| Laccase, a copper-containing polyphenol oxidase, had been widely researched in the treatment of phenolic wastewater, environmental degradation of toxic phenolic, straw biodegradation, bioremediation of soil and feed industries. T. pubescens MB89, a kind of wihte-rot fungi,has a high laccase productivity. In present study, the culture and purification conditions suitable for laccase in T. pubescens MB89 were established first; and then enzymatic properties were examined. In addition, methods and characteristics of immobilized laccase, straw lignin decomposing, chlorophenol and dyes degradation were studied.Results of the above-mentioned studies indicate that:⑴The optimal culture conditions for laccase production were glucose 20 g / L, peptone 5.0 g / L, inoculation volume 6.0 mL, pH5, 25℃, 110 r / min.The to Adding 1.0μmol / L of Cu2 + in mid-fermentation (10 d) can promote laccase activity. All of veratryl alcohol, guaiacol and benzyl alcohol can induce laccase produce.⑵The results of laccase separation and purification showed that about 90% of laccase could be separated from fermentation broth by using 80% ammonium sulfate. After DEAE-Sepharose treatment, two characteristic peaks were detected between 280 to 610 nm. With Sephadex G-100 peak separation and purification of the paint to be an enzyme, the samples from the specific activity of 20.5, dynamic recovery rate reached 6 percent, for the purification of a multiple of 16.7.⑶Laccase characteristics were that:①It is a sugar content of 13.3% of the secreted glycoprotein extracellular protease, the molecular weight was about 60000Da and pI2.8.②The optimum catalytic temperature was 50℃, thermal stability was poor. It was an acid-resistant enzyme which stability was superior in alkaline conditions.③For phenolic substrate with I-or non-phenolic compounds, the most suitable pH range were between 4.0 to 4.5.④NaN3 was a strong inhibitor to laccase while 0.1 mmol / L EDTA and 1.0 mmol / L H2O2 were slight inhibition. The inhibitory of Chatham halogen ions were F-> Cl-> Br-, Cl-and Br- had transient suppression characteristics. Ions of Cu2 +, Co2 +, K +, Ag + could speed laccase activity, but ions of Fe2 +, Fe3 +, Ca2 + slowly enzyme reaction, and other ions such as Mg2 +, Zn2 +, Mn2 + had no significant effect.⑤with ABTS as substrate, the catalytic efficiency was the highest, reaching 48×106 M-1 ? s-1, followed by lilac aldehyde azine, catalytic efficiency up to 47×106, with hydroxy or methoxy - substituted phenols as well as the inorganic substrate of ferrous ions, but also have a high catalytic activity of the iodine ions are basically no catalytic activity.⑷The best methods and fixed conditions for immobilization①chitosan immobilized laccase (IE1): the best concentration of glutaraldehyde 4%, the optimum crosslinking time for 8 h, the best to the enzyme was 1.5 mg / g;②chitosan immobilized copper (IE2): CuSO4 ? 5H2O its adding the best of 0.6 mg, the optimal time for complex 10 h, the best to the enzyme was 0.25 mg / g;③alginate / immobilized chitosan (IE3): 2% sodium alginate, 2% CaCl2, 1% glutaraldehyde, 1.5% chitosan. The volume of the best enzyme to 1.5 mg / g;④TEOB / PEG immobilized (IE4): The optimum PEG molecular weight of 600 800, the best use of the concentration of 1.5%, the best to the enzyme was 1.5mg / g.The optimum temperature of IE1 and IE4 were 60℃, IE2 and IE3 were 30℃. IE1, IE3 and IE4 are the optimum pH value of 4.5, IE2 optimum pH of 4. IE pH stability, thermal stability and storage stability are superior to Lac.⑸The results of lignin degradation were①corn stalk can be used as carbon source for T. pubescens MB89 fermentation. Laccase had good degradation effect on lignin than on the cellulose and hemicellulose degradation.②The degree of polymerization and dispersion of separated straw lignin were reduced after laccase treatment. The degradation was mainly occured in parts of high molecular weight of lignin.③The phenolic hydroxyl contents of lignin increased with laccase treatment, but was decreased while ABTS existed.⑹Degradation of chlorophenol compounds:①The degradation time for DCP, TCP and PCP wer 4h by Lac, and degradation rate can reach about 50%; IE degradation 2h, the degradation rate has exceeded 50%.②With same pH value, IE and Lac efficiency on the degradation of TCP, followed by DCP, PCP degradation effects were the worst. DCP degradation by Lac and IE the best pH of 5.5, IE1 maximum degradation rate of 89.6%, Lac minimize the degradation rate, was 82.9%. TCP best pH for the degradation of 5.5, IE1, IE3 and IE4 are the largest degradation rate of 92.4%, Lac and IE2 close to the maximum degradation rate about 89%. The results show that the PCP degradation Lac, IE2, IE3 and IE4 best pH for the degradation of 5, IE1 degradation rate of the largest in the pH5.5.③Lac on the DCP, TCP and PCP degradation of the optimum temperature of 35℃, 45℃and 45℃, PCP degradation process, IE1 and IE4 the optimal temperature of 45℃, IE2 and IE3 the optimum temperature was 35℃.④Lac degradation of TCP, DCP and PCP the best dynamic for 30 U / mL. IE1 of its adding DCP degradation 40U/mL, its adding the TCP degradation 20U/mL. IE2 degradation of DCP, TCP and PCP its adding the optimum 20U/mL, 20U/mL and 40U/mL. IE3 and IE4 chlorophenol degradation of three compounds were the best addition 30U/mL, 20U/mL and 40U/mL.⑤With the increase of initial concentration of DCP, the degradation rate decreased, but the slow change in the trend; DCP initial concentration of 10 mg / L, the degradation rate was 100%; the initial concentration of less than 10 mg / L, the degradation rate of 100% of the level, the initial concentration is higher than 10 mg / L, the degradation rate was slow downward trend. PCP degradation rates increased with lower initial concentration, 5 mg / L the degradation rate of the highest, reaching 37.8%, high concentrations of PCP (20 mg / L) degradation rate was only 16.9%. IE in the degradation of chlorophenol compounds, the substrate concentration on the degradation rate of less than free laccase on the effect of degradation.⑥laccase added in 5 mmol / L ABTS, the reaction can be within 30 min to remove 69% of the PCP, without ABTS, the reaction after 17 h only 24% of PCP was removed.
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