A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant trichoderma beta-(1-6)-glucanase expressed in Pichia pastoris.
The fungal cell wall is a supramolecular network of glycoproteins and polysaccharides. Its analysis is seriously hampered by the lack of easily available hydrolytic enzymes in a pure form. Here we describe a simple and efficient purification procedure of a recombinant beta-(1-6)-glucanase from Trichoderma harzianum expressed in Pichia pastoris. Transformed cells efficiently secreted the enzyme into the induction medium. We purified the enzyme using a one-step method based on hydrophobic interaction chromatography. The yield was 80%. SDS-PAGE of the purified enzyme revealed a single band with an apparent molecular mass of 43 kDa. The isoelectric point of the enzyme was 5.8, and it showed maximal enzyme activity and stability at pH 5.0. As beta-(1-6)-glucan is an important component of fungal cell walls, the easy availability of pure beta-(1-6)-glucanase will highly facilitate studies of the molecular organization of the fungal cell wall.
Bom
I J
IJ
Unilever Research Laboratories, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands.
Dielbandhoesing
S K
SK
Harvey
K N
KN
Oomes
S J
SJ
Klis
F M
FM
Brul
S
S
D002462:Cell Membrane / Q000737:chemistry; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C020105:endo-1,6-beta-glucanase