偏肿革裥菌锰过氧化物酶的分级纯化

doi:10.11707/j.1001-7488.20170808

摘要/Abstract

摘要： [目的]研究白腐菌偏肿革裥菌（Lenzites gibbosa）锰过氧化物酶（MnP）的分级纯化，以期得到L.gibbosa MnP的纯品和进行后续酶学性质研究。[方法]在对L.gibbosa所产MnPs进行优化培养、获得大量MnPs粗酶液的基础上，通过硫酸铵（NH₄）₂SO₄分级盐析、聚乙二醇（PEG）沉淀进行粗酶液浓缩、在起始缓冲液中进行透析、经DEAE-琼脂糖CL-6B离子交换柱层析进行梯度洗脱和葡聚糖G-75凝胶过滤柱层析等方法对MnPs粗酶液进行纯化，并对分级纯化方法进行优化，对纯化浓缩后的纯酶样品采用SDS-PAGE方法进行酶纯度检验。[结果]在75%、80%和85%这3种（NH₄）₂SO₄饱和度中，85%的（NH₄）₂SO₄是沉淀MnPs的适宜饱和度，在该饱和度下测得上清液中酶活为3.63 U·L^-1，蛋白浓度为0.132 mg·mL^-1，表明MnPs已基本沉淀完全；试管小试法确定离子交换柱层析中上样的最适缓冲体系为pH7的Na₂HPO₄-NaH₂PO₄起始缓冲液；离子交换柱层析后收集的洗脱液检测到3个蛋白吸收峰，其中只在第2个蛋白吸收峰中检测出MnP活性，该MnP活性值变化曲线与蛋白吸光值一致。这个活性峰经凝胶过滤柱层析后，洗脱结果只出现1个明显的蛋白吸收峰，在其中检测出较高的MnP活性；浓缩后的冻干样品经SDS-PAGE电泳检测只在约40 kDa处有1条清晰的蛋白谱带，表明对MnP的纯化已达到电泳纯。[结论]筛选出85%饱和度的（NH₄）₂SO₄盐析法是初期沉淀与纯化L.gibbosa所产MnPs的最适方法；PEG浓缩法简便、易行、有效；初期浓缩后再经过离子交换柱层析和凝胶过滤柱层析可以得到电泳纯的MnP纯化样品。

关键词: 偏肿革裥菌, 锰过氧化物酶, 纯化, 离子交换柱层析, 电泳纯

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

中图分类号:

S718.81

张玉龙, 池玉杰, 冯连荣. 偏肿革裥菌锰过氧化物酶的分级纯化[J]. 林业科学, 2017, 53(8): 64-70.

Zhang Yulong, Chi Yujie, Feng Lianrong. Grading Purification of Manganese Peroxidases from Lenzites gibbosa[J]. Scientia Silvae Sinicae, 2017, 53(8): 64-70.

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