CN1166860A - Method for increasing monosaccharide levels in saccharification of starch and enzymes useful therefor - Google Patents
Method for increasing monosaccharide levels in saccharification of starch and enzymes useful therefor Download PDFInfo
- Publication number
- CN1166860A CN1166860A CN95196461A CN95196461A CN1166860A CN 1166860 A CN1166860 A CN 1166860A CN 95196461 A CN95196461 A CN 95196461A CN 95196461 A CN95196461 A CN 95196461A CN 1166860 A CN1166860 A CN 1166860A
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- CN
- China
- Prior art keywords
- enzyme
- isomeric maltose
- hydrolysis
- maltose
- isomeric
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 112
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- 238000000034 method Methods 0.000 title claims abstract description 45
- 235000019698 starch Nutrition 0.000 title claims abstract description 43
- 239000008107 starch Substances 0.000 title claims abstract description 43
- 150000002772 monosaccharides Chemical class 0.000 title abstract description 3
- 230000007062 hydrolysis Effects 0.000 claims abstract description 54
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 54
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- 238000002360 preparation method Methods 0.000 claims abstract description 25
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 136
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- 239000006188 syrup Substances 0.000 claims description 48
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- NBGXQZRRLOGAJF-UHFFFAOYSA-N Maltulose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)(CO)OCC1O NBGXQZRRLOGAJF-UHFFFAOYSA-N 0.000 abstract description 5
- JCQLYHFGKNRPGE-HFZVAGMNSA-N maltulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-HFZVAGMNSA-N 0.000 abstract description 5
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- 125000003275 alpha amino acid group Chemical group 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
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- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
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- 229930003779 Vitamin B12 Natural products 0.000 description 1
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- 229940092253 ovalbumin Drugs 0.000 description 1
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- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
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- -1 α-D-glucopyranosyl-1 Chemical class 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01022—Alpha-galactosidase (3.2.1.22)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
During the commercial production of dextrose from starch, the disaccharide maltulose is produced which cannot be hydrolysed by the enzymes amyloglucosidase, pullulanase or alpha -amylase. The present invention discloses an enzyme preparation for the hydrolysis of maltulose. Enzymatic hydrolysis of maltulose provides a new and additional method for improvement of the monosaccharide levels in the saccharification of starch.
Description
The present invention relates to the enzymolysis of starch.More particularly, the invention provides a kind of method that from the starch of liquefaction, obtains high-content monose.
Known native starch contains two kinds of macromole of being made up of glucose unit.A kind of is the amylose starch of wire, and glucose unit wherein links to each other with α-1,4 key fully.Second kind is the amylopectin that a lot of side chains are arranged, and it removes and contains α-1, outside the 4-key, also contains α-1,6 key.α-1, total body burden of 6-key generally is less than 5%.
The form of the sugar of being made by starch is spissated glucose syrup, it is made by two step enzyme catalysis methods at present, comprising: (a 1) liquefaction (or dilution) step, comprise with α-Dian Fenmei starch is hydrolyzed to the dextrin that mean polymerisation degree is approximately 7-10, (2), obtained having the syrup (92-96wt% of solid matter) of high glucose content thus with starch (dextrin) saccharification of amyloglucosidase with resulting liquefaction.The glucose syrup of most of commercial production all uses the enzyme isomery to turn to the glucose mixture, i.e. isomery syrup (iso-syrup).
At present two kinds of enzymes that use are α-Dian Fenmei and amyloglucosidase, difference are arranged at two aspect important.At first, α-Dian Fenmei is a kind of restriction endonuclease, and it opens the interior keys of starch molecule randomly.And amyloglucosidase is a kind of excision enzyme, and it scales off glucose unit from the non reducing end of dextrin molecule.Secondly, α-Dian Fenmei acts on α-1 almost single-mindedly, and 4-key, and amyloglucosidase also can cut α-1,6 key is although to compare this ratio quite low with α-1,4 key.
The appropriate title of amyloglucosidase is circumscribed-1,4-alpha-D-glucose glycosides enzyme (EC3.2.1.3), and its system is called α-1,4 dextran glucose hydrolysis enzyme.Amyloglucosidase also is known as AG or glucoamylase, should remember that employed hereinafter these speech all are synonyms.
It is inferior optimum that saccharification step in the commercial dextrose production process is considered to for a long time aspect a lot.Particularly not only catalysis glucose formation reaction of the getable amyloglucosidase of institute at present, also its reversed reaction of catalysis is different isomeric maltose with a kind of speed based on glucose concn with conversion of glucose for example.In this manner, to make the starch hydrolysate saccharification be that the DX value of glucose surpasses about 95% at 33%d.s. (dry-matter that promptly contains 33wt% in syrup at least) Shi Buhui in the formation of by product.It is the weight percent of the glucose of benchmark that the DX value is defined as with the carbohydrate dry-matter.
