Identification of d-Erythro-Dihydroneopterin Triphosphate as a Product of Guanosine Triphosphate Cyclohydrolase from Serratia indica

Guanosine triphosphate cyclohydrolase (EC 3.5.4.16) was previously shown to exist in two forms (GTP cyclohydrolase D-I and D-II) in Serratia indica IFO 3759, and they were homogeneously isolated. The present study deals with the characterization of their reaction products. A fluorescent product formed from guanosine triphosphate by GTP cyclohydrolase D-II was identified as 7,8-dihydroneopterin triphosphate by its absorption spectra, phosphate analysis and gas chromatography-mass spectrometry of the dephosphorylated trimethylsilyl derivative. After oxidation and dephosphorylation, the d-erythro configuration of the side chain was made clear by the elution profile on ECTEOLA-cellulose chromatography, Rf values on thin-layer chromatography and by biological activity to Crithidia fasciculata ATCC 12857. The fluorescent products from GTP cyclohydrolase D-I and D-II were indistinguishable.     

Purification and Some Properties of Lignostilbene-a,/i-dioxygenase Responsible for the Ca—Cp Cleavage of a Diarylpropane Type Lignin Model Compound from Pseudomonas sp. TMY1009

A novel dioxygenase, lignostilbene-a,β-dioxygenase (LSD), which catalyzes cleavage of the interphenyl double bond of lignin-derived stilbenes, was isolated. Four isozymes of LSD were separated from cell-free extracts of Pseudomonas sp. TMY1009 by ion-exchange chromatography on a DEAE- Toyopearl column. The major isozyme, LSD-I, was purified to electrophoretic homogeneity and characterized.

LSD-I cleaved the interphenyl double bond of l,2-bis(4′-hydroxy-3′-methoxyphenyl)ethylene with the optimum pH at 8.5. The Km of LSD-I was 11 μm for the stilbene and 110/iM for oxygen. The molecular weight of LSD-I, which is composed of two identical subunits, was estimated to be 94,000. LSD-I contained 1 g atom of iron per 1 mol of enzyme protein.     

Rapid Gas Chromatographic Determination of Micro-quantities of N-Methyl Carbamates as their 2, 4-Dinitrophenyl Derivatives

A rapid and sensitive method is presented for the determination of micro-quantities (1 to 100 μg) of N-methyl carbamates as dinitrophenyl methylamine (DNP-MA) by electron capture gas liquid chromatography (GLC). The method described is characterized by rapidity and simplicity of procedure due to an improvement made in the present investigation, i.e., hydrolysis of an N-methyl carbamate and dinitrophenylation of the resulting methylamine with 2, 4-dinitro-1-fluorobenzene (DNFB) were accomplished simultaneously in a single reaction mixture. A novel approach was also made to eliminate unreacted DNFB by conversion to dinitrophenyl glycine (DNP-glycine).     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

Formation of Pungent Principles in Fruits of Sweet Pepper,Capsicum annuum L. var. grossum during Post-harvest Ripening under Continuous Light

Pungent principles (Capsaicinoid(s)) were found to be produced in fruits of sweet pepper, Capsicum annuum L. var. grossum, during post-harvest ripening under continuous light. The initial formation was observed after 4 days’ ripening. After 7 days’ ripening, the capsaicinoids content in placenta increased to 12.9 μg per fruit, which was 2.5-fold of that in pericarp. No pungent principles were detected in fruits during ripening in the dark and in seeds under continuous light. In placenta, the formation of dihydrocapsaicin and nordihydrocapsaicin which are the vanillylamides of saturated branched fatty acids was higher than that of capsaicin which is the vanillylamide of an unsaturated one. Remarkable formation and accumulation of carotenoid were also observed during post-harvest ripening under continuous light.     

Food Processing Characteristics of Soybean Proteins

Relation between sulfhydryl groups in soybean proteins and the physical properties of tofu was studied. Changes in the amount of sulfhydryl groups by heating or treatment with urea were more rapid in 11S protein as compared with 7S protein. Moreover, by changing the amount of sulfhydryl groups in proteins by N-ethylmaleimide, 2-mercapto-ethanol and dithiothreitol, the physical properties of tofu from 11S protein were more significantly effected than that from 7S protein. Namely, tofu-gel from 11S. protein got harder and stronger as the amount of sulfhydryl groups increased.

The results may suggest that tofu prepared from IIS protein has more disulfide bonds in its gel than that from 7S protein.     

Studies on Production of Biotin by Microorganisms

The utilization of hydrocarbons by microorganisms was studied in many fields, but the production of biotin vitamers by hydrocarbon-utilizing bacteria has never been reported.

We have screened many hydrocarbon-utilizing bacteria which produce biotin vitamers in the culture broth. The effects of cultural conditions on biotin vitamers production by strain 5–2, tentatively assigned to the genus Pseudomonas, were studied.

More than 98% of biotin vitamers produced from hydrocarbons by strain 5–2 was chromatographically determined as desthiobiotin. As nitrogen source, natural nutrients were more effective than inorganic nitrogen sources. The production of biotin vitamers was increased under the condition of good aeration. Exogenous pimelic or azelaic acid enhanced biotin vitamers production by strain 5–2.

The production of biotin vitamers from n-alkanes, n-alkenes or glucose by an isolated bacterium, strain 5-2, tentatively assigned to the genus Pseudomonas, was investigated. Among these carbon sources, n-undecane was the most excellent for biotin vitamers production.

The biosynthetic pathway of biotin vitamers, especially desthiobiotin, from n-undecane was also studied. It was found by thin-layer and gas-liquid chromatographical methods that pimelic and azelaic acids were the main acid components in n-undecane culture.

This result, together with previously reported enhancement of biotin vitamers production by these acids, suggests that pimelic and azelaic acids may be the intermediates of biotin vitamers biosynthesis from n-undecane.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.