CN105925550A - Method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion - Google Patents
Method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion Download PDFInfo
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- 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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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- 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
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
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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)
- C12Y302/0102—Alpha-glucosidase (3.2.1.20)
Abstract
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion. The half-life t1/2 of the alpha-glucosidase is increased by 3.7 times at 66 DEG C; the proportion of core components (IG2 (Immune Globulin 2)+P+IG3 (Immune Globulin 3)) of the isomaltooligosaccharides, which are prepared by adopting the alpha-glucosidase, high-temperature-resistant alpha-amylase, intermediate-temperature alpha-amylase, beta-amylase and pullulanase to carry out starch liquefaction and through the synchronous saccharification and glucoside conversion, in dry substances reaches 52 to 59 percent.
Description
Technical field:
The invention belongs to technical field of enzyme engineering, be specifically related to acquisition and the height of a kind of heat-resisting alpha-glucosaccharase enzyme mutant
Effect preparation method, starch liquefacation new method, saccharifying turns glycosides and synchronizes to carry out realizing process prepared by high-quality oligomeric isomaltose.
Background technology:
Oligomeric isomaltose (Isomaltooligosaccharides, hereinafter referred to as IMO), also known as bifurcation oligose, different
Fructus Hordei Germinatus oligose etc., be between glucose at least one with α-1,6 glycosidic bonds are combined into, monosaccharide number is not 2～6 not etc.
One class oligosaccharide.Its main component is dextrinose (IG2), panose (P), Isomaltotriose (IG3) and Isomaltotetraose (Gn)
Deng.Oligomeric isomaltose belongs to non-digestible property oligosaccharide, and it cannot be as the direct sources of body energy, but it can select
The probiotics such as bacillus bifidus, lactobacillus in the propagation intestinal of property, the short-chain fatty acid of its metabolism can reduce the pH of intestinal, presses down
The growth of harmful bacteria processed, maintains the balance of intestinal microecology；It addition, these short-chain fatty acid provide to other microorganism
Energy, so also known as functional oligose.The commercial product specification of oligomeric isomaltose mainly has two kinds: IMO-50 type (IG2+P
+IG3+Gn>=50%) and IMO-90 type (IG2+P+IG3+Gn>=90%).Containing a certain amount of glucose, maltose in IMO-50,
And containing less glucose and maltose in IMO-90, product purity is higher, is to produce through nanofiltration separation on the basis of IMO-50
High-purity oligomeric isomaltose.The main method of industrialized production IMO is that to turn glycosides with glucosyl group glycosides enzyme glycolysis be IMO, then
Through decolouring, concentrating, be dried form.
Oligomeric isomaltose has a lot of physiological function.Wherein directly physiological function has: (1) is difficult to be digested by gastric enzyme, heat
Measuring low, sugariness is low, does not the most increase blood pressure and blood lipoid；(2) propagation of probiotic bifidobacteria in human body intestinal canal can be promoted,
All kinds of harmful bacterias and the formation of corrupt substance in suppressing intestinal, increase the content of various vitamin, improves immunity of organisms；
(3) prevention of dental caries function, it is not decomposed by oral cavity enzyme liquid, is not utilized by the streptococcus of dental caries, thus can prevent dental caries, oligomeric different
The panose in the maltose effect to hindering tooth dirt to be formed is the most obvious；(4) it belongs to non-digest oligosaccharide class, functional equivalent in
Water soluble dietary fiber.Indirect physiological effect has: (1) facilitating digestion system, to the digestion of food, absorption, maintains the normal merit of intestinal
Energy；(2) intestinal tract normal flora during recovering antibiotic therapy, radiation cure, chemotherapy falls；(2) constipation and improvement are prevented
Diarrhoea, suppression pathogen and putrefaction bacteria；(4) improve immunity of organisms, be excellent immunomodulator；(5) suppression intestinal is carcinogenic
The generation of material, reduces cholesterol level, improves serum lipids；(6) increase the body absorption to the mineral such as calcium, magnesium, help
Vitamin B group is synthesized in body.
