Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.     

A Novel Low Molecular Weight Endo-xylanase from Streptomyces sp. CS628 Cultivated in Wheat Bran

An extracellular low molecular weight xylanase (Xyn628) from Streptomyces sp. CS628 was isolated from Korean soil sample, produced in wheat bran medium, purified, and biochemically characterized. Xyn628 was purified 4.8-fold with a 33.78 % yield using Sepharose CL-6B column chromatography. The purified xylanase was ~18.1 kDa estimated by SDS-PAGE and xylan zymography. N-terminal amino acid sequences of Xyn628 were AYIKEVVSRAYM. The enzyme was found to be stable in a broad range of pH (5.0–13.0) and up to 60 °C and have optimal pH and temperature of pH 11.0 and 60 °C, respectively. Xyn628 activities were remarkable affected by various detergents, chelators, modulators, and metal ions. The xylanase produced xylobiose and xylotriose as principal hydrolyzed end products from the xylan. It was found to degrade agro-waste materials like corn cob and wheat bran by Xyn628 (20 U/g) as shown by electron microscopy. As being simple in purification, low molecular weight, alkaline, thermostable, and ability to produce xylooligosaccharides show that Xyn628 has potential applications in bioindustries as a biobleaching agent or/and xylooligosaccharides production with an appropriate utilization of agro-waste.     

Purification and Properties of a Psychrotrophic <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K _m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.     

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml^?1 and 45.66 μmol min^?1 mg^?1 and 9.22 mg ml^?1 and 16.05 μmol min^?1 mg^?1, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.     

Determination of xylo-oligosaccharides in enzymatically hydrolysed pulp by liquid chromatography and capillary electrophoresis

Three different commercial β-1,4-endoxylanase preparations were used to hydrolyze bleached kraft pulp. Xylo-oligosaccharides in the produced filtrates were separated and quantified using both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). All the determinations were performed without sample derivatization. The analytical methods were used to highlight the differences between the enzymes behaviour in terms of hydrolysates, but also to estimate the productivity of xylo-oligosaccharides from kraft pulp when the bleached material would be used in biorefining industry. The research showed that the glycosyl hydrolase family 10 enzyme produced by Aspergillus oryzae released xylobiose and xylotriose from the pulp material. The major oligosaccharides released by the family 11 enzyme produced by Bacillus sp. were xylotriose, xylobiose and xylotetraose. On the contrary, another family 11 enzyme produced by A. oryzae produced also xylose. The HPLC results agreed well with the xylose concentrations obtained after acid hydrolysis. The CE data showed the same trend, but much lower concentrations were identified than with HPLC. At the same time the HPLC method was able to separate only small oligosaccharides, whereas CE could be used for separation of all the xylo-oligosaccharides from xylobiose to xylohexaose. The highest xylo-oligosaccharide yield was achieved with Shearzyme at pH 5 corresponding to 22 % of total xylan from bleached birch kraft pulp.     

Purification and Characterization of Novel Halo-Acid-Alkali-Thermo-stable Xylanase from Gracilibacillus sp. TSCPVG

An aerobic xylanolytic moderately halophilic and alkali-tolerant bacterium, Gracilibacillus sp. TSCPVG, produces multiple xylanases of unusual halo-acid-alkali-thermo-stable nature. The purification of a major xylanase from TSCPVG culture supernatant was achieved by hydrophobic and gel permeation chromatographic methods followed by electroelution from preparatory PAGE. The molecular mass of the purified xylanase was 42 kDa, as analyzed by SDS-PAGE, with a pI value of 6.1. It exhibited maximal activity in 3.5 % NaCl and retained over 75 % of its activity across the broad salinity range of 0–30 % NaCl, indicating a high halo-tolerance. It showed maximal activity at pH 7.5 and had retained 63 % of its activity at pH 5.0 and 73 % at pH 10.5, signifying the tolerance to broad acid to alkaline conditions. With birchwood xylan as a substrate, K _m and specific activity values were 21 mg/ml and 1,667 U/mg, respectively. It is an endoxylanase that degrades xylan to xylose and xylobiose and had no activity on p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl acetate, carboxymethylcellulose, and filter paper. Since it showed remarkable stability over different salinities, broad pH, and temperature ranges, it is promising for application in many industries.     

