Approach to a High-Level Production of the Basidiomycetous Ligninolytic Enzymes in Secretory Expression Systems of Genetically Modified Yeast

白腐担子真菌降解木质素是天然高分子材料中最复杂的聚合物。木质素含有大量潜在的有用原料，因此对担子菌木质素分解酶的研究受到鼓励。近年来的研究表明，真菌也能有效地降解人造外源物，如2,4,6-三氯苯酚、多氯联苯（PCBs）和氯化二苯并对二恶英。白腐菌对这些外源物的降解主要依赖于分泌酶的反应，其中主要是木质素过氧化物酶（LiP）、锰过氧化物酶（MnP）和漆酶。由于木质素降解酶的生长活性较低，在真菌中很难高产出木质素降解酶。近年来的研究倾向于利用酵母来克服这一局限性，毕赤酵母是最具特征的真核表达系统之一。因此，本研究旨在提供在转基因巴氏酵母分泌表达系统中高水平生产木质素降解酶的配方数据。从经EcoRIr^+基因转化的巴氏酵母GS115培养的上清液中获得了大量EcoRI。该实验证实了巴氏酵母对外源蛋白生产的有用性。以木质素和双酚A作为人工外源，分别用白腐担子菌、革氏菌IFO4917和Elfvingia applanata SMC700进行处理。两者都被有效地降解。木质素的降解几乎与MnP活性同步。由此克隆了一个编码MnP的基因。该基因由1095 bp的ORF组成，编码364个氨基酸残基。

White-rot basidiomycetous fungi degrade xenobiotic lignin that is the most complex polymer among naturally occurring high-molecular weight materials. Lignin contains large quantities of potentially useful raw materials, and hence the work on the basidiomycetous ligninolytic enzymes is encouraged. Recent studies have shown that fungi also effectively degrade artificial xenobiotics, such as 2,4,6-trichlorophenol, polychlorinated biphenyls (PCBs), and chlorinated dibenzo-p-dioxin, Degradation of these xenobiotics by white-rot fungi is largely dependant on the reaction of secreted enzymes, major of which are ligin peroxidase (LiP), manganese peroxidase(MnP) and laccase.High production of ligninolytic enzymes in fungi is difficult because of their low growth activity. Recent studies prefer to overcome the limitation with the use of yeast Pichia pastor is that is one of the best characterized eukaryotic expression systems. Thus the present study was undertaken to presernt data for a formulation of a high-level production of the basidiomycetous ligninolytic enzymes in secretory expression systems of genetically modified P.pastoris.Large amount of EcoRI was obtained from the supernatant of the culture of P.pastoris GS115 that was transformed with the the EcoRIr^+ gene. This experiment corroborates the usefulness of P.pastoris for foreign protein productions.Lignin and bisphenol A, which is used as an artificial xenobiotics, were then treated by white-rot basidiomycetousfungi, Coriolus hirstus IFO4917 and Elfvingia applanata SMC700. Both were effectively degraded. Degradation of lignin was almost concurrent with MnP activity. Thus a gene encoding a MnP was cloned from E.applanata. TheMnP gene was consisted of 1,095 bp ORF coding for 364 amino acid residues.