Purification and Properties of L-Methionine γ-Lyase from Aeromonassp.

Four types of β-xylosidases from a concentrated culture filtrate of Pénicillium wortmanni IFO 7237, designated as xylosidase-1, -2, -3, and -4 were purified to homogeneity on SDS polyacrylamide gel electrophoresis by an alcohol precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and isoelectric focusing. The molecular weights of xylosidase-1, -2, -3, and -4 were estimated to be 110,000, 195,000, 210,000, and 180,000 respectively and their isoelectric points to be 3.7, 4.28, 4.6, and 4.8. The pH optima of β-xylosidase activities were from 3 to 4.5. The optimum temperature for enzyme activities was from 55°C to 65°C. On the enzymic hydrolysis of phenyl ß-d- xyloside, the reaction product of each enzyme was found to be β-d-xylose with retention of configuration. All the four ß-xylosidases were free of α-xylosidase and ß-glucosidase activities. All the enzyme activities of four β-xylosidases were strongly inhibited by Hg²⁺ and N- bromosuccinimide. With respect to the hydrolysis patterns and HPLC analysis of hydrolyzates from xylooligosaccharides, xylosidase-2 was totally different from other three as a distinct enzyme. Xylosidase-1 was also in a separate group although xylosidase-3 and -4 showed closely related action patterns as a different group.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Acid carboxypeptidase III from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The optimum activity of the enzyme occurred at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, αα′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 61,000. The enzyme hydrolyzed the peptides that possess masked or bulky N-terminal.     

Enzymatic Synthesis of s-Substituted l-Cysteines with Tryptophan Synthase of Escherichia coli

The α₂β₂ complex of tryptophan synthase from Escherichia coli catalyzes β-replacement reactions of l-serine and its derivatives (e.g., β-chloro-l-alanine and O-methyl-Dl-serine) with various alkanethiols. The products from thiobenzyl alcohol and ethanethiol were isolated to demonstrate the enzymatic synthesis of the corresponding S-substituted l-cysteines. Reactivities of various S-substituent donors were examined, and thiols such as thiobenzyl alcohol, 1-propanethiol and 1-butanethiol were found to be much more efficient substituent donors than the physiological substrate, indole. In addition, tryptophan synthase catalyzes β-replacement reactions of l-threonine with thiols to form the corresponding S-substituted β-methylcysteines, which are also produced by β-addition reactions of l-vinylglycine with thiols. These enzymatic reactions facilitate the synthesis of various sulfur-containing amino acids.     

Action of S-Carbamoyl and S-Thiocarbamoyl Derivatives of L-Cysteine on L-Amino Acid Oxidase

Methods are investigated for evaluating the kinetic parameters in a modified Monod’s equation which give the best fit to the growth thermograms for bacterial cultures observed in batch calorimeters. Four mathematical methods were employed as parameter fitting techniques. The growth thermograms observed for soil microbes cultured with glucose as a limiting substrate were used as the objects of the analysis. For the calculation of the heat evolution rate, the Runge-Kutta method, which is commonly used for the numerical analysis, was employed. A comparison of the results obtained by the four methods in terms of closeness of fit to the actual thermograms showed that optimization by direct searching with the Simplex method is the most effective procedure for obtaining the best values of the parameters to reproduce the observed thermograms.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Successive Isolation of Proteinase Hyperproductive Mutants of Aspergillus sojae

Hydrochloric acid treatment of methyl 3-(4-isobutylphenyl)-3-methylglycidate and methyl 2-hydroxy-3-(4-isobutylphenyl)-3-butenoate, a rearrangement product of the former, in acetic acid gave 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-isobutylphenyl)-pro-panal. The same treatment of 2-hydroxy-3-(4-isobutylphenyl)-3-butenoic acid gave 2-(4-isobutylphenyl)-propanal. Both 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-iso-butylphenyl)-propanal were oxidized to 2-(4-isobutylphenyl)-propionic acid.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

The First and Second Dissociation Constants of Subtilisin BPN′-Plasminostreptin Complex Determined by a Polarographic Method,and the Effects of pH on Them

A polarographic method based on the Brdi?ka current (the polarographic catalytic hydrogen evolution current produced by proteins in the presence of cobalt salts) was applied to direct titration of subtilisin BPN′ (S.BPIST) with plasminostreptin (PS) at a concentration level of 10”⁸ m. The first and second dissociation constants of the S.BPN-PS complex were determined by fitting theoretical curves to the titration data, in which the multiple equilibria involving microscopically distinct forms of S.BPN-PS complex were taken into account. The intrinsic free energy change in the first binding of S.BPN′ to dimeric PS was larger than that in the second binding. The dependence of the microscopic dissociation constants of S.BPN′-PS complex on pH indicates the participation of ionizable groups of pK_a 8.0 and 9.4 in the complex formation. The repulsive effect between negatively charged molecules of S.BPN′ and PS in the complex formation at elevated pH is suggested.     

Formation of long-chain N-vanillyl-acylamides from plant oils

Long-chain N-vanillyl-acylamides (LCNVAs) were generated from plant oils and vanillylamine (VA) by nucleophilic amidation without any catalytic reagents. The resulting LCNVAs varied according to the fatty acid composition of the plant oil used. Therefore, the LCNVAs contained in Capsicum oleoresins were products that were spontaneously generated from the oleoresin during storage.