RESUMO
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.
Assuntos
Aminoidrolases/biossíntese , Escherichia coli/metabolismo , Fermentação , Glicerol/química , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/biossíntese , Reatores Biológicos , Carbono/química , Meios de Cultura/metabolismo , Glucose/química , Microbiologia Industrial , Isopropiltiogalactosídeo/química , Plasmídeos/metabolismo , Pseudomonas fluorescens/enzimologiaRESUMO
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.