Purification and Partial Characterization of Alkaline Xylanase from Escherichia coli Carrying pCX311

Xylanase produced by E. coli HB 101 carrying plasmid pCX311, which contains the xylanase A gene of alkalophilic Bacillus sp. strain C-125, was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified enzyme had a molecular weight of 43,000. The pH and temperature optima for its activity were 6~10 and 70°C, respectively. The enzyme retained full activity after incubation at 50°C for 10 min. These enzymatic properties of the xylanase were almost the same as those of xylanase A. But this enzyme was less stable than xylanase A at low pHs. Furthermore, we could purify a larger amount of alkaline xylanase from E. coli than from alkalophilic Bacillus sp. strain C-125.     

Enzymatic clarification of fruit juices (Apple, Pineapple, and Tomato) using purified Bacillus pumilus SV-85S xylanase

The xylanase obtained from a hyper-producer Bacillus pumilus SV-85S was purified and characterized to evaluate its potential in industrial applications. Xylanase was purified to homogeneity 25.3-fold with 63.2% recovery using cation-exchange chromatography through CMSephadex C-50. The purified xylanase showed a single band on Native-PAGE and a single peak in RP-HPLC confirming its homogeneity. The purified enzyme revealed a single band on SDS-PAGE with a molecular mass of 23.6 kDa, which was confirmed with gel filtration chromatography through Sepharose 6B. The K_m and V_max of the purified xylanase was 1.0 mg/mL and 333.3 IU/mL, respectively. The temperature and pH profiles of the purified xylanase revealed that it was thermo and alkali stable. In recent years due to the overall increase in natural fruit juice consumption, juices have become important from a consumption point of view. However, raw juice is turbid and viscous which tends to settle during storage. Therefore, it must be clarified before commercialization. The efficacy of absolutely purified xylanase was studied on juice enrichment of apples (Malus domestica), pineapples (Ananas comosus L.) and tomatos (Lycopersicum esculentum). The treatment with xylanase lead to an increased juice yield by 23.53% (apple), 10.78% (pineapple), and 20.78% (tomato) as well as having a significant effect on juice clarity by an increase of % transmittance of 22.20, 19.80, and 14.30, respectively. The turbidity and viscosity was also decreased without affecting acid neutrality significantly.     

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06

Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had K_m and V_max values of 3.45 mg mL⁻¹ and 387.3 µmol min⁻¹mg⁻¹, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed.     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.     

Color Variants of Aureobasidium pullulans Overproduce Xylanase with Extremely High Specific Activity

Xylanase activity from naturally occurring color variants of Aureobasidium pullulans was associated with extracellular monomeric proteins of 20 to 21 kilodaltons. Xylanase represented nearly half the total extracellular protein, with a yield of up to 0.3 g of xylanase per liter. The specific activity of partially purified xylanase exceeded 2,000 IU/mg. Xylanase from typically pigmented strains appeared similar to that from color variants with respect to molecular weight, pH and temperature optima, and specific activity of purified (but not crude) enzyme. However, xylanase from typical strains made up only about 1.0% of total extracellular protein. Xylanase from strains of Cryptococcus albidus was associated with abundant proteins of about 43 kilodaltons and showed much lower specific activity.     

Production, Partial Purification and Characterization of Extracelluar Xylanase from Chaetomium globosum

Culture conditions for efficient production of extracellular xylanase by fungus, Chaetomium globosum isolate Cg2, have been standardized. Further, xylanase has been partially purified and characterized. Xylanase activity was maximum after 9 days of incubation when amended in medium with 1.5 % xylan as carbon source and 0.6% NH₄H₂PO₄ as nitrogen source. Partial purification of the xylanase was accomplished by ammonium sulphate precipitation, followed by further purification by anion exchange chromatography on DEAE-Sephadex A-50 column. The partially purified enzyme was electrophoresed on SDS-PAGE and a single band produced corresponded to molecular weight, 32 kD. The optimum temperature and pH for maximum activity of purified xylanase were 30°C and 5.5, respectively. Both the purified xylanase and culture filtrate have shown the antifungal activity against Bipolaris sorokiniana, a causal organism of spot blotch of wheat. Purified xylanase at 100 μg ml^?1 concentration caused 100 per cent inhibition of conidia germination of B. sorokiniana, whereas the culture filtrate was able to inhibit germination up to 67.5 per cent.     

Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 ± 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO₃) on enzyme production. A 2³ central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the −α level for KNO₃, along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37°C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50°C, but the enzyme displayed 78% residual activity even at 65°C. The enzyme retained 50% activity after an incubation of 1 h at 60°C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.     

