Haruo Misono

Purification and properties of a novel enzyme, L-α-amino-ϵ-caprolactamase from Cryptococcus laurentii

A novel synthetic process of L-lysine from DL-~-amino-e-caprolactam with almost 100% yield has been established [1 ]. This process is composed of two new enzymatic reactions, the selective hydrolysis of L-a-amino-e-caprolactam to L-lysine, and the racemization of a-amino-e-caprolactam, which proceed in the same vessel. The L-a-amino-e-caprolactam hydrolyzing enzyme (L-~-amino-e-caprolactamase (EC 3.5.2.)) has been found in the cells of Cryptococcus laurentii and other yeasts [2,3]. a-Amino-e-caprolactam racemase has been found in the cells ofAchromobacter obae and other bacteria [4]. Both enzymes were partially purified from C laurentii and A. obae, respectively [5,6]. In this paper, we describe the purification of the new amidohydrolase from C. laurentii to homogeneity and some of its properties.

Crystalline L-leucine dehydrogenase

Abstract The preparation of crystalline L-leucine dehydrogenase from the extract of Bacillus sphaericus is described. The enzyme is homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight is 280,000. The enzyme catalyzes the oxidative deamination of L-leucine, L-valine, L-isoleucine, L-norleucine and L-norvaline, while none of D-leucine, D-valine, L-alanine and L-glutamate is deaminated. NAD only serves as the hydrogen acceptor.

Occurrence of meso-α,ɛ-diaminopimelate dehydrogenase in Bacillus sphaericus

Abstract A new amino acid dehydrogenase catalyzing the oxidative deamination of meso -α,ɛ-diaminopimelate was found in the crude extract of Bacillus sphaericus IFO 3525. This dehydrogenase requiring NADP was specific for meso -diaminopimelate and the other isomers were not substrates. The enzyme was optimally active at about pH 10.5. NAD could not replace NADP.

Purification and crystallization of D-amino acid aminotransferase of Bacillus sphaericus.

Enzymatic transamination of D-amino acids was demonstrated in bacilli [l-4] , Rhodospirillum rubrum [5] , and mammalian liver [6] . D-Alanine aminotransferase was purified to near homogeneity from the extract of Bacillus subtilis to elucidate the enzymological properties [7-91. We found the occurrence of high activity of D-amino acid transaminase in the cell-free extract of Bacillus sphaerlcus IF0 3525 [IO] . The present communication describes the purification and crystallization of the enzyme from B. sphaericus, and some of its properties. reaction system consisted of 25 pmoles of D-amino acids, 25 pmoles of cY-keto acids, 1 pmole, of PLP, 80 E.tmoles of potassium phosphate buffer (pH 8.0) and enzyme in a final volume of 1 .O ml. Enzyme was replaced by water in a blank. After the mixture was, incubated at 37°C for 20 min, the amount of pyruvate or amino acids formed was determine,d with salicylaldehyde [8] or ninhydrin [14] , respectively. Oneunit of enzyme was defined as the amount of enzynie,which catalyzes the formation of 1 pmole of pyruvate or, amino acids per min. Specific activity was expressed as units per mg of protein. Protein was determined by the method of Lowry et al. [16] .

Properties of taurine : α-ketoglutarate aminotransferase of Achromobacter superficialis Inactivation and reactivation of enzyme

The activity of taurine: alpha-ketoglutarate aminotransferase (taurine: 2-oxoglutarate aminotransferase, EC 2.6.1.55) from Achromobacter superficialis is significantly diminished by treatment of the enzyme with (NH4)2SO4 in the course of purification, and recovered by incubation with pyridoxal phosphate at high temperatures such as 60 degrees C. The inactive form of enzyme absorbing at 280 and 345 nm contains 3 mol of pyridoxal phosphate per mol. The activated enzyme contains additional 1 mol of pyridoxal phosphate with a maximum at 430 nm. This peak is shifted to about 400 nm as a shoulder by dialysis of the enzyme, but the activity is not influenced. The inactive form is regarded as a partially resolved form, i.e. a semiapoenzyme. The enzyme catalyzes transamination of various omega-amino aicds with alpha-ketoglutarate, which is the exclusive amino acceptor. Hypotaurine, DL-beta-aminoisobutyrate, beta-alanine and taurine are the preferred amino donors. The apparent Michaelis constants are as follows; taurine 12 mM, hypotaurine 16 mM, DL-beta-aminoisobutyrate 11 mM, beta-alanine 17 mM, alpha ketoglutarate 11 mM and pyridoxal phosphate 5 micron.

Crystalline taurine:α-ketoglutarate aminotransferase from Achromobacter superficialis

Summary The preparation and some properties of crystalline taurine:α-ketoglutarate aminotransferase from Achromobacter superficialis is described. The enzyme exhibits absorption maxima at 280 and 340 mμ. No appreciable spectral change was observed on varing pH. The enzyme is homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight is 156,000, and four moles of a vitamin B 6 compound are bound per mole of the enzyme. The enzyme catalyzes the transamination of DL-β-aminoisobutyrate, β-alanine, taurine, and 3-aminopropanesulfonate to α-ketoglutarate. The enzyme inactivated by treatments with ammonium sulfate is activated by incubation with pyridoxal 5′-phosphate at 45–60° for about 10 min.

Purification and crystallization of L-ornithine:α-ketoglutarate δ-aminotransferase from Bacillus sphaericus

L-Ornithine 6-aminotransferase (L-ornithine:2oxoacid aminotransferase (EC 2.6.1.13)) catalyzes the 6-transamination of L-ornithine with a-ketoglutarate to produce glutamic-y-semialdehyde, which is spontaneously converted to A’-pyrroline-5-carboxylate, and Lglutamate and plays an important role in ornithine and proline metabolism. This enzyme has been purified to homogeneity from rat liver and kidney, and its enzymological properties and physiological functions extensively studied [l-8]. Although microbial ornithine 6-aminotransferases have been demonstrated [9-l 11, little effort has been devoted to their purification and characterization. Here we describe the purification and crystallization of the enzyme from Bacillus sphaericus and some of its properties.