Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion

Abstract

Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α‐hydroxy fatty acids, thus avoiding the use of expensive electron‐donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2O2 toward proteins and cells often results in the failure of the reaction scale‐up when it is directly added as co‐substrate. In order to bypass this problem, we designed a H2O2 self‐producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2O2 donor system, in a unique polypeptide chain, obtaining the P450SPα‐polyG‐MSOX fusion protein. The purified P450SPα‐polyG‐MSOX protein displayed high purity (A417/A280 = 0.6) and H2O2‐tolerance (kdecay = 0.0021 ± 0.000055 min−1; ΔA417 = 0.018 ± 0.001) as well as good thermal stability (Tm: 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2O2. Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass‐representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).

Catalytically self‐sufficient CYP116B5: Domain switch for improved peroxygenase activity

“…To this end, we have recently demonstrated that the use of MSOX in a chimeric system is beneficial for the catalytic activity as it provides H 2 O 2 in a controlled manner and it prevents the rapid degradation of the heme. [ 37 ] Moreover, in the latter chimeric system the catalytic activity of MSOX was comparable to the ones previously reported in the literature. [ 34 ] Indeed, several studies have shown how the catalytic activity of a P450 domain can be modulated by creating functional self‐sufficient fusion proteins via the so‐called “Molecular Lego” approach.…”

“…To this end, we have recently demonstrated that the use of MSOX in a chimeric system is beneficial for the catalytic activity as it provides H 2 O 2 in a controlled manner and it prevents the rapid degradation of the heme. [37] Moreover, in the latter chimeric TA B L E 1 Kinetic properties of CYP116B using NADPH and NADH as reductants and cytochrome c as electron acceptor.…”

“…[ 31 ] The DNA sequence of the MSOX from Bacillus sp. B0618 [ 36 ] was cloned into the expression vector pET30a (+), inserting a GPGGGGGGGPG loop between the two functional units, which was previously designed in our lab, [ 37 ] and a poly‐histidine tag at its C‐terminus. Plasmid construction for the expression of the chimeric enzyme CYP116B5‐SOX was performed by GenScript (Piscataway, NJ, USA).…”

CYP116B5‐SOX: An artificial peroxygenase for drug metabolites production and bioremediation

“…We measured the accumulation of H 2 O 2 in real time coupling the CYP116B5-SOX reaction to the horseradish peroxidase activity, which uses H 2 O 2 to convert ABTS into a colored product (ABTS• + ) (Figure S4). [43,55] The data indicate that the CYP116B5-SOX produced 1.308 mM ± 0.038 mM after 5 min of reaction, that is the time used in our experiment to extrapolate the kinetics parameter for the conversion of p-nitrophenol, and up to 3.650 ± 0.136 mM over 20 min.…”

Cascade reactions with two non‐physiological partners for NAD(P)H regeneration via renewable hydrogen

“…Important improvements can stem from protein engineering and creation of chimeric enzymes based on the modular combination called "Molecular Lego", which has already successfully employed BMR as a fused module to different enzymes. [35][36][37][38][39][40]88,89] AUTHOR CONTRIBUTIONS Conceptualization: Francesca Valetti; Gianluca Catucci; Sheila J.…”