Phosphorylation of Streptomycin by an Intracellular Enzyme of Streptomyces griseus

A zinc-binding protein was purified to homogeneity for the first time from the gonad parts of scallops, Patinopecten yessoensis, kept in filtered seawater to which no heavy metals were added. Based upon the elution profiles in two chromatographic systems, spectrophotometric analysis, and amino acid composition of the purified preparation, the protein met the criteria for classification as a metallothionein; i.e., low molecular weight (about 9000), paucity of both aromatic amino acid residues and absorbance at 280 nm, and abundance of both cysteinyl residues (> 25%) and absorbance at 215 and 254 nm. Furthermore, the results of chromatographies on a Sephacryl S-300 column and electrophoresis with or without SDS suggested that the protein molecules would be in several polymeric forms in vivo. The antiserum prepared with the purified protein as the antigen was shown to have immunocross-reactivity to neither an extract of the surf clam, Pseudocardium sybillae, nor the whelk, Neptunea arthritica, indicating the heterogeneity of the proteins in marine shellfishes. These results suggested that the Zn-binding protein purified in this study was characteristic of scallops and involved in zinc storage of this organism.     

L-Asparaginase Production by Streptomyces griseus

Streptomyces griseus ATCC 10137 synthesizes about 1 IU of L-asparaginase/100 ml of a 4% peptone medium. The enzyme has a pH optimum of 8.5 which is comparable to that of the L-asparaginase derived from Escherichia coli which has antitumor properties.     

The Mitochondrial Toxin Produced by Streptomyces griseus Strains Isolated from an Indoor Environment Is Valinomycin

Actinomycete isolates from indoor air and dust in water-damaged schools and children’s day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC₅₀) were 10 to 63 ng (dry weight) ml of extended boar semen⁻¹. The four strains were identified as Streptomyces griseus strains by 16S ribosomal DNA and chemotaxonomic methods. The four S. griseus strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of ≥5,000 to 72,000 ng ml⁻¹. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of S. griseus. The EC₅₀ of purified toxin from the S. griseus strains were 1 to 3 ng ml of extended boar semen⁻¹, and the EC₅₀ of commercial valinomycin was 2 ng ml of extended boar semen⁻¹. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as S. griseus.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Hexavalent chromate reduction by immobilized Streptomyces griseus

Hexavalent chromium, which is a mutagen and carcinogen, was efficiently reduced by Streptomyces griseus. This activity was associated with the cell. Cr⁶⁺ reduction by free as well as immobilized cells was studied: cells in PVA-alginate had the highest (100%) Cr⁶⁺ removal efficiency in 24 h with reduction rates similar to free cells. Immobilized cells completely reduced 25 mg Cr⁶⁺ l⁻¹ in 24 h. PVA-alginate immobilized cells could be reused four times to completely reduce 25 mg Cr⁶⁺ l⁻¹ in 24 h each time. Chromate in a simulated effluent containing Cu²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ was completely reduced by PVA-alginate immobilized cells within 9 h.     

Polylysine Produced by Streptomyces

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

Novel Biotransformations of 7-Ethoxycoumarin by Streptomyces griseus

Biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of two metabolites which were isolated and identified as 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin. A novel series of biotransformation reactions is implicated in the conversion of the ethoxycoumarin substrate to these products, including O-deethylation, 6-hydroxylation to form a 6,7-dihydroxycoumarin catechol, and subsequent O-methylation. Either 7-hydroxycoumarin or 6,7-dihydroxycoumarin was biotransformed to 7-hydroxy-6-methoxycoumarin by S. griseus. Trace amounts of the isomeric 6-hydroxy-7-methoxycoumarin were detected when 6,7-dihydroxycoumarin was used as the substrate. Efforts to obtain a cell-free catechol-O-methyltransferase enzyme system from S. griseus were unsuccessful. However, [methyl-¹⁴C]methionine was used with cultures of S. griseus to form 7-hydroxy-6-[¹⁴C]methoxycoumarin.     

Phosphorylation of Streptomycin at C6-OH of Streptidine Moiety by an Intracellular Enzyme of Streptomyces griseus

In streptomycin-producing Streptomyces griseus HUT 6037, an enzyme which phosphorylated streptomycin appeared in old mycelium at stationary to autolyzing stage. This enzyme phosphorylated streptomycin with equimolar ATP at C₆-OH in the streptidine moiety. This phosphomonoester of streptomycin was identified with the phosphorylated streptomycin (referred to as L compound) which was previously reported to accumulate in the culture broth when the pH was controlled below neutral.     

Metabolites Produced by Streptomyces mobaraensis

New metabolites, A-, B- and X-substance and piericidin X, were isolated from mycellia of Streptomyces mobaraensis. Structure of A-substance was decided as VII and B-substance was identified as reticulol (VIII).     

灰色链霉菌RX-17溶菌酶R2的纯化及其酶学鉴定

从灰色链霉菌 (Streptomycesgriseus)RX 1 7的发酵液中 ,通过硫酸铵分级沉淀 ,CM SephadexC 5 0和CM SepharoseFastFlow离子交换层析 ,纯化得到了溶菌酶R2 .该酶分子量约为 2 4 8kD ,等电点约为 9 7,N端 1 5个氨基酸的顺序为DTSGVQGIDVSHWQG .R2酶溶解变链球菌Ingbritt(StreptococcusmutansIngbritt)的最适作用温度为 5 5℃ ,最适pH为 7 0 .5 0℃处理 1h ,R2酶残存酶活74 % ,碱性条件 (pH >9)下该酶保持稳定 .Zn2 + 、Cu2 + 、Fe2 + 、Cd2 + 、Pb2 + 可使酶完全失活 ,螯合剂、盐酸羟胺、溴替丁二酰亚胺及离子型去垢剂SDS抑制R2酶的溶菌作用 ,而非离子型去垢剂TritonX 1 0 0等则能促进溶菌 .R2酶溶菌谱广泛 ,能够溶解多种鸡卵清溶菌酶不能作用的革兰氏阳性菌和革兰氏阴性菌 .从对金黄色葡萄球菌 (Staphylococcusaureus)的高活性来看 ,该酶应分类为 β 1 ,4 N ,6 O 二乙酰胞壁质酶 (β 1 ,4 N ,6 O diacetylmuramidase) .     

Properties of Streptomyces griseus aminopeptidase

The paper deals with studying the properties of aminopeptidase isolated from Str. griseus culture fluid. The preparation is characterized by a high specific activity and heat stability, it has no admixtures of carboxypeptidases and proteinases. The enzyme is easily inhibited by EDTA, but the addition of Ca2+ evokes its complete reactivation. A partial recovery of the activity may be also reached under the influence of some other bivalent metals. In hydrolysis of di- and tripeptides it is shown that the enzyme has a preferential effect on the substrates with N-terminal leucine. Peptides with N-terminal alanine, valine and glycine are almost not hydrolyzed. The use of the native insulin and decapeptide with the known amino acidic sequence as substrates shows that aminopeptidase can hydrolyze proteins and peptides with the successive release of some amino acids: phenylalanine, serine triptophane, valine, asparagine, etc. Glycine is difficult for removal and may inhibit the further hydrolysis of the polypeptide chain.