At present, the classical way of production IMO is mainly with starch as raw material both at home and abroad, uses amylase system multienzyme synergism method to turn
Change starch fluid to obtain.Industrial first being liquefied under the effect of high-temperatureα-amylase by starch, liquefying starch is at middle temperature alphalise starch
Generating maltose syrup under enzyme or beta amylase continuation effect, recycling phlorose transglucosidase carries out glycosyl conversion and generates IMO,
End-product carbohydrate components about contains the IMO of 50%～60%, glucose, maltose and the maltotriose of 40%～50%,
Finished product is obtained by filtration, decolouring, desalination, concentration etc..Operation is many, time length, technological parameter to use classical production technology to exist
Control difficulty, be difficult to the shortcomings such as continuous prodution, due in production process use alpha-glucosaccharase enzyme preparation at home
Production field is nearly at blank, relies primarily on import, there is the problems such as expensive, source is unstable, meanwhile, merit in product
The content of energy property sugar is the highest, only accounts for 35% (w/w) of solid content, and these all govern the development of China IMO.
Owing to classical technique produces the deficiency that IMO exists, in recent years, domestic and international researcher is devoted to enzyme catalysis and produces
The research of IMO, finds that in microorganism, antibacterial, yeast, mycete etc. can secrete alpha-glucosidase, and in relevant report, aspergillus is micro-
Bio enzyme is higher, but is endocellular enzyme owing to having the alpha-glucosidase turning glycosides activity, and the apparent vigor of enzyme is relatively low, some
Scholar launches research for the apparent glycosides vigor that turns improving alpha-glucosidase.Permeabilized cells apparent of the preparation such as Chen Gui light
Enzyme live reaches 483.9U/g wet thallus, be the apparent enzyme of intact cell live 193.3%, use Permeabilized cells convert produce oligomeric
Dextrinose, its transformation period is 24h, and more complete cell shortens 24h, and conversion ratio is maintained at more than 70%.Further, he is with black fermented preparation
Mould GXM-3 is that starting strain carries out 60C-gamma-rays and ultraviolet irradiation compound mutation breeding, it was found that mutant D-597
Conversion capability the strongest, in 3L fermentation cylinder for fermentation 80h, in syrup product, oligomeric isomaltose content reaches peak
169.4mg/mL, improves 37.2% than starting strain, and fermentation period shortens 40h.The ion implantation technique pair such as Lin Yongxian
Aspergillus niger starting strain carries out mutagenic treatment, selects a strain high productive mutant AL7, by being optimized condition of enzyme production, makes
Its yield of enzyme is that 9.24U, relatively starting strain improve 4.18 times.Owing to wild Aspergillus niger product enzyme level is low, difficult
With extraction purification, domestic and international researcher starts to build and utilize genetic engineering bacterium production to have height one after another and turns the α-Portugal of glycosides activity
Polyglycoside enzyme.Child film stars etc. express aspergillus niger Aspergillus with Pichia sp. Pichia pastoris KM71 for Host Strains
Niger SG136 alpha-glucosidase, the Yeast expression carrier pPIC9K wherein selected contains coding sequence of secretory signal peptide, beneficially outside born of the same parents
Expressing, the glycosides that turns of the crude enzyme liquid of preparation reacts at pH 5, and temperature 55 DEG C, during reaction 24h, the total content of oligomeric isomaltose reaches
26%.
Visible, still there is production time length and the low problem of conversion ratio in current IMO production technology.
Summary of the invention:
In order to solve above-mentioned technical problem, the present invention, by the compound mode of reasonably combined various amylolytic enzymes, uses
Heat resistance alpha-glucosidase, define can the manufacturing process of a step catalytic production oligomeric isomaltose, simplify life
Production. art.
