芽胞杆菌苎麻脱胶酶—甘露聚糖酶的纯化及酶学性质

Purification and Properties of Ramie Degumming Key Factor-mannanase from Bacillus subtilis No.16A

研究了苎麻高效脱胶菌株Bacillus subtilisNo.16A甘露聚糖酶的产生条件,纯化过程以及酶学性质。其优化的培养基为魔芋胶30 g/L,蛋白胨9 g/L,酵母膏2 g/L,KH2PO45 g/L,MgSO4.7H2O 0.25 g/L。优化的培养条件为起始pH 8.0,装样量50 mL,接种量10 mL,发酵72 h。进一步通过硫酸铵沉淀、阴离子交换层析、凝胶过滤3步从Bacillus subtilisNo.16A发酵液中纯化了甘露聚糖酶。结果表明,该酶分子量约为38.5ku,最适作用pH为7.0,最适作用温度为60℃,在55℃以下、中性pH（6.0～8.0）范围内稳定,Ca^2＋、Mn^2＋和Al^3＋、Mg^2＋对该酶有激活作用,Cu^2＋、Zn^2＋有抑制作用。

In this paper we studied the conditions, purification and properties of mannanase produced by Bacillus subtilis No. 16A. The optimized culture medium composition is： konjac 30 g/L, peptone 9 g/L, yeast extract 2 g/L, KH2PO＋ 5 g/L, MgSO4·7H2O 0. 025 g/L. The optimized fermentation condition is： initial pH8.0,fermentation period 72 h, filling level 50 mL, inoculum size 10 mL. Crude enzymes were treated in turn with ammonium sulfate salting out, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatograph, vacuum freezing desiccation, Sephadex G-75 gel filtration chromatography, and finally SDS-PAGE discontinuous flow electrophoresis detected a singular strip, with the molecular weight of 38.5 ku. The optimal pH and temperature of the enzyme activity were 7.0 and 60℃, it was stable under 55℃ and in the neutral pH（6.0 ～8.0）. The enzyme activity was stimulated by Ca^2＋ , Al^3＋, Mg^2＋ and Mn^2＋ ,while inhibited by Cu^2＋ and Zn^2＋.