一种筛选胞外纤维素酶产生菌的快捷、灵敏、有效的方法

A rapid, sensitive and effective method for screening of the fungi excreting extracellular cellulases

【目的】建立一种能快捷、灵敏、有效筛选高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的方法,用于胞外纤维素酶产生菌检测筛选。【方法】将以微晶纤维素(Avicel)或羧甲基纤维素(CMC)为底物的胞外纤维素酶产生菌平板筛选法常用的刚果红或碘液浸泡染色改为碘液熏染,减少碘液的消耗和对环境的污染;建立以对硝基苯酚-β-1,4-葡萄糖苷(p NPG)为底物的β-葡萄糖苷酶发色底物平板筛选法;将两方法串联用于高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的筛选。【结果】建立了分别以CMC和Avicel为底物结合碘液熏染的胞外纤维素酶产生菌平板筛选法和以p NPG为底物的β-葡萄糖苷酶发色底物平板筛选法,从56株真菌中筛选出了8株纤维素酶活性水平为++++的胞外纤维素酶产生菌,从后者筛选出4株高β-葡萄糖苷酶活性的胞外纤维素酶产生菌。【结论】将以CMC和Avicel为底物结合碘液熏染的平板筛选法和以p NPG为底物的发色底物平板筛选法串联,可快捷、灵敏、有效地用于高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的筛选。

Objective] To establish a rapid, sensitive and effective method for screening of the fungi producing extracellular cellulases with high β-glucosidase activity. [Methods] Iodine vapor staining was used to replace Congo red or iodine dip dyeing in the screening methods of extracellular cellulase producing fungi by agarose plates complemented with avicel or CMC in order to reduce the consumption and pollution of gram＇s iodine, and the screening method of β-glucosidase producing fungi by the agarose plates complemented with 4-Nitrophenyl-β-D-glucopyranoside（pNPG） was established. Finally, these two methods were used successively to screening the fungi producing extracellular cellulases with high β-glucosidase activity. [Results] The screening methods of both the avicel or CMC agarose plates with iodine vapor staining and the pNPG agarose plates were established successfully, and used successively to screening 8 strains of extracellular cellulase producing fungi with level ＋＋＋＋ cellulase activity out of 56 fungus strains, and from which 4 strains of extracellular cellulase producing fungi with high β-glucosidase activity were screening out. [Conclusion] It could be concluded that the method of avicel or CMC agarose plate with iodine vapor staining integrated with pNPG agarose plate assay would be a rapid, sensitive and effective method for screening of the fungi producing extracellular cellulases with high β-glucosidase activity.