title: Chymotrypsin selectively digests β-lactoglobulin in whey protein isolate away from enzyme optimal conditions: potential for native α-lactalbumin purification.
abstract: The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7.0, 7.8 and 8.5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0.1%, 0.5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman-Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8.5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8.5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman-Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations.
authors: Laboratory for Milk Technology and Dairy Products, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia.
authors: Katarina
authors: K
authors: Lisak
authors: Jose
authors: J
authors: Toro-Sierra
authors: Ulrich
authors: U
authors: Kulozik
authors: Rajka
authors: R
authors: Božanić
authors: Seronei Chelulei
authors: SC
authors: Cheison
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007768:Lactalbumin / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000378:metabolism*; D008892:Milk; D008894:Milk Proteins / Q000737:chemistry*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D000067816:Whey Proteins
chemical_list: D007782:Lactoglobulins; D008894:Milk Proteins; D011480:Protease Inhibitors; D000067816:Whey Proteins; D007768:Lactalbumin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin