Abstract

The heterologous expression, isolation, and characterization of a novel xylanase from Paenibacillus brasilensis are described. The xyl1 gene from the Paenibacillus brasilensis strain X1 VKPM B-13092, which consists of 639 nucleotides, encodes a secreted endo-1,4-β-xylanase (EC 3.2.1.8) containing 184 amino acids and 28 residues of the putative signal peptide in the N-terminal region. The nucleotide sequence of the xyl1 gene and the amino acid sequence of the mature Xyll protein have the greatest homology with the Bacillus subtilis endo-1,4-β-xylanase sequences (78 and 83%, respectively). A gene fragment encoding the mature protein was expressed in Pichia pastoris. The purified recombinant Xyl1 enzyme was able to use birch xylan and arabinoxylan as substrates. With birch xylan, the optimal pH for the enzymatic reaction was 6.0, the optimal temperature was 40–50°C, and K_m and V_max, were equal to 1.1288 mg/mL and 5124.3 μmol/(min mg), respectively. The recombinant Xyl1 protein showed high pH and thermal stability, and the resistance to digestive enzymes and xylanase protein inhibitors from cereals. It was also shown that Mn²⁺ and Со²⁺ ions stimulate enzyme activity.

中文翻译：

---

巴西paenibacillus Brasilensis X1的木聚糖酶基因在巴斯德毕赤酵母中的表达及重组酶特性

摘要

描述了来自巴西芽孢杆菌的新型木聚糖酶的异源表达，分离和表征。来自巴西芽孢杆菌X1 VKPM B-13092菌株的xyl 1基因，由639个核苷酸组成，编码一种分泌的内切1,4-β-木聚糖酶（EC 3.2.1.8），含有184个氨基酸和28个推定信号残基N末端区域中的肽。xyl 1基因的核苷酸序列和成熟Xyll蛋白的氨基酸序列与枯草芽孢杆菌内吞1,4-β-木聚糖酶序列具有最大的同源性（分别为78％和83％）。编码成熟蛋白的基因片段在毕赤酵母中表达。纯化的重组Xyl1酶能够使用桦木聚糖和阿拉伯木聚糖作为底物。使用桦木聚糖时，酶促反应的最佳pH为6.0，最佳温度为40–50°C，K _m和V _max分别等于1.1288 mg / mL和5124.3μmol/（min mg）。重组Xyl1蛋白显示出高pH值和热稳定性，并且对谷物的消化酶和木聚糖酶蛋白抑制剂具有抗性。还显示了Mn ²⁺和Со2 ⁺离子刺激了酶的活性。