CN102296032B - 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 with the Aspergillus niger strain that screens from soil (
Aspergillus niger) BLB-16 is starting strain, through mutagenesis, the screening bacterial strain BLB-28 that is optimized, and through fermentation, degerming, concentrated preparation transglucosidase liquid, also relates to the immobilization technology of this transglucosidase.
Background technology
Transglucosidase (Transglucosidase), 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.
Turn heteroside enzyme Main Function and be from the non-reducing end of substrate and cut α-Isosorbide-5-Nitrae glycosidic link, discharge alpha-D-glucose, 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 not decomposing the enzyme of oligomeric isomaltose in human body alimentary canal, therefore, it is after taking in human body, have not digested, promote the intestinal beneficial bacterium group breed, relax bowel, regulate blood fat, low sugariness, the 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 during transglucosidase is produced as oligomeric isomaltose, the zymin of most critical, but never realize suitability for industrialized production at home.At present, the complete dependence on import of this zymin, this not only makes in the production of oligomeric isomaltose, and gordian technique is controlled by external relevant enterprise, more makes in oligomeric isomaltose production, and the zymin use cost can be in any more.
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, the Metabolism of E. coli production of adopting genetic engineering technique to build is generally intracellular enzyme, and its extraction process is complicated, and the enzyme rate of recovery alive is low, also the unfavorable suitability for industrialized production of using.Therefore, be necessary to develop the outer transglucosidase of a kind of born of the same parents, the needs of producing to adapt to oligomeric isomaltose.
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, greatly improve the situation that domestic transglucosidase is limited by the world market, and can significantly reduce oligomeric isomaltose produce in the use cost of enzyme.
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, namely one is to have broken through only to adopt resin to add in the glutaraldehyde cross-linking immobilization technology, because immobilization is firm not, and make immobilized enzyme be easy to come off, reveal, increased that subsequent technique is refining, 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, because hardness is lower, is difficult to use in large-scale packed column, and is added directly in liquid glucose, need filter, increased the difficult problems such as complicacy of technique.
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 screens from soil (
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's common micro-organisms center (CGMCC) on August 16th, 2011, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, in 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.Its Main Function is in the production process take starch as raw material, starch through liquefaction and saccharification after, turn 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 dissociating is with α-1,6 glycosidic links are transferred on another carbohydrate substrate, thereby obtain oligomeric isomaltose; Adopt the transglucosidase in 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, aspergillus niger (Aspergillus niger) BLB-28 that namely optimizes gained take the inventor is as fermentation strain, through seed culture, fermentation, heat sterilization, the concentrated preparation of nanofiltration transglucosidase liquid;
In above-mentioned preparation method, the substratum of said seed culture consists of: 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.
The concentrated concentrated purifying that reaches of organic composite nanofiltration membrane that adopts of said nanofiltration.
With the prepared transglucosidase liquid of aforesaid method, through resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization, namely make the immobilization transglucosidase.
Adopt the immobilization transglucosidase of technique preparation, not only stable high, and also the difficult leakage of zymin, be beneficial to the oligomeric isomaltose purifying products, improve product quality, reduce product cost.Adopt immobilized enzyme to produce oligose, and than adopting the transglucosidase liquid phase ratio for preparing in the present invention, reduce zymin use cost 100-150 unit.
The preparation method of the 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, dilute complete after, go respectively 200ul on the plate isolation base, with the spreading rod coating evenly, be cultured to and grow ripe bacterium colony on flat board, identify and be purified into the aspergillus niger bacterium colony, will but the colony inoculation inclined-plane, cultivation.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.
Method for detecting enzymatic activity: make reaction substrate with colourless p-NP-α-D-glucopyranoside (pNPG), through alpha-glucosidase hydrolyzing alpha-1, discharge p-NP (pNP) after the 4-glucoside bond, by the criterion of the pNP generation under detection 405nm as α-Isosorbide-5-Nitrae-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, then uses filtered through gauze, then adds glucose 20g and agar 20g, dissolves rear moisturizing to 1000ml, the pH nature.
Slant medium is: potato 200g peeling, and stripping and slicing is boiled and is kept 30min, then uses filtered through gauze, then adds glucose 22g and agar 20g, and after it melted, moisturizing was to 1000ml, and pH is natural.
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 screens from soil, through ultraviolet ray+NaN
3Complex mutation, and obtain aspergillus niger BLB-28(CGMCC No 5143 through screening).
Concrete mutagenic processes is: the appropriate 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.Spore suspension is poured into the aseptic plate that contains aseptic pin, kept the plate lid to open, 20cm shines 0s, 20s, 40s, 60s, 80s, 100s, 120s in 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 respectively 10 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 28 ℃ of biochemical cultivation cases and cultivate 4-5d, the statistics lethality rate.
Bacterial screening: the aspergillus niger starting strain BLB-16 before the mutagenesis is as contrast, the bacterial strain after mutagenesis carried out fermentation culture is produced enzyme and transglucosidase is active detects.
Enzymic activity detects: during with bacterial screening, enzyme biopsy survey method is consistent.Filter out high transglucosidase and produce bacterial strain, namely 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: be that enzyme biopsy survey method is identical with bacterial screening.
