Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-04-07
2001-01-09
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S014000, C435S004000, C435S968000, C536S001110, C536S123130, C536S123100
Reexamination Certificate
active
06171810
ABSTRACT:
FIELD OF THE INVENTION
Glycosidases, enzymes which hydrolyze glycosidic bonds, are a diverse group of enzymes widely present in nature. Glycosidases are important in a number of biological processes, and a number of disease states have been linked to defects in these enzymes. For example, an absence of lysosomal &agr;-glucosidase leads to a fatal condition, Pompe disease, and defects in glucocerebrosidase activity lead to Gaucher's disease. In addition, elevated serum levels of some glycosidases are correlated with the presence of cancerous cells. Glycosidases are also important in industrial processes ranging from food processing (fruit juice processing, lactose reduction, production of “invert” sugar) to textile processing, pulp processing in papermaking, and biomass degradation.
Exoglycosidases are a subset of glycosidases that hydrolyze a terminal sugar residue from polysaccharides and/or glyconconjugates. Examples of exoglycosidase include fucosidases, glucosidases, galactosidases, and mannosidases. Due to their involvement in a variety of biological phenomena and potential application in biotechnological and industrial settings, there has been a great deal of interest in understanding the active sites and catalytic mechanisms of these enzymes. In addition, through the use of various mutagenesis strategies, efforts have been made to alter and improve the properties of wild-type exoglycosidases, including their specific activities, thermostabilities, substrate specificities and pH optima.
A powerful mutagenesis strategy for the study of enzymes, including exoglycosidases, is directed evolution. In this technique, clones expressing the gene for the enzyme of interest are subjected to multiple rounds of mutagenesis, functional screening, and amplification. More particularly, cells bearing the gene of interest are first subjected to mutagenesis, then a selected number of clones are individually screened for a desired change in activity. The most desirable clones are retained, and the DNA bearing the gene of interest is then pooled from these clones and used to transform cells for the next round of mutagenesis. In this manner, the desired phenotype, as dictated by the functional screening, is enhanced with each round. Using directed evolution, &bgr;-fucosidase activity was obtained from a &bgr;-galactosidase gene after seven rounds of mutagenesis, screening, and amplification (Zhang et al.,
Proc Natl Acad Sci U S A
94(9):4504-4509 (1997)).
However, such directed evolution methods are generally labor-intensive and time-consuming. For example, in the work described above, 10,000 clones were visually screened in each round. The labor-intensive nature of this method limits the number of initial clones, the number of rounds of mutagenesis and screening, and the number of enzyme activities that can be tested in any series of experiments. In addition, in performing directed evolution, it is crucial to distinguish between those clones which express mutated enzymes, and those clones whose change in enzyme activity are due to alterations in the level of expression of the enzyme gene. This determination requires an additional step to measure the amount of enzyme inside the cell. This additional step increases the time, cost and labor required for the entire process.
Separate measurements of the amount of enzyme in the cell can be avoided by taking advantage of enzyme kinetic parameters that are independent of the amount of enzyme in a cell sample. By screening for changes in these parameters, alterations in the structure of the enzyme, rather than in its expression level, can be easily detected. However, these measurements often require many additional experiments to determine the true enzyme activity. Thus, although they avoid the need to measure the level of enzyme in the cells, these additional experiments also increase the time, cost and labor required for the entire process.
Directed evolution methods are generally paired with high throughput enzyme assay systems to minimize the time, cost and labor required in assaying a large number of samples. High throughput enzyme assay systems utilize robots and automatic data analysis methods to simultaneously analyze many samples with minimal operator interaction. These systems may also utilize other common laboratory supplies, such as 96-well or 384-well microplates and disposable pipet tips, to further lower costs and time associated with assay of the samples. By using a high throughput enzyme assay system, many experiments can be simultaneously performed at a low cost.
4-methylumbelliferyl (hereinafter “4-Mu”)-linked monosaccharide derivatives are commonly used substrates for assays of exoglycosidase activity. The fluorescence intensity of 4-Mu is pH-dependent, and is maximal above a pH of 9. However, a majority of exoglycosidases are most active below neutral pH, that is, below a pH of about 7.0. In conventional in vitro endpoint assays of exogylcosidase activity that employ such a 4-Mu-linked substrate, the enzyme and the substrate are incubated at the pH that is optimal for the activity of the enzyme for a defined period of time. The enzyme cleaves the 4-Mu moiety from the substrate. Thereafter, the reaction is terminated by addition of a high-pH buffer (pH between 9.5 and 10.5). This buffer both terminates the reaction and enhances the fluorescence intensity of the 4-Mu product, thereby increasing the sensitivity of the assay. The amount of the liberated 4-Mu moiety generated is measured by fluorescence spectroscopy, usually using an excitation wavelength around 365 nm and emission wavelength around 440 nm. By varying the amount of substrate or the assay environment, values for various enzyme kinetic parameters can be determined.
In high throughput enzyme assay systems, it is conventional to employ endpoint assays. In endpoint assays, the reaction between the enzyme and its substrate is halted before the amount of product is measured. However, in determining enzyme kinetic parameters, endpoint assays are not desirable, because a separate experiment is required to generate data for each time point, and many such experiments must be run to generate sufficient data to determine the enzyme kinetic parameter values in each sample. In such experiments, kinetic assays are more appropriate than endpoint assays. In a kinetic assay, the amount of the product generated by the reaction of the enzyme with the substrate is measured at various time points while the reaction is in progress. Kinetic assays speed up the measurement of enzyme kinetic parameters by reducing the number of experiments required to obtain the necessary data.
The detection of mutant exoglycosidase enzymes could be accelerated by the use of kinetic enzyme assays in a high throughput enzyme assay system. However, since most exoglycosidases require a much lower pH for optimal activity than that required for maximal fluorescence of the 4-Mu product, kinetic assays have not previously been employed. To date, most exoglycosidase assays have employed either an endpoint assay or have used other compounds to detect activity, e.g., paranitrophenol. Therefore, the lack of a useful kinetic assay for exoglycosidase activity which employs fluorogenic substrates has hampered the application of high throughput enzyme assay systems to the study of these enzymes.
A previous attempt to employ 4-Mu-linked monosaccharide derivatives in a kinetic exoglycosidase assay focused on balancing the pH optima of the enzyme and the product (Hendrikx, P.-J. et al.,
Anal. Biochem
. 222:456-460 (1996)). However, this approach is still unsatisfactory for high throughput screening systems, because the deviation from the optimal pH of the enzyme introduces an additional factor into the measurement of potential mutant enzyme activity. Correction for this deviation may slow down the high throughput screening rate.
It is therefore desirable to have an enzyme activity assay system that is adaptable to high throughput screening of exoglycosidases, so as to be able to screen a large number of individual exogl
Amster Rothstein & Ebenstein
Leary Louise N.
New York Blood Center Inc.
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