The present invention solves one of technical scheme that above-mentioned technical problem used: provide one to pass through genetic engineering means
The alpha-glucosaccharase enzyme mutant that significantly improves of thermostability obtained, its aminoacid sequence as shown in sequence table SEQ ID NO:4,
Its gene order is as shown in sequence table SEQ ID NO:3；
Half-life t at the more original alpha-glucosidase of described mutant 60 DEG C1/2Improve 3.7 times；
The present invention goes out alpha-glucosidase gene with aspergillus niger NRRL3135 total serum IgE for template reverse transcription amplification, utilizes easily
Wrong round pcr carries out molecular improvement to alpha-glucosidase gene agD, to obtain the excellent mutant that thermostability significantly improves.
The present invention solves the two of the technical scheme that above-mentioned technical problem is used: α described in a kind of technical scheme one of offer-
The preparation method of glucoside enzyme mutant:
(1) gene described in SEQ ID NO:3 is carried out enzyme action, and carry out to the plasmid pHY-WZX through corresponding enzyme action even
Connect, construction recombination plasmid；
(2) by recombinant plasmid transformed to Bacillus licheniformis CBB3008 competent cell, recombinant bacterial strain is prepared；
(3) recombinant bacterial strain described in expression, obtains the alpha-glucosaccharase enzyme mutant shown in SEQ ID NO:4；
Described Bacillus licheniformis CBB3008, deposit number CCTCC No.M 208236, special referring specifically to Chinese invention
Profit ZL 200810235368.0, and Dandan Niu, et al.Microbial Cell Factories, 2009,8:58；
Described plasmid pHY-WZX is with reference to Dandan Niu and Zhengxiang Wang.J Ind Microbiol
Biotechnol,2007,34:357-362；
The present invention solves the three of the technical scheme that above-mentioned technical problem is used: utilize α-Portugal that thermostability significantly improves
Polyglycoside enzyme mutant, with starch as raw material, it is provided that a kind of synchronous saccharification turns the method that glycosides efficiently prepares oligomeric isomaltose, tool
Body is as follows:
(1) starch liquefacation
Starch liquefaction can use injection liquefaction or ebuillition of heated two ways to implement；
Injection liquefaction: add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred), by starch
After breast stirs, add water in fermentation tank 18m3, regulate pH to 5.8～6.5；Add Thermostable α-Amylase to fermentation tank
Final concentration 4～5U/mL, through Jet liquefier 107～110 DEG C of injection liquefaction, maintains tank i.e. to can be used for saccharifying and turn glycosides after maintaining.
Ebuillition of heated liquefies: add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred), will
Starch milk is stirring evenly and then adding into a certain amount of water, and in fermentation tank, constant volume is to 18m3, regulate pH to 5.0～6.5.By starch milk
It is heated to 40～50 DEG C, adds Thermostable α-Amylase 3～5U/mL, continue the extremely boiling of heating starch mash, i.e. complete liquefaction
Further, mesophilicα-diastase, final concentration of 0.1～2U/mL are added while adding Thermostable α-Amylase
(2) synchronous saccharification turns glycosides
Mash after above-mentioned liquefaction is cooled to 50 DEG C～60 DEG C, adds mesophilicα-diastase, pullulanase, β-starch
Enzyme, several in alpha-glucosidase, maintain reaction temperature at 50 DEG C～60 DEG C, specifically have following three kinds of schemes:
Scheme one: add fermentation tank final concentration 13～25U/mL beta amylase and 0.2～5U/mL pullulanase, at pH
5.0～5.5 and temperature 50 C～55 DEG C under the conditions of vanguard action 1～4h, be eventually adding fermentation tank final concentration 40～250U/mL
Alpha-glucosidase, under the conditions of pH 5.8～6.5 and temperature 55 DEG C～60 DEG C, continuation effect 9～20h carries out turning glycosides effect；
Scheme two: add fermentation tank final concentration 2～5U/mL mesophilicα-diastase, 13～25U/mL beta amylase and
The pullulanase of 0.2～5U/mL, vanguard action 1～4h under the conditions of pH 5.0～5.5 and temperature 50 C～55 DEG C, then add
Enter the alpha-glucosidase of final concentration 40～250U/mL, act under the conditions of pH 5.5～6.0 and temperature 55 DEG C～60 DEG C 9～
20h；
The priority that three: four enzymes of scheme press pullulanase, mesophilicα-diastase, beta amylase and alpha-glucosidase is suitable
Sequence, according to mesophilicα-diastase final concentration 2～5U/mL, pullulanase final concentration 0.2～5U/mL, beta amylase final concentration 13～
25U/mL, the addition of alpha-glucosidase final concentration 40～250U/mL, in pH5.0～6.5 and temperature 50 C～60 DEG C of conditions
Under the most respectively effect 1～6h complete catalysis；
Further, alpha-glucosidase is as described in technical scheme one；
Further, described starch liquefaction, its DE (degree of hydrolysis) can reach 5～50；
Further, the relevant enzyme catalytic starch in the present invention prepares oligomeric isomaltose (IG2+P+IG3) account for dry
The isomaltooligosaccharide syrup of proportion 52%～59%.
