Abstract
d-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining d-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several d-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic d-amino acids, in particular hydrophobic d-amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and d-Glu, a general substrate for d-aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent kcat and Km values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability (T50 value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.
Highlights
DAAO is an enzyme that is valuable for use in various applications[10,11,12], such as the optical resolution of a racemic mixture of amino acids[13]; detection and quantification of d-amino acids[14]; and production of α-keto acids, which can be used in pharmaceutical materials[15]
Three fungi were obtained at 50 °C from bark and cattle manure composts—strain YA and strains PB and PE, respectively; whereas, no fungi growth was observed at 60 °C. These thermophilic fungi were able to grow on a minimal medium containing d-Ala, d-Asn, d-Gln, or d-His as the sole nitrogen source, which are better substrates of broad substrate specificity DAAOs such as RgDAAO and TvDAAO16 (Fig. 1)
The results suggested that the recombinant protein is DAAO with broad substrate specificity and high catalytic activity
Summary
DAAO is an enzyme that is valuable for use in various applications[10,11,12], such as the optical resolution of a racemic mixture of amino acids[13]; detection and quantification of d-amino acids[14]; and production of α-keto acids, which can be used in pharmaceutical materials[15]. To obtain a highly thermostable DAAO, we have previously identified a DAAO homologous gene in the thermophilic bacterium Rubrobacter xylanophilus (RxDAAO), and the recombinant RxDAAO has been shown to have the highest thermal stability among the known DAAOs17; the substrate specificity of this thermophilic bacterial enzyme is narrow and limited to branched-chain d-amino acids, such as d-Val and d-Ile. the catalytic activity is much lower than those of the fungal RgDAAO and TvDAAO. Rasamsonia emersonii (phylum, Ascomycota), has been found in composts[20] and grows well at 45–50 °C21 This fungus produces several biotechnologically valuable enzymes with high thermal stability, such as β-glucosidase, α-glucuronidase, and FAD-dependent glucose dehydrogenase[20,22,23], suggesting it to be a promising source of various thermostable enzymes including DAAO. ReDAAO, which was the first characterized thermophilic fungal DAAO, showed unique broad substrate specificity and more advantageous properties for practical applications than other DAAOs
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