Enzyme reaction engineering: design of peptide synthesis by stabilized trypsin.
By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS]. In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst.
Instituto de Catálisis, C.S.I.C., Madrid, Spain.
R M
RM
Blanco
G
G
Alvaro
J M
JM
Guisán
D004151:Dipeptides / Q000138:chemical synthesis*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D007700:Kinetics; D012685:Sepharose; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism*
D004151:Dipeptides; D004800:Enzymes, Immobilized; C071467:benzoylarginine leucinamide; D012685:Sepharose; D014357:Trypsin