Design of a H₂ O₂ -generating P450_SPα fusion protein for high yield fatty acid conversion

Sphingomonas paucimobilis' P450_SPα (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 H₂ O₂ 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 H₂ O₂ self-producing enzyme by fusing the P450_SPα to the monomeric sarcosine oxidase (MSOX), as H₂ O₂ donor system, in a unique polypeptide chain, obtaining the P450_SPα -polyG-MSOX fusion protein. The purified P450_SPα -polyG-MSOX protein displayed high purity (A₄₁₇ /A₂₈₀  = 0.6) and H₂ O₂ -tolerance (k_decay  = 0.0021 ± 0.000055 min^-1 ; ΔA₄₁₇  = 0.018 ± 0.001) as well as good thermal stability (T_m : 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450_SPα 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 H₂ O₂ . 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 P450_SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).