Abstract

Aspergillus niger is featured with its copious amount of extracellular β-glucosidase which is generally used to balance the cellulolytic enzyme cocktails for lignocellulose saccharification. However, whether or not A. niger produces any intracellular β-glucosidase remains obscure. In this study, we analyzed a total of fifteen putative β-glucosidase genes (bgls) in A. niger CBS 513.88 genome and the five of them were predicted as intracellular bgls due to the lack of signal peptide of extracellular proteins. After further characterization of these five genes through a Saccharomyces cerevisiae in vivo system, only An03g03740 (designated bgl1B) was confirmed to be a β-glucosidase gene. Western blot and mass spectrometry analysis confirmed BGL1B protein localization inside the cell. BGL1B exhibited the maximal activity at 40 °C and pH 5.6. The K_m for p-nitrophenyl-β-d-glucopyranoside and K_i for glucose were 0.233 ± 0.058 mM and 119.8 ± 4.35 mM, respectively. BGL1B showed a strong transglycosylation activity while hydrolyzing cellodextrins with sophorose, laminaribiose, and cellotriose formed from cellobiose, and sophorose and laminaribiose formed from cellotriose. The confirmation of the intracellular β-glucosidase BGL1B in A. niger further extends our understanding of how A. niger utilizes lignocellulose.

Key points

• Identification of putative genes revealed a novel β-glucosidase in Aspergillus niger.

• Newly identified β-glucosidase BGL1B was an intracellular enzyme of A. niger.

• BGL1B exhibited a strong transglycosylation activity.

中文翻译：

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具有转糖基化活性的黑曲霉细胞内β-葡萄糖苷酶的鉴定。

摘要

黑曲霉的特征在于其大量的细胞外β-葡糖苷酶，通常用于平衡纤维素酶的混合物，以木质纤维素的糖化作用。然而，黑曲霉是否产生任何细胞内β-葡萄糖苷酶仍然不清楚。在这项研究中，我们分析了黑曲霉CBS 513.88基因组中总共15个推定的β-葡萄糖苷酶基因（bgl），由于缺乏细胞外蛋白的信号肽，其中5个被预测为细胞内bgl。在通过酿酒酵母体内系统对这五个基因进行进一步表征后，只有An03g03740（称为bgl1B被确认为β-葡萄糖苷酶基因。蛋白质印迹和质谱分析证实了BGL1B蛋白在细胞内的定位。BGL1B在40°C和pH 5.6下表现出最大的活性。该ķ_米为p硝基苯基β- d吡喃葡萄糖苷和ķ_我葡萄糖为0.233±0.058 mM和119.8±4.35毫米。BGL1B表现出很强的转糖基化活性，同时水解纤维糊精与由纤维二糖形成的槐糖，拉米纳比糖和纤维三糖，以及由纤维三糖形成的槐糖和拉米二糖。在细胞内的β葡萄糖苷酶BGL1B的确认黑曲霉进一步拓展了我们如何理解黑曲霉 利用木质纤维素。

关键点

•推定基因的鉴定揭示了黑曲霉中一种新型的β-葡萄糖苷酶。

•新近鉴定的β-葡萄糖苷酶BGL1B是黑曲霉的胞内酶。

• BGL1B表现出很强的转糖基活性。