Sato Shota, Ohta Kunihiko, Kojima Kaoru, Kozeki Takuya, Ohmachi Tetsuo, Yoshida Takashi
1 Faculty of Agriculture and Life Science, Hirosaki University.
2 The United Graduate School of Agricultural Sciences, Iwate University.
J Appl Glycosci (1999). 2016 Feb 20;63(1):13-18. doi: 10.5458/jag.jag.JAG-2015_012. eCollection 2016.
Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 3.2.1.151).
木葡聚糖是植物细胞壁中一种主要的半纤维素成分。植物病原真菌在感染宿主时会分泌细胞壁降解酶，然而这类真菌的细胞壁裂解酶的性质尚未完全明确。[病原菌名称]是一种土传真菌，可在全球多种具有重要商业价值的作物上引发维管束枯萎病。我们从自然分离的[病原菌名称]菌株2148的培养物中纯化出了两种木葡聚糖酶，即XEG12A和XEG74B。XEG12A的分子大小为23 kDa，在pH 7.5时具有最大活性。XEG12A能特异性水解木葡聚糖，对其他β-葡聚糖无活性。XEG74B的分子大小为110 kDa，最适pH为6.0。XEG74B主要水解木葡聚糖，对β-1,3-1,4-葡聚糖有轻微活性。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）对木葡寡糖（XXXGXXXG）的水解产物进行分析表明，这两种酶都在无分支链的位置切割β-1,4-糖苷键，而XEG74B的切割位点略有波动。两种酶都完全不水解木葡庚糖（XXXG）。对这些酶进行N端和内部氨基酸测序表明，XEG12A和XEG74B分别属于糖苷水解酶（GH）家族12和74。基于这些结果，我们得出结论，XEG12A和XEG74B是木葡聚糖特异性内切β-1,4-葡聚糖酶（EC 3.2.1.151）。
