CN102382770A - Bacterial strain building method of glucoside transferase genetic engineering - Google Patents
Bacterial strain building method of glucoside transferase genetic engineering Download PDFInfo
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Abstract
The invention relates to a bacterial strain building method of glucoside transferase genetic engineering, specifically to a method for screening production bacterial strain that produces glucoside transferase, comprising the steps of: (a) measuring the titer of glucoside transferase as an antigen in a protein simple of each candidate strain by a specific antibody of glucoside transferase; and (b) selecting high titer candidate strain as a production strain for producing the glucoside transferase according to titer value measured. The candidate strain is a bacterial strain which is performed with gene knockout. The invention can efficiently prepare high yield bacterial strain of glucoside transferase and improve the efficiency of catalytically synthesizing isomaltooligosaccharide by glucoside transferase to a great extent.
Description
Technical field
The present invention relates to the fermentation field, more specifically, the present invention relates to glycosyltransferase (Alpha-GTase) construction method of gene engineering strain method, and the application in the synthetic dextrinosan of efficient catalytic.
Background technology
Glycosyltransferase (Transglucosidase; EC 2.4.1.24 alpha-GTase) is a kind of α-D glucuroide, can generate glucose for hydrolysis SANMALT-S; Also can carry out the shift reaction of glucoside bond single-mindedly; Be one of indispensable enzyme preparation of producing dextrinosan, generate non-fermentable oligose such as the isomaltose that has α-1,6 key, panose by SANMALT-S.
Low molecular weight oligomeric sugar such as discovered in recent years isomaltose, panose are a kind of good pair of qi factors, take the photograph behind the people not absorbed by human consumption, also are difficult for being utilized by the most putrefactive bacterium in the large intestine, but can be utilized as the carbon source of bifidus bacillus.Therefore as a kind of functional food ingredient, dextrinosan extensively has been used in the manufacturing of various food, occupies first of the various functional oligoses.
Having had with starch or SANMALT-S at present is raw material, through the technology of Production by Enzymes dextrinosan.Yet present production technique exists transformation efficiency lower, generally at 30%-45%.In order to improve transformation efficiency, need to obtain the higher dextrinosan of highly purified activity of conversion, also need screen the production bacterial strain that obtains corresponding highly active glycosyltransferase.
From industriallization, though China's isomaltose output is very high, the consumption of glycosyltransferase is very big, does not have industriallization, at present the complete dependence on import of this enzyme.In addition, present production technique exists transformation efficiency lower, generally at 30%-45%, therefore will obtain highly purified dextrinosan, needs to separate further, has increased production cost.
Aspergillus tubigensis in the filamentous fungus (Aspergillus) is grown rapidly owing to it has, is easy to cultivate, can produce secreted protein in a large number, also receives people's very big concern.Wherein black-koji mould (Aspergillus niger) is a kind of safe fungi that does not produce Toxins, afla, so it is widely used on the fermentation industry of producing various fungal metabolites and enzyme.
Though have bacterial strain quite a lot all to have to produce the ability of glycosyltransferase, yet people and do not know the active higher of glycosyltransferase that which kind of bacterium produces.
In sum, this area presses for the superior strain of exploitation glycosyltransferase.
Summary of the invention
The object of the invention just provides a kind of method for preparing the superior strain of glycosyltransferase.
Another object of the present invention is to provides the method for preparing glycosyltransferase from described production bacterial strain, and the application of said glycosyltransferase in producing dextrinosan.
In first aspect of the present invention, a kind of method of screening the production bacterial strain of producing glycosyltransferase is provided, comprise step:
(a), measure tiring as antigenic glycosyltransferase in the protein sample of each candidate strain with the specific antibody of anti-glycosyltransferase; With
(b), select high candidate strain of tiring as the production bacterial strain of producing glycosyltransferase according to the valence value of measuring;
Wherein, described candidate strain is the bacterial strain of handling through gene knockout.
Certainly, this method bacterial strain of also can be used for natural bacterial strain or handling without gene knockout.
In another preference, the method that the mensuration in the step (a) is tired is an enzymoimmunoassay.
In another preference, said method also comprises step (c): for selected production bacterial strain, measure the ability that its glycosyltransferase catalysis SANMALT-S is produced dextrinosan.
In another preference, the candidate strain of in step (a), measuring is selected from group down: black mold (Aspergillus niger), smelly aspergillus (Aspergillus foetidus), carbon black aspergillus (Aspergillus carbonarious), cinnamon aspergillus (Aspergillus cinmomeus), Aspergillus usamii (Aspergillus usamii), Aspergillus nidulans (Aspergillus nidulan), aspergillus oryzae (Aspergillus oryzae), penicillium spp (P.chrysogenum) and Aureobasidium pullulans (Aureobasidium pullulans).
In another preference, described candidate strain is to carry out the bacterial strain that gene knockout is handled with the agrobacterium tumefaciens Ti-plasmids.
In another preference, the quantity of the candidate strain of in step (a), measuring is the 5-1000 kind, and preferably the 5-200 kind more preferably is the 10-100 kind.
In another preference, described antibody is monoclonal antibody, polyclonal antibody or antiserum(antisera).
In second aspect of the present invention, provide a kind of glycosyltransferase that filters out with method described in the first aspect present invention to produce bacterial strain.
In another preference, described production bacterial strain is black mold or Aspergillus usamii.
