Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-09-24
2004-08-31
Tate, Christopher R. (Department: 1654)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S173100, C435S173200, C435S183000, C424S094300
Reexamination Certificate
active
06783968
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for sterilizing preparations of glycosidases to reduce the level therein of one or more active biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and/or single or multicellular parasites. The present invention particularly relates to methods of sterilizing preparations of glycosidases, such as alpha-glucosidase or alpha-galactosidase, with irradiation.
2. Background of the Related Art
The principal foods upon which an organism, such as a human, survives can be broadly categorized as carbohydrates, fats and proteins. These substances, however, are useless as nutrients without the process of digestion to break down foods into chemical components that are sufficiently small to be absorbable in the digestive tract.
Digestion of carbohydrates begins in the mouth and stomach. Saliva contains the enzyme ptyalin (an alpha-amylase), which hydrolyses starch into maltose and other small polymers of glucose. The pancreatic alpha-amylase is similar to the salivary ptyalin, but several times as powerful. Therefore, soon after chyme empties into the duodenum and mixes with pancreatic juice, virtually all of the starches are converted into disaccharides and small glucose polymers. These disaccharides and small glucose polymers are hydrolysed into monosaccharides by intestinal epithelial enzymes, such as intestinal sucrase, intestinal maltase, and intestinal lactase.
Digestion of proteins begins in the stomach. The ability of pepsin to digest collagen is especially important because collagen is a major constituent of the intercellular connective tissue of meats. For other glycosidases to penetrate meats and digest various cellular proteins, the collagen fibers must first be partially digested by pepsin. People who lack peptic activity in the stomach will experience poor absorption of ingested meats because there is poor penetration by these other glycosidases.
Most protein digestion results from the actions of the pancreatic proteolytic enzymes. Proteins leave the stomach in the form of proteoses, peptones and large polypeptides, and are digested into dipeptides, tripeptides and the like by pancreatic proteolytic enzymes or polypeptidases. Trypsin and chymotrypsin split protein molecules into smaller polypeptides at specific peptide linkages, whereas carboxypolypeptidase cleaves amino acids from the carboxyl ends of polypeptides. The zymogen proelastase is converted to the active protease elastase, which in turn digests elastin fibers that hold together most meat.
Further digestion of polypeptides takes place in the intestinal lumen. Aminopolypeptidase and several other polypeptidases split large polypeptides into dipeptides, tripeptides and amino acids, which are then transported into enterocytes that line the intestinal villi. Inside the enterocytes, other polypeptidases split any remaining peptides into their constituent amino acids, which then enter the blood.
Digestion of fats first requires emulsification by bile acids and lecithin, which increase the surface area of the fats up to 1000-fold. Because lipases are water-soluble glycosidases that can bind only on the surface of a fat globule, this emulsification process is important for the complete digestion of fat. The most important glycosidase in the digestion of triglycerides is pancreatic lipase, which breaks these down into free fatty acids and 2-monoglycerides. After these free fatty acids and monoglycerides enter the enterocytes, they are generally recombined into new triglyerides. A few monoglycerides, however, are further digested by intracellular lipases into free fatty acids.
Glycosidases are required to digest or break down saccharide units (usually polysaccharides). For example, glycosidases are required to digest or breakdown the saccharide untis that are covalently attached to proteins so that proteases can then gain access to the protein for cleavage of the individual amino acids therefrom, to thereby promote absorption of the resulting amino acids.
A number of glycosidases are present in the human gastrointestinal tract, but, to date, only some have been isolated and sufficiently characterized. Many glycosidases, however, have been isolated and characterized from plant and microbial sources, which, to some extent, has provided a roadmap for studies of animal glycosidases. Thus, glycosidases are known that cleave and remove O-linked sugar units from glycoproteins, and from glycolipids and polysaccharides, for example, alpha-N-acelylgalactosaminidase. Other glycosidases include N-acetylneuraminic acid aldolase, beta(1-4) galactosidase, beta(1-3,6) galactosidase, beta(1-3,4,6) galactosidase, beta(1-6)galactosidase, alpha(1-3,6) galactosidase, beta-glucosaminidase, alpha-mannosidase, alpha(1-3,4) fucosidase, alpha(1-2,3,4) fucosidase, alpha(1-2) fucosidase, beta(1-2) xylosidase, beta(1-4) xylosidase, peptide-N
4
-(acetyl-beta-glucosaminyl)-asparagine amidase (EC 3.5.1.52) hexosaminidase, beta-N-acetylhexosaminidase, alpha(2-3,6,8,9) neuraminidase, various sialidases, such as N-acetylneuraminate glycohydrolase (EC 3.2.1.18), various glycoamidases, alpha-mannosidase, and beta-mannosidase.
The nomenclature of glycosidases typically indicates the type of linkages that are cleaved by the enzyme. For example, beta(1-3,6) galactosidase cleaves only beta(1-3,6) galactose linkages, but not beta(1-4) galactose linkages.
Reaction conditions and substrate specificities vary greatly, depending on the particular glycosidase. For example, a given glycosidase may be ineffective when sialic acid is present on N-linked oligosaccharides. The optimal pH activities of glycosidases also vary according to the particular glycosidase, with some being optimally active at acidic pH values, others at values near neutral pH, and yet others at alkaline pH values.
Preparations of glycosidases may be required for administration to humans and other animals when, for example, there is a genetically caused disease, such as lack of endogenous glycosidases, or lack of active glycosidases. One genetic disease characterized by a partially or completely inactive glycosidase is Fabry disease, which afflicts about one in 40,000 people in the United States. Fabry disease is an X-linked lysosomal disorder in which the patient's body does not have a normal ability to break down a fatty substance, globotriaosylceramide (also known as Gb
3
or ceramidetrihexoside). Gb
3
is present in membranes of many cell types, including the membranes of red blood cells. Roughly 1% of one's red blood cells are replaced each day, which means that a significant amount of Gb
3
requires degradation each day.
One of the major lysosomal enzymes involved in the degradation of Gb
3
is alpha-galactosidase A (“&agr;-gal A”), which is either partially or completely inactive in patients with Fabry disease. As a result, Gb
3
accumulates in lysosomes throughout the patient's body, which impairs (clogs blood vessels with built-up Gb
3
) organs and body parts that depend on proper functioning of small blood vessels, such as kidneys, heart, nervous system, and skin.
The most common symptom of Fabry disease is pain, which may occur in the form of periods of intense burning, or sharp, shooting pain. The pain may be brought on by such events as exercise, fever, fatigue, stress, and/or exposure to temperature changes. In addition, many patients with Fabry disease are unable to perspire, which causes further discomfort with exercise or exposure to high temperatures. Periods of pain are most common in childhood, but also may not present until the 20s, when sufficient Gb
3
has accumulated. In some patients, the pain subsides with increasing age, and in others the pain increases.
Another common symptom of Fabry disease is a spotted, dark-red skin rash that most commonly occur
Burgess Wilson
Drohan William N.
MacPhee Martin J.
Mann David M.
Clearant Inc.
Tate Christopher R.
Winston Randall
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