Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis.
Among the laccases produced by the white-rot fungus Pleurotus ostreatus, there are two closely related atypical isoenzymes, POXA3a and POXA3b. These isoenzymes are endowed with quaternary structure, consisting of two subunits very different in size. The POXA3 large subunit is clearly homologous to other known laccases, while the small subunit does not show significant homology with any protein in data banks. To investigate on the singular structure of the POXA3 complex, a new system for recombinant expression of heterodimer proteins in the yeast Kluyveromyces lactis has been set up. A unique expression vector has been used and the cDNAs encoding the two subunits have been cloned under the control of the same bi-directionally acting promoter. Expression of the large subunit alone and co-expression of both subunits in the same host have been demonstrated and the properties of the recombinant proteins have been compared. Clones expressing the large subunit alone exhibited always notably lower activity than those expressing both subunits. In addition to the activity increase, the presence of the small subunit led to a significant increase of laccase stability. Therefore, a role of the small subunit in POXA3 stabilisation is suggested.
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, via Cintia, 80126, Napoli, Italy. vfaraco@unina.it
Vincenza
V
Faraco
Carmine
C
Ercole
Giovanna
G
Festa
Paola
P
Giardina
Alessandra
A
Piscitelli
Giovanni
G
Sannia
D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D007716:Kluyveromyces / Q000235:genetics*; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D021122:Protein Subunits; D042845:Laccase