CN101070553A - Starch process. - Google Patents

Abstract

The present invention relates to a process for enzymatic hydrolysis of granular starch into a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of said granular starch.

Description

Starch process

The application is a Chinese patent application (denomination of invention: starch process; The applying date: on February 10th, 2003; Application number: the dividing an application of 03803986.9 (international application no: PCT/DK03/00084)).

Invention field

The present invention relates in the single stage method that granular starch hydrolyzing is become the Zulkovsky starch hydrolysate, this method is carried out under the temperature of the initial gelation temperature that is lower than described granular starch.

Background of invention

Described many with starch change into starch hydrolysate, such as maltose, glucose or extraordinary syrupy method, these hydrolysates both can be used as sweetener, also can be as such as other sugared precursor of this class of fructose.Glucose fermentation can also be become alcohol or other tunning.

The high-molecular weight polymer that starch is made up of glucose unit.It is made up of about 80% amylopectin and 20% amylose starch usually.Amylopectin is the side chain polysaccharide, and wherein the straight chain of α-1,4 D-glucosyl residue connects by α-1,6 glycosidic link.

The amylose starch straight-chain polysaccharide that the D-Glucopyranose unit that connects by α-1,4 glycosidic link constitutes of serving as reasons each other.Starch is being changed in the situation of Zulkovsky starch hydrolysate the starch depolymerization.Depolymerization commonly used by gelling step and two successive processing steps, be that liquefaction process and Mashing process are formed.

Granular starch is made up of water-fast microscopic particles at room temperature.When with aqueous starch when heating slurry, particle swelling and finally breaking, thus starch molecule is scattered in the solution.In this " gelling " technological process, viscosity significantly increases.When solids concn is 30-40% in the typical industrial method, must make starch thinning or " liquefaction " so that can operate on it.Mainly viscosity is reduced at present by enzymatic degradation.In the liquefaction step process, α-Dian Fenmei becomes less side chain and straight chain unit's (Star Dri 5) with the long-chain starch degradation.Liquefaction step is generally under about 105-110 ℃ about 5-10 minute, under about 95 ℃ about 1-2 hour subsequently.Make temperature reduce to 60 ℃ then, add glucoamylase or beta-amylase and optional debranching factor, such as isoamylase or Starch debranching enzyme and make the about 24-72 of saccharification step hour.

Obviously can see that from above-mentioned discussion conventional starch conversion method extremely consumes energy because of the demand to temperature in various steps.Whole technology needs thus to select used in the method enzyme so that can be carried out needn't making under the starch agglomerative condition.These class methods are themes of US4591560, US4727026 and US4009074 and EP0171218 patent.

The present invention relates to granular starch is changed into the single stage method of Zulkovsky starch hydrolysate under the temperature of the initial gelation temperature that is lower than this starch.

Summary of the invention

The present invention provides the single stage method that is used to produce the Zulkovsky starch hydrolysate in aspect first, this method comprises the step that makes the aqueous particulate starch slurry carry out effect in the following enzymic activity under the temperature of the initial gelation temperature that is lower than described granular starch: first kind of enzyme, it be the 13rd family's glycosides lytic enzyme the member, to have α-1.4-glycoside hydrolysis active and comprise the 20th family's carbohydrate binding constituents (Carbohydrate-Binding Module Family 20); With second kind of enzyme, it is fungal alpha-amylase (EC 3.2.1.1), beta-amylase (E.C.3.2.1.2) or glucoamylase (E.C.3.2.1.3).

The present invention is in second production method that provides in aspect this based on the syrup (HFSS) of high fructose starch, and this method comprises that the method for using first aspect of the present invention produces the Zulkovsky starch hydrolysate and further comprise this Zulkovsky starch hydrolysate is changed into step based on the syrup (HFSS) of high fructose starch.

The present invention provides fuel alcohol or drinkable production method of alcohol the 3rd in aspect this, this method comprises the method production Zulkovsky starch hydrolysate that uses first aspect of the present invention and further comprises the step that this Zulkovsky starch hydrolysate is fermented into alcohol that wherein the hydrolysing step of fermentation step and granular starch carries out at the same time or separately/successively.

Particularly, the present invention relates to following aspect:

1. be used to produce the single stage method of Zulkovsky starch hydrolysate, this method comprises makes aqueous granular starch slurry carry out the step that acts in the following enzyme under the temperature of the initial gelation temperature that is lower than described granular starch:

First kind of enzyme:

(a) member of the 13rd family's glycosides lytic enzyme;

(b) has α-1.4-glycoside hydrolysis activity; With

(c) comprise the 20th family's carbohydrate binding constituents;

With at least a second kind of enzyme that belongs to fungal alpha-amylase (EC 3.2.1.1), beta-amylase (E.C.3.2.1.2) or glucoamylase (E.C.3.2.1.3).

2. the method described in above-mentioned 1, wherein starch slurry contains the dried solid particle starch of the dried solid particle starch of 20-55%, preferred 25-40%, more preferably the dried solid particle starch, especially about 33% dried solid particle starch of 30-35%.

3. above-mentioned every in any described method, the dried solid of granular starch that wherein will at least 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% changes into the Zulkovsky starch hydrolysate.

4. above-mentioned every in any described method, wherein said first kind of enzyme source is in microorganism and preferably derive from bacterium.

5. above-mentioned every in any described method, wherein said first kind of enzyme is CGTase (EC 2.4.1.19).

6. above-mentioned every in any described method, wherein said first kind of enzyme for have at least 3.5, preferably at least 4,4.5,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 or most preferably at least 23 micromoles/minute/CGTase of the hydrolytic activity of mg.

7. above-mentioned every in any described method, wherein said first kind of enzyme be with Joergensen etc. in (1997), aminoacid sequence has the CGTase of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology shown in Fig. 1 of Biotechnol.Lett.19:1027-1031.

8. above-mentioned every in any described method, wherein said first kind of enzyme maltogenic alpha-amylase enzyme (E.C.3.2.1.133) of making a living.

9. above-mentioned every in any described method, wherein Fructus Hordei Germinatus sugar α-Dian Fenmei derives from bacillus, preferably derives from bacstearothermophilus.

10. above-mentioned every in any described method, wherein said first kind of enzyme is for having the Fructus Hordei Germinatus sugar α-Dian Fenmei of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology with aminoacid sequence shown in the SEQ ID NO:1 of WO9943794.

11. above-mentioned every in any described method, wherein said first kind of enzyme is the Fructus Hordei Germinatus sugar α-Dian Fenmei that contains the variant of disclosed this aminoacid sequence in aminoacid sequence shown in the SEQ ID NO:1 of WO9943794 or the described patent.

12. above-mentioned every in any described method, wherein said first kind of enzyme for have at least 3.5, preferably at least 4,4.5,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,100,200,300,400,500,600 or most preferably at least 700 micromoles/minute/the Fructus Hordei Germinatus sugar α-Dian Fenmei of the hydrolytic activity of mg.

13. above-mentioned every in any described method, the fungal alpha-amylase of wherein said second kind of enzyme for having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology with aminoacid sequence shown in the SEQ ID NO:10 of WO9623874.

14. above-mentioned every in any described method, wherein said second kind of enzyme is barley beta-amylase (E.C.2.4.1.2), such as Spezyme  BBA 1500 or the Spezyme  DBA from Genencor int.

15. above-mentioned every in any described method, wherein said second kind of enzyme is glucoamylase.

16. above-mentioned every in any described method, wherein said second kind of enzyme is the glucoamylase that derives from aspergillus oryzae, such as the glucoamylase that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology with aminoacid sequence shown in the SEQ ID NO:2 of WO00/04136.

17. above-mentioned every in any described method, wherein there is the third enzyme, described the third enzyme is the α-Dian Fenmei that derives from the bacillus kind, such as disclosed enzyme, variant and hybrid among WO99/19467, WO96/23874, WO97/41213 and the WO99/19467.

18. above-mentioned every in any described method, wherein have the third enzyme, described the third enzyme is isoamylase or Starch debranching enzyme.

19. above-mentioned every in any described method, wherein said temperature is at least at 58 ℃, 59 ℃ or more preferably be at least 60 ℃.

20. above-mentioned every in any described method, wherein said pH is in 3.0-7.0, preferred 3.5-6.0 or the more preferably scope of 4.0-5.0.

21. above-mentioned every in any described method, wherein said Zulkovsky starch hydrolysate has at least 94.5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0% or at least 99.5% DX.

22. above-mentioned every in any described method, wherein main sugar is glucose or maltose in the Zulkovsky starch hydrolysate.

23. above-mentioned every in any described method, wherein said granular starch is available from stem tuber, root, stem or full cereal.

24. above-mentioned every in any described method, wherein said granular starch is available from cereal.

25. above-mentioned every in any described method, wherein said granular starch is available from corn, corn cob, wheat, barley, rye, milo, sago, cassava, tapioca (flour), jowar, rice or potato.

26. above-mentioned every in any described method, wherein said granular starch obtains by the full cereal of full cereal or wet-milling of dry grinding.

27. above-mentioned every in any described method, wherein this method in ultrafiltration system, carry out and wherein phegma to have remained on enzyme, given birth to that starch and water exists in the circulation and wherein see through thing be the Zulkovsky starch hydrolysate.

28. above-mentioned every in any described method, wherein this method in having the continuous film reactor of ultra-filtration membrane, carry out and wherein phegma to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate.

29. above-mentioned every in any described method, wherein this method in having the continuous film reactor of microfiltration membrane, carry out and wherein phegma to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate.

30. production method based on the syrup (HFSS) of high fructose starch, Zulkovsky starch hydrolysate in wherein aforesaid right being required in any described method changes into the syrup (HFSS) based on high fructose starch, such as high-fructose corn syrup (HFCS).

31. fuel alcohol or drinkable production method of alcohol wherein are fermented into alcohol with the Zulkovsky starch hydrolysate in arbitrary described method among the item 1-29.

32. 31 a described method, the hydrolysing step of wherein said fermentation step and granular starch carries out at the same time or separately/successively.

33. arbitrary described method among the 31-32, wherein this method is carried out in ultrafiltration system, and wherein to remain on enzyme, give birth in the circulation that starch, yeast, yeast nutrition thing and water exists and wherein see through thing be the liquid that contains alcohol to phegma.

34. arbitrary described method among the 31-33, wherein this method in having the continuous film reactor of ultra-filtration membrane, carry out and wherein phegma to remain on enzyme, give birth in the circulation that starch, yeast, yeast nutrition thing and water exists and wherein see through thing be the liquid that contains alcohol.