Beneficial effect:
1, the invention provides acquisition and the high efficiency preparation method of a kind of heat-resisting alpha-glucosaccharase enzyme mutant, obtained prominent
Become enzyme half-life t at 60 DEG C1/2Improve 3.7 times.
2, the invention provides and a kind of synchronize to turn glycosides with starch for synchronous saccharification and efficiently prepare high-quality oligomeric isomaltose
Manufacturing technology, prepared oligomeric isomaltose core composition (IG2+P+IG3) account for the ratio of dry reach 52%～
59%；The oligomeric isomaltose preparation utilizing alpha-glucosaccharase enzyme mutant provided by the present invention and commercial enzyme combination to be formed is new
Technique, the total content of reaction 13h oligomeric isomaltose can reach more than 52%.
3, Thermostable α-Amylase involved by the manufacturing technology of the oligomeric isomaltose of the present invention, mesophilicα-diastase,
Beta amylase, pullulanase and alpha-glucosidase have employed novel expression and combinational expression mode, have notable catalytic performance；
4, the starch liquefaction of the present invention uses fast and convenient preparation process；
5, the liquefaction syrup of the present invention can quickly realize the preparation of isomaltooligosaccharide syrup simultaneously or under the conditions of substep ground
Process.
Accompanying drawing illustrates:
The PCR amplification of Fig. 1 alpha-glucosidase gene
Wherein, swimming lane 1 is that 1 λ DNA Pst I enzyme action molecular weight is demarcated；The alpha-glucosidase base that swimming lane 2 expands for PCR
Because of fragment；
Fig. 2 SDS-PAGE analyzes the mutant enzyme of purification
Wherein, 1 is fermentation liquid；2 is the component after ion exchange column purification；3 is gel column component after purification；M is egg
White molecular weight standard；
Fig. 3 prepares oligomeric isomaltose sample HPLC and resolves collection of illustrative plates
Wherein, DP1-glucose, DP2-maltose, IG2-dextrinose, DP3-maltotriose, P-panose, IG3-different Fructus Hordei Germinatus
Trisaccharide, IG4-Isomaltotetraose.
Detailed description of the invention:
Below in conjunction with embodiment, the present invention is further described；Following embodiment does not limit the present invention, it is impossible to
Following embodiment limits protection scope of the present invention.
Embodiment 1: the acquisition of alpha-glucosidase encoding gene segment
TRNzol total RNA extraction reagent is used to extract the total serum IgE of aspergillus niger NRRL3135.With total serum IgE as template, reference
RT-PCR kit description, is that primer reverse transcription synthesizes the first chain cDNA, the most respectively with the first chain cDNA with oligo (dT)
For template, carry out PCR amplify aspergillus niger alpha-glucosidase with primers F 1 (SEQ ID NO:5) and R1 (SEQ ID NO:6)
Gene agD (Fig. 1).PCR primer is attached with plasmid pMD19-T simple vector, it is thus achieved that recombiant plasmid pMD-
AgD, Transformed E scherichia coli JM109 competent cell.Screening positive transformant, and extract its plasmid and carry out enzyme action
Checking.Carrying out recombiant plasmid correct for digestion verification checking order, (sequence SEQ ID NO:1, its aminoacid sequence is SEQ ID again
NO:2)。
Embodiment 2: utilize fallibility PCR method to build alpha-glucosidase mutated library
Fallibility round pcr is utilized to introduce coding mutation to alpha-glucosidase gene agD in vitro.Fallibility PCR's is anti-
Answer condition as follows:
PCR amplification condition: 94 DEG C of 3min；94 DEG C of 1min, 58 DEG C of 1min, 72 DEG C of 1.5min, 30 circulations；72℃10min.
