Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1996-03-01
2001-02-20
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S195000, C435S201000
Reexamination Certificate
active
06190898
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is related to the crystallization of cellulase enzymes. More particularly, the present invention relates to selective crystallization using salt of cellulase enzymes in an aqueous solution produced by, for example, the fermentation of microbial organisms such as filamentous fungi, yeast or bacteria.
Intensive research efforts have been directed to the precipitation and crystallization of enzymes as a means of purification and preparation of enzyme products. For example, in U.S. Pat. No. 4,659,667, a process is disclosed for the recovery of an enzyme from solution by concentrating to supersaturation the enzyme-containing solution at pH near the isoelectric point of the enzyme, inducing crystallization and recovering the crystallized final product. Inducing crystallization is achieved by allowing the enzymes to spontaneously crystallize upon concentration or by seeding, sound, stirring or scratching the inner surface of the container. Crystallization of alpha-amylase is exemplified.
In PCT Publication No. WO 89/08703, a process is described for the crystallization of subtilisin by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution of at least about 40 grams per liter at temperatures less than 10° C.
In PCT Publication No. WO 91/09943, a method for the crystallization of enzymes is disclosed which is characterized by using as a starting material an aqueous solution containing liquid with a relatively high enzyme purity and a concentration of enzyme of about at least 5 grams per liter and adding as a crystallization agent an easily soluble salt of the non-halide type to a concentration which is considerably smaller than the amount necessary to precipitate the enzymes in an amorphous form. Crystallization of certain subtilisin enzymes is exemplified.
In EP 549,048, a method for the preparation of highly purified alkaline protease from
Bacillus licheniformis
or
Bacillus alcalophilus
is disclosed which is characterized by adding hydrolytic enzymes and sodium chloride to concentrated alkaline protease, incubating the mixture at a temperature above 20° C. to separate the alkaline protease from hydrolyzed polymeric impurities and collecting the purified precipitate. However, the precipitate was predominantly amorphous.
Methods for preparing cellulase crystals for the purpose of x-ray crystallography have been successful on a small scale. For example, Bergfors et al., J. Mol. Biol., vol. 209, no. 1, pp. 167-169 (1989) illustrate crystallization using the hanging drop method of the core protein of cellobiohydrolase II from
Trichoderma reesei
and subsequent study of the crystals to determine tertiary structure. In Wilson, Crit. Rev. Biochem., vol. 12(1/2), pp. 45-63 (1992) the 30 K catalytic subunit of the E2 cellulase from
T. fusca
was crystallized using ammonium sulfate as a precipitant to determine the tertiary structure. Importantly, Wilson points out that the numerous attempts to crystallize the
T. fusca
cellulases E2, E3 and E5 in the prior art using the hanging drop method with both ammonium sulfate and polyethylene glycol had failed. Thus, although crystallization of certain enzymes has been successful according to the means described above, the crystallization of cellulases has remained problematic with no known method for such crystallization on a large scale basis.
In spite of these advances in the field of enzyme crystallization in general, and occurrences of crystallization of cellulases, reported in the scientific literature, for the purpose of crystallographic studies, inexpensive and efficient crystallization of cellulase enzymes suitable for large scale production has remained problematic in industry. In fact, no commercially feasible process has been described for the crystallization of cellulases in terms of producing a low cost, high yield, rapid and highly purified cellulase enzyme in a simple manner.
To the contrary, Applicants have discovered that the employment of a specific group of salts and a specific temperature range provides the capability of selectively purifying through crystallization a commercially important subgroup of cellulases (i.e., those which lack a distinct cellulose binding domain) over other cellulases which possess a cellulose binding domain.
Further, any protein can be precipitated with enough salt, however, the industry prefers crystalline enzymes for further processing into, i.e., granules or immobilized enzymes. The present invention provides methods of obtaining a cellulase enzyme product.
BACKGROUND OF THE INVENTION
It is an object of the present invention to provide for a simple and low-cost method which selectively crystallizes cellulases which lack a cellulose binding domain using specific salts.
It is an object of the invention to produce a cellulase enzyme.
According to the present invention, a method for the crystallization of cellulase enzyme which lacks a cellulose binding domain is provided comprising (a) preparing an aqueous solution containing the cellulase enzyme; and (b) adding to the aqueous solution a salt comprising an anion selected from the group consisting of sulfate, phosphate, formate, acetate, sorbate, chloride, bromide, fluoride or iodide, and a cation selected from the group consisting of sodium, ammonium, magnesium, potassium or calcium, or a mixture thereof. In a preferred embodiment, the aqueous solution is at a temperature above 4° C. In a preferred embodiment, the anion is selected from the group consisting of sulfate, acetate and chloride, and the cation is selected from the group consisting of sodium, ammonium or magnesium.
Applicants have surprisingly discovered that the crystallization kinetics for cellulase enzymes are improved when the crystallization takes place at a temperature above 4° C. As such, crystallization can take place at room temperature and significant savings are available in terms of equipment and energy.
Through the practice of the present invention, it is possible to obtain in an unexpectedly short period of time a highly purified cellulase enzyme product which has exceptional yield characteristics. In fact, by optimizing conditions according to the present invention, it is often possible to obtain consistent yields of greater than 50%, and in a particularly preferred embodiment, yields of greater than 70-80% in a period of five hours. This result is of great value to the industry.
In practicing the present invention, Applicants have surprisingly discovered that cellulases which lack a cellulose binding domain are characterized by more favorable crystallization kinetics than cellulases which possess a cellulose binding domain. As a result, an unexpected advantage is achieved by the practice of the present invention whereby cellulases which lack a cellulose binding domain can be easily crystallized in comparison with cellulases which possess a cellulose binding domain allowing for selective crystallization out of solution of only the cellulases which possess a cellulose binding domain. In the practice of the present invention, for the first time an easily reproducible method for preparing a highly purified and crystalline product from an industrially important subgroup of cellulase enzymes, i.e., those without cellulose binding domains, has become available. Such a result is a surprising and advantageous advance in the art of crystallization and solves a long standing problem in the field.
Another advantage of the present invention is the surprising discovery that the crystallized cellulase enzymes produced according to the present invention results in reduced backstaining in textile applications, such as stonewashing, over other cellulases produced by other methods. Examples of processes utilizing cellulase which will benefit from the present method of purification includes methods for the treatment of textiles described in PCT Publication No. WO 92/06221.
Yet another advantage of the present invention is that the crystallization process occurs very quickly. In contrast to m
Becker Nathaniel T.
Braunstein Edit L.
Fewkes Robert
Gros Ernst H.
Heng Meng H.
Genencor International Inc.
Genencor International Inc.
Lankford , Jr. Leon B.
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