一株产耐热右旋糖酐酶真菌的筛选、鉴定及其酶学性质

Screening, identification and characterization of thermotolerant dextranase from a fungus

摘要:

[目的] 从土壤中筛选得到1株产耐热右旋糖酐酶的真菌。[方法] 采用营养缺陷型培养基,结合稀释涂布法和平板透明圈法分离筛选出产耐热右旋糖酐酶的菌株。通过观察菌落形态、菌体形态和培养特征,结合ITS rDNA序列分析对菌株进行鉴定。研究菌株所产右旋糖酐酶的酶学性质。[结果] 通过筛选得到1株产耐热右旋糖酐酶的菌株DG001,经鉴定为淡紫拟青霉(Paecilomyces lilacinus)。菌株DG001所产右旋糖酐酶的最佳催化条件为55℃,pH 5.0;最适底物为5% Dextran T70。酶在60℃以下和pH 4.0-7.0之间稳定。urea、Mn²⁺和Mg²⁺对酶活均有促进作用,低浓度的Mn²⁺和urea可使酶活分别提高到116.91%和110.14%,而Cu²⁺则对其有强烈抑制作用。该酶水解右旋糖酐T2000的产物主要是异麦芽糖和异麦芽三糖,被确定为内切右旋糖酐酶。酶对底物的亲和性随底物分子量的增加而增强。[结论] 成功筛选获得1株产耐热右旋糖酐酶的菌株DG001,所产酶在较宽温度范围内具有较高活力,热稳定性好。该酶在制糖工业及不同分子量右旋糖酐的制备中具有很好的应用前景。

Abstract:

[Objective] We attempted to obtain a fungus producing thermotolerant dextranase by screening samples from soil. [Methods] The fungus producing thermotolerant dextranase was isolated and screened by auxotrophic medium, combined with Pour Plate method and Flat Transparent Circle method. The strain was identified by its colony, cell morphology and cultural characteristics, as well as ITS rDNA sequence analysis. The dextranase produced by the strain was characterized. [Results] We obtained the strain DG001 producing thermotolerant dextranase, which was identified as Paecilomyces lilacinus. The optimum catalytic conditions for the dextranase were 55℃, pH 5.0, and the optimum substrate concentration was 5% dextran T70. The dextranase was stable below 60℃ and between pH 4.0 and 7.0. Urea, Mn²⁺ and Mg²⁺ could increase enzyme activity, and the low concentration of Mn²⁺ and Urea could increase enzyme activity to 116.91% and 110.14% respectively, whereas Cu²⁺ had a strong inhibitory effect on the dextranase. The dextranase, identified as endo-dextranase, hydrolyzed dextran T2000 with main products as isomalt and isomaltotriose. The enzyme-substrate affinity increased with the increasing substrate molecular weight. [Conclusion] Strain DG001 producing thermotolerant dextranase was obtained through successful screening, bearing a high activity in a wide temperature range and a good thermal stability. This enzyme shows a promising prospect of application in sugar industry and in the preparation of different molecular weight dextran.