Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2001-07-06
2004-02-03
Patten, Patricia (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S370000, C424S725000, C424S773000, C514S002600
Reexamination Certificate
active
06686456
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to the removal of proteinase inhibitors from plant sources and, more specifically, to the removal of Kunitz and Bowman-Birk trypsin inhibitors and carboxypeptidase inhibitors from proteins extracted from potato tubers.
2. Background of the Prior Art
Proteins that inhibit proteolytic enzymes are often found in high concentrations in many seeds and other plant storage organs. Inhibitor proteins are also found in virtually all animal tissues and fluids. These proteins have been the object of considerable research for many years because of their ability to complex with and inhibit proteolytic enzymes from animals and microorganisms. The inhibitors have become valuable tools for the study of proteolysis in medicine and biology. Protease inhibitors are of particular interest due to their therapeutic potentials in controlling proteinases involved in a number of disorders such as pancreatitis, shock, and emphysema, and as agents for the regulation of mammalian fertilization. Potato tubers are a rich source of a complex group of proteins and polypeptides that potently inhibit several proteolytic enzymes usually found in animals and microorganisms. In particular, potato inhibitors are known to inhibit human digestive proteinases, and thus have application in the control of obesity and diabetes.
Proteinase inhibitors found in plants are typically polypeptides and proteins that are composed entirely of L-amino acids through peptide bonds. These proteinase inhibitors differ significantly in their properties. The association of natural proteinase inhibitors with the proteinases that they inhibit is strong at neutral pH, and association constants are usually in the range of 10
7
-10
14
M
−1
. Such associations are pH-dependent, and they decrease rapidly as the pH is lowered from neutrality to 3 (Ryan, C. A., and Walker-Simmons, M. 1981. Plant Proteinase. In
The Biochemistry of Plants
, V6, pp. 321-350, Academic Press).
Plant proteinase inhibitors generally are quite stable molecules and are often resistant to heat, pH extremes, and proteolysis by proteinases, even by those they do not inhibit. This stability has been attributed in part to the high degree of cross-linking through disulfide bridges. Other, non-covalent interactions also contribute significantly to the stability of the inhibitors. For example, protease inhibitor I from potatoes is a powerful chemotrypsin inhibitor that, while stable in solution at 80° C. for several minutes (Melville, J. C., and Ryan, C. A. Chemotrypsin inhibitor I from potatoes.
J. Microb. Chem
. 247: 3445-3453, 1972), contains only one disulfide bond per monomer unit (MW ~8,300) that can be reduced and carboxymethylated without loss of inhibitory activity (Plunkett, G., and Ryan, C. A. Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities.
J. Biol. Chem
., 255: 2752-2755, 1980).
Several proteinases exhibit substrate specificity, whereas others, such as papain, have broad substrate specificity. Specific proteinase inhibitor families were identified for each of the four mechanistic classes of proteolytic enzymes, i.e., serine, cysteinyl, aspartyl and metallo-proteases (Ryan, C. A. Proteinase Inhibitors. In The Biochemistry of Plants, V6, pp.351-370, Academic Press). In other circumstances, identical abundant proteins were capable of inhibiting enzymes of various families and have very different substrate specificity, such as the inhibitors of both proteinases and &agr;-amylases which were isolated from cereal seeds (Campos, F. A. P., and Richardson, M. The complete amino acid sequence of &agr;-amylases/trypsin inhibitor from seeds of ragi (Indian finger millet;
Eleusine coracana
Goertn.). FEBS Lett., 152: 300-304, 1983; Campos, F. A. P., and Richardson, M. The complete amino acid sequence of &agr;-amylases/trypsin inhibitor from seeds of ragi (Indian finger millet;
Eleusine coracana
Goertn.). FEBS Lett., 167: 221-225, 1984).
Two broad classes of protease inhibitor superfamilies have been identified from soybean and other legumes with each class having several isoinhibitors. Kunitz-type inhibitor is the major member of the first class whose members have 170-200 amino acids, molecular weights between 20,000 and 25,000, and act principally against trypsin. Kunitz-type proteinase inhibitors are mostly single chain polypeptides with 4 cysteines linked in two disulfide bridges, and with one reactive site located in a loop defined by disulfide bridge. The second class of inhibitors contains 60-85 amino acids, has a range in molecular weight of 6000-10,000, has high proportion of disulfide bonds, is relatively heat-stable, and inhibits both trypsin and chemotrypsin at independent binding sites. Bowman-Birk inhibitor is an example of this class.
Kunitz inhibitor is capable of inhibiting trypsin derived from a number of animal species as well as bovine chemotrypsin, human plasmin, and plasma kallikrein. The cationic form of human trypsin, which accounts for a majority of trypsin activity, is only weakly inhibited by the Kunitz inhibitor, whereas the anionic form is fully inhibited.
The Bowman-Birk inhibitor is a 71 amino acid chain protein with 7 disulfide bonds characterized by its low molecular weight of about 8000 (in non-associated monomers), high concentration (about 20%) of cystine, high solubility, resistance to heat denaturation and having the capacity to inhibit trypsin and chymotrypsin at independent inhibitory sites.
The major effects of proteinase inhibitors in animal diets include growth depression and pancreatic hypertrophy. Resistance of raw soybean protein to proteolysis, low levels of sulfur-containing amino acids in soybean proteins, and lower digestibility, absorption, and utilization of available nitrogen from the small intestine due to the presence of proteinase inhibitors, all appear to contribute to growth depression.
Proteinase inhibitors extracted from potatoes have been distinguished into two groups based on their heat stability. The group of inhibitors that is stable at 80° C. for 10 minutes have been identified as inhibitor I (mol. wt. 39,000) (Melville et al.), carboxypeptidase inhibitor (CPI) (mol. wt. 4,100) (Ryan, C. L., Purification and properties of a carboxypeptidase inhibitor from potatoes.
J. Biol. Chem
. 249: 5495-5499, 1974), inhibitors IIa and IIb (mol. wt. 20,700) (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components.
Biochemistry
15: 3418-3424, 1976), and inhibitor A5 (mol. wt. 26,000).
Separation of proteinase inhibitor I by ion exchange chromatography on sulfoethylcellulose in the presence of 0.1 M formic acid in 8 M urea resolved two major and two minor inhibitor protomers. Reassociation by dilution to the tetramer form resulted in two major protomers. The first protomer was shown to be a powerful inhibitor of both chymotrypsin and trypsin. The second protomer was shown to strongly inhibit chymotrypsin but only weakly inhibit trypsin. All four purified promoters resolved from Inhibitor I can be reassociated either individually or hybridized with each other to form tetrameric isoinhibitors. All of the tetrameric inhibitor I species prepared from each of the four protomeric types have glutamic acid at the NH
2
terminal. However, they differ from each other in amino acid composition, electrophoretic mobility, reactivity with chymotrypsin and trypsin, and digestibility with pepsin.
Proteinase inhibitor II, an inhibitor of chemotrypsin and trypsin, which are serine proteases, is also a heat stable protein. It has a dimeric molecular weight of 21,000. Four monomeric isoinhibitor species of molecular weight 10,500 comprise inhibitor II and have been isolated by chromatography in the presence of urea. Upon removal of the urea, each monomeric species dimerized to yield homogenous dimers. The three major pr
Ausich Rod
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Stomp Robert
Davis Law Firm
Herink Kent
Kemin Foods, L.C.
Patten Patricia
Rosenberg Daniel
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