野生茯苓鉴定及其木质纤维素降解酶系研究

Identification of wild Wolfiporia cocos and study on its lignocellulolytic enzymes

关键词:  茯苓, ITS序列, 纤维素酶, 半纤维素酶, 木质素降解酶

Key words:  Wolfiporia cocos, ITS, cellulase, hemicellulase, ligninolytic enzymes

摘要:

为揭示茯苓木质纤维素降解酶系及培养方式对其主要酶系的影响,该研究通过对野生茯苓菌株进行培养特性的显微观察,利用3对引物PCR扩增进行系统发育学的鉴定,经定性培养筛选出优势菌株YX1,采用酶标仪测定不同条件下茯苓纤维素酶、半纤维素酶和木质素降解酶的活力大小。结果表明:(1)茯苓有菌丝体、子实体和菌核3 种形态特征。(2)PCR分别获得rDNA-ITS序列1 652 bp、核糖体大亚基序列 660 bp和翻译延伸因子序列545 bp,提交至 NCBI,登录号分别为 ON129554、ON129553和 ON155840。(3)纤维素酶和半纤维素酶在有松木屑和无松木屑条件下,外切β-葡聚糖酶(CBH)、内切β-葡聚糖酶(EG)、β-葡萄糖苷酶(BGL)最高分泌量分别为16 ～ 17 U·mL^-1、32 ～ 35 U·mL^-1、36 ～ 37 U·mL^-1; 木聚糖酶、甘露聚糖酶和α-葡萄糖苷酶最高分泌量分别为28 ～ 38 U·mL^-1、280 ～ 342 U·mL^-1、9 ～ 11 U·mL^-1; 锰过氧化物酶(MnP)、漆酶(laccase)、木质素过氧化物酶(LiP)这3种木质素降解酶在4种不同培养液中均有微弱的酶活性,木质纤维素酶中的酶活性大小依次为甘露聚糖酶>木聚糖酶>BGL>EG>CBH>α-葡萄糖苷酶>LiP>MnP>Laccase,纤维素酶和半纤维素酶酶活性之间存在显著差异(P<0.05)。综上表明,该研究结合形态学与分子鉴定,明确了野生茯苓YX1的分类地位,与褐腐菌在亲缘关系上既有联系又存在遗传差距,为茯苓产生木质纤维素降解酶系的降解机制提供了基础酶学参考。

Abstract:

In order to reveal the effects of Wolfiporia cocos lignocellulolytic enzymes and culture methods on its main enzymes, the main lignocellulolytic enzymes of W. cocos were determined in this study. The microscopic observation of the culture characteristics of the wild W. cocos strains was carried out, three pairs of primers were used for PCR amplification to carry out phylogenetic identification, and the dominant strain YX1 was screened by qualitative culture and finally the activities of cellulase, hemicellulase and ligninolytic enzymes under different conditions were determined by microplate reader. The results were as follows:(1)W. cocos had mycelium, fruiting body and sclerotium three morphological characteristics.(2)PCR obtained rDNA-ITS sequence of 1 652 bp, ribosomal large subunit sequence of 660 bp and translation elongation factor sequence of 545 bp, and submitted to NCBI,accession numbers were ON129554, ON129553, and ON155840, respectively.(3)The highest secretion of exo-β-glucanase(CBH), endo-β-glucanase(EG)and β-glucosidase(BGL)in the presence or absence of sawdust was 16-17 U·mL^-1, 32-35 U·mL^-1, 36-37 U·mL^-1; The maximum secretion of xylanase, mannanase and α-glucosidase was 28-38 U·mL^-1, 280-342 U·mL^-1, 9-11 U·mL^-1. The three ligninolytic enzymes manganese peroxidase(MnP), laccase, and lignin peroxidase(LiP)had weak enzymatic activities in four different cultures, the magnitude of enzymatic activities in lignocellulases were in the order of mannanase > xylanase > BGL> EG > CBH > α-glucosidase > LiP > MnP > laccase, and there were significant differences between cellulase and hemicellulase enzymatic activities(P<0.05). In conclusion, this study combines morphological and molecular identification to clarify the taxonomic status of YX1, which has both relationship and genetic gap with brown rot fungi, and provides a basic enzymatic reference for the degradation mechanism of the lignocellulolytic enzymes system produced by W. cocos.