A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp MEY-1: gene cloning and overexpression in Pichia pastoris
A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp MEY-1: gene cloning and overexpression in Pichia pastoris
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DOI：
10.1007/s00253-008-1766-x
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发表时间：
2009-03-01
影响因子：
5
通讯作者：
Yao, Bin
中科院分区：
文献类型：
--
作者：
Luo, Huiying;Wang, Yaru;Yao, Bin
利用简并聚合酶链式反应（PCR）和热不对称交叉PCR技术，从嗜酸双孢菌MEY-1中克隆出全长1,347 bp的含有man5A基因的互补DNA片段，该基因编码429个氨基酸的β -甘露聚糖酶，计算质量为46.8 kDa。所得氨基酸序列（催化结构域）与内切- β -1,4-d甘露聚糖酶（糖苷水解酶家族成员）具有最高的一致性（54.1%）。重组MAN5A在毕赤酵母中过表达，其在培养基中的活性达到500 U ml（-1）。该酶为嗜酸酶，在pH 1.0-1.5时活性最高，低于已知的甘露聚糖酶，活性的最佳温度为65℃，具有良好的pH适应性，良好的热稳定性和pH稳定性，对胃蛋白酶和胰蛋白酶均具有较高的抗性。对刺槐豆胶底物的比活性、K (m)和V （max）分别为3373 U mg（-1）、1.56 mg ml（-1）和6587.6 mu mol min(-1) mg（-1）。Ca2+、Cr3+、Co2+、Zn2+、Na+、K+和Mg2+对酶活性影响不显著，Ni2+、Fe3+、Mn2+和Ag+对酶活性有增强作用。这些有利的特性使MAN5A成为各种工业应用的潜在候选者。
Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid beta-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-beta-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml(-1). The enzyme was acidophilic, with highest activity at pH 1.0-1.5, lower than any known mannanases, and optimal temperature for activity was 65A degrees C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K (m), and V (max) for locust bean gum substrate was 3,373 U mg(-1), 1.56 mg ml(-1), and 6,587.6 mu mol min(-1) mg(-1), respectively. The enzymatic activity was not significantly affected by ions such as Ca2+, Cr3+, Co2+, Zn2+, Na+, K+, and Mg2+ and enhanced by Ni2+, Fe3+, Mn2+ and Ag+. These favorable properties make MAN5A a potential candidate for use in various industrial applications.