Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.     

High activity xylanase from Aspergillus sydowii MG49 during growth on jute stalk lignocellulose

Aspergillus sydowil MG49 produced 33.0 U mg^-1 of extracellular xylanase activity when grown in liquid state fermeniation (LSF) with 1% ground jute stalk as the sole carbon source compared to 56.0 U mg^-1 when pure xylan was used. Optimum time-course and pH for maximum enzyme production were 144 h and 4.0 respectively. The culture filtrate was devoid of any cellulase and β-xylosidase activity. The xylanase exhibited optimum activity at 60°C and pH 5.5. Partially-fermented jute stalk could be recycled at least twice for xylanase production, exhibiting 25.8 and 17.4 U mg^-1 activity in two later consecutive cycles respectively.     

Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp

A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively.     

Production of xylanolytic enzymes during growth on pulverized grass by Aspergillus ochraceus-42

Xylanase and β-xylosidase with activity of 6.46 U mg^-1 and 0.500 U mg^-1, respectively, were produced extracellularly by Aspergillus ochraceus during growth on pulverized grass in liquid state fermentation, compared to 9.3 U mg^-1 and 0.74 U mg^-1 when pure xylan was used. The culture filtrate was devoid of any cellulase activity. Xylanolytic enzymes were produced optimally in 144 h of incubation on 1% pulverized grass, pH 6.5. About 8.43% (w/w) sugars were liberated from alkali-treated grass in 6 h by the synergistic effect of xylanolytic enzymes. The half-lives for xylanase and β-xylosidase at 50°C were 210 min and 300 min, respectively, and half-life increased with the increase in protein concentration. Both mono- and divalent cations, especially K⁺ and Zn²⁺, exhibited a profound effect on the rate of enzyme saccharification.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture

The thermophilic, xylanolytic, anaerobic organism, Dictyoglomus sp. B1, was cultivated in batch and continuous cultures in media containing insoluble beech-wood xylan. The extracellular xylanase activity levels obtained for the two cultivation methods were compared. Experiments were performed separately to determine the optimum substrate concentration, dilution rate, pH and temperature for xylanase production. Maximum xylanase activity was found at a substrate concentration of 1.5 g xylan/l, a dilution rate of 0.112 h^–1, pH 8.0 and at 7°C. Different combinations of these optimum values were used in a 2³ factorial experiment to investigate whether an increase in the xylanase production/activity could be achieved. A maximum xylanase activity of 2312 U/l was found when fermentors were operated at 73°C with a substrate concentration of 1.5 g xylan/l, pH 8.0, and a dilution rate of 0.112 h^–1. Thus, the optimum xylanase activity in the factorial experiment was obtained when the conditions that gave the maximum xylanase activities in the individual experiments were combined. Optimum xylanase activity obtained in the 2³ factorial experiment was 6.2 times higher than the activity found in the initial batch culture (373 U/l) and 3.0 times higher than the activity of a batch culture (783 U/l) grown at the same optimum conditions as the factorial experiment. The higher specific xylanase activity (217 U/mg protein) found in the 2³ factorial experiment was 4.1 times higher than the specific activity in the initial batch culture (53 U/mg protein).     

Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan

The mature peptide of Aspergillus niger xylanase A (AnxA) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant AnxA (reAnxA) was secreted into culture medium. After 96-h 0.25% methanol induction, the activity of reAnxA in the culture supernatant reached the peak, 175 U/mg, which was 1.9 times as high as that of the native AnxA (92 U/mg). Studies on enzymatic properties showed that the optimum temperature and optimum pH of reAnxA were 50 degrees C and 5.0, respectively. The reAnxA was very stable in a wide pH range of 3.0-8.0. After incubation at the pH 3.0-8.0, 25 degrees C for 1h, all the residual activities of reAnxA were over 80%. The K(m) and k(cat) values for reAnxA were 4.8 mg/ml and 123.2s(-1), respectively. HPLC analysis showed that xylotriose was the main hydrolysis product of birchwood xylan and bran insoluble xylan by reAnxA.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

嗜热子囊菌JCM12803来源的双功能木聚糖/纤维素酶

纤维素和木聚糖的充分利用对于生物燃料的生产是非常重要的。文中利用PCR的方法从嗜热子囊菌Thermoascus crustaceus JCM12803中克隆到一个新颖的双功能木聚糖/纤维素酶基因Tcxyn10a,并将其在毕赤酵母Pichia pastoris GS115中实现高效异源表达。经过蛋白纯化和酶学性质研究分析,TcXyn10A的最适pH值和最适温度分别为5.0和65-70℃,能够在酸性至碱性(pH 3.0-11.0)条件下和60℃下保持稳定;对榉木木聚糖、小麦阿拉伯木聚糖、羧甲基纤维素钠和地衣多糖均有降解活性,比活分别为(1 480±26)U/mg、(2 055±28)U/mg、(7.4±0.2)U/mg和(10.9±0.4)U/mg;同源建模结构以及分子对接试验表明,双功能酶TcXyn10A只含有单一催化结构域,且木聚糖底物与纤维素底物共用一条催化通道。文中为探索双功能酶结构与其功能的关系提供了很好的素材。     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Thermostable xylanase from Marasmius sp.: purification and characterization

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as 90 degrees C. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of 90 degrees C. When using xylan from birchwood as substrate, it exhibits Km and Vmax values of 2.6 +/- 0.6 mg/ml and 428 +/- 26 U/mg, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to 70 degrees C. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at 70 degrees C for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.     