In another preference, said glycosyltransferase produce bacterial strain production of enzyme be the 2g/1000ml fermented liquid, enzyme is lived and is 50000U/ml.
In the third aspect of the invention, a kind of glycosyltransferase is provided, described glycosyltransferase is to produce with the production bacterial strain described in the second aspect present invention.
In another preference, described glycosyltransferase is that black mold, smelly aspergillus, carbon black aspergillus, cinnamon aspergillus, Aspergillus nidulans, aspergillus oryzae, penicillium spp, Aureobasidium pullulans or Aspergillus usamii produce.
In fourth aspect of the present invention, a kind of method of producing dextrinosan is provided, said method comprises step: use the shift reaction of the described glycosyltransferase catalysis of third aspect present invention glucoside bond, thereby form dextrinosan.
In another preference, described dextrinosan comprises isomaltose, trisaccharide maltose, Isomaltotriose, maltotetrose or panose.
In another preference, the used raw material of shift reaction is a SANMALT-S.
In another preference, the condition of described shift reaction is 30-60 ℃, and preferably 53-57 ℃, the time is 24-72 hour.
In another preference, when being raw material, use the transformation efficiency of the shift reaction formation dextrinosan of described glycosyltransferase catalysis glucoside bond to be 55-65% with SANMALT-S or starch, preferably be 56-63%, more preferably be 60-62%.
In another preference; Described catalyzed reaction comprises: by SANMALT-S weight; The glycosyltransferase that adds 0.05～0.5wt%, and 30 ℃～60 ℃ insulations of temperature 24～72 hours, thereby the dextrinosan slurry that dextrinosan content is 50～65wt% obtained.
Should be understood that within the scope of the present invention each above-mentioned and following technical characterictic of the present invention can mutual combination, form and constitute new or optimized technical scheme.
Description of drawings
Fig. 1 has shown that the Elisa of different strains glycosyltransferase detects colour developing figure.
Wherein, the different sesquialter dilution number of times (tiring) of horizontal 1,2,3,4,5,6,7,8,9,10 representatives;
The corresponding strain number 10Z307GTase1 of vertical 1,2,3,4,5,6 difference, 10Z307GTase2,10Z307GTase5,10Z307GTase6,10Z307GTase8.
Fig. 2 has shown glycosyltransferase fermentation kinetics curve.
Fig. 3 has shown that W-Gum is the HPLC analysis of substrate dextrinosan.
Fig. 4 SANMALT-S is that the HPLC of substrate dextrinosan analyzes.
Fig. 5 has shown the synoptic diagram that carries out gene knockout through T-DNA insertion sudden change.
Fig. 6 has shown the PCR evaluation figure of the Aspergillus niger10ZTGase1 that handles through gene knockout in instance of the present invention.Each swimming lane is following: M-molecular weight standard, the original bacterium of 1-, 2-plasmid pTFMC, the positive muton of 3-8.
Embodiment
The inventor is through extensive and deep research; Be surprised to find that; Can come high-throughput through the specific reaction of Ag-Ab and screen the production bacterial strain of glycosyltransferase on a large scale, wherein used antibody is the antibody (especially antiserum(antisera)) of specific anti glycosyltransferase.Through the specific reaction of Ag-Ab, not only can screen the bacterial strain that produces glycosyltransferase, and unexpectedly, the catalysis of the glycosyltransferase of being produced forms active the tiring with mensuration of oligose and just has positive correlation.
The also further using gene engineering technique of the present invention designs the metabolic flux in the microorganism cells again, thereby obtains the industrial strain of high-yield and high-efficiency.Particularly, the inventor adopts the metabolic bypass of gene knockout technology blocking-up cell, thereby and/or reach through introducing output that the mutational site changes the purpose product or quality and to regulate metabolism stream, optimize the purpose of pathways metabolism.
On this basis, the inventor has accomplished the present invention.
As used herein, term " oligomeric isomaltose " and " dextrinosan " interchangeable use all refer to be selected from down one or more sugar of organizing: isomaltose, trisaccharide maltose, Isomaltotriose, maltotetrose or panose.In addition, should be understood that described term also comprises the mixture of above-mentioned sugar.
Screening method
In screening method of the present invention, its core is to have utilized the antibody of anti-glycosyltransferase to screen corresponding antigen, i.e. glycosyltransferase.
Because screening method of the present invention is based on the exclusive reaction of antigen-antibody, so accuracy is high.
In the present invention, the specificity whether Ag-Ab takes place combines and the method for the size of tiring for being used to measure, not special restriction.This area various mensuration Ag-Ab specificitys commonly used combine and measure the method that combines to tire and all can use.Representational example comprises (but being not limited to): enzyme-linked immunosorbent assay (ELISA), competitive combined techniques etc.
The not special restriction of the specific antibody of the anti-glycosyltransferase that is suitable in the present invention.Can be commercially available, also can prepare with ordinary method.Described antibody can be monoclonal antibody, also can be polyclonal antibody, especially antiserum(antisera).
In screening method of the present invention,, measure tiring as antigenic glycosyltransferase in the protein sample of each candidate strain earlier with the specific antibody of anti-glycosyltransferase; According to the valence value of measuring, select high candidate strain of tiring then as the production bacterial strain of producing glycosyltransferase.
