Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-08-04
2001-10-16
Brunsman, David (Department: 1755)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S098000, C435S099000, C435S105000, C127S040000, C127S055000
Reexamination Certificate
active
06303346
ABSTRACT:
The present invention relates to the production of mono and/or oligosaccharides from starch, including dextrose, trehalose, isomaltooligosaccaharides, cyclodextrins and maltooligosaccharides. In a specific aspect, the invention provides a method of saccharifying a liquefied starch solution, which method comprises a saccharification step during which step one or more enzymatic saccharification stages takes place, and the subsequent steps of one or more high temperature membrane separation steps, and re-circulation of the saccharification enzyme, in which method the membrane separation steps are carried out as an integral part of the saccharification step.
In another specific aspect, the invention provides a method of producing a mono and/or oligosaccharide, such as dextrose, trehalose, isomaltooligosaccaharide, cyclodextrins and maltooligosaccharide preparation, which method comprises an enzymatic saccharification step, and the subsequent steps of one or more high temperature membrane separation steps and re-circulation of the saccharification enzyme.
BACKGROUND ART
Saccharides may be grouped into the following two groups: 1) monosaccharides and 2) saccharides which can be hydrolyzed into monosaccharides. Such saccharides which can be hydrolyzed into from 2-10 monosaccharides are termed “oligosaccharides”, whereas such saccharides which can be hydrolyzed into more than 10 monosaccharides are termed “polysaccharides”.
Starch to Sugar Conversion
In the case of converting starch into a sugar (e.g., the starch is depolymerized. A such depolymerization process consists of a pretreatment step and two or three consecutive process steps, viz. a liquefaction process, a saccharification process and dependent on the desired end product optionally an isomerization process.
Pre-treatment of Native Starch
Native starch consists of microscopic granules which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this “gelatinization” process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typically industrial process, the starch has to be thinned or “liquefied” so that it can be handled. This reduction in viscosity is today mostly obtained by enzymatic degradation.
Liquefaction
During the liquefaction step, the long chained starch is degraded into branched and linear shorter units (maltodextrins) by an &agr;-amylase (e.g., Termamyl™). The liquefaction process is carried out at 105-110° C. for 5 to 10 minutes followed by 1-2 hours at 95° C. The pH lies between 5.5 and 6.2. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions). After this treatment the liquefied starch will have a “dextrose equivalent” (DE) of 10-15.
Saccharification
After the liquefaction process the maltodextrins are converted into dextrose by addition of a glucoamylase (e.g., AMG™, available from Novo Nordisk) and a debranching enzyme, such as an isoamylase (e.g., U.S. Pat. No. 4,335,208) or a pullulanase (e:g., Promozyme™—see U.S. Pat. No. 4,560,651). Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95° C.) to inactivate the liquefying &agr;-amylase to reduce the formation of short oligosaccharide called “panose precursors” which cannot be hydrolyzed properly by the debranching enzyme. The temperature is traditionally lowered to about 60° C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.
Normally, when denaturing the &agr;-amylase after the liquefaction step about 0.2-0.5% of the saccharification product is the branched trisaccharide 6
2
-&agr;-glucosyl maltose (panose) which cannot be degraded by a pullulanase. If active amylase from the liquefaction step is present during saccharification (i.e., no denaturing), this level can be as high as 1-2%, which is highly undesirable as it lowers the saccharification yield significantly.
The above pre-treatment and liquefaction steps may suitably be used for providing the liquefied starch for saccharification or hydrolyzing step.
Dextrose Syrups
Dextrose (D-glucose) syrups may be produced by enzymatic conversion of starch into sugars (e.g., as described above). Enzymatic conversion of starch into sugars involves the subsequent steps of liquefaction and saccharification. In this way a high dextrose syrup, usually of 95-96% DX (DX meaning percentage by weight of dextrose (D-glucose) calculated on the basis of dry substance (DS) of syrup) can be obtained. By-products are, e.g., maltose, isomaltose and panose. If syrups of a higher dextrose content is desired, purification may be accomplished by crystallization.
Maltooligosaccharide Syrups
Maltooligosaccharide syrups are syrups comprising from 40 to above 80% maltose (O-&agr;-D-glucopyranosyl-(1-4)-D-glucopyranose). Maltose is a reducing water soluble disaccharide consisting of two glucose units linked in &agr;-1,4 position.
Maltooligosaccharide syrups are today usually produced enzymatically as will be described further below.
Isomaltooligosaccharide Syrups
Isomaltooligosaccharide syrups are sometimes referred to as “Alo mixtures” and defines a mixture containing isomaltose (O-&agr;-D-glucopyranosyl-(1-6)-D-glucopyranose), panose, isomaltotriose and several other branched oligosaccharides composed of four and five glucose residues. “Alo mixture” syrups may be produced enzymatically from starch using a thermostable bacterial &agr;-amylase in the liquefaction step. In the following step the liquefied starch is hydrolyzed or saccharified using a &bgr;-amylase and a transglucosidase simultaneously.
Trehalose Syrups
Trehalose (&agr;-D-glucopyranosyl &agr;-D-glucopyranoside) is a non reducing disaccharide with two glucose residues bound by a &agr;-1,1 linkage.
Enzymatic processes for producing trehalose from starch or maltooligosaccharides are described by, e.g., Kato et al., (1996), Biosci. Biotech. Biochem., 60 (3), p. 546-550); Kazuhisa et al. (1997), Starch 49, no. 1. p. 26-30; and in EP 764,720.
Cyclodextrin Syrups
Cyclodextrins are oligosaccharides with a closed-ring structure in which the glucose units are joined together by &agr;-1,4 linkages. Cyclodextrins containing 6,7 or 8 glucose units are most common and are known as &agr;, &bgr; and &ggr;-CD, respectively.
Cyclodextrins may be produced enzymatically from starch using the enzyme Cyclodextrin glucanotransferase (E.C. 2.4.1.19), in short termed CGTase. CGTases catalyse the conversion of starch and similar substrates into cyclodextrins via an intramolecular transglycosylation reaction, thereby forming cyclodextrins of various sizes.
JP 3-224493 describes an enzymatic conversion of starch into sugars, in which process the saccharified solution is subjected to membrane fractionation to obtain a starch sugar fraction and a fraction containing high molecular dextrins, and feeding back the dextrin fractions to appropriate upstream points.
JP 1-191693 describes a saccharification process by which glucoamylase enzyme is added to liquefied starch at 50-60° C. to cause reaction, followed by continuous separation and recovery of the resulting glucose through a membrane.
JP 62-272987 describes a saccharification process by which glucoamylase enzyme is added to liquefied starch and the saccharification process is practiced inside a semi-permeable membrane, and the glucose formed is discharged to the outside. The molecular weight distribution of the liquefied starch determines the addition of enzyme and the retention time.
Membrane Separation
Membrane separation processes includes the following four basic processes: reverse osmosis, nanofiltration, ultrafiltration and microfiltration.
Reverse osmosis is the tightest possible membrane process in liquid/liquid separation. Water is in principle the only material passing the membrane. Essentially all dissolved and suspended material is rejected. The more open types of reverse osmosis membranes are sometimes confused
Hendriksen Hanne Vang
Liaw Gin C.
Nielsen Bjarne Rønfeldt
Nielsen Ruby Illum
Pedersen Sven
Brunsman David
Garbell Jason I.
Lambiris Elias J.
Novozymes A/S
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