Production And Purification Of Laccaset For Optical Detection Of 2, 4-dichlorophenol

The laccase is a kind of polyphenoloxidase containing copper and is widely distributed in plant,fungus and bacterium.The laccase can catalyze degradations of many organic and inorganic substrates,and can be widely used in industry and biological repai.The Coriolus varicolor is one kind of white-rot fungus and can produce laccase.2,4-DCP is a kind of environmental endocrine interferon with toxicity and is difficult to degrade.The detection of its concentration is very important to environmental protection and human health.In this thesis,the research work focused on the fermentation production and purification of Coriolus varicolor laccase,the laccase catalysis characteristics and its application in 2,4-DCP biodegradation and optical determination.The main results are as follows:(1) The main raise conditions of laccase production with Coriolus varicolor have been optimized and the best enzyme culture medium composition is confirmed as follows:potato juice 20%,glucose 20 g/L,peptone 4.0 g/L,KH₂PO₄ 3.0g/L,MgSO₄ 1.5 g/L,ZnSO₄ 500 mg/L,MnSO₄ 500 mg/L,CuSO₄ 160 mg/L,ABTS 1.0 mmol/L, Tween-800.10%,pH=5.0,28℃,13 d.(2) In the optimal fermentation conditions,the laccase activity is 92.2 U/mL in the thick enzyme fluid,.The laccase was puirifed from Coriolus versicolor by uhraifltration,Sephadex G-100 and DEAE-Sephrose Fast Flow.After the purification the enzyme activity is 28.4 U/mg.The molecular weight of the puirifed laccase was determined as about 64.4 kD by SDS-PAGE.(3)For the ABTS osidation catalyzed with laccase,the optimal pH and temperature is 2.0 and 55℃,respectively.The Km value is 7.78μM,and Vmax is 5.11×10^-5 M·min^-1 at 25℃.The laccase has good thermostability and storage stability. Mg²⁺,Cu²⁺,Zn²⁺,Ca²⁺ can increase the laccase activity,but Ag⁺,Pb²⁺,Hg²⁺perform the suppression to the laccase activity.Among them,Mg²⁺ has the best promotion ability to the laccase activity and Ag⁺ perform the strongest suppression to the laccase activity.(4) The DCP oxidation catalyzed by laccase is an irreversible redox reaction. The best pH is 5.0 and the optimum temperature is 45℃.Under the optimal catalysis conditions,the early stage of the DCP oxidation catalyzed by laccase can be regarded as first-order dynamic process.(5) The laccase-mediator system was formed using ABTS as the mediator. With this system,the optimal pH and temperature for the DCP oxidation are:4.5 and 50℃,respectively.(6) The optical detection system of DCP concemtration has been constructed based on the catalysis of laccase-mediator system.At 25℃and pH 4.5,the detecting range is 1×10^-7-9×10^-5 M and the response time is 40 s.Mg²⁺,Ca²⁺ can cause the detection results to be higher,which can be eliminated by adding the shielding substance in the system.This detecting method is reliable.