Exclusive overproduction of recombinant versatile peroxidase MnP2 by genetically modified white rot fungus, Pleurotus ostreatus.
By combining a homologous recombinant gene expression system and optimization of the culture conditions, hyper overproduction of Pleurtous ostreatus MnP2 was achieved. Genetically modified P. ostreatus strains with the recombinant mnp2 sequence under the control of sdi1 expression signals, were subjected to agitated culture using media supplemented with wheat bran or its hot-water extract. The best result, whereby 7300 U/l of MnP was produced by a recombinant strain TM2-18, indicated that more than 30-fold overproduction of the recombinant MnP2 compared to the previous result was achieved. On the other hand, no MnP activity was detected for the wild-type strain under the same conditions. Accumulation of the recombinant, but not endogenous, mnp2 transcripts was demonstrated in reverse-transcription PCR experiments. These results indicated that the recombinant MnP2 was exclusively expressed by the recombinant strain. Purified recombinant MnP2 showed almost identical properties to native MnP2 in electrophoresis, spectroscopic and kinetic analyses, including determination of K(m) and V(max) values for Mn(II), H(2)O(2) and veratryl alcohol. Moreover, the recombinant MnP2 directly oxidized a high-molecularweight substrate RNase A in the absence of redox mediators, as does native MnP2. The homologous overproduction system will provide a plat form for exclusive production of mutant or variant peroxidases with a desired property in basidiomycete.
Tsukihara
Takahisa
T
Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan.
Honda
Yoichi
Y
Sakai
Ryota
R
Watanabe
Takahito
T
Watanabe
Takashi
T
D030781:Organisms, Genetically Modified; D010544:Peroxidases / Q000096:biosynthesis* / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D011994:Recombinant Proteins; D010544:Peroxidases; C051129:manganese peroxidase