A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428

A cellulase free thermostable xylanase from Streptomyces sp. CS428 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular xylanase was purified 26 fold with a 55% yield by CM Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428) was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (4 to ～13.6) and to 50 °C and have an optimum temperature of 80 °C. Xyn428 had K_m and V_max values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM, and produced xylose and xylobiose as major products. It was found to degrade agro waste materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being simple in purification, thermo tolerant, pH stability in broad range and ability to produce xylooligosaccharides show that Xyn428 has potential applications in industries as a biobleaching agent and for xylooligosaccharides production.     

Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9

A new xylanase gene, xynAS9, was cloned from Streptomyces sp. S9, which was isolated from Turpan Basin, China. The full-length gene consists of 1,395 bp and encodes 465 amino acids including 38 residues of a putative signal peptide. The overall amino acid sequence shares the highest identity (50.8%) with a putative endo-1,4-beta-xylanase from Streptomyces avermitilis of the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.5 and 60 degrees C, respectively. The enzyme showed broad temperature adaptability, retaining more than 65% of the maximum activity when assayed at 50-80 degrees C. The enzyme also had good thermal and pH stability. The K (m) values for oat spelt xylan and birchwood xylan substrates were 2.85 and 2.43 mg ml(-1), with the V (max) values of 772.20 and 490.87 mumol min(-1) mg(-1), respectively. The hydrolysis products of xylan were mainly xylose and xylobiose. These favorable properties should make XynAS9 a good candidate in various industrial applications.     

Purification and characterization of xylanase from a thermophilic Streptomyces sp. K37

Extracellular xylanase (EC 3.2.1.8) from Streptomyces sp. K37 was purified 33.53 by ultrafiltration and cation exchange chromatography followed by gel filtration chromatography. The optimum pH and temperature for purified xylanase were found to be pH 6.0 and 60 degrees C. The Km and V(max) values of the purified xylanase were 15.4 mg ml(-1) and 0.67 micromole reducing sugar min(-1) ml(-1). High performance liquid chromatography (HPLC) gel filtration of the purified xylanase eluted xylanase activity as a peak corresponding to the molecular weight of about 24.3 kDa while the molecular weight determined by SDS-PAGE was found to be 26.4 kDa. The purified xylanase of Streptomyces sp. K37 was found to be endoxylanase and non arabinose liberating enzyme and was highly glycosylated (73.97%).     

Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library

There is a great interest in xylanases due to the wide variety of industrial applications for these enzymes. We cloned a xylanase gene (xyn8) from an environmental genomic DNA library. The encoded enzyme was predicted to be 399 amino acids with a molecular weight of 45.9 kD. The enzyme was categorized as a glycosyl hydrolase family 8 member based on sequence analysis of the putative catalytic domain. The purified enzyme was thermolabile, had an activity temperature optimum of 20°C on native xylan substrate, and retained significant activity at lower temperatures. At 4°C, the apparent K _m was 3.7 mg/ml, and the apparent k _cat was 123/s.Reference to a company and/or products is only for the purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

链霉菌Strz-6木聚糖酶的纯化和固定化研究

链霉菌胞外木聚糖酶经过盐析、离子交换和分子筛层析纯化,粗酶液被纯化了32.5倍,比活力达498u/mg,活力回收46.6%。纯化后的酶固定在戊二醛交联的壳聚糖上,酶活回收率为42.8%。固定化酶的最适pH为6.0,最适温度为60℃,且固定化酶在65~75℃活力都较高。该酶的耐热性比较强,固定化酶热稳定性优于原酶;以木聚糖为底物,固定化酶的表观米氏常数为0.93×10-2g/L。     

Identification and characterization of a highly alkaline and thermotolerant novel xylanase from Streptomyces sp.

