PubMed · 7346438
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L Narain. 1981. Abnormal conjunctival lithiasis.. https://pubmed.ncbi.nlm.nih.gov/7346438/
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Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.
A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material. Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different.
A noninvasive method to determine the carboxyl content in oxidized celluloses, using solid-state carbon-13 cross-polarization-magic angle spinning nuclear magnetic resonance (13C CP/MAS NMR) spectroscopy, has been developed. Standard samples containing 0, 4, 8, 12, 16, and 20% carboxyl content were prepared by mixing appropriate amounts of powdered cellulose, a non-oxidized cellulose standard prepared from cotton linter by ball-milling for 96 h, and a commercial oxidized cellulose sample that had a 20% carboxyl content. Standard curves were constructed by plotting the percent carboxyl content against the peak area at 171 ppm, normalized with (i) a peak area at 104 ppm and (ii) the sum of peak areas at 171, 62, and 65 ppm. The regression analysis of the curves yielded a linear relationship with correlation coefficient (R2) values of 0.9868 and 0.9863, respectively. To validate the methods, five new samples of oxidized cellulose were prepared and analyzed. The values obtained were comparable to those determined by the calcium acetate method described in the United States Pharmacopoeia (USP), indicating that the solid-state 13C CP/MAS NMR can be used to analyze carboxyl content in oxidized celluloses.