Gene knockout
In the remodeling method to production bacterial strains such as aspergillus tubigensis of the present invention, its core is an agrobacterium tumefaciens T-DNA knockout technique.
With the black mold is example, relates generally to the agrobacterium tumefaciens Ti-plasmids based on the gene knockout system of agrobacterium tumefaciens.Wherein, T-DNA is the part of agrobacterium tumefaciens Ti (tumor-inducing) plasmid, and it can be integrated in the genome through any dna sequence dna that the agrobacterium tumefaciens effect will be inserted between its two border.Do not having under the situation of homologous sequence, T-DNA can carry out non-homogeneous reorganization, inserts sudden change at random and produce, and makes mutator gene obtain the mark (see figure 5).Shortcomings such as the transformation efficiency that protoplasm body had that the conversion that utilizes the agrobacterium tumefaciens method to carry out filamentous fungus has overcome conventional PEG mediation is low, complicated operation, multiple copied.
Applicable to the not special restriction of agrobacterium tumefaciens Ti-plasmids of the present invention, Ti-plasmids that this area is commonly used or the Ti-plasmids that makes up with ordinary method all can be used for the present invention.One type of preferred Ti-plasmids is between the left arm of T-DNA and right arm, to insert the formed plasmid of resistant gene (like hygromycin gene, penicillin resistance gene etc.), so that utilize resistance to screen.
With regard to process, gene knockout comprises: the preparation
of the activation culture of agrobacterium tumefaciens
fungal spore transforms
screening
acquisition muton.
In a preference, the conversion condition of employing is following: incubation time 4-48 hour, and spore number 1-100/ml, inductor Syringylethanone (AS) concentration 5-300mmol/L, Agrobacterium concentration 0.1～2OD, culture temperature is 10～45 ℃ altogether.
Fermentative prepn
The present invention the production bacterial strain that filters out from said antibody screening method also is provided and further handle through gene knockout after the production bacterial strain (being commonly referred to as " the production bacterial strain that filters out ") that filters out, the technology of preparation glycosyltransferase.
The production bacterial strain that the inventive method filters out can be used for preparing glycosyltransferase.When producing glycosyltransferase, can adopt the technology of the conventional preparation zymin in this area.Usually, this method comprises: cultivate described production bacterial strain; And separation obtains glycosyltransferase from described tunning.
In fermentation, adoptable fermentation condition is following:
The not special restriction of carbon source can be chosen glucose, Semen Maydis powder, W-Gum, sucrose, fructose, wood sugar, SANMALT-S, lactose and hydrolysis sugar.The not special restriction of concentration, concentration is between 1%-6% usually.
The not special restriction of nitrogenous source can be chosen wheat bran, yeast powder, peptone, Semen arachidis hypogaeae protein, soybean cake powder, steeping water, soya-bean cake hydrolyzed solution, urea, ammonium sulfate.The not special restriction of concentration, concentration is between 0.1%-4% usually.
Nutritive salt can be selected by the kind of producing bacterial strain is conventional, comprises K usually
3PO
4, MgSO
47H
2O, FeCl
3, zinc sulfate, boric acid, calcium chloride, various constituent concentrations generally arrive the milligram level in the ppm level.
The tensio-active agent tween 80 is between 0.1～1%.
Leavening temperature in 25-35 ℃ scope, fermentation time 3-6 days.
Mixing speed is between 120-300rpm, and ventilation is at 0.5～1.0v/v/m.
PH is controlled between the 5-6 in the fermenting process.
During fermentation, can adopt or not adopt the strategy of ferment middle fed-batch medium.If forming, employing fed-batch medium, then representational feeding culture can be carbon source 30%, nitrogenous source 10%, nutritive salt 10～20%.Stream adds 12 hours-24 hours time, looks the situation of fermentative activity and decides.Flow acceleration requires and adjustment automatically according to the control of pH and dissolved oxygen.
After the fermentation, can be selected from suitable separation method according to glycosyltransferase and the characteristic of producing bacterial strain.
A kind of preferred separation method comprises: fermented liquid is got supernatant through high speed frozen centrifugation (like 7500rpm, 4 ℃, 20 minutes).Adopt hollow fiber membrane ultrafiltration device, ultra-filtration membrane dams molecular weight greater than 100,000 dalton, intake pressure 0.1-0.3MPa; Top hole pressure is 0.40～0.5MPa; Filtrating is 1-2: 2-1 (preferred about 1: 1) with the quantity of reflux ratio, 10 ℃-30 ℃ of service temperatures, and cycles of concentration is 2-10 doubly (as 5 times); The liquid that dams is Liquid Glucose glycosides transferring enzyme preparation, enzyme 80,000～150,000 U/ml alive.
Another kind of preferred separation method comprises: fermented liquid is got supernatant through high speed frozen centrifugation (like 7500rpm, 4 ℃, 20 minutes); Use 60% saturated ammonium sulphate, deposition is dissolved in pure water, adopts hollow fiber membrane ultrafiltration device; Ultra-filtration membrane dams molecular weight greater than 100,000 dalton, and intake pressure 0.1-0.3MPa, top hole pressure are 0.40～0.5MPa; Filtrating is 1-2: 2-1 (preferred about 1: 1), 10 ℃-30 ℃ of service temperatures, 5 times of cycles of concentration with the quantity of reflux ratio; The liquid that dams carries out vacuum spray drying after adding drying protectant (comprising 1%～7% N.F,USP MANNITOL, 5%～10% trehalose or lactose), and drying temperature is lower than 45 ℃.Dry powder enzyme 20～400,000 U/g alive.
