基于酶反应显色的产纤维素酶真菌筛选方法

Screening method of cellulose-producing fungi based on chromogenic enzyme reaction

刚果红显色法及革兰氏碘液显色法是进行产纤维素酶真菌筛选的常用方法,但存在显色不够明显和易受培养基成分干扰等缺点.建立方便、灵敏的产纤维素酶真菌筛选新方法,有助于快捷准确地筛选产纤维素酶真菌的菌株.基于真菌产生的纤维素酶会导致培养基中的纤维素底物水解,产生纤维寡糖,且寡糖产量与纤维素酶活力正相关.利用寡糖氧化酶-辣根过氧化物酶显色方法对寡糖浓度及纤维素酶活力进行分析,发现在一定浓度范围内,光密度值与寡糖浓度及纤维素酶浓度均存在良好的线性关系.将在中国深圳塘朗山的林地采样、浸提的菌液稀释,涂布到改良的马丁氏培养基平板.待长出单菌落后,将这些单菌落在含改良马丁氏培养基的24孔板中培养72 h,离心后取培养上清液加入到寡糖氧化酶-辣根过氧化物酶纤维素体系中进行显色,根据颜色深浅对产纤维素酶菌株进行初筛.采用纤维素酶产酶培养基对初筛菌株进行复筛和初步鉴定,共获得15株具有较高产纤维素酶能力的真菌.研究可为产纤维素酶真菌的筛选提供了一种明确而简单的方法.

Congo red staining and Gram iodine staining methods are common methods for screening of cellulaseproducing fungi,but there are shortcomings such as insufficient color development and being easy to be interfered by medium components.The establishment of a convenient and sensitive screening method is helpful for rapid and accurate screening of cellulase-producing fungi.Firstly,oligosaccharide concentration and cellulase activity were analyzed through the gluco-oligosaccharide oxidase-based horseradish peroxidase colorimetric method.The results showed that within a certain concentration,optical density value was linearly correlated with the oligosaccharide concentration and cellulase activity.Then,soil and woody samples were taken from the forest land of Tanglang Mountain in Shenzhen city,Guangdong province of China,diluted to proper concentration and coated on modified Martin's medium agar plates.After the purification of fungi,the single colonies were picked and inoculated in 24 well plates containing modified Martin's medium and cultivated for 72 hours.After centrifugation,the culture supernatant was added to the oligosaccharide oxidase based horseradish peroxidase colorimetric system for color development,and the cellulase producing strains were screened according to the color depth.Finally,the strains obtained in primary screening were rescreened with a cellulase-producing medium,and 15 fungal strains with relatively higher cellulase producing productivity were obtained and preliminarily identified.This work provides an easy and efficient method for screening cellulase-producing fungi.