Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of investigation of alicyclic hydrocarbon-utilizing microorganisms, five strains of ethylcyclohexane-utilizing bacteria were isolated from soil samples.

Among those bacteria, the strain S6B1 that was identified as Alcaligenes faecalis, showed the best growth in shaking culture.

The strain S6B1 was found to produce 4-ethylcyclohexanol from ethylcyclohexane.

This substance separated from culture broth was purified and identified to be trans-4-ethylcyclohexanol by the use of NMR.     

Studies on γ-Radiolysis of Sulfur Containing Amino Acids

In connection with flavor deterioration accompanied by food irradiation, the effect of γ-irradiation on sulfoxide amino acids in air free aqueous system was investigated. The major radiolysis products from S-n-propyl-l-cysteine sulfoxide (PCSO) were alanine, cysteic acid, dipropyl disulfide, etc., and from S-allyl-l-cysteine sulfoxide (ACSO) were S-allyl-l-cysteine, cystine, cysteic acid, etc., which were isolated chromatographycally and identified by using IR and mass spectrometry. The sulfoxide in ACSO was more easily reduced to sulfide than that of in PCSO, and the bond of S-C (β-carbon in alanine moiety) in ACSO was difficult to cleave. These differences observed between PCSO and ACSO in the radiolysis products and their yields indicate that the radiolysis degradation is considerably influenced by the structure of alkyl group. From the experiments with N₂O or KBr addition during irradiation, principal roles of the active species in irradiated water in the degradation processes were partly elucidated.     

Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Structural Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Structural Elucidation of N-Linked Sugar Chains of Storage Glycoproteins in Mature Pea (Pisum sativum) Seeds by Ion-spray Tandem Mass Spectrometry (IS-MS/MS)

The structures of N-linked sugar chains of the storage glycoproteins in mature pea seeds have been estimated. Nine pyridylaminated (PA-) N-linked sugar chains were derived and purified from the hydrazinolysate of the storage glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping, considering the data of sugar composition analysis or sequential exoglycosidase digestions. The deduced structures were further analyzed by IS-MS/MS analysis. Every relevant fragment ion derived from all PA-sugar chains could be assigned on the basis of deduced structures. The estimated nine structures fell into two categories; the first was a typical oligomannose type (Man_8-3GlcNAc₂; 77.7%) which can be hydrolyzed by endo-β-N-acetylglucosaminidase PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228–232 (1996)], the second was a xylose-containing type (Man_4-3Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 22.3%). Among these structures, Man₈GlcNAc₂ (19.7%), Man₆GlcNAc₂ (24.7%), and Man₃Fuc₁Xyl₁GlcNAc₂ (18.8%) were the dominant structures.     

An Acid- and Heat-stable β-Fructofuranosidase from Corticium rolfsii

We reported previously that an ndhB gene disruptant, ΔndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as ΔndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.     

Damages Caused by Irradiation in the Presence of NaCl in Macromolecular Components of E. coli

Damages in macromolecular components and in their functions of E. coli produced by gamma irradiation in the presence of higher concentration of NaCl were determined with reference to the basic mechanism for NaCl radiosensitization. As the results, inhibitions of DNA repair, of DNA degradation, of macromolecular syntheses and of RNA species formation were found in DNA repair proficient strains. The number of radiation-induced DNA-strand breaks was not increased by the presence of 1 m NaCl during irradiation. It was suggested that inhibition of DNA repair may be of primary importance among these lesions for NaCl radiosensitization.     

Purification and Properties of Penicillin Acylase from Kluyvera citrophila

Penicillin acylase (EC 3.5.1.11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectro-focusing fractionation. The purified enzyme, with an approximate molecular weight of 63,000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4 mM and 3.6 mM, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesacetoxycephalosporanic acid.     

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.     

Purification,cDNA cloning,and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.