Study On The Enzymatic Property Of Lac MP And Its Application Of Waste-paper Deinking

Laccase, containing copper, is a polyphenol oxidase, which belongs to the blue copper oxidase family(MCOs). It is widely distributed in plants, fungi, a small number of bacteria and insects. The activity center of laccase generally contains four copper ions. And its catalytic oxidation reaction is transferring electron, capturing the substrate’s electron, oxidating the substrate to the free radicals form, at the same time, transferring the electron captured from the substrate to oxygen, reducing oxygen to the water, which is going on through the synergy between the copper ions. The substrates of laccase are very widespread, including phenols and their derivatives, aromatic amines and their derivatives, carboxylic acid and its derivatives, steroid hormones and biological pigment, metal organic compounds and other non-phenolic substrates, and the range will be expanded with the presence of the redox mediators. Waste paper is a kind of very important recyclable resources, because of it is accessible, simple process, resource saving and environmental protection, so good economic and social benefits can achieved from it. And because the waste paper contains a lot of lignin, and laccase can oxidize lignin, so we preliminary studied the application of waste paper deinking of Lac MP in this paper.The optimum p H of a laccase derived from Coprinus comatus are 3.0, 6.0, 5.5 and 6.0, when the substrate is ABTS, guaiacol, 2,6-DMP and SGZ respectively, which are all close to neutral. And each of the optimum temperature is 65, 55, 70 and 50℃ respectively, which are relative high. At the same time, this laccase’s decolourization ratio of active deep blue KR is above 90%. So we used its amino acid sequence as template, blast through NCBI, and found many known sequence close to it. Fortunately, we found a high homology protein sequence as we predicted laccase from the unknown function protein, which is derived from Moniliophthora perniciosa FA553, having 64% homology with the laccase derived from Coprinus comatus. We synthesize it through the whole gene synthesis, named Lac MP, and expressed it in pichia pastoris. Through the shaking flask fermentation and the fermented liquid supernatant purification, we researched the enzymology properties using the purified enzyme, and we found: the optimum p H of Lac MP are 6.0, 6.5, 6.5 and 7.5, when the substrate is ABTS, guaiacol, 2,6-DMP and SGZ respectively, and each of the optimum temperature is 60, 45, 55 and 45℃ respectively, which are all close to the laccase derived from Coprinus comatus. The enzyme activity of Lac MP is more than 80% after 6 h under p H4.5-8.0, demonstrating its p H stability is good, and the enzyme activity of Lac MP has hardly fallen at all after 40 min under 30℃, but under 40℃, the enzyme activity is 80% after 15 min, 65% after 30 min, under 50℃, the enzyme activity is only 40% after 10 min, only 20% after 20 min, demonstrating its thermal stability is poor. At the same time, we also studied the Km and Kcat values under the four different substrates, and also know the high metal ion concentration environment has much effect on the enzyme activity, especially heavy metal ions, at the same time, EDTA has a very big impact on the enzyme activity because of it is copper ion oxidase.We mainly studied the effect of waste paper deinking through the amount of Lac MP, the combination use of Lac MP and its redox mediators and the combination use of Lac MP and S7 xylanase. The result is: the addition of Lac MP doesn’t improve the whiteness of paper, on the contrary, the whiteness is reduced, and the more addition, the whiteness is lower, as well the effective residual ink concentration(ERIC). But in terms of the mechanical properties of paper, the bursting index, tearing index and tensile index were improved( highest when the addition is 2U/g, increased by 4.35%, 10.81% and 8.16% respectively). In addition, the paper mechanical properties of the combination use of Lac MP and S7 xylanase are higher than the lonely use of Lac MP, the bursting index, tearing index and tensile index were increased by 3.33%, 5.23% and 2.82% respectively.The research result is: Lac MP has the property of the optimum p H close to neutral, and the p H stability is good in the neutral alkali environment. And the addition of Lac MP can improve the paper’s mechanical properties, and using together with S7 xylanase can improve further. This application of Lac MP in waste paper deinking provides a certain basis, and have a certain significance for the environment protection.