CN105925550B - Alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose - Google Patents
Alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose Download PDFInfo
- Publication number
- CN105925550B CN105925550B CN201610461264.6A CN201610461264A CN105925550B CN 105925550 B CN105925550 B CN 105925550B CN 201610461264 A CN201610461264 A CN 201610461264A CN 105925550 B CN105925550 B CN 105925550B
- Authority
- CN
- China
- Prior art keywords
- alpha
- glycosides
- starch
- oligoisomaltose
- added
- 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.)
- Active
Links
Classifications
-
- 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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- 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
- 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
- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
-
- 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
- C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
-
- 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)
- C12Y302/0102—Alpha-glucosidase (3.2.1.20)
Landscapes
- 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)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to technical field of enzyme engineering, and in particular to a kind of alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose.Half-life period t of the alpha-glucosidase at 66 DEG C1/23.7 times are improved, starch liquefacation is carried out using the alpha-glucosidase and Thermostable α-Amylase, mesophilicα-diastase, beta amylase, Pullulanase and synchronous saccharification turns the composition of oligoisomaltose core prepared by glycosides (IG2+P+IG3) ratio that accounts for dry matter reaches 52%~59%.
Description
Technical field:
The invention belongs to technical field of enzyme engineering, and in particular to a kind of acquisition of heat-resisting alpha-glucosaccharase enzyme mutant and height
Preparation method, starch liquefacation new method are imitated, saccharification turns glycosides and synchronizes the process for carrying out realizing the preparation of high-quality oligoisomaltose.
Background technique:
It is oligoisomaltose (Isomaltooligosaccharides, hereinafter referred to as IMO), also known as bifurcation oligose, different
Malto-oligosaccharide etc., be between glucose at least one with α -1,6 glycosidic bonds are combined into, monosaccharide number is not 2~6 not etc.
A kind of oligosaccharide.Its main component is isomaltose (IG2), panose (P), Isomaltotriose (IG3) and Isomaltotetraose (Gn)
Deng.Oligoisomaltose belongs to non-digestible property oligosaccharide, cannot be as the direct sources of body energy, but it can be selected
Property proliferation enteron aisle in the beneficial bacteriums such as Bifidobacterium, Bacillus acidi lactici, the short chain fatty acids of metabolism can reduce the pH of enteron aisle, suppression
The growth of harmful bacteria processed, to maintain the balance of intestinal microecology；In addition, these short chain fatty acids can be provided to other microorganisms
Energy, so also known as functional oligose.There are mainly two types of the commercial product specifications of oligoisomaltose: IMO-50 type (IG2+P
+IG3+Gn>=50%) and IMO-90 type (IG2+P+IG3+Gn>=90%).Contain 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 on the basis of IMO-50 by nanofiltration separation
High-purity oligoisomaltose.The main method of industrialized production IMO is that turn glycosides with glucosyl group glycosides enzymatic conversion be IMO, then
It is formed through decoloration, concentration, drying.
Oligoisomaltose has many physiological functions.Wherein direct physiological function has: (1) being difficult to be digested by gastric enzyme, heat
Measure low, sugariness is low, does not increase blood pressure and blood lipoid substantially；(2) probiotics in human body intestinal canal can be promoted --- the proliferation of Bifidobacterium,
The formation that can inhibit all kinds of harmful bacterias and corrupt substance in enteron aisle increases the content of various vitamins, improves immunity of organisms；
(3) prevention of dental caries function, it is not decomposed by oral cavity enzyme solution, is not utilized by the streptococcus of saprodontia, thus can prevent saprodontia, oligomeric different
Panose in maltose is extremely obvious to the effect for hindering tooth dirt to be formed；(4) it belongs to non-digest oligosaccharide class, and function is equal to
Water-soluble dietary fiber.Indirect physiological effect has: (1) promoting digestion, absorption of the digestive system to food, maintain the normal function of enteron aisle
Energy；(2) restore the normal bacterium colony of enteron aisle during antibiotic therapy, radiation cure, chemotherapy；(2) it prevents constipation and improves
Diarrhea inhibits pathogen and spoilage organisms；(4) immunity of organisms is improved, is excellent immunomodulator；(5) inhibit enteron aisle carcinogenic
The generation of substance reduces cholesterol level, improves serum lipids；(6) increase absorption of the body to minerals such as calcium, magnesium, help
B family vitamin is synthesized in body.
