CN102296032A - Transglucosidase, its preparation method and immobilization method - Google Patents
Transglucosidase, its preparation method and immobilization method Download PDFInfo
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Abstract
The invention relates to transglucosidase, its preparation method and a immobilization method, which belong to the fields of enzyme engineering and fermentation engineering. More specifically, the invention provides the following processes: 1) Aspergillus niger BLB-16 is screened from soil as an original strain, processes of mutation treatment, optimization and screening are performed to obtain an optimized bacterial strain (BLB-28) for fermenting; 2) a fermentation broth is carried out a heat sterilization to obtain transglucosidase liquid; 3) transglucosidase liquid is performed a nanofiltration to concentrate; 4) processes of resin adsorption, sodium alginate entrapment, immobilization by a glutaraldehyde cross-linking method are carried out for preparing immobilized enzymes. The prepared transglucosidase is suitable for an application in the industrial fields such as foodstuff, medicine, feed and the like, and used for producing isomaltose hypgather.
Description
Technical field
The invention belongs to enzyme engineering and field of fermentation engineering, relate to a kind of transglucosidase and preparation thereof and process for fixation.Specifically be with the Aspergillus niger strain that from soil, screens (
Aspergillus niger) BLB-16 is starting strain, through mutagenesis, the screening bacterial strain BLB-28 that is optimized, and through fermentation, degerming, concentrate preparation transglucosidase liquid, also relate to the immobilization technology of this transglucosidase.
Background technology
Transglucosidase (Transglucosidase), be also referred to as glucose transglucosidase, alpha-glucosidase, α-transfering grape glycosidase, alpha-D-glucose transglucosidase etc., according to " name of enzyme and International Classification numbering ", belong to transferase, be specially EC 2.4.1.24, CAS is 9030-12-0.
According to " foodstuff additive use hygienic standard " criteria for classification (GB2760), transglucosidase is a kind of zymin, belongs to the foodstuffs industry processing aid.
Changeing that the heteroside enzyme mainly acts on is that non-reducing end from substrate cuts α-1,4 glycosidic link, discharges alpha-D-glucose, and the glucosyl residue that maybe will dissociate is transferred on another carbohydrate substrate with α-1,6 glycosidic link, thereby obtains oligomeric isomaltose.
Oligomeric isomaltose is the functional oligose that is made of by α-1,6 glycosidic link 2-10 monose molecule, and main component is the above oligose of isomaltose, panose, Isomaltotriose and tetrose.Natural being present in numerous food such as honey, owing to do not decompose the enzyme of oligomeric isomaltose in the human body alimentary canal, therefore, it is after taking in human body, have not by human consumption absorb, promote that the intestinal beneficial bacterium group breed, relaxes bowel, regulating blood fat, low sugariness, unique effects such as low in calories, especially effect is remarkable aspect promotion intestinal beneficial bacterium group's increment.Milk-product, cocoa products, candy class, bakery product, frozen, beverage, drinks, meat product, jelly, fried food product, puffed food, infant or baby food etc. have been widely used in.At present, consumption was at few hundred thousand tonnes of in domestic year.
But the zymin of most critical but never realized suitability for industrialized production at home during transglucosidase was produced as oligomeric isomaltose.At present, the complete dependence on import of this zymin, this makes not only that in the production of oligomeric isomaltose gordian technique is made more that by external relevant enterprise control the zymin use cost can be in any more in the oligomeric isomaltose production.
Now, at home, transglucosidase has caused a lot of scientific workers' concern, and still, the expression amount of its transglucosidase of wild strain of screening is very low on the one hand, is difficult to realize suitability for industrialized production; On the other hand, it generally is intracellular enzyme that the intestinal bacteria metabolism of adopting genetic engineering technique to make up is produced, its extraction process complexity, and the enzyme rate of recovery alive is low, the also unfavorable suitability for industrialized production of using.Therefore, be necessary to develop the outer transglucosidase of a kind of born of the same parents, to adapt to the needs that oligomeric isomaltose is produced.
