关键词: 偏肿革裥菌, 锰过氧化物酶, 纯化, 酶学性质, 染料脱色

Abstract: [Objective] In order to get a clear picture of the sequence signature, characteristics, and decolorizing ability to four types of dyes of the purified manganese peroxidase (MnP) produced by white-rot basidiomycete Lenzites gibbosa strain CB-1.[Method] A single MnP protein band detected in the SDS-PAGE electrophoresis of the purified sample was cut and sequenced by Nano LC-ESI-MS/MS peptide sequencing technology. The characteristics of the purified MnP were studied, the apparent molecular weight of the purified MnP was calculated by the standard curve of protein molecular weight and mobility according to SDA-PAGE pattern, the optimal reaction temperature and thermal stability, the optimal reaction pH and pH stability were determined by enzymatic activity measuringmethod, Michaelis-constant K_m was solved by lineweaver-Burk double bottom figure. The decolorization ability of culture fluid and purified MnP of L. gibbosa to four types s of dyes, namely, alizarin red, neutral red, congo red, and crystal violet were studied, respectively.[Result] Two conserved amino acid sequences of MnP scanned by Tandem MS perfectly match with Lg-MnP1 (GenBank Accession No. ACO92620) sequence, indicating that Lg-MnP1 represented by two conserved peptides is the main protein in the only electrophoresis band. The apparent molecular weight of Lg-MnP1 is 45.39 kDa. Its optimal reaction temperature is about 35℃, it has some catalytic ability and stability during 40℃ but rapidly becomes inactivated beyond 50℃. Its optimal reaction pH value is 3.5, its catakytic activity rapidly decreases when pH value is above 4.5, and it is the most stable in pH 2-3 when it is kept for more than 10 hours. Its Michaelis-constant K_m is 6.124 mmol·L^-1 at 21℃ with 2,6-DMP as substrate. The decolorization percent of culture fluid is 100% to alizarin, 22.17% to neutral red, and 19.09% to congo red at 1 h, respectively, but low to crystal violet and only 0.018% at 36 h, whereas that of the purified Lg-MnP1 solution at 2 h is 100% to neutral red, 95.55% to congo red, 75.85% to alizarin, and 36.57% to crystal violet.[Conclusion] The MnP in the only band of SDS-PAGE is Lg-MnP1. Lg-MnP1 is a mesophile and partial acidic enzyme and with higher affinity to 2,6-DMP. The culture fluid with mycylia of L. gibbosa has a thoroughly decolorizing ability to anthraquinone dye, while the purified Lg-MnP1 has a higher decolorizing efficiency to neutral red, congo red, and crystal violet than the culture fluid with mycylia, laccases and MnPs.

Key words: Lenzites gibbosa, manganese peroxidases (MnPs), purification, characteristics in enzymology, dye decoloring