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An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH2-terminal region (13 aa), an Arg-cluster region (13 aa), a hydrophobic region (23 aa), and an Ala/Pro-repeat region (12 aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutations in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threoni...

The production of the group of peptide antibiotics called No. 1907 by Paecilomyces lilacinus was stabilized and enhanced by mono-spore isolation followed by strain improvement, and the production seemed to be related to a morphological change of the strain. Unstable and a low productivity (below 10 micrograms/ml) of the antibiotics No. 1907 by P. lilacinus was greatly improved up to 140 micrograms/ml by successive mono-spore isolation along with mutagenization. Alkaline conditions caused by adding Na2CO3, K2CO3, NaHCO3 or KHCO3 were essential to obtain high production of the antibiotics following simultaneous morphological change of the cells from filamentous mycelia to round arthroconidia. Resting cell experiments showed that a significant amount of antibiotics (213 micrograms/ml) was synthesized by the filamentous cells along with their morphological change to arthroconidia in the presence of glucose under the alkaline conditions.

An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH2-terminal region (13 aa), an Arg-cluster region (13 aa), a hydrophobic region (23 aa), and an Ala/Pro-repeat region (12 aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutation in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threonine and proline in the NH2-terminal region enhanced the secretion to a small extent. Deletion of the Ala/Pro repeat region had almost no effect on secretion.

Basidifferquinone is a potent inducer produced by Streptomyces strain B-412 for differentiation of a basidiomycetes Favolus arcularius to form incomplete fruiting bodies. X-Ray crystallography together with MS, IR, 1H-NMR and 13C-NMR spectral analyses revealed the structure of basidifferquinone, C24H16O8, to be (1R)-4,7,9-trihydroxy-10-methoxy-8-methyl-1-phenyl-(1H)-anthra[1,2-C]-furan-3,6,ll-trione (Fig. 1).

Screening was carried out of the inducers for the fruiting-body formation in a basidiomycetes Favolus arcularius from microbial metabolites. Streptomyces strain B-412 similar to Streptmyces rubiginosus was isolated from the soil as the producer of a substance which the induced the formation of incomplete fruiting bodies or stipes without pileus of F. arcularius under dark conditions. The active substance was extracted from the supernatant of the cultured medium with ethyl acetate in the acidic pH range and purified by successive chromatography as orange crystals, having its degradation point of >245–255°C. The absorption spectrum in a neutral methanol solution showed peaks at 211, 242, 270, 341 and 430 nm, which changed depending on the pH value. The active substance, named basidifferquinone, induced stipe formation in F. arcularius at 3 ng/ml, which corresponds to 6.9 × 10−9 M, and the resulting stipes completed differentiation to form pileus upon successive.photoirradiation.

(1990). Construction of a Bacillus subtilis Strain Deficient in Three Proteases. Agricultural and Biological Chemistry: Vol. 54, No. 5, pp. 1307-1309.

A shuttle vector for Gluconobacter suboxydans and Escherichia coli was constructed by ligation of a cryptic plasmid, pMV201, found in G. suboxydans IFO 3130 to E. coli plasmid pACYC177. The chimeric plasmid named pMGlOl carries the ampicillin resistance gene derived from pACYC177 and transforms G. suboxydans var. α IFO 3254 as well as E. coli. The transformation conditions for G. suboxydansvar. α IFO 3254 were examined using pMGlOl DNA. Competent cells were induced efficiently by treatment with LiCl or RbCl CaCl2 which induced the competency of Acetobacter was much less effective. Addition of polyethylene glycol enhanced the transformation efficiency significantly. An efficiency of approximately 102 transformants per μg DNA was finally obtained.

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM-7 a showed specific antiphage activity against the male specific, deoxyribonucleic acidcontaining filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.