The present invention screens and has obtained the aspergillus niger that a strain can the outer transglucosidase of fermentating metabolism production born of the same parents, and obtained aspergillus niger BLB-28 by mutagenesis optimization screening, improved the expression amount of its transglucosidase, and the advanced degerming of employing, purifying, concentration technique, prepare this zymin, and by developing the immobilization technology of transglucosidase, realize the efficient circulation utilization of this zymin, not only realized the production domesticization of this zymin, improve the situation that domestic transglucosidase is limited by the world market greatly, and can significantly reduce the use cost of enzyme in the oligomeric isomaltose production.
Simultaneously, the present invention is in the exploitation of carrying out the zymin immobilization technology, adopted resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization technology, broken through the technology limitation in the transglucosidase immobilization, promptly one is to have broken through only to adopt resin to add in the glutaraldehyde cross-linking immobilization technology, because immobilization is firm inadequately, and make that immobilized enzyme is easy to come off, reveal, increased that subsequent technique is made with extra care, the difficult problems such as difficulty of purifying oligomeric isomaltose; The 2nd, broken through and only adopted sodium alginate to embed to add CaCl
2In the sclerosis immobilization technology,, be difficult in large-scale packed column, use, and be added directly in the liquid glucose, then need filter, increased the difficult problems such as complicacy of technology because hardness is lower.
Summary of the invention
An object of the present invention is to provide a kind of Aspergillus niger strain (
Aspergillus niger) BLB-28, it be with the aspergillus niger that from soil, screens (
Aspergillus niger) BLB-16 is starting strain, through ultraviolet ray+LiCl complex mutation, and obtain through screening; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 16th, 2011, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, in the Institute of Microorganism, Academia Sinica, preserving number CGMCC No 5143.
Another object of the present invention is to provide a kind of transglucosidase, it be use Aspergillus niger strain (
Aspergillus niger) BLB-28 fermentation makes.This zymin is the key enzyme preparation during oligomeric isomaltose is produced.It mainly acts on is to be in the production process of raw material with starch, starch through liquefaction and saccharification after, change glycosides, the non-reducing end that is about to substrate cuts α-1,4 glycosidic links discharge alpha-D-glucose, and the glucosyl residue that of maybe will dissociating is with α-1,6 glycosidic links are transferred on another carbohydrate substrate, thereby obtain oligomeric isomaltose; Adopt the transglucosidase among the present invention to produce oligose, compare with adopting the import zymin, the zymin use cost reduces 300-400 unit.
A further object of the present invention is to provide a kind of preparation method of transglucosidase, the preparation method who comprises transglucosidase liquid and immobilization transglucosidase, promptly aspergillus niger (Aspergillus niger) BLB-28 that optimizes gained with the inventor is a fermentation strain, concentrates preparation transglucosidase liquid through seed culture, fermentation, heat sterilization, nanofiltration;
The substratum of said seed culture consists of among the above-mentioned preparation method: glucose 300-500g/L, yeast extract paste 16g/L, agar powder 18g/L, potassium primary phosphate 1.2 g/L, sal epsom 0.45g/L, urea 1g/L, pH5.5-6.0.
The substratum of said fermentation consists of: glucose 300-500g/L, yeast extract paste 18g/L, agar powder 15g/L, potassium primary phosphate 1.5 g/L, sal epsom 0.5g/L, urea 2g/L, pH5.5-6.0.
Said heat sterilization adopts ceramic composite membrane filtration sterilization degerming and purifying.
Said nanofiltration concentrates adopts organic composite nanometer filter membrane concentration and purifying.
With the prepared transglucosidase liquid of aforesaid method,, promptly make the immobilization transglucosidase through resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.
Adopt the immobilization transglucosidase of this technology preparation, not only stability is high, and the difficult leakage of zymin, is beneficial to the oligomeric isomaltose purifying products, improves product quality, reduces product cost.Adopt immobilized enzyme to produce oligose, and than adopting the transglucosidase liquid phase ratio for preparing among the present invention, reduce zymin use cost 100-150 unit.
