Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-04-06
2002-02-12
Wortman, Donna C. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069200, C435S071100, C435S071200, C435S183000, C435S200000, C435S252330, C435S097000, C435S320100, C536S023100, C536S023200, C536S023700
Reexamination Certificate
active
06346394
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a recombinant thermostable enzyme which releases trehalose from non-reducing saccharides having a trehalose structure as an end unit and a degree of glucose polymerization of at least 3.
2. Description of the Prior Art
Trehalose is a disaccharide which consists of 2 glucose molecules that are linked together with their reducing groups, and, naturally, it is present in fungi, algae, insects, etc., in an extremely small quantity. Having no reducing residue within the molecule, trehalose does not cause an unsatisfactory browning reaction even when heated in the presence of amino acids or the like, and because of this it can advantageously sweeten food products without fear of causing unsatisfactory coloration and deterioration. Trehalose, however, could not have been readily prepared in a desired amount by conventional production methods, so that it has not scarcely been used for sweetening food products.
Conventional production methods are roughly classified into 2 groups, i.e. the one using cells of microorganisms and the other using a multi-enzymatic system where several enzymes are allowed to act on saccharides. The former, as disclosed in Japanese Patent Laid-Open No.154,485/75, is a method which comprises growing microorganisms such as bacteria and yeasts in nutrient culture media, and collecting trehalose mainly from the proliferated cells. The latter, as disclosed in Japanese Patent Laid-Open No.216,695/83, is a method which comprises providing maltose as a substrate, allowing a multi-enzymatic system using maltose- and trehalose-phosphorylases to act on maltose, and recovering the formed trehalose from the reaction system. The former facilitates the growth of microorganisms, but has a demerit that the content in the microorganisms is at most 15 w/w %, on a dry solid basis (d.s.b.). Although the latter can readily separate trehalose, it is theoretically difficult to increase the trehalose yield by allowing such phosphorylases to act on substrates at a considerably-high concentration because the enzymatic reaction in itself is an equilibrium reaction of 2 different types of enzymes and the equilibrium point constantly inclines to the side of forming glucose phosphate.
In view of the foregoing, the present inventors energetically screened enzymes which form saccharides having a trehalose structure from amylaceous saccharides, and have found that microorganisms such as those of the genera Rhizobium and Arthrobacter produce an absolutely novel enzyme which forms non-reducing saccharides having a trehalose structure as an end unit from reducing amylaceous saccharides having a degree of glucose polymerization of at least 3. They disclosed such an enzyme in Japanese Patent Application No.349,216/93. At almost the same time, they also found that these non-reducing saccharides are nearly quantitatively hydrolyzed into trehalose and glucose and/or maltooligosaccharides by other enzymes produced from the same microorganisms of the genera Rhizobium and Arthrobacter.
It was found that the enzymes produced from the aforesaid microorganisms have an optimum temperature of about 40° C., and have some difficulties in their thermostability when actually used to produce trehalose. It is recognized in this field that a recommendable temperature in the saccharification reaction of starch or amylaceous saccharides is one which exceeds 55° C. because bacterial contamination will occur at a temperature of 55° C. or lower and decreasing the pH of the reaction mixtures and inactivating the enzymes used. Thus, a relatively-large amount of substrates remain intact. While the use of enzymes with a poor thermostability, a great care should be taken to control the pH, and, when the pH level lowers to an extremely low level, alkalis should be added to reaction mixtures to increase the pH level as quickly as possible.
In view of the foregoing, the present inventors screened thermostable enzyme with a satisfactory activity and have found that enzymes produced from microorganisms of the genus Sulfolobus including
Sulfolobus acidocaldarius
(ATCC 33909) are not substantially inactivated even when incubated at a temperature exceeding 55° C., and they efficiently release trehalose from non-reducing saccharides having a trehalose structure as an end unit and a degree of glucose polymerization of at least 3. These microorganisms, however, are not sufficient in the enzyme productivity, and this requires a relatively-large scale culture to industrially produce trehalose from those non-reducing saccharides.
Recently, the recombinant DNA technology has made a remarkable progress. At present, even an enzyme whose total amino acid sequence has not been revealed can be readily prepared in a desired amount, if once a gene encoding the enzyme is isolated and the base sequence is decoded, by preparing a recombinant DNA containing a DNA that encodes the enzyme, introducing the recombinant DNA into microorganisms or cells of plants or animals, and culturing the resultant transformants. Under these circumstances, urgently required are to find a gene that encodes the thermostable enzyme and to decode the base sequence.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a recombinant thermostable enzyme which releases trehalose from non-reducing saccharides having a trehalose structure as an end unit and a degree of glucose polymerization of at least 3 by using the recombinant DNA technology.
It is a further object of the present invention to provide a DNA which encodes the recombinant thermostable enzyme.
It is yet another object of the present invention to provide a replicable recombinant DNA which contains the DNA.
It is another object of the present invention to provide a transformant into which the recombinant DNA is introduced.
It is yet another object of the present invention to provide a process for preparing the recombinant thermostable enzyme using the transformant.
It is another object of the present invention to provide an enzymatic conversion method for releasing trehalose from non-reducing saccharides having a trehalose structure as an end unit and a degree of glucose polymerization of at least 3.
The first object of the present invention is attained by a recombinant thermostable enzyme having the following physicochemical properties:
(1) Action
Releasing trehalose from non-reducing saccharides having a trehalose structure as an end unit and a degree of glucose polymerization of at least 3;
(2) Molecular weight
About 54,000-64,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(3) Isoelectric point (pI)
About 5.6-6.6 on isoelectrophoresis; and
(4) Thermostability
Substantially not inactivated even when incubated in an aqueous solution (pH 7.0) at 85° C. for 60 min.
The second object of the present invention is attained by a DNA which encodes the recombinant thermostable enzyme.
The third object of the present invention is attained by a replicable recombinant DNA which contains a self-replicable vector and the recombinant thermostable enzyme.
The fourth object of the present invention is attained by a transformant which is prepared by introducing the replicable recombinant DNA into an appropriate host.
The fifth object of the present invention is attained by a process for preparing the recombinant thermostable enzyme which comprises culturing the transformant in a nutrient culture medium, and collecting the formed recombinant thermostable enzyme from the culture.
The sixth object of the present invention is attained by an enzymatic conversion method of non-reducing saccharides having a trehalose structure as an end unit and a degree of glucose polymerization of at least 3, which contains a step of allowing the recombinant thermostable enzyme to act on the non-reducing saccharides to release trehalose.
REFERENCES:
patent: 4521252 (1985-06-01), Miyake et al.
patent: 5591612 (1997-01-01), Maruta et al.
patent: 0 606 753 (1983-12-01), None
patent: 2106912 (1982-08-01),
Kubota Michio
Mitsuzumi Hitoshi
Sugimoto Toshiyuki
Browdy and Neimark
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Wortman Donna C.
Zeman Mary K.
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