Isolation, identification of pullulanase producing strain and optimization of its fermentation conditions | 一株普鲁兰酶产生菌的分离鉴定及发酵条件的优化

Chinese. 普鲁兰酶是一类淀粉分支酶，在淀粉加工等行业有着广泛的应用。利用平板涂布法从淀粉加工厂附近土壤中分离到一株产普鲁兰酶的细菌H4，通过形态学、生理生化试验及16S rDNA分类鉴定，确定该细菌为一株微小杆菌(Exiguobacterium sp. H4)。对该菌株培养基成分及产酶条件进行了优化，即2%土豆淀粉，1%蛋白胨，0.5%牛肉膏，0.1% KH2PO4，0.05% MgSO4•7H2O和0.0001% FeSO4，初始pH 7.0，发酵温度37℃，摇床转数200 r/min，发酵72 h后胞外产生普鲁兰酶的活力可达6.35 U/mL，比复筛的产量3.02 U/mL提高了110.2%。

English. Pullulanase is considered as a starch debranching enzyme, and is very useful in starch industry. The present study aims at the isolation and identification of bacteria producing pullulanase from the environmental samples, and studies on the optimum growth conditions in fermentative medium. The strains producing pullulanase were obtained by using spread-plate method. A strain with the highest pullulanase activity was identified according to its morphological, physiological, biochemical and 16S rDNA sequence. A series of experiments were conducted for the optimization of fermentative medium and growth conditions. Among the isolated strain, strain H4 showed the highest pullulanse activity from the soil close to a starch processing plants. Strain H4 was identified as Exiguobacterium sp.. The optimum medium for pullulanase was consisted of 2% potato starch, 1% peptone, 0.5% yeast extract, 0.1% KH2PO4, 0.05% MgSO4•7H2O and 0.0001% FeSO4. Under the condition of initial pH 7.0, 37℃ and 200 r/min for 72 h, the yield of pullulanase could reach to 6.35 U/mL, which was 110.2% higher than the control (3.02 U/mL).