Immobilized d-Amino Acid Oxidase Preparation,Some Enzymatic Properties,and Potential Uses

Immobilization of d-amino acid oxidase was investigated by covalently binding the enzyme to cyanogen bromide activated polysaccharides. Among polysaccharides tested, Sepharose 6B was found to be the best carrier.

Some enzymatic properties of the immobilized enzyme were investigated and compared with those of the native enzyme. The optimum pH of the immobilized enzyme was shifted by 0.5 pH units to the acid side in comparison with that of the native enzyme. With regard to substrate specificity, heat stability and effect of temperature, no significant differences were observed between the immobilized and native enzymes.

The immobilized enzyme was conveniently used for a determination of d-amino acids and an analysis of optical purity of l-amino acids.     

Continuous production of L-malic acid by immobilized cells

l-Malic acid is used extensively in the pharmaceutical industry and as a food additive. It is now produced on an industrial scale by the enzymatic conversion of fumaric acid using immobilized cells of Brevibacterium flavum. Recent improvements to this system, especially the use of x-carrageenan supports, have resulted in a continuous process capable of yielding 30 tonnes of l-malic acid per month.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

Norleucine accumulation by a norleucine-resistant mutant of Serratia marcescens.

A norleucine-resistant mutant was derived from an isoleucine-valine auxotroph of a leucine accumulator of Serratia marcescens. The norleucine-resistant mutant could accumulate norleucine from norvaline in the medium without the addition of methionine, which antagonized norleucine. This mutant constitutively formed homoserine-O-transsuccinylase.     

Production of l-Glutamine by a Penicillin-Resistant Mutant of Flavobacterium rigense

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.     

Glutathione production coupled with an ATP regeneration system

Summary Escherichia coli cells possessing glutathione synthetase and acetate kinase activities were immobilized with carrageenan gel. To enhance the operational stability, immobilized cells were treated with hardening agent, glutaraldehyde in the presence of hexamethylenediamine. The continuous production of glutathione was investigated using the column packed with immobilized Escherichia coli cell preparations. Glutathione was continuously produced by this column in the presence of acetyl phosphate and the half-life of this column was calculated to be 8 days at the flow rate of S.V.=0.1 h^–1 at 37°C.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Immobilized Aspartase-Containing Microbial Cells: Preparation and Enzymatic Properties

The immobilization of asparatase-containing Escherichia coli was investigated by various methods, and the most active immobilized cells were obtained by entrapment in a polyacrylamide gel lattice. Other asparatase-containing bacteria were also entrapped by the same method, and the enzymatically active immobilized cells were obtained. The aspartase activity of the immobilized E. coli cells was increased nine- to tenfold by autolysis of the cells entrapped in the gel lattice. Enzymatic properties of the immobilized E. coli cells were investigated and compared with those of the intact cells. The optimal pH was 8.5 for the immobilized cells and 10.5 for the intact cells. The aspartase activities of immobilized and intact cells were not activated by Mn(2+), which can activate the immobilized and native aspartases. The heat stability of the immobilized cells was somewhat higher than that of the intact cells. Bivalent metal ions such as Mn(2+), Mg(2+), Ca(2+) protected against thermal inactivation of the aspartase activity of the immobilized and intact cells.     

Basic Studies for Continuous Production of L-Aspartic Acid by Immobilized Escherichia coli Cells

By using a column packed with immobilized Escherichia coli cells entrapped in a polyacrylamide gel lattice, conditions for continuous production of L-aspartic acid from ammonium fumarate were investigated. When a solution of 1 M ammonium fumarate (pH 8.5) containing 1 mM Mg(2+) was passed through the immobilized cell column at a flow rate of space velocity (SV) = 0.8 at 37 C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in good yield. The immobilized cell column was very stable.     

Continuous production of urocanic acid by immobilized Achromobacter liquidum cells

Several microorganisms having higher L -histidine ammonia-lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat-treated at 70°C for 30 min to inactivate the urocanase. Enzymatic properties of the immobilized A. liquidum cells were investigated and compared with those of the intact cells. No difference was observed between the pH activity curve and optimal temperature for the intact and immobilized cells. The permeability of substrate or product through the cell wall was increased by immobilization of the cells. When an aqueous solution of 0.25M L -histidine (pH 9.0) containing 1mM Mg²⁺ was passed through a column packed with the immobilized A. liquidum cells at a flow rate of SV = 0.06 at 37°C, L -histidine was completely converted to urocanic acid. The L -histidine ammonia-lyase activity of the immobilized cell column was stable over 40 days at 37°C. From the effluent of the immobilized cell column, Urocanic acid was easily obtained in a good yield.