The preparation method of described transglucosidase liquid of technical solution of the present invention and immobilization transglucosidase comprises following concrete steps:
(1) bacterial screening
Aspergillus niger (Aspergillus niger) BLB-16 separates from development area, Yucheng City agricultural land soil, and isolation medium is common nutritional medium.
The separation method of aspergillus niger (Aspergillus niger) BLB-16 is: in sterilisable chamber, and with the soil water solution of sterilized water configuration 10%, and by 10
0, 10
1, 10
2, 10
3, 10
4, 10
5, 10
6Extension rate dilution, after dilution finishes, go 200ul respectively on the plate isolation substratum, with the spreading rod coating evenly, be cultured to and grow ripe bacterium colony on the flat board, identify and be purified into the aspergillus niger bacterium colony, will but the colony inoculation inclined-plane cultivate.With slant culture colony inoculation shake-flask culture base, primary dcreening operation is measured enzyme activity.Filter out the BLB-16 bacterial strain according to the shake-flask culture enzyme activity.
Enzymic activity detection method: make reaction substrate with colourless p-NP-α-D-glucopyranoside (pNPG), through alpha-glucosidase hydrolyzing alpha-1, discharge p-NP (pNP) behind the 4-glucoside bond, by detecting pNP generation under the 405nm as α-1, the criterion of 4-glucosidase activity size.
(annotate: west, substrate pNPG(Shanghai is precious), full name 4-nitrophenyl-2-D-galactopyranoside; In above-mentioned detection method, the enzyme amount that per minute catalysis produces 1 μ mol pNP is a unit of activity (U).）
Isolation medium is: potato 200g peeling, 30min is boiled in stripping and slicing, uses filtered through gauze then, adds glucose 20g and agar 20g again, dissolves the back moisturizing to 1000ml, the pH nature.
Slant medium is: potato 200g peeling, stripping and slicing is boiled and is kept 30min, uses filtered through gauze then, adds glucose 22g and agar 20g again, treat that it melts after, moisturizing is to 1000ml, pH naturally.
The shake-flask culture base is: maltitol powder 300-500g/L, yeast extract paste 15g/L, peptone 15g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.5g/L, urea 5g/L, PH5.5-6.0.
(2) the zymin technological process of production
1) mutagenesis of step 1--bacterial classification
By the aspergillus niger BLB-16 for bacterial classification that from soil, screens, through ultraviolet ray+NaN
3Complex mutation, and through screening acquisition aspergillus niger BLB-28(CGMCC No 5143).
Concrete mutagenic processes is: an amount of aspergillus niger BLB-16 of transfering loop picking slant pore is in aseptic 1.5% NaN that contains
3In the triangular flask of solution, the magnetic force concussion stirs, and cultivates 18-30h, spore germination.Pour spore suspension into contain aseptic pin aseptic plate, keep the plate lid to open, 0s, 20s, 40s, 60s, 80s, 100s, 120s are shone in 20cm place under the ultraviolet lamp of the 15W of preheating in advance, and the bacterium liquid of different irradiation doses is diluted to 10
-1, 1-2h is placed at dark place.Be diluted to 10 respectively with physiological saline
-3, draw the good bacterium liquid 100 μ L of dilution with liquid-transfering gun, the screening of coating malt agar is dull and stereotyped, 3 flat boards of each extent of dilution coating.The plate that coats is wrapped with black cloth, put biochemical incubator and cultivate 4-5d for 28 ℃, the statistics lethality rate.
Bacterial screening: with the aspergillus niger starting strain BLB-16 before the mutagenesis is contrast, the bacterial strain after the mutagenesis is carried out fermentation culture produce enzyme and the active detection of transglucosidase.
