Cloning,Sequencing, and Expression of the Gene Encoding an Intracellular β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50°C.     

Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity.

The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.     

Cloning and expression of an alpha-L-arabinofuranosidase gene (stxIV) from Streptomyces thermoviolaceus OPC-520, and characterization of the enzyme

The gene encoding alpha-L-arabinofuranosidase (STX-IV), located upstream of the previously reported stxI gene, was cloned and sequenced. The gene is divergently transcribed from the stxI gene, and the two genes are separated by 661 nucleotides. The stxIV gene consists of a 1,092-bp open reading frame encoding 363 amino acids. The deduced amino acid sequence of the gene showed that STX-IV was an enzyme consisting of only a catalytic domain, and that the enzyme had significant similarity with alpha-L-arabinofuranosidases belonging to family 62 of glycosyl hydrolases. The stxIV gene was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Arabinoxylan and oat spelt xylan were good substrates for STX-IV, however, the enzyme showed a low activity with p-nitrophenyl alpha-L-arabinofuranoside. The optimum pH and temperature were 5.0 and 60 degrees C, respectively.     

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.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Gene cloning and characterization of alpha-glucuronidase of Bacillus stearothermophilus no. 236

The alpha-glucuronidase gene of Bacillus stearothermophilus No. 236 was cloned, sequenced, and expressed in Escherichia coli. The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34. The alpha-glucuronidase produced by a recombinant E. coli strain containing the aguA gene was purified to apparent homogeneity and characterized. The molecular weight of the alpha-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the alpha-glucuronidase therefore was dimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and 40 degrees C, respectively. The enzyme's half-life at 50 degrees C was 50 min. The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucopyranosyluronic)-D-xylobiose]. The alpha-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan. Nevertheless, synergism in the release of xylose from xylan was found when alpha-glucuronidase was added to a mixture of endoxylanase and beta-xylosidase.     

Sequencing and expression of the gene encoding the Clostridium stercorarium beta-xylosidase Xyl43B in Escherichia coli

The Clostridium stercorarium F-9 xyl43B gene encoding the beta-xylosidase Xyl43B consists of an open reading frame of 1,491 nucleotides that encodes a putative protein, classified in family 43, of 497 amino acids with a predicted molecular weight of 56,355. The deduced amino acid sequence of Xyl43B has sequence similarity with beta-xylosidases from Bacteriodes thetaiotaomicron (57% sequence identity), Prevotella ruminicola (45%), Streptomyces coelicolor (40%), and Clostridium acetobutylicum (36%), all of which have been classified in family 43 of the glycoside hydrolases. Xyl43B was purified from a recombinant Escherichia coli and characterized. The optimum pH of the purified enzyme was 3.5 and it was stable over pH from 3.0 to 8.0. Its optimum temperature was 80 degrees C and it showed thermostability in the temperature range from 50 to 70 degrees C. Xyl43B had a K(m) of 6.2 mM and a V(max) of 15 micromol min(-1) mg(-1) for p-nitrophenyl-beta-D-xylopyranoside.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme.     

Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. k11

The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437 bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-beta-xylanase from Streptomyces coelicolor A3, which belongs to 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 homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and 60 degrees , respectively. The enzyme showed stability over a pH range of 4-10. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by Fe2+ and strongly inhibited by Hg2+ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.     

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80 degrees C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80 degrees C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80 degrees C with a half-life of 4 h at 90 degrees C and 1.5 h at 100 degrees C.     

Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli

The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.     

Cloning,analysis, and expression of the gene for inorganic pyrophosphatase of Aquifex pyrophilus and properties of the enzyme

The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced. The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to Aquifex aeolicus (Aae) pyrophosphatase. The gene exhibits a difference in the codon usage at the third position from Aae pyrophosphatase. The gene was expressed under the control of a tac promoter in E. coli. The recombinant Apy pyrophosphatase was purified 18.7-fold with a 52.8% yield and a specific activity of 26.2 U mg(-1) protein. The native enzyme has a homotetramer of 177 amino acids. The enzyme shows optimal activity in pH 7.5. The optimum temperature was approximately 70 degrees C. A divalent cation was absolutely required for the enzyme activity; Mg2+ was the most effective.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.     

Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium

The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.