Chitin deacetylase preparations

Details Chitin deacetylase preparations Chitin deacetylase preparations

1999-07-12

2001-10-02

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S084000, C435S183000, C536S023100, C536S023200

Reexamination Certificate

active

06297040

ABSTRACT:

BACKGROUND OF THE INVENTION
Next to cellulose, chitin is the world's most abundant, easily obtained, and renewable biological material. It is a natural product synthesized by a wide variety of organisms. Several billion tons of the material are produced annually. Chitin is a carbohydrate polymer, the N-acetylated polymer of &bgr;(1→4) linked N-acetylglucosamine, or poly-N-acetyl glucosamine. In plants, chitin is a cell wall constituent replacing cellulose or sometimes occurring together with cellulose. In animals, chitin is usually organized as a cuticle at one surface of the epithelial tissue. Although structurally similar to cellulose, chitin has distinctly different chemical properties. It is an extremely insoluble material, with limited industrial applicability.
The deacetylated derivative of chitin, chitosan, is a much more tractable material with a broad and impressive array of practical applications. Chitosan is positively charged, thus, it can be used as a protein precipitant and a metal chelating agent. It can be formulated as a solution, gel, membrane, film or fiber. Such formulations are useful, for example, in the areas of precious metal recovery, crop protection, chromatography and enzyme immobilization. Chitosan is a biologically benign, non-immunogenic, material making it ideal for use in the agricultural, food, drug and cosmetic industries. It can form complexes with other natural polymers, such as collagen and keratin, to form materials with unique biomedical properties. For example, such materials can be used as wound healing accelerants, components of artificial skin and blood vessels, anticoagulants, and controlled drug release vehicles.
At present the bulk of the chitosan produced worldwide is prepared from crustacean shell material. Chitin comprises about 20-50% of the dry weight of crustacean cuticles, the balance being primarily calcium carbonate, calcium phosphate and other proteins. Chitin is first isolated by treating ground crustacean shells with dilute acid and alkali to remove proteins and minerals. The raw chitin is then deacetylated by exposure to concentrated alkali at high temperature to generate chitosan. Although the chitosan produced in this manner has many useful features, it is impossible to effectively control the production process, which leads to the production of a material having a broad range of molecular weight and a heterogenous extent of deacetylation. Such a product is not of great value, since many of the potentially important applications, particularly in the biomedical area, require uniform material with very specific physical and chemical properties.
SUMMARY OF THE INVENTION
The subject invention relates to an isolated DNA sequence encoding an enzyme which catalyzes the conversion of chitin to chitosan. Specific embodiments include DNA sequences which are characterized by the ability to hybridize to the DNA sequence represented in SEQ ID NO: 1 under stringent hybridization conditions. The invention also relates to a DNA expression construct encoding an enzyme having the specificity described above, or biologically active portions thereof
The invention also relates to a method for converting chitin to chitosan by contacting chitin with an enzyme having a chitin deacetylase activity. In this method, the enzyme is produced by recombinant DNA techniques in which an isolated DNA sequence encoding the enzyme, or a biologically active portion thereof, is expressed from a DNA expression construct.


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