| Summary: | 碩士 === 逢甲大學 === 化學工程研究所 === 86 === In Escherichia coli, aspartate aminotransferase (encoded by
aspC) and aromatic amino acid aminotransferase (encoded by tyrB)
share overlapping substrate specificity in the synthesis of
aromatic amino acids. Through the transamination reactions
catalyzed by AspC or TyrB, L-phenylalanine can be produced from
phenylpyruvate with aspartic acid as the amino donor.
Consequently, oxaloacetate is produced as the by-product. To
modulate and enhance the production levels of proteins, both
aspC and tyrB were subcloned into a runaway vector. Employing
resting cells of AspC- or TryB-overproducting E. coli K-12
strains for L-phenylalanine productions resulted in molar yields
of 70% and 50%, respectively. Further introduction of
phosphoenolpyruvatecarboxykinase (encoded by pck) into the
bioconversion reactions, the conversion yields were improved to
93% from 70% and 70% from 50% in relatively short time. These
results correspond to 1.68 mg and 1.26 mg of L-phenylalanine
produced per mg dry cell weight per hour, which account for 30-
and 40-fold increase compared to the previous report [13].Pck is
the enzyme capable of converting oxaloacetate to
phosphoenolpyruvate .Accordingly, itsuggests that the increased
yields by additional involvement of Pck may be due to the relief
of the inhibitory effect of oxaloacetate on aminotransferases.A
transcriptional fusion was constructed by placing lacZ gene
downstream of the tyrB promoter. By altering environmental
conditions, the responding activities of LacZ were measured and
used to determine the optimal condit ions for production of TyrB
protein on a runaway vector. It was found that the highest yield
of TyrB protein was obtained when the culture was continued for
7 hours in Luria-Bertani medium or 11 hours in M9 minimal medium
supplemented with 0.2% of glucose after shifting the temperature
from 30℃ to 40℃at 0.5 OD550 mm unit.
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