The main goal of this project is the overproduction of native and recombinant tailor-made lipolytic enzymes from Candida rugosa and Rhizopus oryzae, to be employed in the development of an enzyme technology suited for the production of asymmetric fine chemicals and pharmaceutical intermediates.
Fundamental aspects of yeast physiology, heterologous lipase expression systems improvement, expression and secretion of the recombinant lipases in transformed cells (Pichia pastoris), on-line monitoring and control of fermentation process, and downstream processes considerations for the overproduction of native and recombinant lipases have been improved in a pilot plant scale level.
Characterisation of Candida rugosa lipase produced in the project has showed better performance in terms of catalytic properties in front of the target reactions than commercial ones.
Mutagenesis of lipases has allowed increasing our knowledge on the molecular aspects of the interaction between these enzymes and different substrates, and a proper evaluation of the effects induced by different mutations. On the one hand, the effect of the enzyme glycosylation in its activity has been studied in CRL by constructing a range of partially or fully deglycosylated mutants.
The design of new immobilized enzyme derivatives has also been achieved. Three different immobilized derivatives have been made available: Interfacial adsorption on hydrophobic groups, adsorption on stationary phases, based on vinyl properly functionalised from an aqueous solutions and adsorption on a hydrophobic or an ion exchange resin, for instance EP100 (microporous polypropylene powder).
These new enzymes and derivatives have been applied in the production of diltiazem derivatives, synthesis of chiral amines, production of non-steroidal anti-inflammatory drugs, resolution of mandelic acid derivatives and chiral alcohols as glycidol and trans-2-phenyl.1.cyclohexanol. In terms of lipolytic activity and enantioselectivity the derivatives produced in the project have shown better performance than commercial ones. Finally, the design of an enzymatic plug-flow packed bed reactor in order to scale the process has been made.
