Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji).
BACKGROUND
The flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. Recently, it was shown that P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase (GOx), which traditionally is used in these applications. P2Ox offers several advantages over GOx for this application, e.g., its much broader substrate specificity. Because of this renewed interest in P2Ox, knowledge on novel pyranose oxidases isolated from organisms other than white-rot fungi, which represent the traditional source of this enzyme, is of importance, as these novel enzymes might differ in their biochemical and physical properties.


RESULTS
We isolated and over-expressed the p2ox gene encoding P2Ox from the ectomycorrhizal fungus Lyophyllum shimeji. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in E. coli Rosetta 2. We obtained active, flavinylated recombinant P2Ox in yields of approximately 130 mg per L of medium. The enzyme was purified by a two-step procedure based on anion exchange chromatography and preparative native PAGE, yielding an apparently homogenous enzyme preparation with a specific activity of 1.92 U/mg (using glucose and air oxygen as the substrates). Recombinant P2Ox from L. shimeji was characterized in some detail with respect to its physical and catalytic properties, and compared to the well-characterised enzymes from Phanerochaete chrysosporium and Trametes multicolor.


CONCLUSIONS
L. shimeji P2Ox shows properties that are comparable to those of P2Ox from white-rot fungal origin, and is in general characterised by lower K(m) and k(cat) values both for electron donor (sugar) as well as electron acceptor (ferrocenium ion, 1,4-benzoquinone, 2,6-dichloroindophenol). While L. shimeji P2Ox is the least thermostable of these three enzymes (melting temperature T(m) of 54.9 degrees C; half-life time of activity tau1/2 of 0.12 at 50 degrees C and pH 6.5), P. chrysosporium P2Ox showed remarkable thermostability with T(m) of 75.4 degrees C and tau1/2 of 96 h under identical conditions.
Salaheddin
Clara
C
Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria.
Takakura
Yoshimitsu
Y
Tsunashima
Masako
M
Stranzinger
Barbara
B
Spadiut
Oliver
O
Yamabhai
Montarop
M
Peterbauer
Clemens K
CK
Haltrich
Dietmar
D
D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase