一色齿毛菌锰过氧化物酶(MnP)活性检测与MnP1基因的克隆

Detection on Manganese Peroxidase Activity and Cloning of Cu-mnp1 of Cerena unicolor

锰过氧化物酶(MnP)广泛存在于白腐菌中,是降解木质素的主要酶系。首先对采自长白山的一色齿毛菌菌株CB1降解木质素的过氧化物酶进行检测,结果表明一色齿毛菌可产生MnP,但不产生木质素过氧化物酶(LiP);Mn2+不是其产生MnP的必要因子,在LNAS培养基中加入木屑为底物的条件下,一色齿毛菌MnP最大酶活为18.4 U·L-1。在酶活检测的基础上,为了深入研究MnP降解木质素的功能,根据已知的MnP基因保守区序列设计引物,以一色齿毛菌菌株CB1的基因组DNA为模板,采用染色体步移技术,克隆到该菌株一个编码MnP1蛋白的全长DNA基因(GenBank登录号JQ782578.1),命名为Cu-mnp1。该基因DNA全长4 268 bp,含有16个外显子和15个内含子;其启动子区域含有TATA-Box,CAAT-Box,SP1,AP1,HSE,XRE等多个顺式作用元件;其cDNA基因(GenBank登录号JQ782579.1)编码区长度为1 092 bp,编码363个氨基酸。BLAST比对分析表明,在cDNA水平上Cu-mnp1与猴头菇菌株CB1的mnp2基因的相似性最高为70%。蛋白结构分析表明,Cu-MnP1含有4个二硫键属于短MnP。

Manganese peroxidase （MnP） widely exists in white-rot fungi and it is the main enzyme to degrade lignin. The ligninolytic peroxidase activity of Cerena unicolor stain CB1 collected from Changbai Mountain was firstly detected, result showed that C. unicolor could produce MnP, but no lignin peroxidase （LiP） excreted, and Mn2＋ was not the essential factor for C. unicolor to produce MnP. The highest MnP activity was gained when sawdust was added to LNAS culture solution with Mn2＋ substrate which was 18.4 U· L-1 On the basis of MnP activity determination, a MnP full length DNA gene termed Cu-mnpl （GenBank accession No. JQ782578.1 ） was cloned in order to study the function of MnP to degrade lignin furtherly. Forward and reverse primers for PCR and nested gene-specific primers for genome walking PCR were designed based on the conserved sequences of the known MnP genes and amplified DNA fragment, all the PCR reactions were conducted by the genomic DNA of the C. unicolor stain CB1 as the template. The full length DNA gene Cu- mnpl was 4 268 bp containing 16 exons and 15 introns, its promoter region contained diverse cis-aeting elements such as TATA-Box, CAAT-Box, SP1, API, HSE, and XRE. Its eDNA gene contained 1 092 bp coding region encoding 363 amino acids. BLAST analysis indicated that Cu-mnpl in the eDNA level showed the highest similarity with mnp2 gene （GenBank accession No. JQ248599. 1 ）from Hericium erinaceum strain CB1 which was 70%. Protein structure analysis showed that Cu-Mnpl contained 4 disulfide bonds belonging to short MnP.