The primer is primers F 1 (SEQ ID NO:5) and R1 (SEQ ID NO:6).
Fallibility pcr amplification product (naming with agDX), after DNA purification reclaims kits, uses restricted enzyme
Xba I and Kpn I carries out enzyme action to it, and to plasmid pHY-WZX (the Dandan Niu and through corresponding enzyme action
Zhengxiang Wang.J Ind Microbiol Biotechnol, 2007,34:357-362) it is attached, build restructuring
Plasmid pHY-WZX-agDX.Take 8 μ L recombiant plasmid pHY-WZX-agDX DNA and bacillus licheniformis CBB3008 competent cell
Mixing, moves into the electric revolving cup of pre-cooling, and after electric shock, (LB containing 0.5M sorbitol and 0.5M mannitol cultivates to add recovery medium
Base).37 DEG C, 160r/min recovery cultivate 3h, then coating receives the LB flat board of mycin (0.03mg/mL) containing card, incubated overnight.
According to above-mentioned method, carry out taking turns fallibility PCR with mutant gene group for template more, build mutated library.
Embodiment 3: the screening of Heat Stability Mutations bacterial strain
Single bacterium colony on picking LB flat board, uses 96 orifice plate LB liquid cultures to cultivate 55～70h based on 30～32 DEG C, dress
Liquid measure is 200～250 μ L, simultaneously with initial recombinant bacterium B.licheniformis CBB3008 (pHY-WZX) for comparison.
By 96 orifice plate I centrifugal segregation thalline, take 10 μ L of supernatant liquid from every hole successively and be transferred to another 96 plate II.And should
96 orifice plates are just being placed in 60 DEG C of heat treatment 60min.Substrate o-methoxyphenyl-alpha-D-glucose the glycosides of 0.2% is added in every hole
50 μ L, 30 DEG C of temperature bath 10min.Add 80 μ L 2.5mol/L NaOH solution terminate reaction (matched group is initially charged stop buffer, then
Add substrate).Prepare diazonium salt solution: take 0.2g/L NaNO simultaneously2100mL, is subsequently adding the HCl 5mL of 3mol/L, makes
Solution is acidity, 30 DEG C of temperature bath 5min, adds the aniline 10mL of 50g/L, 30 DEG C of reaction 6min.Take 110 μ L diazonium salt solutions to add
Enter in enzyme reaction system, color development at room temperature 2min.At 450nm, microplate reader measures light absorption value.The enzyme of residual is lived higher than control strain
Bacterial strain for the purpose of i.e., uses the method to screen 6 strain bacterial strains altogether.
Embodiment 4: the THERMAL STABILITY of mutant enzyme
The fermentation liquor Ammonium persulfate. of purpose bacterial strain is precipitated, dialysis, the exchange of DEAE-Sepharose ion and
SuperdexTM200 gel filtrations carry out isolated and purified (Fig. 2).
Use the enzyme activity determination method of embodiment 3, the half-life t at measuring 60 DEG C1/2.Concrete assay method is: 10U is pure
Change enzyme to be put at 60 DEG C, take out through different time intervals, equilibrium at room temperature 5min, measure enzyme and live.After taking-up, enzyme is lived at for not heat
The time of 50% during reason, it is enzyme half-life t at 60 DEG C1/2。
Screen based on the mutated library that fallibility PCR builds, it is thus achieved that the 6 strain enzymes bacterial strain significantly improved alive, measure
Alpha-glucosidase nucleotide sequence, utilizes triplet codon to speculate the aminoacid sequence of alpha-glucosidase, phlorose
Half-life t at the aminoacid replacement of glycosides enzyme mutant and 60 DEG C1/2Improve multiple as shown in table 1.Wherein mutant 2-5's is resistance to
Hot property is the most excellent, and corresponding strain is B.licheniformis BL-25.After the production strain of alpha-glucosidase
Continuous use.Its aminoacid sequence is as shown in sequence table SEQ ID NO:4, and its gene order is as shown in sequence table SEQ ID NO:3.