Currently, the classical way of production IMO is mainly made from starch both at home and abroad, turned using starch enzyme system multienzyme synergism method
Change starch fluid to obtain.It is industrially liquefied under the action of high-temperatureα-amylase by starch first, liquefying starch is in medium temperature alphalise starch
Enzyme or beta amylase continue to act on lower generation malt syrup, recycle phlorose to turn glycosides enzyme and carry out glycosyl conversion generation IMO,
In final product carbohydrate components about containing 50%~60% IMO, 40%~50% glucose, maltose and maltotriose, most
Finished product is obtained by filtering, decoloration, desalination, concentration etc..Using classical production technology, that there are processes is more, the time is long, technological parameter
Control is difficult, is difficult to realize the disadvantages of continuous production, and the alpha-glucosaccharase enzyme preparation as used in production process is at home
Production field is nearly at blank, relies primarily on import, and there are the problems such as expensive, source is unstable, meanwhile, function in product
The content of energy property sugar is not high, only accounts for 35% (w/w) of solid content, these all restrict the development of China IMO.
The deficiency as existing for classical technique production IMO, in recent years, domestic and international researcher is dedicated to enzymatic production
The research of IMO finds that bacterium, yeast, mould etc. can secrete alpha-glucosidase in microorganism, and aspergillus is micro- in relevant report
Biological producing enzyme is higher, but turns the active alpha-glucosidase of glycosides due to having as endocellular enzyme, and the apparent vigor of enzyme is lower, some
Scholar studies for the glycosides vigor expansion that apparently turns for improving alpha-glucosidase.The Permeabilized cells of the preparations such as Chen Gui light it is apparent
Enzyme activity reaches 483.9U/g wet thallus, is the 193.3% of the apparent enzyme activity of intact cell, oligomeric using Permeabilized cells conversion production
Isomaltose, transformation period are that for 24 hours, more complete cell shortens for 24 hours, and conversion ratio is maintained at 70% or more.Also, he is with black song
Mould GXM-3 carries out 60C- gamma-rays for starting strain and ultraviolet light irradiates compound mutation breeding, as a result, it has been found that, mutant strain D-597
Conversion capability it is most strong, oligoisomaltose content reaches peak in 3L fermentation cylinder for fermentation 80h, syrup product
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, and select one plant of high productive mutant AL7 is made by optimizing to condition of enzyme production
Its yield of enzyme is 9.24U, improves 4.18 times compared with starting strain.It is difficult since wild Aspergillus niger producing enzyme level is low
With extraction purification, domestic and international researcher starts building one after another and using genetic engineering bacterium production there is height to turn the active α-Portugal of glycosides
Polyglycoside enzyme.Child film stars etc. express aspergillus niger Aspergillus by host strain of Pichia pastoris Pichia pastoris KM71
Niger SG136 alpha-glucosidase is conducive to extracellular wherein the Yeast expression carrier pPIC9K selected contains coding sequence of secretory signal peptide
Expression, the crude enzyme liquid of preparation turn glycosides reaction in pH 5, and 55 DEG C of temperature, when reacting for 24 hours, the total content of oligoisomaltose reaches
26%.
As it can be seen that current IMO production technology still remains the problem that the production time is long and conversion ratio is low.
Summary of the invention:
In order to solve the above-mentioned technical problem, the present invention is used by the combination of reasonably combined various amylolytic enzymes
Heat resistance alpha-glucosidase, to form can simplify life with the manufacturing process of a step catalytic production oligoisomaltose
Production. art.
The present invention solve above-mentioned technical problem used by technical solution first is that: one kind is provided and passes through genetic engineering means
The alpha-glucosaccharase enzyme mutant that the heat resistance of acquisition significantly improves, amino acid 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 period t at more original 60 DEG C of alpha-glucosidase of the mutant1/2Improve 3.7 times；
The present invention goes out alpha-glucosidase gene by template reverse transcription amplification of aspergillus niger NRRL3135 total serum IgE, using easy
Wrong round pcr carries out molecular improvement to alpha-glucosidase gene agD, to obtain the excellent mutant that heat resistance significantly improves.
The present invention solve technical solution used by above-mentioned technical problem second is that: α-described in a kind of technical solution one is provided
The preparation method of glucoside enzyme mutant:
(1) gene described in SEQ ID NO:3 is subjected to digestion, and is connected to the plasmid pHY-WZX Jing Guo corresponding digestion
It connects, construction recombination plasmid；
(2) by recombinant plasmid transformed to bacillus licheniformis CBB3008 competent cell, preparation and reorganization bacterial strain；
(3) the expression recombinant bacterial strain, obtains alpha-glucosaccharase enzyme mutant shown in SEQ ID NO:4；
The bacillus licheniformis CBB3008, deposit number CCTCC No.M 208236, it is special referring specifically to Chinese invention
Sharp ZL 200810235368.0 and Dandan Niu, et al.Microbial Cell Factories, 2009,8:58；
The plasmid pHY-WZX refers to Dandan Niu and Zhengxiang Wang.J Ind Microbiol
Biotechnol,2007,34:357-362；
The present invention solve above-mentioned technical problem used by technical solution third is that: the α-Portugal significantly improved using heat resistance
Polyglycoside enzyme mutant, is made from starch, and provides a kind of synchronous saccharification and turns the method that glycosides efficiently prepares oligoisomaltose, has
Body is as follows:
(1) starch liquefacation
Injection liquefaction can be used in starch liquefaction or ebuillition of heated two ways is implemented；
Injection liquefaction: 4.5t starch is added to equipped with 10m3In the fermentor of water (starch, side stirring is added in side), by starch
After mixing evenly, in the fermenter plus water is to 18m for cream3, adjust pH to 5.8~6.5；Thermostable α-Amylase is added to fermentor
4~5U/mL of final concentration can be used to the glycosides that is saccharified and turns after maintaining tank to maintain through 107~110 DEG C of injection liquefaction of Jet liquefier.
