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openaire: EC/H2020/648925/EU//BHIVE The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.

Streptomyces thermoviolaceus SRC3, a newly isolated actinobacterial strain from Algerian river sediments, exhibited a broad activity against various bacterial and yeast human pathogens ( Salmonella Typhi ATCC 14028, Vibrio cholerae ATCC 14035, MRSA ATCC 43300 and Candida albicans ATCC 10231). The strain SRC3 was selected from thirty nine actinobacterial isolates and identified as S. thermoviolaceus based on morphology, cultural properties, physiological analyses and 16S rRNA gene sequencing. Culture parameters for the antibiotic production were optimized by sequential statistical strategy including Plackett-Burman design (PBD) and Response Surface Methodology (RSM). In PBD experiments, KCl, K 2HPO4, MgSO4·7H2 O, pH value and incubation time emerged as the most significant in affecting the output of antimicrobial activities . These factors were further optimized using Central Composite Design (CCD). The best achieved conditions were: KCl (0.01%), K2HPO4 (0.1%), MgSO4·7H2O (0.02%) and 9 days incubation for anti-S. Typhi compounds, KCl (0.051%), MgSO4·7H2O (0.05%) and 5 days incubation for C. albicans inhibitors. The metabolite responsible for the bioactivities was purified, structurally characterized (by NMR, MS, UV and IR analyses) and identified as streptazolin, recently reported as a promising antibiotic adjuvant.

Enzymes, specifically those with microbial origin, are considered as the potential biocatalysts for many reactions and have extensive uses in industries. Microbes are alternative source of enzymes which can be grown in large quantities in a less time period by fermentation process and are more efficient as well as active. For the fulfillment of the recent necessities of different enzymes, industries are searching for new microbial strains. Lipases and other polymer-hydrolyzing enzymes such as cellulases, chitinases, and amylases are nowadays used for industrial applications. Some microbes are responsible for the production of thermostable enzymes such as Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis reported to produce thermostable amylase. Members of the Bacillus sp., Streptomyces sp., Thermoascus aurantiacus, and Fusarium proliferatum have been responsible to produce xylanases. For chitinases Bacillus licheniformis, Bacillus sp., and Streptomyces thermoviolaceus were known to be the chief sources. Amylases, proteases or lipases have great marketable potential in many textile industries.

Analyzing the structure of proteins from extremophiles is a promising way to study the rules governing the protein structure, because such proteins are results of structural and functional optimization under well-defined conditions. Studying the structure of chitinases addresses an interesting aspect of enzymology, because chitin, while being the world&rsquo, s second most abundant biopolymer, is also a recalcitrant substrate. The crystal structure of a thermostable chitinase from Streptomyces thermoviolaceus (StChi40) has been solved revealing a &beta, /&alpha, barrel (TIM-barrel) fold with an &alpha, +&beta, insertion domain. This is the first chitinase structure of the multi-chitinase system of S. thermoviolaceus. The protein is also known to refold efficiently after thermal or chemical denaturation. StChi40 is structurally close to the catalytic domain of psychrophilic chitinase B from Arthrobacter TAD20. Differences are noted in comparison to the previously examined chitinases, particularly in the substrate-binding cleft. A comparison of the thermophilic enzyme with its psychrophilic homologue revealed structural features that could be attributed to StChi40&rsquo, s thermal stability: compactness of the structure with trimmed surface loops and unique disulfide bridges, one of which is additionally stabilized by S&ndash, &pi, interactions with aromatic rings. Uncharacteristically for thermophilic proteins, StChi40 has fewer salt bridges than its mesophilic and psychrophilic homologues.

Searching for endophytic actinomycetes, strain NT1 was isolated from surface sterilized stem of Catharanthus roseus (L.) G. Don collected from Paschim Medinipur, India. The strain was identified as Streptomyces thermoviolaceus NT1 on the basis of morphological, biochemical and 16s rDNA based phylogenetic analysis. It showed potential antagonism against several Gram positive and Gram negative bacterial pathogens along with drug resistant Staphylococcus aureus. Maximum antibacterial production was obtained in ISP2 media at pH 7.2, 35 °C for 10 days. The active antibacterial substance was purified by Silica gel column chromatography and activity guided TLC. IR and NMR analysis identified the active compound as granaticinic acid of m/z 463.26 [M+H]. These results suggest that the antimicrobial produced by the isolated endophytic strain will be useful in near future.