Dextrinosan is synthesized in catalysis
The present invention also provides the method with the synthetic dextrinosan of highly active glycosyltransferase catalysis.
Glycosyltransferase for preparation can directly be used for the catalytic production dextrinosan.In the present invention, use glycosyltransferase to produce the not special restriction of condition of dextrinosan, can adopt the conventional processing condition in this area.For example, in production technique, both can use starch to be raw material, and also can use SANMALT-S as raw material.
A kind of preferred production technique comprises: be raw material (concentration 10%～40%) with starch; Through high-temperature (0.05%～0.5%) 90 ℃～115 ℃ DE10 that liquefies～20; Boil 5 minutes～15 minutes enzymes that go out; Be cooled to 60 ℃; Transfer pH to 4～7, add Pullulanase (0.05%～0.5%), α-fungal amylase (0.05%～0.5%) and Maltogenase (0.05%～0.5%), glycosyltransferase of the present invention then (0.05%～0.5%) is incubated conversion (30 ℃～60 ℃ of temperature; 24～72 hours time), thus obtain high level dextrinosan slurry (content 50%～60%).
Another kind of preferred technology comprises: with SANMALT-S (concentration 10%～40%) is substrate, adds glycosyltransferase of the present invention (0.05%～0.5%), is incubated conversion (30 ℃～60 ℃ of temperature then; 24～72 hours time), obtain high level dextrinosan slurry (content 50%～60%).
Major advantage of the present invention is:
(a) antibody screening method of the present invention, based on the exclusive reaction of antigen-antibody, so accuracy is high.
(b) utilize enzyme linked immunological, the sample size of primary treatment is big, and the screening that can make bacterial classification more quick and precisely.
(c) with the synthetic dextrinosan of the inventive method catalysis efficiently, the transformation efficiency of dextrinosan is up to 55-65wt%.
(d), can further improve the output of glycosyltransferase through the metabolic bypass of gene knockout technology blocking-up cell.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.In the present invention, per-cent and umber are weight percentage and parts by weight, unless stated otherwise.
The antiserum(antisera) experiment
1.1 the acquisition of antiserum(antisera) (is anti-)
1.1.1 antigenic preparation
Commercially available commercialization glycosyltransferase is made the SDS-PAGE electrophoresis earlier, cut off the target protein band behind the coomassie brilliant blue staining.
1.1.2 emulsification antigen
To cut off the target protein band and grind the slurry device with tissue and grind, and add the incomplete immunological adjuvant of equivalent Fu Shi, it is sucked in two syringes, the emulsion tube long with about 5cm connects these two syringes, alternately promotes needle tubing back and forth, until formation oyster white emulsion.Inspection emulsification situation, with its in the cold water surface, if indiffusion on the water surface, it is complete to be emulsification.
2.1.3 immunization protocol
The big ear rabbit of Japan is adopted in this experiment, uses the mixed immunity method, immune programme for children such as following table:
Table 1 immunization protocol
A week after the last immunity, can from the rabbit auricular vein, take a blood sample on a small quantity, detect with corresponding antigens, if dissatisfied, can continue injection with booster immunization, until reaching satisfied effect.
After the last immunity the 7th day adopts quiet arterial blood letting method to obtain serum.
1.1.4 the separation of serum and preservation
After placing 37 ℃ of following 1h to make the thorough aggegation of blood the blood, along the bottle wall clot is separated with the bottle wall, put into 4 ℃ again and spend the night, serum is fully separated out with the big rifle head of 1mL of sterilization.Use the rifle head with the serum sucking-off then, as be mixed with red corpuscle, then centrifugal removing.
With immune serum (anti-) aseptic subpackaged after, be put in-80 ℃ of preservations.
Enzyme joint inspection survey method
2.1 crude antigen (glycosyltransferase) preparation
Get the 1ml fermented liquid, 10000rpm is centrifugal, gets supernatant, adds 2 times of volume ice extraction using alcohols, and 10000rpm is centrifugal, gets deposition.It is 1～10 μ g/ml that deposition uses the sodium carbonate buffer of freshly prepared PH9.6 to be diluted to protein contnt, bed board.
2.2 bed board
The antigen bed board that dilution is good, dilute with sodium carbonate buffer: in first hole, add the 50ul antigen liquid, the 50ul sodium carbonate buffer, mixing is got 50ul and is moved to second hole, adds the 50ul sodium carbonate buffer, mixing, put 37 ℃ one hour.
2.2 blocking-up
Pour out raffinate, clap to do, add and newly join the PBS-BSA blocking-up, every hole adds 100ul, put 37 ℃ two hours.
* PBS: PBS (PH7.4)
* BSA: bovine serum albumin
* PBS-BSA:100ml includes 1g BSA, uses according to aequum at every turn and prepares.
2.3 adding one resists
Pour out raffinate, add one and resist every hole 50ul.Put 37 ℃ two hours.
* one is anti-: with 100 times of PBS dilutions, prepare according to aequum.
2.4 wash plate
Pour out and mix liquid, use PBS+Tween20, washing twice is with twice of PBS washing.Clap gently and do.
* add 200ulTween20 in the PBS+Tween20:100mlPBS.