Detailed Description Of The Invention

Definition

Term " granular starch " should be understood the not starch (raw uncooked starch) of heat system of making a living, promptly not carry out agglomerative starch.Starch forms in plant as small water-fast particle.These particles are protected in starch being lower than under the temperature of initial gelation temperature.When putting into cold water, these particles can absorb small amount of liquid.Under up to 50 ℃-70 ℃, swelling is a reversible, and reversible degree depends on specific starch.Under higher temperature, be called the agglomerative irreversible swelling and begin to take place.

Term " initial gelation temperature " should be interpreted as that starch begins the agglomerative minimum temperature.Starch begins gelling between 60 ℃-70 ℃, definite temperature depends on concrete starch.Initial gelation temperature depends on the source of institute's starch producing.The initial gelation temperature of wheat is about 52 ℃, and the initial gelation temperature of potato is about 56 ℃, and the initial gelation temperature of corn is about 62 ℃.Yet starch primary quality changes with growth conditions different and tackles every batch of initial gelation temperature of starch test thus with the specified plant kind.

" Zulkovsky starch hydrolysate " should be interpreted as the soluble product of the inventive method and can comprise monose, disaccharides and oligosaccharides class, such as glucose, maltose, Star Dri 5, cyclodextrin and mixture arbitrarily thereof.Preferably general at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% the dried solid of granular starch changes into the Zulkovsky starch hydrolysate.

Term " special syrup " be term well-known in the art and by DE and carbohydrate spectral characterization (referring to the article of p.50+ going up in the textbook " Molecular Structure and Function of Food Carbohydrate " " new special glucose syrup ", G.G.Birch and L.F.Green edit, AppliedScience Publishers LTD., London).In general, special syrup has the DE of 35-45 scope.

In the context of the present invention, " the 13rd family's glycosides lytic enzyme " is defined as comprises having (beta/alpha) ₈Or the lytic enzyme family of TIM bucket (barrel) structure and the catalyst structure domain that starch and related substrates worked by α-reservation reaction mechanism (Koshland, 1953, Biol.Rev.Camp.Philos.Soc 28,416-436).

In the context of the present invention, the enzyme that will have " α-1.4-glycoside hydrolysis activity " is defined as the Takata (Takata etc. that comprise by definition, 1992, J.Biol.Chem.267,18447-18452) and Koshland (Koshland, 1953, Biol.Rev.Camp.Philos.Soc 28,416-436) catalysis α-1.4-glycosidic link hydrolysis and/or synthetic enzyme family.

In the context of the present invention, " the 20th family's carbohydrate binding constituents " or CBM-20 composition are defined as about 100 the amino acid whose sequences that have at least 45% homology with (1997) carbohydrate binding constituents (CBM) of disclosed polypeptide in Biotechnol.Lett.19:1027-1031 Fig. 1 such as Joergensen.CBM comprises 102 the most last amino acid of this polypeptide, promptly from the subsequence of the 582nd amino acids-the 683rd amino acids.

(a) belonging to the 13rd member of glycosides lytic enzyme family of family, (b), to have α-1.4-glycoside hydrolysis active and (c) comprise the 20th family's carbohydrate binding constituents, and comprise the enzyme that is categorized as EC 2.4.1.19, be cyclodextrin glucanotrasferase enzyme and EC 3.2.1.133, be the selection member in Fructus Hordei Germinatus sugar α-Dian Fenmei and 3.2.1.1 α-Dian Fenmei and the 3.2.1.60 maltotetrose formation amylase for the enzyme of special concern of the present invention.

By in the starch insulating process according to Wind, R.D. etc. 1995 determine " hydrolytic activity " of CGTases and Fructus Hordei Germinatus sugar α-Dian Fenmei in the increase of the described mensuration reducing power of Appl.Environ.Microbiol.61:1257-1265 (reducing power).The dinitrosalicylic acid method of using Bernfield (Bernfield, P.1955.Amylases alpha and beta.Methods Enzymol.1:149-158) is through suitably revising the concentration of measuring reducing sugar.Under 60 ℃ the enzyme of dilution is being incubated the appropriate time time limit with 1% (wt/v) Zulkovsky starch (maybe can select from the Paselli SA2 starch of Dutch Avebe from Merck Zulkovsky starch) in 10mM Trisodium Citrate (pH 5.9) damping fluid.The hydrolytic activity of a unit is defined as the enzyme amount that under standard conditions per minute produces 1 micromole's maltose.

The polypeptide " homology " that relates in this manual is interpreted as that first kind of sequence of expression is from the identity degree between two kinds of sequences of second kind of sequence deutero-.Can suitably measure homology by computer program as known in the art, such as the GAP that provides in the GCG routine package (Wisconsin Package procedure manual, Version 8, August, 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48,443-453.Use following to peptide sequence setting relatively: GAP generation compensation (penalty) 3.0 and GAP expansion are compensated for as 0.1.

Cyclodextrin glucanotrasferase enzyme (CGTases)

As the concrete enzyme of first kind of enzyme in the inventive method can be for cyclomaltodextrin glucanotransferase (CGTASE) (E.C.2.4.1.19), be also referred to as cyclodextrin glucanotrasferase enzyme or Maltose 4-glucosyltransferase, be called CGTase hereinafter, it changes the glycosyl reaction by intramolecularly starch is catalytically converted into the ring Star Dri 5 with similar substrate, forms the ring Star Dri 5 of all size thus.Most of CGTases had not only had the glycosyl of commentaries on classics activity, but also had had the activity of degraded starch.The CGTases that is paid close attention to preferably derives from microorganism and most preferably derives from bacterium.The CGTases of special concern comprises: the CGTases that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology with sequence shown in the 1-679 amino acids of SEQ ID NO:2 among the WO02/06508; With 1997 CGTases that disclosed polypeptid acid sequence has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology in Biotechnol.Lett.19:1027-1031 Fig. 1 such as Joergensen; And the CGTases described in US5278059 and the US5545587.Preferably the hydrolytic activity that has of the CGTase that uses as first kind of enzyme of described method be at least 3.5, preferably be at least 4,4.5,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 or most preferably be at least 23 micromoles/minute/mg.The DS CGTases that can add 0.01-100.0NU/g DS, preferred 0.2-50.0NU/g DS, preferred 10.0-20.0NU/g consumption.

Fructus Hordei Germinatus sugar α-Dian Fenmei

As the another kind of concrete enzyme of first kind of enzyme of the inventive method maltogenic alpha-amylase enzyme (E.C.3.2.1.133) of making a living.Fructus Hordei Germinatus sugar α-Dian Fenmei (dextran 1,4-α-maltose lytic enzyme) can be hydrolyzed into amylose starch and amylopectin the maltose of α-configuration.In addition, Fructus Hordei Germinatus sugar α-Dian Fenmei can hydrolysis trisaccharide maltose and cyclodextrin.The Fructus Hordei Germinatus of special concern sugar α-Dian Fenmei can derive from the kind of bacillus, preferably derive from bacstearothermophilus, most preferably derive from such as the bacstearothermophilus C599 that is described among the EP120.693.This specific Fructus Hordei Germinatus sugar α-Dian Fenmei has the aminoacid sequence shown in the 1-686 amino acids of SEQ ID NO:1 among the US6162628.The 1-686 amino acids of SEQ ID NO:1 has at least 70% identity, preferred at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or particularly at least 99% identity among aminoacid sequence that preferred Fructus Hordei Germinatus sugar α-Dian Fenmei contains and the US6162628.The variant of most preferred Fructus Hordei Germinatus sugar α-Dian Fenmei comprises disclosed variant among the WO99/43794.

Contain and have 714 hydrolytic activity just like the Fructus Hordei Germinatus of the aminoacid sequence shown in the 1-686 amino acids of SEQ ID NO:1 among US6162628 sugar α-Dian Fenmei.The Fructus Hordei Germinatus sugar α-Dian Fenmei that is preferably used as first kind of enzyme of the inventive method have at least 3.5, preferably at least 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,100,200,300,400,500,600 or most preferably at least 700 micromoles/minute/hydrolytic activity of mg.

The Fructus Hordei Germinatus sugar α-Dian Fenmei that can add 0.01-40.0MANU/g DS, preferred 0.02-10MANU/g DS, preferred 0.05-5.0MANU/g consumption.

Fungal alpha-amylase

Concrete enzyme as second kind of enzyme of the inventive method is fungal alpha-amylase (EC 3.2.1.1), adds Mill sample (Fungamyl-like) α-Dian Fenmei such as sweet smell.In this manual, term " sweet smell adds Mill sample α-Dian Fenmei " refers to aminoacid sequence shown in the SEQ ID No.10 that shows with WO96/23874 and has high homology, promptly has a α-Dian Fenmei of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% above homology with aminoacid sequence shown in the SEQ ID No.10 of WO96/23874.The fungal alpha-amylase that can add 0.001-1.0AFAU/g DS, preferred 0.002-0.5AFAU/g DS, preferred 0.02-0.1AFAU/g DS consumption.

Beta-amylase

Another kind of concrete enzyme as second kind of enzyme in the inventive method is beta-amylase (E.C 3.2.1.2).Beta-amylase is the title that generally malt amylase that plays circumscribed effect is given, 1 on its catalysis amylose starch, amylopectin and the relevant glucose polymer, the hydrolysis of 4-α-glycosidic link.

From each kind of plant and microorganism, separated beta-amylase (W.M.Fogarty and C.T.Kelly, Progress in Industrial Microbiology, vol.15, pp.112-115,1979).These beta-amylases are characterised in that the optimum temps of 40 ℃ of-65 ℃ of scopes of tool and the best pH of 4.5-7.0 scope.The beta-amylase of paying close attention to comprises from barley Spezyme  BBA 1500, Spezyme  DBA and Optimalt ^TMThe beta-amylase of ME, from the Optimalt of Genencor int ^TMBBA and from the Novozym of Novozymes A/S ^TMWBA.

Glucoamylase

Also can be as the another kind of concrete enzyme of second kind of enzyme in the inventive method for deriving from microorganism or deriving from the glucoamylase (E.C.3.2.1.3) of plant.Preferably derive from the glucoamylase of fungi or bacterium, they are selected from Aspergillus glucoamylase, particularly black aspergillus G1 or G2 glucoamylase (Boel etc. (1984), EMBO is (5) J.3, p.1097-1102) or it is such as the variant, Aspergillus awamori glucoamylase (WO84/02921), the aspergillus oryzae (Agric.Biol.Chem. (1991) that are disclosed among WO92/00381 and the WO00/04136,55 (4), p.941-949) or the group formed of its variant or fragment.

