Mutanda T, Wilhelmi B, Whiteley C G
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, South Africa.
Appl Biochem Biotechnol. 2009 Oct;159(1):65-77. doi: 10.1007/s12010-008-8479-6. Epub 2009 Jan 6.
An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg(-1)(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 degrees C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K (m) and V (max) values were 4.75 mM and 833.3 micromol min(-1) ml(-1), respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 degrees C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 degrees C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.
已从市售的泡盛曲霉发酵液中分离、纯化并表征了一种外切菊粉酶。经透析、硫酸铵分级分离、Sephacryl S - 200 尺寸排阻色谱和离子交换色谱后，该酶的比活性为 12300 U mg⁻¹（蛋白质），纯化了 4.2 倍，产率为 21%。通过 SDS - PAGE 估计该酶的分子量为 63 kDa。它的最适 pH 和温度分别为 5.4 和 50℃，在此条件下，酶分别在孵育 12 小时和 72 小时后仍保持稳定，残余活性分别为 96%和 63.8%。各自的 Km 和 Vmax 值分别为 4.75 mM 和 833.3 μmol min⁻¹ ml⁻¹。采用响应面法对纯商业菊苣菊粉水解产生果糖的最大产量进行了统计分析。在静态分批条件下，透析后的粗外切菊粉酶（100 U/ml，48 小时，50℃，150%菊粉，pH 5.0）孵育产生的果糖量最高（106.4 mg/ml）。对纯化的外切菊粉酶进行了果糖产量评估，在 50℃、150%菊粉、pH 5.0 条件下孵育 12 小时后产生的果糖量最高（98 mg/ml）。使用粗外切菊粉酶制剂在经济上是可取的，并且通过菊粉水解进行果糖的工业生产在生物技术上是可行的。
