Purification and properties of a xylanase from Streptomyces lividans.

An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).     

Purification and characterization of a novel extracellular Streptomyces lividans 66 enzyme inactivating fusidic acid.

The wild-type strain Streptomyces lividans 66 is resistant against the steroid-like antibiotic fusidic acid. Comparative studies of the wild-type strain and a fusidic acid-sensitive mutant allowed the identification of an extracellular enzyme which inactivates fusidic acid. With the help of a combination of ultrafiltration and chromatographies with Phenyl-Sepharose and an anion exchanger, the enzyme was highly purified. Its apparent molecular mass is 48 kDa, its optimal activity ranges between 45 and 55 degrees C, and its optimal pH is 6.0 to 9.0. It is stimulated by neither monovalent nor divalent ions. The enzyme acts as a specific esterase which removes the acetyl group at C-16 from fusidic acid. The resulting intermediate is unstable, and spontaneous lactonization between C-21 and C-16 occurs rapidly.     

Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans

Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.     

Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 microM for parathion, 46 microM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 microM for methyl parathion, and 357 microM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45 degrees C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CuSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CuSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.     

Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 microM for parathion, 46 microM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 microM for methyl parathion, and 357 microM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45 degrees C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CuSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CuSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.     

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.     

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.     

Purification and characterization of chitinase produced by Streptomyces erythraeus

A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.     

Cloning of the xylanase gene of Streptomyces lividans

The xylanase (xln) gene of Streptomyces lividans 1326 was cloned by functional complementation of the xylanase-negative and beta-1,4-glucan-glucanohydrolase-negative double mutant of S. lividans using the multicopy plasmid pIJ702. Three clones had a common 2-kb DNA fragment as determined by restriction mapping and Southern hybridization. These clones secreted a xylanase of Mr 43,000 which reacted with specific anti-xylanase antibodies and corresponded exactly to the enzyme previously isolated from the wild-type strain. The DNA fragment likely carried the full structural gene, the xln promoter and also the regulatory sequence, since the xylanase activity was inducible by xylan. Enzyme levels of up to 380 IU/ml of culture filtrate were obtained.     

Characterization of cellulase and xylanase activities of Streptomyces lividans

Summary The production of cellulases and of xylanase by Streptomyces lividans 1326 was studied under different growth conditions. The strain grew between 18°C and 46°C and is therefore thermotolerant. Submerged cultures of the microorganism, when grown on a defined salt medium containing xylan as main carbon source, exhibited an overall cellulolytic activity as determined by the filter paper test. S. lividans produced optimal levels of extracellular -1,4-glucan-glucanohydrolase (1 IU/ml) and large amounts of -1,4-xylanxylanohydrolase (50 IU/ml) at 40°C. A better production of both enzymes was observed when xylan instead of cellulose was used as substrate.The stability of the enzyme was found to be significantly greater than those of the cellulases and xylanases produced by other streptomycetes. The optimal incubation temperatures for the enzyme assays were 55°C and 60°C for CM-cellulase and xylanase respectively and optimal pH values were found in the range of pH 6–7.     

Purification and characterization of the Streptomyces lividans initiator protein DnaA.

The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as a His-tagged fusion protein. The purified protein (isoelectric point, 5.7) has a weak ATPase activity. By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity. Whether the acidic domain II interacts with accessory proteins remains to be elucidated.     

Purification and characterization of lysozyme produced by Streptomyces erythraeus.

A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcbeta(1 leads to 4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.     

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

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

Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32

Streptomyces cyaneus SN32 was used in this study to produce extracellular xylanase, an important industrial enzyme used in pulp and paper industry. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by anion exchange chromatography using DEAE-Sepharose column, with 43.0% yield. The enzyme was found to be a monomer of 20.5 kDa as determined by SDS gel electrophoresis and has a pI of 8.5. The optimum pH and temperature for purified xylanase activity was 6.0 and 60-65 degrees C, respectively. The half-lives of xylanase at 50 and 65 degrees C were approximately 200 and 50 min, respectively. The xylanase exhibited K(m) and V(max) values of 11.1 mg/ml and 45.45 micromol/min/mg. The 15 residue N-terminal sequence of the enzyme was found to be 87% identical up to that of endoxylanases from Steptomyces sp. Based on the zymogram analysis, sequence similarity and other characteristics, it is proposed that the purified enzyme from S. cyaneus SN32 is an endoxylanase and belongs to Group 1 xylanases (low molecular weight - basic proteins). The purified enzyme was stable for more than 20 week at 4 degrees C. Easy purification from the fermentation broth and its high stability will be highly useful for industrial application of this endoxylanase.     

Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage HAU3 resistance (HAU3^r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

DNA deletions in spontaneous chloramphenicol-sensitive mutants of Streptomyces coelicolor A 3(2) and Streptomyces lividans 66

Summary A mutant of Streptomyces coelicolor A3(2) highly resistant to chloramphenicol was selected. It had amplified some chromosomal DNA fragments to a copy number of 20–50. Some of the amplified fragments were cloned and used as hybridisation probes to investigate the spontaneous chloramphenicol-sensitive mutants which occur at high frequency in this species and the closely related species Streptomyces lividans 66. These investigations demonstrated that chloramphenicol sensitivity in both species is associated with large deletions that are at least 40 kb in length.