Abstract
Pyruvic acid is a key intermediate of microbial
carbohydrate metabolism. It can
be used as a starting
material for production of amino acids, pharmaceuticals
and pesticides. It is
also possible to utilize
pyruvic acid itself as a flavoring agent. In this work
the enhancement of
pyruvic acid secretion by
Candida utilis IFO 0396 was studied with reference to
coenzyrne regeneration
and metabolic
modifications. The coenzyme dependent conversion of
pyruvate to L alanine was
investigated with
immobilized and free enzymes.
High glucose concentration was shown to be beneficial for
pyruvic acid
production. Oxygen availability,
affecting the redox balance in the cells, was a critical
factor. In microbial
metabolism pyruvic acid is
formed at the end of the glycolytic sequence, which
operates only in the
presence of sufficient amounts of
the oxidized coenzyrne NAD+. The pathway of coenzyme
regeneration corresponded
with the
extracellular redox potentials measured. The highest
pyruvic acid yield
observed in this study, 0.33 g (g
glucose)~l, was obtained using a decreasing aeration
profile. In these
conditions pyruvic acid was also
produced independently of cell growth. Ammonium was shown
to be a better
nitrogen source for pyruvic
acid production than nitrate. By addition of optimal
concentrations of arsenate
or dinitrophenol
(uncouplers of oxidation and phosphorylation) or arsenite
(blocking pyruvate
degradation by the
pyruvate dehydrogenase complex) pyruvic acid production
could be enhanced. The
most efficient
metabolic modifier was acetoin, which was
stoichiometrically reduced to 2,3
butanediol.
Acetoin reductase, which catalyzes this reaction, was
shown to be predominantly
NADH dependent. The
highest pyruvic acid productivity, 0.5 g l lh ', was
obtained by acetoin
addition. Ethanol was the
dominant by product in pyruvic acid production. Glycerol
was produced under
strongly reducing
conditions. Minor amounts of other carboxylic acids,
amino acids and volatile
compounds were also
produced. Pyruvate decarboxylase, catalyzing the first
step of pyruvate
degradation to ethanol, was
shown to be present even in conditions where no ethanol
could be detected.
Clear correlation between
ethanol concentration and the specific activity of
pyruvate decarboxylase was
not observed.
The amination of pyruvate to L alanine, catalyzed by
alanine dehydrogenase,
requires NADH. NADH
could be oxidized in the production of carbon dioxide
from formate by formate
dehydrogenase. Two
different methods, droplet gel entrapment and covalent
binding to tresyl
chlorideactivated agarose, were
examined for the coimmobilization of these two
dehydrogenases. The kinetic
constants as well as
optimal conditions for continuous reactors applying these
coimmobilized enzymes
were determined. In
long term operation (30 days) the highest L alanine
productivity per unit of
alanine dehydrogenase,
conversion and total turnover number of the coenzyme
(TIN) were obtained using
free enzymes.
However, using the droplet gel immobilized biocatalyst
the decrease of both the
conversion ratio and the
TTN value were smallest during long term operation
indicating enchanced
stability of the enzymes. In
this system 60 % of the maximum production of L alanine
remained after 30 days,
in comparison with 10
% when covalently boud enzymes were employed. Using free
enzymes 80 % of the
alanine produced was
obtained during the first five days, after which the
production rate slowed
down considerably.
Original language | English |
---|---|
Qualification | Doctor Degree |
Awarding Institution |
|
Award date | 11 Jun 1991 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 951-38-3946-X |
Publication status | Published - 1991 |
MoE publication type | G4 Doctoral dissertation (monograph) |
Keywords
- pyruvates
- metabolism
- candida utilis
- coenzymes
- enzymes