CN110004128A - Compounded saccharifying enzyme preparation and amylolytic method - Google Patents
Compounded saccharifying enzyme preparation and amylolytic method Download PDFInfo
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- CN110004128A CN110004128A CN201910205311.4A CN201910205311A CN110004128A CN 110004128 A CN110004128 A CN 110004128A CN 201910205311 A CN201910205311 A CN 201910205311A CN 110004128 A CN110004128 A CN 110004128A
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- C—CHEMISTRY; METALLURGY
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- 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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
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- C12P19/02—Monosaccharides
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- 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)
- C12Y302/01041—Pullulanase (3.2.1.41)
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Abstract
The present invention relates to production starch sugar fields, and in particular to compounded saccharifying enzyme preparation and amylolytic method.The preparation contains the first carbohydrase and Pullulanase, and first carbohydrase is the carbohydrase of the aspergillus niger secretion after inactivating agdB gene, and the enzyme activity ratio of the first carbohydrase and Pullulanase is (7.5-80): 1.Back reaction can be effectively reduced in compounded saccharifying enzyme preparation of the present invention, obtains the DX higher of product, while can substantially shorten saccharification period, extends saccharification window.
Description
Technical field
The present invention relates to production starch sugar fields, and in particular to compounded saccharifying enzyme preparation and amylolytic method.
Background technique
Starch sugar is the starch that is rich in using cereal, potato etc. as raw material, under the catalysis of enzyme, by liquefying, being saccharified etc. one
Serial reaction, the bulk product of production.There are many function of starch sugar, both can be used as food additives, can also be used as
The important raw material of industry, moreover it is possible to by the products such as deep processing functional oligosaccharide, vitamin, amino acid again.
Starch sugar sugar refining technology generally uses two enzymes method at present, that is, includes enzyme solution and Mashing process.Wherein be saccharified work
Skill refers in starch slurry after being liquefied as dextrin, and glucoamylase (also known as carbohydrase), Pullulanase etc. is added, and grasps addition
Enzyme amount and enzyme concentration control reaction temperature, time, the process of final production dextrose syrup.Wherein glucoamylase is one
Class excision enzyme cuts α-Isosorbide-5-Nitrae glucoside bond from the non reducing end of starch molecule one by one, generates glucose, and glucose forms sediment
Powder enzyme effect is bigger than short-chain active in long-chain, and with the progress of hydrolysis, dextrin chain can be smaller and smaller, so as to cause hydrolysis rate change
Slowly.In addition, glucoamylase can be with hydrolyzing alpha -1,6 key, but hydrolysis rate is very slow.And amylopectin in cornstarch
Content is up to 70% or more, and contained α -1,6 glycosidic bonds are the main reason for causing the cornstarch saccharification later period to slow.
Commercialized carbohydrase is to be got by fermentation of Aspergillus niger, but aspergillus niger can also express a small amount of protease, form sediment during the fermentation
Powder enzyme and alpha-glucosidase (also referred to as turning glycosides enzyme) etc..Alpha-glucosidase influences maximum to the quality of carbohydrase, it can be urged
Change glucosyl group to be transferred on another glucose or maltose etc., turn glycosides site mostly on 6-OH, generates isomaltose, panose
Deng；It can also be transferred on 2-OH, 3-OH of another glucosyl group, kojibiose and nigerose be generated, to reduce saccharification
The DX value of product glucose syrup (DX value refers to that glucose accounts for the mass percent of syrup).
And in traditional starch sugar refining technology, generally concentration of substrate is used to be saccharified for the liquefier of 25%-35%.
This typical sugar refining technology benefit is general.Two aspects are mainly reflected in, first is that glucose yield is not high, current highest DX
It is worth production level (in the case where 32% substrate) only 96.5% or so, has very large space further to be promoted；Another
It is the technique water consume, energy consumption is high.Actually enzymatic reaction only needs to consume minimal amount of water, since slurry concentration is dilute, under causing
Have to evaporate a large amount of water in trip technique liquid glucose concentration process, expends a large amount of energy, production cost is caused to increase.Some
In fermentation industry, the sugar concentration for mending sugar is usually required 40% or more；In fructose production, ingredient initial concentration is on 35% left side
The right side needs to be concentrated to 42-45% or so and carries out isomerization, if ingredients concentration can be increased to 40-45%, so that it may save
Evaporization process before isomery reduces energy consumption to reduce fixed investment, simplifies process.And in saccharifying, with concentration of substrate
Raising, the viscosity of reaction solution increases, and will lead to saccharification reaction rate and the decline of glucose yield；In addition in high concentration substrate item
Under part, product purity also will receive influence.
