CN101812435A - Preparation and extraction process of extracellular lactase - Google Patents

Preparation and extraction process of extracellular lactase Download PDF

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Publication number
CN101812435A
CN101812435A CN 201010187278 CN201010187278A CN101812435A CN 101812435 A CN101812435 A CN 101812435A CN 201010187278 CN201010187278 CN 201010187278 CN 201010187278 A CN201010187278 A CN 201010187278A CN 101812435 A CN101812435 A CN 101812435A
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preparation
solid medium
extraction process
extracellular lactase
extracellular
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刘鼎阔
王立红
董惠峰
张勇
张凤洪
张俊霞
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Dingzheng Animal Pharmaceutical Tianjin Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01108—Lactase (3.2.1.108)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)

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Abstract

The invention relates to a preparation and extraction process of extracellular lactase. The process comprises the following steps of: (1) vaccinating activated actinomucor elegans into a solid culture medium for culturing; (2) adding an inorganic flocculant to a crude enzyme solution after culturing for precipitating, and then filtering to obtain a semi-finished product; and (3) carrying out salting-out, chromatograph and purification on the semi-finished product to obtain a finished lactase product. Compared with the enzyme production in cells in the prior art, the invention has simple production process, is suitable for large-scale production and also has high heat resistance, good stability, safety, stability and no pollution, and the acting pH is more suitable for the in vivo biotic environment of organisms.