Table 1 protoenzyme and mutant enzyme half-life at 60 DEG C
Under embodiment 5:15L fermentation system, BL-25 strain prepares alpha-glucosidase
Strain BL-25 is inoculated into seed culture medium, and (yeast extract 3%, peptone 3.8%, glucose 10%, remaining is
Water, pH 7.0) cultivate exponential phase, in 15L fermentation tank, carry out fermenting experiment further, fermentation medium is (w/v):
Yeast extract 1%, peptone 3%, lactose 10%, calcium carbonate 1.4%, surplus is water, controls with sulphuric acid or ammonia in sweat
PH is 7.0, with 5% inoculum concentration inoculate, 37 DEG C, fermentation 120h after, this bacterial strain produce alpha-glucosidase fermentation broth enzyme live
Level can reach 11000U/mL.Under embodiment 6:15L fermentation system, BL-25 strain prepares alpha-glucosidase
Strain BL-25 is inoculated into seed culture medium, and (yeast extract 5%, peptone 6.2%, glucose 20%, remaining is
Water, pH 7.0) cultivate exponential phase, in 15L fermentation tank, carry out fermenting experiment further, fermentation medium is (w/v):
Yeast extract 1%, peptone 3%, lactose 10%, calcium carbonate 1.4%, surplus is water, controls with sulphuric acid or ammonia in sweat
PH is 7.0.Inoculum concentration with 5% is inoculated, 37 DEG C, after fermentation 150h, and this bacterial strain produces that the enzyme running water of alpha-glucosidase is flat can
Reach 13000U/mL.
Embodiment 7: at 30m3Under system, alpha-glucosidase is prepared in strain BL-25 fermentation
It is 30m by the technique adjustment of embodiment 63The ratio that fermentation system is corresponding.It is respectively completed seed culture, inoculates 15L
First class seed pot, subcultivation 3m3Secondary seed tank, after main fermentation tank subcultivation etc. operates, cultivates thalline, after feed supplement growth stage,
Enzymatic production 90～120h after fermentation terminates.Fermentation liquor plate-and-frame filtration remove thalline, ultrafilter membrane concentrate enzyme liquid to suitable concn,
After adding auxiliary agent, fine straining prepares alpha-glucosidase liquid end product, or adds spray drying preparation after appropriate food-grade starches
Powder form alpha-glucosidase finished product, finished product enzyme is lived as 350000U/g～370000U/g.
Embodiment 8:30m3Sugaring system prepares oligomeric isomaltose technique one
Add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred), by starch milk stirring all
After even, add water in fermentation tank 18m3, regulate pH to 6.1；Add Thermostable α-Amylase 4U/mL, through Jet liquefier 109
DEG C injection liquefaction, maintains tank to maintain after 6h and i.e. can be used for saccharifying and turn glycosides.
As the mash after above-mentioned liquefaction is cooled to 54 DEG C by scheme one, regulation pH be 5.5 addition pullulanase 2U/mL, β-
After amylase 14U/mL, effect 3h, pH is adjusted to 6.0, adds embodiment 6 gained alpha-glucosidase 220U/mL, maintains reaction
Temperature is at 57 DEG C, and reaction 13h i.e. can get oligomeric isomaltose (IG2+P+IG3) and accounts for the sugar that dry matter content is 38%～49%
Slurry.(addition of above-mentioned enzyme is the final concentration in fermentation tank).
