Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-12-28
2001-08-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S100000, C435S183000, C435S200000
Reexamination Certificate
active
06274355
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
[Not Applicable.]
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[Not Applicable.]
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to an immobilised maltogenic &agr;-amylase and the method of manufacturing same. It relates further to a method of manufacturing a syrup which is rich in maltose. It relates also to a method of manufacturing a syrup which is rich in maltitol from a maltose-rich syrup obtained by the method according to the present invention. It relates equally to a method of manufacturing maltitol crystallised from a maltose-rich syrup obtained by the method according to the present invention.
2. Description of the Prior Art
Methods allowing the production of maltose-rich syrups are already known. Amongst these methods it is possible to quote in particular the one described by HODGE and Coll. in “Cereal Chemistry” No. 25, pages 19-30, January 1948, and which contains a stage of precipitation of the dextrin limits by alcohol solutions, and the one described by WOLFROM and THOMPSON in “Methods in carbohydrate chemistry”, 1962, pages 334-335.
Other methods of manufacturing maltose-rich syrups have also been proposed comprising a stage of adsorption on charcoal of the dextrins (U.S. Pat. No. 4,194,623), a stage of chromatography on zeolites or cationic or anionic resins (FR-A-2.510.581), a stage of ultrafiltration of maltose syrups (U.S. Pat. No.4,429,122), the combined use of several different enzymes, that is to say an &agr;-amylase, a &bgr;-amylase and an isoamylase or a pullulanase (FR-A-2.012.831).
This last technique presents, in relation to the preceding ones, numerous advantages. It suffers nevertheless from certain disadvantages, including in particular the one residing in the fact that the saccharifications have to be carried out with very low contents of dry matter, of the order of 20 g/l, in order to obtain a maximum hydrolysis efficiency of the enzymes.
The document FR-A-2.000.580 describes a method of preparing a syrup with a high content of maltitol by hydrogenation of a syrup with a high content of maltose which is obtained by liquefaction of a starch milk with a low content of dry matter to a dextrose equivalent lower than 2, the product thus obtained being saccharified under the action of specific enzymes.
This process is expensive, gives a mediocre yield and gives rise to problems of bacterial contamination and to occurrences of retrogradation of the amylose. In addition, the syrup obtained contains proportions of polymers with a degree of polymerisation (DP in the rest of the specification) greater than or equal to 4, which are a nuisance.
More recently, the document U.S. Pat. No. 5,141,859 proposed a method of manufacturing a syrup with a high maltose content, using two successive stages of saccharification. This document advocates in fact a method comprising a first stage of saccharification in the presence of a &bgr;-amylase and a subsequent stage of saccharification in the presence of a maltogenic &agr;-amylase. According to this document, the maltogenic &agr;-amylase is used, after the first stage of saccharification with the &bgr;-amylase, to hydrolyse the oligosaccharides (from DP3 to DP7) and essentially the maltotriose (trisaccharide) into maltose and glucose.
In a surprising and unexpected manner, the Applicant has noted that syrups with a maltose content as high as those described in the document U.S. Pat. No. 5,141,859 could be obtained by saccharifying a starch milk liquefied by means of an immobilised maltogenic &agr;-amylase.
To the knowledge of the Applicant only document FR-A-2.356.665 has proposed immobilising an &agr;-amylase termed maltogenic, but on a very specific substrate constituted by casein granules essentially covered with a proteinic layer permeable to the liquids in which the enzyme is cross-linked in common with egg albumin by glutaric aldehyde. Furthermore, according to this document, the use (of which no example was given) of maltogenic &agr;-amylase immobilised on a porous substrate for processing a starch hydrolysate proves theoretically impossible for reasons of steric inhibition, the diffusion of the oligosaccharides of DP6 to DP10 contained in such a hydrolysate towards the enzyme enclosed within the porous structure being inhibited in view of the size of the molecules.
Against all expectation and contrary to the teaching of the document FR-A-2.356.665, the Applicant has highlighted on the one hand that it was possible to immobilise a maltogenic &agr;-amylase on a porous substrate, and, on the other hand, that such a maltogenic &agr;-amylase was capable of hydrolysing oligosaccharides of DP3 to DP7.
DETAILED DESCRIPTION OF THE INVENTION
The invention proposes, therefore, an immobilised maltogenic &agr;-amylase, characterised by the fact that it is adsorbed on particles of at least one porous substrate. Advantageously, such a porous substrate is selected from the group made up of phenolic resins, acrylic resins and polystyrene resins.
The invention also proposes a method of immobilising a maltogenic &agr;-amylase according to the invention, characterised by the fact that it comprises the stages consisting of:
(a) putting a maltogenic &agr;-amylase into solution;
(b) putting particles of at least one porous substrate into suspension;
(c) bringing said solution into contact with said suspension at ambient temperature with a view to obtaining an immobilised maltogenic &agr;-amylase.
The invention also proposes a method of manufacturing a maltose-rich syrup, comprising the successive stages consisting of:
(a) carrying out liquefaction of a starch milk;
(b) carrying out saccharification of the liquefied starch milk in the presence of a &bgr;-amylase and at least one debranching enzyme chosen from the group made up of pullulanases and isoamylases;
(c) carrying out molecular sieving of the liquefied and saccharified starch milk so as to collect a fraction enriched with maltose and a fraction depleted of maltose;
(d) bringing said fraction enriched with maltose into contact with an immobilised maltogenic &agr;-amylase according to the invention with a view to obtaining a syrup which is rich in maltose.
Thus the invention relates firstly to an immobilised maltogenic &agr;-amylase. In the sense of the present invention, what is meant by “&agr;-amylase” is an &agr;-amylase capable of hydrolysing oligosaccharides (from DP3 to DP7) and essentially maltotriose (trisaccharide), into maltose and glucose. According to the present invention, maltogenic &agr;-amylase is advantageously one of those marketed by the company NOVO, under the names Maltogénase® 4000 L and NOVAMYL®.
The Applicant recommends, however, proceeding to a purification of the commercial maltogenic &agr;-amylase, before carrying out the immobilisation of same.
It has indeed been noted that the commercial enzymatic preparations could be contaminated by parasitic enzymatic activities, such as protease or glucosidase activities.
This supplementary purification makes it possible, moreover, to increase the specific activity of the maltogenic &agr;-amylase thus prepared, which increases accordingly the potential for converting the maltotriose by the enzyme in its immobilised form. The purification of the commercial maltogenic &agr;-amylase can be carried out by any means known by the person skilled in the art.
Advantageously, this purification can be carried out by the succession of stages consisting in dialysing the commercial preparation (using, for example, a dialysis membrane supplied by DISLAB, with the trade name ROTH®, cut-off threshold between 10 and 20,000 dalton), precipitating the commercial solution thus dialysed with 50% ammonium sulphate, processing said solution enriched with maltogenic &agr;-amylase by gel filtration, dialysing (using a membrane of the same type as the one mentioned above) then subjecting it to processing by ion-exchange chromatography of the type CM-Sephadex C-50. It is then possible to increase the specific activity of the said en
Duflot Pierrick
Fouache Catherine
Henderson & Sturm LLP
Prouty Rebecca E.
Rao Manjunath N.
Roquette Freres
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