Abstract: [Objective] This study aims to obtain pure manganese peroxidase produced by white-rot basidiomycete Lenzites gibbosa strain CB-1 and study its characterizations in enzymology.[Method] On the basis of optimizing the culture of L. gibbosa and obtaining massive crude manganese peroxidases (MnPs) solutions which were salted out by (NH₄)₂SO₄, concentrated by polyethylene glycol (PEG) precipitation, and dialyzed against the initial buffer. The DEAE-Sepharose CL-6B ion exchange column chromatography, and Sephadex G-75 gel filtration column chromatography were used to purify MnP, and the purity of the purified MnP sample was detected by SDS-PAGE.[Result]Among the three (NH₄)₂SO₄ concentrations of 75%, 80%, and 85%, 85% (NH₄)₂SO₄ was the optimum saturation for precipitation of MnPs. With 85% (NH₄)₂SO₄ precipitation, the MnP activity and protein concentration in the supernatant were only 3.63 U·L^-1 and 0.132 mg·mL^-1, respectively, indicating that most MnPs had been precipitated. The initial buffer of Na₂HPO₄-NaH₂PO₄ (pH=7) was fixed to be the optimal buffer system by small test tube method. Two higher and one lower protein absorption peaks were detected in the samples after ion exchange column chromatography, and the MnP activity was detected only in the second higher peak. After the second higher peak sample went through Sephadex G-75 gel filtration column and was eluted, one distinct protein absorption peak was detected in which there was a high MnP activity. A single and distinct MnP protein band around 40 kDa in concentrated lyophilized sample was detected by SDS-PAGE, indicating that the purity of purified MnP had come up to the electrophoresis pure.[Conclusion] The salting out method with 85% (NH₄)₂SO₄ was the optimal method for precipitating and purifying MnPs produced by L. gibbosa in initial stage. PEG concentration method was proved simple, easy and effective. The purified MnP samples could be come up to the electrophoresis pure by the means of ion exchange chromatography and gel filtration chromatography after initial concentration.

Key words: Lenzites gibbosa, manganese peroxidases(MnPs), purification, ion exchange chromatography, electrophoresis pure