Recombinant production and characterization of an N-acyl-d-amino acid amidohydrolase from Streptomyces sp. 64E6

N-Acyl-d-amino acid amidohydrolases (d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)d-Phe (0.05–6.32 μmol min^?1 mg^?1) under conditions without CoCl₂. Addition of 1 mM CoCl₂ enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min^?1 mg^?1 for N-Ac-d-Phe and N-Ac-d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0–9.0. It was stable at pH 5.5–9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-d-Phe was 2.1-fold higher than that toward N-Ac-d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.     

Peptidoglycan of Pseudomonas aeruginosa

A practical method for preparing peptidoglycan from Ps. aeruginosa and E. coli was devised. After bacterial cells were dissolved in boiling 4% SDS solution, peptidoglycan was collected and washed with water by centrifugation. Peptidoglycan was treated further with pronase and lyophilized. The final preparation of peptidoglycan from Ps. aeruginosa appeared as a filmy coagulation in electron micrograph and its amino acid composition was determined as follows: Glu/Ala/A₂pm/Mur/GlcN (100/183/104/61/98). The lysozyme digest showed the same pattern as that of E. coli peptidoglycan. N-Terminal analysis suggested that about half of the peptide chains was interbridged by the peptide bond between Ala and A₂pm. The probable ratio of muropeptides in the peptidoglycan was estimated.     

Effect of Various Inhibitors on Lipase Action of Thermophilic Fungus Humicola lanuginosa S-38

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.     

Possible Mechanism of Oxygen Activation in Linoleate Peroxidation by Fusarium Lipoxygenase

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 evolved could be responsible for the peroxidation of linoleate. H₂O₂ produced endogenously in the enzyme reaction might act as an activating factor for the enzyme. This possible mechanism of oxygen activation can explain the absence of a need for exogenous cofactors with Fusarium lipoxygenase in contrast to an other heme-containing dioxygenase, tryptophan pyrrolase, which requires an exogenous activating factor, such as H₂O₂.     

Studies on the Lipoprotein Lipases of Microorganisms

About 2000 strains of microorganisms were examined for lipoprotein lipase producibilities and some microorganisms were found to produce lipases similar to animal lipoprotein lipases.

Microorganisms were cultured on solid media containing a serum-activated olive oil emulsion, and strains which formed a clear zone around the colony were collected. The collected microorganisms were cultured on liquid media containing 0.5% of olive oil by shaking and the culture filtrates were tested for lipoprotein lipase activity by a turbidity method. The superior lipoprotein lipase producers obtained belonged to genera of Serratia, Pseudomonas, Mucor, and Streptomyces.     

Effect of Exogenous Fatty Acids on the Cellular Fatty Acid Composition and Neomycin Formation in a Mutant Strain of Streptomyces fradiae

A mutant of Streptomyces fradiae which requires oleic acid for neomycin formation was isolated and the effects of exogenous fatty acids and other additives on the formation of neomycin were studied. Palmitic acid and high concentration of sodium ions could replace oleic acid in neomycin formation. The fatty acid spectrum of the mutant strain ST–5B was quite different from that of the parent strain 3123. The major fatty acid components of the mutant and the parent were anteiso 15:0 and iso 16: 0, respectively. However the fatty acid composition of the mutant was changed from the anteiso 15: 0-type to the parental iso 16: 0-type by the supplement of oleic acid or high concentration of sodium ions in the medium. In the case of palmitic acid, the major fatty acid component of the mutant cells was changed from anteriso 15: 0 to normal 16:0. The role of these additives in neomycin formation by the mutant is discussed.     

Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

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 small amounts of AFS without any cell lysis.     

A New Method for Estimation of the Mycelial Weight in Koji

A New method was devised for the estimation of the mycelial weight in rice-koji. In this method, the content of glucosamine in koji was used for the calculation of mycelial weight. The content of glucosamine in the mycelia of Aspergillus oryzae, koji, and rice was determined by a colorimetry after hydrolysis of these materials with sulfuric acid and purification of glucosamine fraction with a Dowex 50 W column. And the values of glucosamine were 114.5 mg/g in mycelia, 3.03 in the koji for amazake,* 1.34 in the koji for sake, and 0.0 in rice. The mycelial contents calculated from these data were 2.6% dry weight in amazake-koji and 1.2% in sake-koji.     

Structure of Bovine κ-Casein: Spectrophotometric Titration and Molecular Size Changes in Alkaline Solution

The ionization of tyrosyl groups in bovine κ-casein and S-carboxyamidomethyl-κ-casein (CAM-κ) was studied by spectrophotometric titration at 295 mµ. In the denaturing solvent 8 m urea, the titration curves are reversible and the pK_app values of eight tyrosyl groups both in κ-casein and in CMA-κ-casein are 10.7. In 0.2 m KCl solution, κ-casein has six tyrosyl groups with normal pK_app value of 10.5 and two groups with higher pK_app value of 11.4. CAM-κ-casein has eight tyrosyl groups with pK_app value of 10.6 in 0.2 m KCl solution. These observations suggest that -S-S- bondings in κ-casein are concerned with the ‘masking’ of the tyrosyl groups. The evidence of the rupture of intermolecular -S-S- bondings of κ-casein in alkaline solution was shown by the study of gel Chromatograph y on Sephadex G–150. One of the possible explanation is that the ionization of tyrosyl groups with higher pK_app value is associated with the destruction of hydrophobic regions, and this destruction is due to the rupture of intermolecular -S-S- bondings under alkaline conditions.