Hideo Hirohara

Studies on hydrolysis of chiral, achiral and racemic alcohol esters with Pseudomonas cepacia lipase: mechanism of stereospecificity of the enzyme

Steady-state kinetics of Pseudomonas cepacia nlipase-catalysed hydrolysis of five analogous chiral and achiral nsubstrates, i.e. (R)- and n(S)-1-methyl-2-(4-phenoxyphenoxy)ethyl acetates (R)- nand (S)-1a, (R)- and n(S)-2-methyl-2-(4-phenoxyphenoxy)ethyl acetates (R)- nand (S)-1b and 2-(4-phenoxyphenoxy)ethyl acetate 1c, were ninvestigated in sufficiently emulsified reaction mixtures of nwater-insoluble substrates. The apparent Michaelis constant nKm values were identical for all the esters, and no nnonproductive binding was observed in these substrates. The apparent ncatalytic constants kcat were found to reflect the nleaving abilities of the alcoholate ions for the fast-reacting nenantiomers. These observations, based on the findings that acyl-enzyme nintermediate formation was rate-determining in the overall reaction, nstrongly suggested that all the substrates are bound to the enzyme in nthe same manner whether or not the alcohol moiety has a medium-sized nsubstituent LM at the stereocentre and that the breakdown of na tetrahedral intermediate is rate-determining in the acylation of the nenzyme. Time courses were also studied for the hydrolysis of racemic n1-ethyl-2-(4-phenoxyphenoxy)ethyl acetate 1d together with 1a, 1b and n1c. The enzyme distinguished (R)-1d from its antipode perfectly nand hydrolysed only the (R)-enantiomer. These results were ninterpreted to indicate that LM of the slow-reacting nenantiomer is positioned close to the imidazole ring of the catalytic nHis and hinders NIµ2 of the residue from forming a weak ninteraction with O1 of the leaving alcohol and that the nbreakdown of the tetrahedral intermediate is thus inhibited.

Studies on enantioselective hydrolysis of the acetic ester of a secondary alcohol with Arthrobacter lipase

SummaryCharacteristics of the enantioselective hydrolysis of the acetic ester of 4-hydroxy-3-methyl-2-(2-propynyl)-2-cyclopentenone (HMPC) by Arthrobacter lipase were investigated in a water/oil biphasic reaction mixture. Kinetic studies revealed that the strict enantioselectivity was entirely due to a difference in the catalytic constants for the enantiomeric substrates and that (S)-HMPC acetate acted as a competitive inhibitor. The comparison of enantioselectivity for the acetates of HMPC analogues indicated that hydrophobic substituents in the HMPC molecule were essential for the strict enantioselectivity.

Continuous Hydrolysis of Lactose in Skim Milk and Acid Whey by Immobilized Lactase of Aspergillus Oryzae

We report here the development of a stable immobilized lactase to hydrolyze lactoses in both skim milk and acid whey. Aspergillus oryzae lactase was used. This fungal lactase has good thermal stability and effective activity in the pH ranges of both milk and acid whey (1,2).