Satoshi Hanzawa

Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding α-keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol−1) yield from 180 mM NPA and 360 mM Glu.

Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation

The effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures <300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (ΔV ‡) were about −75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (ΔV ‡∼−60 ml/mol). The intrinsic fluorescence of TLN changed at pressures >300 MPa, and the latter two mutants showed a smaller ΔG app and ΔV app of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region.

Preparative-scale Enzyme-catalyzed Synthesis of (R)-α-Fluorophenylacetic Acid

A preparative-scale asymmetric synthesis of (R)-α-fluorophenylacetic acid, a useful chiral derivatizing reagent, is described. Starting from ethyl α-bromophenylacetate, α-fluorophenylmalonic acid dipotassium salt was prepared in three steps (54% yield), including nucleophilic substitution by the fluoride ion as the keystep. Both the purified form and crude preparation of arylmalonate decarboxylase in E. coli worked well on this substrate, and (R)-α-flurophenylacetic acid (>99% e.e.) was prepared in a quantitative yield.

Construction of affinity changeable antibody in response to Ca2

Immunoaffinity chromatography is a powerful method for purification of proteins because of the high selectivity and avidity of antibodies. Due to the strength of antigen–antibody binding, however, elution of proteins bound to antibodies that are covalently immobilized on the column is performed by temporary denaturation of the antibody. Therefore, the development of milder elution conditions could improve the recovery of the antibodies and prolong the life of the immunoaffinity column. We describe the design and construction of an antibody that changes its affinity in response to external stimuli. The heavy chain and light chain of a single chain Fv of the D1.3 antibody against hen egg-white lysozyme (HEL) were fused at the N- and C-termini, respectively, of the calmodulin-M13 fusion protein. The affinity of this fusion protein for HEL could be modulated by changing the Ca2+ concentration.

The first synthesis of both enantiomers of [α-2H]phenylacetic acid in high enantiomeric excess

Arylmalonate decarboxylase (EC. 4. 1. 1. 76) catalysed decarboxylation followed by enantioface-differentiating protonation of [α-2H]- and [α-1H]phenylmalonic acid in 1H2O and 2H2O respectively, gave highly enantiomerically enriched (R)- and (S)-[α-2H]phenylacetic acid in quantitative yields.

Pressure-induced activation and pressure-tolerance of mutated thermolysin at 119

Abstract The pressure activation of thermolysin substitution at the 119th site was studied for four mutants and the wild type (Q119Q. Q119N, Q119R, Q119E and Q119D). The highest activation recorded over 30 fold and the activation volumes (ΔV‡) were about -75ml/mol for Q119Q, Q119N and Q119R, while Q119E and Q119D showed only a10 fold activation and ΔV‡ of around -6Oml/mol. The pressure-tolerance of these enzymes were investigated through the in situ observation of their intrinsic fluorescence. Q119E and Q119D showed smaller ΔG app and ΔG app of transition than the wild type.