Performance of cross‐linked enzyme crystals of engineered halohydrin dehalogenase HheG in different chemical reactor systems

Abstract

Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β‐substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross‐linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross‐linked with bis‐maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space‐time yield of 23.9 kgproduct gCLEC−1 h−1 Lreactor volume−1 along with the highest achieved product enantiomeric excess [64%] for application in a packed‐bed reactor. Additionally, lyophilization of those CLECs yielded a storage‐stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature.

Application of Immobilized Enzymes in Flow Biocatalysis for Efficient Synthesis

“…In the cross-linking strategy, enzymes are cross-linked to the support matrices with covalent bonds by using bifunctional reagents . This strategy can also be used to make carrier-free immobilized enzymes, such as cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs). − Although cross-linked enzymes usually have improved reusability and stability, the catalytic activities might be lost during the cross-linking process …”

Synthesis and Characterization of Cross-Linked Aggregates of Peroxidase from Megathyrsus maximus (Guinea Grass) and Their Application for Indigo Carmine Decolorization

“…Immobilization can be performed using very different strategies, that can be divided into using pre-existing solids (that is, using a solid matrix as support) or generating an ex novo solid, without using a pre-existing support. Among these last ones, we can remark nanoflowers [ 31 , 32 , 33 ], crosslinked enzyme crystals (CLECs) [ 34 , 35 , 36 ], even though cross-linked enzyme aggregates (CLEAS) stand out as the best option [ 37 , 38 , 39 ]. This is a carrier-free enzyme immobilization methodology developed by Prof Sheldon [ 37 , 38 , 39 ].…”