The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-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 beta-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-beta-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 degrees C.

The chiS and chiR genes located upstream of the chitinase locus (chi40) on the chromosome of Streptomyces thermoviolaceus OPC-520 were cloned and sequenced. The deduced amino acid sequences revealed that ChiS (390 amino acids, 40.9 kDa) and ChiR (213 amino acids, 22 kDa) show significant sequence similarities to histidine kinases and response regulators, respectively, of typical prokaryotic two-component regulatory systems. The extracellular chitinase activity of Streptomyces lividans 66 (pTSR2 (bearing chiS, chiR and chi40)) was significantly enhanced by a high dosage of the chiS and chiR genes.

Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).

We purified and characterized an intracellular beta-N-acetylglucosaminidase (NagC) from a cytoplasmic fraction of Streptomyces thermoviolaceus OPC-520. The molecular mass of NagC was estimated to be 60 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the enzyme were 6.0 and 50 degrees C respectively. Purified NagC hydrolyzed chitin oligosaccharides from N,N'-diacetylchitobiose (GlcNAc)(2) to chitopentaose (GlcNAc)(5), hydrolyzed N,N'-diacetylchitobiose especially rapidly, and showed a tendency to decrease with increases in the degree of polymerization. But, NagC didn't hydrolyze chitohexaose (GlcNAc)(6). The gene encoding NagC was cloned and sequenced. The open reading frame of nagC encoded a protein of 564 amino acids with a calculated molecular mass of 62,076 Da. The deduced amino acid sequence of NagC showed homology with several beta-N-acetylglucosaminidases belonging to glycosyl hydrolase family 20. The expression plasmid coding for NagC was constructed in Escherichia coli. The recombinant enzyme showed pH and temperature optima and substrate specificity similar to those of the native enzyme. The gene arrangement near the nagC gene of S. thermoviolaceus OPC-520 was compared with that of S. coelicolor A3(2). Three genes, which appear to constitute an ABC transport system for sugar, were missing in the vicinity of the nagC gene.

Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an alpha-L-arabinofuranosidase (StxIV) in the presence of xylan. Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular beta-xylosidase (BxlA). A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA). The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA. Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA. The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon. The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides. The protein showed high-level affinity for xylobiose (K(d) = 8.75 x 10(-9) M) and for xylotriose (K(d) = 8.42 x 10(-8) M). Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S. thermoviolaceus membranes. The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins. These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters. In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli. The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators. The purified BxlR protein specifically bound to a 4-bp inverted sequence overlapping the -10 region of the bxl operon. The binding of BxlR to the site was inhibited specifically by low concentrations of xylobiose. This site was also present in the region located between stxI and stxIV and in the upstream region of stxII. BxlR specifically bound to the regions containing the inverted sequence. These results suggest that BxlR might act as a repressor of the genes involved not only in the uptake system of xylan degradation products but also in xylan degradation of S. thermoviolaceus OPC-520.

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 �\-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60�XC and the ability to cleave �\-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl �\-l-arabinofuranoside were also detected. After selective removal of �\-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining �\-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove �\-l-Araf residues that are (1��2) and (1��3) linked to monosubstituted �]-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed �]-propeller fold and revealed a molecular Velcro in blade V between the �]1 and �]21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the �] propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.

A chitinase was purified from the culture filtrate of Streptomyces thermoviolaceus OPC-520. The enzyme showed a high optimum temperature (70 to 80 degrees C), a high optimum pH level (8.0 to 10.0), and heat stability. This enzyme showed high sequence homology with chitinases from Serratia marcescens QMB1466 and Bacillus circulans WL-12.

The gene (chi40) encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 was cloned in Escherichia coli JM109 using pUC18. The nucleotide (nt) sequence of chi40 has been determined. A single open reading frame (ORF) encoded a protein consisting of 414 amino acids (aa) with a M(r) of 43,838. The deduced aa sequence of the cloned chitinase (Chi40) showed striking homology (74%) with Chi63 from Streptomyces plicatus. Comparison with other chitinases revealed that Chi40 contained the two conserved regions common to microbial and plant chitinases. Furthermore, the putative promoter region of chi40, which might be involved in the repression and induction of such catabolite-controlled genes, was detected by the DNA sequence alignments.

The alpha-amylase (Amy)-encoding gene (amy) of Streptomyces thermoviolaceus CUB74, previously cloned in Escherichia coli and S. lividans and localised on a 1.7-kb BamHI-SphI genomic DNA fragment, has been sequenced. A single open reading frame of 1380 bp, which could encode an Amy protein of 460 amino acids (aa), was identified. The deduced aa sequence of the thermophilic Amy is similar (up to 69.5%) to the mesophilic Amy of S. griseus, S. limosus, S. venezuelae and S. hygroscopicus. A 40% sequence similarity was found between the extracellular forms of the S. thermoviolaceus and the pig pancreatic Amy. In addition, the activity of the S. thermoviolaceus Amy is strongly inhibited by tendamistat, a potent inhibitor of mammalian Amy. The nucleotide sequence at the 5' end of amy was able to initiate transcription in S. lividans and contains a promoter whose sequence is identical to the promoters of the S. limosus, S. venezuelae and S. griseus amy.

Previous studies have shown that an Actinomycetes strain produces a novel protein with a molecular weight of 87,000 which stimulated the growth of murine erythroid progenitors in vitro, and we purified the protein. In this study, we have isolated and sequenced a clone encoding a polypeptide which had the equivalent activity. The clone contained an open reading frame of 334 amino acids. The activity was confirmed by the expression in E. coli as a fusion protein with glutathion S-transferase (GST). Based on the differences of molecular weight and N-terminal amino acid sequences, this protein was considered to be different from that previously reported and was named "ESA36".

The nagB gene encoding beta-N-acetylglucosaminidase from S. thermoviolaceus OPC-520 was cloned and sequenced. The nagB gene could encode a protein of 541 amino acids with a calculated molecular mass of 58274. NagB revealed significant similarities to beta-N-acetylhexosaminidases and chitobiases from bacteria, which are classified into family 20 glycosyl hydrolases. NagB effectively hydrolyzed all of the chitin oligosaccharides from dimer to hexamer.

A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66, 329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned beta-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical beta-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable beta-glucosidase activity, revealed homology with microbial beta-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of beta-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60 degreesC and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-beta-N-acetylglucosamine were 425.7 microM and 24.8 micromol min-1 mg of protein-1, respectively.