Abstract

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and β-glucosidases when cultivated on cellulose. In order to investigate its effect on cellulose, a highly purified preparation of CDH was obtained from the culture supernatant of the fungus cultivated on cellulose. A combination of ultrafiltration, ion-exchange and gel-filtration chromatography was used to purify CDH by ∼172-fold to a high specific activity of ∼324 U/mg protein on lactose which was used for routine measurement of enzyme activity. The enzyme displayed a pH optimum of 5.0 and stability between pH 5.0 and 8.0 with maximum catalytic efficiency (k_cat/K_m) of 397 mM⁻¹ s⁻¹ on cellobiose. Incubation of microcrystalline cellulose with the purified CDH led to production of reducing sugars which was accelerated by the addition of FeCl₃ during the early stages of incubation. A mass spectrometric analysis revealed fragmentation products of cellulose which were concluded to be cellodextrins, sugars, and corresponding aldonic acids suggesting that CDH can release reducing sugars in the absence of externally added lytic polysaccharide monooxygenases. Polymerized products of glucose were also detected at low intensity.

摘要

Termitomyces sp. OE 147 是生态圈中的活性纤维素降解剂之一，在纤维素上培养时会产生大量纤维二糖脱氢酶 (CDH) 和 β-葡萄糖苷酶。为了研究其对纤维素的影响，从在纤维素上培养的真菌的培养上清液中获得了高度纯化的 CDH 制剂。超滤、离子交换和凝胶过滤色谱的组合用于将 CDH 纯化约 172 倍，达到约 324 U/mg 蛋白质对乳糖的高比活性，用于酶活性的常规测量。该酶的最适 pH 值为 5.0，在 pH 5.0 和 8.0 之间具有稳定性，最大​​催化效率 ( k _cat / K _m ) 为 397 mM ^-1 s^-1纤维二糖。微晶纤维素与纯化的 CDH 的孵育导致还原糖的产生，这在孵育的早期阶段通过添加 FeCl ₃加速。质谱分析揭示了纤维素的碎裂产物，其被推断为纤维糊精、糖和相应的醛糖酸，表明 CDH 可以在没有外部添加的裂解多糖单加氧酶的情况下释放还原糖。在低强度下也检测到葡萄糖的聚合产物。