Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-03-04
2001-09-25
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C536S123130, C514S053000, C514S777000, C425SDIG121
Reexamination Certificate
active
06294360
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a saccharide composition containing trehalulose, its preparation and uses, more particularly, it relates to a saccharide composition containing trehalulose obtained by allowing a maltose/trehalose converting enzyme to act on a sucrose solution to produce trehalulose, a process for producing a saccharide composition comprising a step of allowing a maltose/trehalose converting enzyme to act on a sucrose solution to produce trehalulose, and a composition containing the saccharide composition.
2. Description of the Prior Art
Trehalulose, a disaccharide consisting of glucose and fructose, is a reducing saccharide represented by the chemical formula of 1-O-&agr;-D-glucopyranosyl-D-fructose. In the natural world, honey contains only a minimal amount of trehalulose. Because trehalulose is a non-crystalline saccharide which readily dissolves in water, does not substantially have cariogenicity, and has an about 40% sweetening power of sucrose, it is greatly expected to be used in food products, especially, in foods enriched with sweeteners such as jams, “an” (bean jams), and sweet jellies of beans. As is disclosed in “
Seito
-
Gijutsu
-
Kenkyu
-
Kaishi
”, Vol.34, pp.37-44 (1985), it is known that trehalulose is produced from sucrose by a method using an &agr;-glucosidase from
Protaminobacter rubrum
with a saccharide-transferring activity. In such an enzymatic reaction, trehalulose is produced as a by-product of crystalline palatinose or 6-O-&agr;-D-glucopyranosyl-D-fructose as a main product. Although it is proposed a method for producing saccharide compositions containing trehalulose which produces trehalulose from sucrose using immobilized cells of
Pseudomonas mesoacidophila
MX-45 as disclosed in Japanese Patent Laid-Open No.169,190/92 or those of
Agrobacterium radiobacter
MX-232 as disclosed in Japanese Patent Laid-Open No.130,886/93, it has not yet been actually used on an industrial scale because of their insufficient enzymatic activity and thermal stability.
SUMMARY OF THE INVENTION
The present invention provides a process for producing trehalulose, which produces trehalulose from sucrose in a satisfactorily high yield and is readily feasible on an industrial scale, and provides a saccharide composition containing trehalulose obtained by the process, and uses thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors energetically studied on processes for producing trehalulose. As a result, they unexpectedly found that the maltose/trehalose converting enzyme, as disclosed in Japanese Patent Laid-Open No.170,977/95 applied by the present inventors, converts maltose into trehalose and readily produces trehalulose from sucrose in a satisfactorily high yield, established a process for producing a saccharide composition containing trehalulose, characterized in that it comprises a step of allowing a maltose/trehalose converting enzyme to act on a sucrose solution, and established a saccharide composition containing trehalulose obtained by the process, and compositions containing the saccharide composition in the form of a food, cosmetic or pharmaceutical. Thus, the present inventors accomplished this invention.
The maltose/trehalose converting enzymes usable in the present invention are intramolecular saccharide-transferring enzymes which are produced by microorganisms of the species Pimelobacter sp. R48 (FERM BP-4315) and
Pseudomonas putida
H262 (FERM BP-4579) as disclosed in Japanese Patent Laid-Open No.170,977/95, and those of the genus Thermus, and convert maltose into trehalose and vice versa. For example, these enzymes have the following physicochemical properties:
(1) Action
Converting maltose into trehalose and vice versa.
(2) Molecular weight
About 57,000-120,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(3) Isoelectric point (pI)
About 3.8-5.1 on isoelectrophoresis using ampholyte;
(4) Inhibition of activity
Being inhibited by one mM Cu
++
, Hg
++
and Tris-HCl buffer; and
(5) Origin
Originated from microorganisms.
The equilibrium point of the reaction system of the maltose/trehalose converting enzymes inclines to the side of trehalose formation, and the trehalose yield increases up to an about 80 w/w % (the wording “w/w %” will be abbreviated as “%” unless specified otherwise) when used maltose as a substrate.
In addition to the above microorganisms, other strains and mutants, which produce such maltose/trehalose converting enzymes and belong to the microorganisms of the genera Pimelobacter, Pseudomonas, and Thermus, can be selectively used. Microorganisms of the genus Thermus such as
Thermus aquaticus
ATCC 25104
, Thermus aquaticus
ATCC 27634
, Thermus aquaticus
ATCC 33923
, Thermus filiformis
ATCC 43280
, Thermus ruber
ATCC 35948, Thermus sp. ATCC 43814, and Thermus sp. ATCC 43815 can be arbitrarily used.
Any synthetic and natural nutrient culture media for culturing the above microorganisms can be used in the present invention as long as the microorganisms can grow therein and produce the maltose/trehalose converting enzymes. The carbon sources used in the present invention include those which are utilized by the microorganisms: For example, saccharides such as glucose, fructose, molasses, trehalose, lactose, sucrose, mannitol, sorbitol, and partial starch hydrolysates, and organic acids such as citric acid and succinic acid, as well as their salts, can be used. The concentration of the carbon sources in nutrient culture media is appropriately selected depending on the type of carbon sources. In the case of using glucose as a carbon source, a preferable concentration is 40 w/v % or lower, especially, a concentration of 10 w/v % or lower is suitably used in view of the growth of microorganisms. Inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract, and beef extract can be used in the present invention as nitrogen sources. Salts of calcium, magnesium, potassium, sodium, phosphate, manganese, zinc, iron, copper, molybdenum, and cobalt can be selectively used as inorganic ingredients.
The culture conditions for the microorganisms are those under which the microorganisms grow and produce the maltose/trehalose converting enzymes. Usually, the microorganisms are cultured under aerobic conditions at temperatures of about 4-80° C., preferably, 20-75° C., and pHs of 5-9, preferably, 6-8.5. Preferable cultivation time is set to those under which the microorganisms can grow, preferably, about 10-100 hours. The concentration of dissolved oxygen (DO) is not specifically restricted to, but usually it is preferably set to about 0.5-20 ppm. The DO is kept within the range by regulating the aeration and stirring ratios, feeding oxygen, and increasing the inner pressure of fermenters. Cultivation methods in a batch-wise and a continuous manner can be used in the present invention.
After completion of the culture of microorganisms, desired enzymes are collected from the culture. Since the activity of maltose/trehalose converting enzyme is found intra- and extra-cellularly, cell-free cultures and cells can be collected as a crude enzyme. Intact cultures can be also used as a crude enzyme. Examples of the methods for separating cultures into cells and cell-free cultures include conventional solid-liquid phase separation methods: For example, centrifugation methods to separate intact cultures, filtration methods comprising a step of adding filtration agents to the cultures or treating intact cultures with precoat filters, and membrane filtration methods using plain filters and hollow fibers. Cell-free cultures can be used as a crude enzyme, preferably, they are concentrated in conventional manner before use: For example, salting out using ammonium sulfate, sedimentation method using acetone and alcohols, and concentration methods using membrane filters such as plain filters and hollow fibers can be used for suc
Chaen Hiroto
Fukuda Shigeharu
Miyake Toshio
Nishimoto Tomoyuki
Browdy and Neimark
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Prats Francisco
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