| Nowadays, researches on laccase are more and more attractive. Laccase (EC.1.10.3.2) is one of Cu-contained polyphenol oxidases produced by fungin, especially white rot fungi. Laccase can interact with various of substrates such as polyphenol and aromatic diamines, which properties were widely applied in many industrial fields. In this paper, a mutant Ganoderma lucidum UIM-281 with high yield of laccase was obtained by Low-energy N+ ion beam implantation technique from the original strain U60.The conditions for enzyme-producing was optimizatized and the purification, characterization of laccase were investigated respectively. The main results are described as following.1. The mycelium of G. lucidum U60 was implanted by Low-energy N+ ion beam technique to obtain the laccase high-yield strains. The dose of implanted ion with the higher proportion of positive mutation proportion was confirmed from 2.6×1015 to 3.9×1015 ions/cm2 by investigating the relationship between the implantation dose under 15 keV energy and the suvival rates and the mutation rates of G. lucidum. The implanted ion dose of 3.12×1015 ions/cm2 was choosen for the next step of multiple screening, and finally a laccase high-yield mutant UIM-281 with stable genetic character was obtained. The laccase-producing fermentation characteristics indicated that the peak values of laccase activity in mutant UIM-281 was 1.7-fold and 2.28-fold higher than that of the original strain U60, moreover the fermentation time of the mutant was shortened as 24 h, which suggest the mutant UIM-281 be the efficient and economic laccase-yielding G. lucidum strain in industrial fermentation.2. Key factors effecting laccase production in fermentation medium of G. lucidum UIM-281 were screened and optimized by single factor experiments and Plackett-Burman (PB) design. The key factors which significantly affected laccase yield were corn meal wheat bran and ABTS. After steepest ascent experiment approaching the optimal level of the three factors, the center combination design and response surface analysis were applied to establish the second-order equation model for the yield of laccase, The optimal fermentation medium were obtained as following(g/L):corn meal 17.56, wheat bran 17.91, ABTS 0.0274, glucose 10, soybean powder 10, KH2PO4 4.5, and the verification test value of the laccase activity was 6325.3 U/L, which had been increased 1.27 fold compared with chemical synthetic medium. This research provided theoretics base for the industrial fermentation of laccase.3. The main cultivation factors effecting laccase yield of G. lucidum UIM-281 was firstly screened by (PB) design. After steepest ascent experiment approaching the optimal level of the three factors, the center combination design and response surface analysis were applied to establish the second-order equation model for the yield of laccase. The optimal fermentation conditions were obtained as following:52 mL of broth content, 158 r/min of rotating speed,172 h of culture time,10% of inoculation volume, and 96 h of seed age at initial pH 5.5 and at 28℃. The verification test value of the laccase activity was 8342.6 U/L, which was nearly in agreed with the theoretical prediction value 8387.7 U/L. The results suggested the predicted model was reliable and available for the optimization of laccase fermentation conditions.4. The result of Native-PAGE for laccase crude extract showed that there is no isoenzyme of laccase existing in the UIM-281. The laccase was purified by acetone fractionatiom and filtrated with millipore filtration. The molecular weight of purified laccase determined by SDS-PAGE was about 46KDa. The optimum temperature and pH for the laccase activity were 50℃and 6 respectively. The enzyme was stable under 10℃at pH 4.8. The activity of laccase was enhanced by Co2+, Cu2+, EDTA and DMSO, whereas it was inhibited by the metal ions Mn2+, Ba2+, Mg2+, Ca2+, Fe2+,β-ME, SDS and NaN3. The activity of laccase was completely inhibited by Fe2+,β-ME, SDS and NaN3, while Zn2+and Na+ did not strongly effect on laccase activity. Michaelis constant of the laccase was 1.552 mmol/L...
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