产β-甘露聚糖酶内生菌的筛选、鉴定及酶学性质研究

Isolation and Identification of Endophyte Strain Producing β-mannanase and the Studies of Its Characterization

试验从萌发的花魔芋实生种子中筛选出一株产β-甘露聚糖酶的内生菌,对该菌株进行鉴定并研究所产β-甘露聚糖酶的酶学性质。采用透明圈法初筛,DNS法测酶活力,摇瓶发酵复筛获得产酶最高的菌株,利用16SrDNA序列分析对该菌株进行鉴定并对所产酶的基本酶学性质进行研究。结果显示,该高产β-甘露聚糖酶的内生菌株与路德维希肠杆菌(Enterobacter ludwigii)同源性达99%。所产β-甘露聚糖酶的最适温度为60℃,在30~50℃条件下较稳定;最适pH为6.0,在pH 4.0~9.0的条件下较稳定;Zn^(2+)(117.84%)、EDTA(115.80%)、Cu^(2+)(113.76%)对β-甘露聚糖酶有较强的激活作用,Mn^(2+)(22.02%)对其有强烈的抑制作用;以魔芋粉为底物时,Km值为26.65mg/mL。该菌摇瓶发酵72h后,β-甘露聚糖酶酶活力达9.48U/mL,具有良好的酶学性质,在动物饲料工业中具有广阔的应用前景。

An endophyte strain producingβ-mannanase was isolated and identified from sprouting konjac seeds,then enzymatic characteristics ofβ-mannanase was studied.The highestβ-mannanase producing strain was obtained by transparent circle preliminary screening and shake flask fermentation secondary screening,and the enzyme activity was evaluated according to dinitrosalicylic acid（DNS）method,and then the strain was identified based on 16 SrDNA sequence.The results showed that it was most closely related to Enterobacter ludwigii（99%sequence similarity）.The optimal temperature of theβ-mannanase was 60 ℃,and it had a good stability under temperature from 30 to 50℃.The optimal pH of theβ-mannanase was 6.0,and it was stable over a range of pH 4.0 to 9.0.The enzyme was slightly activated by Zn^（2＋）（117.84%）,EDTA（115.80%）,Cu^（2＋）（113.76%）.On the contrary,Mn^（2＋）（22.02%）could strongly inhibit the enzyme activity.The Km of theβ-mannanase towards konjac flour was 26.65 mg/mL.Furthermore,after 72h shaking-flask fermentation,theβ-mannanase activity achieved 9.48U/mL,and it had excellent enzymatic characteristics.These superior properties made theβ-mannanase a broad application prospects in feed industry.