Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity
Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity
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具有高比活性的 Talaromyces leycettanus JCM12802 嗜热 β-甘露聚糖酶的生化特征
DOI：
10.1007/s00253-014-5979-x
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发表时间：
2015-02-01
影响因子：
5
通讯作者：
Yao, Bin
中科院分区：
文献类型：
--
作者：
Wang, Caihong;Luo, Huiying;Yao, Bin
嗜热β-甘露聚糖酶对于广泛的工业应用具有越来越重要的意义。本文报道了嗜热TalaromycesleycettanusJCM 12802嗜热β-甘露聚糖酶（Man 5A）的基因克隆、在毕赤酵母（Pichiapastoris）中的功能表达及酶学性质。推测的Man 5A与来自Talaromyces stipitatus ATCC 10500的推定的β-甘露聚糖酶（70.3%）表现出最高的同一性，并且由N-末端信号肽、家族1的真菌型碳水化合物结合模块（CBM）和C-末端的糖基水解酶（GH）家族5的催化结构域组成。纯化后鉴定出两种具有不同糖基化水平的重组蛋白，称为Man 5A 1（72 kDa）和Man 5A 2（60 kDa）。这两种酶都是嗜热的，在85-90摄氏度表现出最佳活性，在70摄氏度高度稳定。Man 5A 1和Man 5A 2的最适pH值分别为4.5和4.0，并且在3.0-10.0的宽pH范围内高度稳定。大多数金属离子和十二烷基硫酸钠（SDS）对酶活性没有影响。Man 5A 1和Man 5A 2在以刺槐豆胶为底物时表现出较高的比活性（分别为2，160和1，800 U/mg）。发现CBM 1和两个关键残基D191和R286影响Man 5A的热稳定性。Man 5A显示出经典的四位点结合模式，将甘露寡糖水解成更小的单元，将半乳甘露聚糖水解成甘露糖和甘露二糖，将葡甘露聚糖分别水解成甘露糖、甘露二糖和甘露五糖。所有这些特性使Man 5A成为生物转化、纸浆漂白、纺织、食品和饲料工业中广泛应用的良好候选者。
Thermophilic beta-mannanases are of increasing importance for wide industrial applications. In the current study, gene cloning, functional expression in Pichia pastoris, and characterization of a thermophilic beta-mannanase (Man5A) from thermophilic Talaromyces leycettanus JCM12802 are reported. Deduced Man5A exhibits the highest identity with a putative beta-mannanase from Talaromyces stipitatus ATCC10500 (70.3 %) and is composed of an N-terminal signal peptide, a fungal-type carbohydrate-binding module (CBM) of family 1, and a catalytic domain of glycosyl hydrolase (GH) family 5 at the C-terminus. Two recombinant proteins with different glycosylation levels, termed Man5A1 (72 kDa) and Man5A2 (60 kDa), were identified after purification. Both enzymes were thermophilic, exhibiting optimal activity at 85-90 degrees C, and were highly stable at 70 degrees C. Man5A1 and Man5A2 had a pH optimum of 4.5 and 4.0, respectively, and were highly stable over the broad pH range of 3.0-10.0. Most metal ions and sodium dodecyl sulfate (SDS) had no effect on the enzymatic activities. Man5A1 and Man5A2 exhibited high specific activity (2,160 and 1,800 U/mg, respectively) when using locust bean gum as the substrate. The CBM1 and two key residues D191 and R286 were found to affect Man5A thermostability. Man5A displays a classical four-site-binding mode, hydrolyzing mannooligosaccharides into smaller units, galactomannan into mannose and mannobiose, and glucomanman into mannose, mannobiose, and mannopentaose, respectively. All these properties make Man5A a good candidate for extensive applications in the bioconversion, pulp bleaching, textile, food, and feed industries.