2.5 adding two resists
Every Kong Erkang 50ul is put 37 ℃ of half a hour.
* two is anti-: goat-anti rabbit (HRP), with 2500 times of PBS dilutions, prepare according to aequum.
2.6 wash plate
Pour out and mix liquid,,, clap gently and do with PBS washing twice with 1%SDS washing twice.
2.7 add the substrate colour developing
A liquid (0.1M acetate buffer pH5.0+0.05%～0.1%H2O2)+B liquid (0.1% hydrochloric acid soln (PH≤3.0)+TMB 0.4mg/ml+0.5mM EDTA), as required, can be half the with the PBS dilution, every hole 50ul.Colour developing.
2.8 reading
According to the height of ELIASA at 450nm place reading, judge that enzyme is lived what.
The screening of glycosyltransferase superior strain
3.1 bacterial strain
Produce bacterial strains such as saccharifying enzyme, aspartic protease, Hydrocerol A, polygalacturonase and glucono-surplus choosing 100 in the strain industry; These bacterial strains belong to black mold (Aspergillus niger), smelly aspergillus (Aspergillus foetidus); Carbon black aspergillus (Aspergillus carbonarious), cinnamon aspergillus (Aspergillus cinmomeus), Aspergillus usamii (Aspergillus usamii); Aspergillus nidulans (Aspergillus nidulan); Aspergillus oryzae (Aspergillus oryzae), penicillium spp (P.chrysogenum), Aureobasidium pullulans (Aureobasidium pullulans) etc.
3.2 substratum
SANMALT-S 2%, yeast powder 0.5%, K
3PO
40.1%, MgSO
47H
2O 0.05%, FeCl
30.03%
3.3 fermentation condition
The bottled liquid 100ml of 500ml triangle, initial pH5.5,30 ℃, 250rpm, rotating and culturing 72 hours.
3.4 enzyme linked immunosorbent detection result
Crude antigen (glycosyltransferase) is according to 2.1 method among the embodiment 2, and testing process is according to embodiment 2 described methods, and representative result such as table 2 are with shown in Figure 1.
Tire (reaction enzymes is lived just) of table 2 different strains glycosyltransferase
1Absorbance ratio (OD
450nm/ contrast OD
450nmBacterial strain malaga FscM is explained in)>=2 expression positive (+); Sample OD
450nm/ contrast OD
450nmBacterial strain malaga FscM is not basically explained in＜2 expressions negative (-).
2The methyl glucoside detection method is seen 4.5 among the embodiment 4.
3The TLC method.-,+, ++, +++, ++ ++ represent the dextrinosan growing amount respectively from low to high.Thin layer chromatography board: Silica Gel 60 (Merck, Germany); Developping agent: 2-propyl alcohol (2-propanol): butanols (1-butanol): water (water)=12: 3: 4 (volume ratio); Developer and condition: acetate includes the sulfuric acid of 1% aubepine (p-Anisaldehyde) and 2%, 110 ℃ of heating colour developing in 10 minutes.
The above results shows that the methyl glucoside enzyme is lived high, and it is tired and the dextrinosan growing amount may not be certain high.10Z307GTase4 bacterial strain for example, its methyl glucoside enzyme is lived high, but dextrinosan growing amount very low (being merely+).
In contrast, tire when high, the catalytic dextrinosan growing amount of glycosyltransferase is just high, demonstrates positive correlation.For example, tiring is 2
8Bacterial strain, dextrinosan growing amount the highest (do ++ ++) is 2 and tire
7Bacterial strain, dextrinosan growing amount also very high (do +++).This shows that screening method of the present invention is particularly suitable for high-throughput ground screening glycosyltransferase and produces bacterial strain.
Glycosyltransferase zymotechnique and preparation
4.1 bacterial strain
The 10Z307GTase1 (black mold) that chooses among the embodiment 3 to be filtered out is a fermentation strain.
4.2 substratum
Carbon source is chosen glucose, Semen Maydis powder, W-Gum, sucrose, fructose, wood sugar, SANMALT-S, lactose and hydrolysis sugar respectively, and concentration is between 1%-6%.Nitrogenous source is chosen wheat bran, yeast powder, peptone, Semen arachidis hypogaeae protein, soybean cake powder, steeping water, soya-bean cake hydrolyzed solution, urea, ammonium sulfate respectively, and concentration is between 0.1%-4%.Nutritive salt mainly comprises K
3PO
4, MgSO
47H
2O, FeCl
3, zinc sulfate, boric acid, calcium chloride, various constituent concentrations arrive the milligram level in the ppm level.The tensio-active agent tween 80 is between 0.1～1%.
4.3 technological condition for fermentation
4.3.1 leavening temperature and time
In 25-35 ℃ scope, fermentation time 3-6 days.
4.3.2 ventilate and stirring
Because glycosyltransferase is an aerobic fermentation, therefore ventilating must have influence on substrate and oxygen mixing and transmission in fermented liquid with stirring, thereby influences the growth of thalline and the output of enzyme.Thalline exists with the form of bacterium ball in the fermentation of Aspergillus niger process; Owing to influence the absorption transmission of matrix; And the secretion of glycosyltransferase, therefore, suitable mixing speed and the air flow of control is very important during the fermentation; Usually mixing speed is between 120-300rpm, and ventilation is at 0.5～1.0v/v/m.