Ebuillition of heated liquefaction: 4.5t starch is added to equipped with 10m3It, will in the fermentor of water (starch, side stirring is added in side)
Starch milk is stirring evenly and then adding into a certain amount of water, in the fermenter constant volume to 18m3, adjust pH to 5.0~6.5.By starch milk
40~50 DEG C are heated to, 3~5U/mL of Thermostable α-Amylase is added, continues heating starch mash to boiling, that is, completes liquefaction
Further, mesophilicα-diastase, final concentration of 0.1~2U/mL is added while Thermostable α-Amylase is added
(2) synchronous saccharification turns glycosides
Mash after above-mentioned liquefaction is cooled to 50 DEG C~60 DEG C, mesophilicα-diastase, Pullulanase, β-starch is added
Enzyme, several in alpha-glucosidase, maintaining reaction temperature specifically has following three kinds of schemes at 50 DEG C~60 DEG C:
Scheme one: addition 13~25U/mL of fermentor final concentration beta amylase and 0.2~5U/mL Pullulanase, in pH
5.0~5.5 and temperature 50 C~55 DEG C under the conditions of 1~4h of vanguard action, be eventually adding 40~250U/mL's of fermentor final concentration
Alpha-glucosidase continues 9~20h of effect under the conditions of pH 5.8~6.5 and 55 DEG C~60 DEG C of temperature and carries out turning glycosides effect；
Scheme two: the addition mesophilicα-diastase of 2~5U/mL of fermentor final concentration, 13~25U/mL beta amylase and
The Pullulanase of 0.2~5U/mL, 1~4h of vanguard action under the conditions of pH 5.0~5.5 and temperature 50 C~55 DEG C, then plus
The alpha-glucosidase for entering 40~250U/mL of final concentration, under the conditions of pH 5.5~6.0 and 55 DEG C~60 DEG C of temperature act on 9~
20h；
Three: four enzymes of scheme are successive suitable by Pullulanase, mesophilicα-diastase, beta amylase and alpha-glucosidase
Sequence, according to 2~5U/mL of mesophilicα-diastase final concentration, 0.2~5U/mL of Pullulanase final concentration, beta amylase final concentration 13~
The additive amount of 25U/mL, 40~250U/mL of alpha-glucosidase final concentration, in pH5.0~6.5 and temperature 50 C~60 DEG C condition
It is lower that 1~6h of effect completes catalysis respectively in succession；
Further, alpha-glucosidase is as described in technical solution one；
Further, the starch liquefaction, DE (degree of hydrolysis) can achieve 5~50；
Further, the relevant enzyme catalytic starch in the present invention prepares oligoisomaltose (IG2+P+IG3) account for dry matter
The isomaltooligosaccharide syrup of specific gravity 52%~59%.
The utility model has the advantages that
1, it the present invention provides a kind of acquisition of heat-resisting alpha-glucosaccharase enzyme mutant and high efficiency preparation method, is dashed forward
Become half-life period t of the enzyme at 60 DEG C1/2Improve 3.7 times.
2, the present invention provides one kind is synchronized using starch as synchronous saccharification and turns glycosides and efficiently prepare high-quality oligoisomaltose
Manufacturing technology, prepared oligoisomaltose core forms (IG2+P+IG3) ratio that accounts for dry matter reaches 52%~
59%；It is new that the oligoisomaltose to be formed preparation is combined with commercial enzyme using alpha-glucosaccharase enzyme mutant provided by the present invention
The total content of technique, reaction 13h oligoisomaltose can reach 52% or more.
3, Thermostable α-Amylase, mesophilicα-diastase involved in the manufacturing technology of oligoisomaltose of the invention,
Beta amylase, Pullulanase and alpha-glucosidase use novel expression and combinational expression mode, have significant catalytic performance；
4, starch liquefaction of the invention uses fast and convenient preparation process；
5, liquefaction syrup of the invention can fast implement the preparation of isomaltooligosaccharide syrup simultaneously or under the conditions of substep ground
Process.
