Rapidly and Precisely Cross-Linked Enzymes Using Bio-Orthogonal Chemistry from Cell Lysate for the Synthesis of (S)-1-(2,6-Dichloro-3-fluorophenyl) Ethanol

Abstract

To develop a method for preparing rapidly, precisely, and bio-orthogonally cross-linked enzymes (RP-CLEs), nonstandard amino acids (NSAAs) were inserted into the enzyme protein, and microwave irradiation was used to accelerate its site-specific linkage through Cu-free strain-promoted alkyne–azide cycloaddition (SPAAC). To this end, we selected aldehyde ketone reductase (AKR) as model enzyme, and AKR mutants were obtained by five-point insertion of p-azido-l-phenylalanine (pAzF) which were subsequently cross-linked to form RP-CLEs from cell lysate supernatant under microwave irradiation. The AKR five-point mutant and corresponding RP-CLEs were characterized using MALDI-TOF MS, SEM, and FT-IR, respectively. The specific activities of RP-CLEs of three-point and five-point AKR were 1.27 and 2.06 U·mg–1, 1.21- and 2.16-fold those of the corresponding free enzymes, respectively. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol, the yield was up to 90.8%, and the ee was 99.98%. Moreover, after 6 consecutive 12 h reaction cycles, AKR five-point RP-CLEs still retained 80% of their initial activity. Thus, depending on the enzyme structure analysis, different numbers of NSAAs could be reasonably incorporated into the protein to accurately guide and control the covalent linkage to form RP-CLEs. This green method could be further developed both to generate bio-orthogonally cross-linked enzyme and separate them from nontarget proteins for industrial biocatalysis.

New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy

“…Alternatively, genetic engineering can enable site-specific incorporation of nonstandard amino acids (NSAAs) containing functional groups which enable crosslinking of enzyme aggregates in a highly controlled manner, thus avoiding undesirable reactions of glutaraldehyde which lead to loss of enzyme activity. 42 The complementarity of PE and EI is readily apparent from a comparison of their main features (Table 2). For example, EI focuses on enzyme reuse and controlling the microenvironment whereas PE focuses on genetic variability but both techniques contribute to improving stability.…”

Streamlining Design, Engineering, and Applications of Enzymes for Sustainable Biocatalysis

“…However, some enzymes have a paucity of surface lysine residues, and PE can be used to create multiple attachment points by inserting additional surface lysine residues. Alternatively, genetic engineering can be used to incorporate non-standard amino acids (NSAAs) possessing functional groups that enable highly controlled generation of CLEAs, thereby eliminating undesirable side reactions that can in some cases cause loss of activity …”

“…Alternatively, genetic engineering can be used to incorporate non-standard amino acids (NSAAs) possessing functional groups that enable highly controlled generation of CLEAs, thereby eliminating undesirable side reactions that can in some cases cause loss of activity. 136 In Vivo vs In Vitro Immobilization. Even when EI is integrated in the screening protocol of directed evolution, overall the production of the free enzyme is performed in vivo, while the immobilization is conducted separately in vitro.…”

Multicatalytic Hybrid Materials for Biocatalytic and Chemoenzymatic Cascades—Strategies for Multicatalyst (Enzyme) Co-Immobilization

“…CLEA's surface morphology can be characterized through electronic scanning microscopy (SEM) [152] and atomic force microscopy (AFM) [157]. Fourier transform infrared spectroscopy (FTIR) can be used to analyze the efficiency of bounds formation in CLEAs [158], to verify the presence of functional groups for covalent bond formation between enzymes and functionalized magnetic nanoparticles [150] as well as identify the presence of some bounds in magnetic nanoparticles used as additives in CLEAs [159].…”