The Aspergillus glucoamylase variant of other concern comprise the variant that improves thermostability: G137A and G139A (Chen etc. (1996), Prof. Engng.9,499-505); D257E and D293E/Q (Chen etc. (1995), Prot.Engng.8,575-582); N182 (Chen etc. (1994), Biochem.J.301,275-281); Disulfide linkage A246C (Fierobe etc. (1996), Biochemistry, 35,8698-8704; With the Pro residue that imports on A435 and the S436 position (Li etc. (1997), Protein Engng.10,1199-1204.In addition, Clark Ford is at the ENZYMEENGINEERING 14 on October 17th, 1997, Beijing/China October, and 12-17,97, the summary collected works provide in p.0-61 and have been paper.Proposed in this summary to improving thermostability sudden change on G137A, N20C/A27C and the S30P position in the Aspergillus awamori glucoamylase.The glucoamylase of other concern comprises the Talaromyces glucoamylase, particularly derive from Talaromycesemersonii (WO99/28448), Talaromyces leycettanus (US patent no.Re.32,153), Talaromyces duponti, Talaromyces thermophilus (US patent no.4,587,215) glucoamylase.The bacterium glucoamylase of paying close attention to comprises the glucoamylase from Clostridium, particularly C.thermoamylolyticum (EP135,138) and the hot anaerobic bacillus(cillus anaerobicus) of hot sulfurization hydrogen (WO86/01831).Preferred glucoamylase comprises the glucoamylase that derives from aspergillus oryzae, such as the glucoamylase that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% identity with aminoacid sequence shown in the SEQ ID NO:2 of WO00/04136.That pay close attention in addition is commodity AMG 200L, AMG 300L, SAN ^TMSUPER and AMG ^TME (from Novozymes), OPTIDEX ^TM300 (from Genencor Int.), AMIGASE ^TMAnd AMIGASE ^TMPLUS (from DSM), G-ZYME ^TMG900 (from EnzymeBio-Systems), G-ZYME ^TMG990 ZR (black aspergillus glucoamylase and low protease content).

Can add 0.02-2.0AGU/g DS, preferred 0.1-1.0AGU/g DS, such as the glucoamylase of 0.2AGU/gDS consumption.

Other enzyme

Can also under the situation that has the third enzyme to exist, implement method of the present invention.The third concrete enzyme can be the α-Dian Fenmei (so-called " saturating A Mier sample (Termamyl-like) α-Dian Fenmei ") of bacillus.Well-known A Mier sample α-Dian Fenmei comprises the α-Dian Fenmei that derives from Bacillus licheniformis (being purchased as saturating A Mier), bacillus amyloliquefaciens and bacstearothermophilus bacterial strain α-Dian Fenmei.Other saturating A Mier sample α-Dian Fenmei comprise the α-Dian Fenmei that derives from bacillus kind NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375 bacterial strains of whole specific descriptions in WO95/26397 and by Tsukamoto etc. at Biochemical and Biophysical ResearchCommunications, 151 (1988), the α-Dian Fenmei of describing among the pp.25-31.In the context of the present invention, saturating A Mier sample α-Dian Fenmei is that page 3 the 18th walks to the α-Dian Fenmei that the 6th page of the 27th row upward defines among the WO99/19467.Variant and the hybrid paid close attention to are described among WO96/23874, WO97/41213 and the WO99/19467.Special concern contains sudden change I181 ^*+ G182 ^*The reorganization bacstearothermophilus alpha-amylase variants of+N193F.The bacillus α-Dian Fenmei that can add the well-known significant quantity of those skilled in the art.

The third concrete enzyme of another kind in the described method can be debranching factor, such as isoamylase (E.C.3.2.1.68) or Starch debranching enzyme (E.C.3.2.1.41).α on isoamylase hydrolysis amylopectin and the β-limit dextrin-1,6-D-glucosides branching key and can be able to not and aspect the limited action of α-limit dextrin, be different from Starch debranching enzyme with Aureobasidium pullulans glycan generation chemical reaction.The debranching factor that can add the well-known significant quantity of those skilled in the art.

Embodiment of the present invention

The starch slurry that is used to implement the inventive method can contain the dried solid particle starch of the dried solid particle starch of 20-55%, preferred 25-40%, the more preferably dried solid particle starch of 30-35%.

After implementing the method for the present invention aspect first, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferred 99% dried solid particle starch is converted to the Zulkovsky starch hydrolysate.

The method of the present invention first and second aspect is carried out being lower than under the temperature of initial gelation temperature.The preferred temperature of implementing this method is at least 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ or preferably at least 60 ℃.

The pH that implements first aspect of the present invention can be in 3.0-7.0, preferred 3.5-6.0 or the more preferably scope of 4.0-5.0.

The definite composition of the product Zulkovsky starch hydrolysate in the method for first aspect of the present invention depends on the combination of used enzyme and the type of the granular starch of being processed.Preferred solubility hydrolysate is the maltose with at least 85%, 90%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5,99.0% or 99.5% purity.Even more preferably the Zulkovsky starch hydrolysate be glucose and most preferably starch hydrolysate have at least 94.5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5,99.0% or 99.5% DX (per-cent of glucose in the dried solid of dissolved).But, focus attentions equally on be that product Zulkovsky starch hydrolysate in the inventive method is the method for special syrup (specialty syrup), the special syrup that is used for ice-creams, cake, candy, fruit can of described special syrup such as the mixture that contains glucose, maltose, DP3 and DPn.

The granular starch of processing in the inventive method especially can be available from stem tuber, root, stem, beans, cereal or full cereal.More particularly, granular starch can be available from corn, corn cob (cobs), wheat, barley, rye, milo (milo), sago (sago), cassava (cassava), tapioca (flour) (tapioca), jowar, rice, pea, beans, banana or potato.Special concern wax class and non-wax class corn and barley.The granular starch of being processed can have for highly refined starch quality, preferred purity be higher than 90%, 95%, 97% 99.5% or its can be for containing the thicker starch of the material that comprises the full cereal of ground, the full cereal of described ground comprises non-starch part, such as plumule nubbin and fiber.Grinding such as the so thick material of full cereal is in order to make structure opening and further to process.The preferred two kinds of polishings of the present invention: wet-milling and dry grinding.In the dry grinding method, grind complete grain and use.Wet milling process can make plumule and meal (starch granules and protein) obtain good separation and this method can be used for the situation that any production syrup except that a few cases uses starch hydrolysate.Dry grinding and wet-milling are well-known and focus attentions equally on methods of the present invention in the starch manufacture field.The method of first aspect of the present invention can be carried out in ultrafiltration system, and wherein phegma has remained on enzyme.Give birth in the circulation that starch (raw starch) and water exists and be the Zulkovsky starch hydrolysate wherein through thing.The method that focus attentions equally on carries out in having the continuous film reactor of ultra-filtration membrane and wherein phegma to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate.Also focus on the method for carrying out in the continuous film reactor that has microfiltration membrane and wherein phegma to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate.

In the method aspect second of the present invention, the Zulkovsky starch hydrolysate in first aspect method of the present invention is changed into syrup (HFSS) based on high fructose starch, such as high fructose corn syrup (HFCS).Preferably use glucose isomerase and more preferably carry out this conversion by the fixed glucose isomerase of supporting on the solid support.The isomerase of paying close attention to comprises the commodity Sweetzyme from Novozymes A/S ^TMIT, from the G-zyme of Rhodia ^TMIMGI and G-zyme ^TMG993, Ketomax ^TMAnd G-zyme ^TMG993, from the G-zyme of Genemcor Int ^TMG993 liquid and GenSweet ^TM

In the present invention the 3rd method aspect this, the Zulkovsky starch hydrolysate in first aspect method of the present invention is used to produce fuel alcohol or drinkable alcohol.In the method aspect the 3rd, fermentation can be carried out at the same time or separately/successively with the hydrolysis of granular starch slurry.When fermentation and hydrolysis were carried out simultaneously, temperature was preferably at 30 ℃-35 ℃ and more preferably at 31 ℃-34 ℃.The method of third aspect of the present invention can be carried out in ultrafiltration system, in the circulation that wherein phegma remains on enzyme, gives birth to starch, yeast, yeast nutrition thing and water exist and wherein to see through thing be the liquid that contains alcohol.In method that focus attentions equally on carries out in having the continuous film reactor of ultra-filtration membrane and the circulation that wherein phegma remains on enzyme, gives birth to starch, yeast, yeast nutrition thing and water exist and wherein to see through thing be the liquid that contains alcohol.

Material and method

Alpha-amylase activity (KNU)

Use yam starch to measure amylolytic activity as substrate.This method is mixed with iodine solution to the decomposition of the yam starch of modification and at this reaction back slurry starch/enzyme solution sample based on enzyme.Begin to form black-and-blue, but in amylolytic process, blueness shoal and progressively become pale red brown, with itself and tinted shade standard substance relatively.

A Kilo Novo α-Dian Fenmei unit (KNU) is defined as under standard conditions (promptly 37 ℃+/-0.05; 0.0003M Ca ²⁺With pH 5.6 times) make the enzyme dosage of 5.26g Zulkovsky starch dry-matter dextrinization.

The file AF 9/6 that more specifically describes this analytical procedure is available from Novozymes A/S, and Denmark is included in this document herein as a reference.

CGTase activity (KNU)

Measure the CGTase alpha-amylase activity by using Phadebas  sheet as substrate.Phadebas sheet (Phadebas  amylase test is provided by Pharmacia Diagnostic) contains and bovine serum albumin(BSA) and the crosslinked insoluble blue starch polymer of buffering material blended.

For each single mensuration, a slice is suspended in contains 5ml 50mM Britton-Robinson damping fluid (50mM acetate, 50mM phosphoric acid, 50mM boric acid, 0.1mMCaCl ₂, with NaOH with pH regulator to significant value) pipe in.Carry out in the water-bath of this test under the concern temperature.50mM Britton-Robinson damping fluid with x ml dilutes the α-Dian Fenmei of being tested.This α-Dian Fenmei solution of 1ml is joined in the 5ml 50mM Britton-Robinson damping fluid.Use α-Dian Fenmeishuixie starch, obtain the soluble blue color part.The optical density of the gained blue solution at the 620nm place by spectrophotometry is the function of alpha-amylase activity.