Detailed description of the invention:
The PCR amplification of Fig. 1 alpha-glucosidase gene
Wherein, swimming lane 1 is the calibration of 1 λ DNA Pst I digestion molecular weight；Swimming lane 2 is the alpha-glucosidase base of PCR amplification
Because of segment；
The mutant enzyme of Fig. 2 SDS-PAGE analysis purifying
Wherein, 1 is fermentation liquid；2 be the component after ion exchange column purification；3 be the component of gel column after purification；M is egg
White molecular weight standard；
Fig. 3 prepares oligoisomaltose sample HPLC parsing map
Wherein, DP1- glucose, DP2- maltose, IG2Isomaltose, DP3- maltotriose, P- panose, IG3Different malt
Trisaccharide, IG4Isomaltotetraose.
Specific embodiment:
The present invention will be further described below in conjunction with the embodiments；Following embodiments do not limit the present invention, cannot be with
Following embodiments limit the scope of protection of the present invention.
Embodiment 1: the acquisition of alpha-glucosidase encoding gene segment
The total serum IgE of aspergillus niger NRRL3135 is extracted using TRNzol total RNA extraction reagent.Using total serum IgE as template, reference
RT-PCR kit specification is that primer reverse transcription synthesizes the first chain cDNA with oligo (dT), then respectively with the first chain cDNA
For template, PCR amplification is carried out with primers F 1 (SEQ ID NO:5) and R1 (SEQ ID NO:6) and goes out aspergillus niger alpha-glucosidase
Gene agD (Fig. 1).PCR product and plasmid pMD19-T simple vector are attached, recombinant plasmid pMD- is obtained
AgD, Transformed E scherichia coli JM109 competent cell.Positive transformant is screened, and extracts its plasmid and carries out digestion
Verifying.The correct recombinant plasmid of digestion verification is sequenced to (sequence SEQ ID NO:1, amino acid sequence are SEQ ID again
NO:2)。
Embodiment 2: alpha-glucosidase mutated library is constructed using fallibility PCR method
Coding mutation is introduced to alpha-glucosidase gene agD using fallibility round pcr 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 (being named with agDX) uses restriction enzyme after DNA purification and recovery kits
Xba I and Kpn I carry out digestion to it, and to plasmid pHY-WZX (the Dandan Niu and Jing Guo corresponding digestion
Zhengxiang Wang.J Ind Microbiol Biotechnol, 2007,34:357-362) it is attached, building recombination
Plasmid pHY-WZX-agDX.Take 8 μ L recombinant plasmid pHY-WZX-agDX DNA and bacillus licheniformis CBB3008 competent cell
It mixes, moves into the electric revolving cup of pre-cooling, after electric shock, the recovery medium (LB of sorbierite containing 0.5M and 0.5M mannitol culture is added
Base).37 DEG C, 160r/min recovery culture 3h, then coating receives the LB plate of mycin (0.03mg/mL) containing card, is incubated overnight.
According to above-mentioned method, more wheel fallibility PCR are carried out by template of mutant gene group, construct mutated library.
Embodiment 3: the screening of Heat Stability Mutations bacterial strain
Single colonie on picking LB plate, using 96 orifice plate LB liquid mediums in 30~32 DEG C of 55~70h of culture, dress
Liquid measure is 200~250 μ L, while being control with initial recombinant bacterium B.licheniformis CBB3008 (pHY-WZX).
96 orifice plate I are centrifuged removal thallus, successively take 10 μ L supernatants to be transferred in another 96 plate II from every hole.And it should
96 orifice plates are just being placed in 60 DEG C of heat treatment 60min.0.2% substrate o-methoxyphenyl-alpha-D-glucose glycosides is added into every hole
50 μ L, 30 DEG C of warm bath 10min.Be added 80 μ L 2.5mol/L NaOH solution terminate reaction (terminate liquid is first added in control group, then
Substrate is added).It prepares diazonium salt solution simultaneously: taking 0.2g/L NaNO2Then 100mL is added the HCl 5mL of 3mol/L, makes
The aniline 10mL, 30 DEG C of reaction 6min of 50g/L is added in acidity, 30 DEG C of warm bath 5min in solution.110 μ L diazonium salt solutions are taken to add
Enter in enzyme reaction system, color development at room temperature 2min.Microplate reader measures light absorption value at 450nm.Remaining enzyme activity is higher than control strain
It is purpose bacterial strain, screens 6 plants of bacterial strains altogether using this method.
Embodiment 4: the THERMAL STABILITY of mutant enzyme
By the fermentation liquid of purpose bacterial strain by ammonium sulfate precipitation, dialysis, DEAE-Sepharose ion exchange and
SuperdexTM200 gel filtrations are isolated and purified (Fig. 2).
Half-life period t using the enzyme activity determination method of embodiment 3, at 60 DEG C of measurement1/2.Specific measuring method are as follows: 10U is pure
Change enzyme to be put at 60 DEG C, be taken out through different time intervals, equilibrium at room temperature 5min, measures enzyme activity.Enzyme activity is at not hot after taking-up
The half-life period t of 50% time when reason, as enzyme at 60 DEG C1/2。
It is screened based on the mutated library of fallibility PCR building, obtains the bacterial strain that 6 plants of enzyme activity significantly improve, measured
Alpha-glucosidase nucleotide sequence speculates the amino acid sequence of alpha-glucosidase, phlorose using triplet codon
Half-life period t at the amino acid substitution of glycosides enzyme mutant and 60 DEG C1/2It is as shown in table 1 to improve multiple.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
It is continuous to use.Its amino acid sequence is as shown in sequence table SEQ ID NO:4, and gene order is as shown in sequence table SEQ ID NO:3.
