Purification and characterization of recombinant endoglucanases from the pine wood nematode Bursaphelenchus xylophilus
Purification and characterization of recombinant endoglucanases from the pine wood nematode Bursaphelenchus xylophilus
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DOI：
10.1271/bbb.70819
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发表时间：
2008-05-01
影响因子：
1.6
通讯作者：
Kikuchi, Taisei
中科院分区：
文献类型：
--
作者：
Shibuya, Hajime;Kikuchi, Taisei
从松材线虫中分离到一个苷水解酶家族（GHF） 45内切葡聚糖酶。本文描述了在毕赤酵母中表达的重组酶Bx-ENG-1、2和3的纯化和表征。纯化后的Bx-ENG-1、2和3的SDS-PAGE分子量分别为18、33-39和100-140kDa，凝胶过滤分子量分别为18、67和252 kDa，表明Bx-ENG-1以未糖基化的单体形式存在，Bx-ENG-2和Bx-ENG-3以糖基化的二聚体形式存在。重组酶的酶学性质彼此相似：与GHF45的其他内切葡聚糖酶一样，在60℃、pH 6.0左右具有最佳活性。重组酶对地衣纤维素的活性最高，对羧甲基纤维素和无定形纤维素的活性较低。线虫酶还能水解葡甘露聚糖，这是软木细胞壁中含量最多的半纤维素。这些底物特异性表明，B. xylophilus内切葡聚糖酶作用于细胞壁中的纤维素-半纤维素复合物，导致细胞壁的机械强度减弱，从而促进线虫对植物细胞的取食。
A family of endoglucanases belonging to glycoside hydrolase family (GHF) 45 have been isolated from the pine wood nematode Bursaphelenchus xylophilus. Here we describe the purification and characterization of the recombinant enzymes, named Bx-ENG-1, 2, and 3, expressed in Pichia pastoris. The respective molecular masses of purified Bx-ENG-1, 2, and 3 were estimated to be 18, 33-39, and 100-140kDa by SDS-PAGE, and 18, 67, and 252 kDa by gel filtration, suggesting that BxENG-1 existed in an unglycosylated monomeric form and Bx-ENG-2 and Bx-ENG-3 in a glycosylated dimeric form. The enzymatic properties of the recombinant enzymes were similar to each other: optimal activity at 60 degrees C at about pH 6.0, like other endoglucanases of GHF45. The recombinant enzymes displayed the highest activity toward lichenan, and lower activities were observed on carboxymethyl cellulose and amorphous cellulose. Nematode enzymes also hydrolyzed glucomannan, the most abundant hemicellulose in the cell walls of softwood. These substrate specificities suggest that B. xylophilus endoglucanases acted on the cellulose-hemicellulose complex in the cell walls, resulting in a weakening of the mechanical strength of the cell walls to facilitate the nematode's feeding on plant cells.