木质素降解菌的筛选及其纤维素酶基因克隆表达研究

Screening and cloning ligninolytic strain and cellulase gene expression

以高效降解木质素为指标,进行木质素降解菌的筛选和纤维素酶处理纤维材料的研究。通过测定14株白腐菌菌株在愈创木酚、苯胺兰和鞣酸培养基上生长状况和酶活分泌能力,得到8株能产生阳性反应的菌株。以木质素和综纤维素失重的比值(SF指数)为指标,对这几株菌进行复筛,从中筛选出具有生长优势和强酶分泌能力的菌株平菇10969和侧耳WP1。与黄胞原毛平革菌Phanerochaete chrysosporium RP78和P.chrysosporium BKM-F-1767两株模式菌相比,白腐菌具有良好的生长优势、强酶系分泌能力和降解的优势。从分离的白腐菌中克隆纤维素酶基因(egl2),表达蛋白并测定酶活。用粗酶液处理不同的纤维材料,结果表明,其还原糖产量为综纤维素(酸解)>菌草(白腐菌处理)>未处理菌草。白腐菌的研究对草质资源的充分利用、污染物的降解、燃料乙醇的开发以及我国生态农业的持续发展等都有着重要意义。

In order to effectively biodegrade lignin,lignin-degrading bacteria screening and fiber material treatment with cellulase were conducted.Eight strains producing positive reaction were selected by detecting the growth status and enzyme activity of 14 strains on the media with guaiacol,aniline and tannic acid.The strains were re-screened by using the ratio of Klason lignin degradation rate to holocellulose degradation rate （SF index） as indicator.In comparison to Phanerochaete chrysosporium RP78 and P.chrysosporium BKM-F-1767,strains Pleurotus ostreatus Po 10969 and Ganoderma applanatum WP1 were selected because their higher growth rate,enzyme secretion and degradation ability.Meanwhile,cellulase gene （egl2） was cloned,expressed and enzyme activity determined.Reducing-sugar produced by different treatments was determined by soaking the materials with crude enzymes solution.The results show that sugar-reduction ability of the materials is in the order of：Juncao Penniusetum sinese Roxb treated with NaClO2Juncao P.sinese Roxb treated with Po10969untreated Juncao P.sinese Roxb.Research on white rot fungus is significant for making full use of plant resources,safe degradation of pollutants and fuel ethanol exploration,as well as the sustainable development of eco-agriculture in China.