Soden D M, O'Callaghan J, Dobson A D W
National Food Biotechnology Centre1 and Microbiology Department2, University College, National University of Ireland, Cork, Ireland.
Microbiology (Reading). 2002 Dec;148(Pt 12):4003-4014. doi: 10.1099/00221287-148-12-4003.
The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. The native Ple. sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic. pastoris. The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4.38. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3.3, 6, 6.5 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 degrees C and pH 3.5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.
来自凤尾菇的Psc lac4基因已被克隆，并在AOX1甲醇诱导型启动子的控制下，在异源宿主毕赤酵母中表达。天然凤尾菇漆酶信号序列在指导毕赤酵母中表达的lac4分泌方面是有效的。发现控制培养基的pH值和温度对于获得足够量的蛋白质以进行纯化和后续的生化表征很重要。重组Psc Lac4被纯化至电泳纯，并显示与刺芹侧耳Lac1有免疫相关性。纯化的漆酶估计分子量约为59 kDa，碳水化合物含量约为7%，计算得到的pI为4.38。该酶氧化底物2,2-联氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)、2,6-二甲氧基苯酚、丁香醛连氮和愈创木酚，分别表现出最佳pH值为3.3、6、6.5和7。以ABTS为底物时，该酶在35℃和pH 3.5下表现出最佳活性。该酶受到叠氮化钠和巯基乙酸的强烈抑制，但不受EDTA的抑制。
