Laccase gene of Agrocybe sp. CU43 was cloned and expressed in E. coli. Agrocybe sp.CU43 was cultured in N-limit media containing 500 ppm fluorene and total RNA was extracted from dry mycelia harvested at day 21 which gave 111.11 U/ml of laccase activity. First strand cDNA was synthesized and used as template for cDNA of laccase gene amplification by PCR using lacC-F1 และ lacC-R5 primers. PCR product obtained was 1,584 bp and was cloned into pGEM®-T easy vector. The recombinanant plasmid was designated as pTTLC. pTTLC was used as a template to amplify laccase gene with and without fungal signal sequence. The PCR products were cloned into pET2126_4 vector for expressing in E. coli Rosetta-Gami B (DE3) pLysS. Laccase gene was expressed under the control of T7 promoter, the 5’ end was adjacent to pelB coding sequence, and the 3’ end was fused with six His Tag coding sequences. Transformants cultured on LB agar supplemented with guaiacol did not show any laccase activity. Selected transformants without signal sequence of fungi were cultured in LB liquid medium and induced by IPTG. SDS PAGE of insoluble protein extracted by denature condition revealed additional protein band at approximately 55 KDa. The optimum conditions for recombinant laccase expression are cultivation at 37℃ with 400 µM IPTG induction for 3 hours. Affinity column chromatography followed by stepwise refolding method generated 228.51 µg/ml of protein with no laccase activity and was not able to degrade fluorene.