CN103981239B discloses the ratio for only passing through when ingredients concentration is 50% and changing carbohydrase and Pullulanase
Come improve final product DX value method, but saccharification result is general, only up to 93.65%.
Therefore, need it is a kind of can be effectively reduced in saccharifying be especially high concentration substrate saccharifying in it is inverse
Reaction, improve product DX enzyme and amylolytic method.
Summary of the invention
The purpose of the invention is to overcome the problems such as saccharification product purity of the existing technology is not high, process is tedious,
Provide compounded saccharifying enzyme preparation and amylolytic method.
The inventors found that influencing the factor of high concentration substrate saccharification mainly has following two points: (1) turning glycosides enzyme activity
Otherwise power, carbohydrase must can aggravate the generation of back reaction, generate more isomaltose, panose etc. without glycosides enzyme activity is turned,
Influence the DX value of final syrup；(2) the proportion needs of the proportion of carbohydrase and Pullulanase, Pullulanase increase substantially.
To achieve the goals above, first aspect present invention provides a kind of compounded saccharifying enzyme preparation, and the preparation contains
First carbohydrase and Pullulanase, first carbohydrase are the carbohydrase of the aspergillus niger secretion after inactivating agdB gene.
Second aspect of the present invention provides a kind of amylolytic method, which comprises
(1) feed liquid is configured using starch；
(2) it is saccharified in the presence of compounded saccharifying enzyme preparation described in first aspect present invention to feed liquid.
Compounded saccharifying enzyme preparation of the present invention in saccharifying particularly in the saccharifying of high concentration substrate, can
To efficiently reduce back reaction, the DX higher of product is obtained, while can substantially shorten saccharification period, extends saccharification window.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
Term is explained
Term " carbohydrase ": also known as glucoamylase [Glucoamylase, (EC.3.2.1.3.)], can by starch from
Irreducibility end hydrolyzing alpha-Isosorbide-5-Nitrae glucoside bond generates glucose, also can slow hydrolyzing alpha -1,6 glucoside bond, be converted into
Glucose；Simultaneously also can hydrolyse dextrin, the non-reducing end of glycogen discharges β-D-Glucose.Carbohydrase with glucose for being sent out
The fermentation of the various antibiotic, organic acid, amino acid, vitamin of ferment culture medium is also used to produce the glucose of various specifications, all
It is all applicable in the process to starch, dextrin progress enzyme hydrolysis.
Term " alpha-glucosidase ": α-glucosidase, abbreviation AGD enzyme, EC 3.2.1.20 also known as alpha-D-glucose
Glycosides hydrolase, it can cut α-Isosorbide-5-Nitrae glycosidic bond from the non-reducing end of oligosaccharides substrate, release glucose；Or it will trip
The glucose residue separated out is transferred to formation α -1 on another saccharide substrates, 6 glycosidic bonds, to form the function with non-fermented
It can property oligosaccharide --- oligoisomaltose (abbreviation IMOs mainly includes isomaltose, panose, Isomaltotriose etc.).Term
" saccharification ", which refers to the process of, is decomposed into glucose for short chain dextrin.
Term " DX value " refers to that glucose accounts for the mass percent of syrup.
Term " DE value " refers to that reduced sugar accounts for the mass percent of syrup.
Term " weight (or DS) based on starch on dry basis " refers to be calculated on the basis of the weight of starch on dry basis.
Term " about " refers to ± the 10% of fiducial value.
Term " enzyme activity ratio " refers to the ratio between enzyme activity (unit U/g or U/mL), wherein carbohydrase and Propiram
The enzyme activity determination of enzyme is according to national standard GB 1886.174-2016.
First aspect present invention provides a kind of compounded saccharifying enzyme preparation, and the preparation contains the first carbohydrase and Propiram
Enzyme, first carbohydrase are the carbohydrase of the aspergillus niger secretion after inactivating agdB gene.
Here, the agdB gene is a kind of gene for expressing alpha-glucosidase.
According to the present invention, first carbohydrase is the carbohydrase of the aspergillus niger secretion after inactivating agdB gene.Here,
AgdB gene can turn glycoside enzyme gene for one kind well known in the art, such as can be gene order be described in SEQ ID NO:1
Gene.
