We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Morarella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%). Various halogenated organic compounds have been synthesized and are now used as pesticides, herbicides, solvents, and so on in a large scale. Several halogen compounds such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2-bis(chlorophenyl)ethane (DDT), and 1,1,1-trichloroethane have accumulated in the environment and cause environmental pollution. Various enzymes involved in the degradation of halogenated compounds have been found, purified, and characterized. They are categorized into reductive, oxygenative, and hydrolytic dehalogenases (15, 31, 32). 2-Halo acid dehalogenases (DEXs) catalyze the hydrolytic dehalogenation of 2-halo acids and are further classified into four groups based on their stereospecificities. L-DEX acts on L-2-halo acids to give D-2-hydroxy acids (16, 18). D-DEX catalyzes the conversion of D-2-halo acids into L-2-hydroxy acids (27). There are two different types of DL-DEXs, which act on both Land D-2-halo acids: one catalyzes the dehalogenation of Land D-2-halo acids with retention of the C2-configuration of the substrates, and the other catalyzes
The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.
In general, Saccharomyces cerevisiae is a main microorganism in beer brewing, because this microbe has potent ability to produce alcohol dehydrogenase. Recently, we discovered that some genera of mushroom produce alcohol dehydrogenase, and made a beer-like drink using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this drink was achieved with Tricholoma matsutake (1069 mM, 4.6%). This beer-like drink contained about 0.17% ß-D-glucan, which is known to have preventive effects against cancer. The drink showed thrombosis preventing activity: prolonged thrombin clotting time 2.3 fold that of control. Thus, the beer-like drink made using mushroom seems to be a healthful alcoholic beverage.
SummaryWe found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP+ -dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K. pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive. Key Words aspartic acid, dehydrogenase, vitamin B12, Klebsiella pneumoniaeWe have previously reported on vitamin B12 production by Klebsiella pneumoniae IFO 13541, and we pointed out that the growth of the organism and the amount of vitamin B12 produced depended exclusively on the concentration of yeast extract added to the medium. The yeast extract components required were identified as aspartic acid and pyrroloquinoline quinone (PQQ). We have also reported the effects of aspartic acid and PQQ on the production of vitamin B12 by K pneumoniae IFO 13541 and the metabolism of aspartic acid in the vitamin B12 production (1-3).On the other hand, a variety of L-amino acid dehydrogenases have been extensively studied, as reviewed by Ohshima and Soda (4), and used for the synthesis or measurement of L-amino acids (4,5). However, the specific enzyme for L-aspartic acid dehydrogenation has not yet been identified.We found a new reaction of aspartic acid dehydrogenation, a novel NADP+ -dependent aspartate dehydrogenase, in crude extract of vitamin B12-producing K. pneumoniae IFO 13541.We describe here a characterization of the novel NADP+ dependent aspartate 483
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.