An extracellular exo-β-(1,3)-glucanase from Pichia pastoris: Purification, characterization, molecular cloning, and functional expression

Abstract An extracellular exo -β-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47 kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent K m , 3.5 mg/ml; V max , 192 μmole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p -nitrophenyl-β- d -glucopyranoside and p -nitrophenyl-β- d -xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; p I , 4.46) that shares 36–64% amino acid identity with other yeast exo -β-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.