Studies on the Photolysis of Sulfur Containing Compounds in Foods

The aqueous solutions of 2-alkyl-thiazolidine-4-carboxylic acids and mercaptals of l-cysteine were exposed to sunlight in the presence of a small amount of riboflavin. Hydrogen sulfide, ammonia, carbon dioxide and carbonyl compounds were the photolysis products.

As for the carbonyl compounds, formaldehyde and acetaldehyde were identified from thiazolidine-4-carboxylic acid and djenkolic acid, acetaldehyde from 2-methyl-thiazolidine-4-carboxylic acid and l-cysteine-mercaptal of acetaldehyde, and acetaldehyde and n-butyraldehyde from 2-n-propyl-thiazolidine-4-carboxylic acid and l-cysteine-mercaptal of n-butyraldehyde.     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

Cysteine-aldehyde compounds were prepared by the reactions of l-cysteine with formaldehyde, acetaldehyde, n-butyraldehyde, benzaldehyde and furfural in 50% ethanol solutions. Hydrogen sulfide and ammonia liberated from cysteine-aldehyde compounds in heated aqueous solutions (oil bath : 120°C) were determined. Although thiazolidine derivatives were stable generally in boiling aqueous solution, l-cysteine-furfural compound was unstable and a large amount of hydrogen sulfide compared with other compounds was released.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

Studies on the Synthesis of l-Amino Acids

l-Homoserine was prepared by the reduction of l-aspartic acid β-methyl ester with sodium borohydride in water solution without any racemization. The yield of l-homoserine was about 25% of the theoretical amount, and no product other than l-homoserine, l-aspartic acid and l-aspartic acid β-methyl ester was present in the reaction mixture. The low yield of l-homoserine was ascribed to the hydrolysis of the ester.

l-Azetidine-2-carboxylic acid could not be detected in the reaction mixture. In contrast with the reduction of l-glutamic acid γ-esters, the reduction of l-aspartic acid β-ester was not accompanied by the cyclization.     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

Production of l-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met^?) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met^?, AHV^R, s-Lys^R) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose).

Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.     

Studies on the Photolysis of l-Cysteine and l-Cystine

In the presence and absende of riboflavin, the aqueous solutions of l-cysteine and l-cystine were exposed to sunlight. The solutions in the presence of riboflavin developed the typical flavor of cooked rice. Hydrogen sulfide, ammonia, carbon dioxide and acetaldeohyde were found from the solutions that formed the flavor.

The component responsible for the flavor was not a single one but consisted of three compounds, such as hydrogen sulfide, ammonia and acetaldehyde. An aqueous solution of the mixture containing these three compounds developed organoleptically the typical flavor of cooked rice. These compounds were also present in the vapor of cooked rice.     

Polyethylene Glycol–Modified Papain Catalyzed Oligopeptide Synthesis from the Esters of l-Aspartic and l-Glutamic Acids in Benzene

Comparative studies were made of the polymerization of l-aspartic and l-glutamic acid dialkyl esters using polyethylene glycol–modified papain as a catalyst in phosphate buffer (pH 7.5) and in benzene. Changes in the substrate specificity of papain and in the composition of oligomerized products were observed. In the buffer, the diethyl and di-n-propyl esters of l-glutamic acid were sufficiently converted to high molecular weight oligomers with the accumulation of dimer and trimer, but l-aspartic acid esters were very poor substrates. In benzene, l-aspartic acid esters became more reactive than L-glutamic acid esters. In particular, from l-aspartic acid dimethyl ester the product, which was mainly composed of heptamer to decamer, was obtained in a 90% yield. The reaction in benzene required desalted substrates and a small amount of water to proceed extensively.     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

2-Keto-l-gulonic Acid Fermentation

l-Sorbose metabolism in Pseudomonas aeruginosa IFO 3898 was studied. When the strain was cultivated in l-sorbose medium, l-idonic and 2-keto-l-gulonic acids were detected in the culture broth.

