Macromolecular Interactions in Food Technology - American Chemical

Chapter 14

Characteristics of the Products of Limited Proteolysis of β-Lactoglobulin Harold E, Swaisgood, Xiaolin L. Huang, and George L. Catignani

Downloaded by FUDAN UNIV on February 21, 2017 | http://pubs.acs.org Publication Date: November 19, 1996 | doi: 10.1021/bk-1996-0650.ch014

Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624

Application of limited proteolysis to β-lactoglobulin (β-Lg) using immobilized trypsin allows precise control of the extent of reaction resulting in the release offragmentsof the central core β-barrel domain. These 4- or 5-stranded β-barrel fragments have β-structure that reversibly unfolds in a transition centered at 3.88 M urea. Conditions yielding 35-55% disappearance of intact protein produced 13-15% of the protein as domainfragments,the majorfragmentbeing β-Lg(f41100 + 149-162) which is linked by a disulfide bond. Membrane fractionation of a limited proteolysate of β-Lg yielded a protein ingredient, consisting of domain fragments, in the fraction passing through a 30 kDa retention membrane and retained by a 3 kDa retention membrane. The Emulsifying Activity Index (EAI) of this fraction was two-fold larger than that of the intact protein throughout the pH range 3 to 9 and also was greater than that for egg white protein. Furthermore, the emulsified oil droplets were smaller when the domain fraction was used and the emulsions appeared to be more stable. Treatment of whey protein isolate (WPI) under conditions that should yield 10-15% of the domain fragments produced a protein ingredient that gave particulate gels, whereas the untreated WPI gave fine-stranded gels.

The functional properties of a protein are determined by its structure, structural stability and flexibility, its surface topology and the chemical characteristics of its surface. Therefore, any changes in the primary structure that affect these characteristics will also affect its functional properties. Limited proteolysis obviously alters the primary structure thus changing the stability and flexibility of the resulting oligopeptides, often exposing new protein surface, and altering the chemical properties of the surface. Many proteins are composites of multiple domains (7,2), where a domain is a region of tertiary structure with many interactions between residues within

0097-6156/96/0650-0166S15.CK)^ © 1996 American Chemical Society

Parris et al.; Macromolecular Interactions in Food Technology ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


14. SWAISGOOD ET AL.

Products of Limited Proteolysis of P-Lactoglobulin

the structure but few interactions with residues outside the domain. Hence, a domain possesses a compact, rather stable structural motif that is resistant to proteolysis; whereas, the protein chain linking domains is flexible and thus susceptible to proteolysis (2,5). Hence, proteinases are commonly used to probe the structure of proteins for the presence of multiple domains. There are a number of important advantages derived by using immobilized proteinases rather than soluble enzyme for limited proteolysis, especially for the production of functional peptides (2-4), The most significant advantages are: 1) the extent of reaction is easily and precisely controlled so the proteolysis is reproducibly limited, 2) an enzyme denaturation step is not required to stop the reaction which would cause destruction of functional structure of the oligopeptides produced, and 3) enzyme autolysis does not occur so autolysis products are not present and the enzyme activity remains constant. The x-ray crystal structure of the whey protein P-lactoglobulin (p-Lg) reveals a central structural motif consisting of an eight-stranded anti-parallel P-barrel with another P-strand and an a-helix lying on its surface (5,6). The p-barrel forms a calyx which is the most likely site of the high affinity binding of retinol (5,7,8) or the retinyl moiety (9,10). We have shown that limited proteolysis of P-Lg with immobilized trypsin removes the a-helix and some of the P-strands yielding, as major peptides, fractions of the central core calyx structure (7,11). Limited Proteolysis of Whey Protein Immobilization of Trypsin. The enzyme was covalently immobilized on succinamidopropyl-glass or succinamidopropyl-Celite using either the simultaneous or the sequential activation/immobilization procedure with water-soluble carbodiimide to activate carboxyl groups (12,13). Using p-tosyl-L-arginine methyl ester as substrate, either immobilization procedure typically yields a biocatalyst with a specific activity of 20-40 pmol/min/mL of beads. Titration of active sites with p-nitrophenyl-/?'quanidinobenzoate indicated that more than 20% of the immobilized trypsin molecules had competent active sites. For most of the studies described here, a 100-mL fixed-bed bioreactor of trypsin-Celite was used. This bioreactor has been used for more than one year without significant loss of activity. Conditions for Limited Proteolysis. The rate of proteolysis depends upon the temperature, pH and the ratio of activity to substrate protein. To optimize the concentration of functional domain peptides, we have observed that conditions that result in 35-55% disappearance of the intact P-Lg appear to be optimal. We have used conditions rangingfrom4°C to 24°C at pH 7.6 and 8.0 and activity/substrate ratios of 500 to 2400 U/g with various time periods of treatment to yield the desired degree of proteolysis. Membrane Fractionation of the Limited Proteolysate. A membrane fractionation process was developed to obtain the oligopeptidefractionof the limited proteolysate for potential use as a value-added protein ingredient (14). The proteolysate was fractionated with a 30-kDa membrane (YM30, Amicon, Inc., Beverly, MA) and the