Case study on implementation 9:30m3Sugaring system prepares oligomeric isomaltose technique two
Starch liquefacation and saccharifying: add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred),
Starch milk is stirring evenly and then adding into a certain amount of water, and in fermentation tank, constant volume is to 18m3, regulate pH to 6.0.Starch milk is added
Heat, to 45 DEG C, adds Thermostable α-Amylase 4.5U/mL, continues the extremely boiling of heating starch mash, i.e. completes liquefaction.By above-mentioned liquid
Mash after change is cooled to 59 DEG C, adds pullulanase 1.8U/mL, beta amylase 14.2U/mL, embodiment 7 gained phlorose
Glycosides enzyme 165U/mL, maintains reaction temperature at 59 DEG C, and reaction 10h i.e. can get oligomeric isomaltose (IG2+P+IG3) and accounts for dry
Content is the syrup of 35%～45%.(addition of above-mentioned enzyme is the final concentration in fermentation tank).
Case study on implementation 10:30m3Sugaring system prepares oligomeric isomaltose technique three
Starch liquefacation and saccharifying: add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred),
Starch milk is stirring evenly and then adding into a certain amount of water, and in fermentation tank, constant volume is to 18m3, regulate pH to 6.0.Starch milk is added
Heat, to 48 DEG C, adds Thermostable α-Amylase 4.3U/mL and mesophilicα-diastase 1.2U/mL, continues heating starch mash to boiling
Rise, i.e. complete liquefaction.Mash after above-mentioned liquefaction is cooled to 59 DEG C, adds pullulanase 1.8U/mL, beta amylase 14.2U/
ML, embodiment 7 gained alpha-glucosidase 165U/mL, maintain reaction temperature at 59 DEG C, and reaction 10h i.e. can get oligomeric different wheat
Bud sugar (IG2+P+IG3) accounts for the syrup that dry matter content is 40%～50%.(addition of above-mentioned enzyme is the end in fermentation tank
Concentration).
Case study on implementation 11:30m3Sugaring system prepares oligomeric isomaltose technique four
Starch liquefacation and saccharifying: add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred),
Starch milk is stirring evenly and then adding into a certain amount of water, and in fermentation tank, constant volume is to 18m3, regulate pH to 6.0.Starch milk is added
Heat, to 49 DEG C, adds Thermostable α-Amylase 4.8U/mL and mesophilicα-diastase 1.1U/mL, continues heating starch mash to boiling
Rise, i.e. complete liquefaction.
As the mash after above-mentioned liquefaction is cooled to 55 DEG C by scheme two, regulation pH be 5.5 addition pullulanase 1.6U/mL,
Beta amylase 13.5U/mL, mesophilicα-diastase 2.0U/mL, after effect 3h, pH is adjusted to 6.0, adds embodiment 7 gained α-Portugal
Polyglycoside enzyme 186U/mL, maintains reaction temperature at 55 DEG C, and reaction 13h i.e. can get oligomeric isomaltose (IG2+P+IG3) and accounts for dry
Content of material is the syrup of 52%～59%.(addition of above-mentioned enzyme is the final concentration in fermentation tank).
Case study on implementation 12:30m3Sugaring system prepares oligomeric isomaltose technique five
Starch liquefacation and saccharifying: add 4.5t starch to equipped with 10m3In the fermentation tank of water (limit adds starch, and limit is stirred),
Starch milk is stirring evenly and then adding into a certain amount of water, and in fermentation tank, constant volume is to 18m3, regulate pH to 6.0.Starch milk is added
Heat, to 49 DEG C, adds Thermostable α-Amylase 4.8U/mL and mesophilicα-diastase 1.1U/mL, continues heating starch mash to boiling
Rise, i.e. complete liquefaction.
As the mash after above-mentioned liquefaction is cooled to 54 DEG C by scheme three, regulation pH is that 5.3 addition pullulanase 1.7U/mL make
After 3h, after adding mesophilicα-diastase 2.2U/mL continuation effect 4h, after adding beta amylase 15.8U/mL continuation effect 2h,
PH is adjusted to 6.0, adds embodiment 7 gained alpha-glucosidase 186U/mL, maintains reaction temperature to react 4h at 55 DEG C, amounts to
Reaction 13h i.e. can get oligomeric isomaltose (IG2+P+IG3) and accounts for the syrup that dry matter content is 49%～56%.(above-mentioned enzyme
The final concentration that is in fermentation tank of addition).