Enzymic activity detects: enzyme biopsy survey method unanimity during with bacterial screening.Filter out high transglucosidase and produce bacterial strain, promptly aspergillus niger (
Aspergillus niger) BLB-28.
2) step 2--actication of culture
With the Aspergillus niger strain of cryopreservation (
Aspergillus niger) BLB-28, activating through the first order seed cultivation, secondary seed is cultivated and is carried out enlarged culturing.Culture condition is: pH5.5-6.0, culture temperature is 25-40 ℃, incubation time 18-24h.
Wherein first order seed cultivation, secondary seed medium are: glucose 300-500g/L, yeast extract paste 16 g/L, agar powder 18g/L, potassium primary phosphate 1.2 g/L, sal epsom 0.45g/L, urea 1g/L, pH5.5-6.0
3) step 3--fermentation
To be inoculated in fermention medium through the seed of enlarged culturing, carry out fermentation culture.Wherein, inoculum size is 5-12%(V/V), fermentation condition is: pH5.5-6.0, culture temperature is 25-40 ℃, dissolved oxygen is 28-30%, fermentation time 96-120h.
Fermention medium consists of: glucose 300-500g/L, yeast extract paste 18 g/L, agar powder 15g/L, potassium primary phosphate 1.5 g/L, sal epsom 0.5g/L, urea 2g/L, pH5.5-6.0.
Fermentation broth enzyme biopsy survey method: with bacterial screening is that enzyme biopsy survey method is identical.
4) step 4--heat sterilization
Adopt ceramic membrane ultrafitration, remove the fermentation thalline, preparation transglucosidase liquid.The condition of heat sterilization is: pressure 1.5-2.5MPa, temperature 4-25 ℃; The ceramic membrane molecular weight cut-off is 150-200 kDa, heat sterilization and macromolecular substance thereof, preliminary purification transglucosidase.
5) step 5--nanofiltration concentrates
Adopt organic composite nanometer filtering film to concentrate.Operational condition is: pressure 2.0-3.0MPa, temperature 4-25 ℃; Organic composite nanometer filter retaining molecular weight 200-2000Da, removes inorganic salt and small-molecule substance thereof, preparation transglucosidase liquid at the spissated while.
Concentrate back enzyme biopsy survey method: enzyme biopsy survey method is identical during with bacterial screening.
6) immobilization of step 6--enzyme
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization, the preparation immobilized enzyme.Fixing condition is: under the room temperature, add a certain amount of D301(R in transglucosidase liquid) the type resin, leach behind the vibration absorption 8-12h; After using 3% sodium alginate to embed again, be soaked in 0.1mol/L CaCl
2The 4h that hardens in the solution leaches, and at last it is soaked in the glutaraldehyde solution 1.5h of 1.5-2.0%, leaches, i.e. being fixed enzyme.
7) step 7-activity of the immobilized enzyme and Detection of Stability
Immobilized enzyme is adorned post, and the Fructus Hordei Germinatus liquid glucose of preparation pH5.0-6.0 is crossed post by certain flow velocity, flow velocity 0.6-1.8 times column volume/h, and temperature 50-60 ℃, uninterruptedly experimentize, sampling regularly detects the oligomeric isomaltose product component.
Activity of the immobilized enzyme, the index that reaches the oligomeric isomaltose (IMO50) of market sale with the oligomeric isomaltose product component is a standard, the activity of reaction immobilized enzyme.
Immobilized enzyme stability: to produce the total amount of qualified IMO50 in immobilized enzyme is during activated as the investigation standard.
The present invention optimizes gained strain fermentation enzyme 6-8U/ml alive, and the fermenting enzyme work of wild bacterium only is 0.08-0.12U/ml, and fermenting enzyme is lived and improved greatly.
