Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-08-05
2001-09-04
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252330, C435S320100, C435S441000, C435S212000, C536S023200
Reexamination Certificate
active
06284511
ABSTRACT:
The present invention relates to the field of biotechnology and concerns heat-stable prolylcndopeptidase, recombinant DNA coding for heat-stable prolylendopeptidase, and processes for the production of heat-stable prolylendopeptidase and of recombinant DNA coding therefor, a host transformed with said recombinant DNA and a process for the production of said transformed host.
DESCRIPTION OF THE RELATED ART
Prolylendopeptidase was first found in human uterus as a specific endopeptidase which cleaves a peptide at the carboxyl-terminal side of a proline Tesidue. The unique substrate selectivity of the enzyme drew much attention to the study of its physiological functions. An endopeptidase that shows the same substrate specificity as mammalian prolylendopeptidase was found also in a bacterium,
Flavobacterium meningosepticum
. This finding made prolylendopeptidase commercially available and enabled its use as a biochemical reagent for the specific cleavage of peptides. The preparation of prolylendopeptidase from F. meningosepticum, however, has the following two crucial drawbacks arising from the bacterium. The bacterium is pathogenic and it produces not only prolylendopeptidase but also significant amounts of other specific or non-specific peptidases. The commercial preparations are thus contaminated with significant amounts of trypsin and aminopeptidase, which in fact, severely diminishes the utility of the commercial products as specific biochemical reagent.
Prolylendopeptidase catalyzes selective hydrolytic cleavage of peptides at the C-terminal side of a proline residue under physiological conditions. The enzyme can also catalyze the coupling of peptide fragments by condensation or transpeptidation, depending on reaction conditions and substrates. In the production of pharmaceutically active peptides, prolylendopeptidase can thus be used to catalyze i) selective cleavage of precursor peptides in order to liberate the pharmaceutically active peptide, ii) in vitro modification of peptides including amidation of C-termini and iii) coupling of peptides. The term peptide used herein shall not indicate that only short peptides are meant but that the molecules in question are composed of amino acids linked via peptide bonds. Peptides may be short peptides, oligopeptides or polypeptides.
Of the three reactions mentioned above, the former two are especially important for downstream processing in production processes of the peptides with recombinant DNA technology. The recombinant peptides are often expressed in the form of a precursor or fusion protein, which is then subjected to in vitro processing for the conversion to active or mature forms. Prolylendopeptidase is for example useful for C-terminal amidation of biologically active peptides such as ACTH, cholecystolin, calcitonin, endorphin, insulin, LH-RH, oxytocin and vasopressin, or the like. The characteristic substrate specificity of prolylenopeptidase makes it very useful for the cleavage of the precursors at specific sites and the in vitro modification of peptides without side reactions that are often associated with non-specific peptidases.
However, prolylendopeptidase is quite susceptible to inhibition or inactivation by conditions usually applied in peptidase catalyzed reactions. It is susceptible to denaturing and/or solubilizing agents in buffers used for cleavage of precursor peptides or also to conditions commonly used in peptidase catalyzed coupling reactions in order to make the formation of coupling products favourable over hydrolysis, e.g. the presence of high concentrations of organic solvents such as 1,4-dioxane, DMF or DMSO, extremes of pH and/or high temperature. Therefore, it is desirable to improve the stability of prolylendopeptidase in order to make it more versatile as a catalyst for the industrial production of peptides.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide prolylendopeptidases which are more heat-stable than the corresponding wild type enzymes. Such a prolylendopeptidase is hereinafter named “heat-stable prolylendopeptidase”.
A heat-stable prolylendopeptidase is particularly useful for an industrial production process of peptides, because its superior stability prolongs the life of the enzyme in the catalytic reactions and thus improves a total turnover of the endopeptidase. It is useful as a stable and selective catalyst for production of the biologically active peptides that contain proline residues. In other words, a heat-stable prolylendopeptidase can be used in much lower ratio to the substrates than that of the wild-type enzyme, decreasing costs of the catalyst which is often a critical factor for commercial feasibility of the production process. The high heat-stability of heat-stable prolylendopeptidases of the invention also enables the use thereof under the severe conditions that improve efficiencies, yields or conversions of the catalytic reactions, e.g., higher reaction temperature, extreme pH or the presence of a high concentration of organic solvent. A heat-stable prolylendopeptidase created in the present invention is also more resistant to other forms of protein denaturation, i.e. to other physical stress than heat inactivation and more resistant to treatment with chemicals. Thus, it is more-stable than the corresponding wild type enzyme in solutions containing organic solvents, denaturing agents or extreme pH.
Another object is to provide a method for the generation of heat-stable prolylendopeptidase starting from DNA coding for a wild-type enzyme and a method for the improvement of heat-stability by a “molecular evolution” method comprising multiple cycles of mutagenization and screening.
A further object of the present invention is to provide recombinant DNA coding for heat-stable prolylendopeptidase, a process for the production of such recombinant DNA, a host transformed with such recombinant DNA, and a process for the production of a heat-stable prolylendopeptidase by means of a transformed host.
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Gagnon et al. GenBank Accession No. M81461, Apr. 26, 1993.*
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Inaoka Tetsuya
Kokubo Toshio
Ohkuma-Soyejima Toyomi
Fronda Christian L.
Japat Limited
Nashed Nashaat T.
Wildman David E.
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