Expression and Characterization of Hyperthermotolerant Xylanases,SyXyn11P and SyXyn11E,in Pichia pastoris and Escherichia coli

Both Syxyn11P and Syxyn11E, two codon-optimized genes encoding glycoside hydrolase (GH) family 11 hyperthermotolerant xylanases (designated SyXyn11P and SyXyn11E), were synthesized and inserted into pPIC9K^M and pET-28a(+) vectors, respectively. The resulting recombinant expression vectors, pPIC9K^M-Syxyn11P and pET-28a(+)-Syxyn11E, were transformed into Pichia pastoris GS115 and Escherichia coli BL21, respectively. The maximum activities of two recombinant xylanases (reSyXyn11P and reSyXyn11E) expressed in P. pastoris and E. coli reached 30.9 and 17.8 U/ml, respectively. The purified reSyXyn11P and reSyXyn11E displayed the same pH optimum at 6.5 and pH stability at a broad range of 4.5–9.0. The temperature optimum and stability of reSyXyn11P were 85 and 80 °C, higher than those of reSyXyn11E, respectively. Their activities were not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of reSyXyn11P toward birchwood xylan were 4.3 mg/ml and 694.6 U/mg, whose K _m was close to that (4.8 mg/ml), but whose V _max was much higher than that (205.6 U/mg) of reSyXyn11E. High-performance liquid chromatography analysis indicated that xylobiose and xylotriose as the major products were excised from insoluble corncob xylan by reSyXyn11P.     

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T _1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.     

Expression and Characterization of Recombinant GH11 Xylanase from Thermotolerant Streptomyces sp. SWU10

Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-β-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5–6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6–10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.     

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence‐assisted carbohydrate electrophoresis

The structure of xylan, which has a 1,4‐linked β‐xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence‐assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA²Xyl₃ and MeGlcA²Xyl₄, respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family‐specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β‐xylosidase degraded MeGlcA²Xyl₄ into xylose and MeGlcA²Xyl₃ but further degradation of MeGlcA²Xyl₃ required additional enzymes. Synergy between xylanases and β‐xylosidase was also found to significantly accelerate the conversion of XOS into xylose.     

Thermostable Hemicellulases of a Bacterium,Geobacillus sp. DC3, Isolated from the Former Homestake Gold Mine in Lead,South Dakota

A thermophilic strain, Geobacillus sp. DC3, capable of producing hemicellulolytic enzymes was isolated from the 1.5-km depth of the former Homestake gold mine in Lead, South Dakota. The DC3 strain expressed a high level of extracellular endoxylanase at 39.5 U/mg protein with additional hemicellulases including β-xylosidase (0.209 U/mg) and arabinofuranosidase (0.230 U/mg), after the bacterium was grown in xylan for 24 h. Partially purified DC3 endoxylanase exhibited a molecular mass of approximately 43 kDa according to zymography with an optimal pH of 7 and optimal temperature of 70 °C. The kinetic constants, K _m and V _max, were 13.8 mg/mL and 77.5 μmol xylose/min·mg xylan, respectively. The endoxylanase was highly stable and maintained 70 % of its original activity after 16 h incubation at 70 °C. The thermostable properties and presence of three different hemicellulases of Geobacillus sp. DC3 strain support its potential application for industrial hydrolysis of renewable biomass such as lignocelluloses.     

Lactose-inducted production of a complete lignocellulolytic enzyme system by a novel bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. BS-5 and its application for saccharification of alkali-pretreated corn cob

In this study, with combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assay, a novel cellulase and xylanase-producing strain identified as Bacillus sp. was isolated. Using lactose as the only carbon source, a complete and balanced lignocellulolytic enzyme system containing at least endoglucanase (9.6 U/ml), exoglucanase (0.8 U/ml), Fpase (1.4 U/ml), xylanase (3.8 U/ml) and β-glucosidase (1.2 U/ml) was produced. Interestingly, a zymogram of the crude culture supernatant displayed a multifunctional lignocellulolytic enzyme system including at least four bonds with both endoglucanase activity and xylanase activity at 21.2, 23.8, 28.9 and 31.2 kDa, respectively, indicating that these enzymes might be bifunctional. More gratifyingly, according to the binding affinity analysis and scanning electron microscopy, the crude enzyme complex produced by strain BS-5 was capable of hydrolyzing not only pure insoluble polysaccharides, but also agricultural residues such as corn cob. At 5% substrate concentration and 20 FPU/g enzyme loading, the reducing sugar was 350.8 mg/g of alkali-pretreated corn cob after 72 h enzymatic hydrolysis. These results suggested that this strain could be a good candidate for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail for the saccharification of lignocellulosic biomass.     

A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (TBS2) Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and Its pH and Temperature Stability

A gene encoding a highly thermostable β-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable β-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0–8.0 and was active at 20–100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other β-mannanase reported up to now, suggesting its potential for industrial applications.     

