D-Amino acid production from racemic amino acids by a microbial asymmetric degradation

To obtain D-amino acid from a mixture of racemic amino acids, Proteus vulgaris, having the ability of selective degradation of L-isomer in various DL-amino acids mixtures, was used as a biocatalyst. D-Isomer crystals of valine, leucine, isoleucine, histidine and ornithine were isolated at a good yield (25~41%) from the reaction mixture after 24~72 hours.     

Novel molecular identification methods for the larger black flour beetle,Cynaeus angustus (Coleoptera: Tenebrionidae)

Two new methods were developed for identifying Cynaeus angustus (LeConte) (Coleoptera: Tenebrionidae) by DNA amplification using simplex and real-time PCR targeting the cytochrome c oxidase subunit I (COI) sequence reported previously. The specificities of the PCR primers and probe were also confirmed by the two PCR methods using the 22 main stored-product insect species, including DNA samples from nine tenebrionid beetle species. The results showed that the newly developed simplex and real-time PCR-based methods have sufficient specificity for analysis. The limits of detection for C. angustus total DNA by the simplex and multiplex PCR were 320 fg and 20 pg, respectively.

     

Identification of protein functions from a molecular surface database,eF-site

A bioinformatics method was developed to identify the protein surface around the functional site and to estimate the biochemical function, using a newly constructed molecular surface database named the eF-site (electrostatic surface of Functional site. Molecular surfaces of protein molecules were computed based on the atom coordinates, and the eF-site database was prepared by adding the physical properties on the constructed molecular surfaces. The electrostatic potential on each molecular surface was individually calculated solving the Poisson–Boltzmann equation numerically for the precise continuum model, and the hydrophobicity information of each residue was also included. The eF-site database is accessed by the internet (http://pi.protein.osaka-u.ac.jp/eF-site/). We have prepared four different databases, eF-site/antibody, eF-site/prosite, eF-site/P-site, and eF-site/ActiveSite, corresponding to the antigen binding sites of antibodies with the same orientations, the molecular surfaces for the individual motifs in PROSITE database, the phosphate binding sites, and the active site surfaces for the representatives of the individual protein family, respectively. An algorithm using the clique detection method as an applied graph theory was developed to search of the eF-site database, so as to recognize and discriminate the characteristic molecular surfaces of the proteins. The method identifies the active site having the similar function to those of the known proteins.     

Scale-up of D-lysine production from L-lysine by successive chemical racemization and microbial asymmetric degradation

In order to develop an industrial production process of D-lysine from L-lysine, successive chemical racemization and a microbial asymmetric degradation were investigated in a pilot scale. The racemization of L-lysine proceeded quantitatively. The cultivation conditions of Comamonas testosteroni for L-lysine degradation were optimized in a 30L jar fermenter and scaled-up to 5m tank. The L-lysine-degrading reaction was performed by using racemized lysine crystals as substrate and C. testosteroni IAM 1048 intact cells as biocatalysts. Crystalline D-lysine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 36% from the reaction mixture by simple purification. On the basis of these results, we have designed a process for a large scale production of D-lysine.     

n-acylation of β-amino alcohol by acyl migration following enzyme-catalyzed esterification

Lipase-catalyzed n-acylations of β-amino alcohols such as ethanolamine and l-serine were investigated. To prepare n-acyl derivatives by taking advantage of the acyl migration, we first carried out a screening of suitable enzymes for the desired reaction. As a result, we found a higher activity for n-acylation with Lipase L. This lipase had higher hydrolytic activity for the o-acyl compound but not the n-acyl compound. The observation shows that n-acylation results from the esterification and successive acyl migration into the amino group. Using Lipase L, we then investigated the n-acylation of ethanolamine or l-serine with fatty acids as acyl donors. The reaction parameters for the n-acylation were clarified.     

d-Methionine preparation from racemic methionines by Proteus vulgaris IAM 12003 with asymmetric degrading activity

The microbial degradation of l-methionine was investigated in order to develop a practical process for d-methionine production from racemic methionines. Among the 1000 culture strains tested, microorganisms belonging to the Achromobacter, Bacillus, Micrococcus, Morganella, Proteus, Providencia, Pseudomonas and Sarcina genera exhibited a high l-methionine-degrading activity. Proteus vulgaris IAM 12003 was determined to be the best strain and was used as a biocatalyst for eliminating the l-isomer. The degradation of l-isomer in this P. vulgaris IAM 12003 cell was assured by the action of l-amino acid oxidase. The maximum rate of l-isomer degradation was obtained at 30 °C and pH 8.0. Under these optimal conditions, the l-isomer in a 100 g/l mixture of racemic methionines was almost degraded within 20 h, with 46.5 g d-methionine/l remaining in the reaction mixture. Crystalline d-methionine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 30% from the reaction mixture by simple purification. Received: 17 June 1996 / Received last revision: 11 September 1996 / Accepted: 29 September 1996     

Direct activation of phospholipase A2 by GTP-binding protein in human peripheral polymorphonuclear leukocytes.

In human peripheral polymorphonuclear leukocyte (PMN), 10% of PLA2 activity was found in the particulate fraction. In the particulate fraction, the activity of phospholipase A2 was enhanced 270% by 100 microM guanosine 5'-[gamma-thio]triphosphate, a hydrolysis-resistant analog of GTP. In the soluble fraction, such enhancement was not observed. Guanosine 5'-[beta-thio]diphosphate (2 mM), which irreversibly inactivates GTP-binding protein, blocked the enhancement in the particulate fraction. Membrane-binding phospholipase A2 activity of PMN would thus appear to be regulated directly by GTP-binding protein.     

Evidence of a role for phosphatidylinositol synthesis in human amnion cell proliferation.

Phosphatidylinositol (PtdIns) is the key precursor of phosphoinositide-derived intracellular mediators. The effects of changing the rate of PtdIns synthesis on mitogenic activity of human amnion-derived WISH cells were investigated. Incubation of the cells with [3H]inositol caused a time- and dose-dependent PtdIns labeling. Exogenous Ca2+ inhibited [3H]inositol incorporation in a dose-dependent fashion; half-maximal inhibition occurred with 0.3-1.0 mM Ca2+. In contrast, removal of cytosolic Ca2+ by ionophore A23187 and 1 mM EGTA induced enhancement of the PtdIns labeling as a function of A23187 concentration, perhaps through release of inhibitory effects of endogenous Ca2+. The A23187-stimulated PtdIns labeling with [3H]inositol was not abolished by additional unlabeled inositol, suggesting that [3H]inositol labeling of PtdIns occurred mainly through de novo synthesis catalyzed by PtdIns synthase (EC 2.7.8.11). In cells with PtdIns synthase activity decreased by exogenous Ca2+, [3H]thymidine incorporation was also inhibited, while A23187 caused dose-dependent enhancement of thymidine incorporation. The changes in PtdIns synthase activity occurred in parallel with changes in mitogenic activity caused by increasing the dose of exogenous Ca2+ or A23187. A similar lowering of mitogenic activity was observed upon suppression of PtdIns synthase by pemirolast potassium (9-methyl-3-1H-tetrazol-5yl-4H-pyrido[1,2-a]pyridin-4-one potassium) via a Ca(2+)-independent mechanism. These data demonstrate that changes in PtdIns synthase activity by some agents acting via different mechanisms are associated with parallel changes in thymidine incorporation, and suggest that PtdIns production is tightly coupled to cell proliferation in human amnion cells.