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.

Robust ω-Transaminases by Computational Stabilization of the Subunit Interface

“…In view of the instability of dimeric class III TAs 9 and the importance of enzyme robustness for biocatalytic applications, we investigated the possibility to enhance the stability of the homodimeric Pj TA by protein engineering. For this we chose the FRESCO workflow, which was used earlier by us and others to increase the thermostability of dimeric limonene epoxide hydrolase (Δ T m app = 35 °C), 30 monomeric haloalkane dehalogenase (Δ T m app = 23 °C), 31 monomeric peptide amidase (Δ T m app = 23 °C), 32 monomeric xylanase (Δ T m app = 14 °C), 33 monomeric HMF oxidase (Δ T m app = 11 °C), 34 monomeric cyclohexanone monooxygenase (Δ T m app = 13 °C), 35 and a tetrameric halohydrin dehalogenase (Δ T m app = 28 °C). 36 First, all possible point mutations were subjected to folding energy calculations.…”

Structure-Based Redesign of a Self-Sufficient Flavin-Containing Monooxygenase towards Indigo Production

“…In an ideal scenario, one would like to have an as stable as possible starting enzyme. mFMO falls into the category of moderately stable enzymes with an apparent melting temperature of 43.3 • C. The recently developed FRESCO protocol [16,17] is an effective tool for the stabilization of enzymes, including flavin-containing enzymes [19]. Through this computational protocol, mutations are predicted that should render the target protein more (thermo) stable.…”

Pseudomonas taiwanensis biofilms for continuous conversion of cyclohexanone in drip flow and rotating bed reactors

“…The Baeyer‐Villiger (BV) oxidation of ketones to the corresponding esters or lactones is an important reaction for the production of pharmaceutical compounds (steroids), fine chemicals (antibiotics, terpenoids), and bulk chemicals (polyester monomers) [1, 2]. Chemically catalyzed BV oxidations often suffer from unstable or explosive chemical oxidants, generate hazardous waste products, and show limited selectivity [3]. Biological BV oxidations, in contrast, performed by flavin‐dependent enzymes, categorized as Baeyer‐Villiger monooxygenases (BVMOs) have attracted considerable attention to overcome these challenges.…”

“…Biological BV oxidations, in contrast, performed by flavin‐dependent enzymes, categorized as Baeyer‐Villiger monooxygenases (BVMOs) have attracted considerable attention to overcome these challenges. BVMOs utilize the ‘green’ oxidant oxygen, operate at ambient reaction conditions, generate water as a sole byproduct, and mediate high product selectivity [1, 3, 4]. Yet, industrial applications of BVMOs are restricted due to low enzyme stability and form inhibitory effects of substrate and/or product [3].…”

“…BVMOs utilize the ‘green’ oxidant oxygen, operate at ambient reaction conditions, generate water as a sole byproduct, and mediate high product selectivity [1, 3, 4]. Yet, industrial applications of BVMOs are restricted due to low enzyme stability and form inhibitory effects of substrate and/or product [3]. The only known industrial example that involves a BVMO catalyzed reaction is the production of a pharmaceutical compound, esomeprazole, on the grams scale (Patent WO2011071982A2).…”