A Highly Thermostable Alkaline Cellulase-Free Xylanase from Thermoalkalophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. JB 99 Suitable for Paper and Pulp Industry: Purification and Characterization

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0–10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K _m and V _max of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 μM min⁻¹ mg⁻¹, respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.     

Purification and Characterization of a Low Molecular Weight Endo-xylanase from Mushroom <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

A low molecular weight endo-xylanase (EC 3.2.1.8) was purified from an edible mushroom Termitomyces clypeatus grown in submerged medium with oat spelt xylan. Xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. Its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kDa. The enzyme was found to be most active at 50°C and pH 5.0, being most stable at pH 6.5. The K_m for oat spelt xylan was determined to be 10.4 mg/ml. The specificities of the enzyme was observed to be highly specific towards oat spelt xylan and was inhibited by mercuric chloride (HgCl₂), N-bromosuccinimide, and trans-1,2-diaminocyclohexane-N′,N′,N′,N′-tetraacetic acid strongly. The inhibitory action of N-bromosuccinimide on enzyme confirmed the presence of one tryptophan residue in its substrate-binding site. Amino acid analysis for xylanase showed the presence of high amount of hydrophobic serine, glycine, threonine, and alanine residues. The N-terminal sequencing study for the previously purified and characterized 56 kDa xylanolytic amyloglucosidase reveal the presence of 33.30% identity with glucoamylase chain A from Aspergillus awamori. The N-terminal sequence analysis of the present 12 kDa enzyme showed highest similarity (72.22% identity) towards xylanase from Neurospora crassa.     

Purification and Biochemical Characterization of Two Xylanases from <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> SBS 45

Two xylanases were isolated and purified from crude culture filtrate of Aspergillus sydowii SBS 45 after 9 days of growth on wheat bran containing 0.5% (w/v) birch wood xylan as the carbon source under solid-state fermentation. After a three-step purification scheme involving ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200), and anion exchange chromatography (DEAE-Sephadex A-50), xylanase I was purified 93.41 times, and xylanase II was purified 77.40 times with yields of 4.49 and 10.46, respectively. Molecular weights of xylanase I and II were 20.1 and 43 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature was 50 degrees C, and optimum pH was 10.0 for both xylanase I and II. The Km value of xylanase I for birch wood xylan was 3.18 mg ml(-1) and for oat spelt xylan 6.45 mg ml(-1), while the Km value of xylanase II for birch wood xylan was 6.51 mg ml(-1) and for oat spelt xylan 7.69 mg ml(-1). Metal ions like Al3+, Ba2+, Ca2+, Na+, and Zn2+ enhanced the activity of xylanase I and II at 10 mM concentration. Among the additives, L-tryptophan enhanced the activity of xylanase I and II at 10-, 20-, and 30-mM concentrations. Both xylanases appeared to be glycoproteins.     

Purification and Biochemical Characterization of a Highly Thermostable Xylanase from <Emphasis Type="Italic">Actinomadura</Emphasis> sp. Strain Cpt20 Isolated from Poultry Compost

An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of 90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively. The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan. This enzyme obeyed the Michaelis–Menten kinetics, with the K _m and k _cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min⁻¹, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn²⁺, Ca²⁺, and Cu²⁺, it was, strongly inhibited by Hg²⁺, Zn²⁺, and Ba²⁺. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.     

Production of extracellular xylanases byPenicillium janthinellum

Xylanase production byPenicillium janthinellum using 10–100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 Μmol/ min (11.4 U/mL or 84.4 U/mg) of Β-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). Β-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 Β- xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No Β-xylosidase or Β- glucosidase was induced until d 5. The Β-xylanases were rapidly inactivated at 50‡C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, theΒ-xylosidase andΒ-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.     

Gene Cloning,Overexpression, and Characterization of a Xylanase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml⁻¹. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K _m, and V _max for oat spelt xylan substrate was 7,988 U mg⁻¹, 22.2 mg ml⁻¹, and 15,105.7 μmol min⁻¹ mg⁻¹, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.     