The half-life period of 1 protoenzyme of table and mutant enzyme at 60 DEG C
BL-25 strain prepares alpha-glucosidase under embodiment 5:15L fermentation system
Strain BL-25 is inoculated into seed culture medium, and (yeast extract 3%, peptone 3.8%, glucose 10%, remaining is
Water, pH 7.0) it cultivates and arrives logarithmic growth phase, fermenting experiment is further carried out in 15L fermentor, fermentation medium is (w/v):
Yeast extract 1%, peptone 3%, lactose 10%, calcium carbonate 1.4%, surplus are water, are controlled in fermentation process with sulfuric acid or ammonium hydroxide
PH is 7.0, with 5% inoculum concentration be inoculated with, 37 DEG C, fermentation 120h after, this bacterial strain generate alpha-glucosidase fermentation liquid enzyme activity
Level can reach 11000U/mL.BL-25 strain prepares alpha-glucosidase under embodiment 6:15L fermentation system
Strain BL-25 is inoculated into seed culture medium, and (yeast extract 5%, peptone 6.2%, glucose 20%, remaining is
Water, pH 7.0) it cultivates and arrives logarithmic growth phase, fermenting experiment is further carried out in 15L fermentor, fermentation medium is (w/v):
Yeast extract 1%, peptone 3%, lactose 10%, calcium carbonate 1.4%, surplus are water, are controlled in fermentation process with sulfuric acid or ammonium hydroxide
PH is 7.0.With 5% inoculum concentration be inoculated with, 37 DEG C, fermentation 150h after, this bacterial strain generate alpha-glucosidase enzyme activity level can
Reach 13000U/mL.
Embodiment 7: in 30m3Strain BL-25 fermentation prepares alpha-glucosidase under system
It is 30m by the technique adjustment of embodiment 63The corresponding ratio of fermentation system.It is respectively completed seed culture, is inoculated with 15L
First class seed pot, culture transferring 3m3Secondary seed tank cultivates thallus after the operation such as main fermentation tank culture transferring, after feed supplement growth phase,
Enzymatic production 90~120h post-fermentation terminates.Fermentation liquid removes thallus through plate-frame filtering, and enzyme solution is concentrated to suitable concentration in ultrafiltration membrane,
Refined filtration is spray-dried preparation after preparing alpha-glucosidase liquid end product, or the appropriate food-grade starches of addition after addition auxiliary agent
Powder form alpha-glucosidase finished product, finished product enzyme activity are 350000U/g~370000U/g.
Embodiment 8:30m3Sugaring system prepares oligoisomaltose technique one
4.5t starch is added to equipped with 10m3In the fermentor of water (starch, side stirring is added in side), starch milk is stirred equal
After even, in the fermenter plus water is to 18m3, adjust pH to 6.1；Thermostable α-Amylase 4U/mL is added, through Jet liquefier 109
DEG C injection liquefaction, maintain tank maintain 6h after can be used to the glycosides that is saccharified and turns.
If the mash after above-mentioned liquefaction is cooled to 54 DEG C by scheme one, adjusting pH is 5.5 addition Pullulanase 2U/mL, β-
PH is adjusted to 6.0 after amylase 14U/mL, effect 3h, and 6 gained alpha-glucosidase 220U/mL of embodiment is added, maintains reaction
At 57 DEG C, reaction 13h can be obtained oligoisomaltose (IG2+P+IG3) and accounts for the sugar that dry matter content is 38%~49% temperature
Slurry.(additive amount of above-mentioned enzyme is the final concentration in fermentor).
Case study on implementation 9:30m3Sugaring system prepares oligoisomaltose technique two
Starch liquefacation and saccharification: 4.5t starch is added to equipped with 10m3In the fermentor of water (starch, side stirring is added in side),
Starch milk is stirring evenly and then adding into a certain amount of water, in the fermenter constant volume to 18m3, adjust pH to 6.0.By starch milk plus
Thermostable α-Amylase 4.5U/mL is added to 45 DEG C in heat, continues heating starch mash to boiling, that is, completes liquefaction.By above-mentioned liquid
Mash after change is cooled to 59 DEG C, and Pullulanase 1.8U/mL, beta amylase 14.2U/mL, 7 gained phlorose of embodiment is added
Glycosides enzyme 165U/mL, at 59 DEG C, reaction 10h can be obtained oligoisomaltose (IG2+P+IG3) and accounts for dry matter maintaining reaction temperature
The syrup that content is 35%~45%.(additive amount of above-mentioned enzyme is the final concentration in fermentor).
