Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli.

Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32 °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process.

Temperature-dependent interaction of the bovine hippocampal serotonin(1A) receptor with G-proteins.

The ligand binding and G-protein coupling of the bovine hippocampal 5-HT1A receptor as a function of temperature was monitored. There is an almost complete and irreversible loss in agonist binding at 50 degrees C. However, the antagonist binding is reduced only by 50%, and this could be reversed if the temperature is lowered to 25 degrees C. Interestingly, the agonist binding of the 5-HT1A receptor in membranes exposed to 50 degrees C is inhibited to a much lesser extent by GTP-gamma-S, a non-hydrolysable analogue of GTP, indicating uncoupling of the 5-HT1A receptor to G-proteins at 50 degrees C. We propose that high temperature selectively and irreversibly inactivates G-proteins thereby affecting G-protein-receptor interaction and agonist binding of the 5-HT1A receptor.