东祁连山高寒草地土壤产漆酶真菌的筛选、鉴定及产酶条件的初步研究  

Study on a laccase-producing fungus from alpine grassland soil in eastern Qilian Mountains:screening,identification,and activity analyses

作　　者：芦光新[1] 王军邦[2] 陈秀蓉[3] 杨成德[3] 薛莉[3] 

机构地区：[1]青海大学农牧学院草业科学系,青海西宁810016 [2]中国科学院地理科学与资源研究所,北京100094 [3]甘肃农业大学草业学院草业生态系统教育部重点实验室中-美草地畜牧业可持续发展研究中心,甘肃兰州730070

出　　处：《草业学报》2014年第2期243-252,共10页Acta Prataculturae Sinica

基　　金：国家自然科学基金"青藏高原草地耐低温纤维素分解真菌多样性研究"(41261064);青海大学高层次人才基金"产漆酶真菌的筛选及多酶系菌群的构建"(2012-QGC-9)资助

摘　　要：以高寒草地土壤分离出的５６个真菌菌株为研究对象，经愈创木酚-ＰＤＡ培养基和α- 萘酚-ＰＤＡ 选择性培养基初步筛选，再根据菌株在愈创木酚、邻苯二酚、邻苯甲苯胺为底物的选择性培养基上菌体生长及菌落大小、漆酶催化氧化还原反应产生的变色圈直径大小及其变色圈颜色深浅程度，以及测定油菜秸秆诱导的液体发酵产漆酶活力，筛选出１株产漆酶酶活真菌菌株（编号为３１０ｂ）。经ｒＤＮＡ-ＩＴＳ基因序列分析，初步鉴定为Marasmius tricolor 。对菌株３１０ｂ产漆酶的条件进行了初步研究，结果表明，菌株３１０ｂ在２５℃ 培养条件下产漆酶活力最大，初始ｐＨ 值为４．０时，漆酶活力最大，蔗糖、蛋白胨分别为诱导菌株产漆酶活力最高的碳、氮源。几种非营养有机物对菌株产漆酶活力大小不同，α- 萘酚和吐温-８０对菌株产漆酶没有明显影响，愈创木酚、单宁酸、吲哚乙酸抑制菌株产漆酶，其中吲哚乙酸抑制作用最强烈（P＜０．０１）。在Ｃｕ２＋ 浓度为０．００１～０．０２５ｇ／Ｌ 范围内，随Ｃｕ２＋ 增加，产漆酶活力增加，０．０２５ｇ／Ｌ 时产酶活力最大。随着接种量的增加，诱导漆酶活力增加。在６０～１８０ｒ／ｍｉｎ的转速范围内，随着转速的增加，漆酶活力增加。油菜秸秆粉的量在０～１ｇ范围内，随着秸秆添加量的增加，漆酶活力增加；１～２ｇ范围内，随着秸秆添加量的增加，漆酶活力减小。The sixty-five fungi strains that were isolated from the alpine grassland soil in the eastern Qilian Mountains were researched as the object in this paper. The fungi initially selected through culturing on the me dium of guaiacol-PDA and α naphthol-PDA medium. The fungi with the function to produce laccases was screened （identified as No. 310b） based on the growth and colony sizes on the culture mediums of guaiacol, caechol, and o-benzyl aniline, the activities of redox reaction catalyzed by the laccases in these fungi grown, as well as the activity of laccases in these fungi cultured on the stems of oil rape. By rDNA ITS sequence analysis, the fungus was identified as Marasmius tricolor. Then elementary study on the conditions under which laccases were produced by the fungus （No. 310b） was carried out. These results showed that the activity of laccases in the fungus reached the peak when it was cultured under the condition： 25C , initial pH 4.0, sucrose and pep tone that were used as carbon and nitrogen resources, respectively. Few non-trophic organic chemicals showed different effects on laccase activity in the fungus. α-naphthol and Tween-80 did not have significant effects on laccase activity in the fungus, but guaiacol, tannic acid, and indole acetic acid inhibited the activity of laccascs, especiall indole acetic acid, which significantly inhibited laccase activity （P〈0.01）. When Cu2＋ concentrations ranged from 0.001--0.025 g/L, laccase activity increased as the concentrations of Cu2＋ increased, reaching the peak at 0. 025 g/L. With the increase of inoculum sizes, laccase activity increased. Ranging from 60 to 180 r/min, laccase activity increased. When the amount of the powder of oil rape stems ranged from 0 to 1 g, lacca se activity increased, but when the amount was higher than 1, the activity decreased

关 键 词：高寒草地土壤 漆酶 真菌 鉴定 产酶条件 

分 类 号：Q93[生物学—微生物学]