Production Of Alkali-tolerant Laccase And Its Utilizition In The Degradation Of Dyeing Waste Water

Laccase (EC1.10.3.2) belongs to a group of polyphenol oxidases that contain copper atoms in their catalytic centers; thus, they are typically referred to as multicopper oxidases. Laccase is able to catalyze an array of organic and inorganic substrates, including mono-, di-and poly-phenols, aminophenols, methoxyphenols as well as metal complexes coupled to the reduction of molecular oxygen to water with the one-electron oxidation mechanism. The rather broad substrate specificity of laccase has attracted great scientific interest for the biotechnological applications of laccase. Laccase may be used in bioremediation, biobleaching and biopulping, synthesis of green organic substances, food processing and construction of biosensor. In these application fields, many processes, such as pulp bleaching, decolorization of textile dye wastewaters, etc., involve high pH condition. It is necessary for application of laccases in these processes to produce laccases with high activity and stability under alkaline conditions. However, most laccases have their highest activity at acidic pH. Once the pH value is above7, their activities are greatly reduced, which largely limits the applications of laccases under neutral or alkaline condition. Therefore, one of the goals in the paper is breeding strains to produce laccase with high activity at higher pH.In the thesis, as the original strain, strain Monilia sp. M5-3was screened to produce alkali-resistant laccase and optimum pH of7.5. The strain was mutated by UV, ARTP, which was used as a mutation regent, screened from compost was isolated by PDA (potato dextrose agar) plate containing guaiacol as indicator compound, and a genetic stable mutant DM5-5. Laccase activity of the crude enzyme from the mutant DM5-5was3.33IU/mL. Protoplast mutagenesis technology was also used for screening of strains to obtain laccase overproducing strains in the thesis. ZMpl-17produced crude laccase with an activity of8.79IU/mL. Construction of expression vector of laccase genes from Monilia sp. M5-3. Two plasmids were tranformed into the Pencillium decumbens,and expression a low laccase activity after liquid fermentation.The crude laccase produced by DM5-5was used to stuies on synthetic dye decolorization.The results showed that the laccase could be directly used for decolorizing a variety of dyes, and had a effective decolorizing results.