Erschienen:
[Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2006
Sprache:
Englisch
Entstehung:
Hochschulschrift:
Dissertation, 2006
Anmerkungen:
Beschreibung:
The enzymatic production of R-phenylacetylcarbinol (R-PAC), with either whole cells or partially purified pyruvatedecarboxylase (PDC) as the biocatalyst, requires high PDC activity and an inexpensive source of pyruvate for aneconomical feasible biotransformation process.Microbial pyruvate produced by a vitamin auxotrophic strain of Candida glabrata was selected as a potential substratefor biotransformation. With an optimal thiamine concentration of 60 µg/l, a pyruvic acid concentration of 43 g/l and yield of0.42 g/g glucose consumed were obtained. Using microbially-produced unpurified pyruvate resulted in similar PACconcentrations to those with commercial pure substrate confirming its potential for enzymatic PAC production.To obtain high activity yeast PDC, Candida utilis was cultivated in a controlled bioreactor. Optimal conditions for PDCproduction were identified as: fermentative cell growth at initial pH at 6.0 followed by pH downshift to 3.0. Averagespecific PDC carboligase activity of 392 ± 20 U/g DCW was achieved representing a 2.7-fold increase when compared to aconstant pH process. A mechanism was proposed in which the cells adapted to the pH decrease by increasing PDC activityto convert the accumulated internal pyruvic acid via acetaldehyde to ethanol thereby reducing intracellular acidification.The effect of pH shift on specific PDC activity of Saccharomyces cerevisiae achieved a comparable increase of specificPDC carboligase activity to 335 U/g DCW. The effect of pyruvic acid at pH 3.0 on induction of PDC activity wasconfirmed by cultivation at pH 3 with added pyruvic acid.Using microarray techniques, genome-wide transcriptional analyses of the effect of pH shift on S. cerevisiae revealed atransient increased expression of PDC1 after pH shift, which corresponded to the increase in specific PDC activity(although the latter was sustained for a longer period). The results showed significant gene responses to the pH shift withapproximately 39 % of the yeast genome involved. The induced transcriptional responses to the pH shift were distinctiveand showed only limited resemblance to gene responses reported for other environmental stress conditions, namelyincreased temperature, oxidative conditions, reduced pH (succinic acid), alkaline pH and increased osmolarity.