Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on pro-teins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel-Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (>=83%; e.e. >99.9%) were achieved in one pot with this artificial multifunc-tional metalloenzyme.
Title: Effects of macroporous resin size on Candida antarctica lipase B adsorption, fraction of active molecules, and catalytic activity for polyester synthesis Chen B, Miller EM, Miller L, Maikner JJ, Gross RA Ref: Langmuir, 23:1381, 2007 : PubMed
Methyl methacrylate resins with identical average pore diameter (250 A) and surface area (500 m2/g) but with varied particle size (35 to 560-710 microm) were employed to study how immobilization resin particle size influences Candida antarctica Lipase B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed more rapidly on smaller beads. Saturation occurred in less than 30 s and 48 h for beads with diameters 35 and 560-710 microm, respectively. Linearization of adsorption isotherm data by the Scatchard analysis showed for the 35 microm resin that: (i) CALB loading at saturation was well below that required to form a monolayer and fully cover the support surface and (ii) CALB has a high affinity for this resin surface. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560-710 and 120 microm. In contrast, CALB appears evenly distributed throughout 35 microm resins. By titration with p-nitrophenyl n-hexyl phosphate (MNPHP), the fraction of active CALB molecules adsorbed onto resins was <50% which was not influenced by particle size. The fraction of active CALB molecules on the 35 microm support increased from 30 to 43% as enzyme loading was increased from 0.9 to 5.7% (w/w) leading to increased activity for epsilon-caprolactone (epsilon-CL) ring-opening polymerization. At about 5% w/w CALB loading, by decreasing the immobilization support diameter from 560-710 to 120, 75, and 35 microm, conversion of epsilon-CL % to polyester increased (20 to 36, 42, and 61%, respectively, at 80 min). Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid.
A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.
