Abstract

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis are described. The endo-1,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino-acid sequence of the mature protein has the greatest homology (84%) with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-1,4-β-xylanase. A synthetic nucleotide sequence encoding the mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme was active with birch xylan and arabinoxylan as substrates. The optimal pH and temperature for enzyme activity were established as 5.0 and 50°C, respectively, with the use of birch xylan. The specific activity of xylanase was 4700 U/mg protein, and K_M and V_max were equal to 0.51 mg/mL and 7395.3 μmol/(min mg), respectively. The recombinant XylP protein showed moderate thermal and high pH stability, as well as resistance to digestive enzymes and protein xylanase inhibitors from cereals. It was also shown that Mg²⁺, Co²⁺ and Li⁺ ions have a positive effect on enzyme activity.

中文翻译：

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毕赤酵母木聚糖酶基因在毕赤酵母中的表达及重组蛋白的鉴定

摘要

描述了来自毕赤酵母的新木聚糖酶的异源表达和特征。1,4-β-木聚糖内切酶XylP（EC 3.2.1.8）在N端区域由223个氨基酸和19个假定信号肽残基组成。成熟蛋白的氨基酸序列与新Callimastix patriciarumendo -1,4-β-木聚糖酶的天然催化N末端结构域的序列具有最大的同源性（84％）。编码成熟XylP蛋白的合成核苷酸序列在巴斯德毕赤酵母中表达。纯化的重组酶以桦木木聚糖和阿拉伯木聚糖为底物具有活性。用桦木木聚糖将酶活性的最佳pH和温度分别设定为5.0和50℃。木聚糖酶的比活为4700 U / mg蛋白，K _M和V _max分别等于0.51 mg / mL和7395.3μmol/（min mg）。重组XylP蛋白显示出中等的热稳定性和高pH稳定性，以及对谷物中的消化酶和蛋白质木聚糖酶抑制剂的抗性。还显示出Mg 2 ⁺，Co ²⁺和Li ⁺离子对酶活性具有积极作用。