title: Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme.
abstract: Previous studies on Melanocarpus albomyces laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus Trichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin-laccase fusion protein. Both proteins were expressed in T. reesei under the cbh1 promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l(-1)). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin-laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l(-1). The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.
authors.affiliation: VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland.
authors.forename: Laura-Leena
authors.initials: LL
authors.lastname: Kiiskinen
authors.affiliation: VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Kruus
authors.affiliation: VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland.
authors.forename: Michael
authors.initials: M
authors.lastname: Bailey
authors.affiliation: VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland.
authors.forename: Erkko
authors.initials: E
authors.lastname: Ylösmäki
authors.affiliation: VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland.
authors.forename: Matti
authors.initials: M
authors.lastname: Siika-Aho
authors.affiliation: VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Saloheimo
mesh_terms: D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004795:Enzyme Stability; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020037:Sordariales / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D042845:Laccase; D043366:Cellulose 1,4-beta-Cellobiosidase; D010206:Papain