The Isolation of Collagenase and Its Enzymological and Physico-chemical Properties

A collagenolytic enzyme specific for native collagen and gelatin was isolated from Pseudomonas marinoglutinosa by DEAE-cellulose column chromatography, Sephadex G–150 gel filtration and by disc electrophoresis on polyacrylamide gel.

The molecular weight of the enzyme was approximately 74,000 and its isoelectric point was found to be around 4.5. The optimum pH and temperature for Z–GPLGP hydrolysis were around 7.6 and 38°C, respectively. The enzyme was rather stable up to 50°C and in the range between pH 5.0 and 10.0, and was stabilized by Ca²⁺ to some extent. Some chelating agents and metal ions such as Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ inactivated the enzyme, but diisopropyl phosphofluoridate, sulfhydryl agents and some trypsin inhibitors did not affect the activity.

The EDTA-inactivated enzyme was restored its activity by added Ca-salt to almost completely and very slightly by Co-, Mn- and Sr-salt.

Metal analysis showed the enzyme contained 1 g atom of zinc and 4 g atoms of calcium per mole.     

Purification and Some Properties of Cyclo(Gly-Gly) Hydrolase from a Strain of Bacillus sp. No. 106

Bacillus sp. No. 106, which was isolated from soil, secreted an enzyme that hydrolyzed cyclo(Gly-Gly). The enzyme was purified to the ultracentrifugally homogeneous state and an activity more than 450-fold that of culture broth. The enzyme was activated by Na⁺, Mg²⁺, Ca²⁺, and Sr²⁺, and strongly inhibited by Ni²⁺, Cu²⁺, p-chloromercuribenzoate, and monoiodoacetic acid. The Km value for cyclo(Gly-Gly) was estimated to be 11.1 mm. The enzyme hydrolyzed only cyclo(Gly-Gly) among various diketopiperazines tested. Aslo, the enzyme was inert toward Gly-Gly, milk casein, and hemoglobin.     

Lignin-Carbohydrate Complexes Containing Phenolic Acids Isolated from the Culms of Bamboo

Lignin-carbohydrate complexes containing phenolic acids (LCC-W) were isolated from Moso-bamboo (Phyllostachys pubescens Mazel) and characterized. LCC-W was separated into three fractions (W-l, 2, and 3) by gel filtration on Sepharose 4B. Of these three LCCs W-2 and W-3 were included in the gel matrices. W-2 consists of 34.7% neutral sugar, 1.6% uronic acid, 52.1% lignin, and 6.1% phenolic acid, and W-3, 67.9%, 3.6%, 22.3%, and 1.1%, respectively. Neutral sugar residues of W-2 and W-3 were mainly l-arabinose and d-xylose in the ratios of 5.5:94.3 in W-2, and 4.5:95.0 in W-3, respectively. Methylation, periodate degradation, and NMR analyses indicated that the carbohydrate moiety of LCC-W is composed of a linear backbone of β-(l→4)-linked d-xylopyranose residues with approximately every thirty residues carrying one 4-O-methyl-d-glucuronic acid and one or two arabinofuranose residues. Saponified phenolic acids were composed of trans-p-coumaric and trans-ferulic acids, which seems to be esterified to carbohydrate and lignin, respectively. Alkaline treatment, periodate degradation, and hydrophobic interaction chromatography suggested the presence of alkali labile and stable lignin–carbohydrate linkages.     

Solubilities Studies of Basic Amino Acids

The solbilities of l-basic amino acids in the type of free, monohydro-chloride and dihydrochloride in water were determined, and the results were formulated as follows.

l-Arginine: log S = 0.9770+0.01345t (t is from 0°~70°C)

l-Histidine: log S = 0.3627+0.00905t (t is from 0°~70°C)

l-Arginine monohydrochloride: log S = 1.6532+0.01301t (t is from 0°~70°C)

l-Lysine monohydrochloride dihydrate: log S = 1.6990+0.01294t (t is from 0°~55°C)

l-Lysine monohydrochloride monohydrate: log S = 1.7404+0.01256t (t is from 55°~70°C)

l-Lysine dihydrochloride: log S = 2.2138+0.00409t (t is from 0°~70°C)

l-Histidine dihydrochloride: log S = 1.9085+0.00265t (t is from 0°~70°C)

Three component systems (basic amino acid, hydrochloric acid, water) were studied and the solubilities in mixed solution system of alcohol-water were also investigated.     