Immobilization technology that the present invention adopts is resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization technology, enzyme transformation efficiency alive reaches more than 85%, this technological breakthrough the limitation in traditional transglucosidase immobilization: on the one hand adopting resin is carrier, the immobilization transglucosidase, guaranteed the hardness of immobilization finished product, can in large-scale packed column, use, simplify production technique; On the other hand, after the resin immobilization,, place CaCl with sodium alginate to embed
2Harden in the solution, adopt glutaraldehyde cross-linking at last, strengthened the stability of immobilized enzyme, avoided the leakage of zymin, thereby transglucosidase can reuse, and has improved the utilization ratio of enzyme greatly, has reduced the products production cost.Adopt this immobilization technology, with only adopt the resin immobilization and compare with the process for fixation of glutaraldehyde cross-linking, enzyme stability is higher, as to produce batch coming of oligomeric isomaltose, the former can produce 8-10 batch of qualified product, and the latter only can produce the 2-3 batch products.Adopt this immobilization technology, and only adopt sodium alginate to embed and with CaCl
2The sclerosis immobilization technology is compared, and the latter need be added directly to the immobilized enzyme preparation in the liquid glucose, after reaction finishes, needs to filter and removes immobilized enzyme altogether; And the latter then can make the direct post of crossing of liquid glucose, does not need to filter, and has simplified production technique.
The prepared transglucosidase main application fields of the present invention is industrial circles such as food, medicine, feed, is applied to the production of oligomeric isomaltose.
The preparation of a kind of transglucosidase of the present invention and immobilization technology thereof have the following advantages:
1) the fermenting enzyme work of optimization back bacterial strain can reach 6-8U, compares with wild aspergillus niger 0.08-0.12U, and enzyme work has obtained significantly promoting, and is easy to realize industrialization.
2) adopt ceramic membrane ultrafitration degerming and the concentration integrated technology of nanofiltration, realized degerming, concentrate, one step of purifying finishes, and reduces enzyme loss in process of production, shortened the production cycle, improved production efficiency.
3) adopt resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization, at home, realize the immobilization of transglucosidase first, improved the utilization ratio of enzyme, help the oligomeric isomaltose production technique and simplify, and improve the oligomeric isomaltose product quality.
Embodiment
Embodiment 1
Preparation pH5.4, the Fructus Hordei Germinatus liquid glucose of sugar dense 30%; Get import transglucosidase liquid (enzyme work is about 57-58U/ml), press 0.85L/ ton dry-matter and add in the Fructus Hordei Germinatus liquid glucose, place 58-60 ℃ of water-bath, insulation 30h; Sampling places boiling water bath 15-20min, and the enzyme that goes out adopts the liquid chromatographic detection technology, analysed preparation oligomeric isomaltose component.
Embodiment 2
With the Aspergillus niger strain of cryopreservation (
Aspergillus niger) BLB-28, under pH6.0,32 ℃ of conditions, cultivate 20h, carry out first order seed and cultivate activation, secondary seed enlarged culturing; To be 10%(V/V by inoculum size through the seed of enlarged culturing), be inoculated in fermention medium, at pH5.5-6.0, ferment under 30-34 ℃ the condition, fermentation time is 110h, after the fermentation ends, adopt molecular weight cut-off 150-200 kDa ceramic membrane, at 1.5-2.5MPa, 10 ℃, fermentation thalline and other macromolecular substance are removed in ultrafiltration; The nanofiltration membrane of molecular weight 200-2000Da is stayed in employing, and at 2.0-3.0MPa, 10 ℃ of ultrafiltration and concentration are removed inorganic salt and small-molecule substance thereof simultaneously, make transglucosidase liquid, and enzyme is lived and is 60-65U.
Preparation pH5.4, the Fructus Hordei Germinatus liquid glucose of sugar dense 30%; Get the transglucosidase liquid after concentrating, press 0.72L/ ton dry-matter and add in the Fructus Hordei Germinatus liquid glucose, place 55-58 ℃ of water-bath, insulation 30h; Sampling places boiling water bath 15-20min, and the enzyme that goes out adopts the liquid chromatographic detection technology, analysed preparation oligomeric isomaltose component.