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α- l -arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l -arabinofuranose ( l -Ara f ) from singly substituted xylopyranosyl (Xyl p ) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α- l -arabinofuranoside were also detected. After selective removal of α-1,3 l -Ara f substituents from disubstituted Xyl p residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l -Ara f substituents, confirming the ability of SthAbf62A to remove α- l -Ara f residues that are (1→2) and (1→3) linked to monosubstituted β- d -Xyl p sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l -arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 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 β 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.

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 A resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins.

Streptomyces thermoviolaceus NT1, an endophytic isolate, was studied for optimization of granaticinic acid production. It is an antimicrobial metabolite active against even drug resistant bacteria. Different media, optimum glucose concentration, initial media pH, incubation temperature, incubation period, and inoculum size were among the selected parameters optimized in the one-variable-at-a-time (OVAT) approach, where glucose concentration, pH, and temperature were found to play a critical role in antibiotic production by this strain. Finally, the Box-Behnken experimental design (BBD) was employed with three key factors (selected after OVAT studies) for response surface methodological (RSM) analysis of this optimization study.RSM analysis revealed a multifactorial combination; glucose 0.38%, pH 7.02, and temperature 36.53 °C as the optimum conditions for maximum antimicrobial yield. Experimental verification of model analysis led to 3.30-fold (61.35 mg/L as compared to 18.64 mg/L produced in un-optimized condition) enhanced granaticinic acid production in ISP2 medium with 5% inoculum and a suitable incubation period of 10 days. So, the conjugated optimization study for maximum antibiotic production from Streptomyces thermoviolaceus NT1 was found to result in significantly higher yield, which might be exploited in industrial applications.

An α-l-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3–5 (37–80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-l-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu188) and base (Asp28) catalysts, and the general acid pK a modulator (Asp136) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp23 and Tyr44, situated about 30 A from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4–7-folds, but lost 77–96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp23 is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp23 and Tyr44 belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.

Anionic polymers of the cell surface of a thermophilic streptomycete were investigated. The cell wall of Streptomyces thermoviolaceus subsp. thermoviolaceus VKM Ac-1857(T) was found to contain polymers with different structure: teichoic acid--1,3-poly(glycerol phosphate), disaccharide-1-phosphate polymer with repeating unit -6)-alpha-Galp-(1--6)-alpha-GlcpNAc-P-, and polysaccharide without phosphate with repeating unit --6)-alpha-GalpNAc-(1--3)-beta-GalpNAc-(1--. Disaccharide-1-phosphate and polysaccharide without phosphate have not been described earlier in prokaryotic cell walls.

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.

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.

A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described. Chi40 was overproduced in the cytosolic and secreted forms. The cytosolic form (Chi40c) was highly overproduced and purified by metal-affinity and ion-exchange chromatography in large amounts. The protein was highly active and thermostable but not homogeneous, since a considerable proportion of the Chi40c protein was not correctly folded as determined by native polyacrylamide gel electrophoresis. The Chi40 protein secreted into the culture medium (Chi40s) was purified by hydrophobic interaction and ion-exchange chromatography and high amounts of correctly folded and active Chi40 protein could be recovered in a short time. The enzymatic activity of Chi40s on a synthetic and on its natural substrate, chitin, was studied. Thermostability measurements showed that Chi40 has a T m of 60.7°C at neutral pH. 13 C- 15 N double-labeled recombinant Chi40s was also produced and purified from the pECHChi40-9 construct introduced into BL21 trx B(DE3) cells grown in minimal medium in the presence of the paramagnetic elements [ 13 C]glucose and 15 NH 4 Cl. The presented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectroscopy.

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.

Keratinolytic activity was detected in the culture broth of feather-degrading thermophilic Streptomyces thermoviolaceus SD8. The crude enzyme was concentrated by precipitation with 80% saturation of ammonium sulphate and desalted by SephadexG-10–120 gel chromatography followed by lyophilization. The specific activity of the enzyme was enhanced 50-fold. PAGE analysis indicated a monomeric form with a molecular weight of 40 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 °C, respectively. The enzyme was stable at a pH range of 6·5–8·5 and up to 65 °C. The enzyme could hydrolyse fibrin, muscle, collagen, nail and hair and could produce leucine, threonine and tyrosine from feather. It could be a useful enzyme for waste treatment by promoting hydrolysis of the above substances in the sewage, or it could be used for animal feed preparation.

