Bioconversion of ethyl 4-chloro-3-oxobutanoate by permeabilized fresh brewer’s yeast cells in the presence of allyl bromide

Abstract

Ethyl(R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) are obtained by cetyltrimetylammonium bromide (CTAB) permeabilized fresh brewer's yeast whole cells bioconversion of ethyl 4-chloro-3-oxobutanoate (COBE ) in the presence of allyl bromide. The results showed that the activities of alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in CTAB permeabilized brewer's yeast cells increased 525 and 7.9-fold, respectively, compared with that in the nonpermeabilized cells and had high enantioselectivity to convert COBE to (R)-CHBE. As one of co-substrates, glucose-6-phosphate was preprepared using glucose phosphorylation by hexokinase-catalyzed of CTAB permeabilized brewer's yeast cells. In a two phase reaction system with n-butyl acetate as organic solvent and with 2-propanol and glucose-6-phosphate as co-substrates, the highest (R)-CHBE concentration of 447 mM was obtained with 110-130 g/l of the CTAB permeabilized cells at optimized pH, temperature, feeding rate and the shake speed of 125 r/min. The yield and enantiomeric excess (ee) of (R)-CHBE reached 99.5 and 99%, respectively, within 6 h.

Effects of industrial storage on the bioreduction capacity of brewer’s yeast

“…Yeast suspension at 0.5 ml (1 £ 10 7 cells per ml suspension) was mixed with 0.5 ml of methylene blue and examined microscopically after 5 min to count the living and dead cells as before. The yeast cells were then permeabilized with 0.2% w/v CTAB according to the method described previously [13].…”

“…Recently, a new, simple and eYcient method has been developed for bioreduction using permeabilization pretreated microorganism cells containing either ADH and glucose dehydrogenase [11] or ketoreductase and a G6PDH [12]. In our laboratory, CTAB permeabilized brewer's yeast cells were successfully used as the whole cell biocatalysts for bioreduction of ethyl-4-chloro-3-oxobutanoate to ethyl(R)-4-chloro-3-hydroxybutanoate [13]. Nevertheless, yeast cell has a limited lifespan, usually within 30 divisions, before entering a non-productive state of senescence, followed by cell death and autolysis [14].…”

Characterization of Alcohol Dehydrogenase from Permeabilized Brewer's Yeast Cells Immobilized on the Derived Attapulgite Nanofibers

“…ketobutyric acid [1][2][3][4]. However, with permeabilized cells being reused, the activities of the intracellular enzymes and the productivity of chiral alcohol was significantly decreased, respectively [3].…”

Asymmetric Reduction of Heteroaryl Methyl Ketones Using <i>Daucus carota</i>

“…The present process developed has the advantage over the currently known chemical and biological methodologies in obtaining value added chiral aromatic hetero alcohols in higher yields and enantioselectivity [28,29]. To study the steric-orientation of the enzyme active site in enantioselective reduction of the heteroaryl methyl ketones to respective chiral alcohols, it is planned to carry out chemical additive studies like allyl bromide and ethylenediamine tetra acetic acid (EDTA) that influence the stereoselectity in enzymatic bioreduction processes [30][31][32]. On addition of allyl bromide (0.2 mM solution) to the incubation medium the process of bio-reduction of pyridyl ketones to chiral alcohols using D.carota, was found increased by 20% -40%, whereas no difference was observed in the enzyme dehydrogenase activity on addition of EDTA (0.5 mM) (results not presented).…”