Embodiment 3
With the Aspergillus niger strain of cryopreservation (
Aspergillus niger) BLB-28, under pH5.6,31 ℃ of conditions, cultivate 20h, carry out first order seed and cultivate activation, secondary seed enlarged culturing; To be 8%(V/V by inoculum size through the seed of enlarged culturing), be inoculated in fermention medium, at pH5.5-6.0, ferment under 30-35 ℃ the condition, fermentation time is 100h; After the fermentation ends, adopt molecular weight cut-off 150-200 kDa ceramic membrane, at 1.5-2.5MPa, 20 ℃, fermentation thalline and other macromolecular substance are removed in ultrafiltration; The nanofiltration membrane of molecular weight 200-2000Da is stayed in employing, and at 2.0-3.0MPa, 20 ℃ of ultrafiltration and concentration are removed inorganic salt and small-molecule substance thereof simultaneously, make transglucosidase liquid, and enzyme liquid is concentrated 10 times.
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.Under the room temperature, in 1L transglucosidase liquid, add 1.2kgD301(R) the type resin, leach behind the vibration absorption 8h; Use 3% sodium alginate 1.5L embedding again, be soaked in 0.1mol/L CaCl
2The 4h that hardens among the solution 5L leaches, and at last it is soaked 1.5h in 1.5% glutaraldehyde solution 3L, leaches, i.e. being fixed enzyme.
Immobilized enzyme is adorned post, preparation pH5.4, the Fructus Hordei Germinatus liquid glucose of sugar dense 30% under 52 ℃, with 1 times of column volume/h of flow velocity, uninterruptedly experimentizes, and sampling regularly detects the oligomeric isomaltose product component, and adds up qualified IMO50 liquid ultimate production.
Embodiment 4
With the Aspergillus niger strain of cryopreservation (
Aspergillus niger) BLB-28, under pH5.8,33 ℃ of conditions, cultivate 22h, carry out first order seed and cultivate activation, secondary seed enlarged culturing; To be 10%(V/V by inoculum size through the seed of enlarged culturing), be inoculated in fermention medium, at pH5.5-6.0, ferment under 28-32 ℃ the condition, fermentation time is 110h; After the fermentation ends, adopt molecular weight cut-off 150-200 kDa ceramic membrane, at 1.5-2.5MPa, 15 ℃, fermentation thalline and other macromolecular substance are removed in ultrafiltration; The nanofiltration membrane of molecular weight 200-2000Da is stayed in employing, and at 2.0-3.0MPa, 15 ℃ of ultrafiltration and concentration are removed inorganic salt and small-molecule substance thereof simultaneously, make transglucosidase liquid, and enzyme liquid is concentrated 10 times.
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.Under the room temperature, in 1L transglucosidase liquid, add 1.2kgD301(R) the type resin, leach behind the vibration absorption 10h; Use 3% sodium alginate 1.5L embedding again, be soaked in 0.1mol/L CaCl
2The 4h that hardens among the solution 5L leaches, and at last its glutaraldehyde solution 3L in 2% is soaked 1.5h, leaches, i.e. being fixed enzyme.
Immobilized enzyme is adorned post, preparation pH5.8, the Fructus Hordei Germinatus liquid glucose of sugar dense 30% under 55 ℃, with 1.5 times of column volume/h of flow velocity, uninterruptedly experimentizes, and sampling regularly detects the oligomeric isomaltose product component, and adds up qualified IMO50 liquid ultimate production.
Embodiment 5
With the Aspergillus niger strain of cryopreservation (
Aspergillus niger) BLB-28, under pH6.0,33 ℃ of conditions, cultivate 20h, carry out first order seed and cultivate activation, secondary seed enlarged culturing; To be 12%(V/V by inoculum size through the seed of enlarged culturing), be inoculated in fermention medium, at pH5.5-6.0, ferment under 32-36 ℃ the condition, fermentation time is 120h; After the fermentation ends, adopt molecular weight cut-off 150-200 kDa ceramic membrane, at 1.5-2.5MPa, 10 ℃, fermentation thalline and other macromolecular substance are removed in ultrafiltration; The nanofiltration membrane of molecular weight 200-2000Da is stayed in employing, and at 2.0-3.0MPa, 10 ℃ of ultrafiltration and concentration are removed inorganic salt and small-molecule substance thereof simultaneously, make transglucosidase liquid, and enzyme liquid is concentrated 10 times.