Together with the integral membrane proteins BxlF and BxlG, BxlE isolated from Streptomyces thermoviolaceus OPC-520 forms an ATP-binding cassette (ABC) transport system that mediates the uptake of xylan. To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 44.63, b = 63.27, c = 66.40 A, beta = 103.05 degrees, and contained one 48 kDa molecule per asymmetric unit (V(M) = 1.96 A3 Da(-1)). Diffraction data collected to a resolution of 1.65 A using a rotating-anode X-ray source gave a data set with an overall R(merge) of 2.6% and a completeness of 91.3%. A data set from a platinum derivative is being used for phasing by the SAD method.

Streptomyces thermoviolaceus extracellular xylanase preparations were used to treat birchwood Kraft pulp prior to chlorine bleaching. Xylanase concentrations in the range 5–100 IU g −1 dry weight pulp released reducing sugars and chromophores that absorb at 280 nm. At enzyme doses ≥10 IU, further removal of chromophoric material was not significant although reducing sugar yields probably from oligosaccharide hydrolysis continued to increase. Pulp Kappa number was reduced by enzyme treatment with and without subsequent alkali extraction, and the pulp brightness after CEDED bleaching at 4% chlorine charge was boosted without significant reductions in fiber strength or interfiber bonding. Scanning electron microscopy revealed marked disruption and separation of pulp fibers even at low (5 IU g −1 dry weight pulp) xylanase doses, and a 30–35% saving in the chlorine charge required to obtain pulp brightness comparable to controls could be achieved. Comparative and standardized treatments of pulp with the commercial bleach-boosting enzyme preparations Cartazyme and Pulpzyme demonstrated that the crude S. thermoviolaceus xylanase preparation was at least as effective in enhancing brightness with concomitant preservation of paper strength properties; however, S. thermoviolaceus xylanase does have the advantage of being active and stable at 65°C and, like Pulpzyme but not Cartazyme, at neutral to alkaline pH values appropriate for applications in paper pulp processing.

Numerous recombinant proteins of industrial and pharmaceutical importance are secreted to the periplasmic space of Escherichia coli; yet, very few generic periplasmic recovery methods with the potential for scaleup exist. This work describes the development of an enzymatic method for the release of recombinant proteins from the periplasm of E. coli. The simple two-step method involving resuspension of the cells in a fractionation buffer, followed by recovery of the periplasmic fraction, has been shown to be feasible for use in both the laboratory and 5-l scale. For the efficient release of a recombinant Streptomyces thermoviolaceus α-amylase from the E. coli periplasm, it has been shown that an osmotic shock must immediately follow lysozyme treatment to obtain high yields. The phase of growth and level of recombinant protein expression has an affect on the recovery from the E. coli periplasm. Yields of 70–90% of recombinant α-amylase were recovered from the periplasm of stationary phase E. coli cells in both laboratory and 5-l scale experiments.

Streptomyces thermoviolaceus was grown in a glutamate salts medium and the applicability of DNA and a number of intracellular dehydrogenases as indicators for growth in a particulate rapemeal salts medium were assessed. Only NADH dehydrogenase proved unsuitable but analysis of the rates of increase of activities of this enzyme during batch culture suggested that energy metabolism was an important factor in defining the onset of secondary metabolism and as a causal element of the biphasic pattern of growth exhibited by this streptomycete. Growth, substrate utilization and production of extracellular products could readily be analysed in the particulate rapemeal salts medium. Free sugars and carbohydrates were utilized predominantly in early exponential phase but thereafter the rapemeal proteins were metabolized as observed by rises in protease activities and levels of ammoniacal nitrogen. Growth in rapemeal-derived media produced titres of the antibiotic granaticin which were at least as high (usually higher) as most of the growth substrates tested suggesting that rapemeal may have considerable potential for the exploitation of natural products. Particulate rapemeal medium also resulted in high yields of commercially important extracellular enzymes.

