Expression of a laccase cDNA from Trametes sp AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation
Expression of a laccase cDNA from Trametes sp AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation
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DOI：
10.1111/j.1574-6968.2006.00209.x
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发表时间：
2006-05-01
影响因子：
2.1
通讯作者：
Yu, ZL
中科院分区：
文献类型：
--
作者：
Hong, YZ;Xiao, YH;Yu, ZL
将栓菌AH 28 -2漆酶cDNA在毕赤酵母中进行表达，最高表达量为4.0 mg L-1（1360 U mg（-1））。重组漆酶A（rLacA）的ABTS（2，2 ′-连氮双[3-乙基苯并噻唑-唑啉-6-磺酸]）的表观Km（24.6 μ M）和碳水化合物含量与天然LacA（nLacA）的表观Km和碳水化合物含量大致相同。然而，当ABTS和愈创木酚作为底物时，这两种酶的最适pH不同。rLacA酶稳定性的最适pH为5.5。还研究了热稳定性。利用低能氮离子注入对rLacA进行诱变，得到一株漆酶产量为7.7 mg L ~（-1）（1085 U mg ~（-1））的酵母克隆，比未辐照的对照（4.0 mg L ~（-1））高92.5%。与rLacA相比，突变体LacA（mLacA）的催化活性略有变化，但热稳定性上级。
A laccase cDNA from Trametes sp. AH28-2 was expressed in Pichia pastoris, with the highest expression level of 4.0 mg L-1 (1360 U mg(-1)). The apparent K-m (24.6 mu M) for ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) and the carbohydrate content of the recombinant laccase A (rLacA) are approximately identical to those of the native LacA (nLacA). However, the two enzymes differed in the pH optimum when both ABTS and guaiacol served as substrates. The optimum pH for enzyme stability is 5.5 for rLacA. Thermal stability was also investigated. The mutagenesis of rLacA utilizing low-energy nitrogen ion implantation resulted in the isolation of a yeast clone that produced 7.7 mg L-1 (1085 U mg(-1)) of laccase, 92.5% more than the nonirradiated control (4.0 mg L-1). Compared with rLacA, the mutant LacA (mLacA) with five amino-acid residue changes in the coding sequence showed a slight change in its catalytic ability but superior thermal stability.