Stabilization of cyclohexanone monooxygenase by computational and experimental library design

Abstract

Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature-sensitive enzymes catalyzing the Baeyer-Villiger reaction, which display excellent stereo- and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI-31, a substrate-promiscuous enzyme that efficiently catalyzes the production of the nylon-6 precursor ε-caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein-stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half-life at 30°C. In contrast to the wild-type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications.

Keywords: Baeyer-Villiger monooxygenase; computational design; cyclohexanone; stabilization; thermostability.

Figure 1

T _m increase of RhCHMO for the first (a) and second (b) set of point mutants. Stabilizing mutations with a T _m increase of at least 1°C are shaded pink, stabilizing mutations not considered for mutant combinations are indicated by a striped pattern. RhCHMO, cyclohexanone monooxygenase from Rhodococcus sp. HI‐31 [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Method to create the second shuffled library without bias. The wild‐type plasmid was used as a template in PCRs with mutated primers to generate DNA fragments containing two mutations on each end. The purified fragments were assembled by Gibson cloning to obtain a plasmid with all mutations. An equal mix of this mutated and the wild‐type plasmid was used in the next round of PCR where primers were used that bound outside the mutated region to generate a mix of fragments with and without mutations. Upon Gibson assembly of this mix, the shuffled library was obtained. PCR, polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

T _m increase of RhCHMO by a combination of mutations to a highly stabilized multiple mutant. RhCHMO, cyclohexanone monooxygenase from Rhodococcus sp HI‐31 [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Comparison of the stabilized mutants with wild‐type RhCHMO. (a) Temperature optimum of wild‐type RhCHMO and M8B mutant. (b) Activity over time upon incubation at 30°C. (c) Comparison of T _m and half‐life. RhCHMO, cyclohexanone monooxygenase from Rhodococcus sp. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Mutations introduced in RhCHMO to increase thermostability. The crystal structure of RhCHMO (PDB ID 4RG3) shown in cartoon representation and colored yellow, green and cyan for FAD, NADP, and helical domain, respectively. FAD is shown in yellow and NADP⁺ as green sticks. (a–c) show the close up view of important residues, where the computationally predicted mutations are superimposed and shown as sticks with pink carbons. Alpha carbons are indicated by balls, hydrogen bonds as yellow dashed lines, and water molecules from the crystal structures as red balls. In (a) a transparent sphere indicates a cavity observed in the wild‐type, which is filled by the tyrosine mutation. (d) shows the overall protein structure and all mutations experimentally shown to be stabilizing are shown as balls in shades of red indicating the degree of stabilization. FAD, flavin adenine dinucleotide; RhCHMO, cyclohexanone monooxygenase from Rhodococcus sp HI‐31 [Color figure can be viewed at http://wileyonlinelibrary.com]