Abstract Immobilized metal ion affinity chromatography (IMAC) enables the fractionation of commercial fungal enzyme preparations. Pectin‐lyase showed no affinity with the chromatographic matrix at pH 7. Endo‐glucanase and cellobiohydrolase were both eluted in almost all fractions but predominantly in three fractions when pH decreased. This suggests that IMAC can discriminate between different forms of cellulase isoenzymes. Polygalacturonase and pectinesterase were released with last fraction, showing their strongest affinity for the matrix. Following an experimental design, IMAC fractions containing separately pectin‐lyase and cellulase have been used to formulate an optimal enzymatic preparation for liquefaction of passion fruit pulp. It has been found that for every liter of passion fruit juice, at least 85 units of pectin‐lyase, 100 units of cellobiohydrolases and 700 units of endo‐glucanases are required for convenient liquefaction after 1 hour of incubation at 30 °C.