Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior
Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior
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DOI：
10.1016/j.carres.2019.04.006
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发表时间：
2019-05-15
影响因子：
3.1
通讯作者：
Chadha, Bhupinder S.
中科院分区：
文献类型：
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作者：
Basotra, Neha;Dhiman, Saurabh Sudha;Chadha, Bhupinder S.
从嗜热真菌Malbranchea cinnamomea中克隆了一个新的裂解多糖单加氧酶（LPMO）家族AA 9（PMO9A_MALCI）蛋白，并在毕赤酵母中表达。表达的蛋白质用离子交换和疏水相互作用层析纯化至均一。SDS-PAGE分析显示PMO9A_MALCI与27 kDa蛋白相似。高效阴离子交换色谱和质谱法证实，纯化的蛋白质对一系列纤维素（微晶纤维素，羧甲基纤维素）和半纤维素（桦木木聚糖，小麦阿拉伯木聚糖和黑麦阿拉伯木聚糖）底物具有活性，分别释放氧化和未氧化的纤维寡糖和低聚木糖产物。在质谱分析以及计算机模拟分析期间双氧化产物的存在证实表达的蛋白属于3型LPMO家族。分子动力学模拟进一步证实了对于纤维素和半纤维素底物的催化保守的共同氨基酸残基的共享，这进一步表明两种底物是同样优选的。通过用PM 0 9A_MALCI（9：1/8：2）替代一部分商业纤维素酶CellicCTec 2构成的酶混合物导致酸和碱预处理的生物质的糖化的协同改进。这是第一次从M. cinnamomea，表现出纤维素和纯木聚糖的催化作用。
A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be similar to 27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.