Importantly the 620nm place optical density of measuring in 10 or 15 minutes (test duration) of insulation back is 0.2-2.0 optical density scope at the 620nm place.In this optical density scope, linear between activity and the optical density (Lambert-Beer law).Necessary thus regulatory enzyme diluent is to be fit to this standard.Under one group of actual conditions (temperature, pH, reaction times, buffer conditions), the specified α-Dian Fenmei of 1mg can a certain amount of substrate of hydrolysis and generation blueness.Measure colour intensity at the 620nm place.The optical density of measuring is directly proportional under a specified set condition with the activity specific (the pure α-Dian Fenmei albumen of activity/mg) of described α-Dian Fenmei.

The file EAL-SM-0351 that more specifically describes this analytical procedure is available from Novozymes A/S, and Denmark is included in this document herein as a reference.

Fructus Hordei Germinatus sugar alpha-amylase activity (MANU)

A malt amylase Novo unit (MANU) is defined as the enzyme amount of per minute cracking 1 micromole's trisaccharide maltose under standard conditions.Standard conditions are 10mg/ml trisaccharide maltose, 37 ℃, pH 5.0 and 30 minute reaction times.(GlucDH Merck) will change into glucono-lactone with the glucose that forms, and pass through spectrophotometry at the 340nm place with Hexose phosphate dehydrogenase under the condition that forms NADH.The file (EAL-SM-0203.01) of more specifically describing this analytical procedure is available from Novozymes A/S, and Denmark is included in this document herein as a reference.

Glucoamylase activity (AGU)

Novo glucose starch unit of enzyme (AGU) is defined as 37 ℃ of enzyme amounts with 4.3 times per minute cracking of pH, 1 micromole's maltose.

With using from Boehringer Mannheim, the method that revise (AEL-SM-0131 obtains from Novozymes as required) of 124036 glucose GOD-Perid test kit back is AGU/ml with determination of activity.Standard substance: AMG-standard substance, lot number 7-1195,195AGU/ml.Under 37 ℃ 375microL substrate (1% maltose in the 50mM sodium acetate, pH 4.3) is being incubated 5 minutes.The 25microL enzyme that adding is diluted with sodium acetate.Make reaction terminating by adding 100microL 0.25MNaOH after 10 minutes.Change 20microL over to 96 hole microtiter plates and add 200microLGOD-Perid solution (124036, Boehringer Mannheim).After at room temperature 30 minutes, in 650nm place mensuration optical density and according to the activity of AMG-standard substance calculating by AGU/ml.The file (AEL-SM-0131) of more specifically describing this analytical procedure is as required available from Novozymes A/S, and Denmark is included in this document herein as a reference.

Fungal alpha-amylase activity (FAU)

Measure alpha-amylase activity with FAU (fungal alpha-amylase activity unit).The FAU of a unit (1) is for per hour decomposing 5260mg solid starch (Amylum solubile, enzyme amount Merck) under standard conditions (promptly 37 ℃ and pH 4.7 times).The file AF9.1/3 that more specifically describes this analytical procedure is as required available from Novozymes A/S, and Denmark is included in this document herein as a reference.

Acid alpha-amylase activity (AFAU)

Measure the acid alpha-amylase activity with AFAU (acid fungal alpha-amylase activity unit), their contrast enzyme standard substance are measured.

Used standard substance are AMG 300 L (from Novozymes A/S, glucoamylase wild-type black aspergillus G1 also is disclosed in (1984) such as Boel, and EMBO is (5) J.3, p.1097-1102WO92/00381 in).Neutral α-Dian Fenmei in this AMG drops to below the 0.05FAU/mL from about 1FAU/mL after at room temperature storing for 3 weeks.

Measure acid alpha-amylase activity in this AMG standard substance according to following description.In this method, 1 AFAU is defined as the enzyme amount that under standard conditions, per hour makes the dried solid degraded of 5.26mg starch.

Iodine and starch and do not form blue mixture with its degraded product.Colour intensity is directly proportional with starch concentration thus.Use anti-phase colorimetry amylase activity to be determined as the decline of starch concentration under the concrete analysis condition.

α-Dian Fenmei

Starch+iodine → dextrin+oligosaccharides class

40℃，pH 2.5

Blueness/purple t=23 decolours second

Standard conditions/reaction conditions: (per minute)

Substrate starch, about 0.17g/L

The damping fluid Citrate trianion, about 0.03M

Iodine (I): 0.03g/L

CaCl ₂： 1.85mM

pH： 2.50-0.05

Soaking time: 40 ℃

Reaction times: 23 seconds

Wavelength: λ=590nm

Enzyme concn: 0.025AFAU/mL

Enzyme working range: 0.01-0.04AFAU/mL

If preferably other particular content then can find them and they are being incorporated herein by reference as required from the EB-SM-0259.02/01 available from Novozymes A/S.

Beta-amylase activity (DP °)

The activity of representing SPEZYME  BBA 1500 with saccharogenic power (DP) degree.It is to produce to be enough to reduce the enzyme amount that contains in the 0.1ml 5% enzyme preparation sample solution of recuding sugars of 5ml fehling's solution when under 20 ℃ sample being incubated 1 hour with the 100ml substrate.

Amylopectin enzymic activity (new amylopectin unit of enzyme Novo (NPUN)

Aureobasidium pullulans glycan substrate is measured the amylopectin enzymic activity relatively.The Aureobasidium pullulans glycan is mainly by passing through 1, the straight chain D-glucose polymer that the maltotriose glycosyl unit that 6-α-key connects forms.Inscribe-Starch debranching enzyme random hydrolysis 1,6-α-key discharges trisaccharide maltose, 6 ³-α-maltotriose glycosyl-trisaccharide maltose, 6 ³-α-(6 ³-α-maltotriose glycosyl-maltotriose glycosyl)-trisaccharide maltose.

A new amylopectin unit of enzyme Novo (NPUN) is that inscribe-amylopectin unit of enzyme activity and contrast Novozymes A/S Promozyme D standard substance are measured.Standard conditions be under 40 ℃ and pH 4.5 30 minute reaction times and with 0.7% Aureobasidium pullulans glycan as substrate.Going out amount by the red degradation of substrates product of spectrophotometry and this amount at 510nm is directly proportional with inscribe-amylopectin enzymic activity in the sample.The file (EB-SM.0420.02/01) of more specifically describing this analytical procedure is as required available from Novozymes A/S, and Denmark is included in this document herein as a reference.

Under standard conditions, a NPUN approximates to discharge has the enzyme amount of the reducing sugar that equals 2.86 micromole's glucose/minute reducing power.

The mensuration of CGTase hydrolytic activity

By as Wind etc. 1995 measure described in the Appl.Environ.Microbiol.61:1257-1265 with Paselli SA2 starch (from Avebe, The Netherlands) insulating process together in the increase of reducing power determine the hydrolytic activity of CGTase.

The dried solid of sugar cloth and dissolved is measured

Measure that sugar in the starch hydrolysate is formed and be DX with the glucose calculation of yield subsequently by HPLC.Determine the dried solid of dissolved in the starch hydrolysate (solubility) ° BRIX by measuring specific refractory power.

Material

Use following enzymic activity.Fructus Hordei Germinatus sugar α-Dian Fenmei contains the aminoacid sequence shown in the SEQ ID No:1 of WO9/943794.Glucoamylase derives from the aspergillus oryzae of one of aminoacid sequence shown in the SEQ ID No:2 that contains WO00/04136 or disclosed variant.Acid fungal alpha-amylase derives from black aspergillus.The bacillus α-Dian Fenmei is for containing sudden change I181 ^*+ G182 ^*The reorganization bacstearothermophilus variant of+N193F.Fungal alpha-amylase derives from aspergillus oryzae.CGTase O contains just like the sequence shown in this paper SEQ ID NO 2.CGTase T contains the aminoacid sequence shown in disclosed and this paper SEQ ID NO 3 among (1997) Fig. 1 in Biotechnol.Lett.19:1027-1031 such as Joergensen.CGTase A contains just like the sequence shown in this paper SEQ ID NO 4.

Conventional corn starch (C x PHARM 03406) is available from Cerestar.

Embodiment 1

Present embodiment has explained that use CGTase T and glucoamylase and acid fungal amylase change into glucose with granular starch.Join by W-Gum under agitation condition that 247.5g is commonly used that preparation has 33% dried solid (DS) granular starch slurry in the 502.5ml water.With HCl with pH regulator to 4.5.The granular starch slurry is distributed in the blue lid of the 100ml flask, and every flask contains 75g.When magnetic stirs, flask is incubated in 60 ℃ water-bath.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 1.Draw samples after 24,48,72 and 96 hours.

The used enzyme activity level of table 1. is:

CGTase T KNU/kg DS	Glucoamylase AGU/kg DS	Acid fungal alpha-amylase AFAU/kg DS
			12.5 25.0 100.0	200 200 200	50 50 50

Use following method to measure total solids starch.By adding excessive α-Dian Fenmei (the dried solid of 300KNU/Kg) and subsequently sample being placed the oil bath under 95 ℃ to make the starch complete hydrolysis in 45 minutes.By behind the 0.22microM membrane filtration, determine dried solid by measuring specific refractory power.

By behind the 0.22microM membrane filtration to the dried solid of the solubility in the sample determination starch hydrolysate.Determine the dried solid of solubility and measure sugar cloth by measuring specific refractory power by HPLC.The amount of glucose is calculated as DX.The result is as shown in table 2 and 3.

Table 2. dried solid of solubility under three kinds of CGTase activity levels accounts for the per-cent of total dry matter.

KNU/kgDS	24 hours	48 hours	72 hours	96 hours
					12.5 25.0 100.0	68 76 83	82 89 96	89 93 98	94 97 99

The DX of table 3. solubility hydrolysate under three kinds of CGTase activity levels.

KNU/kgDS	24 hours	48 hours	72 hours	96 hours
					12.5 25.0 100.0	92.6 92.4 92.7	94.5 94.8 94.9	95.1 95.4 95.4	95.3 95.5 95.4

Embodiment 2

Present embodiment has explained that use CGTase T, glucoamylase, acid fungal alpha-amylase and genus bacillus α-Dian Fenmei change into glucose with granular starch.

Be ready to contain the flask of 33%DS granular starch and preparation insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 4.

The used enzyme activity level of table 4. is.

CGTase T KNU/kg DS	Glucoamylase AGU/kg DS	Acid fungal alpha-amylase AFAU/kg DS	Genus bacillus α-Dian Fenmei KNU/kg DS
				5.0	200	50	300

Draw samples and analysis as described in example 1 above after 24,48,72 and 96 hours.The result is as shown in table 4 and 5.