The biobleaching effect of cellulase-free thermophilic xylanase preparations from Streptomyces thermoviolaceus on birchwood kraft pulp was determined. Enzyme pretreatment was found to be most affective at high pulp consistencies and resulted in the hydrolysis of hemicellulose and the release of chromophoric material from the pulp. Removal of residual lignin was indicated by a reduction in Kappa number. The application of cyclic enzyme treatments coupled with intermediate alkaline extraction stages increased the biobleaching effect; however, more than two cycles resulted in a decrease in the physical strength of the paper. The enzyme treatment process was optimized and found to be most effective at 65°C and pH 6.0, with the crude enzyme preparation blended to a pulp consistency of 5%. A 3 h enzyme pretreatment of pulp followed by single-stage peroxide bleaching yielded pulp with a brightness of >70 ISO units for the production of paper handsheets in which the physical strength of the fibers had been preserved.

A thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus isolate 76T-2 that can degrade poly(ε-caprolactone) (PCL) was isolated from soil in Taiwan. Isolate 76T-2 grew well in urea fructose oatmeal medium and exhibited clear zones on agar plates containing PCL, indicating the presence of extracellular PCL depolymerases. The PCL powder present in culture medium was completely degraded within 6 h of culture at 45°C. Two PCL-degrading enzymes were purified to homogeneity from the culture supernatant. The molecular weights of these two enzymes were estimated to be 25 kDa and 55 kDa, respectively. A portion of the N-terminal region of the 25-kDa protein was determined, and the sequence Ala-Asn-Phe-Val-Val-Ser-Glu-Ala thus obtained was identical to that of A64-A71 of the Chi25 chitinase of Streptomyces thermoviolaceus OPC-520. The 25-kDa protein was shown to also degrade chitin, suggesting that isolate 76T-2 has the ability to degrade both PCL and chitin.

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".

Streptomyces thermoviolaceus is a thermophilic actinomycete that was found to produce relatively large amounts of extracellular peroxidase activity when grown on xylan as primary carbon source. The activity was due to multiple isoforms of peroxidase, of which two, designated P-3 and P-5, were predominant. The two proteins were purified to homogeneity by a combination of ultrafiltration, ammonium sulphate precipitation, anion-exchange chromatography, gel filtration and preparative gel electrophoresis. The peroxidases were found to be haemoproteins that catalysed the oxidation of a range of substrates in the presence of hydrogen peroxide. Both are monomeric acidic proteins (P-3: 82 kDa, pl 5.0; P-5: 60 kDa, pl 4.75) but with some differences in substrate specificity, P-3 exhibiting the broader substrate range. Peroxidase activity was optimal at pH values close to neutrality, and both enzymes were robust, exhibiting activity at elevated temperatures in the presence of denaturing agents such as SDS or 8 M urea. Peroxidase P-3 was stable at 50° for more than 24 h and had a half-life of 70 min at 70°. Polyclonal antibodies prepared against each isoform cross-reacted, indicating that the proteins were antigenically related. No cross-reactions were detected against horseradish peroxidase or crude peroxidase preparations from two other thermophilic streptomycetes.

A pH-stable and protease-resistant xylanase (XynB119) was identified from Streptomyces sp. TN119, a strain isolated from the gut luminal contents of longhorned beetle (Batocera horsfieldi) larvae. Using the GC TAIL-PCR method, the 1,026-bp coding gene (xynB119) with 67.3% GC content was successfully cloned and expressed in Escherichia coli. It encodes a 341-residue polypeptide with a calculated molecular mass of 35.9 kDa, including a putative 41-residue signal peptide, a catalytic domain of glycosyl hydrolase (GH) family 11, a short Gly/Pro-rich linker, and a family 2 cellulose-binding domain (CBM 2). The deduced amino acid sequence is most similar to (61.9% identity) an endo-1,4-β-xylanase from Streptomyces thermoviolaceus OPC-520. Purified recombinant XynB119 exhibited peak activity at 50°C and pH 7.0, remained stable over a broad pH range (retaining >70% activity after incubation at pH 1.0–11.0 for 1 h at 37°C without substrate), had strong protease resistance (retaining >90% activity after proteolytic treatment at 37°C for 1 h) and SDS resistance (at 100 mM). These properties make XynB119 promising for application in the feed industry and valuable for basic research. Compared to r-XynB119, the r-XynB119 derivative without CBM 2 and linker region (r-XynB119d) exhibited a decreased pH stability of >25% at extreme pHs (pH 1.0–3.0 and pH 11.0–12.0).