For improving the DX value, the someone proposes debranching factor and amyloglucosidase are used jointly, more effectively will be present in the oligosaccharides that has side chain (containing α-1, the 6 key) hydrolysis in the liquefying starch.European patent application EP-A1-0063909 has described a kind of debranching factor that belongs to Starch debranching enzyme, and it is made by the genus bacillus of a kind of Bacillus of being called acidopullulyticus.It seems that this method the DX value has been improved 0.4-0.6.
Further improve in the method for DX value at another kind, the someone proposes to use a kind of amyloglucosidase (having the side chain vigor in the AG preparation) that has improved the acid starch enzyme level that has.European patent EP B1-0140410 is described this.DX value when this method makes 33%d.s. has improved 0.5.
In the starch liquefacation process, use high temperature usually.This high temperature and applied pH value impel the glucose unit isomery that is present in dextrin molecule reducing end under neutral to turn to fructose jointly.The hydrolysis of dextrin, be included in the reducing end under neutral isomerization glucose (fructose), caused free glucose and a kind of generation that is called the disaccharide of isomeric maltose (maltulose, glucose-α-1,4-fructose).This disaccharide, promptly therefore isomeric maltose can not will be retained in the glucose syrup of making by AG enzyme, Starch debranching enzyme and the hydrolysis of acid starch enzyme institute.The difference of condition during according to liquefaction, the concentration of isomeric maltose can be up to 2% of total reducing sugar.When producing glucose syrup and isomery syrup, the hydrolysis of isomeric maltose will provide a kind of new additional method of the DX of raising value at starch saccharification.Because isomeric maltose can not be utilized by microorganism during the fermentation, isomeric maltose is converted into fermentable sugar, as glucose and fructose, will improve the output of fermentable sugars in the mashing process, and will improve main metabolites such as alcoholic acid output.
The purpose of this invention is to provide the enzyme in a kind of method that can be used for hydrolysis not fermentable sugars, especially isomeric maltose, to produce fermentable sugar, such as glucose and fructose.
Another object of the present invention provides a kind of low cost method, and this method can improve the output of glucose in the mashing process.
A further object of the invention provides a kind of low cost method, and this method can improve main metabolites in the fermenting process, for example alcoholic acid output.
The invention provides a kind of zymin, it comprise can the hydrolysis isomeric maltose enzyme.In a preferred embodiment, zymin comprises isomeric maltose enzyme purifying or enrichment.In a more preferred, the isomeric maltose enzyme extracts from fungi, most preferably is to extract from Aspergillus (Aspergillus) or Trichoderma (Trichoderma).In a specific embodiment, the isomeric maltose enzyme extracts from aspergillus niger (Aspergillus niger), is approximately 132kD with its molecular weight of gel filteration determining.
A kind of method of enzymolysis isomeric maltose is provided in embodiments of the present invention.The present invention also provides a kind of method of syrup saccharification, and it can use zymin malaga in next life syrup of the present invention, has so just improved the syrupy DX value of being produced.The invention also discloses dextrin and glucose syrup, comprise the isomery syrup, they are made by starch and do not contain isomeric maltose.
Fig. 1. the gel-filtration spectrogram of alpha-galactosidase on Sephacryl S 200HR.The relation of OD value (280nm place) and elution time.
The present invention is based on a surprising discovery: a kind of enzyme that exists in the zymotic fluid that obtains with microbe fermentation method can be hydrolyzed to isomeric maltose glucose and fructose. For instance, the enzyme that is applicable to the isomeric maltose hydrolysis can extract from the commodity enzyme preparation, preferably extracts from fungal cellulase and alpha-galactoside enzyme preparation. Have isomeric maltose hydrolysis vigor, namely have isomeric maltose is hydrolyzed to for example enzyme of the ability of glucose and fructose, be called hereinafter the isomeric maltose enzyme. The isomeric maltose enzyme can be purified from zymotic fluid, also can from enzyme preparation, be purified (for example referring to P.K.Scopes 1987 by the purify protein method of those skilled in the art's Application standard, " Protein Purification; Principles and Practice " Springer Verlag, New York, 2nd edn).
Just as shown here, although unexpectedly in the commercial fungal cellulase of respectively or aspergillus mould by wood preparation and alpha-galactosidase, detected the isomeric maltose enzyme activity, very possible fungi from other, or even also can obtain the isomeric maltose enzyme in the microorganism of other kind, thereby microorganism isomeric maltose enzyme all falls within the scope of the present invention usually. If this technology can be by being hydrolyzed instant open the obtaining of vigor to examining and determine isomeric maltose, those skilled in the art just can examine and determine and purification isomeric maltose enzyme from many commercial enzyme preparations or the zymotic fluid that makes from being cultivated by many microorganisms.