Nucleotide sequence shown in SEQ ID NO:1 are as follows: atgttggggt ctttgctttt actcttaccc
cttgtgggcg ctgctgtcat tggacccagg 60
gcaaacagtc agagttgccc agggtataag gcgtccaacg tccaaaagca ggctaggtca 120
ctgactgcgg atctgactct agctggtacg ccttgtaata gctatggcaa ggatttggaa 180
gacctcaagc tgcttgtgga atatcagact ggtgagtgtt ggcttgtgtg aatcaagagt 240
tcctgactaa atgcttgctc agatgaacgg ttacatgtta tgatctacga tgccgacgag 300
gaagtctatc aagttcctga atcagtcctt cctcgcgtgg gtagtgacga ggactctgag 360
gacagtgttt tggaatttga ctatgtggaa gaaccgtttt cattcaccat ctccaaggga 420
gatgaggtcc tgtttgactc ttcggcatca ccactagttt ttcagtcgca atatgtgaac 480
cttcgcacct ggttgcccga tgatccctat gtgtatggtc tcggagagca ttctgaccct 540
atgcgcttgc caacatacaa ttacacgcgg accctttgga accgcgacgc gtatggcact 600
ccaaacaaca ccaacttgta cggtagtcat cctgtctact atgatcaccg tggaaagtcc 660
ggaacttatg gagtcttcct gctgaactct aatggtatgg acatcaagat caaccaaacg 720
acagatggaa agcagtactt ggaatacaat cttctcggcg gtgttctgga cttctacttc 780
ttctacggag aagatcctaa gcaagcgagc atggaatact caaagattgt cggtctcccg 840
gcaatgcaga gttactggac tttcggcgta tgccccccac cccctaatcc cataacagtc 900
cgagttgtat gctgactctt cagttccatc aatgccgtta tggataccgc gatgtgtatg 960
aacttgccga ggtggtctac aactacagcc aggcaaagat tcctctggag acgatgtgga
1020
cagatatcga ctacatggac aagagaaggg tgtttaccct tgatcctcag aggttcccgc
1080
tcgaaaagat gcgggagttg gtaacctacc tgcacaatca tgatcagcat tacattgtca
1140
tggttgaccc ggctgtgagc gtaagcagtg agtgacttga cgattcccca tccttgcaac
1200
tttcagctaa tggatacttt ctagataaca cggcatatat caccggcgtg agagacgatg
1260
ttttccttca caatcagaac ggtagcctat acgagggtaa gtatatacac atctcatatc
1320
tctcaacacg agctaaacta tgcaggtgct gtttggcctg gtgtcactgt tttcccagac
1380
tggttcaatg agggtactca ggattactgg actgcgcaat ttcaacagtt ctttgatccc
1440
aagtccggag tcgatattga cgccctgtgg attgacatga acgaagcctc caatttctgc
1500
ccttatcctt gtctggaccc agcggcatac gcgatctccg ccgacctccc accggcagca
1560
ccacctgttc ggccaagcag cccgatccca ctgcccggat tccccgcgga ctttcagcct
1620
tcgtctaagc gatctgttaa aagagcgcaa ggagataaag ggaagaaggt tgggttgccc
1680
aatcgcaacc tcactgaccc gccctacacc attcggaatg ccgcaggtgt ccttagtatg
1740
agcactatcg agacggatct cattcatgcg ggtgaagggt atgccgagta tgatactcac
1800
aatctctatg gaacaagtaa gtctttcaaa tatttgcata gatgatttgc cattgacagg
1860
gttagtgatg agctctgctt cccgcacggc tatgcaggcc cgccgtcccg atgtgaggcc
1920
tttggtcatc actcgcagta cgtttgcagg cgctggagca cacgtaggac actggtaagt
1980
tgaccgatag ccttcgctag cacatcgctg attcgtacag gctgggcgac aactttagcg
2040
attgggttca ctaccggatc tccatcgcgc agatcctctc cttcgcgtcc atgttccaga
2100
ttccaatggt cggggctgac gtgtgtgggt ttggtagcaa cacgacggag gaattgtgtg
2160
cccgatgggc gtcacttggt gccttctata cgttctaccg caatcataac gagctgggcg
2220
acatatcgca agagttctac cgctggccta cggttgccga gtccgcgcgt aaggccattg
2280
acatccggta caagctcctc gattatatct acactgctct tcaccggcaa agccagaccg
2340
gcgagccatt cctgcagcct caattctacc tgtaccctga ggattcgaac acctttgcga
2400
acgaccggca gttcttctat ggtgacgccc ttcttgtcag ccccgtgttg aatgagggat
2460
ccacctcagt cgacgcatac ttcccggacg acatcttcta cgattggtac acaggggcag
2520
tggtgcgtgg gcacggagaa aacatcacgc tcagcaacat caacatcacc cacatccctc
2580
tgcacatccg cggtggaaat atcatacctg tcaggacatc cagcggcatg acaaccactg
2640
aggttcgtaa gcagggcttc gagctgatca tcgcgccaga cttggatgac accgcatcgg
2700
gcagtctata tttggatgat ggagactcgt tgaacccgtc atctgtgaca gagctcgagt
2760
tcacgtacag caaaggggag ttgcacgtga agggtacatt cggacagaag gccgtcccca
2820
aggtggagaa atgtaccttg ctggggaagt cagcacggac gttcaagggc tttgcactcg
2880
atgcgccggt gaactttaag ctgaagtag
According to the present invention, the method for inactivating agdB gene can be known in the art any method, such as can be
Method described in CN104962594A.
