栓菌漆酶在毕赤酵母中高效表达及重组酶的性质

High Output of a Trametes Laccase in Pichia pastoris and Characterization of Recombinant Enzymes

摘要:

栓菌420（Trametes sp. 420）漆酶基因lacD以两种方式在巴斯德毕赤酵母（Pichia pastoris）进行异源表达，产生两种重组漆酶：rLacDx（具有天然N-末端）和rLacDe（N-末端带有8个额外的氨基酸残基）。摇瓶发酵18d，rLacDx和rLacDe的产量分别为1.21×10⁵u/L、7.38×10⁴u/L ［以2,2′-连氮-3-乙苯-二噻唑-6磺酸（ABTS）为底物］。在高密度发酵条件下，rLacDx的产量增加到2.39×10⁵u/L，同时其生产周期降至7.5 d。两种重组酶对愈创木酚底物的氧化特性相似，且在50℃和pH3～10的范围内均稳定。然而，rLacDx对底物ABTS的比活力（1761u/mg）高于rLacDe （1122u/mg），其表观K_m值(427μmol/L)低于rLacDe (604μmol/L)。

Abstract:

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21×10⁵u/L and 7.38×10⁴u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39×10⁵u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50℃ and at a pH range from 3 to 10. However, the specific activity of rLacDx (1,761u/mg) for ABTS was higher than that of rLacDe (1,122u/mg), and the apparent K_m value of rLacDx (427 μM) was less than that of rLacDe (604 μM).