From the results on the metabolism of various sugars and sugar acids with the cell suspension and the metabolites accumulated, the following pathway was proposed for the l-sorbose metabolism in Ps. aeruginosa IFO 3898.

l-Sorbose → l-idose → l-idonic acid → 2-keto-l-gulonic acid.     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

Pyrolysis of Sulfur-containing Amino Acids

Sulfur-containing amino acids (l-cysteine, l-cystine and dl-methionine) were pyrolyzed. From pyrolyzed cysteine and cystine were identified 7~8 volatile compounds including 2-methylthiazolidine which is considered to be the product of the reaction of acetaldehyde with mercaptethylamine, and from pyrolyzed methionine were identified 11 volatiles. At the same time, besides these volatile compounds, alanine, cystine and isoleucine, and alanine, isoleucine and methionine were detected in the pyrolyzed products of cysteine and cystine, respectively, but no amino acid was detected from that of methionine. The mixture of seven identified volatiles generated from l-cystine developed a pop-corn like aroma with a roasted sesame like one, and methylmercaptane seemed to be the main contributor to the pickled radish like odor produced from pyrolysis of dl-methionine. Degradation schemes of cystine and methionine were proposed.     

Microbial Production of l-Phenylalanine from n-Alkanes

A tyrosine auxotroph derived from a hydrocarbon utilizing bacterium, Corynebacterium sp. KY 4309, was found to accumulate a large amount of l-phenylalanine in the broth. The cultural conditions for l-phenylalanine production were studied. The pH value during cultivations exhibited a remakable effect on l-phenylalanine production. The addition of l-tryptophan enhanced the l-phenylalanine accumulation. Shikimic acid and phenylpyruvic acid are possible precursors of phenylalanine biosynthesis in this bacterium. Production of l-phenylalanine attained to a level of 10 mg per ml for 68 hr under optimal conditions.     

Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol

During the course of studies on the oxidative metabolism of d-sorbitol by acetic acid bacteria, it was found that d-sorbitol was almost quantitatively converted to 5-keto-d-fructose via l-sorbose by a certain strain of Gluconobacter suboxydans. In addition to 5-keto-d-fructose, three γ-pyrone compounds, kojic acid, 5-oxymaltol, and 3-oxykojic acid, 2-keto-l-gulonate, and several organic acids such as succinic, glycolic, and glyceric acids were confirmed in the culture filtrate of this bacterium.

• The most suitable carbon source for 5-ketofructose fermentation by Gluconobacter suboxydans Strain 1 was confirmed to be d-sorbitol or l-sorbose using growing and resting cells. d-Fructose had little effect on the formation of this dicarbonylhexose.

• The optimal pH for the formation from l-sorbose by intact cells was found to be at 4.2.

• The activity of the pentose phosphate cycle in the resting cells was calculated as 13~17 μatoms/hr/mg of dry cells by the use of the manometric techniques.

• There was no strain tested so far which could accumulate a large amount of 5- keto-d-fructose from d-sorbitol except this bacterium.

• The experimental results shown in this paper makes the prediction that a certain dehydrogenating system of l-sorbose is functional in the organism, and the metabolic pathways of d-sorbitol via l-sorbose and 5-keto-d-fructose is proposed.

     

Pectin Isolated from the Midrib of Leaves of Nicotiana tabacum

A pectin isolated from tobacco midrib contained residues of d-galacturonic acid (83.7%), L-rhamnose (2.2%), l-arabinose (2.4%) and d-galactose (11.2%) and small amounts of d-xylose and d-glucose. Methylation analysis of the pectin gave 2, 3, 5-tri- and 2, 3-di-O-methyl-l-arabinose, 3, 4-di- and 3-O-methyl-l-rhamnose and 2, 3, 6-tri-O-methyl-d-galactose. Reduction with lithium aluminum hydride of the permethylated pectin gave mainly 2, 3-di-O-methyl-d-galactose and the above methylated sugars. Partial acid hydrolysis gave homologous series of β-(1 → 4)-linked oligosaccharides up to pentaose of d-galactopyranosyl residues, and 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, and di- and tri-saccharides of α-(1 → 4)-linked d-galactopyranosyluronic acid residues.

These results suggest that the tobacco pectin has a backbone consisting of α-(1 → 4)-linked d-galactopyranosyluronic acid residues which is interspersed with 2-linked l-rhamnopyranosyl residues. Some of the l-rhamnopyranosyl residues carry substituents on C-4. The pectin has long chain moieties of β-(1 → 4)-linked d-galactopyranosy] residues.     