In compounded saccharifying enzyme preparation of the present invention, the enzyme activity ratio of first carbohydrase and Pullulanase is
(7.5-80): 1, such as can be about 7.5:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1,
About 35:1, about 40:1, about 45:1, about 50:1, about 55:1, about 60:1, about 65:1, about 70:1 or about 80:1 and aforementioned proportion
Any one value in range in any two institute compositing range, the enzyme activity ratio of preferably described first carbohydrase and Pullulanase
For (10-60): 1, further preferably (20-60): 1, more preferably 40:1.
In a kind of preferred embodiment of compounded saccharifying enzyme preparation of the present invention, the preparation is by described first
Carbohydrase and Pullulanase are combined according to aforementioned proportion.
Second aspect of the present invention provides a kind of amylolytic method, which comprises
(1) feed liquid is configured；
(2) it is saccharified in the presence of compounded saccharifying enzyme preparation described in first aspect present invention to feed liquid.
Here, the starch is selected from but not limited at least one of cornstarch, tapioca, wheaten starch.
It, can be according to existing by the method for starch configuration feed liquid in step (1) in amylolytic method of the present invention
There is technology to select.Here, the feed liquid mainly contains dextrin, also containing a small amount of protein, liposome etc..Preferably, institute
Feed liquid is stated to obtain by the starch solution that mass concentration is 28%-65% through α-amylaseliquefied.It is highly preferred that the feed liquid is logical
The starch solution that mass concentration is 38-55% is crossed to obtain through α-amylaseliquefied.It is further preferred that the feed liquid passes through quality
The starch solution that concentration is 45% is obtained through α-amylaseliquefied.
In amylolytic method of the present invention, in step (2), it is preferable that the use of the compounded saccharifying enzyme preparation
Amount is 20-80U/g DS, preferably 30-60U/g DS, more preferably 50U/g DS.
In amylolytic method of the present invention, it is preferable that the saccharification carries out under conditions of pH is 3-5, excellent
Selection of land carries out under conditions of pH is 4-5.
In amylolytic method of the present invention, it is preferable that the condition of the saccharification includes: that temperature is 40-70 DEG C,
Preferably from about 50-60 DEG C, saccharificatinn period 24-96h, preferably 24-48h, even more preferably about 36-48h.
The back reaction of compounded saccharifying enzyme preparation of the present invention is lower than conventional carbohydrase, and the DX of products therefrom is apparently higher than
Conventional carbohydrase, and with the extension of reaction time, the DX of products therefrom is almost without being decreased obviously trend；And with routine
Carbohydrase is compared, and compounded saccharifying enzyme preparation of the present invention can substantially shorten saccharification period, such as shorten 8-10h, in addition,
Extend saccharificatinn period to have little effect the saccharification result of compounded saccharifying enzyme preparation of the present invention, is added significantly to saccharification behaviour
The elasticity of work expands the window phase (30-48h) of saccharification.
The present invention will be described in detail by way of examples below.
The following are analysis involved in each embodiment, comparative example and test methods:
(1) HPLC analytical method:
Instrument: Shimadzu LC-20A, chromatographic column: Aminex HPX-87H pillar, mobile phase are 5mmol/L sulfuric acid, and flow velocity is
0.6ml/min, detector RID-20A.
(2) the OD detection method of saccharification terminal sample are as follows:
0.25mL saccharification terminal sample is taken to move in spectrophotometer after addition 4.75mL dehydrated alcohol mixes well 30s
Detect the absorbance of 420nm.
