假单孢菌PKE117木素过氧化物酶的酶学性质及基因克隆

北京大学学报（自然科学版）

假单孢菌PKE117木素过氧化物酶的酶学性质及基因克隆

杨金水¹,袁红莉¹,李宝珍¹,倪晋仁²

¹中国农业大学生物学院，北京，100094；²,京大学环境工程系，水沙科学教育部重点实验室，北京，100871，通讯作者，E-mail:nijinren@iee.pku.edu.cn

收稿日期:2006-08-13 出版日期:2007-07-20 发布日期:2007-07-20

Lignin Peroxidase Characteristics and Its Gene Clone from Pseudomonas sp. PKE117

YANG Jinshui¹,YUAN Hongli¹,LI Baozhen¹,NI Jinren²

¹College of Biological Science, China Agricultural University, Beijing, 100094; ²The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Department of Environmental Engineering, Peking University, Beijing， 100871，　Corresponding Author, E-mail: nijinren@iee.pku.edu.cn

Received:2006-08-13 Online:2007-07-20 Published:2007-07-20

摘要/Abstract

摘要： 对纯化得到的假单孢菌PKE117的胞外木素过氧化物酶的酶学性质进行了初步研究，发现其最适pH为5.0，最适温度为30 ℃，以藜芦醇为底物，在25 ℃，pH3.0的琥珀酸钠缓冲液中与底物反应时的 K _m值为(0.277±0.000 8) mmol/L，v_max为(0.049±0.001) mmol?mg^-1 protein?min^-1。根据LiP已知保守序列设计引物，提取质粒，扩增得到一条199 bp的DNA片段lip1和一条563 bp的DNA片段lip2，序列比对结果显示与已报道的白腐真菌过氧化物酶基因的同源性不是很高。核苷酸翻译后的氨基酸序列比对结果发现， LiP1与 Pycnoporus sanguineus的漆酶的同源性达到100％，与Mycobacterium bovis 的木素过氧化物酶的同源性达到80％；LiP2与 Arabidopsis thaliana 的过氧化物酶同源性达到100％，与 Hordeum vulgare 的过氧化物酶1同源性为75％。

关键词: 假单孢菌, 木素过氧化物酶, 基因片段

Abstract: The present study was undertaken to investigate the enzyme characteristics of purified lignin peroxidase (LiP) from Pseudomonas sp. PKE117. The PKE117 LiP catalyzed veratryl alcohol oxidase activity at 25 ℃ showed that the optimum pH was 5.0, the optimum temperature at 30 ℃,　the K_m of the LiP was (0.277±0.000 8) mmol/L and the v_max was (0.049±0.001) mmol?mg^-1 protein?min^-1.The authors designed two special primers according to conservative sequences of lignin peroxidase and got a 199 bp DNA fragment (lip1) and a 563 bp DNA fragment (lip2). Sequence comparison demonstrated that lip1 and lip2 had low homology with known white rot fungi's lip genes. After translation, LiP1 had 100% homology with Pycnoporus sanguineus laccase, 80% homology with Mycobacterium bovis LiP. LiP2 had 100% homology with Arabidopsis thaliana LiP and 75% homology with Hordeum vulgare peroxidase 1.

Key words: Pseudomonassp., lignin peroxidase, gene fragment

中图分类号:

X 172
Q 785

杨金水,袁红莉,李宝珍,倪晋仁. 假单孢菌PKE117木素过氧化物酶的酶学性质及基因克隆[J]. 北京大学学报（自然科学版）.

YANG Jinshui,YUAN Hongli,LI Baozhen,NI Jinren. Lignin Peroxidase Characteristics and Its Gene Clone from Pseudomonas sp. PKE117[J]. Acta Scientiarum Naturalium Universitatis Pekinensis.