Transferase and amylase, process for producing the enzymes,...

Details Transferase and amylase, process for producing the enzymes,... Transferase and amylase, process for producing the enzymes,...

1999-04-26

2002-05-21

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091530, C435S097000, C435S183000, C435S194000, C435S200000

Reexamination Certificate

active

06391595

ABSTRACT:

TECHNICAL FIELD
The present invention relates to:
I. a novel transferase, a process for producing the same, a process for producing an oligosaccharide by using the enzyme, a gene coding for the enzyme, and use thereof; and
II. a novel amylase, a process for producing the same, a process for producing &agr;,&agr;-trehalose by using the enzyme, a gene coding for the enzyme, and use thereof. More specifically, as follows.
I. The present invention relates to a novel transferase which acts on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are &agr;-1,4-linked, so as to transfer the &agr;-1,4 linkages to &agr;1,&agr;-1 linkages; and a process for producing the transferase. More particularly, the present invention relates to the above-mentioned enzyme produced from archaebacteria belonging to the order Sulfolobales, for example, bacteria of the genus Sulfolobus or Acidianus.
Further, the present invention relates to a novel process for producing trehaloseoligosaccharides or the like by using the above-mentioned novel enzyme, and more particularly, relates to an efficient and high-yield process for producing trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehaloses by using a maltooligosaccharide or the like as a raw material.
Moreover, the present invention relates to a DNA fragment coding for the above-mentioned novel transferase and to the use of the DNA fragment in genetic engineering.
II. The present invention relates to a novel amylase which acts on a substrate saccharide, the saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end are glucose residues, so as to liberate principally monosaccharides and/or disaccharides by hydrolyzing the substrate from the reducing end; and a process for producing the amylase. More particularly, the present invention relates to a novel amylase which has an principal activity of acting on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end side are glucose residues and the linkage between the first and the second glucose residues from the reducing end side is &agr;-1,&agr;-1 while the linkage between the second and the third glucose residues from the reducing end side is &agr;-1,4, so as to liberate &agr;,&agr;-trehalose by hydrolyzing the &agr;-1,4 linkage between the second and the third glucose residues; and a process for producing the amylase. The novel amylase also has another activity of endotype-hydrolyzing one or more &agr;-1,4 linkages within the molecular chain of the substrate, and can be produced by bacteria belonging to the genus Sulfolobus. This enzyme is available for the starch sugar industry, textile industry, food industry, and the like.
Further, the present invention relates to a process for producing &agr;,&agr;-trehalose, characterized by using the above novel amylase in combination with the above novel transferase. In detail, the present invention relates to a process for producing &agr;,&agr;-trehalose in a high yield by using, as a raw material, any one of starch, starch hydrolysate and maltooligosaccharides, or a mixture of maltooligosaccharides, and as enzymes, the novel transferase and amylase of the present invention.
Moreover, the present invention relates to a DNA fragment coding for the above novel amylase, and use of the DNA fragment in genetic engineering.
BACKGROUND ART
I. Background Art of Transferase
Hitherto, in relation to glycosyltransferase acting on starch and starch hydrolysates such as maltooligosaccharides, various glucosyltransferases, cyclodextringlucanotransferases (CGTase), and others have been found [c.f. “Seibutsu-kagaku Jikken-hou” 25 (“Experimental Methods in Biochemistry”, Vol. 25), ‘Denpun.Kanren Toushitsu Kouso Jikken-hou’ (‘Experimental Methods in Enzymes for Starch and Relating Saccharides’), published by Gakkai-shuppan-sentah, Bioindustry, Vol. 9, No. 1 (1992), p. 39-44, and others]. These enzymes transfer a glucosyl group to the &agr;-1,2, &agr;-1,3, &agr;-1,4, or &agr;-1,6 linkage. However, an enzyme which transfers a glucosyl group to the &agr;-1,&agr;-1 linkage has not been found yet. Though trehalase has been found as an enzyme which acts on the &agr;-1,&agr;-1 linkage, trehalose is absolutely the only substrate for the enzyme, and the equilibrium or the reaction rate lies to the degrading reaction.
Recently, oligosaccharides were found to have physicochemical properties such as moisture-retaining ability, shape-retaining ability, viscous ability and browning-preventive ability, and bioactivities such as a low-calorigenetic property, an anticariogenic property and a bifidus-proliferation activity. In relation to that, various oligosaccharides such as maltooligosaccharides, branched-chain oligosaccharides, fructooligosaccharide, galacto-oligosaccharide, and xylooligosaccharide have been developed [c.f. “Kammiryo” (“Sweetener”) (1989), Medikaru-risahchi-sha (Medical Research Co.) (1989), Gekkan Fuhdokemikaru (
Monthly Foodchemical
) (1993), February p. 21-29, and others].
Among oligosaccharides, the oligosaccharides which have no reducing end may include fructooligosaccharide having a structure composed of sucrose which is not reductive, and being produced by fructosyltransferase. Meanwhile, among starch hydrolysates such as maltooligosaccharides, the oligosaccharides which have no reducing end may include cyclodextrins produced by the above-mentioned CGTase, &agr;,&bgr;-trehalose (neotrehalose), and reduced oligosaccharides chemically synthesized by hydrogenating the reducing end (oligosaccharide alcohol). These oligosaccharides having no reducing end have various physicochemical properties and bioactivities which are not possessed by conventional starch syrups and maltooligosac-charides. Accordingly, among maltooligosaccharides, the oligosaccharides the reducing ends of which are modified with an &agr;-1,&agr;-1 linkage may be also expected to have the similar physicochemical properties and bioactivities to those possessed by the above-mentioned oligosaccharide having no reducing end, since such oligosaccharides also have no reducing end.
Here, the oligosaccharides the reducing ends of which are modified with an &agr;-1,&agr;-1 linkage as described above may be recognized as a trehaloseoligosaccharide in which &agr;,&agr;-trehalose is linked with glucose or a maltooligoshaccharide. Accordingly, such a trehaloseoligosaccharide may be expected to have the physicochemical properties and bioactivities which are possessed by the oligosaccharide having no reducing end, and in addition, may be expected to have the specific activities as exhibited by &agr;,&agr;-trehalose (c.f. Japanese Patent Laid-open Publication No. 63-500562).
Though it was reported that a trace amount of trehaloseoligosaccharides could be detected in yeast [
Biosci. Biotech. Biochem.,
57(7), p. 1220-1221 (1993)], this is the only report referring to its existence in nature. On the other hand, as to its synthesis by using an enzyme, though there has been a report of such synthesis [Abstracts of “1994 Nihon Nougei-kagaku Taikai” (“Annual Meeting of the Japan Society for Bioscience, Biotechnology and Agrochemistry in 1994”), p. 247], the method described in the report uses trehalose, which is expensive, as the raw material. Therefore, production at low cost has not yet been established.
Recently, Lama, et al. found that a cell extract from the
Sulfolobus solfataricus
strain MT-4 (DSM 5833), a species of archaebacteria, has a thermostable starch-hydrolyzing activity [
Biotech. Forum. Eur.
8, 4, 2-1 (1991)]. They further reported that the activity is also of producing trehalose and glucose from starch. The above-mentioned report, however, does not at all refer to the existence of trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehalose. Moreover, no investigation in ar

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