The dried solid of table 5. solubility accounts for the per-cent of total dry matter.

24 hours	48 hours	72 hours	96 hours
				82.8	93.0	96.3	98.7

The DX of table 6. solubility hydrolysate.

24 hours	48 hours	72 hours	96 hours
				92.8	94.9	95.5	95.8

Embodiment 3

Present embodiment has explained that use Fructus Hordei Germinatus sugar α-Dian Fenmei, glucoamylase and acid fungal alpha-amylase change into glucose with granular starch.

Be ready to contain the flask of 33%DS granular starch and insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 6.

The used enzyme activity level of table 6. is:

	Fructus Hordei Germinatus sugar α-Dian Fenmei MANU/kg DS	Glucoamylase AGU/kg DS	Acid fungal alpha-amylase AFAU/kg DS
				Flask 1 flask 2	5000 20000	200 200	50 50

Draw samples and analysis as described in example 1 above after 24,48,72 and 96 hours.The result is as shown in table 7 and 8.

Table 7. dried solid of solubility under the activity level of two kinds of Fructus Hordei Germinatus sugar α-Dian Fenmei accounts for the per-cent of total dry matter.

MANU/kgD S	24 hours	48 hours	72 hours	96 hours
					5000 20000	63.1 67.0	75 77.9	79.3 82.7	85.3 88.1

The DX of table 8. solubility hydrolysate under the activity level of two kinds of Fructus Hordei Germinatus sugar α-Dian Fenmei.

MANU/kgD S	24 hours	48 hours	72 hours	96 hours
					5000 20000	95.2 93.8	95.4 94.9	95.3 94.9	95.5 94.8

Embodiment 4

Present embodiment has explained that use glucoamylase and acid fungal alpha-amylase only change into glucose with the part granular starch.

Be ready to contain the flask of 33%DS granular starch and insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 9.Draw samples after 24,48,72 and 96 hours.Analytic sample as described in example 1 above.The result is as shown in table 10 and 11.

The used enzyme activity level of table 9. is:

Glucoamylase AGU/kg DS	Acid fungal alpha-amylase AFAU/kg DS
		200	50

The dried solid of table 10. solubility accounts for the per-cent of total dry matter.

24 hours	48 hours	72 hours	96 hours
				28.5	36.3	41.6	45.7

The DX of table 11. solubility hydrolysate.

24 hours	48 hours	72 hours	96 hours
				27.7	34.9	39.2	42.2

Embodiment 5

Present embodiment has been explained as what being measured appearred in the dried solid of solubility and DX and has been used CGTase and glucoamylase granular starch to be changed into the dependency between the 4 kinds of different CGTases (CGTase A, CGTase N, CGTase O and CGTase T) and productive rate in the process of glucose syrup.

Be ready to contain the flask of 33%DS granular starch and insulation as described in example 1 above.In the time of 0 hour, all give the CGTases of 100KNU/kg DS and the glucoamylase of 200AGU/kg DS.Draw samples is also analyzed as described in example 1 above in the time of 48 hours.To the results are shown in the table 12.

Table 12. hydrolytic activity (micromole/minute/mg protein) and dried solid of solubility (DS) and DX after 48 hours

CGTase	Hydrolytic activity	Solubility DS	DX
				CGTase N CGTase A CGTase O CGTase T	0.27 0.38 1.62 4.59	37.4 49.9 60.9 97.9	35.1 46.7 57.1 91.2

Embodiment 6

Present embodiment has been explained the described method of carrying out in ultrafiltration system, wherein to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate to phegma.In the ultrafiltration system in batches (PCI type) that has tubular film assembly (PU 120 types), handle and comprise the 100kg granule corn starch that is suspended in 233 L city tap-waters and the slurry of CGTase T (12.5KNU/kg starch), genus bacillus α-Dian Fenmei (300KNU/kg starch) and glucoamylase (200AGU/kg starch).Stir this slurry with 100rpm, use the 30%HCl of 170mL to be set in 57 ℃ with pH regulator to 4.5 and with temperature of reaction.

Analyze the dried solids content and the sugared composition that see through in thing and the phegma sample.

The correction factor of insoluble substance is: q=(100-S%)/(100-BRIX).The centrifugal index of sugar is: ciS%=BRIX/S% (not calibration).The theoretical yield S of sugar (glucose) _{Productive rate}=ciS%*q*100/111*100%.Thus 100kg starch is proofreaied and correct, obtained about 111kg glucose dry-matter as the result of hydrolysis reaction.

Use identical enzyme system in one pass systems, to test as film test.As a comparison, show among table 15a and the b that the film system is prior to reaching the maximum stripping of starch.

Table 13. phegma and see through and to do solids content in the thing and sugar is formed

Sample	Hour	Reactor volume, L	％DS	％DP 1	％DP 2	％DP 3	％DP 4
								Reaction	3	207	16.1	75.3	10.3	2.6	11.5

Device
								Reactor	28	123	28.3	95.0	2.7	0.8	1.5
Reactor	53	123	31.4	95.2	3.4	0.5	0.9
								See through thing	3	207	12.1	71.2	17.4	2.9	8.5
See through thing	28	123	21.8	94.9	2.9	0.8	1.3

Table 14.3, the dried solids distribution 28,53 and 77 hours the time in the phegma.

	3 hours	28 hours	53 hours	77 hours
					The total DS of solubility DS	16 38	28 37	31 42	39 45

The glucose theoretical yield and the time relation of table 15a. film system

Hour	Total DS% in the reactor	°BRIX	q＝(100-S％)/(100 -°BRIX)	cis％＝°Brix /S％	Theoretical yield sis=cis*q*10 0/111%
						0	27.0	2.2	0.75	0.08	5
24	35.9	27.3	0.88	0.76	73
						48	41.2	30.0	0.84	0.73	89
72	41.2	33.1	0.88	0.80	98
						94	41.2	34.8	0.90	0.85	103

The theoretical yield and the time relation of the glucose in the table 15b. batch reactor system.

Hour	Total DS% in the reactor	°BRIX	q＝(100-S％)/(100-° BRIX)	cis％＝°Brix/ S％	Theoretical yield sis=cis*q*100/111%
						0	29.7	2.0	0.72	0.07	4

24	29.7	25.6	0.95	0.86	74
						48	29.7	28.8	0.99	0.97	86
72	29.7	29.8	1.00	1.00	91
						94	29.7	29.8	1.00	1.00	91

Conclusion is: when keeping substrate in the saccharifying in the film system when saturated, solubleness be used for comparing and improve to giving birth to simple batch reactions system that starch carries out cold mashing.

Embodiment 7

Present embodiment has been explained cold liquefaction of the present invention and the saccharifying that carries out simultaneously in the non-stop run microfiltration membrane reactor that uses ceramic component.

200L entry mixers jar is connected with the 200L reactor tank that has temperature control by the reactor feed pump.The pump that use has 0-20I/ hour capacity makes the mixture of autoreactor by being used for the APV pottery microfiltration assembly recirculation of separating glucose.Hole size is that the area of 0.2 micron and film is 0.2m ²

About 200 hours of the dosage work of the glucoamylase of reactor use 100KNU/kgDS CGT-ase T and 300AGU/kg DS.Use the mean holding time in 35-45 hour the reactor, system produces the DP1=93% glucose syrup with the whole time limit of quiet rum, and productive rate is near 100%.

Under agitation load the 60kgCerestar C x PHARM 03406 type W-Gum that is suspended in 58 ℃ of city tap-waters of 140L to reactor tank.Make steam heated cover with temperature regulation to 60 ℃.Use 30%HCl to make pH reduce to 4.5 from 6.1.Check pH (pH=4.5) after 15 minutes once more.In the time of 0 hour, add the preceding sampling immediately of enzyme CGTase T (100KNU/kg starch) and glucoamylase (300AGU/kg starch) and be used for being determined at desk centrifuge with the sludge volume % of 3000rpm after centrifugal 3 minutes.In addition, the use refractometer is measured the BRIX in the supernatant liquor.Regularly measure the BRIX in sludge volume and the supernatant liquor after the reaction process as mentioned above.

Load cold city tap-water of 186L and 80kg Cerestar C x PHARM03406 type W-Gum for the entry mixers jar.Maintenance to the slow stirring of entry mixers and with 30%HCl with pH regulator to 4.5.Use water coolant that temperature is remained on 7-8 ℃ and add enzyme CGTase T (100 KNU/kg starch) and glucoamylase (300AGU/kg starch).Guarantee low temperature so that do not react.

Open continuously suddenly ° Brix-value stabilization behind (upstart) reactor to 30 hour about 27.Use the pressure drop of 0.15Bar then and guarantee that the maximum phegma of this pressure flow to start microfiltration.Make filtrate be recycled to reactor tank first 5.7 hours.After this in separating tank, collect filtrate and be the function of time stereometry.Start the reactor feed pump at this time point place and be adjusted to flow velocity and be equal to flow of filtrate (L/ minute).Make the volume in the reactor tank keep constant by implementing this step.

Make the starch slurry charging continuously, simultaneously sampling as mentioned above.In addition, get filtrate sample.By increasing the decline that the phegma flow compensates any flow of filtrate, the filter cake on the broken thus film.Thereby pressure drop is also increased.Sample is regarded as the function of the time of HPLC and BRIX filtrate and also measured the volume of collecting.Sampling is used to measure total DS, sludge, ° Brix and sugar is formed and carries out HPLC from reactor simultaneously.

This test duration 220 hours.When this time point, pressure drop increases to about 0.4Bar.

The enzyme system of being made up of CGTase and glucoamylase as the flow of filtrate (based on unitary determination) of function mensuration process period and average flow of filtrate value (integration) demonstration is kept separately in process period and is guaranteed regime flow growing.This results highlight the potential industrial advantages of this stable system.

Result and material balance are listed among the table 16-18.

The analysis of the filtrate of table 16. pair collection.

Date and time	Hours from beginning	The filtrate of collecting, L	％ DS w/w	Density, kg/L	The quality kg of DS	Average discharge mL/ minute
							13/03/0 216:05,14/03/0 216:50,16/03/0 216:00,18/03/0 213:02,19/03/0 216:45 totally collects	30 * 55 102 147 174	- 142 187 200 100 629.0	- 25.8 25.6 28.7 29.6 27.3	- 1.12 1.12 1.14 1.14 1.13	- 41.1 53.7 65.2 33.8 193.7	- 95.6 66.1 74.0 60.1 -

* begin to give continuously reactor feed

The syrupy composition that table 17. produces

％DP1	％DP2	％DP3	％DP4
				93	5	1	2

The material balance of table 18. embodiment 7 tests

	Quality, kg	％DS	The quality of DS, kg	DS *Productive rate %
					Open reactor
Starch	60	90	54	25
					Water	140	0	0
Reactor is opened	200	27.0	54	25
					Continuous production
Consumption of starch (t=28.75 hour-t=174.5 hour)	235.48	90	212	100
					Water consumption (t=28.75 hour-t=174.5 hour)	548.12	0	0
Base consumption	783.6	27.0	212	100
					Syrup output	629.0	27.3	172	81
Reactor during end
					Total content	200	35	70	33
Unconverted starch	18	50	9	4
					Mud，L	18	50	9	4
Glucose syrup	164	30	49	23

^*Basic base consumption during continuous production.

Compare with the batch test of carrying out in the simple jar that stirs, use the set(ting)value of said hydrolyzed granular starch that the reaction times is significantly reduced.If use 30%DS not run into viscosity problem, think so to make DS increase to 40%, and even 45% is high and still to keep stable operation be practicable.

Embodiment 8

Present embodiment has compared the inventive method and the ordinary method that is used for producing from the living starch Yellow Dent No.2 of dried ground corn form fuel alcohol or drinkable alcohol.

In the blue lid of 250ml flask, make thick W-Gum contact cold liquefaction and saccharification simultaneously with the 30%DS slurry of tap water preparation dry grinding corn and with the inventive method.This slurry is heated to 60 ℃ in the water-bath that magnetic stirs, use 30%HCl is with pH regulator to 4.5 and add CGTase T (75KNU/kgDS) and glucoamylase (500AGU/kg DS).After 48 hours, flask is cooled to 32 ℃ in water-bath.

Make 30%DS slurry liquefaction in advance in the conventional continuous processing of forming by pre-liquefaction vessel, jet-type pulp digester, flasher (flash) and liquefaction back container of dry grinding corn.Add genus bacillus α-Dian Fenmei (10KNU/kgDS) in the pre-liquefaction process under 70-90 ℃ and after about 85-90 ℃ liquefaction, add genus bacillus α-Dian Fenmei (20KNU/kg DS) in the process again.Under 115-120 ℃, carry out the jet-type boiling.Under stirring, magnetic carries out premashing by in the lid of the indigo plant in the water-bath flask mash being heated to 60 ℃.After using 30%HCl, add the glucoamylase that dosage equals 500AGU/kg DS with pH regulator to 4.5.After 48 hours, flask is cooled to 32 ℃ in water-bath.

In being installed, the indigo plant lid flask that charges into soybean oil of yeast lock directly ferments.Add the Bakers yeast (Saccharomyces cerevisiae) of the consumption equal 1,000 ten thousand/mL living yeast and the yeast nutrition thing of 0.25% urea form is joined in each flask.Handle all by carrying out in triplicate at every turn.

By the CO that measures as the flask of weighing in regular intervals of time ₂Consume the monitoring fermentation.Use following formula to calculate L EtOH/100kg cereal dry-matter (DS) then:

Mash contains 30%w/w cereal dry-matter.0.79g/mL be alcohol density.

Shown bipartite fermentation result in the table 19 and 20, comprised statistical calculations (by the disappearance result of interpolation technique estimation) two types of pretreated thick materials.

Table 19. uses the fermentation result of the inventive method of CGTase T (75KNU/kgDS) and glucoamylase (500AGU/kg DS).

Hour	L EtOH/100kg cereal	STDEV
			0 25.5	- 28.3	- 0.9

48 69 79 97

35.4 37.1 *37.5 38.3

0.6 0.2 - 0.2

^*Estimated value

Table 20. uses the fermentation result of the ordinary method of genus bacillus α-Dian Fenmei (10+20KNU/kg DS) and glucoamylase (500AGU/kg DS)

Hour	L EtOH/100kg cereal	STDEV
			0 25.5 48 69 79 97	- 22.5 33.9 *37.2 38.8 40.5	- 1.3 0.7 - 0.4 0.5

^*Estimated value

The ethanol production that the mash that uses simulation industrial fermentation time at about 48-70 hour interval to produce from the inventive method obtains is equal to or higher than can be from by consuming the more pre-liquefaction of hot slurry of two steps of multipotency and the productive rate of the mash acquisition that the jet-type cooking process is produced.

Embodiment 9

Present embodiment has been explained at 60 ℃ and has been used CGTase, glucoamylase and acid fungal alpha-amylase that particle wheat and conventional corn starch are changed into glucose down.

Be ready to contain the flask of 33%DS conventional corn or wheat grain starch and insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 20.Draw samples and analytic sample as described in example 1 above after 24,48,72 and 96 hours.The result is as shown in table 21 and 22.

The enzyme activity level that table 20. is used:

CGTase NU/g DS 100.0

Glucoamylase AGU/g DS 0.2

Acid fungal alpha-amylase AFAU/g DS 0.05

Table 21. uses the dried solid of solubility of two kinds of different starch type to account for the per-cent of total dry matter.

Starch	24 hours	48 hours	72 hours	96 hours
					The conventional corn wheat	85.9 95.7	96.2 98.9	99.4 99.6	100.0 100.0

Table 22. uses the DX of the solubility hydrolysate of two kinds of different starch type.

Starch	24 hours	48 hours	72 hours	96 hours
					The conventional corn wheat	76.2 86.2	89.2 92.4	93.4 93.6	94.7 94.4

Sequence table

＜110〉Novozymes Company (Novozymes)

＜120〉starch process

<130>10270-WO

<160>6

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＜213〉bacillus

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Val Phe Leu Lys Asn Leu Thr Val Leu Leu Lys Thr Ile Pro Leu Ala

-25 -20 -15

Leu Leu Leu Phe Ile Leu Leu Ser Leu Pro Thr Ala Ala Gln Ala Asp

-10 -5 -1 1

Val Thr Asn Lys Val Asn Tyr Thr Arg Asp Val Ile Tyr Gln Ile Val

5 10 15 20

Thr Asp Arg Phe Ser Asp Gly Asp Pro Ser Asn Asn Pro Thr Gly Ala

25 30 35

Ile Tyr Ser Gln Asp Cys Ser Asp Leu His Lys Tyr Cys Gly Gly Asp

40 45 50

Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly Tyr Leu Thr Asp Leu

55 60 65

Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val Glu Asn Val Tyr Ala

70 75 80

Leu His Pro Ser Gly Tyr Thr Ser Tyr His Gly Tyr Trp Ala Arg Asp

85 90 95 100

Tyr Lys Arg Thr Asn Pro Phe Tyr Gly Asp Phe Ser Asp Phe Asp Arg

105 110 115

Leu Met Asp Thr Ala His Ser Asn Gly Ile Lys Val Ile Met Asp Phe

120 125 130

Thr Pro Asn His Ser Ser Pro Ala Leu Glu Thr Asp Pro Ser Tyr Ala

135 140 145

Glu Asn Gly Ala Val Tyr Asn Asp Gly Val Leu Ile Gly Asn Tyr Ser

150 155 160

Asn Asp Pro Asn Asn Leu Phe His His Asn Gly Gly Thr Asp Phe Ser

165 170 175 180

Ser Tyr Glu Asp Ser Ile Tyr Arg Asn Leu Tyr Asp Leu Ala Asp Tyr

185 190 195

Asp Leu Asn Asn Thr Val Met Asp Gln Tyr Leu Lys Glu Ser Ile Lys

200 205 210

Leu Trp Leu Asp Lys Gly Ile Asp Gly Ile Arg Val Asp Ala Val Lys

215 220 225

His Met Ser Glu Gly Trp Gln Thr Ser Leu Met Ser Asp Ile Tyr Ala

230 235 240

His Glu Pro Val Phe Thr Phe Gly Glu Trp Phe Leu Gly Ser Gly Glu

245 250 255 260

Val Asp Pro Gln Asn His His Phe Ala Asn Glu Ser Gly Met Ser Leu

265 270 275

Leu Asp Phe Gln Phe Gly Gln Thr Ile Arg Asp Val Leu Met Asp Gly

280 285 290

Ser Ser Asn Trp Tyr Asp Phe Asn Glu Met Ile Ala Ser Thr Glu Glu

295 300 305

Asp Tyr Asp Glu Val Ile Asp Gln Val Thr Phe Ile Asp Asn His Asp

310 315 320

Met Ser Arg Phe Ser Phe Glu Gln Ser Ser Asn Arg His Thr Asp Ile

325 330 335 340

Ala Leu Ala Val Leu Leu Thr Ser Arg Gly Val Pro Thr Ile Tyr Tyr

345 350 355

Gly Thr Glu Gln Tyr Leu Thr Gly Gly Asn Asp Pro Glu Asn Arg Lys

360 365 370

Pro Met Ser Asp Phe Asp Arg Thr Thr Asn Ser Tyr Gln Ile Ile Ser

375 380 385

Thr Leu Ala Ser Leu Arg Gln Ser Asn Pro Ala Leu Gly Tyr Gly Asn

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Thr Ser Glu Arg Trp Ile Asn Ser Asp Val Tyr Ile Tyr Glu Arg Ala

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Phe Gly Asp Ser Val Val Leu Thr Ala Val Asn Ser Gly Asp Thr Ser

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Tyr Thr Ile Asn Asn Leu Asn Thr Ser Leu Pro Gln Gly Gln Tyr Thr

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Asp Glu Leu Gln Gln Leu Leu Asp Gly Asn Glu Ile Thr Val Asn Ser

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Asn Gly Ala Val Asp Ser Phe Gln Leu Ser Ala Asn Gly Val Ser Val

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Trp Gln Ile Thr Glu Glu His Ala Ser Pro Leu Ile Gly His Val Gly

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Pro Met Met Gly Lys His Gly Asn Thr Val Thr Ile Thr Gly Glu Gly

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Phe Gly Asp Asn Glu Gly Ser Val Leu Phe Asp Ser Asp Phe Ser Asp

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Val Leu Ser Trp Ser Asp Thr Lys Ile Glu Val Ser Val Pro Asp Val

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Thr Ala Gly His Tyr Asp Ile Ser Val Val Asn Ala Gly Asp Ser Gln

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Ser Pro Thr Tyr Asp Lys Phe Glu Val Leu Thr Gly Asp Gln Val Ser

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Ile Arg Phe Ala Val Asn Asn Ala Thr Thr Ser Leu Gly Thr Asn Leu

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Tyr Met Val Gly Asn Val Asn Glu Leu Gly Asn Trp Asp Pro Asp Gln

