Cloned gene expression in recombinant Saccharomyces cerevisiae 20B-12 containing three different plasmids was compared in batch and fed-batch cultures. The plasmids pNA3, pNA7, and pNA9 contain the alpha-amylase gene under the control of SUC2, PGK, and GAL7 Promoters, respectively. The synthesis of alpha-amylase was therefore induced by low glucose concentration for the SUC2 and PGK promoters, and by galactose for GAL7 promoter. The specific cell growth rates were similar among cells harboring the three different plasmids; they decreased from 0.35 to 0.38 h(-1) during the cell growth phase to 0.03 to 0.06 h(-1) during the production phase. The secretory alpha-amylase activity of cells harboring plasmid pNA7 was 129 U/mL in fed-batch culture, which was 1.4 and 2 times as high as the activities of cells harboring plasmids pNA3 and pNA9, respectively. The secretion ratios (amount of extracellular alpha-amylase activity/amounts of total alpha-amylase activity) of cells harboring plasmids pNA3, pNA7, and pNA9 were 91.4%, 94.5%, and 95.3%, respectively.
Anbydrotrypsin, a derivative of bovine trypsin, immobilized on Sepharose tightly adsorbs various peptides containing L-arginine at the carboxyl termini, such as bradykinin and tuftsin. These peptides correspond to the specific products of the action of trypsin-like enzymes. Native trypsin immobilized on Sepharose does not show such strong affinity. Fragment 2, a peptide with 41 amino acid residues, which has been released together with bradykinin from bovine high-molecular-weight kininogen by the action of plasma kallikrein, is also adsorbed on the immobilized anhydrotrypsin. When only the carboxyl-terminal arginine is removed with carboxy-peptidase B, however, the peptide loses its adsorptive ability. Immobilized anhydrochymotrypsin, on the other hand, exerts specific affinity for the peptides which correspond to the products of chymotrypsin. These results suggest that the anhydro-derivatives of serine-pr0 teases in general may be of great use in the affinity chromatography of respective series of various naturally occurring peptides.
Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b . . . . }, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When "medium" concentrations of ethanol and glucose in fedbatch culture of baker's yeast were set at 2 g/1 and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67 + 0.35 g/l and 0.27 + 0.25 g/l, respectively, ethanol production was reduced from 26 g/1 to 3-4 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/1.
The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the alpha-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. The increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy controller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 395 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled.
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