The following are raw materials involved in each embodiment, comparative example:
Hundred this outstanding carbohydrase HighDEX Ultra3.0, are purchased from hundred Si Jie bioengineering Co., Ltd of Nanjing.Conventional saccharification
Enzyme (by fermentation of Aspergillus niger, being turned glycosides enzymatic process purifying gained) compounds, conventional carbohydrase and Pullulanase with Pullulanase
Enzyme activity ratio is about 80:1；
Novi believes carbohydrase Dextrozyme 1.5Plus, believes (China) Investment Co., Ltd purchased from Novi, wherein conventional
Carbohydrase and Pullulanase enzyme activity ratio are about 80:1；
First carbohydrase is obtained according to method described in CN104962594A embodiment 6.
Pullulanase is purchased from hundred Si Jie bioengineering Co., Ltd of Nanjing
Glucoamylase in normal starch enzyme a, b is purchased from hundred Si Jie bioengineering Co., Ltd of Nanjing.
Compounded saccharifying enzyme preparation A, wherein only the first carbohydrase, is free of Pullulanase, is denoted as the first carbohydrase and general Shandong
Blue enzyme enzyme activity ratio is 1:0；
Compounded saccharifying enzyme preparation B, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 100:1；
Compounded saccharifying enzyme formulation C, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 80:1；
Compounded saccharifying enzyme preparation D, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 60:1；
Compounded saccharifying enzyme preparation E, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 40:1；
Compounded saccharifying enzyme preparation F, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 20:1；
Compounded saccharifying enzyme preparation G, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 10:1；
Compounded saccharifying enzyme preparation H, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 8.5:1；
Compounded saccharifying enzyme preparation I, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 7.5:1；
Compounded saccharifying enzyme preparation J, wherein the first carbohydrase and Pullulanase enzyme activity ratio are 5:1；
Conventional group synthase a: glucoamylase and Pullulanase enzyme activity ratio are 10:1,
Conventional group synthase b: glucoamylase and Pullulanase enzyme activity ratio are 7.5:1.
Embodiment 1
Embodiment 1 is for illustrating different composite saccharidase preparation to the activity of alpha-glucosidase
The fresh glucose feed liquid of configuration 32% adjusts pH to 4.3 using 1mol/L HCl.Weigh the above-mentioned feed liquid of 50g in
In 200ml triangular flask, 50U/gDS (with glucoamylase enzyme work) carbohydrase is added (respectively using hundred this outstanding carbohydrase HighDEX
Ultra3.0, compounded saccharifying enzyme preparation A, B, C, D, E, F, G), it places after mixing and is reacted in shaking table, react 48h at 60 DEG C.It is right
Sample after reaction carries out efficient liquid phase chromatographic analysis, and the results are shown in Table 1.
Table 1
DP1 in table 1 and following table indicates that monosaccharide, DP2 indicate that disaccharides, DP3 indicate that trisaccharide, DP4 indicate tetrose or more.
Liquid phase analysis composition produces in table 1 and following table to account for overall reaction according to each component of the conversion of area obtained by efficient liquid phase chromatographic analysis
The weight percent of object (dry weight).Here, DP1 content is DX value.
As shown in Table 1, the glycosylation back reaction of compounded saccharifying enzyme formulation C-G of the present invention is lower than conventional carbohydrase
(this can find out that the content of DP2 is higher by the content of DP2, and the degree of back reaction is bigger), and compounded saccharifying enzyme preparation E
Back reaction it is minimum, improve the ratio of Pullulanase again on this basis, back reaction reduces unobvious.
Embodiment 2
By content be 45% starch solution prepare fresh dextrin (DE12) feed liquid, using 1mol/L HCl adjusting pH to
4.3.The above-mentioned feed liquid of 50g is weighed in 200mL triangular flask, 50U/gDS (with glucoamylase enzyme work) carbohydrase is added and (uses respectively
Hundred this outstanding carbohydrase HighDEX Ultra3.0, compounded saccharifying enzyme preparation A, B, C, D, E, F, G, H, I, J), it places and shakes after mixing
It is reacted in bed, reacts 48h at 60 DEG C.Efficient liquid phase chromatographic analysis is carried out to the sample after saccharification, while detecting saccharification
The OD of terminal sample, the results are shown in Table 2.