Preferably the isomeric maltose enzyme being purified is homogeneous substance, can determine biochemistry, physics and the kinetic parameter of enzyme by it, for example specific activity, molecular weight or have the part or all of amino acid sequence of the polypeptide of isomeric maltose enzyme activity. Resulting part or all of amino acid sequence is used for the design oligonucleotides probe, it can carry out molecular cloning (referring to Sambrook et al.1989 to the gene of coding isomeric maltose enzyme, " Molecular Cloning:a laboratory manual " Cold Spring Harbour Laboratories, Cold Spring Harbour, New York). On the other hand, the isomeric maltose enzyme after the purification also can be used for cultivating antibody, and this antibody capable carries out Gene cloning by expression vector. The isomeric maltose enzyme gene of cloning can be used for making up the overexpression bacterial strain of industrial microorganism, described industrial microorganism is aspergillus, trichoderma, bacillus, streptomyces (Streptomyces), Blastocystis (Saccharomyces) for example, perhaps Kluyveromyces (Kluyveromyces) is (referring to Bennett and Lasure eds., 1991 " More Gene Manipulations in Fungi " Academic Press Inc., New York). The bacterial strain of these high yields provides cheaply a kind of and the pure isomeric maltose enzyme in unlimited source has been arranged actually. No matter be by purifying or the bacterial strain by high yield, resulting isomeric maltose enzyme preparation all can be used for existing enzyme preparation concentrated, perhaps is used for containing the sugar juice of isomeric maltose with its quite pure form. With regard to saccharification, this quite pure isomeric maltose enzyme preferably only has other small enzymatic activity.
The isomeric maltose enzyme preparation of " concentrating " of the present invention is the preparation that extracts from the zymotic fluid that is made by the naturally occurring microbial fermentation that can produce the isomeric maltose enzyme, and the isomeric maltose enzyme concentration of this preparation is higher than unearned concentration from microbial fermentation. On the other hand, concentrated isomeric maltose enzyme preparation can prepare by the purification to the isomeric maltose enzyme, this enzyme comes from the zymotic fluid of microorganism natural or that obtained by genetic engineering, like this, just " has concentrated " the isomeric maltose enzyme with respect to the impurity of removing. Similarly, the isomeric maltose enzyme of having purified can be added in the mixture of naturally occurring enzyme, for instance, comprise amylopectase, glucoamylase or acid starch enzyme in the mixture, at this moment the enzyme concentration that obtains in the zymotic fluid of naturally occurring microorganism of needed enzymatic mixture of the concentration specific energy of isomeric maltose enzyme is high. In addition, concentrated isomeric maltose enzyme preparation can be extracted by the fermentation of genetically modified microorganism, has used recombinant technique in order to increase the expression of isomeric maltose enzyme in the zymotic fluid in this microorganism.
As mentioned above, it is believed that and in many different enzyme preparations, to find a small amount of isomeric maltose enzyme. Yet, the amount of cultivating the isomeric maltose enzyme of producing by microorganism under the condition of producing commodity enzyme preparations (being the commodity alpha-galactosidases) is not enough to that usually saccharifying is had actual promotion, and perhaps this kind of enzyme is present in the saccharification stage and usually is not used in the enzyme preparation of starch matrix (being commercial fibre element enzyme). In a particularly preferred embodiment of the present invention, concentrated the isomeric maltose enzyme content in a kind of enzyme preparation, in order to isomeric maltose hydrolysis vigor is brought up to the level of remarkable commercial value. The isomeric maltose enzyme that adds should be enough to the most of isomeric maltose in the hydrating solution. The amount of the isomeric maltose enzyme that certainly, adds according to the present invention depends on the amount of the isomeric maltose in starch or the sugar juice. For example, contain 2% isomeric maltose containing 40% in the sugar juice of material, the amount of isomeric maltose approximately is 8g/kg sugar. Like this, if 1 unit (U) equals the isomeric maltose that per minute is hydrolyzed 1 μ mol, the isomeric maltose in the hydrating solution just needs with 6U/kg syrup effect 72 hours so. Perhaps, the isomeric maltose in the hydrating solution just needs with 432U/kg effect 1 hour. Therefore, it is reasonable that the isomeric maltose hydrolysis vigor of enzymatic compositions of the present invention is higher than about 1U/kg sugar d.s., be more preferably 50 and 5000U/kg sugar d.s. between, most preferably be 50 and 1000U/kg sugar d.s. between.