Protein Extraction and Enzymatic Hydrolysis of Ammonia-Treated Cassava Leaves (Manihot esculenta Crantz)

In the present work, cassava leaves were treated with 0.5 kg ammonia/kg dry matter at 78 °C and 30% moisture content in a 2-kg reactor. Protein extraction was carried out with a calcium hydroxide solution (pH 10) for 30 min at several temperatures (30 °C, 45 °C, 60 °C, 75 °C, and 90 °C) and solid/liquid ratios (1:10 and 1:15) in a thermostatized bath. Soluble protein content of the extracts was determined by Lowry’s method. Dry substrate concentrations of 5%, 7.5%, and 10% and enzyme doses of 2 and 5 IU/g dry matter were used for the enzymatic hydrolysis in an orbital incubator at 50 °C and 100 rpm. Both cellulase and xylanase were used. Reducing sugars produced were determined with the dinitrosalicylic acid method. The highest protein extraction yield for the ammonia-treated leaves was 29.10%, which was 50% higher than with the untreated leaves (20%), and was obtained at 90 °C with a 1:10 solid/liquid ratio. The concentrate had a protein content of 36.35% and the amino acid profile was suitable for swine and poultry. The highest sugar yield was 54.72% with respect to theoretical and was obtained with 5% solids and an enzyme dose of 5 IU/g dry matter. This yield was 3.4 times higher than the yield of the untreated leaves (16.13%). These results indicate that cassava leaves have a great potential for animal feeding and ethanol production. Both protein extraction and sugar yields may be enhanced by optimizing the ammonia treatment.     

Cloning,Expression, and Characterization of a New <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S27 Xylanase for Which Xylobiose is the Main Hydrolysis Product

A xylanase gene, xynBS27, was cloned from Streptomyces sp. S27 and consisted of 693 bp encoding a 230-residue protein, including a putative 41-residue signal peptide. Belonging to the glycoside hydrolase family 11, XynBS27 exhibits the maximum identity (75.9%) to the xylanase from Streptomyces sp. zxy19. Recombinant XynBS27 was overexpressed in Pichia pastoris, and the xylanase activity was 7624.0 U/ml after high-cell-density fermentation in 3.7-L fermenter. The purified recombinant XynBS27 had a high specific activity of 3272.0 U/mg. The optimum temperature and pH for XynBS27 activity was 65 °C and pH 6.5, respectively. XynBS27 showed good pH stability and retained more than 80% of the maximum activity after incubation in buffers with pH ranging between 4.0 and 12.0 at 37 °C for 1 h. The main hydrolysis product of xylan by XynBS27 was xylobiose (>75%), which was good for human health derived from its ability to modulate the intestinal function. The attractive biochemical characteristics of XynBS27 suggest that it may be a good candidate in a variety of industrial applications.     

Purification and Characterization of an Ethanol-Tolerant β-Glucosidase from Sporidiobolus pararoseus and Its Potential for Hydrolysis of Wine Aroma Precursors

An extracellular ethanol-tolerant β-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-βgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-βgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-βgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement.     

Purification and Characterization of the Xylanase Produced by Jonesia denitrificans BN-13

Jonesia denitrificans BN-13 produces six xylanases: Xyl1, Xyl2, Xyl3, Xyl4, Xyl5, and Xyl6; the Xyl4 was purified and characterized after two consecutive purification steps using ultrafiltration and anion exchange chromatography. The xylanase-specific activity was found to be 77 unit (U)/mg. The molecular weight of the Xyl4 estimated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) revealed a monomeric isoenzyme of about 42 kDa. It showed an optimum pH value of 7.0 and a temperature of 50 °C. It was stable at 50 °C for 9.34 h. The enzyme showed to be activated by Mn⁺², β-mercaptoethanol, and dithiothreitol (DTT) with a high affinity towards birchwood xylan (with a K _m of 1 mg ml^?1) and hydrolysis of oat-spelt xylan with a K _m of 1.85 mg ml^?1. The ability of binding to cellulose and/or xylan was also investigated.     

A Xylanase Gene Directly Cloned from the Genomic DNA of Alkaline Wastewater Sludge Showing Application Potential in the Paper Industry

A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.     

Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from <Emphasis Type="Italic">Hypocrea orientalis</Emphasis>

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7–22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19–29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K _m and V _max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.     

Improvement of Strain Penicillium sp. EZ-ZH190 for Tannase Production by Induced Mutation

In the search for an efficient producer of tannase, Penicillium sp. EZ-ZH190 was subjected to mutagenesis using heat treatment and strain EZ-ZH290 was isolated. The maximum tannase in this mutant strain was 4.32 U/mL with an incubation period of 84 h as compared to wild strain EZ-ZH190 where the incubation period was 96 h with a maximum enzyme activity of 4.33 U/mL. Also, the Penicillium sp. EZ-ZH290 tannase had a maximum activity at 40 °C and pH 5.5. Then, the spores of strain EZ-ZH290 were subjected to γ irradiation mutagenesis and strain EZ-ZH390 was isolated. Strain EZ-ZH390 exhibited higher tannase activity (7.66 U/mL) than the parent strain EZ-ZH290. It was also found that Penicillium sp. EZ-ZH390 tannase had an optimum activity at 35 °C and a broad pH profile with an optimum at pH 5.5. The tannase pH stability of Penicillium sp. EZ-ZH390 and its maximum production of tannase followed the same trend for five generations confirming the occurrence of stable mutant. This paper is shown that γ irradiation can mutate the Penicillium sp. leading to increase the tannase production.