Production, purification, and determination of the biochemical properties of β-glucosidase in Trichoderma koningii via solid substrate fermentation

Selma Çelen Yücetürk, Ayşe Dilek Azaz
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Abstract

The β-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The β-glucosidase was obtained from T. koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 °C and 6 days. The purification efficiency of the obtained β-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of β-glucosidase activity, p-nitrophenyl-β-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which β-glucosidase showed high activity were determined to be pH 3.0 and 75 °C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K m and V max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. It was also found that d-(+)-glucose and δ-gluconolactone inhibitors exhibited competitive inhibition of β-glucosidase in the presence of pNPG.
通过固体底物发酵法生产、纯化和测定科宁毛霉中的β-葡萄糖苷酶的生化特性
β-葡萄糖苷酶是以玉米芯为底物,在最佳条件下通过固体底物发酵(SSF)从 Trichoderma koningii Oudem.NRRL 54330 中获得。该酶通过硫酸铵沉淀和头孢糖-4B-l-酪氨酸-1-萘胺疏水相互作用色谱两步法纯化,然后进行生化和动力学表征。以磨碎的玉米芯为底物,以 pH 值为 9 的 Na2HPO4 为加湿介质,从 T. koningii 中获得了 β-葡萄糖苷酶。SSF 产酶的最佳条件是 30 °C 和 6 天。经计算,所获得的 β-葡萄糖苷酶的纯化效率为 22.56 倍,产率为 73.51%。在测定β-葡萄糖苷酶活性时,使用了对硝基苯基-β-d-吡喃葡萄糖苷(pNPG)底物,并确定了β-葡萄糖苷酶显示高活性的最佳 pH 值和温度值为 pH 3.0 和 75 ℃。使用两种不同的电泳方法(SDS-PAGE 和 NATIVE-PAGE 电泳方法)检测了酶的纯度和亚基的存在/数量。经测定,纯化酶的 K m 和 V max 值分别为 0.16 mM 和 2000 EU。研究还发现,在 pNPG 的存在下,d-(+)-葡萄糖和 δ-葡萄糖酸内酯抑制剂对 β-葡萄糖苷酶有竞争性抑制作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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