In this study, yeast strains were screened and immobilized in a form preserving the multifaceted biocatalytic activity. Im-mobilization of the silica-supported whole-cells of various yeasts – such as Lodderomyces elongisporus, Pichia carsonii, Candida norvegica and Debaryomyces fabryi – by sol-gel entrapment resulted in easy-to-handle biocatalysts that mediated efficiently different types of synthetic reactions. In the present study, the enantiotope selective reduction of prochiral ke-tones 1a-d and the acyloin condensation of benzaldehyde 3 were studied representing two remarkably diverse types of bio-transformation. The yeast cell biocatalysts – in the presence of fresh or recovered NADH cofactor – could be applied for continuous-flow bioreduction of ketones 1a,b with moderate to good yields (20 to 92%) and excellent enantiomeric purity (>99%). Immobilized L. elongisporus and P. carsonii cells could also mediate acyloin condensation of benzaldehyde 3 in batch as well as in continuous-flow mode. The switchable biocatalytic activity of the immobilized yeast cells was demon-strated by consecutive biotransformations under continuous-flow conditions involving reduction of phenylacetone 1a to (S)-phenylpropane-2-ol (S)-2a first, followed by conversion of benzaldehyde 3 to (R)-1-hydroxy-1-phenylpropan-2-one (R)-4 and reduction of 1a to (S)-2a again by using the same packed bed bioreactor.

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag.

Nanostructured but micro‐sized biocatalysts were created by bottom‐up technology using multi‐functionalized silica nanoparticles (NPs) as nano‐sized building blocks to form cross‐linked enzyme‐adhered nanoparticles (CLEANs) as robust micro‐sized particles with beneficial internal structure and good mechanical properties. Systematic surface modification of NPs with a grafting mixture consisting of organosilanes with reactive (aminopropyl) and inert (e. g., vinyl, propyl, phenyl, or octyl) functions resulted in functional NPs enabling cross‐linking agents, such as glutardialdehyde or bisepoxides (glycerol diglycidyl ether, neopentylglycol diglycidyl ether, and poly(propylene glycol) diglycidyl ether), to bind and cross‐link enzymes covalently and to form macroporous microparticles. These CLEANs were able to diminish several weaknesses of traditional cross‐linked enzyme aggregates as biocatalysts, such as poor mechanical resistance, difficult recovery, and storage, strengthening their use for packed‐bed enzyme reactors. Lipase B from Candida antarctica (CaLB) was selected as model enzyme for development of robust CLEANs, which were successfully tested for various industrially relevant applications including a kinetic resolution of a racemic alcohol and the production of various natural fragrance compounds under continuous‐flow conditions.

This article provides a method to upgrade epoxy-functionalized carriers for covalent enzyme immobilization to selective carriers suitable for covalent immobilization of metal affinity-tagged enzymes without the need of preliminary enzyme purification. Affinity function doping of the epoxy-functionalized surface introduces an advanced possibility to avoid the costly and time-consuming downstream processes required for efficient immobilization on non-selective epoxy carriers. Our approach is based on the partial functionalization of surface epoxides via a proper diamine-derived linker and an ethylenediaminetetraacetic dianhydride-based chelator charged with cobalt ions. The solid macroporous carriers, doped with metal affinity functions, have both coordinative binding ability (rapid anchoring the metal affinity-tagged enzymes to the surface) and subsequent covalent bond-forming ability (preferred binding of the tagged enzyme to the surface after proper washing by the residual epoxide functions), enabling a single operation for the enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag. The immobilized PcPAL was applied in the ammonia elimination of racemic phenylalanine, 4-chlorophenylalanine, and 4-bromophenylalanine to produce the corresponding d-phenylalanines, in addition to the formation of (E)-cinnamates, as well as in ammonia addition reactions to (E)-cinnamates, yielding the corresponding enantiopure l-phenylalanines.