HEXOSE OXIDASE FROM THE RED ALGA CHONDRUS-CRISPUS - PURIFICATION, MOLECULAR-CLONING, AND EXPRESSION IN PICHIA-PASTORIS

Hexose oxidase from Chondrus crispus catalyzes the oxidation of a vari ety of mono- and disaccharides including D-glucose, D-galactose, malto se, and lactose, The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein w ith a relative molecular mass of approximately 130,000 (Sullivan, J, D ,, and Ikawa, M, (1973) Biochim, Biophys, Acta 309, 11-22). We report here the purification to homogeneity of hexose oxidase from C, crispus . The purified enzyme was cleaved with cyanogen bromide and endoprotei nase Lys-C and the peptide fragments were subjected to amino acid sequ ence analysis, Oligonucleotides were designed on the basis of the pept ide sequences and a cDNA clone encoding C, crispus hexose oxidase was obtained using polymerase chain reaction on reverse transcribed cDNA, The nucleotide sequence of the hexose oxidase cDNA contained an open r eading frame of 546 amino acid residues with a predicted relative mole cular mass of 61,898, No significant sequence similarity was found bet ween hexose oxidase and other protein sequences available in data base s. Expression of the hexose oxidase cDNA in Pichia pastoris as an acti ve enzyme confirmed the identity of the DNA sequence, Native hexose ox idase from C, crispus was characterized and compared with purified, re combinant enzyme.