Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis.

The xynA structural gene from the extremely thermophilic anaerobe Dictyoglomus thermophilum Rt46B.1 was fused in frame with the secretion signal of the Kluyveromyces lactis killer toxin in episomal expression vectors based on the Kluyveromyces plasmid pKD1. XynA was secreted predominantly as an unglycosylated 35-kDa protein which comprised up to 90% of the total extracellular proteins and reached a concentration of 130 micrograms/ml in shake-flask cultures grown under selective conditions.     

极端耐热木聚糖酶基因在枯草芽孢杆菌中的整合表达

目的:为了在枯草芽孢杆菌中整合表达极端耐热木聚糖酶。方法:将嗜热网球菌(Dictyoglomus thermophilum)Rt46B.1的极端耐热木聚糖酶基因xynB通过穿梭载体pDL整合到B.subtilis168染色体上,使其实现表达。结果:极端耐热木聚糖基因在枯草芽孢杆菌中成功整合并表达。结论:基因工程菌B.subtilis168-xynB能外泌表达极端耐热木聚糖酶,且表达水平为0.732IU/mL,比在大肠杆菌中的高。酶学性质表明,此酶分子量约为24kD,其最适反应温度为85℃,最适反应pH值为6.5,且在弱碱性条件下稳定。     

Cloning of the xynB Gene from Dictyoglomus thermophilum Rt46B.1 and Action of the Gene Product on Kraft Pulp

A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from Dictyoglomus thermophilum Rt46B.1. Family 11 xylanase consensus fragments (GXCFs) were amplified from Rt46B.1 genomic DNA by using different sets of consensus PCR primers that exhibited broad specificity for conserved motifs within fungal and/or bacterial family 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (xynB) was identified. The entire gene sequence was obtained in the second step by using genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstream and downstream of the xynB GXCF region. The putative XynB peptide (M_r, 39,800) encoded by the Rt46B.1 xynB open reading frame was a multidomain enzyme comprising an N-terminal catalytic domain (M_r, 22,000) and a possible C-terminal substrate-binding domain (M_r, 13,000) that were separated by a short serine-glycine-rich 23-amino-acid linker peptide. Seven xylanases which differed at their N and C termini were produced from different xynB expression plasmids. All seven xylanases exhibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85°C. Pretreatment of Pinus radiata and eucalypt kraft-oxygen pulps with XynB resulted in moderate xylan solubilization and a substantial improvement in the bleachability of these pulps.     

Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. strain Rt69B.1

Three family 10 xylanase genes (xynA, xynB, and xynC) and a single family 11 xylanase gene (xynD) were identified from the extreme thermophile Caldicellulosiruptor strain Rt69B.1 through the use of consensus PCR in conjunction with sequencing and polyacrylamide gel electrophoresis. These genes appear to comprise the complete endoxylanase system of Rt69B.1. The xynA gene was found to be homologous to the xynA gene of the closely related Caldicellulosiruptor strain Rt8B.4, and primers designed previously to amplify the Rt8B.4 xynA gene could amplify homologous full-length xynA gene fragments from Rt69B.1. The complete nucleotide sequences of the Rt69B.1 xynB, xynC, and xynD genes were obtained using genomic walking PCR. The full-length xynB and xynC genes are more than 5 kb in length and encode highly modular enzymes that are the largest xylanases reported to date. XynB has an architecture similar to the family 10 xylanases from Thermoanaerobacterium saccharolyticum (XynA) and Clostridium thermocellum (XynX) and may be cell wall associated, while XynC is a bifunctional enzyme with an architecture similar to the bifunctional β-glycanases from Caldicellulosiruptor saccharolyticus. The xynD gene encodes a two-domain family 11 xylanase that is identical in architecture to the XynB family 11 xylanase from the unrelated extreme thermophile Dictyoglomus thermophilum strain Rt46B.1. The sequence similarities between the Rt69B.1 xylanases with respect to their evolution are discussed. Received: May 13, 1998 / Accepted: October 22, 1998     

Purification, characterization, and molecular cloning of acidophilic xylanase from penicillium sp.40

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family xylanases from fungi.     

Purification,Characterization, and Molecular Cloning of Acidophilic Xylanase from Penicillium sp.40

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family11 xylanases from fungi.     

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 characterization of two xylanases from Trichoderma longibrachiatum.

Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl-cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while xylobiose appeared to inhibit xylanase B, but not xylanase A.     

Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23.

Xylanase A from the recently isolated Bacillus sp. strain BP-23 was purified to homogeneity. The enzyme shows a molecular mass of 32 kDa and an isoelectric point of 9.3. Optimum temperature and pH for xylanase activity were 50 degrees C and 5.5 respectively. Xylanase A was completely inhibited by N-bromosuccinimide. The main products of birchwood xylan hydrolysis were xylotetraose and xylobiose. The enzyme was shown to facilitate chemical bleaching of pulp, generating savings of 38% in terms of chlorine dioxide consumption. The amino-terminal sequence of xylanase A has a conserved sequence of five amino acids found in xylanases from family F.     

Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in lambda EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5.5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and beta-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.     

Two Extremely Thermostable Xylanases of the Hyperthermophilic Bacterium Thermotoga maritima MSB8

During growth with xylose or xylan as the source of carbon, xylanase production by Thermotoga maritima MSB8 was enhanced about 10-fold compared with growth with glucose or starch. Two extremely thermostable endoxylanases (1,4-(beta)-d-xylan-xylanohydrolase, EC 3.2.1.8), designated XynA and XynB, were identified and purified from cells of this organism. XynA and XynB occurred as proteins with apparent molecular masses of about 120 and 40 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum activity at the optimal pH (pH 6.2 and pH 5.4 for XynA and XynB, respectively) was measured at about 92(deg)C for XynA (10-min assay) and at about 105(deg)C for XynB (5-min assay). XynB activity was stimulated twofold by the addition of 500 mM NaCl, while XynA displayed maximum activity without the addition of salt. Both xylanases were tolerant of relatively high salt concentrations. At 2 M (about 12% wt/vol) NaCl, XynA and XynB retained 49 and 65%, respectively, of their maximum activities. In contrast to XynB, XynA was able to adsorb to microcrystalline cellulose. Antibodies raised against a recombinant truncated XynA protein cross-reacted with XynB, indicating that the enzymes may have sequence or structural similarities. Part of the xylanase activity appeared to be associated with the outer membrane of T. maritima cells, since more than 40% of the total xylanase activity present in the crude cellular extract was found in the membrane fraction after high-speed centrifugation. Most of the membrane-bound activity appeared to be due to the 120-kDa xylanase XynA.     

Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.     

Xylanase and acetyl xylan esterase activities of XynA,a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation

The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.     

Xylanase IV, an Exoxylanase of Aeromonas caviae ME-1 Which Produces Xylotetraose as the Only Low-Molecular-Weight Oligosaccharide from Xylan

A novel xylanase (xylanase IV) which produces xylotetraose as the only low-molecular-weight oligosaccharide from oat spelt xylan was isolated from the culture medium of Aeromonas caviae ME-1. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the xylanase IV molecular weight was 41,000. Xylanase IV catalyzed the hydrolysis of oat spelt xylan, producing exclusively xylotetraose. The acid hydrolysate of the product gave d-xylose. The enzyme did not hydrolyze either p-nitrophenyl-(beta)-d-xyloside, small oligosaccharides (xylobiose and xylotetraose), or polysaccharides, such as starch, cellulose, carboxymethyl cellulose, laminarin, and (beta)-1,3-xylan.     

Cloning of a Bacillus sp. BP-23 gene encoding a xylanase with high activity against aryl xylosides

Abstract The xynB gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-23 has been cloned and expressed in Escherichia coli . The enzyme produced in this host shows a molecular size of 41 kDa and a pI of 4.5. The pH and temperature at which the highest activity was found were 5.5 and 50°C respectively. Crude xylanase B showed activity on xylan, aryl xylosides, xylotetraose and xylotriose, while xylobiose was not hydrolyzed by the enzyme. Xylanase B showed high specific activity on aryl xylosides, probably as a result of the transxylosidase activity detected.     

Fractionation and purification of endo-1,4-beta-xylanases and exo-1,4-beta-xylosidases of Aspergillus niger]

Two endo-1,4-beta-zylanases (m. w. 24,000 and 41,000) and six exo-1,4-beta-xylosidases, differing in their molecular weights and isoelectric points, were found in a xylanase preparation from Aspergillus niger, using different methods of fractionation. An electrophoretically homogeneous exo-1,4-beta-xylosidase (m. w. 30,000) purified 120-fold, with pI 4.6, having optimal effect on methyl-beta-D-xyloside at pH 3.0 was obtained. Exo-1,4-beta-xylosidase splits off xylose from the ends of the xylan chains at xylotriose, xylobiose and methyl-beta-D-xyloside and is characterized by a high transglycosilase activity. An electrophoretically homogeneous endo-1,4-beta-xylanase (m. w. 24,000) purified 250-fold, with pI 4.2 and optimal effect on carboxymethylxylan at pH 4.2 was isolated. Endo-1,4-beta-xylanase splits arabinoglucuronoxylan to form xylooligosaccharides; however, it does not hydrolyze xylobiose.     

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.     

Purification and Characterization of Aeromonas caviae ME-1 Xylanase V, Which Produces Exclusively Xylobiose from Xylan

A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37 degrees C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37 degrees C. When soluble birch xylan was used as the substrate, the enzyme had a K(m) and V(max) of 2 mg/ml and 182 mumol of xylose equivalent liberated . min . mg of protein, respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither beta-xylosidase, alpha-l-arabinofuranosidase, cellulase, nor beta-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.