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-30 -25 -20

Ile Val Phe Val Val Phe Leu Ala Leu Ala Asn Asp Val Tyr Ala Ala

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Asn Gln Leu Asn Lys Val Asn Tyr Ala Lys Asp Thr Ile Tyr Gln Ile

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Val Thr Asp Arg Phe Leu Asp Gly Asp Pro Ser Asn Asn Pro Asp Gly

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Ala Leu Tyr Ser Glu Thr Asp Leu His Lys Tyr Met Gly Gly Asp Trp

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Lys Gly Ile Thr Glu Lys Ile Glu Asp His Tyr Phe Thr Asp Leu Gly

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Ile Thr Ala Leu Trp Ile Ser Gln Pro Val Glu Asn Val Tyr Ala Val

70 75 80

His Pro Glu Gly Tyr Thr Ser Tyr His Gly Tyr Trp Ala Arg Asp Tyr

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Lys Lys Thr Asn Pro Phe Tyr Gly Asn Phe Asn Asp Phe Asp Glu Leu

100 105 110

Ile Ser Thr Ala His Ser His Gly Ile Lys Ile Ile Met Asp Phe Thr

115 120 125

Pro Asn His Ser Ser Pro Ala Leu Lys Thr Asp Ser Asp Tyr Val Glu

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Asn Gly Ala Ile Tyr Asp Asn Gly Ser Leu Ile Gly Asn Tyr Ser Asn

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Asp Leu Asp Ile Phe His His Asn Gly Gly Thr Asp Phe Ser Ser Tyr

165 170 175

Glu Asp Gly Ile Tyr Arg Asn Leu Tyr Asp Leu Ala Asp Tyr Asp Leu

180 185 190

Gln Asn Gln Thr Ile Asp Gln Tyr Leu Lys Glu Ser Ile Glu Leu Trp

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Leu Asp Lys Gly Ile Asp Gly Ile Arg Val Asp Ala Val Lys His Met

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Ser Gln Gly Trp Gln Glu Thr Leu Thr Asn His Ile Tyr Ser Tyr Gln

230 235 240

Pro Val Phe Thr Phe Gly Glu Trp Phe Leu Gly Glu Asn Glu Ile Asp

245 250 255

Pro Arg Asn His Tyr Phe Ala Asn Glu Ser Gly Met Ser Leu Leu Asp

260 265 270

Phe Gln Phe Gly Gln Gln Ile Arg Gly Val Leu Met Ser Gln Glu Asp

275 280 285

Asp Trp Thr Asp Phe His Thr Met Ile Glu Asp Thr Ser Asn Ser Tyr

290 295 300 305

Asn Glu Val Ile Asp Gln Val Thr Phe Ile Asp Asn His Asp Met Ser

310 315 320

Arg Phe His Lys Glu Asp Gly Ala Lys Thr Asn Thr Asp Ile Ala Leu

325 330 335

Ala Val Leu Leu Thr Ser Arg Gly Val Pro Thr Ile Tyr Tyr Gly Thr

340 345 350

Glu His Tyr Leu Thr Gly Glu Ser Asp Pro Glu Asn Arg Lys Pro Met

355 360 365

Pro Ser Phe Asp Arg Ala Thr Thr Ala Tyr Gln Ile Ile Ser Lys Leu

370 375 380 385

Ala His Leu Arg Gln Ser Asn Pro Ala Leu Gly Tyr Gly Thr Thr Thr

390 395 400

Glu Arg Trp Leu Asn Glu Asp Val Tyr Ile Phe Glu Arg Lys Phe Gly

405 410 415

Asp Asn Val Val Val Thr Ala Val Asn Ser Gly Glu Gln Ser Tyr Thr

420 425 430

Ile Asn Asn Leu Gln Thr Ser Leu Leu Glu Gly Thr His Pro Asp Val

435 440 445

Leu Glu Gly Leu Met Gly Gly Asp Ala Leu Gln Ile Asp Gly Lys Gly

450 455 460 465

Gln Ala Ser Thr Phe Glu Leu Lys Ala Asn Glu Val Ala Val Trp Glu

470 475 480

Val Thr Ala Glu Ser Asn Thr Pro Leu Ile Gly His Val Gly Pro Met

485 490 495

Val Gly Gln Ala Gly Asn Glu Ile Thr lle Ser Gly Glu Gly Phe Gly

500 505 510

Glu Gly Gln Gly Thr Val Leu Phe Gly Ser Asp Gln Ala Ser Ile Val

515 520 525

Ser Trp Gly Asp Ser Glu Ile Val Val Asn Val Pro Asp Arg Pro Gly

530 535 540 545

Asn His Tyr Asn Ile Glu Val Val Thr Asn Asp Asn Lys Glu Ser Asn

550 555 560

Pro Tyr Ser Asp Phe Glu Ile Leu Thr Asn Lys Leu Ile Pro Val Arg

565 570 575

Phe Ile Val Glu Glu Ala Val Thr Asp Tyr Gly Thr Ser Val Tyr Leu

580 585 590

Val Gly Asn Thr Gln Glu Leu Gly Asn Trp Asp Thr Asp Lys Ala Ile

595 600 605

Gly Pro Phe Phe Asn Gln Ile Ile Ala Gln Tyr Pro Thr Trp Tyr Tyr

610 615 620 625

Asp Ile Ser Val Pro Ala Asp Ser Thr Leu Glu Tyr Lys Phe Ile Lys

630 635 640

Lys Asp Ala Leu Gly Asn Val Val Trp Glu Ser Gly Thr Asn Arg Ser

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Tyr Glu Thr Pro Thr Glu Gly Thr Asp Thr Leu Thr Ser Thr Trp Arg

660 665 670

Asn

<210>3

<211>683

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<213>Thermoanaerobacter sp.

<220>

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<222>(1)..(683)

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Ala Pro Asp Thr Ser Val Ser Asn Val Val Asn Tyr Ser Thr Asp Val

1 5 10 15

Ile Tyr Gln Ile Val Thr Asp Arg Phe Leu Asp Gly Asn Pro Ser Asn

20 25 30

Asn Pro Thr Gly Asp Leu Tyr Asp Pro Thr His Thr Ser Leu Lys Lys

35 40 45

Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly

50 55 60

Tyr Leu Thr Gly Met Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val

65 70 75 80

Glu Asn Ile Tyr Ala Val Leu Pro Asp Ser Thr Phe Gly Gly Ser Thr

85 90 95

Ser Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro Phe

100 105 110

Phe Gly Ser Phe Thr Asp Phe Gln Asn Leu Ile Ala Thr Ala His Ala

115 120 125

His Asn Ile Lys Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro

130 135 140

Ala Ser Glu Thr Asp Pro Thr Tyr Gly Glu Asn Gly Arg Leu Tyr Asp

145 150 155 160

Asn Gly Val Leu Leu Gly Gly Tyr Thr Asn Asp Thr Asn Gly Tyr Phe

165 170 175

His His Tyr Gly Gly Thr Asn Phe Ser Ser Tyr Glu Asp Gly Ile Tyr

180 185 190

Arg Asn Leu Phe Asp Leu Ala Asp Leu Asp Gln Gln Asn Ser Thr Ile

195 200 205

Asp Ser Tyr Leu Lys Ala Ala Ile Lys Leu Trp Leu Asp Met Gly Ile

210 215 220

Asp Gly Ile Arg Met Asp Ala Val Lys His Met Ala Phe Gly Trp Gln

225 230 235 240

Lys Asn Phe Met Asp Ser Ile Leu Ser Tyr Arg Pro Val Phe Thr Phe

245 250 255

Gly Glu Trp Tyr Leu Gly Thr Asn Glu Val Asp Pro Asn Asn Thr Tyr

260 265 270

Phe Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln

275 280 285

Lys Val Arg Gln Val Phe Arg Asp Asn Thr Asp Thr Met Tyr Gly Leu

290 295 300

Asp Ser Met Ile Gln Ser Thr Ala Ala Asp Tyr Asn Phe Ile Asn Asp

305 310 315 320

Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Tyr Thr Gly

325 330 335

Gly Ser Thr Arg Pro Val Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser

340 345 350

Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr Gly

355 360 365

Asn Gly Asp Pro Tyr Asn Arg Ala Met Met Thr Ser Phe Asp Thr Thr

370 375 380

Thr Thr Ala Tyr Asn Val Ile Lys Lys Leu Ala Pro Leu Arg Lys Ser

385 390 395 400

Asn Pro Ala Ile Ala Tyr Gly Thr Gln Lys Gln Arg Trp Ile Asn Asn

405 410 415

Asp Val Tyr Ile Tyr Glu Arg Gln Phe Gly Asn Asn Val Ala Leu Val

420 425 430

Ala Ile Asn Arg Asn Leu Ser Thr Ser Tyr Tyr Ile Thr Gly Leu Tyr

435 440 445

Thr Ala Leu Pro Ala Gly Thr Tyr Ser Asp Met Leu Gly Gly Leu Leu

450 455 460

Asn Gly Ser Ser Ile Thr Val Ser Ser Asn Gly Ser Val Thr Pro Phe

465 470 475 480

Thr Leu Ala Pro Gly Glu Val Ala Val Trp Gln Tyr Val Ser Thr Thr

485 490 495

Asn Pro Pro Leu Ile Gly His Val Gly Pro Thr Met Thr Lys Ala Gly

500 505 510

Gln Thr Ile Thr Ile Asp Gly Arg Gly Phe Gly Thr Thr Ala Gly Gln

515 520 525

Val Leu Phe Gly Thr Thr Pro Ala Thr Ile Val Ser Trp Glu Asp Thr

530 535 540

Glu Val Lys Val Lys Val Pro Ala Leu Thr Pro Gly Lys Tyr Asn Ile

545 550 555 560

Thr Leu Lys Thr Ala Ser Gly Val Thr Ser Asn Ser Tyr Asn Asn Ile

565 570 575

Asn Val Leu Thr Gly Asn Gln Val Cys Val Arg Phe Val Val Asn Asn

580 585 590

Ala Thr Thr Val Trp Gly Glu Asn Val Tyr Leu Thr Gly Asn Val Ala

595 600 605

Glu Leu Gly Asn Trp Asp Thr Ser Lys Ala Ile Gly Pro Met Phe Asn

610 615 620

Gln Val Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser Val Pro

625 630 635 640

Ala Gly Thr Thr Ile Glu Phe Lys Phe Ile Lys Lys Asn Gly Ser Thr

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Val Thr Trp Glu Gly Gly Tyr Asn His Val Tyr Thr Thr Pro Thr Ser

