Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-08-11
2001-05-22
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S072000, C435S074000
Reexamination Certificate
active
06235505
ABSTRACT:
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The subject of this invention is a process for the production of cyclodextrin from amylopectin potato starch by reaction with cyclodextrin glycosyltransferase (CGTase, also known as cyclodextrin transglycosylase, EC No. 2.4.1.19).
The name cyclodextrins refers to a group of substances whose molecules consist of several anhydroglucose units with cyclically linked alpha-1,4-glucosidic bonds, whereby the smallest cyclodextrin, alpha-cyclodextrin, contains 6 glucose units. A distinction is also made between beta- and gamma-cyclodextrin according to the number of anhydro-glucose units in the ring which is formed. Beta-cyclodextrin is the thermodynamically favored product in the conversion of starch to cyclodextrin by means of CGTase. The higher-ring cyclodextrins are preferred for technical applications.
Up to now the following starches have been used as the main starting materials in technical processes for producing cyclodextrins:
Potato starch: the potato can be grown with high yields per hectare even in unfavorable locations. It has low protein and lipid contents and therefore affords a very pure starch.
Maize and waxy maize starch: maize requires a warmer climate. Waxy maize ripens in high proportions. It must be grown in favorable locations with adequate separation from normal maize. Low yields per hectare lead to a further cost increase.
A considerable disadvantage for starch from maize and waxy maize is the high protein and lipid content (necessitating complicated and costly purification of the starch).
Wheat starch: is a poorer substrate than potato or maize starch since the yield of cyclodextrin that can be produced from it is much lower.
The usual natural starches are a mixture of the two forms of starch, amylopectin and amylose. Amylose and amylopectin are not single substances but mixtures of polymers with different molecular weights and different glucose linkages. Amylose consists mainly of straight-chain polysaccharides in which the glucose units are joined by alpha-1,4-linkages. Amylopectin, on the other hand, is a highly branched glucose polymer in which the glucose units are joined through 1,6-linkages at the branching positions in addition to the alpha-1,4-linkages. It has been found that amylopectin forms more stable solutions than amylose because amylose has a tendency towards unwanted retrogradation, i.e. the recombination of chains which have already been separated from one another.
The usual natural starches contain 15% to 30% amylose irrespective of the type of plant they have been obtained from. Only maize varieties of the so-called waxy type provide a starch which consists almost exclusively of amylopectin. In rare cases a starch rich in amylopectin can also be obtained from so-called waxy rice or waxy barley.
Amylose and amylopectin can be separated from each other by fractionation. These processes are very complicated and cost-intensive and are hardly ever used except on a laboratory scale. Furthermore, fractionation of natural starches leads to uncontrolled degradation and damage to the starch fractions with impairment of the properties of the final products.
For this reason amylopectin starch is hardly ever used for technical purposes. The only use in practice involves a certain amount of waxy maize starch in the food industry because this generates a more pleasant feeling in the mouth than does usual starch.
The production of cyclodextrin from starch is the subject of many publications in the literature. Thus U.S. Pat. No. 3,425,910 describes a process for the production of cyclodextrin from a starch hydrolysate. The use of potato starch as starch starting material is mentioned. At the time of filing of the above US patent (1966) potato starch is a usual starch with an amylose content of approximately 20% by weight.
In PCT application WO 93/10255 the production of cyclodextrin from a starch containing at least 90% amylopectin is described whereby it is stated that the cyclodextrin obtained gives a clear solution when dissolved in water. Preferred starch starting materials contain 95% amylopectin or more, preferably around 99%. Waxy maize starch, waxy rice starch and waxy barley starch are expressly stated to be the starting materials with waxy maize starch being preferred. Potato starch and maize starch with normal amylose content are named in Example 1 as comparison starches for demonstrating the positive effects of using waxy maize starch.
Among the process conditions listed in the above PCT application is the use of a complexing agent for cyclodextrin for the purpose of better separation of same from the reaction medium. Toluene, 1-decanol, cyclodecanol, cyclohexane, trichloroethylene, tetrachloroethane, bromobenzene, 2,3-cyclododenopyridine, naphthalene, 1-naphthol, 2-naphthol and dimethylphenol are named as complexing agents.
An overview of the industrial production of cyclodextrins is provided in J. Szejtli and T. Ose, Comprehensive Supramolecular Chemistry, Vol. 3, Cyclodextrins, 1996, Pergamon, Oxford, UK, in Article 3 on Page 41, Preparation and Industrial Production of Cyclodextrins, G. Schmid, Wacker-Chemie GmbH, Munich, Germany.
A description is given of the different conversion conditions and their influence on the ratio of alpha-, beta- and gamma-cyclodextrin. In this connection the addition of a special complexing agent during the production process can also modify the relative proportions of the three kinds of cyclodextrin.
It is also mentioned that the alpha-1,6-glucoside linkages at the branching positions in amylopectin block the effect of CGTase. If amylopectin is treated with debranching enzymes such as pullulanase or isoamylase before the addition of CGTase the level of conversion of the starch into cyclodextrin is increased by several percent.
It is also subsequently mentioned in the above Szejtli and Ose publication that amylopectin is a better substrate than amylose for the production of cyclodextrin because the reaction with CGTase begins at the non-reducing end of the starch molecule. Since amylopectin has considerably more non-reducing ends than amylose, the level of conversion is higher when amylopectin is used. It is therefore recommended that potato starch be used instead of maize starch because potato starch has an intrinsically higher amylopectin content than maize starch (approximately 79% for potato compared with approximately 72% for maize).
Finally, the attempt is also described to produce cyclodextrin directly in the tubers of transgenic potato plants through construction of a chimeric gene by means of the CGTase gene from Klebsiella oxytoca. It did in fact prove possible to detect small amounts of cyclodextrin in the potato tubers. Extraction of the tuber tissue was performed using a C18 Sep-pak column which binds the cyclodextrin but not the starch.
U.S. Pat. No. 4 477 568 mentions among other things the use of fractionated amylopectin starch from a wide variety of crops, e.g. maize, wheat, sorghum, potato, tapioca, sago and rice, for the production of cyclodextrin.
However, since the starch fractionation processes have not been generally accepted for the above reasons, the search is still going on for a cyclodextrin starting material which does not have the disadvantages listed.
SUMMARY OF THE INVENTION
It is accordingly an object of this invention to provide a process for the production of cyclodextrin that overcomes the above-mentioned disadvantages of the prior art methods and enables cyclodextrin to be produced economically for technical applications.
With the foregoing and other objects in view there is provided, in accordance with the invention, a process in which amylopectin potato starch obtained from potatoes with amylose formation inhibited as a result of breeding or of molecular biological/genetic engineering procedures is used as the starting material in a process for the production of cyclodextrin from amylopectin potato starch by reaction with cyclodextrin glycosyltransferase.
Recent years have seen the successful development of genetic modifica
Grüll Dietmar
Stifter Ulrich
Achutamurthy Ponnathapu
Greenberg Laurence A.
Lerner Herbert L.
Monshipouri Maryam
Stemer Werner H.
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