Production of secondary metabolites was investigated in the thermophilic streptomyceteStreptomyces thermoviolaceus grown at 45°C in a fermenter. Extracellular protein was secreted into the culture medium at the same time as an antibiotic granaticin; both were synthesized during the second slower phase of biphasic growth, which is most apparent at 45°C for this organism. Protease, assayed as azocaseinase, was identified as one component of, and marker for, the excreted protein. The effects of different growth temperatures revealed that the synthesis of extracellular protein, like that of the antibiotic, was maximal in cultures grown between 37° and 45°C, whereas protease activity was greatest in 50°C grown cultures. A method was devised, based on acetone precipitation, for concentrating the protease activity from culture supernatants. Characterization of the concentrated activity using inhibitors suggested the presence of a serine and a metallo-type protease. A peptide substrate specific for the metallo-protease showed that it had a pH optimum for activity of 6.5–7.0. Approximately 50% of the activity was lost after 80 min of incubation at 70°C. Although calcium (5 mM) promoted increased thermotolerance such that around 65% of the activity remained after 100 min at 70°C, it seems that manganese and/or zinc may be more important for enzyme activity.

Goldberg, J.D. & Edwards, C. 1990. Purification and characterization of an extracellular amylase from a thermophilic streptomycete. Journal of Applied Bacteriology69, 712–717. A single extracellular alpha-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) from Streptomyces thermoviolaceus subsp. apingens was purified to homogeneity by a starch adsorption method. SDS-PAGE indicated that the enzyme had an apparent M, of 57 kDa and activity was optimal at a pH of 7–2 and a temperature of 55dC. It employed an endo-active mechanism to liberate predominantly maltose, as well as smaller amounts of higher oligosaccharides when incubated with starch. EDTA inhibited enzyme activity, suggesting an involvement of a divalent cation in activity. The enzyme was also stabilized by divalent cations when heated and the results suggested a major role for Ca2+ ions for both activity and thermostability. The alpha-amylase from S. thermoviolaceus displayed some similarities with commercially-used streptomycete alpha-amylases.

Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an α- 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 β-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 × 10 −9 M) and for xylotriose ( K d = 8.42 × 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.

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70 degrees C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.

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.

We have examined induction and repression by various sugars and carbon sources of the synthesis of a thermostable α-amylase in its natural host, S thermoviolaceus CUB74. The smallest molecule capable of inducing synthesis of the enzyme was maltotriose whereas maltose had no effect which might suggest a different control system from that found in other streptomycete amylases. Addition of mannitol to the growth medium impeded the α-amylase induction whereas glucose had no effect. After cloning of its gene into a new streptomycete host, S lividans TK24, the S thermoviolaceus α-amylase could not be induced by any of the sugars tested.

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.

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).

An expression plasmid for a thermostable chitinase gene from S. thermoviolaceus OPC-520 in E. coli was constructed. A cloned chitinase (Chi40) was purified from the periplasmic space of E. coli harboring the expression plasmid. The N-terminal sequence of Chi40 was 11 amino acids longer than that of chitinase from S. thermovilaceus OPC-520 (ST chitinase), however, a loss or addition of the amino acid residues did not affect the enzymatic properties. The mutations of Asp-145 and Glu-147 drastically decreased the specific activity of chitinase from the wild type, indicating that both amino acid residues are the best candidates for the essential catalytic residues of Chi40.

The article entitled "Novel Biocatalysts Will Work Even Better for Industry" published recently in this Journal (1) was informative and interesting. However it touched only briefly on the application of peroxidase as catalyst. Here, we would like to mention in more detail the novel applications of peroxidase in agricultural, paper pulp, water treatment, pharmaceutical, and medical situations. Firstly, the peroxidase isolated from Phanerochaete chyrosporium has been shown to detoxify herbicides such as atrazine to less toxic compounds and would certainly find potential application in agriculture (2). Secondly, the peroxidase produced by Streptomyces thermoviolaceus may find application in the paper pulp industry as a delignifying agent (3). Thirdly, it has been shown that extracellular peroxidase produced by Streptomyces avermitilis can remove the intense color from paper-mill effluent obtained after semichemical alkaline pulping of wheat straw (4), and thus this enzyme might find application as a catalyst...

A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.