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.Under the room temperature, in 1L transglucosidase liquid, add 1.2kgD301(R) the type resin, leach behind the vibration absorption 12h; Use 3% sodium alginate 1.5L embedding again, be soaked in 0.1mol/L CaCl
2The 4h that hardens among the solution 5L leaches, and at last its glutaraldehyde solution 3L in 2.0% is soaked 1.5h, leaches, i.e. being fixed enzyme.
Immobilized enzyme is adorned post, preparation pH6.0, the Fructus Hordei Germinatus liquid glucose of sugar dense 30% under 60 ℃, with 1.8 times of column volume/h of flow velocity, uninterruptedly experimentizes, and sampling regularly detects the oligomeric isomaltose product component, and adds up qualified IMO50 liquid ultimate production.
Embodiment 6
Get 1L transglucosidase liquid (enzyme activity 61U/ml), add 1000g D301(R) the type resin, room temperature concussion absorption 9h; With 80 order filter sieve, elimination supernatant; Resin after fixing with 3% sodium alginate 1.5L embedding, under room temperature, is placed on 0.1mol/L CaCl
2Solution 5L in harden time 4h; Filter with 80 order filter sieve; Immobilized enzyme after will hardening then places 1.5% glutaraldehyde solution 3L to soak, carry out crosslinked, behind the 1.5h, filtering, immobilized enzyme 1800g.
Immobilized enzyme is through enzyme activity determination, measuring method during with strain screening the enzyme activity determination method identical.After measured, every 50g immobilized enzyme, its enzyme are lived and are 1475U, and enzyme immobilization transformation efficiency alive is 87% as calculated.
Immobilized enzyme is adorned post, preparation pH6.0, sugar dense 30% Fructus Hordei Germinatus liquid glucose, in 60 ℃ with 1.5 times of column volume/h of flow velocity, uninterruptedly experimentize, sampling regularly detects the oligomeric isomaltose product component, and adds up qualified 500 liquid ultimate productions; Final production oligomeric isomaltose liquid (IMO50 output is 75% product in dry substance concentration) 17900L.
More than the preparation and the immobilization technology thereof of a kind of transglucosidase provided by the present invention is described in detail, used specific case herein principle of the present invention and embodiment are set forth, above example just helps to understand bright method of this law and central principle.For those skilled in the art,, in concrete enforcement, can change as required each condition according to central principle of the present invention.In sum, this specification sheets embodiment should not be construed as limitation of the present invention.
Claims (9)
- An Aspergillus niger strain ( Aspergillus niger) BLB-28, it is characterized in that with the aspergillus niger that from soil, screens ( Aspergillus niger) BLB-16 is starting strain, through ultraviolet ray+LiCl complex mutation, and obtain through screening; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCC No 5143 on August 16th, 2011.
- 2. transglucosidase, it is characterized in that using Aspergillus niger strain ( Aspergillus niger) BLB-28, fermentation makes.
- 3. as the preparation method of transglucosidase as described in the claim 2, it is characterized in that with aspergillus niger (Aspergillus niger) BLB-28 be fermentation strain, concentrate, preparation transglucosidase liquid through seed culture, fermentation, heat sterilization, nanofiltration.
- 4. as the preparation method of transglucosidase as described in the claim 3, the substratum that it is characterized in that said seed culture consists of: glucose 300-500g/L, yeast extract paste 16 g/L, agar powder 18g/L, potassium primary phosphate 1.2 g/L, sal epsom 0.45g/L, urea 1g/L, pH5.5-6.0.