Claims (10)
1. an alpha-glucosaccharase enzyme mutant, it is characterised in that its aminoacid sequence of described mutant such as SEQ ID No.4 institute
Show.
2. the encoding gene of alpha-glucosaccharase enzyme mutant described in claim 1.
3. the encoding gene of alpha-glucosaccharase enzyme mutant as claimed in claim 2, it is characterised in that described encoding gene is such as
Shown in SEQ ID No.3.
4. alpha-glucosaccharase enzyme mutant described in claim 1 or the purposes of the gene described in claim 2, it is characterised in that
For preparing oligomeric isomaltose.
5. utilize mutant synchronous saccharification described in claim 1 to turn the method that glycosides efficiently prepares oligomeric isomaltose, specifically
As follows:
(1) starch liquefacation
(2) synchronous saccharification turns glycosides
Mash after liquefaction is cooled to 50 DEG C～60 DEG C, adds the beta amylase and 0.2～5U/mL of final concentration 13～25U/mL
Pullulanase vanguard action 1～4h under the conditions of pH 5.0～5.5 and temperature 50 C～55 DEG C, be eventually adding final concentration 40～
The continuation effect 9～20h under the conditions of pH 5.8～6.5 and temperature 55 DEG C～60 DEG C of the alpha-glucosidase of 250U/mL carries out turning
Glycosides.
A kind of synchronous saccharification the most as claimed in claim 5 turns the method that glycosides efficiently prepares oligomeric isomaltose, it is characterised in that
Described starch liquefacation uses injection liquefaction；Add 4.5t starch to equipped with 10m3In the fermentation tank of water, starch milk is stirred
After, add water in fermentation tank 18m3, regulate pH to 5.8～6.5；Add Thermostable α-Amylase to fermentation tank final concentration 4～
5U/mL, through Jet liquefier 101～110 DEG C of injection liquefaction, maintains tank i.e. to can be used for saccharifying and turn glycosides after maintaining.
A kind of synchronous saccharification the most as claimed in claim 5 turns the method that glycosides efficiently prepares oligomeric isomaltose, it is characterised in that
Described starch liquefacation uses ebuillition of heated liquefaction: add 4.5t starch to equipped with 10m3In the fermentation tank of water, starch milk is stirred
Adding a certain amount of water after uniformly, in fermentation tank, constant volume is to 18m3, regulate pH to 5.0～6.5；Starch milk is heated to 40～
50 DEG C, add Thermostable α-Amylase 3～5U/mL, continue the extremely boiling of heating starch mash, i.e. complete liquefaction.
A kind of synchronous saccharification the most as claimed in claim 1 turns the method that glycosides efficiently prepares oligomeric isomaltose, it is characterised in that
The mesophilicα-diastase of final concentration of 0.1～2U/mL is added while adding Thermostable α-Amylase.
A kind of synchronous saccharification the most as claimed in claim 5 turns the method that glycosides efficiently prepares oligomeric isomaltose, it is characterised in that
While synchronous saccharification adds beta amylase and pullulanase during turning glycosides, add the middle temperature alphalise starch of final concentration 2～5U/mL
Enzyme, common effect 1～4h, add final concentration 40～250U/mL alpha-glucosidase afterwards in pH 5.5～6.0 and temperature 55
DEG C～60 DEG C under the conditions of act on 9～20h.
A kind of synchronous saccharification the most as claimed in claim 5 turns the method that glycosides efficiently prepares oligomeric isomaltose, and its feature exists
In, it is as follows that synchronous saccharification turns glycosides process: the mash after liquefaction is cooled to 50 DEG C～60 DEG C, and four enzymes press pullulanase, middle temperature
The sequencing of α-amylase, beta amylase and alpha-glucosidase, respectively according to final concentration 0.2～5U/mL, 2～5U/mL,
The addition of 13～25U/mL, 40～250U/mL acts under the conditions of pH 5.0～6.5 and temperature 50 C～60 DEG C the most respectively
1～6h completes catalysis.
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