Cellulases and Hemicellulases from Endophytic <Emphasis Type="Italic">Acremonium</Emphasis> Species and Its Application on Sugarcane Bagasse Hydrolysis

The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing l-arabinose, d-xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using d-xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.     

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6

An extracellular, endo-??-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS?CPAGE with an apparent molecular weight of 30?kDa. The xylanase activity was optimal at pH?3.0 and 65?°C. The specific activity measured for oat spelt xylan was 2,368?U?mg^?1. The apparent K _m and V _max values were 8.33?mg?ml^?1 and 58.82???mol?min^?1?ml^?1, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962?bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88?kDa.     

Cloning of a New Xylanase Gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TN119 Using a Modified Thermal Asymmetric Interlaced-PCR Specific for GC-Rich Genes and Biochemical Characterization

A bacterial strain, Streptomyces sp. TN119, was isolated from the gut of Batocera horsfieldi larvae and showed xylanolytic activity. A degenerate primer set was designed based on the base usage of G and C in Actinobacteria xylanase-coding sequences belonging to the glycosyl hydrolases family 10 (GH 10), and used to clone the partial xylanase gene from Streptomyces sp. TN119. A modified thermal asymmetric interlaced (TAIL)-PCR specific for high-GC genes, named GC TAIL-PCR, was developed to obtain the full-length xylanase gene (xynA119; 1089 bp). Rich in GC content (67.8%), xynA119 encodes a new GH 10 xylanase (XynA119), which shares highest identity (48.8%) with an endo-1,4-β-xylanase from Cellulosimicrobium sp. HY-12. Recombinant XynA119 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 6.5 and 60 °C, was stable at pH 4.0 to 10.0 and 50 °C, was resistant to most chemicals (except for Cu²⁺, Mn²⁺, Ag⁺, Hg²⁺ and SDS) and trypsin, and produced simple products. The specific activity, K _m, V _max, and k _cat using oat-spelt xylan as substrate were 57.9 U mg⁻¹, 1.0 mg ml⁻¹, 74.8 μmol min⁻¹ mg⁻¹, and 49.2 s^–1, respectively.     

A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4?kDa with an NH₂-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40?°C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5?mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-??-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K _m and V _max values toward beechwood xylan were 3.24?mg?ml^?1 and 58.21???mol?min^?1?mg^?1 protein, respectively.     

Isolation,Purification and Characterisation of Low Molecular Weight Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> SSP-34

Low molecular weight endo-xylanase from Bacillus pumilus SSP-34 was purified to homogeneity using ion exchange and size exclusion chromatographies. Xylanases were isolated by novel purification protocol which includes the use of anion exchange matrix such as DEAE Sepharose CL 6B with less affinity towards enzyme protein. The purified B. pumilus SSP-34 have a molecular weight of 20 kDa, with optimum pH and temperature at 6.0 and 50 °C, respectively. The enzyme was stable at 50 °C for 30 min. It showed remarkable stability at pH values ranging from 4.5 to 9 when the reaction was carried out at 50 °C. K _m and V _max values, determined with oats spelts xylan were 6.5 mg ml⁻¹ and 1,233 μmol min⁻¹ mg⁻¹ protein, respectively, and the specific activity was 1,723 U mg⁻¹     

Specificity and mode of action of a thermostable xylanase from bacillus amyloliquefaciens on-line monitoring of hydrolysis products

A thermostable xylanase purified from a strain of Bacillus amyloliquefaciens MIR 32 was characterized with respect to its substrate specificity and mode of hydrolytic action. The enzyme was highly specific for xylans as substrate and displayed no activity toward other polysaccharides, including cellulose. The enzyme exhibited K_m and V_max of 4.5 mg/mL and 0.58 mmol/min/mg, respectively, with birchwood xylan as the substrate. Microdialysis sampling with anion exchange chromatography and integrated pulsed electrochemical detection were used for rapid on-line monitoring of products during hydrolysis of oat spelt and bagasse xylan, and xylooligosaccharides. Xylobiose and xylotriose were the main end products. Xylotetraose was the smallest oligosaccharide to be acted on by the xylanase. The product pattern confirmed that the enzyme was an endoxylanase.     

