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.     

Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O₂. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent K_m for IDA was 4.0 mM with a k_cat of 5.3 s⁻¹. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Enrichment of new alkane oxidizing bacterial strains for human drug metabolite production

An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.     

Purification and partial characterisation of a thermostable xylanase from salt-tolerant Thermobifida halotolerans YIM 90462T

ThxynA, an extracellular xylanase of T. halotolerans YIM 90462^T, was purified to homogeneity from a fermentation broth by ultra-filtration, ammonium sulphate precipitation, hydrophobic chromatography and ion exchange chromatography. The purified xylanase has a molecular mass of 24 kDa and is optimally active at 80 °C and pH 6.0. The enzyme is stable over a broad pH range (pH 6.0–10.0) and shows good thermal stability when incubated at 70 °C for 1 h. The K_m and V_max values of the enzyme are 11.6 mg/mL and 434 μmol mg^?1 min^?1, respectively, using oat spelt xylan as a substrate. Moreover, the enzyme seemingly has both xylanase activity and cellulase activity. These unique properties suggest that it may be useful for industrial applications.     

Production and purification of high levels of cellulase-free bacterial xylanase by Bacillus sp. SV-34S using agro-residue

Xylanase produced from the isolated bacterial strain Bacillus sp. SV-34S showed a 8.74-fold increase in enzyme activity under optimized submerged fermentation conditions. Cultivation using wheat bran as the carbon source and beef extract and (NH₄)H₂PO₄ as the nitrogen source resulted in productivity of 3,454.01 IU/mL xylanase. Xylanase was purified by 12.94-fold, with a recovery of 13.4 % and a specific activity of 3417.2 IU/mg protein, employing ammonium sulphate fractionation followed by cation-exchange chromatography using CM-Sephadex C-50 column chromatography, with a product of 27 kDa. The purified xylanase showed an optimum temperature and pH of 50 °C and 6.5, respectively although it was active even at pH 11.0. The thermostability study revealed that Bacillus sp. SV-34S was thermotolerant, being stable up to 50 °C; the residual activity at 55 and 60 °C was 96 and 93 %, respectively. The enzyme was stable between pH 6.0 and 8.0, although it retained >100 % activity at pH 8.0 and 9.0, respectively, following pre-incubation for 24 h. Xylanase activity was inhibited by various metal ions added to the assay mixture, with maximum inhibition observed in the presence of HgCl₂. The K_m and V_max values of the purified xylanase using birch wood xylan as substrate were 3.7 mg/mL and 133.33 IU/mL, respectively. The isolated bacterial strain produced high levels of extremophilic cellulase-free xylanase. The fact that it can be used in crude form and that it can be produced cheaply with renewable carbon sources make the process economically feasible. The characteristics of the purified enzyme suggest its potential application in industries such as the paper and pulp industry.     

Purification,characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant K_m of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 mol min^–1 (mg^–1protein)^–1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.     

Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938

An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K _m and k _cat values of the enzyme were obtained as 1.7 mg/ml and 66 s^?1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.     

Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre

An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The K _m and k _cat values of the PG were 2.7 mg/mL and 2.23 × 10³ s^?1, respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50°C and the enzyme does not loose activity below 30°C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag⁺, Hg²⁺ and KMnO₄, while it was stimulated to some extent by Co²⁺. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.     

Purification and characterization of low molecular weight alkali stable xylanase from Neosartorya spinosa UZ-2-11

Alkaliphilic xylanase from Neosartorya spinosa UZ-2-11 was purified using a three-step of purification scheme of ammonium sulphate precipitation followed by Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange chromatography, and compared its properties with N. tatenoi KKU-CLB-3-2-4-1 of our previous report. The purified xylanase from N. spinosa UZ-2-11 exhibited maximum activity at pH 9.0 and 45 °C which was similar to endo-xylanase from N. tatenoi KKU-CLB-3-2-4-1. However, this enzyme was stable in a range of pH 6.0–11.0. It was also more stable at a high temperature of 50 °C where the activity was still up to 50% after heating for 120 min. The xylanase was purified 7.89-fold with 3.0% of yield to obtain a specific activity of 11.88 U/mg. The molecular weight of xylanase from this fungus was 27.68 kDa. The K_m and V_max values of the purified xylanase were 0.24 mg/mL and 15.85 μmol/min/mg, respectively. The xylanase activity was moderately inhibited by Hg²⁺ at a concentration of 10 mM, which was different to the case of N. tatenoi KKU-CLB-3-2-4-1 where Hg²⁺ was a strong inhibitor. In addition, the hydrolysed birchwood xylan was obtained mailnly xylobiose, xylotriose, xylotetraose and xylopentaose as end products, suggesting that it was an endo-xylanase.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Purification and characterization of highly thermostable α-amylase from thermophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

In this study, the production of extracellular thermostable α-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60°C. This extracellular α-amylase was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The α-amylase was purified to 8.138 fold homogeneity with a final recovery of 58% and a specific activity of 3,239 U/mg proteins. The purified α-amylase appeared as a single protein band on SDS-PAGE with a molecular mass of 94.5 kDa. Non-denaturing PAGE analysis showed one major band associated with enzyme activity, indicating the absence of isoenzymes. A TLC analysis showed maltose as major end product of the enzyme. The optimum assay temperature and pH for enzyme activity were 60°C and 6.0 respectively; however, the enzyme activity was stable over a wide range of pH and temperatures. The α-amylase retained its activity in the presence of the denaturing agents — SDS, Triton X-100, Tween-20, Tween-80, and was significantly inhibited by EDTA and urea. Calcium ions increased the enzyme activity, while Hg²⁺, Zn²⁺, and Co²⁺ had inhibitory effects. The K _m and V _max values were found to be 2.9 mg/mL and 7936 U/mL respectively.     