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 interception molecular weight is 150-200 kDa, heat sterilization and macromolecular substance thereof, preliminary purification transglucosidase.
5) step 5--nanofiltration is concentrated
Adopt organic composite nanofiltration membrane to concentrate.Operational condition is: pressure 2.0-3.0MPa, temperature 4-25 ℃; Organic composite nanofiltration membrane molecular weight cut-off 200-2000Da in the time of concentrated, removes inorganic salt and small-molecule substance thereof, preparation transglucosidase liquid.
Enzyme biopsy survey method after concentrated: during with bacterial screening, enzyme biopsy survey method is identical.
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 room temperature, add a certain amount of D301(R in transglucosidase liquid) the type resin, leach after vibration absorption 8-12h; After using again 3% sodium alginate to embed, be soaked in 0.1mol/L CaCl
2The 4h that hardens in 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 filled 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 to test, regularly sampling detects the oligomeric isomaltose product component.
Activity of the immobilized enzyme reaches the index of oligomeric isomaltose (IMO50) of market sale as standard take the oligomeric isomaltose product component, 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 mushroom is only 0.08-0.12U/ml, and fermenting enzyme is lived and greatly improved.
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 use in large-scale packed column, simplify production technique; On the other hand, with sodium alginate to embed, be placed in CaCl after the resin immobilization
2Harden in solution, adopt at last glutaraldehyde cross-linking, strengthened the immobilization Enzymic stability, avoided the leakage of zymin, thereby transglucosidase can reuse, and has greatly improved the utilization ratio of enzyme, 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 to be added directly to the immobilized enzyme preparation in liquid glucose, after reaction finishes, needs to filter and removes altogether immobilized enzyme; And the latter 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 the 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) after optimization, the fermenting enzyme work of 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 that degerming, concentrated, one step of purifying complete, reduce 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 first the immobilization of transglucosidase, improved the utilization ratio of enzyme, be conducive to 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), add in the Fructus Hordei Germinatus liquid glucose by 0.85L/ ton dry-matter, be placed in 58-60 ℃ of water-bath, insulation 30h; Sampling is placed in 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 the condition of 30-34 ℃, fermentation time is 110h, after 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, add in the Fructus Hordei Germinatus liquid glucose by 0.72L/ ton dry-matter, be placed in 55-58 ℃ of water-bath, insulation 30h; Sampling is placed in 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 the condition of 30-35 ℃, fermentation time is 100h; After 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 with concentrated 10 times of enzyme liquid.
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.Under room temperature, add 1.2kgD301(R in 1L transglucosidase liquid) the type resin, leach after vibration absorption 8h; Use again 3% sodium alginate 1.5L embedding, be soaked in 0.1mol/L CaCl
2The 4h that hardens in 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 filled 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, is uninterruptedly tested, and regularly sampling 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 the condition of 28-32 ℃, fermentation time is 110h; After 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 with concentrated 10 times of enzyme liquid.
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.Under room temperature, add 1.2kgD301(R in 1L transglucosidase liquid) the type resin, leach after vibration absorption 10h; Use again 3% sodium alginate 1.5L embedding, be soaked in 0.1mol/L CaCl
2The 4h that hardens in 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 filled 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, is uninterruptedly tested, and regularly sampling 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 the condition of 32-36 ℃, fermentation time is 120h; After 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 with concentrated 10 times of enzyme liquid.
Resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization.Under room temperature, add 1.2kgD301(R in 1L transglucosidase liquid) the type resin, leach after vibration absorption 12h; Use again 3% sodium alginate 1.5L embedding, be soaked in 0.1mol/L CaCl
2The 4h that hardens in 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 filled 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, is uninterruptedly tested, and regularly sampling 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; Then the immobilized enzyme after hardening is placed in 1.5% glutaraldehyde solution 3L and soaks, and carries out crosslinkedly, and after 1.5h, filtering gets 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 filled post, preparation pH6.0, the Fructus Hordei Germinatus liquid glucose of sugar dense 30%, is uninterruptedly tested with 1.5 times of column volume/h of flow velocity in 60 ℃, and regularly sampling detects the oligomeric isomaltose product component, and adds up qualified 500 liquid ultimate productions; Final production oligomeric isomaltose liquid (IMO50 output is take dry substance concentration as 75% product) 17900L.
Above preparation and immobilization technology thereof to 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 the bright method of this law and central principle.For those skilled in the art, according to central principle of the present invention, can change as required each condition in concrete enforcement.In sum, this specification sheets embodiment should not be construed as limitation of the present invention.
Claims (2)
- An Aspergillus niger strain ( Aspergillus niger) BLB-28, it is characterized in that with the aspergillus niger that screens from soil ( 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's 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; Take aspergillus niger (Aspergillus niger) BLB-28 as fermentation strain, concentrate preparation transglucosidase liquid through seed culture, fermentation, heat sterilization, nanofiltration; The substratum of 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; The substratum of 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; Said heat sterilization adopts ceramic composite membrane filtration sterilization and purifying; The concentrated concentrated purifying that reaches of organic composite nanofiltration membrane that adopts of said nanofiltration; Transglucosidase liquid through resin absorption-sodium alginate to embed-glutaraldehyde cross-linking method immobilization, is prepared the immobilization transglucosidase.
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