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MACROMOLECULAR INTERACTIONS IN FOOD TECHNOLOGY


resulting permeate was fractionated with a 3-kDa membrane (YM3) to yield the oligopeptides in the retentate (Figure 1). For these studies, a 100-mL bioreactor of trypsin-Celite was used to treat 2g of P-Lg at pH 7.6 for 60 min at 24°C. The P-Lg was prepared as described by Huang et al(12). Because P-Lg exists as a 36-kDa dimer under these conditions, the intact protein is retained by the 30-kDa membrane. The functional oligopeptide fraction retained by the 3-kDa membrane contains 50% of an 8.4-kDa oligopeptide which is probably P-Lg(f41-100 + 149-162) (see Figure 2). This fraction also contains small amounts of a large oligopeptide, which may represent the dimer with a small peptide removed, and several other fragments. Most of the very small peptides are removed in the permeate of the 3-kDa membrane. Characteristics of P-Lg(f41-100 + 149-162). This oligopeptide has native-like secondary and tertiary structure represented by 5 strands of the calyx motif of the intact protein and undergoes reversible unfolding in urea (7). However, the structure of this domain fragment is considerably less stable than that of the intact protein as indicated by measurements of their structural transitions in urea by CD spectroscopy and their thermal denaturation by DSC (Table I). The pH-dependent binding of the domain fragment to retinyl-Celite was similar to that of the intact protein, which also supports the conclusion that the oligopeptide exhibits the calyx motif (7).

1

Protein P-Lg A & B Domain fragments

Table I. Summary of Thermodynamic Data CD Spectra AGn (kcal/mol) [Urea]i/2 11.4 5.51 6.82 3.88

DSC T (°C) 81.1 56.9 m

Emulsification Characteristics of P-Lg Domain Peptides Emulsifying Properties of the Oligopeptide Fraction. The emulsifying activity index (EAI)(75) for the oligopeptide fraction is compared with that for the intact protein and for egg white protein at several pHs in the range of pH 3 to 9 in Figure 3. The emulsifying activity of both intact p-Lg and the oligopeptide fraction increased with increasing pH, possibly due to increasing flexibility of the structure; whereas, the activity of egg white protein decreased with increasing pH. More importantly, the activity for the oligopeptide fraction was two-fold greater than that of the intact protein throughout the pH range and became increasingly greater than that of egg white protein as the pH increased. The stabilities of peanut oil emulsions prepared with intact P-Lg and the oligopeptide fraction were also compared. Using a turbidometric method based on turbidity measurements at 500 nm (16), similar stabilities were indicated by a similar dependence of the relative turbidity on time. However, after standing the two emulsions were visually distinct (Figure 4). The emulsion formed with intact protein


14.

Products of Limited Proteolysis of (3-Lactoglobulin

SWAISGOOD ET AL.

P-lactoglobulin

(^l^psin-bioreart^


Proteolysate 30 kDa retention membrane >30 kDa protein

3 kDa oligopeptides Functional oligopeptides

Figure 1. Schematic illustration of the bioprocess for obtaining functional oligopeptides by limited proteolysis.

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Macromolecular Interactions in Food Technology - American Chemical

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