Use the isomeric maltose enzyme that isomeric maltose is hydrolyzed to monose, promptly glucose and fructose will improve the DX value in the production of glucose syrup, and this syrup can directly be used subsequently or be used to produce the isomery syrup.The isomery syrup that the enzymically hydrolyse of isomeric maltose will cause glucose syrup or isomeric maltose level to reduce, better be to be lower than 0.5% isomeric maltose (per-cent of isomeric maltose in total reducing sugar), but perhaps preferably be lower than detection level, be approximately 0.1% (promptly being substantially free of isomeric maltose).The hydrolysis of isomeric maltose should preferably be carried out between pH is approximately 3 to 7, more preferably carries out between pH is approximately 4 to 5; Preferred temperature range is about 15 ℃ to 70 ℃, is more preferably about 20 ℃ to 60 ℃.
The method of hydrolysis isomeric maltose provided by the present invention is preferably in to be used the latter stage of conventional saccharifying.Yet because isomeric maltose can not be by AG enzyme, Starch debranching enzyme and acid starch enzymic hydrolysis, the further embodiment of the inventive method provides the application of the isomeric maltose that combines with the employed method of the state of the art.Therefore, the common use of Starch debranching enzyme, glucoamylase or acid starch enzyme and isomeric maltose enzyme will further improve the DX value in the saccharifying.The common use of isomeric maltose enzyme, Starch debranching enzyme and acid starch enzyme makes height when the DX value ratio of prepared glucose syrup is only common to use Starch debranching enzyme and acid starch enzyme.In fact, method of the present invention can be at an easy rate improves 2 units with the DX value of glucose syrup.
In a preferred embodiment, in the starch solution of liquefaction, use isomeric maltose enzymic hydrolysis isomeric maltose.Therefore, for instance, the isomeric maltose enzyme just be introduced into α-Dian Fenmei and passes through in the made liquefying starch of steam ejection liquefaction starch.Perhaps, the isomeric maltose enzyme can add in saccharification step simultaneously with glucoamylase.In another method, the isomeric maltose enzyme can add after handling liquefying starch with glucoamylase, with the DX value of further raising starch saccharification.At last, isomerization the glucose syrup can handle with the concentration of further raising glucose and fructose and reduce the amount of isomeric maltose impurity with the isomeric maltose enzyme.Above-mentioned each method all will benefit from enzyme of the present invention by the output that increases fermentable sugars.Yet, in a given process, select to use which kind of method will depend on the special parameter of this process operation.And those skilled in the art is easy to determine which kind of method of given starch treatment process to be optimum.The hydrolysing step of isomeric maltose is preferably in the saccharifying or carries out behind the saccharifying or in the fermenting process, because the concentration of isomeric maltose is the highest in these steps.
The isomeric maltose hydrolysis will cause the high yield of fermenting process, and glucose syrup is used as the carbon source and/or the energy in the described fermentation.The example that this point is described is by the glucose syrup fermentative production of ethanol.Because by starch being hydrolyzed to the existence of isomeric maltose in the glucose syrup that glucose obtains,, will be improved based on the alcohol yied of glucose syrup consumption along with the hydrolysis of isomeric maltose.When isomeric maltose can be hydrolyzed to fermentable sugars, the productive rate of the biomass in other fermenting process, (elementary and secondary) meta-bolites, medicine (penicillin) or enzyme can be improved equally.Thereby in a version of the present invention, glucose syrup or isomery syrup are used as the energy in the fermentation process or the source of primary metabolite.
The following examples only are to explanation of the present invention, they can not be interpreted as limitation of the present invention.
Embodiment
Embodiment 1
The syrupy analysis of commodity glucose syrup and isomery