Studies on Plant Growth Regulators

The auxin activities of the homologs of racemic and enantiomeric α-alkylphenylacetic acids were estimated by pea straight growth test. The α-methyl, -ethyl and -propyl acids were moderately active whereas the longer and branched alkyl chain were found to make the molecule inactive. The more active enantiomers were shown to have the same configuration as the more active enantiomers in the other series of the optical active synthetic auxins.

The auxin activities of the cyclic homologs of 1, 2, 3, 4-tetrahydro- and 3, 4-dihydro-1- naphthoic acids were determined by pea straight growth test. In the tetrahydro-acid series, it was observed that the alicyclic ring expansion from the 6-membered to the 7-membered made the molecule inactive. In the 3, 4-dihydro-acid series, on the other hand, the activity remained almost unchanged by such a structural change. Structure-activity relationships were discussed in terms of their molecular structures, in particular, the configuration of the carboxyl group.     

Substrate Specificity of the Protease from Streptomyces cellulosae

The substrate specificity and the mode of action of the protease from Streptomyces cellulosae were investigated, using many kinds of peptides and proteins as substrates. The protease hydrolyzed peptides consisting of hydrophobic amino acids such as L-Phe-L-Leu-NH₂, L-Pro-L-Phe-NH₂, l-Leu-L-Met, L-Leu-L-Leu, Gly-L-Ile, L-Phe-L-Phe, L-Pro-L-Leu-Gly-NH₂, etc. The protease hydrolyzed zein best among the proteins tested, but weakly hydrolyzed gelatin, myoglobin, bovine serum albumin, γ-globulin, and collagen. The protease mainly hydrolyzed Ser¹²-Leu¹³, Leu¹³-Tyr¹⁴, and Tyr¹⁴-Gln¹⁵ bonds in the oxidized A-chain of insulin and at least the Leu¹⁵-Tyr¹⁶ bond in the oxidized B-chain of insulin.     

Comparison of Compositions of Odor Components of Natto and Cooked Soybeans

Natto, a traditional Japanese food product, was prepared from cooked soybeans by fermentation and its odor concentrate was obtained with a simultaneous distillation and extraction system. It was compared to those obtained from soybeans cooked for 0 ~ 3, 3 ~ 5.5 and 5.5 ~ 8 hr, respectively, by gas chromatography and gas chromatography-mass spectrometry. In the odor concentrates of the cooked soybeans, hexanal, (E)-2-hexenal and hexanol contributing to the green and grassy odor of soybeans disappeared or decreased while the cooking was in progress. 2-Pentylfuran and 1-octen-3-ol contributing to the beany odor remained even if the soybeans were cooked for 8 hr and were fermented into Natto. In the odor concentrate of Natto, pyrazines and sulfur containing compounds were important contributors to the characteristic odor of Natto. As the beany odor was not detected for Natto, it was concluded that the pyrazines and sulfur containing compounds cause the characteristic odor of Natto and mask the beany odor.     

Evaluation of Soy Sauce Flavor by Stepwise Multiple Regression Analysis of Gas Chromatographic Profiles

The linear correlation was found by the stepwise multiple regression analysis between the sensory test of soy sauce flavor and the gas Chromatographic (GLC) data transformed with arc-sine and logarithm. GLC data will possibly be used for objective evaluation of soy sauce flavor. A multiple correlation coefficient or of a determination coefficient of more than 0.9 was respectively obtained at the 5 th or 10th of 47 steps. The fact that the minimum standard error of an estimate was found at the 24th step suggests the importance of selecting proper peaks from the whole gas chromatogram. High estimated accuracy was acquired by application of GLC data to the calculated multiple regression model.     

Stereochemical Structure–Activity Relationship of N-(2,3-Epoxypropyl)-N-(α-methylbenzyl)benzenesulfonamide Derivatives

The absolute configurations of two asymmetric centers in four stereoisomers of N-(2,3-epoxypropyl)-N-(α-methylbenzyl)benzenesulfonamide were determined and their biological activities were tested. Consequently, N-[(R)-2,3-epoxypropyl]-N-[(R)-α-methylbenzyl]benzenesulfonamide was found to be the most active isomer and the stereochemistry of the benzyl position was found to be more important than that of C₂ in the epoxypropyl group for biological activity.     

Structure-Herbicidal Activity Relationship of α-Phenylsulfonyl Propanamide

α-Phenylsulfonyl alkanamides were synthesized, and their herbicidal activities were tested under paddy conditions. Some of the α-phenylsulfonyl propanamides showed a high herbicidal activity against paddy weeds with no significant effect on rice plants. The activity of the sulfonyl compound was superior to those of the sulfinyl and thio compounds.