Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1998-09-01
2001-04-10
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C510S114000, C510S395000
Reexamination Certificate
active
06214596
DESCRIPTION:
Me-trypsin may be prepared by the following synthesis:
The following solutions are first prepared:
1. Borate buffer: 0.2 M sodium borate buffer, pH 9.2 containing 2 mg/ml benzamidine hydrochloride and a trace amount of n-octanol.
2. Trypsin: 1 g in 150 ml borate buffer.
To the 150 ml solution of trypsin, 10 ml of 1 M sodium borohydride is added followed quickly by 10 ml of 2.4 M formaldehyde. Three more volumes of sodium borohydride and formaldehyde are added at 10 minute intervals. The reaction solution is then acidified with glacial acetic acid to approximately pH 4.2 and then dialyzed extensively against 2 mM HCl at 4° C. (8 changes of 2 L each within 24 hours). The dialyzed solution is finally lyophylized for over 20 hours.
The above reactions are further described in Rice, R H, Means, G E and Brown, W D. Stabilization of bovine trypsin by reductive methylation, Biochimica et Biophysica Acta, volume 492, pages 316-321 (1977); and Means, G E and Feeney, R E. Reductive alkylation of amino groups in proteins, Biochemistry, volume 7, pages 2192-2210 (1968). Me-trypsin is also available from commercial sources such as Sigma Chemical Co. and Promega Corp. (Madison, Wis.).
Other Al-trypsins may be prepared by methods analogous to Example 1, wherein formaldehyde is replaced by other alkylating reagents. For example, ethyl trypsin (“Et-trypsin”) may be synthesized by an analogous method described in Example 1 and Scheme 1 above, wherein acetaldehyde is used as the alkylating reagent in place of formaldehyde.
The liquid enzyme compositions of the present invention will have an enzyme concentration sufficient to provide an effective amount of enzyme to remove substantially or to reduce significantly deposits of proteins, lipids, mucopolysaccharides and other materials typically found on human-worn contact lenses when a small amount of a composition is added to a diluent. As used herein, such a concentration is referred to as “an amount effective to clean the lens.” The amount of enzyme used in the liquid enzyme compositions of the present invention will generally range from about 0.05 to 5% w/v. The selection of a specific concentration will depend on various factors, such as: the enzyme or combination of enzymes selected; the purity, specificity and efficacy of the enzyme(s) selected; the type of lenses to be cleaned; the intended frequency of cleaning (e.g., daily or weekly); and the intended duration of each cleaning.
During storage, some of the activity of the enzyme may be lost, depending on length of storage and temperature conditions. Thus, the liquid enzyme compositions of the present invention may be prepared with initial amounts of enzyme that exceed the concentration ranges described herein. The preferred compositions of the present invention will generally contain one or more enzymes in an amount of about 300-6000 PAU/mL. The compositions will most preferably contain about 900-3000 PAU/mL, which corresponds to pancreatin in the range of about 1 to 3% w/v; subtilisin in a range of about 0.1 to 0.5% w/v; trypsin in the range of about 0.1 to 0.5% w/v; and Me-trypsin in the range of about 0.1 to 0.5% w/v. For purposes of this specification, a “proteolytic activity unit” or “PAU” is defined as the amount of enzyme activity necessary to generate one microgram (mcg) of tyrosine per minute (“mcg Tyr/min”), as determined by the casein-digestion, colorimetric assay described below.
Casein-digestion assay
A 5.0 mL portion of casein substrate (0.65% casein w/v) is equilibrated for 10 minutes (min)±5 seconds (sec) at 37° C. A 1.0 mL portion of enzyme solution (0.2 mg/ml) is then added to the casein substrate and the mixture vortexed, then incubated for 10 min±5 sec at 37° C. After incubation, 5.0 mL of 14% trichloroacetic acid is added and the resultant mixture immediately vortexed. The mixture is incubated for at least another 30 min, then vortexed and centrifuged for 15-20 min (approx. 2000 rpm). The supernatant of the centrifuged sample is filtered into a serum filter sampler and a 2.0 mL aliquot rem
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Asgharian Bahram
Hong Bor-Shyue
Alcon Laboratories Inc.
Brown Gregg C.
Mayo Michael C.
Weber Jon P.
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