Case study on implementation 10:30m3Sugaring system prepares oligoisomaltose technique three
Starch liquefacation and saccharification: 4.5t starch is added to equipped with 10m3In the fermentor of water (starch, side stirring is added in side),
Starch milk is stirring evenly and then adding into a certain amount of water, in the fermenter constant volume to 18m3, adjust pH to 6.0.By starch milk plus
Thermostable α-Amylase 4.3U/mL and mesophilicα-diastase 1.2U/mL is added to 48 DEG C in heat, continues heating starch mash to boiling
It rises, that is, completes liquefaction.Mash after above-mentioned liquefaction is cooled to 59 DEG C, Pullulanase 1.8U/mL, beta amylase 14.2U/ is added
ML, 7 gained alpha-glucosidase 165U/mL of embodiment, for maintaining reaction temperature at 59 DEG C, oligomeric different wheat is can be obtained in reaction 10h
Bud sugar (IG2+P+IG3) accounts for the syrup that dry matter content is 40%~50%.(additive amount of above-mentioned enzyme is the end in fermentor
Concentration).
Case study on implementation 11:30m3Sugaring system prepares oligoisomaltose technique four
Starch liquefacation and saccharification: 4.5t starch is added to equipped with 10m3In the fermentor of water (starch, side stirring is added in side),
Starch milk is stirring evenly and then adding into a certain amount of water, in the fermenter constant volume to 18m3, adjust pH to 6.0.By starch milk plus
Thermostable α-Amylase 4.8U/mL and mesophilicα-diastase 1.1U/mL is added to 49 DEG C in heat, continues heating starch mash to boiling
It rises, that is, completes liquefaction.
If the mash after above-mentioned liquefaction is cooled to 55 DEG C by scheme two, adjust pH be 5.5 be added Pullulanase 1.6U/mL,
Beta amylase 13.5U/mL, mesophilicα-diastase 2.0U/mL, pH is adjusted to 6.0 after acting on 3h, and 7 gained α of embodiment-Portugal is added
Polyglycoside enzyme 186U/mL, for maintaining reaction temperature at 55 DEG C, oligoisomaltose (IG2+P+IG3) Zhan Gan is can be obtained in reaction 13h
The syrup that content of material is 52%~59%.(additive amount of above-mentioned enzyme is the final concentration in fermentor).
Case study on implementation 12:30m3Sugaring system prepares oligoisomaltose technique five
Starch liquefacation and saccharification: 4.5t starch is added to equipped with 10m3In the fermentor of water (starch, side stirring is added in side),
Starch milk is stirring evenly and then adding into a certain amount of water, in the fermenter constant volume to 18m3, adjust pH to 6.0.By starch milk plus
Thermostable α-Amylase 4.8U/mL and mesophilicα-diastase 1.1U/mL is added to 49 DEG C in heat, continues heating starch mash to boiling
It rises, that is, completes liquefaction.
If the mash after above-mentioned liquefaction is cooled to 54 DEG C by scheme three, adjusting pH is that 5.3 addition Pullulanase 1.7U/mL make
After 3h, mesophilicα-diastase 2.2U/mL is added and continues after acting on 4h, beta amylase 15.8U/mL is added and continues after acting on 2h,
PH is adjusted to 6.0, and 7 gained alpha-glucosidase 186U/mL of embodiment is added, and maintaining reaction temperature amounts in 55 DEG C of reaction 4h
Reaction 13h can be obtained oligoisomaltose (IG2+P+IG3) and account for the syrup that dry matter content is 49%~56%.(above-mentioned enzyme
Additive amount be final concentration in fermentor).
Claims (10)
1. a kind of alpha-glucosaccharase enzyme mutant, which is characterized in that its amino acid sequence of the 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, which is characterized in that the encoding gene is such as
Shown in SEQ ID No.3.
4. the purposes of alpha-glucosaccharase enzyme mutant or gene as claimed in claim 2 described in claim 1, which is characterized in that
It is used to prepare oligoisomaltose.
5. a kind of turn the method that glycosides efficiently prepares oligoisomaltose using mutant synchronous saccharification described in claim 1, specifically
It is 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 13~25U/mL of final concentration
Pullulanase under the conditions of pH 5.0~5.5 and temperature 50 C~55 DEG C 1~4h of vanguard action, be eventually adding final concentration 40~
The alpha-glucosidase of 250U/mL continues 9~20h of effect under the conditions of pH 5.8~6.5 and 55 DEG C~60 DEG C of temperature and is turned
Glycosides.
6. a kind of synchronous saccharification as claimed in claim 5 turns the method that glycosides efficiently prepares oligoisomaltose, which is characterized in that
The starch liquefacation is using injection liquefaction；4.5t starch is added to equipped with 10m3In the fermentor of water, starch milk is stirred evenly
Afterwards, in the fermenter plus water is to 18m3, adjust pH to 5.8~6.5；Be added Thermostable α-Amylase to fermentor final concentration 4~
5U/mL can be used to the glycosides that is saccharified and turns after maintaining tank to maintain through 101~110 DEG C of injection liquefaction of Jet liquefier.