Xylanases constitute an important industrial enzyme, which hydrolyzes the polysaccharide xylan. In this work, a novel Streptomyces strain producing cellulase-free xylanase was isolated from the soil samples collected from the mangrove forest of Kadalundi, Kerala, India. The strain produced unique enzyme, which exhibited optimal activity at pH 9.0 and tolerance up to pH 12.0. Media engineering was carried out to improve the enzyme production, which showed best enzyme production at 30°C, medium pH 9.0 and incubation time of 48 h. Enzyme was highly thermo-tolerant up to 70°C and alkaline tolerant. Partial gene amplification as well as partial purification of enzyme was carried out to characterize the enzyme. The unique features of the enzyme make it an ideal candidate for industrial application for paper and pulp industry.     

Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3

Streptomyces sp. QG-11-3, which produces a cellulase-free thermostable xylanase (96 IU ml⁻¹) and a pectinase (46 IU ml⁻¹), was isolated on Horikoshi medium supplemented with 1% w/v wheat bran. Carbon sources that favored xylanase production were rice bran (82 IU ml⁻¹) and birch-wood xylan (81 IU ml⁻¹); pectinase production was also stimulated by pectin and cotton seed cake (34 IU ml⁻¹ each). The partially purified xylanase and pectinase were optimally active at 60°C. Both enzymes were 100% stable at 50°C for more than 24 h. The half-lives of xylanase and pectinase at 70, 75 and 80°C were 90, 75 and 9 min, and 90, 53 and 7 min, respectively. The optimum pH values for xylanase and pectinase were 8.6 and 3.0, respectively, at 60°C. Xylanase and pectinase were stable over a broad pH range between 5.4 and 9.4 and 2.0 to 9.0, respectively, retaining more than 85% of their activity. Ca²⁺ stimulated the activity of both enzymes up to 7%, whereas Cd²⁺, Co²⁺, Cr³⁺, iodoacetic acid and iodoacetamide inhibited xylanase up to 35% and pectinase up to 63%; at 1 mM, Hg²⁺ inhibited both enzymes completely. Journal of Industrial Microbiology & Biotechnology (2000) 24, 396–402. Received 29 September 1999/ Accepted in revised form 02 February 2000     

链霉菌发酵麦草产木聚糖酶的试验研究

通过正交设计试验 ,找出利用链霉菌和麦草基质发酵生产木聚糖酶的试验条件。培养基 (g/L) :麦草粉 ,4 5 ;(NH4 ) 2 SO4 ,7.5 ;酵母膏 ,8;K2 HPO4 ·3H2 O ,1;MgSO4 ·7H2 O ,0 .5 ;NaCl,0 .3。接种量为 5 .0× 10 8个孢子 / 5 0mL培养基 ,振荡培养 (12 0r/min) 5d     

Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana

A beta-xylanase (XynIII) of Acrophialophora nainiana was purified to homogeneity from the culture supernatant by ultrafiltration and a combination of ion exchange and gel filtration chromatographic methods. It was optimally active at 55 degrees C and pH 6.5. XynIII had molecular masses of 27.5 and 54 kDa, as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The purified enzyme hydrolyzed preferentially xylan as the substrate. The half-lives of XynIII at 50 and 60 degrees C were 96 and 1 h, respectively. It was activated by L-tryptophan, dithiothreitol, 5,5-dithio-bis(2-nitrobenzoic acid, L-cysteine and beta-mercaptoethanol and strongly inhibited by N-bromosuccinimide. The presence of carbohydrate was detected in the pure XynIII.     

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.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

Stereoselective reduction towards pharmaceutically potent products with multi‐chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH‐dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha‐hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl₂. Detailed analysis of the pH‐dependent activity and stability yielded a broad pH‐optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half‐life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio‐ and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2‐hydroxypropiophenone in more detail. Biotechnol. Bioeng. 2013; 110: 1838–1848. © 2013 Wiley Periodicals, Inc.     

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.     