4.3.3pH
The pH scope of black mold mycelial growth is 3～8, some growth retardation in pH＞7.Because pH influences mycelial permeability, to the activity of trace elements absorbed and enzyme, it is considerable therefore selecting suitable fermentation pH.Therefore pH is controlled between the 5-6 in the fermenting process.
4.3.4 feed supplement control fermentation
Flat for improving enzyme running water to a greater extent, the strategy of employing ferment middle fed-batch medium.Consisting of of fed-batch medium: carbon source 30%, nitrogenous source 10%, nutritive salt 10～20%.Stream adds 12 hours-24 hours time, looks the situation of fermentative activity and decides.Flow acceleration requires and adjustment automatically according to the control of pH and dissolved oxygen.
Glycosyltransferase generates in the fermenting process zymetology kinetic curve such as Fig. 2.
4.4 glycosyltransferase extracts and is refining
Liquid dosage form
Fermented liquid is got supernatant through high speed frozen centrifugation (7500rpm, 4 ℃, 20 minutes).Adopt hollow fiber membrane ultrafiltration device, ultra-filtration membrane dams molecular weight greater than 100,000 dalton, intake pressure 0.1-0.3MPa; Top hole pressure is 0.40～0.5MPa, and filtrating is 1: 1 with the quantity of reflux ratio, 10 ℃-30 ℃ of service temperatures; 5 times of cycles of concentration; The liquid that dams is Liquid Glucose glycosides transferring enzyme preparation, enzyme 80,000～150,000 U/ml that live, dilute 4 times after enzyme active valency can reach 2
9
The solid, powdery formulation
Fermented liquid is got supernatant through high speed frozen centrifugation (7500rpm, 4 ℃, 20 minutes); Use 60% saturated ammonium sulphate, deposition is dissolved in pure water, adopts hollow fiber membrane ultrafiltration device; Ultra-filtration membrane dams molecular weight greater than 100,000 dalton, and intake pressure 0.1-0.3MPa, top hole pressure are 0.40～0.5MPa; Filtrating is 1: 1 with the quantity of reflux ratio, 10 ℃-30 ℃ of service temperatures, 5 times of cycles of concentration; The liquid that dams carries out vacuum spray drying after adding drying protectant (comprising 1%～7% N.F,USP MANNITOL, 5%～10% trehalose or lactose), and drying temperature is lower than 45 ℃.Dry powder enzyme 20～400,000 U/g that live, dilute 4 times after enzyme active valency reach 2
10
4.5 enzyme activity determination
According to general in the world Alpha-methyl glucoside method.Get 2% methyl glucoside solution, each 1ml of 0.02M acetate buffer solution, 40 ℃ of preheating 5min; Add suitable dilution enzyme liquid 0.5ml; Behind 40 ℃ of reaction 60min, heating 5min deactivation is got reaction solution 0.5ml and is added P-FAD reagent 3ml in 100 ℃ of boiling water baths after the cooling; 40 ℃ of reaction 20min, the absorption value (A1) of 500nm is measured in the cooling back.Other gets damping fluid 1ml, enzyme liquid 0.5ml, heating 5min deactivation in 100 ℃ of boiling water baths earlier, add substrate 1ml (the same) insulation 60min again after, take out the absorption value (A2) that reaction solution 0.5ml (the same) measures 500nm.
Enzyme live definition be under these conditions among the 60min substrate to generate the required enzyme amount of 1 μ g glucose be a unit.
Enzyme activity (U/g)=(A1-A2) * K * 50 * N
The glucose amount (μ g) that the every degree of K=A value institute is suitable, N=enzyme liquid extension rate.
Dextrinosan is synthesized in glycosyltransferase catalysis
5.1 technical process and condition
The key technical indexes of estimating dextrinosan production technique level height is the height of the content of dextrinosan in the finished product; Just change the height of glycosides level; Present technology level of conversion is mostly between 30%-45%; This index gets the height that height depends primarily on the glycosyltransferase catalytic efficiency (, but has four kinds with dextrinosan production involved enzyme: comprise that AMS, Pullulanase, SANMALT-S generate enzyme and glycosyltransferase.Wherein SANMALT-S generation enzyme comprises that beta-amylase, fungal alpha-amylase and actinomycetic SANMALT-S generate enzyme (Maltogenase).The mechanism that dissimilar SANMALT-S generates enzymic hydrolysis starch is different, wherein between product also different, even with a kind of the same enzyme in different action time, under the different consumptions, the polymerization degree of its hydrolysate is also different.While different raw materials, its transformation efficiency are also different.The factor of visible influences transformation efficiency is multiple, takes all factors into consideration these factors, and transformation system is optimized, and will improve transformation efficiency.Preferred technical process and condition are as follows:
A. with starch raw material
Starch (10% to 40%) solution
adding high α-amylase (0.05% ~ 0.5%)
90 ℃ ~ 115 ℃ liquefied to DE10 ~ 20
boiled for 5 minutes to 15 minutes enzyme inactivation
cooled to 60 ℃
adjusted to pH 4-7
Gap Rouland enzyme (0.05% ~ 0.5%)
plus maltose producing enzyme (Maltogenase) (0.05% ~ 0.5%)
add glucosidase transferase (0.05% ~ 0.5%)
thermal conversion (temperature 30 ℃ ~ 60 ℃; Time 24 ~ 72 hours)
isomaltooligosaccharide syrup (containing 50% to 60%)
B. with SANMALT-S raw material
SANMALT-S; (10%～40%) solution
adds glycosyltransferase; (0.05%～0.5%)
is incubated conversion; (30 ℃～60 ℃ of temperature; 24～72 hours time)
dextrinosan slurry (content 50%～60%).
