Rapid Purification and Crystallization of Thermostable Malate Dehydrogenase Isolated from a Thermophilic Bacterium,Thermus flavus AT 62

A structural study of the water-soluble dextran made by Leuconostoc mesenteroides strain C (NRRL B-1298) was conducted by enzymic degradation and subsequent ¹³C-NMR analysis of the native dextran and its limit dextrins. The α-l,2-debranching enzyme removed almost all of the branched D-glucose residues, and gave a limit dextrin having a much longer sequence of the internal chain length (degree of linearity: n = 24.5 compared with the value of n = 3.3 for the native dextran). The degree of hydrolysis with debranching enzyme corresponded to the content of α-1,2-linkages determined by chemical methods, which suggested that most of the α-l,2-linkages in the dextran B-1298 constituted branch points of a single D-glucose residue. A synergistic increase of susceptibility of the dextran B-1299 was observed by simultaneous use of debranching enzyme and endodex-tranase. ¹³C-NMR spectral analysis indicated the similarity of structure of dextran B-1298 to that of B-1396, rather than that of B-1299. Occurrence of α-l,3-linkages in the limit dextrin was supported by a newly visualized chemical shift at 83.7 ppm.     

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)].     

Leucine Dehydrogenase from Corynebacterium pseudodiphtheriticum: Purification and Characterization

Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain l-amino acids, l-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for l-leucine, and the k_cat/Km value for l-leucine was higher than that for l-valine. The enzyme required NAD⁺ as a natural coenzyme. The NAD⁺ analogs 3-acetylpyridine-NAD⁺ and deamino-NAD⁺ were much better coenzymes than NAD ⁺. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5′-phosphate. d-Enantiomers of the substrate amino acids competitively inhibited the oxidation of l-valine.     

Isolation and Sequences of Peptic Peptides,and the Complete Sequence of Ile Chain of Ricin D

Sixteen peptic peptides, which contain arginine(s) or lysine, were isolated from cyanogen bromide fragments CB I and CB II of Ile-chain. Sequence determination has been performed on most of these peptides to provide overlaps for the tryptic peptides. Thus, complete amino acid sequence of Ile-chain consisting of 265 amino acid residues was determined. Some structural characteristics of the protein are also discussed.     

Flavor Activity of Sulfur-containing Compounds Related to Flavor Nucleotides

Substance B, the major component, isolated from rice plant treated with probenzaole and inoculated, having anti-conidial germination activity against blast fungus, was found to be a mixture of fatty acids, including palmitic acid, linoleic acid and linolenic acid. The main compound of substance B was linolenic acid, having strong anti-conidial germination activity. It was determined as α-linolenic acid by gas chromatographic analysis. The minor components showed little or no anti-conidial germination activity.     

Red Pigment Formation by the Reaction of Oxidized Ascorbic Acid and Protein in a Food Model System of Low Moisture Content

Ascorbic acid-protein mixtures of low moisture content stored for 2~3 days in aerobic conditions at 60°C produced a red coloration, which was shown to have resulted from an amino-carbonyl reaction of oxidized ascorbic acid (dehydroascorbic acid, DHA) by the facts that DHA-casein or ovalbumin systems yielded a rapid red coloration in low moisture conditions as well as in an ethanol suspension. Zein showed only a weak red coloration with DHA. An apparent decrease in the free amino group, as determined by the TNBS method, was observed to be associated in parallel with the red pigment formation. The electrophoretic pattern of ovalbumin changed significantly upon incubation with DHA, together with the formation of the red pigment. The red pigment extracted from a DHA-casein system showed an absorption spectrum, TLC Rf value, and hydrolyzed products (DHA and scorbamic acid) identical to those of 2,2′-nitrilo di-2(2)-deoxy-l-ascorbic acid mono ammonium salt, produced from DHA and amino acid. Formation of the red pigment was also observed in the reaction of DHA with N_α-acetyl lysine. These results indicate that the ε-amino group of lysine in protein can be attributed to the formation of a red pigment identical with NDA.     

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.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

Biosynthesis of Leaf Alcohol Formation of 3Z-Hexenal from Linolenic Acid in Chloroplasts of Thea sinensis Leaves

The synthetic activity for 3Z-hexenal, an important precursor of 3Z-hexenol (leaf alcohol), was localized in chloroplasts of Thea sinensis leaves. 3Z-Hexenal, which is easily isomerized to 2E-hexenal (leaf aldehyde), was formed from linolenic acid in the presence of oxygen. 13-l-Hydroperoxy-linolenic acid also served as a precursor, but the triglyceride and methyl ester of linolenic acid did not. This enzyme system appeared to be tightly bound to the lamellae membranes of chloroplasts.