Glucose syrup and the glucose isomery syrup made by starch contain isomeric maltose.The enforcement of analyzing is by providing the different syrup that is made by the saccharification of commodity dextrin mixture to carry out.The implementation condition of saccharification is 60 ℃, and pH4.2 was with amyloglucosidase (Amigase fromGist-brocades) effect 48 hours.From the glucose syrup that obtains, take a sample, analyze wherein isomeric maltose content with HPLC by following description.These analyses the results are shown in table 1 and table 2.As shown in Table 1 and Table 2, the general saccharification step that is applied to commodity dextrin mixture will cause containing in the syrup isomeric maltose.Sugar concentration usable highly effective liquid chromatographic detection.A suitable method is used following condition: post: Bio-Rad HPX 87C elutriant: distilled water column temperature: 85 ℃ of flow velocitys: 0.6ml/min detection method: RI (refractive index) detection method
Table 1
| The syrup sample | Fructose % | Glucose % | Maltose % | Isomeric maltose % | ????DP3 ????％ | ???DP4+ ????％ |
| Derive from the saccharification Maldex 15 of Amylum | ????0.0 | ????92.51 | ????2.34 | ????0.62 | ????0.58 | ????3.92 |
| Derive from the saccharification MD 03 of Roquette | ????0.0 | ????91.42 | ????2.06 | ????1.75 | ????0.73 | ????4.03 |
| Dormamix 98/70(Pfeiffer& Langen) | ????0.1 | ????93.7 | ????2.1 | ????0.5 | ????2.1 | ????1.4 |
| Levudex?42 (IMASA) | ???41.9 | ????52.5 | ????3.3 | ????0.3 | ????0.7 | ????1.3 |
| Levudex?55 (IMASA) | ???53.5 | ????41.8 | ????2.7 | ????0.3 | ????0.5 | ????1.1 |
| Glucose syrup (Barendse) | ????0.0 | ????87.0 | ????6.7 | ????1.2 | ????2.8 | ????2.3 |
| Glucose syrup (Cerestar) | ????0.0 | ????94.3 | ????3.8 | ????0.5 | ????0.8 | ????0.7 |
| Gelastin T58 (Cerestar) after the saccharification | ????0.0 | ????89.4 | ????5.9 | ????0.8 | ????2.1 | ????1.8 |
| Glucose syrup (Amylum) | ????0.0 | ????96.6 | ????2.2 | ????0.2 | ????0.4 | ????0.6 |
| Glucose syrup (cargill) | ????0.0 | ????94.7 | ????2.3 | ????1.0 | ????0.6 | ????1.3 |
Embodiment 2
The check and analysis of isomeric maltose enzyme
(A) preparation of isomeric maltose
Isomeric maltose (maltulose) is a disaccharide, i.e. α-D-glucopyranosyl-1,4-α-Fu Nan fructose.This disaccharide can pass through maltose (α-D-glucopyranosyl-1,4-α-pyranoglucose) alkali isomerization of the glucosyl residue of the reducing end under neutral of this disaccharide makes, method is as follows: the maltose solution of 2g aluminum oxide with 100ml 40% (w/v) mixed, with sodium hydroxide the pH value is adjusted to 11.5.Reaction mixture was preserved 24 hours down at 60 ℃.Then the pH value is transferred to 4.5 and add the 5g bread yeast, purpose is to make maltose and other fermentable sugars fermentation (isomeric maltose can not be fermented by yeast) that is produced under alkaline culture condition.At last reaction mixture is filtered to obtain clear soln, it is concentrated under vacuum to remove ethanol (producing during the fermentation) and to obtain a kind of isomeric maltose solution with high dry matter content.
(B) enzymically hydrolyse of isomeric maltose
Investigated the isomeric maltose hydrolysis vigor of many commercial enzyme preparations.Zymin is mixed with 5% isomeric maltose solution in distilled water.For each enzyme, all be that the 5mg enzyme is joined in the isomeric maltose solution of 5ml.Mixture is incubated under the listed condition of table 2.Reaction mixture is analyzed with the HPLC described in the embodiment 1.These analyses the results are shown in table 2.
Table 2
| Group number | Zymin | Temperature, ℃ | ??pH | Soaking time, hour | Fructose % | Glucose % | Isomeric maltose % | Other % |
| ???1 | Initial substance | ???0.0 | ??0.0 | ????87.0 | ???13.0 | |||
| ???1 | T.reesei cellulase MAXAZYME CL 2,000 (Gist-brocades) | ????50 | ??4.5 | ????40 | ??38.0 | ??29.4 | ????24.0 | ????8.6 |
| ???1 | The Kluyveromyces lactis beta-galactosidase enzymes; MAXILACTLX 5,000 (Gist-brocades) | ????37 | ??6.5 | ????40 | ??0.0 | ??0.0 | ????86.9 | ???13.1 |
| ???1 | Yeast invertase; MAXINVERTL 10,000 (Gist-brocades) | ????50 | ??4.5 | ????40 | ??0.0 | ??0.0 | ????91.5 | ????8.5 |
| ???2 | Initial substance group 2 | ??0.0 | ??0.0 | ????56.2 | ???43.8 | |||
| ???2 | Aspergillus niger alpha-galactosidase SUMIZYME AGS (Shin Nihon) | ????60 | ??4.2 | ????4 | ??15.5 | ??14.6 | ????11.5 | ???58.4 |
Embodiment 3
With cellulase preparation after the AG saccharification to the influence of monose level
Cellulase preparation joins the 20mg cellulase in the 10ml glucose syrup the influence of sugar soln monose level after the saccharification in order to be presented at.This glucose syrup is with AG the dextrin solution saccharification of 33%w/w to be made.When reaching maximum, the DX value place 100 ℃ water-bath to make the AG inactivation in 10 minutes glucose syrup.At the adding cellulase and at pH=4.5, insulation with 0.1ml reaction mixture 3ml distilled water diluting, placed boiling water bath that reaction is stopped the diluted mixture thing after 1 hour under 50 ℃ the condition.At last, sample is analyzed with HPLC by the method that embodiment 1 describes.The results are shown in table 3.