7. a kind of synchronous saccharification as claimed in claim 5 turns the method that glycosides efficiently prepares oligoisomaltose, which is characterized in that
The starch liquefacation is liquefied using ebuillition of heated: 4.5t starch is added to equipped with 10m3In the fermentor of water, starch milk is stirred
A certain amount of water is added after uniformly, in the fermenter constant volume to 18m3, adjust pH to 5.0~6.5；Starch milk is heated to 40~
50 DEG C, 3~5U/mL of Thermostable α-Amylase is added, continues heating starch mash to boiling, that is, completes liquefaction.
8. a kind of synchronous saccharification as claimed in claim 7 turns the method that glycosides efficiently prepares oligoisomaltose, which is characterized in that
The mesophilicα-diastase of final concentration of 0.1~2U/mL is added while Thermostable α-Amylase is added.
9. a kind of synchronous saccharification as claimed in claim 5 turns the method that glycosides efficiently prepares oligoisomaltose, which is characterized in that
While synchronous saccharification turns that beta amylase and Pullulanase are added during glycosides, the medium temperature alphalise starch of 2~5U/mL of final concentration is added
Enzyme, 1~4h of collective effect add 40~250U/mL of final concentration alpha-glucosidase in pH 5.5~6.0 and temperature 55 later
DEG C~60 DEG C under the conditions of act on 9~20h.
10. a kind of synchronous saccharification as claimed in claim 5 turns the method that glycosides efficiently prepares oligoisomaltose, feature exists
In it is as follows that synchronous saccharification turns glycosides process: the mash after liquefaction being cooled to 50 DEG C~60 DEG C, four enzymes press Pullulanase, medium temperature
The sequencing of alpha-amylase, beta amylase and alpha-glucosidase, respectively according to 0.2~5U/mL of final concentration, 2~5U/mL,
13~25U/mL, 40~250U/mL additive amount acted on respectively in succession under the conditions of pH 5.0~6.5 and temperature 50 C~60 DEG C
1~6h completes catalysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610461264.6A CN105925550B (en) | 2016-06-23 | 2016-06-23 | Alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610461264.6A CN105925550B (en) | 2016-06-23 | 2016-06-23 | Alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105925550A CN105925550A (en) | 2016-09-07 |
| CN105925550B true CN105925550B (en) | 2019-06-04 |
Family
ID=56832063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610461264.6A Active CN105925550B (en) | 2016-06-23 | 2016-06-23 | Alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105925550B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164345B (en) * | 2017-07-06 | 2019-09-03 | 江南大学 | A kind of beta amylase mutant that thermal stability improves |
| CN107384985A (en) * | 2017-09-20 | 2017-11-24 | 无锡甜丰食品有限公司 | A kind of Mashing process of high concentration maltodextrin |
| CN109055461B (en) * | 2018-08-28 | 2021-12-17 | 广州双桥股份有限公司 | Production method of isomaltooligosaccharide |
| CN109207458B (en) * | 2018-10-31 | 2021-06-25 | 福州大学 | Heat-resistant medium-temperature alpha-amylase and application thereof |
| JP7002624B1 (en) | 2020-11-12 | 2022-01-20 | 群栄化学工業株式会社 | Grain saccharified liquid and its manufacturing method |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101031643A (en) * | 2004-06-29 | 2007-09-05 | 诺维信股份有限公司 | Polypeptides having alpha-glucosidase activity and polynucleotides encoding same |
| CN103443273A (en) * | 2011-03-16 | 2013-12-11 | 天野酶株式会社 | Modified alpha-lucosidase and applications of same |
| CN103497983A (en) * | 2013-10-14 | 2014-01-08 | 无锡健特药业有限公司 | Method of using alpha-glucosidase for preparing isomaltooligosaccharide |
| CN104131051A (en) * | 2014-08-08 | 2014-11-05 | 山东百龙创园生物科技有限公司 | Preparation method of isomaltooligosaccharide |
| CN104152512A (en) * | 2014-08-08 | 2014-11-19 | 山东百龙创园生物科技有限公司 | Preparation method of isomaltooligosacharide |
| CN104328096A (en) * | 2014-10-29 | 2015-02-04 | 青岛蔚蓝生物集团有限公司 | Alpha-glucosidase and application of alpha-glucosidase |
| CN104877978A (en) * | 2015-02-12 | 2015-09-02 | 广西南宁智天生物科技有限公司 | Preparation method of aspergillus niger-source alpha-glucosidase solid preparation and application thereof |
| CN105219665A (en) * | 2015-10-29 | 2016-01-06 | 天津科技大学 | A kind of manufacture method of oligomeric isomaltose and catalyzer thereof |
| CN105671105A (en) * | 2016-04-11 | 2016-06-15 | 江南大学 | Method for producing malt syrup and isomaltooligosaccharide in enzyme-adding, extruding and liquefying mode |