5.2 the HPLC of dextrinosan slurry analyzes
Instrument: Waters 1525; Analytical column: Carbohydrate HP; Model specification: Part No.WAT044355,4 μ m, 4.6 * 250mm; Detector: Waters 2420 ELS Detector.
Sample preparation: get the dextrinosan slurry 1ml after the conversion, get final product through the disposable filtering degerming of 0.22 μ m.
Testing conditions: sample size 10 μ l: moving phase: acetonitrile 75%, pure water 25%, 30 ℃ of detected temperatures, flow velocity 0.8ml/min.
5L fermentative prepn glycosyltransferase and application thereof
6.15L NBS fermentor tank glycosyltransferase zymotechnique and preparation
6.1.1 bacterial strain
Choosing the 10Z307GTase1 that filters out among the embodiment 3 is fermentation strain.
6.1.2 substratum
Seed culture medium: SANMALT-S 2%, yeast powder 0.5%, K
3PO
40.1%, MgSO
47H
2O 0.05%, FeCl
30.03%.
Fermention medium: Semen Maydis powder 6%, alpha-amylase 0.05%, yeast powder 2%, nutritive salt 10ml, tween 80 0.5%.
Feed supplement liquid: the consisting of of fed-batch medium: SANMALT-S 30%, yeast powder 10%, nutritive salt 10%.
6.1.3 technological condition for fermentation
6.1.3.1 seed culture
3 500ml triangular flasks, each dress liquid 100ml, initial pH5.5,30 ℃, 250rpm, rotating and culturing 48 hours.
6.1.3.2 ferment tank
The 5L fermentor tank, dress liquid medium 2.5L, inoculum size 300ml, initial pH6; 32 ℃ of leavening temperatures, pH is controlled at 5.5, and dissolved oxygen amount is greater than 5%, and mixing speed is between 120-300rpm; Ventilation is at 0.5～1.0v/v/m, and feed supplement finished by the 92nd hour, altogether feed supplement 700ml beginning in the 70th hour.
6.1.4 the liquid dosage form glycosyltransferase extracts and is refining
Fermented liquid is through high speed frozen centrifugation (7500rpm, 4 ℃, 20 minutes), supernatant 2500ml, the enzyme 22000U/ml that lives, and tiring reaches 2
8Adopt hollow fiber membrane ultrafiltration device, ultra-filtration membrane dams molecular weight greater than 100,000 dalton, intake pressure 0.15MPa; Top hole pressure is 0.45MPa, and filtrating is 1: 1 with the quantity of reflux ratio, 25 ℃ of service temperatures; Be concentrated to 500ml; Be Liquid Glucose glycosides transferring enzyme preparation, enzyme 8.8 ten thousand U/ml that live, dilute 4 times after enzyme active valency reach 2
9
6.2 the conversion process and the condition that with the W-Gum are the synthetic dextrinosan of substrate glycosyltransferase catalysis are following:
Corn starch (25%)
high α-amylase (0.1%)
115 ℃ liquefied to DE15
boiled for 5 minutes enzyme inactivation
cooled to 60 ℃
adjusted to pH 5.5
Gap Rouland enzyme (0.05%)
add Maltogenase (0.1%)
plus glucose glycoside transferase enzyme (0.3%)
thermal conversion (temperature 55 ℃; 48 hours)
isomaltooligosaccharide syrup (content 55.85%)
The result is as shown in Figure 3, and in product, glucose content is 44.15%, and comprises that the total content of the dextrinosan of isomaltose, trisaccharide maltose, Isomaltotriose, maltotetrose and panose is 55.85%.
6.3 with SANMALT-S is the synthetic dextrinosan of substrate glycosyltransferase catalysis
Enzymatic conversion method flow process and condition are following:
SANMALT-S; (30%)
adds glycosyltransferase; (0.05%～0.5%)
is incubated conversion; (30 ℃～60 ℃ of temperature; 24～72 hours time)
dextrinosan slurry (content 62%).
The result is as shown in Figure 4, and in product, glucose content is 37.96%, and comprises that the total content of the dextrinosan of isomaltose, trisaccharide maltose and panose is about 61.04%.
5L fermentative prepn glycosyltransferase and application thereof
The result shows that in product, glucose content is about 40%, and comprises that the total content of the dextrinosan of isomaltose, trisaccharide maltose and panose is about 60%.
Gene knockout
8.1 bacterial strain:
Black mold 10Z307GTase1 (seeing embodiment 3), the microbial characteristic of this bacterium is:
Solid culture initial stage white mycelium produces spore in logarithmic phase and is light/dark balance; In balance period spore black in color; Just brown gradually in decline phase spore color.
The growth medium carbon source has glucose, sucrose, fructose, wood sugar, SANMALT-S, lactose and hydrolysis sugar; Nitrogenous source has wheat bran, yeast powder, peptone, Semen arachidis hypogaeae protein, soybean cake powder, steeping water, soya-bean cake hydrolyzed solution, urea, ammonium sulfate; Nutritive salt mainly comprises K
3PO
4, MgSO
47H
2O, FeCl
3, zinc sulfate, boric acid, calcium chloride etc.