Table 3
(DP represents the polymerization degree; DP4+ represents that the polymerization degree is 4 or higher)
| Sample | ????DP1％ | ???DP2％ | ???DP3％ | ???DP4％ |
| Initial substance (glucose syrup) | ????94.5 | ???3.97 | ???0.69 | ???0.84 |
| With T.reesei MaxazymeCL 2000 cellulose treatment; Gist-brocades | ????95.06 | ???3.61 | ???0.63 | ???0.69 |
The listed result of table 4 shows that the adding of cellulase preparation makes the monose level improve 0.5-0.6DX.
Should be realized that, to have only following claims just to limit scope of the present invention although the description of front and embodiment are to explanation of the present invention.
Embodiment 4
The purify hydrolysis vigor of isomeric maltose and residual sugar of part from Sumizyml AGS
(Sumizyml AGS, ShinNihon Japan) carry out partial purification to the alpha galactosides zymin to use gel filtration chromatography.To being described below of this step.Screen each fraction of the different vigor of collected demonstration.Interested fraction collection is used to determine compare vigor.Chromatrographic separation step: post: 58 * 2.5cm.Carrier substance: Sephacryl S 200HR.Elution buffer: the 50mM acetate buffer solution, pH=4.5 comprises 0.02% sodiumazide.Flow velocity: 2ml/min.Detect: UV-light 280nm.Fraction is collected: 2 minutes fractions.Sample: concentration is the Sumizyme AGS (lot60902-02) of 30mg/ml in the 4ml elution buffer.Vigor detects (screening of fraction): 1. the detection of alpha galactosides enzyme activity.
To be dissolved in the collected fraction of the p-nitrophenyl-α-D-semi-lactosi of 100 μ l 1mM in the 50mM acetate buffer solution of pH5.5 and 100ml is incubated jointly.After at room temperature being incubated 3 minutes, the sodium borate buffer liquid of 0.0625M that adds 100 μ l pH9.7 is with stopped reaction.
The activity of the yellow metering alpha-galactosidase that produces with p-NP.The result with the naked eye judges.2. isomeric maltose hydrolysis vitality test.
With the distilled water diluting of isomeric maltose preparation with 4 times.100 these solution of μ l are mixed with fraction and the 700 μ l distilled water that 200 μ l have isomeric maltose hydrolysis vigor.This mixture is incubated 3 hours down at 33 ℃.Then place boiling water bath to make enzyme deactivation in mixture.Use Bio-Rad HPX 87C post on HPLC, mixture to be analyzed at last.Vigor is directly measured in increase with the fructose peak area.3. residual sugar hydrolysis vigor
50 μ l are had the fraction of residual sugar hydrolysis vigor and 50 μ l beer, and (from wet-milling alcohol fuel preparation, Pekin Energy Pekin.Illinois) mixes, then 33 ℃ of insulations 16 hours down.The sulfuric acid that adds 900 μ l 0.006N then is also centrifugal on an Eppendorf whizzer with this reaction mixture.Supernatant liquor is analyzed with HPLC on BIO-RAd HPX 87H post.Vigor is directly measured in minimizing with the residual sugar peak area.
Mensuration than vigor:
Be expressed as the energy value in every mg protein per minute reaction times than vigor.1. the mensuration of protein content.
Measuring Protein content with the BCA method, is standard with the bovine serum albumin.2. the vigor of alpha-galactosidase.
With 10mM right-nitrophenols is dissolved in the acetate buffer solution of 50mM, pH value 5.5 and makes solution.This solution diluted respectively be 240-160-80-40mM.These solution of 1ml are joined in the right-oil of mirbane-α-D-galactopyranoside of 0.8mM in the 2ml acetate buffer solution respectively.The borate buffer (terminator) that in this mixed solution, adds the 625mM of 5ml pH9.7.With water is with reference to the OD value (typical curve) of measuring these solution under 405nm.
The insulation of enzyme: the enzyme solution with the 1ml dilution replaces right-nitrophenols.Mixture is incubated 15 minutes down at 37 ℃.Adding the 5ml boric acid solution stops reaction.Measure the OD value with above-mentioned method.
The definition of vigor: the alpha-galactosidase of 1 unit is meant the enzyme amount of per minute hydrolysis 1 μ mol p-NPGal under standard conditions.3. isomeric maltose hydrolysis vigor.
The isomeric maltose solution of 100 μ l is mixed with 200 μ l enzyme solution and 700 μ l distilled water.Mixture is incubated down at 33 ℃.Sampling and it is analyzed amount with the isomeric maltose of measuring hydrolysis when different soaking times with HPLC.4. the hydrolysis vigor of residual sugar.