A new acidophilic endo-β-1,4-xylanase from Penicillium oxalicum: cloning,purification, and insights into the influence of metal ions on xylanase activity

A new acidophilic xylanase (XYN11A) from Penicillium oxalicum GZ-2 has been purified, identified and characterized. Synchronized fluorescence spectroscopy was used for the first time to evaluate the influence of metal ions on xylanase activity. The purified enzyme was identified by MALDI TOF/TOF mass spectrometry, and its gene (xyn11A) was identified as an open reading frame of 706 bp with a 68 bp intron. This gene encodes a mature protein of 196 residues with a predicted molecular weight of 21.3 kDa that has the 100 % identity with the putative xylanase from the P. oxalicum 114-2. The enzyme shows a structure comprising a catalytic module family 10 (GH10) and no carbohydrate-binding module family. The specific activities were 150.2, 60.2, and 72.6 U/mg for beechwood xylan, birchwood xylan, and oat spelt xylan, respectively. XYN11A exhibited optimal activity at pH 4.0 and remarkable pH stability under extremely acidic condition (pH 3). The specific activity, K _m and V _max values were 150.2 U/mg, 30.7 mg/mL, and 403.9 μmol/min/mg for beechwood xylan, respectively. XYN11A is a endo-β-1,4-xylanase since it release xylobiose and xylotriose as the main products by hydrolyzing xylans. The activity of XYN11A was enhanced 155 % by 1 mM Fe²⁺ ions, but was inhibited strongly by Fe³⁺. The reason of enhancing the xylanase activity of XYN11A with 1 mM Fe²⁺ treatment may be responsible for the change of microenvironment of tryptophan residues studied by synchronous fluorescence spectrophotometry. Inhibition of the xylanase activity by Fe³⁺ was first time demonstrated to associate tryptophan fluorescence quenching.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

Biochemical characterization,cloning and molecular modeling of a detergent and organic solvent-stable family 11 xylanase from the newly isolated Aspergillus niger US368 strain

New β-1,4-d-xylan xylanohydrolase (XAn11) belonging to the xylanase 11 family was purified to homogeneity from a newly soil-isolated Aspergillus niger US368 strain. The pure xylanase is a glycosylated monomer having a molecular mass of about 26 kDa. The N-terminal sequence of the purified enzyme was determined and compared to some Aspergillus xylanases N-terminal ones. The gene encoding the XAn11 was cloned and sequenced.The maximal xylanase activity was obtained at pH 5.0 and 55 °C. The XAn11 was found to be stable in a wide range of pH (3–9) and in presence of some detergents and organic solvents. A specific activity of about 805.6 U/mg or 334 U/mg was measured using birchwood xylan or oatspelt xylan as substrate, respectively. A structural explanation of the difference between experimental and theoretical molecular mass as well as the stability of the enzyme against acidic pH was proposed by molecular modeling.     

Production of high level of cellulase-free and thermostable xylanase by a wild strain of Thermomyces lanuginosus using beechwood xylan

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was studied for production of high level of cellulase-free thermostable xylanase at 50°C using xylan. Optimization of the medium composition was carried out on shake-flask level using Graeco-Latin square technique. This increased xylanase production from 527 nkat ml⁻¹ in the original medium to 9168–9502 nkat ml⁻¹ in the optimized medium under optimized culture conditions e.g. initial medium pH (6.0–6.5), culture temperature (50°C) and time (5–6 d). The lag phase was very much shorter in the laboratory reactor compared to which existed in the shake cultures and 7111 nkat of xylanase activity were obtained per ml of culture filtrate at 60 h of cultivation. With a 15 min reaction time, the optimal pH and temperature for the xylanase activity were at 6.5 and 65°C, respectively. The enzyme was almost stable over a broad range of pH 3–9 at 20°C, with an optimum stability at pH 6.5. After 51 h heating at 50°C the enzyme retained 60%, 100% and 90% activity at pH 5.0, 6.5 and 8.0, respectively. The crude enzyme could hydrolyse xylan effectively and in only 6 h 67.3%, 54.0% and 49.2% saccharifications were achieved for 2%, 5% and 10% substrate levels, respectively. The principal product of hydrolysis was xylobiose together with smaller amounts of xylooligosaccharides (degree of polymerization 3–7) and xylose.