An enzymatic method using L-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to L-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of L-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of Lphenylalanine per ml of reaction mixture. The conversion yield from transcinnamic acid was about 70%. The method may provide a rapid and practical way to produce L-phenylalanine useful as an essential amino acid. L-Phenylalaninle ammonia-lyase (PAL; EC 4.3.1.5), an enzyme found in a variety of plants,

Enzymatic production of dihydroxyacetone (DHA) was studied by immobilization of the whole cells of acetic acid bacteria capable of oxidizing glycerol to DHA. Acetobacter xylinum A-9 cells immobilized in a polyacrylamide gel were selected as the most favorable enzyme preparation. The enzymatic properties of immobilized cells converting glycerol to DHA were investigated and compared with those of intact cells. The optimum pH for the immobilized cells was broad (4.0 to 5.5), whereas the intact cells had a narrow pH optimum at 5.5. The thermal stability of the immobilized cells was somewhat higher than that of the intact cells. Apparent Km values for glycerol with both intact and immobilized cells were about equal, 6.3 x 10-2 to 6.5 x 10-2 M. The complete conversion of glycerol to DHA was achieved within 40 h under optimum conditions, and pure crystalline DHA was readily isolated from the reaction mixture with over 80% yield.

To establish a practical method for the fermentative production of l -glutamine, cultural conditions for the accumulation of a large amounts of l -glutamine were investigated by using Flavobacterium rigense 703, which was previously reported by us as a l -glutamine-producing mutant. As a result, a yield of 25 mg of l -glutamine per ml was obtained after a 48-h cultivation in a medium containing glucose, yeast extract, (NH 4 ) 2 -fumarate, KH 2 PO 4 , K 2 HPO 4 , MgSO 4 .7H 2 O, and CaCO 3 (pH 6.4). Accumulation of l -glutamine was dependent upon the concentration of (NH 4 ) 2 -fumarate, and a suboptimum growth at a relatively high concentration of (NH 4 ) 2 -fumarate was essential for the maximum production of l -glutamine. At the optimum conditions, glutamic acid was formed as a by-product at a concentration of less than 1 mg/ml, but accumulation of the other amino acids was negligible. The product was isolated from the culture broth and readily purified by anion-exchange chromatography. The pure crystals of l -glutamine obtained in an 80% yield were optically and chromatographically pure.

A penicillin-resistant mutant of Flavobacterium rigense designated as strain 703, FERM-P no. 3628, was obtained after ultraviolet treatment of F. rigense FERM-P no. 3556. The parent strain produces 0-2-hydroxypropylhomoserine from 1,2-propanediol. The mutant was found to be a good producer ofL-glutamine. The physiological characteristics of strain 703 were different from the general group of L-glutamic acid-producing bacteria. Strain 703 required L-tryptophan and thiamine but not biotin for its growth. L-Glutamine formation on a specific basis, however, was independent of L-tryptophan and thiamine. Biotin and penicillin were also not effective. Only ammonium fumarate acted as an effective factor on L-glutamine formation. Accumulation of L-glutamine by strain 703 was 10 mg/ml at 300C for 48 h in a chemically defined medium containing 3% diammonium fumarate.

During an investigation of microorganisms utilizing petrochemicals, a strain identified as Flavobacterium rigense was found to accumulate a new amino acid in a medium containing 1,2-propanediol as the sole carbon source. Cultural conditions for the accumulation of the product were investigated, and, as a result, the yield was increased to 2.8 mg/ml after a 5-day incubation in a medium containing 8% 1,2-propanediol. The pure amino acid was isolated, and its structure was investigated. Elemental analysis and infrared, nuclear magnetic resonance, and mass spectral analyses indicated that the amino acid is 0-2-hydroxypropylhomoserine.

The amounts of extracellular D-alanine accumulated by Corynebacterium fascians ATCC 21950 in a medium containing glycerol as the sole carbon source is increased to almost 12 mg/ml by adding pyruvate to the medium. Cell-free extracts of C. fascians were shown to possess both L-alanine dehydrogenase and alanine racemase activities. These results indicated that a mechanism exists that allows this microorganism to synthesize D-alanine from pyruvate. A study comparing the optical purity of the intracellular alanine and the extracellular alanine suggested that the cell membrane possesses the stereospecific permeability for D-alanine. Thus, it may be concluded that L-alanine is first formed from pyruvate by L-alanine dehydrogenase and then converted to D-alanine by racemase inside the cells. Subsequently, only D-alanine leaks out stereospecifically through the cell membrane, and large amounts of D-alanine accumulate in the extracellular medium.