以来源于变色栓菌的同工酶LacA、LacB、LacC,杂色云芝的同工酶Lcc1、Lcc2,以及细菌ARA等6种重组漆酶为研究对象,比较了它们的最适反应温度、最适反应pH、温度稳定性、pH稳定性、动力学常数Km,以及Cu2+对漆酶酶活的影响等酶学特性。结果表明:以邻联甲苯胺为底物时,LacA、LacB、LacC、Lcc1、Lcc2、ARA的最适反应温度分别为60、60、70、60、55和75 ℃; 以愈创木酚为底物时,6种漆酶的最适反应pH分别为5.0、5.0、4.5、5.0、4.5、7.0。LacA在催化ABTS、愈创木酚、邻联甲苯胺、丁香醛连氮以及2,6-二甲基苯酚时,其最适反应温度分别为55、70、60、20、65 ℃。此外,真菌漆酶在30～60 ℃,pH为2.5～7.0范围内都有较好的稳定性,细菌来源的漆酶在80 ℃下保持60 min,在pH=7.0时保持6 h仍具有较高的酶活力。Cu2+对细菌漆酶的激活作用要明显高于真菌漆酶。同时,以ABTS为底物时,Km值最小,且不同来源漆酶的Km值相差不大。因此,6种不同来源的重组漆酶在催化同一种底物时,酶学特性存在着差异性,同一种重组漆酶在催化不同底物时,其酶学特性也有较大差异。
Abstract
In order to illuminate the importance of revising the methods for determining laccase activity according to the porperties and purpose in the actual use,the physical and chemical properties of isoenzymes LacA, LacB and LacC from Trametes versicolor and the Lcc1 and Lcc2 from Coriolus versicolor and ARA from bacterium were compared in this paper. The results showed that the enzymatic characteristics changed with the types of the laccases and substrates. When otolidine as the substrate, the optimal temperatures of LacA, LacB, LacC, Lcc1, Lcc2, ARA were 60,60,70,60,55 and 75 ℃, respectively. While the guaiacol as the substrate, the optimal pH values of the six enzymes were 5.0,5.0,4.5,5.0,4.5 and 7.0,respectively. The optimal temperatures of LacA were 55,70,60,20 and 65 ℃ for the ABTS, guaiacol, o-tolidine, syringaldazine and 2, 6-xylenol as the substrates, respectively. The five fungal laccase enzymes had good thermal stability between 30 ℃ and 60 ℃ and pH stability between 2.5 and 7.0, while the bacterial laccase had strong stability under high temperature and neutral pH. According to the Km, it could be concluded that the specificity for ABTS was better than the four other substrates. All the results indicated that the enzymatic characteristics of laccases catalysis from different sources under the same substrate were different. Meanwhile, the enzymatic characteristics of same recombinant laccase under different substrates were also large differences.
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基金
收稿日期:2013-12-03 修回日期:2014-02-25
基金项目:国家林业局“948”项目(2011-4-15); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:李琦,硕士生。*通信作者:赵林果,教授。E-mail:lgzhao@njfu.edu.cn。
引文格式:李琦,赵东霞,刘世萍,等. 不同来源重组漆酶的酶学特性[J]. 南京林业大学学报:自然科学版,2014,38(3):93-97.
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