Conversion of the Extracellular Dextransucrase Isoenzymes from Leuconostoc mesenteroides NRRL B-1299

Effect of oxygen tension on l-lysine, l-threonine and l-isoleucine accumulation was investigated. Sufficient supply of oxygen to satisfy the cell’s oxygen demand was essential for the maximum production in each fermentation. The dissolved oxygen level must be controlled at greater than 0.01 atm in every fermentation, and the optimum redox potentials of culture media were above ?170 mV in l-lysine and l-threonine and above ?180 mV in l-isoleucine fermentations. The maximum concentrations of the products were 45.5 mg/ml for l-lysine, 10.3 mg/ml for l-threonine and 15.1 mg/ml for l-isoleucine. The degree of the inhibition due to oxygen limitation was slight in the fermentative production of l-lysine, l-threonine and l-isoleucine, whose biosynthesis is initiated with l-aspartic acid, in contrast to the accumulation of l-proline, l-glutamine and l-arginine, which is biosynthesized by way of l-glutamic acid.     

2-Keto-l-gulonic Acid Fermentation

A number of bacterial strains from type culture collections and natural sources were examined in their metabolic characteristics toward sorbitol and l-sorbose.

Paper chromatographic analyses of sorbitol and l-sorbose metabolites obtained from the cultures of various bacteria revealed that the organisms producing 2-keto-l-gulonic acid from sorbitol were merely found in the genera Acetobacter, Gluconobacter and Pseudomonas, whereas those producing the acid from l-sorbose were distributed in the twelve genera of bacteria: Acetobacter, Alcaligenes, Aerobacter, Azotobacter, Bacillus, Escherichia, Gluconobacter, Klebsiella, Micrococcus, Pseudomonas, Serratia and Xanthomonas.

G. melanogenus, which was characterized by excellent production of 2-keto-l-gulonic acid from sorbitol, also formed several other sugars and sugar acids as the sorbitol metabolites. These compounds were identified to be d-fructose, l-sorbose, d-mannonic acid, L-idonic acid, 2-keto-d-gluconic acid and 5-keto-d-mannonic acid, respectively, by means of two-dimensional paper chromatography.

Bacteria producing 2-keto-l-gulonic acid from sorbitol were usually isolated from fruits but not from soil.     

Metabolism of l-Theanine,d-Theanine and the Related Compounds in Bacteria

Some strains of Pseudomonas was found capable of utilizing l-theanine or d-theanine as a sole nitrogen and carbon source. The cell-free extract catalyzes the hydrolysis of the amide group of the compounds and the hydrolase activity was influenced remarkably by the nitrogen source in the medium. l-Theanine and d-theanine were hydrolyzed to yield stoichiometrically l-glutamic acid and d-glutamic acid, respectively, and ethylamine, which were isolated from the reaction mixture and identified.

The theanine hydrolase of Pseudomonas aeruginosa was purified approximately 200-fold. It was shown that the activities of l-theanine hydrolase, d-theanine hydrolase and the heat-stable l-glutamine hydrolase and d-glutamine hydrolase are ascribed to a single enzyme, which may be regarded as a γ-glutamyltransferase from the point of view of the substrate specificity and the properties. This theanine hydrolase catalyzed the transfer of γ-glutamyl moiety of the substrates and glutathione to hydroxylamine. l-Glutamine and d-glutamine were hydrolyzed by the theanine hydrolase and also by the heat-labile enzyme of the same strain of Pseudomonas aeruginosa, whose properties resembled the common glutaminase.     

On the Mechanism of l-Prolyl Diketopiperazine Formation by Streptomyces

Since l-prolyl diketopiperazines, l-prolyl-l-valine anhydride and l-leucyl-l-proline anhydride, had been isolated from the culture filtrate of Streptomyces sp. S-580, the mechanism of l-prolyl diketopiperazine formation by Streptomyces has been studied. These two l-prolyl diketopiperazines were not formed from their constituent amino acids incubated with intact cell or cell free homogenate of this strain in buffered salt solution containing energy source. However, from milk casein, poly peptone or gelatin, the former two were components of the culture medium of this strain, hydrolyzed with the pure streptomyces-protease, these l-prolyl diketopiperazines were obtained (only from gelatin, glycyl-l-proline anhydride were obtained in addition to these two). Furthermore, in hydrolysis of some synthetic l-prolyl peptides with this enzyme, l-prolyl diketopiperazine formation were also studied, and as the result, glycyl-l-proline anhydride was obtained from glycyl-l-prolyl-l-leucine but no l-prolyl diketopiperazine was formed from l-prolyl-l-leucyl-glycine. From these evidences, the possible route of l-prolyl diketopiperazine formation by Streptomyces has been discussed.