| Laccase (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) is a multicopper oxidase widely distributed among plants, fungi and bacteria. It catalyzes the oxidation of a broad range of organic and inorganic substrates, and has been widely used in industry and bioremediation. Pycnoporus sanguineus is a saprophytic fungus belonging to the Basidiomycetes of the family Polyporaceae, it can secret laccase constitutively. In this paper, laccase from Pycnoporus sanguineus was purified and the laccase gene was expressed in Pichia pastoris.Laccase from Pycnoporus sanguineus was purified using ultrafiltration, anion-exchange chromatography and gel filtration. The purification fold was 3.61 and recovery of total laccase activity was 36.53%. The molecular mass of the enzyme was 61.4 kDa as determined by SDSPAGE. The absorption spectrum of the purified protein did not show absorbance peak at around 600 nm, typical for a type-1 copper signal of blue laccases. The N-terminal sequence of the P. sanguineus laccase was identical with laccase from P. cinnabarinus and a high similarity to other laccases from wood-degrading fungi. The pH profile for laccase activity against ABTS showed a peak of maximum activity at pH 3.0. The enzyme was stable at 25℃for 1 h in the pH range from 2.0 to 5.0. The optimum temperature of the purified enzyme was observed at 65℃. There was little loss of activity during preincubation of the enzyme for 1 h at temperature up to 40℃, but the activity decreased rapidly beyond 50℃.The purified laccase showed activity against various substrates, including 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate), 2,6-dimethoxyphenol and syringaldazine. The lowest Km value (0.077 mmol/L) was found for ABTS. The highest catalytic efficiency was also obtained on ABTS, followed by syringaldazine and 2,6-dimethoxyphenol. The purified enzyme was strongly inhibited by 0.1 mmol/L sodium azide, 1 mmol/L L-cysteine and DTT, whereas the metal ion chelator EDTA showed only a slight inhibitory effect.In the present study, the purified laccase from P. sanguineus was able to decolorize RBBR efficiently in the absence of any redox mediators, it was able to decolorize about 90% of RBBR in only 10 rain with an enzyme activity of 5 U/mL. The decolorization ability of P. sanguineus laccase demonstrated its potential applications in dye decolorization.A cDNA encoding for laccase gene was isolated and amplified from P. sanguineus by RTPCR. The cDNA was cloned into pMD18-T simple vector and sequenced, the result showed that three nucleotides in the laccase cDNA were not consistent with the former laccase gene, this change resulted in three discrepancies in deduced amino acids sequence. Laccase cDNA was cloned into the vector pPICZB to construct the recombinant expression vector pPICZB/lac. Pichia pastoris SMD1168H was then transformed with pPICZB/Iac by LiCl. The selection of transformants was carried out on YPD plates containing Zeocin, transformants were further identified by colony PCR.Transformants were cultured in BMMY medium under inducing conditions to express laccase gene. The cell density increased gradually while laccase activity from the culture supernatant changed little after 72 hour's cultivation, and the maximum laccase activity was 10.9 U/L. The low activity of recombinant laccase might have something to do with the selection of signal sequence, codon bias in Pichia pastoris and the choice of culture conditions.
|
PDF Full Text Request