Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-09-14
2002-03-05
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06352850
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel chitinase and a method for preparing the same.
2. Description of the Related Art
Chitin is an insoluble polysaccharide consisting of &bgr;-(1,4)-linked N-acetylglucosamine and is present widely in the bacterial kingdom, plant kingdom and animal kingdom. Chitin is at a yearly production comparative to that of cellulose and is considered as one of the most abundant biomass resources.
Compounds in various chain lengths as recovered by hydrolysis of chitin have increasingly been used, together with chitosan as a deacetylated product of chitin, in a wide variety of fields including medicine, food, cosmetics, agriculture and garments. Particularly, chitin-derived low-molecular N-acetylglucosamine oligomer is known to have useful physiological activities such as immunopotentiation, anti-bacterial activity and anti-vial activity.
Conventionally, the N-acetylglucosamine oligomer has been commonly produced by thermally treating the shell of crab and shrimp in sodium hydroxide solution thereby removing protein, and removing ash with hydrochloric acid to recover chitin, and then preparing the resulting chitin into low-molecular substances by the limited hydrochloride hydrolysis method. Additionally, a method using hydrogen fluoride has been proposed recently.
However, any of these methods is disadvantageous in that the disposal of the equipment and liquid waste requires high cost. Furthermore, the essential bonds are readily cut so that the resulting production efficiency is unavoidably low due to the resulting byproducts.
Because chitinase as chitin decomposition enzyme acts only on certain predetermined sites in chitin under mild reaction conditions owing to the substrate specificity so that less byproducts are generated, cost reduction is possible. Therefore, industrially applicable methods for producing chitinase have been demanded. Currently, chitinase species derived from microorganisms of genera
Bacillus, Serratia
, and streptomyces are-commercially available for the purpose of using the chitinase species for academic research works but are so expensive. Additionally, these chitinase species are at optimum pH in acidity (pH 2 to 6). Thus, these chitinase species are not suitable for use at a range of pHs above neutrality. Additionally, the temperature range in which these chitinase species exert their action is narrow (the active temperature range of 30° C. to 45° C.).
The optimum pH in an acidic region requires corrosion-resistant equipment, disadvantageously, leading to the burden of cost. The narrow active temperature range requires laborious works for temperature control.
It is also reported that microorganisms of the genus Vibrio generate chitinase, but the microorganisms are all mesophilic microorganisms derived from the genus Vibrio [Akio Ohtakara, Masaru Mitsutomi and Yasushi Uchida, J. Ferment. Technol., 57, 169-177 (1979)]. It has never been known that psychrotrophic microorganisms of the genus Vibrio generate chitinase. Mesophilic microorganisms of the genus Vibrio at the optimum temperature at 30° C. to 40° C. require strict temperature control; and additionally, the thermal stability thereof reaches the threshold at 30° C. to 35° C. Thus, the mesophilic microorganisms of the genus Vibrio are likely to be inactivated, disadvantageously.
SUMMARY OF THE INVENTION
It is a purpose of the invention to provide a novel chitinase with the optimum pH principally around neutrality and in alkalinity, a wide active temperature range and useful functions and a method for producing the same.
Based on the fact that a vast amount of chitin is generated in ocean and is then decomposed with microorganisms, which serves for the ecological system in ocean, the present inventors have made investigations about chitinase production with marine bacteria. Consequently, the inventors have found that a marine psychrotrophic bacterial strain of the genus Vibrio generates a novel chitinase species with functions advantageous for industrial use, at high efficiency. Based on the finding, the invention has been achieved.
More specifically, the invention relates to chitinase with the following physico-chemical properties.
1. Action: Random cleavage of chitin &bgr;-1,4 bond to generate the tetramer and dimer of N-acetylglucosamine.
2. Optimum pH: 6.5 to 10.4.
3. Stable pH: 7.0 to 9.0.
4. Optimum temperature: 37° C.
5. Active temperature range: 4 to 60° C.
6. Thermal stability: 60% or more of the initial activity is retained even after heating at 40° C. and pH 8.0 for 30 minutes.
The invention; furthermore relates to a method for producing the chitinase, comprising culturing a chitinase-generating bacterium reacting with chitin to generate N-acetylglucosamine oligomer and collecting the generated chitinase from the culture, wherein the chitinase-generating bacterium is a marine psychrotrophic bacterium of the genus Vibrio.
The chitinase as a novel enzyme in accordance with the invention can be used for modifying chitin into low molecular substances by allowing the chitinase to react with chitin. Particularly, the inventive chitinase can preferably be used for industrial production of N-acetylglucosamine oligomer.
REFERENCES:
patent: 5587292 (1996-12-01), Laine et al.
patent: 5744325 (1998-04-01), Fujishima et al.
patent: 6121420 (2000-09-01), Laine
Computer Derwent Abstract 1994-172744 Mercian Corp JP 06113846 Pub Apr. 1994.*
Computer Caplus Abstract 2000:559302 Suginta et al “Chitinases from Vibrio. . . ” J.Appl.Microbiol (2000) 89(1) 76-84.*
Computer Caplus Abstract 2000:363759 Babenko et al. “The isolation of endochitinase from Vibrio SP X and Some characteristics of the Enzyme” Biotek. (2000) (1) 39-45.*
Computer Caplus Abstract 1996:8198 Osawa et al “An investigation of Aquatic Bacteria Capable of Utilizing Chitin as the Sole Source of Nutrients” Lett Appl. Microbiol. (1995) 21 (5) 288-91.
Fujishima Shizu
Higashihara Takanori
Maruyama Akihiko
Yamano Naoko
Agency of Industrial Science & Technology
Lilling Herbert J.
Rader & Fishman & Grauer, PLLC
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