Eighty-seven strains of acetic acid bacteria were surveyed for plasmids by CsCl-ethidium bromide equilibrium centrifugation of cell lysates. Twenty-seven of the 33 strains of Acetobacter were found to harbor plasmid DNA and most strains contained multiple species of low-molecular-weight plasmids. On the other hand, plasmid DNAs were detected in 23 of the 36 strains of Gluconobacter and most of them had molecular weights of more than 5 megadaltons. Of the 18 strains newly isolated from a vinegar factory, some of which were examined taxonomically, 17 contained plasmid DNA. The molecular weights of plasmids detected in this study were in the range of about one to over 17 megadaltons. The plasmids with molecular weights of less than 5 megadaltons were characterized with restriction endonucleases. The physical maps of two plasmids designated as pMV1O1 and pMV102, which were found in the isolated strains, were constructed.

An alkalophilic Streptomyces strain, KSM-9, producing extracellular cellulases was isolated from soil. Three kinds of cellulases that preferentially hydrolyzed carboxymethylcellulose (CMC) were purified from the strain and designated as CMCase I, II and III. The optimum pH of CMCase I (Mr, 32, 000) is 8.5 while those of CMCase II (Mr, 32, 500) and III (Mr, 92, 000) are at around pH 6.0. CMCase I hydrolyzed CMC in a more random fashion than the other two enzymes.

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.

Extracellular asparaginase from Candida utilis was partially purified by precipitation with acetone and by column chromatography on DEAE Sephadex A-50 and Sephadex G-200. The specific activity of the enzyme preparation was 3900 units per mg of protein. Candida asparaginase characteristically had deaminating activity for d-asparagine as well as for l-asparagine. But this enzyme was not able to hydrolyzed l- or d-glutamine. SH inhibitor, chelating agents and metal ions did not show any inhibition or activation of l-asparaginase activity. Optimum pH was about 6 for both l- and d-asparagine. This asparaginase was stable between pH 4 and pH 10 in heating for 10 min at 50°C.

The proline auxotrophic strain of Acetobacter aceti No. 1023 treated with CaCl2 solution was transformed to the Pro+ phenotype at a frequency of up to 102/μg DNA using chromosomal DNA prepared from the wild type prototrophic strain. The CaCl2-treated cells of A. aceti No. 1023 could also be rendered competent for uptake of plasmid DNA. In an attempt to produce an appropriate cloning vector for A. aceti, the restriction patterns of the cryptic plasmids, pTA5001 A (23.5 Kb) and pTA5001B (23 Kb), found in A. aceti No. 1023 were determined. A selectable marker (ampicillin resistance) was introduced onto these cryptic plasmids by fusing them to vector pACYC177 from E. coli, using their single restriction site for XhoI. The hybrid plasmids generated could replicate in and confer ampicillin resistance to both A. aceti and E. coli. The maximum transformation frequency for A. aceti No. 1023 with these vectors was 103/μg DNA.

Physicochemical and immunochemical comparisons were made on the two acid proteases Mucor pusillus rennin (MPR) and Mucor miehei rennin (MMR). The molecular weight and isoelectric points of the two enzymes were similar: MPR (M.W., 38,500 and I.P., 3.9) vs. MMR (M.W., 42,000 and I.P., 4.1). d-Galactose and d-glucosamine, which had been found in MMR, were also detected in the carbohydrate moiety of MPR with gas-chromatography and amino acid analyses. The structural similarity between MPR and MMR was investigated utilizing peptide mapping techniques, which showed a close correlation between the two enzymes. Furthermore, immunodiffusion test using antisera prepared against each enzyme suggested the presence of a common antigenic structure. The results confirm that a high similarity exists between MPR and MMR, which has been supposed from the morphological identity of the two enzyme-producing fungal strains.

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of smal...

Purified Candida asparaginase was proved to be homogeneous by gel filtration, ultra-centrifugation and disc electrophoresis. The enzyme was found to have properties as glycoprotein containing mannose. The ratio of mannose to protein was 1 to 2 in purified enzyme. Specific activity was 5500 units per nag of protein. Isoelectric point was pH 4 to 4.5 and sedimentation coefficient was found to be about 8.2 S. Antitumor activity of Candida asparaginase was inferior to E. coli enzyme. It was thought as the reason why the Candida asparaginase had less affinity to l-asparagine and it was cleared faster from the blood than E. coli asparaginase.

