南京林业大学学报(自然科学版)
南京林業大學學報(自然科學版)
남경임업대학학보(자연과학판)
JOURNAL OF NANJING FORESTRY UNIVERSITY(NATURAL SCIENCE EDITION)
2014年
3期
93-97
,共5页
李琦%赵东霞%刘世萍%柴常升%赵林果
李琦%趙東霞%劉世萍%柴常升%趙林果
리기%조동하%류세평%시상승%조림과
重组漆酶%酶学性质%酶活力测定
重組漆酶%酶學性質%酶活力測定
중조칠매%매학성질%매활력측정
recombinant laccase%enzymatic characteristic%determination of enzyme activity
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。
以來源于變色栓菌的同工酶LacA、LacB、LacC,雜色雲芝的同工酶Lcc1、Lcc2,以及細菌ARA等6種重組漆酶為研究對象,比較瞭它們的最適反應溫度、最適反應pH、溫度穩定性、pH穩定性、動力學常數Km ,以及Cu2+對漆酶酶活的影響等酶學特性。結果錶明:以鄰聯甲苯胺為底物時,LacA、LacB、LacC、Lcc1、Lcc2、ARA的最適反應溫度分彆為60、60、70、60、55和75℃;以愈創木酚為底物時,6種漆酶的最適反應pH分彆為5.0、5.0、4.5、5.0、4?5、7.0。 LacA在催化ABTS、愈創木酚、鄰聯甲苯胺、丁香醛連氮以及2,6-二甲基苯酚時,其最適反應溫度分彆為55、70、60、20、65℃。此外,真菌漆酶在30~60℃,pH為2?5~7?0範圍內都有較好的穩定性,細菌來源的漆酶在80℃下保持60 min,在pH=7?0時保持6 h仍具有較高的酶活力。 Cu2+對細菌漆酶的激活作用要明顯高于真菌漆酶。同時,以ABTS為底物時,Km值最小,且不同來源漆酶的Km值相差不大。因此,6種不同來源的重組漆酶在催化同一種底物時,酶學特性存在著差異性,同一種重組漆酶在催化不同底物時,其酶學特性也有較大差異。
이래원우변색전균적동공매LacA、LacB、LacC,잡색운지적동공매Lcc1、Lcc2,이급세균ARA등6충중조칠매위연구대상,비교료타문적최괄반응온도、최괄반응pH、온도은정성、pH은정성、동역학상수Km ,이급Cu2+대칠매매활적영향등매학특성。결과표명:이린련갑분알위저물시,LacA、LacB、LacC、Lcc1、Lcc2、ARA적최괄반응온도분별위60、60、70、60、55화75℃;이유창목분위저물시,6충칠매적최괄반응pH분별위5.0、5.0、4.5、5.0、4?5、7.0。 LacA재최화ABTS、유창목분、린련갑분알、정향철련담이급2,6-이갑기분분시,기최괄반응온도분별위55、70、60、20、65℃。차외,진균칠매재30~60℃,pH위2?5~7?0범위내도유교호적은정성,세균래원적칠매재80℃하보지60 min,재pH=7?0시보지6 h잉구유교고적매활력。 Cu2+대세균칠매적격활작용요명현고우진균칠매。동시,이ABTS위저물시,Km치최소,차불동래원칠매적Km치상차불대。인차,6충불동래원적중조칠매재최화동일충저물시,매학특성존재착차이성,동일충중조칠매재최화불동저물시,기매학특성야유교대차이。
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 otoli-dine 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 recom-binant laccase under different substrates were also large differences.