Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium
Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium
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DOI：
10.1016/j.jbiosc.2019.05.012
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发表时间：
2019-12-01
影响因子：
2.8
通讯作者：
Shirasaka, Norifumi
中科院分区：
文献类型：
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作者：
Onuma, Hiroki;Hara, Kento;Shirasaka, Norifumi
从外生菌根真菌松茸(TmEgl5A)的固体培养中分离到一种内切葡聚糖酶。该酶经硫酸铵分级沉淀、离子交换、疏水、凝胶过滤等步骤得到纯化。经SDS-PAGE分析，TmEg15A的相对分子质量约为40 kDa。用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)进行多肽指纹图分析，并将胰酶消化的多肽序列与JGI松茸945 v3.0基因组数据库中推测的内切葡聚糖酶序列(蛋白质ID1465229)进行匹配。根据序列信息，克隆了编码TmEg1的基因，并在毕赤酵母KM71H中进行了表达。推导的氨基酸序列与担子菌GH5家族内切葡聚糖酶相似。该酶作用于大麦的β-葡聚糖、地衣多糖和CMC-Na。这些底物的水解物经薄层层析鉴定为低聚糖和极少量的二糖。这些结果表明，松茸在固态培养中产生一种典型的内切葡聚糖酶，该真菌具有降解β-连接多糖的潜力。(C)2019年，日本生物技术学会。版权所有。
An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEg15A showed a molecular mass of approximately 40 kDa as determined by SDS PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley beta-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade beta-linkage polysaccharides. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.