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Abstract:The halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) catalyzes nucleophilic ring opening of epoxides with cyanide and azide. In the case of 2,2-disubstituted epoxides, this reaction proceeds with excellent enantioselectivity (E values up to > 200), which gives, by kinetic resolution, access to various enantiopure epoxides and bsubstituted tertiary alcohols (ee up to 99 %). Since the enzyme has a broad substrate range and because these tertiary alcohols are difficult to prepare in other ways, HheC is an attractive biocatalyst for the production of b-cyano and b-azido tertiary alcohols.

The crystal structure of the "ene" nicotinamide-dependent cyclohexenone reductase (NCR) from Zymomonas mobilis (PDB ID: 4A3U) has been determined in complex with acetate ion, FMN, and nicotinamide, to a resolution of 1.95 Å. To study the activity and enantioselectivity of this enzyme in the bioreduction of activated α,β-unsaturated alkenes, the rational design methods site- and loop-directed mutagenesis were applied. Based on a multiple sequence alignment of various members of the Old Yellow Enzyme family, eight single-residue variants were generated and investigated in asymmetric bioreduction. Furthermore, a structural alignment of various ene reductases predicted four surface loop regions that are located near the entrance of the active site. Four NCR loop variants, derived from loop-swapping experiments with OYE1 from Saccharomyces pastorianus, were analysed for bioreduction. The three enzyme variants, P245Q, D337Y and F314Y, displayed increased activity compared to wild-type NCR towards the set of substrates tested. The active-site mutation Y177A demonstrated a clear influence on the enantioselectivity. The loop-swapping variants retained reduction efficiency, but demonstrated decreased enzyme activity compared with the wild-type NCR ene reductase enzyme.

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Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: () identification of conserved and highly variable regions; () protein sequence design by substituting residues in the highly variable regions, and gene synthesis; () in vitro DNA recombination of synthetic genes; and () screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

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