Purification and characterization of recombinant endoglucanases from the pine wood nematode Bursaphelenchus xylophilus.
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 100-140 kDa by SDS-PAGE, and 18, 67, and 252 kDa by gel filtration, suggesting that Bx-ENG-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.
Shibuya
Hajime
H
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan. bushi@ffpri.affrc.go.jp
Kikuchi
Taisei
T
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D017187:Tylenchida / Q000201:enzymology* / Q000469:parasitology
D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase; C109691:microcrystalline cellulose