Purification and biochemical characterization of two major thermophilic xylanase isoforms (T70 and T90) from xerophytic Opuntia vulgaris plant spp.

Thermostable xylanase isoforms T₇₀ and T₉₀ were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T₇₀ and T₉₀ isoforms yielded a final specific activity 134.0 and 150.8 U mg^?1 protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T₇₀ and T₉₀ xylanase isoforms was 5.0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T₇₀ and T₉₀ enzyme isoforms was 3.49, 2.1 mg ml^?1, respectively when oat spelt xylan was used as a substrate. The T₇₀ had a Vmax of 10.4 μmol min^?1 mg^?1, and T₉₀ had a Vmax of 8.9 μmol min^?1 mg^?1, respectively. In the presence of 10 mM Co²⁺, and Mn²⁺ the activity of T₇₀ and T₉₀ isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg²⁺, Cd²⁺, Cu²⁺, Zn²⁺ while partial inhibition was observed in the presence of Fe³⁺, Ni²⁺, Ca²⁺and Mg²⁺. The T₇₀ and T₉₀ isoforms retained nearly 50 % activity in the presence of 2.0 M urea, while use of 40 mM SDS lowered the activity nearly 38–41 %. The substrate specificity of both T₇₀ and T₉₀ isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T₉₀ isoform with polyclonal anti-T₉₀ antibody raised in rabbit, as well as cross-reactivity of the antibody with the T₇₀ xylanase isoform.     

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.     

Immobilization of <Emphasis Type="Italic">Aspergillus niger</Emphasis> Xylanase on Chitosan Using Dialdehyde Starch as a Coupling Agent

Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS) beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed to be 5 °C higher than that of free enzyme (50 °C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability as compared to the CS-GA-immobilized and free xylanase. The apparent K _m and V _max values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 μmol/min/mg protein, respectively. The CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose, as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase immobilization application.     

Expression and Characterization of the Dictyoglomus thermophilum Rt46B.1 Xylanase Gene (xynB) in Bacillus subtilis

To obtain extracellular and high-level expression of the Dictyoglomus thermophilum Rt46B.1 xylanase B gene, this gene was integrated into the α-amylase gene site of a host strain of Bacillus subtilis WB800. The extreme thermophile xylanase gene was successfully integrated and expressed in the host, measured at 24 ± 0.4 XUs/mL in the Luria broth medium supernatant. The recombinant enzyme was purified by ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. The molecular mass and pI value of xylanase were estimated to be 24 kDa and 4.3, respectively. The optimal pH level and temperature of the purified enzyme were 6.5 and 85 °C, respectively. Xylanase showed reasonable activity at temperatures up to 95 °C and remained stable at 4 °C for 1 week. The purified enzyme retained most of its activity in 1 mM ethylenediaminetetraacetic acid or dithiothreitol and 0.1% Tween-20 or Triton X-100. However, strong inhibition was observed in the presence of 5 mM Mn²⁺, 0.5% sodium dodecyl sulfate, Tween-20, or Triton X-100; a strong stimulating effect was also observed in the presence of Fe²⁺. The K _m and V _max values of the recombinant xylanase for birchwood xylan were calculated to be 2.417 ± 0.36 mg/mL and 325 ± 41 μmol/min mg, respectively. Xylanase was found to be useful in the prebleaching process of paper pulps.     

Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (K_m = 26.06 mg·mL⁻¹ and V_max = 5.647 U·mg⁻¹). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.     

Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Purification and characterization of a xylanase from the thermophilic ascomyceteThielavia terrestris 255b

Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II). The latter enzyme could be purified to > 99% homogeneity using anion-exchange chromatography and gel filtration. Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6–4.0 and 60–65°C, respectively. The ratio of the enzyme’s activity against xylan and carboxymethylcellulose was 500–1000 to 1, indicating a possible application of this enzyme in biobleaching processes. The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi.     

Improvement of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> Endo-β-1,4-Xylanase Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by Codon Optimization and Analysis of the Enzymic Characterization

The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml⁻¹, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na₂HPO₄–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn²⁺. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.