Hereditary feature: bacterial strain disappearance saccharifying enzyme (EC3.2.1.3).
8.2 method
Ti-plasmids is available from deriving from the cas gene storehouse.This Ti-plasmids is with formed Ti-plasmids between the left arm of HYG resistant gene (from the pBR322 plasmid) insertion T-DNA and the right arm.
With the Agrobacterium is mediation, and the segmental Ti-plasmids of T-DNA (Fig. 5) that will have hygromycin gene changes black mold 10Z307GTase1 over to.Optimization (incubation time 24 hours, 6/ml of spore number, inductor Syringylethanone (AS) concentration 80mmol/L through conversion condition; Agrobacterium concentration 0.6OD; Culture temperature is 30 ℃ altogether), utilize the antibody screening technology, in conjunction with the height of diastatic activity; Successfully made up the T-DNA that contains 3865 transformants and inserted the sudden change storehouse, explained that the circulation ratio of the inventive method is very high.
Polymerase chain reaction (PCR) shows that these mutons all have the T-DNA fragment to insert (see figure 6).Behind the succeeding transfer culture in 5 generations, muton has good stability.
Through fermentation optimization, the engineering bacteria enzyme work that has strain more than 20 to handle through gene knockout is significantly increased, and has reached 35000U/ml or higher, wherein has three strain engineering bacteria enzyme work to reach about 50000U/ml.
Gene knockout
The result shows: the work of smelly aspergillus enzyme is brought up to 2000U/ml by 8000U/ml; The work of carbon black aspergillus enzyme is brought up to 9000U/ml by 3200U/ml; The work of cinnamon aspergillus enzyme is brought up to 10000U/ml by 6000U/ml; The mould enzyme work of Aspergillus usamii is brought up to 25000U/ml by 7000U/ml; The mould enzyme work of Aspergillus nidulans is brought up to 30000U/ml by 5000U/ml, and the mould enzyme work of aspergillus oryzae is brought up to 500U/ml by 320U/ml, does not see obvious raising; The mould enzyme of penicillium spp is lived and is not seen raising, and the mould enzyme work of Aureobasidium pullulans is brought up to 32000U/ml by 4500U/ml.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a method of screening the production bacterial strain of producing glycosyltransferase is characterized in that, comprises step:
(a), measure tiring as antigenic glycosyltransferase in the protein sample of each candidate strain with the specific antibody of anti-glycosyltransferase; With
(b), select high candidate strain of tiring as the production bacterial strain of producing glycosyltransferase according to the valence value of measuring;
Wherein, described candidate strain is the bacterial strain of handling through gene knockout.
2. the method for claim 1 is characterized in that, also comprises step (c): for selected production bacterial strain, measure the ability that its glycosyltransferase catalysis SANMALT-S is produced dextrinosan.
3. the method for claim 1; It is characterized in that the candidate strain of in step (a), measuring is selected from group down: black mold (Aspergillus niger), smelly aspergillus (Aspergillus foetidus), carbon black aspergillus (Aspergillus carbonarious), cinnamon aspergillus (Aspergillus cinmomeus), Aspergillus usamii (Aspergillus usamii), Aspergillus nidulans (Aspergillus nidulan), aspergillus oryzae (Aspergillus oryzae), penicillium spp (P.chrysogenum) and Aureobasidium pullulans (Aureobasidium pullulans).
4. the method for claim 1 is characterized in that, described candidate strain is to carry out the bacterial strain that gene knockout is handled with the agrobacterium tumefaciens Ti-plasmids.
5. the method for claim 1 is characterized in that, described antibody is monoclonal antibody, polyclonal antibody or antiserum(antisera).
6. a glycosyltransferase that filters out with the said method of claim 1 is produced bacterial strain.
7. a glycosyltransferase is characterized in that, described glycosyltransferase is to produce with the described production bacterial strain of claim 6.
8. glycosyltransferase as claimed in claim 7; It is characterized in that described glycosyltransferase is that black mold, smelly aspergillus, carbon black aspergillus, cinnamon aspergillus, Aspergillus nidulans, aspergillus oryzae, penicillium spp, Aureobasidium pullulans or Aspergillus usamii produce.
9. a method of producing dextrinosan is characterized in that, comprises step: use the shift reaction of the described glycosyltransferase catalysis of claim 7 glucoside bond, thereby form dextrinosan.
10. method as claimed in claim 9 is characterized in that described dextrinosan comprises isomaltose, trisaccharide maltose, Isomaltotriose, maltotetrose or panose.
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| CN104878054A (en) * | 2015-02-12 | 2015-09-02 | 广西南宁智天生物科技有限公司 | Carrier-free immobilization aspergillus niger production method of isomaltose hypgather |
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| CN103695323A (en) * | 2013-12-26 | 2014-04-02 | 青岛蔚蓝生物集团有限公司 | Stable and high-yield strain for alpha-transglucosidase |
| CN104878054A (en) * | 2015-02-12 | 2015-09-02 | 广西南宁智天生物科技有限公司 | Carrier-free immobilization aspergillus niger production method of isomaltose hypgather |
| CN104878054B (en) * | 2015-02-12 | 2018-02-13 | 广西南宁智天生物科技有限公司 | A kind of method of carrier-free immobilization aspergillus niger production oligoisomaltose |
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