100 μ l are mixed from the beer of temperature mill alcohol product and 200 μ l enzyme solution be incorporated in 33 ℃ and be incubated down.Sulfuric acid with 700 μ l 8mM joins in the sample that different soaking times are taken out respectively.With HPLC sample is analyzed amount with the residual sugar of measuring hydrolysis.Definition than vigor: 1. alpha-galactosidase: the alpha galactosides unit of enzyme in every mg protein.2. isomeric maltose hydrolysis vigor: the mg number of every mg protein per minute hydrolysis isomeric maltose.3. residual sugar hydrolysis vigor: the μ g number of every mg protein per minute hydrolysis residual sugar.The mensuration of molecular weight:
Use standard merchandise protein mixture (Bio-Rad) as the molecular weight marker thing, as follows: thyroglobulin (MW=670kD), gamma globulin (MW=158kD), ovalbumin (MW=44kD), myohaemoglobin (MW=17kD), vitamin B12 (MW=13.5kD).Gel-filtration result to marker compares with the isomeric maltose enzyme that carries out subsequently and the gel-filtration result of residual sugar lytic enzyme, and the result shows that the roughly molecular weight of isomeric maltose enzyme is respectively about 132kD and 120kD.The result:
The spectrogram that is obtained by gel-filtration alpha galactosides zymin is shown in Fig. 1.Analytical results to alpha galactosides enzyme activity, isomeric maltose hydrolysis vigor and residual sugar hydrolysis vigor in each fraction is listed in the following table 4.This result is used to collect each fraction.Alpha-galactosidase concentrates on (elution time=55-60 minute) in the 8-10 fraction.Residual sugar and isomeric maltose hydrolysis vigor concentrate on (elution time=63-68 minute) in the 12-14 fraction.Collected fraction and parent material are compared vitality test.The results are shown in table 5.
Table 4
| The fraction number | The fraction elution time | Alpha galactosides enzyme activity (yellow) | Isomeric maltose hydrolysis vigor | Residual sugar hydrolysis vigor (reducing of peak) |
| ??????2 | ?????43-44 | |||
| ??????4 | ?????47-48 | ????????+ | ????????2 | |
| ??????6 | ?????51-52 | ????????++ | ????????3 | ???????11 |
| ??????8 | ?????55-56 | ???????+++ | ???????33 | ???????37 |
| ??????10 | ?????59-60 | ???????+++ | ??????100 | ???????63 |
| ??????12 | ?????63-64 | ????????++ | ???????93 | ??????100 |
| ??????14 | ?????67-68 | ????????+ | ???????91 | ??????100 |
| ??????16 | ?????71-72 | ???????67 | ???????58 | |
| ??????18 | ?????75-76 | ????????9 | ???????14 | |
| ??????20 | ?????79-80 | ????????2 | ||
| ??????22 | ?????83-84 | |||
| ??????24 | ?????87-88 | |||
| ??????26 | ?????91-92 | |||
| ??????28 | ?????95-96 | |||
| ??????30 | ?????99-100 | |||
| ??????32 | ????103-104 | |||
| ??????34 | ????107-108 | |||
| ??????36 | ????111-112 | |||
| ??????38 | ????115-116 | |||
| ??????40 | ????119-120 | |||
| ??????42 | ????123-124 | |||
| ??????44 | ????127-128 | |||
| ??????46 | ????131-132 | |||
| ??????48 | ????135-136 |
Table 5
Discussion/conclusion
| Material | Alpha-galactosidase is than vigor, unit/mg | Ratio | The isomeric maltose hydrolysis is than vigor, mg isomeric maltose/mg.min | Ratio | The residual sugar hydrolysis is than vigor, mg residual sugar/mg.min | Ratio |
| Initial substance | ??????8100 | ???????4.82 | ???????0.52 | |||
| The 8-10 fraction | ??????184000 | ???22.7 | ???????87.0 | ???18.1 | ???????12.9 | ???24.8 |
| The 12-14 fraction | ??????16900 | ????2.1 | ??????163.8 | ???34.0 | ???????28.4 | ???54.6 |
Shown in table 4 and table 5, isomeric maltose and residual sugar hydrolysis vigor are that the pair of alpha galactosides zymin belongs to vigor, and this is not to be produced by alpha-galactosidase self.In addition, it seems the ratio vigor that can improve two kinds of enzymes by the single step purification step significantly.
Embodiment 5
The heat inactivation of alpha galactosides enzyme activity
With the alpha galactosides enzyme solution 65 ℃ of following thermal treatments 30 minutes.Measure the ratio vigor of the solution after initial substance and the thermal treatment then.
This result of experiment is listed in table 6.
Table 6
| Material | The ratio vigor of alpha-galactosidase, unit/mg | Ratio | The isomeric maltose hydrolysis is than vigor, mg isomeric maltose/mg.min | Ratio | The residual sugar hydrolysis is than vigor, mg residual sugar/mg.min | Ratio |
| Initial substance | 8100 | 4.82 | 0.52 | |||
| Heat treated material | 18.5 | .002 | 4.19 | 0.9 | 0.42 | 0.8 |
The result shows that alpha galactosides enzyme activity and residual sugar come from different enzymes with isomeric maltose hydrolysis vigor.
