Characterization and purification of lactase (β-galactosidase) and acid-stable, raw-starch hydrolyzing amylase from jackfruit (Artocarpus heterophyllus) seeds

Abstract Lactase is important to treat lactose intolerance that results from the lack of intestinal lactase leading to the inability to digest lactose. Management of the disorder by external enzymes, especially with phyto-enzyme supplementation has not yet been much attempted. The present study reports the recovery of appreciable amount of lactase (900 ± 30U/100 g fresh wt.) from the seeds of jack fruit. The ethanol precipitated fraction of the seed extract could hydrolyze 89 ± 2% and 78 ± 2% of 2% (w/v) and 5% (w/v) lactose respectively at pH 5.0 in 3h at 50°C. Amylase was also detected in the extract (3200 ± 200U/100 g fresh wt.) and the ethanol precipitated fraction hydrolyzed various starches, gelatinized cereal flour (DE value = 43–50) and raw starch (DE value = 16–25). 200KDa lactase (specific activity = 14 ± 0.05 U/mg protein) was purified from the seed extract by ethanol precipitation, followed by ammonium sulphate precipitation, ion exchange chromatography (DEAE Sephadex), size exclusion chromatography (Sephacryl S-300HR) and SDS PAGE. Activity staining by x-gal on Native gel was also performed. Purified lactase was stable in the pH range of 4–8 upto 60°C and displayed a Km and Vmax of 5mM (lactose) and 0.044µmole/min respectively. Purified lactase was unaffected by the action of proteolytic enzymes. Hg2+, Ag+ inhibited the enzyme activity completely (at 1mM conc.) while Ca2+ enhanced the activity by 15 ± 1%. The purified lactase effectively hydrolyzed 1%, 2%, 5% (w/v) lactose solution and pasteurized milk. Lyophilized seed powder and extract remained stable till 18 months of the study period when stored at 0-4°C.