Spheroplasts of auxotrophic mutants derived from Acetobacter aceti subsp. aceti No. 1023 were efficiently prepared by treatment with lysozyme, using sucrose as an osmotic stabilizer, and regenerated on an agar plate containing sorbitol and gelatin. In addition, spheroplast fusion between the several auxotrophic mutants was achieved in the presence of polyethylene glycol and CaCl2. The frequency of fusion was found to be about 5 × 10 5. Spheroplast fusion between A. aceti subsp. aceti No. 2 with the ability to grow at high temperature and A. aceti subsp. xylinum NBI1002 with high resistance to acetic acid was also achieved by the same method, with a frequency of 6.0 × 10 6. The fusants showed various degrees of resistance to acetic acid and ability to grow at high temperature. One of the fusants, named No. 116, could produce acetic acid from ethanol continuously under conditions under which both parent strains were unable to grow. This suggests that spheroplast fusion is applicable to the breeding of strai...

Co2+-requiring heme protein having lipoxygenase activity, obtained from Fusarium oxysporum (FUSARIUM lipoxygenase) was extensively purified by ammonium sulfate precipitation, ion exchange chromatography on SP-Sephadex and gel filtration with Sephadex G–100. The final preparation achieved homogeneity by ultracentrifugation and SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated at 12,000 to 13,000 on the basis of ultracentrifugation, SDS-polyacrylamide gel electrophoresis and gel filtration. FUSARIUM lipoxygenase contained 1 mole protoheme IX per mole enzyme, required Co2+ as a stabilizing factor and lost activity by treatment with heat or proteases. FUSARIUM lipoxygenasecatalyzed oxidation was proved to be differrent from the well-known soybean lipoxygenasecatalyzed oxidation and hemeprotein or cobalt-catalyzed oxidations in various respects including reaction velocity, substrate specificity pI and activation energy.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD+ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.

The Oxygen activating mechanism of Fusarium lipoxygenase, a heme-containing dioxygenase, was studied. The enzyme did not require any cofactors, such as H2O2, however, both superoxide dismutase and catalase inhibited linoleate peroxidation by Fusarium lipoxygenase. A low concentration of H2O2 caused a distinct acceleration in enzymatic peroxidation. These results indicate that both O2− and H2O2 are produced as essential intermediates of oxygen activation during formation of linoleate hydroperoxides by Fusarium lipoxygenase. This peroxidation reaction was also prevented by scavengers of singlet oxygen (1O2), but not by scavengers of hydroxy 1 radical (OH). Generation of O2− in the enzyme reaction was detected by its ability to oxidize epinephrine to adrenochrome. Moreover, the rate of peroxide formation was greater in the D2O than in the H2O buffer system. These results suggest that the Haber–Weiss reaction (O2−+H2O2→OH−+OH·+1O2) is taking part in linoleate peroxidation by Fusarium lipoxygenase, and the 1O2...

The production of prochymosin directed by a cloned cDNA under the control of a trp promoter was examined in E. coli C600 and HB101. The latter host exhibited a higher degree of expression as to the production of prochymosin in the form of inclusion bodies, which accounted for more than 15 ~ 20% of the total cellular protein. The conditions for the processing of prochymosin in the inclusion bodies to active chymosin were determined. Several enzymatic properties of the processed bacterial chymosin, such as its specific activities as to milk-clotting and proteolysis, heat stability and Ca2 + dependence of the clotting activity, were almost identical to those of authentic chymosin. However, a slight difference was observed with regard to the immunological reactivity with anti-prochymosin antibody.

A lytic enzyme was isolated from the lysate of Ps. aeruginosa infected with a new strain of bacteriophage, phage 95. The enzyme, LE95, was purified by chromatography in twice on IRC50 column and by gel filtration in twice on Sephadex G-75 column. The molecular weight was estimated as 21, 000. The optimal condition for the hydrolysis of acetone-dried cells of Ps. aeruginosa was determined to be following: the optimal pH was between 6.5 and 7.0, the temperature about 70°C and the concentration of phosphate buffer about 5mM. The enzyme was strongly inhibited by Ag+, Hg2+, Ni2, Fe2+ and Cu2+ ions. When peptideglycan obtained from Ps. aeruginosa was digested by LE95, free amino groups were liberated without release of reducing sugars. The enzyme was suggested to be amidase or peptidase.