Table 2
As shown in Table 2, it compared with compounded saccharifying enzyme preparation A, B, J, is catalyzed through compounded saccharifying enzyme preparation E-I of the present invention
Products therefrom DX is significantly higher, up to 96.84% or more, or even up to 97.21% or more.
Embodiment 3
Referring to method for saccharifying described in embodiment 2, unlike, the carbohydrase used be respectively compounded saccharifying enzyme formulation C,
D, E, F, G, I, conventional group synthase a, conventional group synthase b, hundred this outstanding carbohydrase HighDEX Ultra3.0, Novi believe carbohydrase
Dextrozyme 1.5Plus, and reaction for 24 hours, the separately sampled detection OD value of 30h, 40h, 48h, 72h, 96h and utilization
Efficient liquid phase chromatographic analysis.The results are shown in Table 3.
Table 3
As shown in Table 3, carbohydrase Dextrozyme is believed with hundred this outstanding carbohydrase HighDEX Ultra3.0, Novi
1.5Plus, conventional group synthase a, conventional group synthase b are compared, obtained by compounded saccharifying enzyme formulation C of the present invention, D, E, F, G, I
The DX of product is significantly higher；And when reacting 30h, DX value reaches highest, is further continued for extending the reaction time, there is no obvious for DX value
Decline illustrates that turning glycosides reacts very limited.It can be seen that compounded saccharifying enzyme formulation C of the present invention, D, E, F, G, I can be substantially
Shorten saccharification period, shortens at least 8-10 hours, and extend saccharificatinn period and have little effect to saccharification result, be significantly increased
The elasticity of mashing operation, expands the window phase of saccharification, and saccharification window phase is extended to 30-72 hours.
Embodiment 4
It is saccharified according to method as described in example 2, unlike, fresh dextrin feed liquid (DE12) is by mass concentration
28%, obtained by 38%, 55%, 65% starch solution liquefies, the carbohydrase used is respectively compounded saccharifying enzyme formulation C, G, hundred
This outstanding carbohydrase HighDEX Ultra3.0, Novi believe carbohydrase Dextrozyme 1.5Plus, and OD value is detected after saccharification
And efficient liquid phase chromatographic analysis is utilized, the results are shown in Table 4.
Table 4
As shown in Table 4, carbohydrase Dextrozyme is believed with hundred this outstanding carbohydrase HighDEX Ultra3.0, Novi
1.5Plus is compared in different concentration of substrate, compounded saccharifying enzyme formulation C of the present invention, G products therefrom DX obviously compared with
Height, back reaction is also less, illustrates that compounded saccharifying enzyme formulation C, G are saccharified suitable for highly concentrated substrate.
Comparative example 1
The starch solution for configuring 50 mass %, is liquefied to obtain feed liquid, adjusts pH to 4.3 using 1mol/L HCl.Claim
It takes the above-mentioned feed liquid of 50g in 200ml triangular flask, is saccharified by condition described in embodiment 1 in CN103981239B, adds 34U/
GDS (with glucoamylase enzyme work) enzyme (respectively compounded saccharifying enzyme preparation I, conventional group synthase b, wherein conventional group synthase b according to
" under the premise of 4ASPU/g Pullulanase is added, 30U/g glucoamylase is added " described in CN103981239B).It mixes
It places and is reacted in shaking table afterwards, react 48h at 60 DEG C.Efficient liquid phase chromatographic analysis is carried out to the sample after saccharification, as a result
As shown in table 5.
Table 5
Such as table 5 it is found that the DX of compounded saccharifying enzyme preparation I products therefrom of the present invention is substantially better than conventional group synthase b, say
Bright compounded saccharifying enzyme preparation I of the present invention has a clear superiority in high concentration substrate saccharifying.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
SEQUENCE LISTING
<110>Cofco Group Co., Ltd.
Jilin COFCO Biochemical Co., Ltd.
