Sphingomonas paucimobilis' P450 (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 H O 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 H O self-producing enzyme by fusing the P450 to the monomeric sarcosine oxidase (MSOX), as H O donor system, in a unique polypeptide chain, obtaining the P450 -polyG-MSOX fusion protein. The purified P450 -polyG... More
Sphingomonas paucimobilis' P450 (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 H O 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 H O self-producing enzyme by fusing the P450 to the monomeric sarcosine oxidase (MSOX), as H O donor system, in a unique polypeptide chain, obtaining the P450 -polyG-MSOX fusion protein. The purified P450 -polyG-MSOX protein displayed high purity (A /A = 0.6) and H O -tolerance (k = 0.0021 ± 0.000055 min ; ΔA = 0.018 ± 0.001) as well as good thermal stability (T : 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450 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 H O . 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 TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).
