Achaetomium sp.Xz8甘露聚糖酶在毕赤酵母中的表达及其酶学性质分析

Expression and Characterization of a Novel Mannanse From Achaetomium sp. Xz8 in Pichia pastoris

甘露聚糖酶作为最主要的半纤维素酶类现已被广泛地应用在饲料、造纸、洗涤剂、食品及石油开采等领域。本研究从高温真菌Achaetomium sp.Xz8菌株中利用兼并引物和Thermal asymmetric interlaced PCR（Tail-PCR）的方法克隆获得一个新的糖苷水解酶5家族的甘露聚糖酶基因（man5Xz8）。基因全长1 239bp,序列分析发现其编码412个氨基酸和1个终止密码子,预测的信号肽序列为N端的20个氨基酸。将成熟蛋白序列克隆到毕赤酵母分泌型表达载体p PIC9中,利用甲醇诱导重组酵母菌表达目的蛋白,经SDS-PAGE电泳分析,表达蛋白分子量约55.0 k Da。对其酶学性质进行测定,Man5Xz8的最适p H值为5.0,在p H值9.0可以保持40%以上的酶活性,在p H值5.0~9.0具有良好的p H稳定性。最适作用温度为50℃,并对SDS具有较高的耐受性。以角豆胶为底物,Man5Xz8的比活、Km及Vmax值分别为101.6 U·mg-1、4.4 mg·m L-1、128.2μmol·min-1·mg-1。因此,Man5Xz8为后期研究甘露聚糖酶的工业应用和酸碱催化机制提供了良好的材料。

Mannanase,one of the main hemicellulose-degrading enzymes,is widely used in animal feed,paper,detergent,food,oil exploration and other fields. In this study,a glycoside hydrolase family 5 mannanse gene,man5Xz8,was cloned by degenerate primers and Tail-PCR from thermophilic fungus Achaetomium sp. Xz8. The fulllength gene consists of 1 239 bp and encodes 412 amino acids,including a putative signal peptide of 20 residues at Nterminus. The mature protein sequence was successfully expressed in pichia pastoris GS115. The recombinant protein expression was induced by methanol,and the molecular weight of Man5Xz8 was about 55. 0 k Da analyzed by SDS-PAGE analysis. Man5Xz8 showed the optimal activity at p H 5. 0,it retained 40. 0% of the maximal activity at p H 9. 0,and showed high stability over the p H range 5. 0 to 9. 0. The optimum temperature was 50℃,and it has strong resistance to SDS. The specific activity,Kmand Vmaxvalues towards beechwood xylan were 101. 6 U·mg- 1,4. 4 mg·m L- 1and 128. 2μmol·（ min·mg）- 1,respectively. This study provides an excellent mannanse as candidate favorable for various industries and primarily determines the acid-base catalytic mechanism.