- 5. as the preparation method of transglucosidase as described in the claim 3, the substratum that it is characterized in that said fermentation consists of: glucose 300-500g/L, yeast extract paste 18 g/L, agar powder 15g/L, potassium primary phosphate 1.5 g/L, sal epsom 0.5g/L, urea 2g/L, pH5.5-6.0.
- 6. as the preparation method of transglucosidase as described in the claim 3, it is characterized in that said heat sterilization adopts ceramic composite membrane filtration sterilization and purifying.
- 7. as the preparation method of transglucosidase as described in the claim 3, it is characterized in that said nanofiltration concentrates to adopt organic composite nanometer filter membrane concentration and purifying.
- 8. as the process for fixation of transglucosidase as described in the claim 2, it is characterized in that with transglucosidase liquid through resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization preparation immobilization transglucosidase.
- 9. as the preparation method of transglucosidase as described in claim 3 or 8, it is characterized in that comprising following concrete steps:1) mutagenesis screening of step 1--bacterial classification;By the aspergillus niger that from soil, screens ( Aspergillus niger) BLB-16, through ultraviolet ray+LiCl complex mutation, and through screening obtain Aspergillus niger strain ( Aspergillus niger) BLB-28;2) step 2--actication of culture;With the Aspergillus niger strain of cryopreservation ( Aspergillus niger) BLB-28, activating through the first order seed cultivation, secondary seed is cultivated and is carried out enlarged culturing; Culture condition is: pH5.5-6.0, and culture temperature is 25-40 ℃, incubation time 18-24h;3) step 3--fermentation;To be inoculated in fermention medium through the seed of enlarged culturing, carry out fermentation culture; Wherein, inoculum size is 5-12%(V/V), fermentation condition is: pH5.5-6.0, culture temperature is 25-40 ℃, dissolved oxygen is 28-30%, fermentation time 96-120h;4) step 4--heat sterilization;Adopt ceramic membrane ultrafitration, remove the fermentation thalline, preparation transglucosidase liquid;Operational condition: 0.5-1.0MPa, 4-25 ℃; The ceramic membrane molecular weight cut-off is 150-200 kDa;5) step 5--nanofiltration concentrates;Adopt organic composite nanometer filtering film to concentrate, operational condition is: pressure 0.5-2.0MPa, temperature 4-25 ℃, preparation transglucosidase liquid; Organic composite nanometer filter retaining molecular weight 200-2000Da;6) immobilization of step 6--enzyme;Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization, the preparation immobilized enzyme; Fixing condition is: under the room temperature, add a certain amount of D301(R in transglucosidase liquid) the type resin, leach behind the vibration absorption 8-12h; Use 3% sodium alginate to embed again, be soaked in 0.1mol/L CaCl 2The 4h that hardens in the solution leaches, and at last it is soaked in the glutaraldehyde solution 1.5h of 1.5-2%, leaches, i.e. being fixed enzyme;7) step 7-activity of the immobilized enzyme and Detection of Stability;Immobilized enzyme is adorned post, and the Fructus Hordei Germinatus liquid glucose of preparation pH5.0-6.0 is crossed post by certain flow velocity, and flow velocity 0.6-1.8 times column volume/h uninterruptedly experimentizes, and sampling regularly detects the oligomeric isomaltose product component;Polyglucosidase is applied to food, medicine, fodder industry field, is used for the production of oligomeric isomaltose.
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| CN109554302A (en) * | 2018-12-26 | 2019-04-02 | 江苏牧之歌生态农业科技有限公司 | A method of utilizing immobilized cell technology fermenting and producing fodder enzyme preparation |
| CN112680367A (en) * | 2021-01-28 | 2021-04-20 | 天津科技大学 | Aspergillus niger strain for producing transglucosidase |
| CN114262703A (en) * | 2021-12-31 | 2022-04-01 | 保龄宝生物股份有限公司 | Method for enriching D-psicose 3-epimerase by using membrane and application thereof |
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