660 665 670

Gly Thr Ala Thr Val Ile Val Asp Trp Gln Pro

675 680

<210>4

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<212>PRT

＜213〉bacillus

<220>

<221>mat-peptide

<222>(28)..()

<400>4

Met Lys Arg Phe Met Lys Leu Thr Ala Val Trp Thr Leu Trp Leu Ser

-25 -20 -15

Leu Thr Leu Gly Leu Leu Ser Pro Val His Ala Ala Pro Asp Thr Ser

-10 -5 -1 1 5

Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val Ile Tyr Gln Ile Phe

10 15 20

Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn Asn Pro Thr Gly Ala

25 30 35

Ala Phe Asp Gly Ser Cys Thr Asn Leu Arg Leu Tyr Cys Gly Gly Asp

40 45 50

Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly Tyr Leu Thr Gly Met

55 60 65

Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val Glu Asn Ile Tyr Ser

70 75 80 85

Val Ile Asn Tyr Ser Gly Val Asn Asn Thr Ala Tyr His Gly Tyr Trp

90 95 100

Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr Gly Thr Met Gln Asp

105 110 115

Phe Lys Asn Leu Ile Asp Thr Ala His Ala His Asn Ile Lys Val Ile

120 125 130

Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala Ser Ser Asp Asp Pro

135 140 145

Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn Gly Asn Leu Leu Gly

150 155 160 165

Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His His Tyr Gly Gly Thr

170 175 180

Asp Phe Ser Thr Ile Glu Asn Gly Ile Tyr Lys Asn Leu Tyr Asp Leu

185 190 195

Ala Asp Leu Asn His Asn Asn Ser Ser Val Asp Val Tyr Leu Lys Asp

200 205 210

Ala Ile Lys Met Trp Leu Asp Leu Gly Val Asp Gly Ile Arg Val Asp

215 220 225

Ala Val Lys His Met Pro Phe Gly Trp Gln Lys Ser Phe Met Ala Thr

230 235 240 245

Ile Asn Asn Tyr Lys Pro Val Phe Thr Phe Gly Glu Trp Phe Leu Gly

250 255 260

Val Asn Glu Ile Ser Pro Glu Tyr His Gln Phe Ala Asn Glu Ser Gly

265 270 275

Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys Ala Arg Gln Val Phe

280 285 290

Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys Ala Met Leu Glu Gly

295 300 305

Ser Glu Val Asp Tyr Ala Gln Val Asn Asp Gln Val Thr Phe Ile Asp

310 315 320 325

Asn His Asp Met Glu Arg Phe His Thr Ser Asn Gly Asp Arg Arg Lys

330 335 340

Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser Arg Gly Val Pro Ala

345 350 355

Ile Tyr Tyr Gly Ser Glu Gln Tyr Met Ser Gly Gly Asn Asp Pro Asp

360 365 370

Asn Arg Ala Arg Leu Pro Ser Phe Ser Thr Thr Thr Thr Ala Tyr Gln

375 380 385

Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Ser Asn Pro Ala Ile Ala

390 395 400 405

Tyr Gly Ser Thr His Glu Arg Trp Ile Asn Asn Asp Val Ile Ile Tyr

410 415 420

Glu Arg Lys Phe Gly Asn Asn Val Ala Val Val Ala Ile Asn Arg Asn

425 430 435

Met Asn Thr Pro Ala Ser Ile Thr Gly Leu Val Thr Ser Leu Arg Arg

440 445 450

Ala Ser Tyr Asn Asp Val Leu Gly Gly Ile Leu Asn Gly Asn Thr Leu

455 460 465

Thr Val Gly Ala Gly Gly Ala Ala Ser Asn Phe Thr Leu Ala Pro Gly

470 475 480 485

Gly Thr Ala Val Trp Gln Tyr Thr Thr Asp Ala Thr Thr Pro Ile Ile

490 495 500

Gly Asn Val Gly Pro Met Met Ala Lys Pro Gly Val Thr Ile Thr Ile

505 510 515

Asp Gly Arg Gly Phe Gly Ser Gly Lys Gly Thr Val Tyr Phe Gly Thr

520 525 530

Thr Ala Val Thr Gly Ala Asp Ile Val Ala Trp Glu Asp Thr Gln Ile

535 540 545

Gln Val Lys Ile Pro Ala Val Pro Gly Gly Ile Tyr Asp Ile Arg Val

550 555 560 565

Ala Asn Ala Ala Gly Ala Ala Ser Asn Ile Tyr Asp Asn Phe Glu Val

570 575 580

Leu Thr Gly Asp Gln Val Thr Val Arg Phe Val Ile Asn Asn Ala Thr

585 590 595

Thr Ala Leu Gly Gln Asn Val Phe Leu Thr Gly Asn Val Ser Glu Leu

600 605 610

Gly Asn Trp Asp Pro Asn Asn Ala Ile Gly Pro Met Tyr Asn Gln Val

615 620 625

Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser Val Pro Ala Gly

630 635 640 645

Gln Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln Gly Ser Thr Val Thr

650 655 660

Trp Glu Gly Gly Ala Asn Arg Thr Phe Thr Thr Pro Thr Ser Gly Thr

665 670 675

Ala Thr Val Asn Val Asn Trp Gln Pro

680 685

<210>5

<211>719

<212>PRT

<213>Bacillus stearothermophilus

<220>

<221>mat_peptide

<222>(34)..(719)

<400>5

Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile

-30 -25 -20

Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu

-15 -10 -5

Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln Ile Ile

-1 1 5 10 15

Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys

20 25 30

Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp

35 40 45

Gly Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln

50 55 60

Leu Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asp Asn Leu Asp

65 70 75

Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg

80 85 90 95

Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp

100 105 110

Thr Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp

115 120 125

Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe

130 135 140

Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr

145 150 155

Phe Asp Asp Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp Ile Ser

160 165 170 175

Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro

180 185 190

Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala

195 200 205

Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Ala Asp

210 215 220

Gly Leu Arg Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys

225 230 235

Ser Leu Ala Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly

240 245 250 255

Glu Trp Tyr Gly Asp Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val

260 265 270

Arg Tyr Ala Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn

275 280 285

Thr Val Ile Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp

290 295 300

Leu Asn Asn Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu

305 310 315

Asn Leu Ile Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser

320 325 330 335

Val Asn Ser Asn Lys Ala Asn Leu His Gln Ala Leu Ala Phe Ile Leu

340 345 350

Thr Ser Arg Gly Thr Pro Ser Ile Tyr Tyr Gly Thr Glu Gln Tyr Met

355 360 365

Ala Gly Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp

370 375 380

Thr Thr Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg

385 390 395

Arg Asn Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile

400 405 410 415

Asn Asn Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val

420 425 430

Leu Val Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly

435 440 445

Leu Gln Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly

450 455 460

Leu Leu Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser

465 470 475

Phe Thr Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser

480 485 490 495

Ala Ser Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro

500 505 510

Gly Asn Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly

515 520 525

Thr Val Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser

530 535 540

Asn Arg Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp

545 550 555

Val Lys Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn

560 565 570 575

Ile Leu Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala

580 585 590

Pro Pro Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro

595 600 605

Glu Leu Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala

610 615 620

Gln Gly Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe

625 630 635

Ser Val Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg

640 645 650 655

Ala Asp Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr

660 665 670

Thr Pro Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn

675 680 685

<210>6

<211>534

<212>PRT

＜213〉aspergillus niger

<220>

<221>mat-peptide

<222>(25)..(534)

<400>6

Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly

-20 -15 -10

Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser

-5 -1 1 5

Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala

10 15 20

Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser

25 30 35 40

Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser

45 50 55

Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr

60 65 70

Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val

75 80 85

Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu

90 95 100

Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp

105 110 115 120

Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile

125 130 135

Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr

140 145 150

Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln

155 160 165

Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser

170 175 180

Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser

185 190 195 200

Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln

205 210 215

Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe

220 225 230

Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr

235 240 245

Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp

250 255 260

Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu

265 270 275 280

Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser

285 290 295

Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr

300 305 310

Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu

315 320 325

Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr

330 335 340

Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr

345 350 355 360

Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala

365 370 375

Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala

380 385 390

Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu

395 400 405

Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr

410 415 420

Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr

425 430 435 440

Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly

445 450 455

Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr

460 465 470

Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr

475 480 485

Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr

490 495 500

Arg Ser Gly Met Ser Leu

505 510

Claims (10)

1. be used to produce the single stage method of Zulkovsky starch hydrolysate, this method comprises makes aqueous granular starch slurry carry out the step that acts in the following enzyme under the temperature of the initial gelation temperature that is lower than described granular starch:

First kind of enzyme:

(a) member of the 13rd family's glycosides lytic enzyme;

(b) has α-1.4-glycoside hydrolysis activity; With

(c) comprise the 20th family's carbohydrate binding constituents;

With at least a second kind of enzyme that belongs to beta-amylase (E.C.3.2.1.2) or glucoamylase (E.C.3.2.1.3).

2. the method described in aforesaid right requires, wherein starch slurry contains the dried solid particle starch of the dried solid particle starch of 20-55%, preferred 25-40%, more preferably the dried solid particle starch, especially about 33% dried solid particle starch of 30-35%.

3. any described method during aforesaid right requires, wherein general at least 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% the dried solid of granular starch changes into the Zulkovsky starch hydrolysate.

4. any described method during aforesaid right requires, wherein said first kind of enzyme source are in microorganism and preferably derive from bacterium.

5. any described method during aforesaid right requires, wherein said first kind of enzyme is CGTase (EC 2.4.1.19).

6. any described method during aforesaid right requires, wherein said first kind of enzyme for have at least 3.5, preferably at least 4,4.5,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 or most preferably at least 23 micromoles/minute/CGTase of the hydrolytic activity of mg.

7. any described method during aforesaid right requires, wherein said first kind of enzyme be with Joergensen etc. in (1997), aminoacid sequence has the CGTase of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology shown in Fig. 1 of Biotechnol.Lett.19:1027-1031.

8. any described method during aforesaid right requires, the wherein said first kind of enzyme maltogenic alpha-amylase enzyme (E.C.3.2.1.133) of making a living.

9. any described method during aforesaid right requires, wherein Fructus Hordei Germinatus sugar α-Dian Fenmei derives from bacillus, preferably derives from bacstearothermophilus.

10. any described method during aforesaid right requires, wherein said first kind of enzyme is for having the Fructus Hordei Germinatus sugar α-Dian Fenmei of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology with aminoacid sequence shown in the SEQ ID NO:1 of WO9943794.