We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.

The development of a green-by-design, two-step, three-enzyme process for the synthesis of a key intermediate in the manufacture of atorvastatin, the active ingredient of the cholesterol lowering drug Lipitor R , is described. The first step involves the biocatalytic reduction of ethyl-4-chloroacetoacetate using a ketoreductase (KRED) in combination with glucose and a NADP-dependent glucose dehydrogenase (GDH) for cofactor regeneration. The (S) ethyl-4-chloro-3-hydroxybutyrate product is obtained in 96% isolated yield and >99.5% e.e. In the second step, a halohydrin dehalogenase (HHDH) is employed to catalyse the replacement of the chloro substituent with cyano by reaction with HCN at neutral pH and ambient temperature. The natural enzymes were highly selective but exhibited productivities that were insufficient for large scale application. Consequently, in vitro enzyme evolution using gene shuffling technologies was employed to optimise their performance according to predefined criteria and process parameters. In the case of the HHDH reaction, this afforded a 2500-fold improvement in the volumetric productivity per biocatalyst loading. This enabled the economical and environmentally attractive production of the key hydroxynitrile intermediate. The overall process has an E factor (kg waste per kg product) of 5.8 when process water is not included, and 18 if included.

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Sterically hindered secondary alkylcobalamins carrying hydrogen in the @-position decompose in neutral aqueous solutions spontaneously by way of @-elimination. The cleavage of the Co-C bond in these compounds is caused by "upward" distortions of the corrin ligand in response to the attachment of the axial base, 5,6-dimethylbenzimidazole, as well as by thermal motions of the corrin ligand system. Organocobalamins carrying organic groups which lack hydrogen in the @-position cannot decompose by way of the /3-elimination mechanism. It is demonstrated on the basis of experiments with the prototype compounds neopentyl-and benzylcobalamin that such cobalamins decompose spontaneously with CwC bond homolysis. Under strictly anaerobic conditions, the resulting vitamin B12r and organic radicals recombine with high efficiency to regenerate the Co-C bond. Aerobically, vitamin B12 and the organic radicals are rapidly oxidized, rendering these cobalamins highly oxygen sensitive in neutral aqueous solution. This is in significant contrast to the behavior of secondary alkylcobalamins undergoing spontaneous, concerted @-elimination. Further insight into the mechanism of Co-C bond cleavage reactions is provided by the results of measurements of the activation parameters [m*, Sa*, and AGa*] of C O X bond thermolysis in neopentyl-, benzyl-, isopropyl-, and isobutylcorrins. From the AHa* values the Co-C bond dissociation energies of these organocorrins were estimated to range between 20 and 32 kcal-mol-'Until recently, secondary alkylcobalamins ( Figure 1) were considered too unstable to be isolated due to steric restrictions imposed by the corrin ligand. We have found: however, that such organocorrins are capable of existence because the corrin ligand can adopt "downward" distorted configurations in response to the steric demands of the cobalt-bound secondary alkyl group. These distortions can become sufficient to rupture the coordinative bond between the axial base (5,6-dimethylbenzimidazole, DMBZ) and the corrin cobalt atom, causing all acyclic secondary alkylcobalamins to exist predominantly in the "base-off' form. In neutral solutions, the unprotonated, appended DMBZ seeks to reattach itself to the corrin cobalt atom because of its affinity for the 6+ charged Co(II1) ion. Reattachment of the DMBZ to cobalt causes "upward" configurational adjustments of the corrin ligand which promote the spontaneous cleavage of the Co-C bond. If the DMBZ is protonated, upward distortions of the corrin ligand can still occur through thermal motions, but these cause Co-C bond cleavage with much lower probability. It is therefore possible to isolate many secondary alkylcobalamins in the protonated base-off form.In alkylcobalamins which carry hydrogen in the 6-position, spontaneous dealkylation produces olefins and vitamin B12s. In