降解生淀粉真菌的筛选、鉴定及其产生淀粉酶的性质研究

Screening and identification of raw starch hydrolyzing fungal strains and characteristics of their raw starch-degrading enzyme

以生木薯粉为唯一碳源,对收集的土壤样品悬浮液进行培养、I2/KI溶液染色,经摇瓶复筛和菌株发酵上清液RSDE活力测定,筛选出10株RSDE活力较高的真菌;对RSDE活力最高的真菌菌株1-31进行形态学观察和ITS(Internaltranscribed spacer)序列分析,将其鉴定为青霉属。对青霉1-31的酶学性质进行测定,结果发现其RSDE对生木薯淀粉的最适作用pH和温度分别为5.0和55℃;分别以玉米和大米为底物时,其RSDE的生淀粉分解活力比(RDA)较高,为59.0%和58.8%;青霉1-31 RSDE对生木薯粉的吸附率约为37.0%。HPLC检测发现,以该菌株RSDE水解生木薯粉4 h,仅释放出葡萄糖,说明青霉1-31产生的RSDE主要为生淀粉糖化酶。电镜观察结果发现,经青霉1-31 RSDE处理,可使生木薯粉颗粒光滑完整的表面变得粗糙,形成无数小坑,说明青霉1-31 RSDE对生淀粉具有较强的水解作用。因此,青霉1-31 RSDE在各种生淀粉如木薯、玉米、大米和马铃薯生淀粉等加工业中具有一定的应用潜力。

The suspensions of collected soil samples were cultured in isolated plates by using raw starch as sole carbon source,and stained by I2/KI solution after culturing for 5 days.The strain with significant hydrolyzing zone was isolated and purified.Ten fungal strains with higher raw starch-degrading enzyme（RSDE） activity to raw cassava starch were screened and RSDE activity was determined in fermented supernatant.Among 10 strains,a fungal strain 1-31 showing higher RSDE activity was identified on the basis of morphological observation and internal transcribed spacer（ITS） region sequence analysis,as a Penicillium sp.The RSDE of Penicillium 1-31 showed maximum activity at pH 5.0 and 50℃.The values of raw starch-degrading ability（RDA） of the RSDE were about 59.0 and 58.8% when corn and rice were used as the substrate,respectively.The adsorption rate of RSDE in Penicillium 1-31 to raw cassava starch was about 37.0%.HPLC analysis revealed that the RSDE could release glucose only from raw cassava starch in 4 hours,indicating that the RSDE from Penicillium sp.1-31 was mainly raw starch-degrading glucoamylase.The scanning electron microscope revealed that the smooth surface of raw cassava starch granules turned into rough when treated by RSDE,thus confirming that RSDE had strong hydrolyzing effects to raw starch.The RSDE of Penicillium sp.1-31 may be used in processing industry of raw starch for cassava,maize,rice and potato,etc.