Production of alkaliphilic, halotolerent, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agro waste: single step purification and characterization

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular alkaliphilic, thermostable and halotolerent xylanase. The culture conditions for xylanase production were optimized with respect to pH, temperature, NaCl and inexpensive agro waste as substrates. Xylanase yield was enhanced more than four fold in the presence of 1% corn husk and 0.5% peptone or feather hydrolysate at pH 11 and 37°C. Xylanase was purified to 11.8-fold with 8.7% yield by using traditional chromatographic methods whereas the same enzyme purified to 20-fold with 72% yield by using corn husk as ligand. Its molecular mass was estimated to be 24 kDa by SDS–PAGE. The xylanase had maximal activity at pH 11 and 70°C. The enzyme was active over broad range, 0–20% sodium chloride. The enzyme was thermostable retaining 100% of the original activity at 70°C for 3 h. The apparent K _m values for oat spelt xylan and brichwood xylan were 4.1 and 4.4 mg/ml respectively. The deduced internal amino acid sequence of PPKS-2 xylanase resembled the sequence of β-1,4-endoxylanase, which is member of glycoside hydrolase family 11.     

Xylanase from a soil isolate, <Emphasis Type="Italic">Bacillus pumilus</Emphasis>: gene isolation,enzyme production,purification, characterization and one-step separation by aqueous-two-phase system

A xylanase producer, Bacillus pumilus SB-M13, was isolated from soil and identified using various tests based on carbohydrate fermentation preferences and fatty acid analysis. Xylanase gene, isolated using PCR amplification, was partially sequenced and it showed 89–94% sequence similarity to the xylanase genes of other B. pumilus strains. Xylanase with very low level of cellulase was produced on agricultural byproducts. The enzyme has been purified 186-fold by hydrophobic interaction chromatography and biochemically characterized. It has a molecular weight of 24.8 kDa and pI of 9.2. Xylanolytic activity is stable at alkaline pH and highest activity is observed at 60 °C and pH 7.5. Enzyme K _m and k _cat values were determined as 1.9 mg/mL and 42,600 U/mg, respectively. In aqueous-two-phase system, xylanase always partitioned to the top phase. Basic pH, low PEG concentration, salt addition, and presence of microbial cells enhanced xylanase partitioning. A maximum sevenfold purification, 10-fold concentration and 100% xylanase recovery were obtained, separately, by adjusting system parameters. A fourfold concentrated xylanase was obtained with 70% enzyme recovery only in one step ATPS process without cell harvesting.     

Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99 %) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70 °C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60 °C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 μg/ml streptomycin-containing plate showed 14 % higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Summary Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The K_m and V_max values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.     

Direct production of feruloyl oligosaccharides and hemicellulase inducement and distribution in a newly isolated Aureobasidium pullulans strain

Studies were carried out to screen and identify strains that are able to directly produce ferulic oligosaccharides (FOs) from wheat bran (WB). The inducement and distribution of hemicellulases from strain 2012, which was identified as a non-melanin secreting strain of Aureobasidium pullulans (A. pullulans), were also determined. In a 60 g/L WB solution, A. pullulans could produce 545 nmol/L FOs, 64.12 IU/mL xylanase and 0.14 IU/mL ferulic acid esterase (FAE). A. pullulans was cultivated in media with WB, glucose, xylose, sucrose, lactose or xylan as the carbon source, and hemicellulases were mainly induced by xylan and WB and inhibited by glucose and sucrose. Xylanase and FAE were mainly present in the culture filtrate, xylosidase in the hyphal filaments and arabinofuranosidase was a membrane-bound enzyme. The yield of FOs was positively correlated to the hemicellulases activity, and significantly positively (P < 0.05) correlated to the xylanase activity (r = 0.992).     

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,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5–8.5. The enzyme was thermostable at 50–60 °C and retained an activity at pH 3.0–7.0. Xylanase XSC738 was activated by Mn²⁺, Co²⁺ and largely inhibited by Cd²⁺, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.     

Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135

An xylanase producing alkaliphilic Micrococcus sp was isolated from an alkaline soda lake. Xylose and xylan induced enzyme production but no activity was detected when it was grown using other carbohydrate sources. The level of xylanase production was higher in the presence of xylose than in the presence of xylan. The enzyme was purified to homogeneity and its molecular weight was estimated to be 56 kD on SDS-PAGE. The optimum temperature and pH for xylanase activity were 55°C and 7.5–9.0, respectively. Sixty per cent of the maximum activity was displayed at pH 11. The enzyme was very stable in the pH range of 6.5–10 and up to a temperature of 40°C. Xylanase activity was inhibited by Cu²⁺ and Hg²⁺. Received 03 October 1997/ Accepted in revised form 03 February 1998     

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.