A laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists of a 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11, 972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18, 123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50°C with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0–7.0. The Km and the Vmax value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

Nanoclay is a natural mineral that has great potential as a reinforcing filler in wood adhesives. In order to investigate the reinforcing mechanism more clearly, the crystalline structure, chemical properties, morphology, and thermal stability of pure PF and organic nanoclay-reinforced phenol formaldehyde (PF-OMMT) adhesive were characterized. The comparative mechanical properties of pure PF adhesive and PF-OMMT in the bondlines of plywood were analyzed by nanoindentation (NI) under different service environments and the shear strain distribution on the interphase was also measured by a digital image correction technique (DIC). X-ray diffraction (XRD), Fourier transform infrared (FTIR) and transmission electron microscopy (TEM) results indicated good dispersion of the clay in the PF matrix. The modified adhesive showed greater thermal stability than did the control adhesive, as evaluated by thermo-gravimetric analysis (TGA). The good dispersion of nanoclay and the positive effects of the nanoclay on the adhesive's water and heat resistance may have contributed to the improved mechanical properties of adhesives in an accelerated durability test. Compared to pure PF, the reduced elastic modulus and hardness of PF-OMMT in the bondline increased significantly and the strain distribution was much more uniform, resulting in an observed increase of macro-bonding strength of plywood, especially under conditions of severe cyclic water saturation and drying.