Claims (14)
1. zymin, it comprise can the hydrolysis isomeric maltose enzyme, wherein this kind of enzyme preparation concentrates with this enzyme that can the hydrolysis isomeric maltose.
2. according to the zymin of claim 1, enzyme that wherein can the hydrolysis isomeric maltose obtains from microorganism.
3. according to the zymin of claim 2, wherein microorganism is a fungi.
4. according to the zymin of claim 3, fungi wherein is the bacterial strain of Aspergillus or Trichoderma.
5. according to the zymin of claim 4, wherein this kind of enzyme preparation contains the isomeric maltose enzyme that obtains from aspergillus niger, measures with gel filtration method, and this enzyme molecular weight approximately is 132kD.
6. the application of zymin in the hydrolysis isomeric maltose.
7. according to the application of zymin in the hydrolysis isomeric maltose of claim 1.
8. the method for a hydrolysis isomeric maltose is characterized in that using a kind of zymin that is used for the hydrolysis isomeric maltose.
9. produce the method for glucose from a kind of solution, this solution contains glucose, isomery syrup and/or contains the dextrin of isomeric maltose, and this method comprises:
A) a kind of aqueous solution that contains glucose, isomery syrup and/or dextrin of preparation;
B) add the zymin contain enzyme that can the hydrolysis isomeric maltose in above-mentioned solution, wherein this kind of enzyme preparation concentrates with this enzyme that can the hydrolysis isomeric maltose; And
C) solution that obtains in (b) is incubated for some time under the condition that is suitable for the hydrolysis isomeric maltose.
10. the method according to claim 9 further comprises the application of Starch debranching enzyme, acid starch enzyme or its composition.
11. a fermentation process comprises the application of the glucose syrup of producing according to claim 7.
12. a kind of fermentation process according to claim 11 is used for fermentative production of ethanol.
13. one kind that obtain from starch, do not contain the glucose syrup of isomeric maltose substantially.
14. one kind that obtain from starch, do not contain the isomery syrup of isomeric maltose substantially.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94203124 | 1994-10-27 | ||
| EP94203124.6 | 1994-10-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1166860A true CN1166860A (en) | 1997-12-03 |
Family
ID=8217318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95196461A Pending CN1166860A (en) | 1994-10-27 | 1995-10-26 | Method for increasing monosaccharide levels in saccharification of starch and enzymes useful therefor |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0787202A1 (en) |
| JP (1) | JPH10512741A (en) |
| KR (1) | KR970707294A (en) |
| CN (1) | CN1166860A (en) |
| AU (1) | AU4278896A (en) |
| CA (1) | CA2203812A1 (en) |
| FI (1) | FI971782A0 (en) |
| WO (1) | WO1996013602A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017120170A1 (en) * | 2016-01-05 | 2017-07-13 | Cargill, Incorporated | Method for fermenting sugars |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3475209D1 (en) * | 1983-09-11 | 1988-12-22 | Gist Brocades Nv | Novel enzyme product and its use in the saccharification of starch |
| US4717662A (en) * | 1985-01-31 | 1988-01-05 | Miles Laboratories, Inc. | Thermal stabilization of alpha-amylase |
| ATE80657T1 (en) * | 1986-07-09 | 1992-10-15 | Novo Nordisk As | MIXTURES OF ALPHA-AMYLASE FOR LIQUEFYING STARCH. |
| US5231017A (en) * | 1991-05-17 | 1993-07-27 | Solvay Enzymes, Inc. | Process for producing ethanol |
-
1995
- 1995-10-26 EP EP95941339A patent/EP0787202A1/en not_active Withdrawn
- 1995-10-26 CA CA002203812A patent/CA2203812A1/en not_active Abandoned
- 1995-10-26 CN CN95196461A patent/CN1166860A/en active Pending
- 1995-10-26 JP JP8514742A patent/JPH10512741A/en active Pending
- 1995-10-26 WO PCT/US1995/013879 patent/WO1996013602A1/en not_active Application Discontinuation
- 1995-10-26 KR KR1019970702751A patent/KR970707294A/en not_active IP Right Cessation
- 1995-10-26 AU AU42788/96A patent/AU4278896A/en not_active Abandoned
-
1997
- 1997-04-25 FI FI971782A patent/FI971782A0/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CA2203812A1 (en) | 1996-05-09 |
| JPH10512741A (en) | 1998-12-08 |
| FI971782A (en) | 1997-04-25 |
| WO1996013602A1 (en) | 1996-05-09 |
| EP0787202A1 (en) | 1997-08-06 |
| AU4278896A (en) | 1996-05-23 |
| KR970707294A (en) | 1997-12-01 |
| FI971782A0 (en) | 1997-04-25 |
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