Cloning of the Genes Concerned in Phenylalanine Biosynthesis in Corynebacterium glutamicum and its Application to Breeding of a Phenylalanine Producing Strain

The chorismate mutase and prephenate dehydratase genes of phenylalanine producing Corynebacterium glutamicum K38, which is resistant to p-fluorophenylalanine and m-fluorophenylalanine, were cloned into plasmid pCE53 in C. glutamicum KY9456, which lacks chorismate mutase and prephenate dehydratase. One of the resultant plasmids, pCmB4, contained a 9.4kb BamHI DNA fragment inserted into the unique BamHl site of pCE53. Plasmid pCmB4 complemented a phenylalanine and tyrosine double auxotroph of C. glutamicum KY9456. Introduction of pCmB4 into C. glutamicum RRL5 resulted in an about ten times increase in chorismate mutase activity. C. glutamicum K38 carrying the plasmid accumulated 19.0mg/ml of phenylalanine (50% increase over the yield of K38).     

Isolation and Structure of Neoannonin,a Novel Insecticidal Compound from the Seeds of Annona squamosa

Two insecticidal compounds were isolated from the seeds of Annona squamosa. One was identified as annonin (squamocin) and the other was characterized as a novel dihydroxy-bistetrahydrofuran fatty acid lactone (acetogenin) with 35 carbons. These two compounds were toxic to eggs, larvae and adults of the fruitfly.     

Biosynthesis of Cercosporin

¹³C NMR spectra were measured for 19 pyrethroids and their related compounds including allethrin, tetramethrin, resmethrin, furamethrin, phenothrin and permethrin. Complete assignment of chemical shifts was accomplished by relative spectral pattern, single-frequency off-resonance decoupling, benzene substituent effects, proton selective decoupling and use of shift reagents. The use of shift reagent was found to be especially efficient for assignment of ¹³C resonances. In the case of allethrin, the splittings of some resonance peaks were observed originating from diastereomerism.     

Synthesis of 9-β-d-Glucopyranosyl-Adenine-6′-Phosphate

Synthesis of 9-β-d-glucopyranosyl-adenine-6′-phosphate is described. The method developed here involves the process of condensation of base (chloromercuri-6-benzamidopurine) (I) with phosphorylated sugar (2,3,4-tri-O-acetyl-6-diphenylphosphoryl-α-d-glucopyranosyl bromide) (II). This reaction gives crystalline 6-benzamido-9-(2′,3′,4′-tri-O-acetyl-6′-diphenylphosphoryl-β-d-glucopyranosyl)-purine (III) in high yield, which is converted to the desired nucleotide by alkaline hydrolysis.     

Chemical Studies on Pyrethroids

Allethrin-(E)-Ol (IV), allethrin-(E)-al (V) and allethrin-(E)-acid (VI), the metabolites of allethrin (III) in the insect body, were synthesized. Their low toxicities to houseflies seem to support the hypothesis that they are products of the detoxication process of allethrin.     

Growth-Promoting Factors for Lactobacillus bifidus var. pennsylvanicus Produced by the Amino-Carbonyl Reaction of l-Lysine and d-Glucose

l-Lysine monohydrochloride and d-glucose were allowed to react in a bicarbonate buffer of pH 8.8 under refluxing. The reaction mixture was then chromatographed on a thin-layer plate of Kiesel Gel using n-propanol ethyl-acetate water 25% aqueous ammonia (6: 1: 2: 1 v/v) as a developing agent. Elson-Morgan-reactive spots on the chromatogram were eluted individually, and each of the eluates was incubated with L. bifidus var. pennsylvanicus in a defined medium. A certain fraction on the chromatogram showed remarkably promoting effect on both the acid productivity and the growth of the organism. And such effect of the fraction was much stronger than that of N-acetyl-d-glucosamine which had been known as a “Bifidus Factor”     

Changes in Flavor Components of Onion by γ-Irradiation

The effect of γ-irradiation on the flavor of Onions (Senshu Yellow) associated with sprout-inhibition has been investigated. The relative amounts of propionaldehyde, n-propyl mercaptan and di-n-propyl disulfide in onions stored for 0, 5, 10, 20 and 30 days, respectively, after irradiation were estimated by measurement of peak areas of gas chromatograms. It was observed that the lachrymatory character and the pungent flavor had been decreased by γ-irradiation (remarkably at 70 and 700 Krads). In this connection, the amounts of propionaldehyde and di-n-propyl disulfide were decreased with increasing radiation dose and storage period at room temperature (20 to 25°C) and at low temperature (4°C). Moreover, it was observed that the sweetness had been decreased by γ-irradiation, but the amount of n-propyl mercaptan was increased with radiation dose and storage period. Therefore it seems that there is no correlation between the sweetness of onion and the amount of n-propyl mercaptan.     

The Heterogeneity of the 7S Soybean Protein by Sepharose Gel Chromatography and Disc Gel Electrophoresis

Two kinds of N-acetylmuramidase, M-1 and M-2 enzymes, that were isolated from the cultural broth of Stm. globisporus 1829, were remarkably different in amino acid composition, immunological properties and modes of lytic action from each other. The M-1 enzyme was composed of 186 amino acid residues of which two moles were of half cystine, while the M-2 enzyme was composed of 99 amino acid residues with no cysteine. The hydrolyzing action of the M-2 enzyme was suppressed by the presence of an O-acetyl group on muramic acid residues in the peptidoglycan moiety, while that of the M-l enzyme was independent of the presence of O-acetyl groups. However, the hydrolyzing activity of both enzymes was enhanced when some muramic acid residues were substituted with stem peptides containing alanine, isoglutamine and lysine.     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.