Production,isolation and partial purification of xylanases from anAspergillus sp.

AnAspergillus sp., isolated from a rubbish dump, produced 10.6 IU ml^-1 xylanase activity. Two xylanases were recognized and each was purified to homogeneity by two-stage chromatography on DEAE-and CM-Sepharose. Xylanase I had a pI of 7.2 and anM _r of 26 kDa whereas xylanase II had a pI of 4.7 and anM _r of 21 kDa. At 50°C, xylanase I was stable for 2.5 h but xylanase II was only stable for 1 h.P. Khanna is with the National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440 020, India. S. Sivakami Sundari and N. Jothi Kumar are with the National Environmental Engineering Research Institute, Madras Zonal Laboratory, CSIR Madras Complex, Taramani 600 113, India.     

Adenylosuccinate synthetase from Azotobacter vinelandii: purification, properties and steady-state kinetics.

Adenylosuccinate synthetase has been purified to homogeneity from Azotobacter, vinelandii. The purification method involves affinity chromatography on blue dextran-Sepharose, and hydrophobic chromatography, in addition to heat treatment, ammonium sulfate fractionation, and ion-exchange chromatography. The purified enzyme displays a single protein band after electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Molecular weights of 110,000 and 54,000 are estimated by gel filtration and SDS gel electrophoresis, respectively.Steady-state kinetic measurements of the forward and reverse reactions and of the reaction in which arsenate replaces phosphate reveal a sequential mechanism with a fully random order of substrate addition in all cases. The maximal velocities of the reverse reaction and arsenolysis are virtually identical, and are approximately 10% of the maximal velocity for the forward reaction. In common with this enzyme from other sources, hadacidin is a potent competitive inhibitor with respect to aspartate (K_i = 0.3 μm). Specific anions, e.g. nitrate and thiocyanate, are competitive inhibitors with respect to GTP; their effectiveness follows the Hofmeister series. Anion inhibition is synergized by GDP, but binding is exclusive with respect to guanylylimidodiphosphate, suggesting binding of the anions at the site normally occupied by the transferable phosphoryl group of GTP.     

Biochemical and Biophysical Characterization of Purified Thermophilic Xylanase Isoforms in Cereus pterogonus Plant Spp

Two thermostable xylanase isoforms T₆₀ and T₈₀ were purified to homogeneity from the cladodes of the xerophytic Cereus pterogonus plant species. After three consecutive purification steps, the specific activity of T₆₀ and T₈₀ isoforms were found to be 178.6 and 216.2 U mg⁻¹ respectively. The molecular mass of both isoforms was determined to be 80 kDa. The optimum temperature for T₆₀ and T₈₀ xylanase isoforms were 60 and 80 °C respectively. The pH was 5.0 for both isoforms. The presence of divalent metal ions (10 mM Co²⁺) showed stimulatory effects of both catalytic activities, where as in the presence of Hg²⁺, Cd²⁺, Cu²⁺ showed inhibitory effect on these activities at all concentrations studied. The thermodynamic analysis of xylanase activity using denaturation kinetics and the presence divalent cations at 30–100 °C, showed lower ΔH, ΔS, and ΔG values at all the temperatures investigated. The melting temperature of purified T₈₀ xylanase isoform as determined by TG/DTA analysis and it showed the unfolding temperature was 80 °C. The g value and hyperfine (A) value purified xylanase T₈₀ isoform was 2.017 and 10.80 respectively. Immunoblot analysis with antiserum raised against the purified T₈₀ xylanase isoforms revealed single immunolgically related polypeptides of 80 kDa, identical with the polypeptide band produced on SDS-PAGE. The results of double immunodiffusion against the T₈₀ isoforms showed a single precipitin line indicating that the serum used was specific to these xylanase isoforms. The kinetic and thermodynamic properties suggested that xylanase from C. pterogonus may have a potential usage in various industries.     

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M_r 30,000). The enzyme had a K_m for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH₄)₂SO₄ fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M_r 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour.     

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.     

Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Study of the intracellular xylanolytic activity of the phytopathogenic fungus Sporisorium reilianum

The present study demonstrates that Sporisorium reilianum, a phytopathogenic fungus of corn, produces intracellular xylanolytic activity during submerged fermentation. Production reached its highest levels in a medium containing glucose, corn hemicellulose and yeast extract. An intracellular xylanase was purified by a process that included precipitation with ammonium sulfate, ion exchange chromatography and gel filtration. Optimal pH and temperature values were 5.0 and 60 °C, respectively. The enzyme showed activity through a broad pH range. The molecular weights of pure xylanase were 36 and 37 kDa, determined by SDS PAGE and gel filtration, respectively. K_m and V_max were 0.160 mg/mL and 1.564 μmol/min/mg, respectively, on a substrate of birchwood xylan. SDS, EDTA, β-Mercaptoethanol, Tween 80, Triton and Mn²⁺ and Ca²⁺ strongly inhibited activity. The purified enzyme hydrolyzed xylan, releasing xylotriose and xylobiose. Sequence protein analysis showed 95% similarity with the theoretical protein encoded by the sr14403 gene of S. reilianum, which encodes a putative endo-β-1,4-xylanase. The enzyme is an isoform of the extracellular xylanase SRXL1 of this basidiomycete.     

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.     

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.