Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme

VTT Biotechnology, PO Box 1500, Fin-02044 VTT, Finland Correspondence Markku Saloheimo Markku.Saloheimo{at}vtt.fi 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. Abbreviations: ABTS, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); CBHI, cellobiohydrolase I; 2,6-DMP, 2,6-dimethoxyphenol; ER, endoplastic reticulum; HFBI, hydrophobin I; UPR, unfolded protein response Present address: Finnzymes Oy, Keilaranta 16 A, Fin-02150 Espoo, Finland.