all strains revealed the presence of a single 24.7 kDa and pI 3.9 xylanase. Phylogenetic analysis by PCR amplification and sequencing of the internal transcribed spacer of nuclear rRNA repeat units and 5.8S rDNA revealed no strain diversity. However, random amplified polymorphic DNA analysis pointed to greater diversity and with one primer (5'-GCCCGACGCG-3'), a relationship was established between xylanase levels and the RAPD pattern.     

Thermomyces lanuginosus: properties of strains and their hemicellulases

The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free beta-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of beta-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50-80 degrees C and over a broad pH range (3-12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two beta-sheets and one alpha-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the beta-strand and the alpha-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.     

Optimizing Xylanase Production by a Newly Isolated Strain CAU44 of the Thermophile Thermomyces lanuginosus

Summary Thermomyces lanuginosus CAU44, a newly isolated thermophilic fungus strain, was used for the production of extracellular xylanase on various lignocellulosic materials under shake flask conditions. High-level production of xylanase by the strain was enhanced by optimizing the type of carbon sources, substrate concentration, particle size and surfactants in the culture medium. The titre of xylanase activity obtained of up to 4156 U ml⁻¹ was the highest ever reported.     

绵毛嗜热丝孢菌木聚糖酶的纯化与性质

研究了绵毛嗜热丝孢菌Thermomyces lanuginosus W205胞外木聚糖酶的纯化与性质。粗酶液经硫酸铵沉淀和Q-Sepharose FF离子交换层析即可得到电泳纯木聚糖酶,回收率为46.6%,比酶活为1396.9U/mg。该酶的最适pH和最适温度分别为pH7.0和75℃,pH稳定范围为5.5-10.8,70℃处理30min残存酶活在70%以上。薄层层析结果显示该酶水解桦木木聚糖的主要产物是木二糖和木三糖,并且能够通过转糖苷作用将木三糖转化为木二糖。该木聚糖酶易于纯化并且具有较宽的pH稳定性及良好的热稳定性,具有较大的潜在工业应用价值。     

Production and properties of hemicellulases by a Thermomyces lanuginosus strain

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.     

A comparison of thermal characteristics and kinetic parameters of trehalases from a thermophilic and a mesophilic fungus

Trehalases from a thermophilic fungus Thermomyces lanuginosus (M(r) 145 kDa) and a mesophilic fungus Neurospora crassa (M(r) 437 kDa) were purified to compare their thermal characteristics and kinetic constants. Both trehalases were maximally active at 50 degrees C, had an acidic pH optimum and were glycoproteins (20% and 43%, w/w, carbohydrate content for T. lanuginosus and N. crassa, respectively). At their temperature optimum, their K(m) was similar (0.57 and 0.52 mM trehalose, for T. lanuginosus and N. crassa, respectively) but the V(max) of N. crassa enzyme was nine times higher than of T. lanuginosus enzyme. The catalytic efficiency, k(cat)/K(m), for N. crassa trehalase was one order of magnitude higher (6.2 x 10(6) M(-1) s(-1)) than of T. lanuginosus trehalase (4 x 10(5) M(-1) s(-1)). At their T(opt) (50 degrees C), trehalase from both sources exhibited similar thermostability (t(1/2)6 h). The energy of activation, E(a), for T. lanuginosus trehalase was 15.12 kcal mol(-1) and for N. crassa trehalase it was 9.62 kcal mol(-1). The activation energy for thermal inactivation for the N. crassa enzyme (92 kcal mol(-1)) was two-fold higher than for the T. lanuginosus enzyme (46 kcal mol(-1)). The present study shows that the trehalase of N. crassa is not only more stable but also a better catalyst than the T. lanuginosus enzyme.     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7

Ten different strains of Thermomyces lanuginosus, isolated from composting soils were found to produce phytase when grown on PSM medium. The wild type strain CM was found to produce maximum amount ofphytase (4.33 units/g DW substrate). Culturing T. lanuginosus strain CM on medium containing wheat bran and optimizing other culture conditions (carbon source, media type, nitrogen source, level of nitrogen, temperature, pH, inoculum age, inoculum level and moisture), increased the phytase yield to 13.26 units/g substrate. This culture was further subjected to UV mutagenesis for developing phytase hyperproducing mutants. The mutant (TL-7) showed 2.29-fold increase in phytase activity as compared to the parental strain. Employing Box-Behnken factor factorial design of response surface methodology resulted in optimized phytase production (32.19 units/g of substrate) by mutant TL-7. A simple two-step purification (40.75-folds) ofphytase from mutant TL-7 was achieved by anion exchange and gel filtration chromatography. The purified phytase (approximately 54 kDa) was characterized to be optimally active at pH 5.0 and temperature 70 degrees C, though the enzyme showed approximately 70% activity over a wide pH and temperature range (2.0-10.0 and 30-90 degrees C, respectively). The phytase showed broad substrate specificity with activity against sodium phytate, ADP and riboflavin phosphate. The phytase from T. lanuginosus was thermoacidstable as it showed up to 70% residual activity after exposure to 70 degrees C at pH 3.0 for 120 min. The enzyme showed Km 4.55 microM and Vmax 0.833 microM/min/mg against sodium phytate as substrate.     

Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus

The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8-4.0, 5.0 and 70 degrees C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s(-1). The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.     

Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus.

A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47 degrees C for 2 h. After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside. The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively.     

Characterization and biobleaching effect of hemicellulases produced by thermophilic fungi

The culture supernatant of Thermomyces lanuginosus strains MED 2D and MED 4B1 had high activities of xylanase with low inducible activities of -xylosidase. The crude xylanase was optimally active at 70 °C and at pH 6.0 to 6.5. Subsequently Eucalyptus kraft pulp was treated with MED 2D supernatant at 10 IU per gram pulp resulting in a 10.5% reduction in Kappa number. XECEDED-Refined bleached pulp resulted in handsheets with increased brightness compared to the control (X = xylanase treatment; E = alkaline extraction; C = Cl2 treatment; D = ClO2 treatment).     

One-step purification of chitinase from Serratia marcescens NK1, a soil isolate

AIMS: A simple single step technique of gel filtration was developed for the purification of chitinase from Serratia marcescens NK1. METHODS AND RESULTS: Chitinase from Ser. marcescens NK1 was purified to homogeneity by gel filtration chromatography with 9.2% recovery. The enzyme had a pH optimum of 6.2 and a temperature optimum of 47 degrees C. It was stable in a wide pH range of 3.0 to 10.0, retaining 60% activity at pH 3.0 and 65% activity at pH 10.5. It retained 70% activity at 28 degrees C after 72 h and nearly 50% activity at 50 degrees C up to 24 h. CONCLUSION: The chitinase from Ser. marcescens NK1 can be efficiently purified in a single step by gel filtration chromatography. The chitinase of Ser. marcescens NK1, a soil isolate, is highly stable and as active as that of other reported isolates of Ser. marcescens. SIGNIFICANCE AND IMPACT OF THE STUDY: This purification scheme is advantageous because of its simplicity and can therefore be applied for the purification of other enzymes. The yield is sufficient for initial characterization studies of the enzyme, and an improved resolution can be obtained if the chromatography is done under fast flow systems.     

High-level expression of the xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.     

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn²⁺, Sn²⁺, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-d-xylopyranoside. The apparent K _m value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.     

Xylanase production by Thermomyces lanuginosus wild and mutant strains

Thermomyces lanuginosus strains from different culture collections, namely ATCC 26909, ATCC 22083, DEN 1457, IMI 84400 and BS1 were compared for xylanase production, and isozyme profile. Of all the strains of T. lanuginosus, BS1 a soil isolate produced the largest amount of xylanase. All strains were found to produce two forms of xylanase (I & II) with molecular mass corresponding to 25.0 and 54.0 KDa. The u.v/NTG mutagenesis of T. lanuginosus BS1 aleurospores/protoplasts resulted in xylanase-hyperproducing mutants. A morphological colour mutant RB 524 produced approximately 2.5-fold higher xylanase (2506.0 units/ml) as compared to the parent strain (1018.1 units/ml).     

Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.     

Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32

Streptomyces cyaneus SN32 was used in this study to produce extracellular xylanase, an important industrial enzyme used in pulp and paper industry. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by anion exchange chromatography using DEAE-Sepharose column, with 43.0% yield. The enzyme was found to be a monomer of 20.5 kDa as determined by SDS gel electrophoresis and has a pI of 8.5. The optimum pH and temperature for purified xylanase activity was 6.0 and 60-65 degrees C, respectively. The half-lives of xylanase at 50 and 65 degrees C were approximately 200 and 50 min, respectively. The xylanase exhibited K(m) and V(max) values of 11.1 mg/ml and 45.45 micromol/min/mg. The 15 residue N-terminal sequence of the enzyme was found to be 87% identical up to that of endoxylanases from Steptomyces sp. Based on the zymogram analysis, sequence similarity and other characteristics, it is proposed that the purified enzyme from S. cyaneus SN32 is an endoxylanase and belongs to Group 1 xylanases (low molecular weight - basic proteins). The purified enzyme was stable for more than 20 week at 4 degrees C. Easy purification from the fermentation broth and its high stability will be highly useful for industrial application of this endoxylanase.