Production of Anticapsin by Streptomyces griseoplanus

Anticapsin is a new fermentation product which inhibits formation of the hyaluronic acid capsule of Streptococcus pyogenes. Production of this metabolite in a complex medium by S. griseoplanus is enhanced by high levels of carbohydrate. A number of carbon sources support biosynthesis but sucrose is most effective, the optimum level being 150 g/liter. Neither glucose nor fructose, alone or in combination, serves as an equivalent substitute for sucrose. The addition of dibasic potassium phosphate to the medium further increases anticapsin production. Dissolved oxygen levels are important for synthesis and stability of the metabolite. Anticapsular activity diminishes rapidly in previously aerated broths which are held under static conditions. This decrease does not occur in pasteurized broths or unpasteurized filtrates.     

Regioselective hydroxylation of adamantane by Streptomyces griseoplanus cells

Hydroxylation of adamantane using whole cells of bacteria, actinomyces, and molds was examined. The structure of the product was determined using gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectroscopy (MS). Among 470 strains tested, Streptomyces griseoplanus was highly regioselective to give 1-adamantanol (0.096 mmol) from adamantane (0.3 mmol) in a 32% molar conversion yield after 72-h cultivation in the presence of 3% (v/v) Tween 60. A P450 inhibitor such as 0.5 mM 1-aminobenzotriazole or menadione significantly inhibited the hydroxylation activity. These results suggested that a P450 oxidation system might be involved in this hydroxylation reaction.     

The structure of anticapsin, a new biologically active metabolite of Streptomyces griseoplanus

1. Physical and analytical data obtained on crystalline anticapsin indicated the empirical formula C(9)H(13)NO(4). Spectral data (u.v., i.r. and proton magnetic resonance) and formation of l-tyrosine on hydrolysis revealed the functionalities and carbon skeleton of the new epoxy keto amino acid. 2. The optical properties of anticapsin (optical rotatory dispersion and circular dichroism) permitted assignment of absolute configuration to the new metabolite. 3. Treatment of anticapsin with hot methanolic hydrochloric acid followed by acetylation gave C(18)H(19)NO(5), the alpha-alkoxycyclohexenone derivative. Analysis of the nuclear-magnetic-resonance and mass spectra of the latter allowed its structure to be determined and confirmed the assigned structure of anticapsin.     

Pactamycin Production by Streptomyces pactum

The optimal fermentation conditions for the production of pactamycin, a new antitumor antibiotic, by Streptomyces pactum var. pactum were investigated. The optimal pH range for growth was 6.5 to 7.0. The optimal temperature for the growth of the culture and the production of the antibiotic was investigated in a medium containing Cerelose, blackstrap molasses, Pabst yeast, Kay Soy, CaCO₃, and KCl. Since maximal growth and maximal production efficiency was obtained at 32 C, all subsequent fermentations were conducted at this temperature. Pactamycin was bound to the mycelium in different amounts, depending on the fermentation conditions, and could be extracted with acetone. Good yields (216 μg/ml) of pactamycin could be obtained in a medium containing Cerelose, soy-peptone, calcium carbonate, and potassium chloride. Analysis of the biochemical changes during fermentation indicated that pactamycin was produced during the later autolytic phase.     

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.     

Production of xylanolytic enzymes by Streptomyces flavogriseus

Summary Cultures of Streptomyces flavogriseus produced considerable amounts of xylanase when grown on xylan containing media. Comparatively lower yields of this enzyme were obtained when hay or avicel served as main carbon source, -xylosidase was synthesized intracellularly and appeared less dependent on the fermentation substrate. The strain produced simultaneously various enzymes of the cellulase complex and the xylose induced glucose isomerase.     

Production of Hydrogen Sulfide by Streptomyces odorifer

By use of various trapping systems, as well as lead acetate papers, Streptomyces odorifer was shown to produce hydrogen sulfide. Other sulfur-containing compounds may be produced by S. odorifer, but the amounts obtained were too small for detailed analysis. It was suggested that hydrogen sulfide might be a part of the earthy-odor complex produced by S. odorifer.     