Hundred Si Jie bioengineering Co., Ltd of Nanjing
<120>compounded saccharifying enzyme preparation and amylolytic method
<130>fast I55910COF
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2909
<212> DNA
<213>agdB gene
<400> 1
atgttggggt ctttgctttt actcttaccc cttgtgggcg ctgctgtcat tggacccagg 60
gcaaacagtc agagttgccc agggtataag gcgtccaacg tccaaaagca ggctaggtca 120
ctgactgcgg atctgactct agctggtacg ccttgtaata gctatggcaa ggatttggaa 180
gacctcaagc tgcttgtgga atatcagact ggtgagtgtt ggcttgtgtg aatcaagagt 240
tcctgactaa atgcttgctc agatgaacgg ttacatgtta tgatctacga tgccgacgag 300
gaagtctatc aagttcctga atcagtcctt cctcgcgtgg gtagtgacga ggactctgag 360
gacagtgttt tggaatttga ctatgtggaa gaaccgtttt cattcaccat ctccaaggga 420
gatgaggtcc tgtttgactc ttcggcatca ccactagttt ttcagtcgca atatgtgaac 480
cttcgcacct ggttgcccga tgatccctat gtgtatggtc tcggagagca ttctgaccct 540
atgcgcttgc caacatacaa ttacacgcgg accctttgga accgcgacgc gtatggcact 600
ccaaacaaca ccaacttgta cggtagtcat cctgtctact atgatcaccg tggaaagtcc 660
ggaacttatg gagtcttcct gctgaactct aatggtatgg acatcaagat caaccaaacg 720
acagatggaa agcagtactt ggaatacaat cttctcggcg gtgttctgga cttctacttc 780
ttctacggag aagatcctaa gcaagcgagc atggaatact caaagattgt cggtctcccg 840
gcaatgcaga gttactggac tttcggcgta tgccccccac cccctaatcc cataacagtc 900
cgagttgtat gctgactctt cagttccatc aatgccgtta tggataccgc gatgtgtatg 960
aacttgccga ggtggtctac aactacagcc aggcaaagat tcctctggag acgatgtgga 1020
cagatatcga ctacatggac aagagaaggg tgtttaccct tgatcctcag aggttcccgc 1080
tcgaaaagat gcgggagttg gtaacctacc tgcacaatca tgatcagcat tacattgtca 1140
tggttgaccc ggctgtgagc gtaagcagtg agtgacttga cgattcccca tccttgcaac 1200
tttcagctaa tggatacttt ctagataaca cggcatatat caccggcgtg agagacgatg 1260
ttttccttca caatcagaac ggtagcctat acgagggtaa gtatatacac atctcatatc 1320
tctcaacacg agctaaacta tgcaggtgct gtttggcctg gtgtcactgt tttcccagac 1380
tggttcaatg agggtactca ggattactgg actgcgcaat ttcaacagtt ctttgatccc 1440
aagtccggag tcgatattga cgccctgtgg attgacatga acgaagcctc caatttctgc 1500
ccttatcctt gtctggaccc agcggcatac gcgatctccg ccgacctccc accggcagca 1560
ccacctgttc ggccaagcag cccgatccca ctgcccggat tccccgcgga ctttcagcct 1620
tcgtctaagc gatctgttaa aagagcgcaa ggagataaag ggaagaaggt tgggttgccc 1680
aatcgcaacc tcactgaccc gccctacacc attcggaatg ccgcaggtgt ccttagtatg 1740
agcactatcg agacggatct cattcatgcg ggtgaagggt atgccgagta tgatactcac 1800
aatctctatg gaacaagtaa gtctttcaaa tatttgcata gatgatttgc cattgacagg 1860
gttagtgatg agctctgctt cccgcacggc tatgcaggcc cgccgtcccg atgtgaggcc 1920
tttggtcatc actcgcagta cgtttgcagg cgctggagca cacgtaggac actggtaagt 1980
tgaccgatag ccttcgctag cacatcgctg attcgtacag gctgggcgac aactttagcg 2040
attgggttca ctaccggatc tccatcgcgc agatcctctc cttcgcgtcc atgttccaga 2100
ttccaatggt cggggctgac gtgtgtgggt ttggtagcaa cacgacggag gaattgtgtg 2160
cccgatgggc gtcacttggt gccttctata cgttctaccg caatcataac gagctgggcg 2220
acatatcgca agagttctac cgctggccta cggttgccga gtccgcgcgt aaggccattg 2280
acatccggta caagctcctc gattatatct acactgctct tcaccggcaa agccagaccg 2340
gcgagccatt cctgcagcct caattctacc tgtaccctga ggattcgaac acctttgcga 2400
acgaccggca gttcttctat ggtgacgccc ttcttgtcag ccccgtgttg aatgagggat 2460
ccacctcagt cgacgcatac ttcccggacg acatcttcta cgattggtac acaggggcag 2520
tggtgcgtgg gcacggagaa aacatcacgc tcagcaacat caacatcacc cacatccctc 2580
tgcacatccg cggtggaaat atcatacctg tcaggacatc cagcggcatg acaaccactg 2640
aggttcgtaa gcagggcttc gagctgatca tcgcgccaga cttggatgac accgcatcgg 2700
gcagtctata tttggatgat ggagactcgt tgaacccgtc atctgtgaca gagctcgagt 2760
tcacgtacag caaaggggag ttgcacgtga agggtacatt cggacagaag gccgtcccca 2820
aggtggagaa atgtaccttg ctggggaagt cagcacggac gttcaagggc tttgcactcg 2880
atgcgccggt gaactttaag ctgaagtag 2909
Claims (10)
1. a kind of compounded saccharifying enzyme preparation, the preparation contains the first carbohydrase and Pullulanase, and first carbohydrase is to lose
The carbohydrase of aspergillus niger secretion after agdB gene living.