-
2016
- 2016-06-23 CN CN201610461264.6A patent/CN105925550B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101031643A (en) * | 2004-06-29 | 2007-09-05 | 诺维信股份有限公司 | Polypeptides having alpha-glucosidase activity and polynucleotides encoding same |
| CN103443273A (en) * | 2011-03-16 | 2013-12-11 | 天野酶株式会社 | Modified alpha-lucosidase and applications of same |
| CN103497983A (en) * | 2013-10-14 | 2014-01-08 | 无锡健特药业有限公司 | Method of using alpha-glucosidase for preparing isomaltooligosaccharide |
| CN104131051A (en) * | 2014-08-08 | 2014-11-05 | 山东百龙创园生物科技有限公司 | Preparation method of isomaltooligosaccharide |
| CN104152512A (en) * | 2014-08-08 | 2014-11-19 | 山东百龙创园生物科技有限公司 | Preparation method of isomaltooligosacharide |
| CN104328096A (en) * | 2014-10-29 | 2015-02-04 | 青岛蔚蓝生物集团有限公司 | Alpha-glucosidase and application of alpha-glucosidase |
| CN104877978A (en) * | 2015-02-12 | 2015-09-02 | 广西南宁智天生物科技有限公司 | Preparation method of aspergillus niger-source alpha-glucosidase solid preparation and application thereof |
| CN105219665A (en) * | 2015-10-29 | 2016-01-06 | 天津科技大学 | A kind of manufacture method of oligomeric isomaltose and catalyzer thereof |
| CN105671105A (en) * | 2016-04-11 | 2016-06-15 | 江南大学 | Method for producing malt syrup and isomaltooligosaccharide in enzyme-adding, extruding and liquefying mode |
Non-Patent Citations (1)
| Title |
|---|
| Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous;LucíaFernández-Arrojo et al.;《Process Biochemistry》;20071130;1530-1536 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105925550A (en) | 2016-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105925550B (en) | Alpha-glucosidase and synchronous saccharification turn the method that glycosides prepares oligoisomaltose | |
| CN104726523B (en) | A kind of method that enzyme process prepares Rebaudiodside A M | |
| BR112016019823B1 (en) | GLUCANE SYNTHESIS METHOD AND SOLUBLE SACCHARIDE BY-PRODUCT HYDROLYSIS | |
| CN106244647B (en) | Method for simultaneously preparing trehalose and gluconolactone | |
| CN103068997A (en) | Neutral ph saccharification and fermentation | |
| CN104212854A (en) | Method for producing isomalto-oligosaccharide by using high-concentration starch | |
| CN106636256A (en) | Xylooligosaccharide with low DP (degree of polymerization) as well as preparation method and application of xylooligosaccharide | |
| CN111662942A (en) | Method for producing rebaudioside-A through double-enzyme fermentation catalysis | |
| CN112852796A (en) | Cellobiose epimerase mutant and application thereof in preparation of lactulose | |
| CN105039374B (en) | A kind of starch induction type recombined bacillus subtilis and preparation method and application | |
| CN105177084B (en) | A kind of method of inulin enzyme mutant fermenting and producing oligofructose | |
| CN108949713B (en) | Preparation method of aspergillus oryzae fermentation broth and application of aspergillus oryzae fermentation broth in production of fructo-oligosaccharide | |
| CN113186141B (en) | Method for efficiently synthesizing rebaudioside M by one-pot method | |
| CN104480075B (en) | Bio-enzyme for synthesis of N(2)-L-alanyl-L-glutamine by catalysis as well as preparation method and application thereof | |
| WO2024113827A1 (en) | Oligosaccharide debranching enzyme mutant and use thereof in glucose mother liquor | |
| AU2002225387B2 (en) | Polypeptide having alpha-isomaltosylglucosaccharide synthase activity | |
| CN109207458B (en) | Heat-resistant medium-temperature alpha-amylase and application thereof | |
| CN111593062A (en) | Method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis | |
| Schoutens et al. | Butanol from whey ultrafiltrate: batch experiments with Clostridium beyerinckii LMD 27.6 | |
| CN106674360A (en) | Purifying method of resistant dextrin | |
| CN111455003A (en) | Method for preparing D-psicose from microalgae | |
| CN113025590A (en) | Optimization method for improving secretion expression of cyclodextrin glucosyltransferase and application thereof | |
| CN114150031A (en) | Method for preparing rebaudioside D by utilizing fermentation catalysis of bacillus subtilis | |
| CN107916282A (en) | A kind of method that bioanalysis prepares L citrulling and L ornithines | |
| CN101012469A (en) | Method for preparing 5'-nucleotides using malt root complex phospho-esterase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |