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毕赤酵母高产木聚糖酶:纯化、特性分析、动力学评价及木寡糖生产。

High-activity production of xylanase by Pichia stipitis: Purification, characterization, kinetic evaluation and xylooligosaccharides production.

机构信息

College of Food Science and Technology, Henan University of Technology, Zhengzhou, China.

Department of Biological Systems Engineering, The University of Nebraska-Lincoln, Lincoln, USA.

出版信息

Int J Biol Macromol. 2018 Oct 1;117:72-77. doi: 10.1016/j.ijbiomac.2018.05.128. Epub 2018 May 21.

Abstract

As an industrially important biological macromolecule, xylanase hydrolyzes xylan to produce xylooligosaccarides (XOS). XOS, with a degree of polymerization (DP) 2 to 4, are important prebiotics used as food ingredients. In this study, xylanase (5536 U/g substrate) was produced by Pichia stipitis using corncob and wheat bran mixture under solid state fermentation. Crude xylanase were purified and biochemically characterized. XOS hydrolyzed by crude and purified xylanases were quantified. Molecular weight of the purified enzyme was around 31.6 kDa on SDS-PAGE. Enzyme kinetics showed Km and Vmax values of 4.52 mg/mL and 9.17 μmol/min/mL, respectively. The optimal conditions were pH 6.0 and 50 °C. Xylanase was stable at pH 5-8 for 60 min by retaining 57% activity and at 50 °C for 80 min by retaining 65% activity. Cooper and potassium had no inhibitory effect on xylanase activity. Xylan hydrolysates produced by purified xylanase contained 92% XOS consisting of 14% xylotetroase (DP 4), 49% xylotriose (DP 3) and 29% xylobiose (DP 2). These findings indicate the potential of applying purified xylanase for industrial XOS production.

摘要

木聚糖酶作为一种重要的工业用生物大分子,能够将木聚糖水解为低聚木糖(XOS)。聚合度为 2 到 4 的 XOS 是一种重要的益生元,可用作食品成分。本研究采用固态发酵法,以玉米芯和麦麸混合物为底物,利用毕赤酵母生产木聚糖酶(5536 U/g 底物)。粗木聚糖酶经过纯化并进行了生化特性分析。对粗酶和纯化酶水解产生的 XOS 进行了定量分析。SDS-PAGE 结果显示,纯化酶的分子量约为 31.6 kDa。酶动力学研究表明,Km 和 Vmax 值分别为 4.52 mg/mL 和 9.17 μmol/min/mL。最佳条件为 pH 6.0 和 50°C。该木聚糖酶在 pH 5-8 条件下稳定 60 min,保留 57%的活性;在 50°C 条件下稳定 80 min,保留 65%的活性。Co2+和 K+对木聚糖酶活性没有抑制作用。纯化木聚糖酶水解木聚糖产生的产物中含有 92%的 XOS,其中包括 14%木二糖水解酶(DP 4)、49%木三糖(DP 3)和 29%木二糖(DP 2)。这些发现表明,纯化木聚糖酶具有在工业上生产 XOS 的潜力。

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