Production of Glucose Isomerase by Streptomyces flavogriseus

A microorganism that produces glucose isomerase was isolated from soil and identified as a strain of Streptomyces flavogriseus. The organism produced a large quantity of glucose isomerase when grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of ryegrass straw. The organism produced glucose isomerase both intra- and extra-cellularly. The highest level of intracellular glucose isomerase (3.5 U/ml) was obtained in about 36 h by a culture grown on straw hemicellulose; the extracellular enzyme (1.5 U/ml) appeared in cultures grown for about 72 h. About equal levels of enzyme were produced in cultures grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of straw, but production of the enzyme was drastically reduced when the organism was grown on other carbon sources. As a nitrogen source, corn steep liquor produced the best results. Soy flour extract, yeast extract, and various peptones also were adequate substrates for glucose isomerase production. Addition of Mg²⁺, Mn²⁺, or Fe²⁺ to the growth medium significantly enhanced enzyme production. The organism, however, did not require Co²⁺, which is commonly required by microorganisms used in the production of glucose isomerase.     

Improved Production of Mannanase by Streptomyces lividans

Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.     

Production of Aminopeptidase and Carboxypeptidase by Streptomyces peptidofaciens

Streptomyces peptidofaciens KY 2389, a new species isolated from a soil sample, exhibited the highest potencies in production of both aminopeptidase and carboxypeptidase among about 1,300 strains tested.

Optimum pH values of both aminopeptidase and carboxypeptidase for Leu-β-naphthyl-amide and Cbz-Gly-Leu were 8.0. Aminopeptidase was thermostable and the activity was not lost by treatment at 70°C for 1 hr, in the presence of Ca²⁺. Carboxypeptidase was heat-labile and over 50% of the activity was lost by treatment at 60°C for 1 hr. Of the synthetic peptides tested, Leu-Gly-Gly and Cbz-Gly-Leu were the most suitable substrates for amino-peptidase and carboxypeptidase, respectively.     

Production of Thermostable Alkaline Proteases by Thermophilic Streptomyces

Conditions for the production of thermostable proteases (alkaline proteinase and carboxypeptidase) by a thermophilic streptomycete (Streptomyces rectus var. proteolyticus) were investigated in 20-liter volumes. Proteinase production was affected by the concentration of defatted soybean powder, its optimum being 1.2% in medium containing 2.0% soluble starch. Relatively high concentration of phosphate (0.3 to 0.4% K(2)HPO(4)) was needed for the maximum enzyme production. A large inoculum size (5 to 10%) was favorable, but the inoculum age did not significantly influence the production. The yield increase of 20 to 30% was obtained by feeding of medium during fermentation. The optimal temperature for proteinase production was 50 C, at which the maximal rate of production was 66.2 proteinase units per ml per hr, whereas at 40 C it was 9.0. Production at 50 C reached the maximum within 12 to 16 hr. The optimal agitation rate was different for the production of proteinase and carboxypeptidase, 400 rev/min for the former and 500 rev/min for the latter. The optimal aeration for proteinase production was 20 to 30 liters/min at 400 rev/min, whereas carboxypeptidase production was not markedly affected by aeration rate. The possibility that carboxypeptidase production was correlated with the shear of mycelium was discussed.     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Production of 3-methylthioacrylic acid by Streptomyces kasugaensis

Production of 3-methylthioacrylic acid (3-MTAA) by Streptomyces kasugaensis SK 619 was controlled by methionine and by quality and concentration of the carbon source. Maltose (5%) and methionine (5 gl^-1) added to the production medium positively influenced production of 3-MTAA. 3-Methylthioacrylic acid, a potential herbicide, inhibited growth of Photobacterium phosphoreum at concentrations of 50–200 μg per disc.     

Production of a Sporulation Pigment by Streptomyces venezuelae

Streptomyces venezuelae S13 produced a pH-indicating sporulation pigment on a glucose-salts-agar medium consisting of glucose, KNO(3), MgSO(4), and Na(2)HPO(4), pH 7. Pigmentation on this medium appeared to be closely associated with sporulation, which normally required 5 to 7 days at 30 C. The pigment was soluble in water as well as in a number of organic solvents. Butanol-extracted pigment exhibited absorption maxima at 430 and 520 nm at pH 3 and 12, respectively. Although many salts of organic acids and amino acids could replace glucose as the sole carbon source in basal salts-agar medium for growth and pigmentation, most sugars that were tested supported good growth but negligible pigmentation. Among the nitrogenous substances tested, KNO(3) was most desirable for pigmentation. The organism did not exhibit any specific requirements for divalent cations with respect to growth and pigmentation. In the absence of MgSO(4), however, glucose-salts-agar prepared by autoclaving all components together failed to support growth. The production of the sporulation pigment on glucose-salts-agar was comparable to that obtained on tomato paste-oatmeal-agar medium. Incorporation of partially purified pigment material into broth medium that did not normally support sporulation induced sporulation, and amino acid-salts-agar medium could induce vegetative mycelia to pigment when transferred from medium that did not support either pigmentation or sporulation.