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

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

An extremely alkaline novel chitinase from Streptomyces sp. CS495

An extremely alkaline chitinase from Streptomyces sp. CS495 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase was purified 7.0 fold with a 33.9% yield by Sepharose Cl-6B column. The molecular mass of the enzyme (Ch495) was approximately 41 kDa. Ch495 was found to be stable over a broad pH range (5–12.5) and to 50 °C and have an optimum temperature of 60 °C. Ch495 had K_m and V_max values of 1.34 ± 2.9 mg/mL and 889 ± 3.6 mmol/min, respectively using different concentrations of colloidal chitin. N-terminal sequence of Ch495 was APREKINLLYFLGYF. HPLC and TLC analysis of Ch495 shows the production of produced N-acetyl d-glucosamine (GlcNAc) as minor and diacetylchitobiose (GlcNAc)₂ as major products. Ch495 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis which can be used for the biological control of fungus. As being simple in purification, extreme alkalophilic, stable in broad range of pH, ability to produce oligosaccharides, and antifungal activity shows that Ch495 has potential applications in industries as for chitooligosaccharides production used as medical prebiotics or/and for the biological control of plant pathogens in agriculture.     

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

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

Purification of xylanase from alkaliphilic Bacillus sp. K-8 by using corn husk column

The objective of this work was to apply low cost materials, agricultural residues, to the purification of xylanase. The results showed that crude extracellular, cellulase-free xylanase of an alkaliphilic Bacillus sp. strain K-8 could be purified in a single step by affinity adsorption–desorption on a corn husk column using a high flow rate, under the conditions 25 mM acetate buffer, pH 4.0, 4 °C, which prevented the hydrolysis of xylan by xylanase. After adsorption, the xylanase was eluted from the enzyme–corn husk complex with 500 mM Urea. The enzyme was purified 5.3-fold to homogeneity from culture supernatant. The molecular weight of the purified enzyme was 24 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity and recovery yield after purification were 25.4 U/mg protein and 42.3%, respectively.     

Cloning and characterization of a xylanase,KRICT PX1 from the strain Paenibacillus sp. HPL-001

The KRICT PX1 gene (GB: FJ380951) consisting of 996 bp encoding a protein of 332 amino acids (38.1 kDa) from the recently isolated Paenibacillus sp. strain HPL-001 (KCTC11365BP) has been cloned and expressed in Escherichia coli. The xylanase KRICT PX1 showed high activity on birchwood xylan, and was active over a pH range of 5.0 to 11.0, with two optima at pH 5.5 and 9.5 at 50 °C with K_m value of 5.35 and 3.23, respectively. The xylanase activity was not affected by most salts, such as NaCl, LiCl, KCl, NH₄Cl, CaCl₂, MgCl₂, MnCl₂, and CsCl₂ at 1 mM, but affected by CuSO₄, ZnSO₄, and FeCl₃. One mM of EDTA, 2-mercaptoethanol, and PMSF did not affect the xylanase activity. TLC analysis of the catalyzed products after reaction with birchwood xylan revealed that xylobiose was the major product with smaller amounts of xylotriose and xylose. A similarity analysis of the amino acids in KRICT PX1 resulted 72% identity with xylanase from Geobacillus stearothermophilus (GB: ZP_03040360), 70% identity with intracellular xylanase from an uncultured bacterium (GB: AAP51133), 68% identity with endo-1-4-xylanse from Paenibacillus sp. (GB: ZP_02847150). In addition, the amino acid alignment of KRICT PX1 with glycosyl hydralase (GH) family 10 xylanases revealed a high degree of homology in highly conserved regions including the catalytic sites, and this was confirmed through PROSITE scan. These results imply that KRICT PX1 is a new xylanase gene, and this alkaline xylanase belongs to GH family 10.     

Purification and characterization of an alkaline keratinase from Streptomyces sp

A protease producing bacterial culture ('S7') was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Streptomyces sp. on the basis of biochemical properties and 16S rRNA gene sequencing. Purification of the protease present in the culture medium supernatant on sephacryl S-100 indicated that it contains a keratinase with 67% recovery, 2.5-fold purification and an estimated molecular mass of approximately 44,000 Da. Keratinase showed an optimal activity at 45 degrees C and pH 11. Keratinase activity increased substantially in presence of Ca(2+) and was inhibited in presence of PMSF and EDTA identifying it as a serine metalloprotease. Stability in the presence of detergents, surfactants and solvents make this keratinase extremely useful for biotechnological process involving keratin hydrolysis or in the leather industry.