2. compounded saccharifying enzyme preparation according to claim 1, wherein the enzyme activity ratio of first carbohydrase and Pullulanase
Example is (7.5-80): 1.
3. compounded saccharifying enzyme preparation according to claim 1, wherein the enzyme activity ratio of first carbohydrase and Pullulanase
Example is (10-60): 1.
4. a kind of amylolytic method, which comprises
(1) feed liquid is configured；
(2) it is saccharified in the presence of compounded saccharifying enzyme preparation described in any one of claim 1-3 to feed liquid.
5. according to the method described in claim 4, wherein, the feed liquid is the starch solution of 28%-65% by mass concentration
It is obtained through α-amylaseliquefied.
6. method according to claim 4 or 5, wherein the starch is selected from cornstarch, tapioca, wheaten starch
At least one of.
7. method according to claim 4 or 5, wherein the weight based on starch on dry basis, the compounded saccharifying enzyme preparation
Dosage is 20-80U/g starch, preferably 30-60U/g starch.
8. method according to claim 4 or 5, wherein the saccharification carries out under conditions of pH is 3-5, preferably exists
PH is carried out under conditions of being 4-5.
9. method according to claim 4 or 5, wherein the condition of the saccharification includes: that temperature is 40-70 DEG C, and the time is
24-48h。
10. method according to claim 4 or 5, wherein the condition of the saccharification includes: that temperature is 50-60 DEG C, the time
For 36-48h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391369A (en) * | 2020-11-24 | 2021-02-23 | 吉林中粮生化有限公司 | Compound saccharifying enzyme capable of remarkably improving saccharifying effect, and preparation method and application thereof |
| CN112501226A (en) * | 2020-11-27 | 2021-03-16 | 广州双桥（重庆）有限公司 | Preparation method of syrup for medical lovastatin |
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|---|---|---|---|---|
| WO2012124520A1 (en) * | 2011-03-16 | 2012-09-20 | 天野エンザイム株式会社 | Modified αlpha-glucosidase and applications of same |
| CN103981239A (en) * | 2014-05-13 | 2014-08-13 | 江南大学 | Method of improving saccharification product purity of high-concentration starch |
| CN104962594A (en) * | 2015-07-21 | 2015-10-07 | 南京百斯杰生物工程有限公司 | Method for enhancing glucose conversion rate of Aspergillus niger saccharifying enzyme |
| CN105969675A (en) * | 2016-05-10 | 2016-09-28 | 华南理工大学 | Saccharifying enzyme high-yield strain gene knockout recombinant bacteria with low trans-glycoside enzyme background as well as construction method and application thereof |
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2019
- 2019-03-18 CN CN201910205311.4A patent/CN110004128A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012124520A1 (en) * | 2011-03-16 | 2012-09-20 | 天野エンザイム株式会社 | Modified αlpha-glucosidase and applications of same |
| CN103981239A (en) * | 2014-05-13 | 2014-08-13 | 江南大学 | Method of improving saccharification product purity of high-concentration starch |
| CN104962594A (en) * | 2015-07-21 | 2015-10-07 | 南京百斯杰生物工程有限公司 | Method for enhancing glucose conversion rate of Aspergillus niger saccharifying enzyme |
| CN105969675A (en) * | 2016-05-10 | 2016-09-28 | 华南理工大学 | Saccharifying enzyme high-yield strain gene knockout recombinant bacteria with low trans-glycoside enzyme background as well as construction method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391369A (en) * | 2020-11-24 | 2021-02-23 | 吉林中粮生化有限公司 | Compound saccharifying enzyme capable of remarkably improving saccharifying effect, and preparation method and application thereof |
| CN112501226A (en) * | 2020-11-27 | 2021-03-16 | 广州双桥（重庆）有限公司 | Preparation method of syrup for medical lovastatin |
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