Description

The preparation of extracellular lactase and extraction process
Technical field
The present invention relates to the preparation technology of proteolytic enzyme, especially a kind of preparation of extracellular lactase and extraction process.
Background technology
Sumylact L, (Latin title: β-Galactosidase), be mainly used in dairy industry can make the lactose of low sugariness and low solubility change glucose and semi-lactosi sweeter, that solubleness is bigger into to the beta-galactosidase enzymes that is otherwise known as; Make ice-creams, concentrate the possibility reduction that lactose crystn is separated out in breast, the evaporated milk, increase sugariness simultaneously.But for various reasons; lack above-mentioned Sumylact L in most people's the body; cause a lot of people to suffer from lactose intolerance; be that people regular meeting behind milk drink causes the main carbohydrate in the milk---the maldigestion of lactose; occur that abdomen rises, symptoms such as borborygmus, acute abdominal pain even diarrhoea, in China, according to scientific research personnel's result of study; the incidence of lactose malabsorption is up to 86.7% behind grownup's milk drink, and not tolerating index is 0.9.And the meeting of lactose intolerance causes gastrointestinal disorder, causes valuable protein and mineral loss, even has influence on infant's intelligence growth, so the many states in the Europe daily life of a family adds milk with Sumylact L, drinks for the baby.
The industrialization of present domestic Sumylact L is produced and is also existed very big problem, and Sumylact L is not used widely, and its major cause is: the unit output of Sumylact L is lower, the Sumylact L of market sale mostly is intracellular enzyme, need smudge cells just can obtain its extraction and purification process complexity, cost height; The operative temperature of enzyme is low, and poor heat resistance is easy to dye assorted bacterium, narrow application range in the production process.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of and utilize graceful radiation Mucor bacterial strain to carry out preparation and the extraction process that born of the same parents produce the extracellular lactase of enzyme outward.
A kind of preparation of extracellular lactase and extraction process, this technology may further comprise the steps:
(1) radiation of the grace after will activating mucormycosis is seeded in the solid medium and cultivates;
(2) crude enzyme liquid after the cultivation adds the inorganic flocculating agent precipitation, filters then and obtains work in-process;
(3) work in-process through saltout and chromatography purification after obtain the Sumylact L finished product.
Grace radiation Mucor bacterial strain inoculum size after the activation described in the step (1) is 10～15% of a solid medium gross weight.
Solid medium comprises in the step (1):
(1) in wheat bran, analysis for soybean powder or the Semen Maydis powder a kind of, two or three;
(2) a kind of in maltose, sucrose or the lactose;
(3) a kind of in extractum carnis, peptone or the yeast extract paste;
(4) MnSO 4, KH 2PO 4, NaH 2PO 4Or CaCl 2In one or both;
(5) water.
Preferred solid medium is wheat bran, lactose, peptone, KH 2PO 4, NaH 2PO 4And water, wherein the weight ratio of wheat bran and water is 1: 1.0～2.0, and the lactose addition is 0.05～0.1g/mL, and the peptone addition is 0.04～0.08g/mL, KH 2PO 4Addition is 0.05～0.15% of a solid medium gross weight, NaH 2PO 4Addition is 0.10～0.2% of a solid medium gross weight.
When above-mentioned solid medium was selected for use through the solid medium after 2～3 fermentation culture, born of the same parents produced the better effects if of enzyme outward.
Culture condition in the step (1) is: culture temperature is 30～50 ℃, and incubation time is 60～80h, and per 24～36h turns over Qu Yici in the culturing process.
Inorganic flocculating agent in the step (2) is a kind of in alum, Tai-Ace S 150 or the magnesium chloride.
What saltouing in the step (3) used is the ammonium sulfate precipitation method, and chromatography purification comprises Sephadex G-100 gel chromatography and DEAE-Mierocrystalline cellulose-52 ion exchange chromatography purifying.
Advantage of the present invention and positively effect are:
The present invention is a starting strain with grace radiation mucormycosis, cultivates in solid medium, has determined that by single factor and orthogonal test best solid medium is wheat bran, lactose, peptone, KH 2PO 4, NaH 2PO 4And water; the crude enzyme liquid that obtains after the cultivation is after inorganic flocculating agent Tai-Ace S 150 is handled; pass through ammonium sulfate precipitation again; Sephadex G-100 gel chromatography and DEAE-Mierocrystalline cellulose-52 ion exchange chromatography purifying make the Sumylact L finished product; this finished product is after the check of polyacrylate hydrogel acid amides electrophoresis; it is finally than vigor 512.68 ± 0.25U/mg; the Sumylact L yield is 44.38%; the purification multiple is 35.82; the action pH of Sumylact L is 4.5～5.5; at 30～60 ℃ higher tolerance is arranged; under 4 ℃ of refrigerated conditions, has satisfactory stability; compare with producing enzyme in the born of the same parents of the prior art; its production technique is simple; raw materials cost is low; be fit to large-scale production; the Sumylact L thermotolerance height that makes; good stability; safety and stability is pollution-free, coenocorrelation in the body of action pH body preferably.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Graceful radiation Mucor used in the present invention is available from China Committee for Culture Collection of Microorganisms common micro-organisms center, and the Latin name of this bacterial classification is called: Actinomucor elegans, deposit number is: CGMCC3.2178.
A kind of preparation purification process of extracellular lactase may further comprise the steps:
(1) radiation of the grace after will activating Mucor bacterial strain is seeded in the solid medium and cultivates;
The activatory step is as follows:
Directly get frozen grace radiation Mucor bacterial strain with transfering loop,, cultivated 36～42 hours in 25～32 ℃ in the line of LB culture medium flat plate surface.
Single bacterium colony of picking forwards in 3～5ml LB substratum, cultivates 36～42 hours in 25～32 ℃.
Nutrient solution all forwarded in the 100ml LB substratum cultivated 36～42 hours, to OD600=0.2～0.4.
Grace radiation Mucor bacterial strain inoculum size after the activation in the step (1) is 10～15% of a solid medium gross weight.
Solid medium in the step (1) comprises:
1. any one in wheat bran, analysis for soybean powder or the Semen Maydis powder;
2. any one in maltose, sucrose or the lactose;
3. any one in extractum carnis, peptone or the yeast extract paste;
4. MnSO 4, KH 2PO 4, NaH 2PO 4Or CaCl 2In any one or two kinds;
5. water.
Determined that through single factor and orthogonal test preferred solid medium is wheat bran, lactose, peptone, KH 2PO 4, NaH 2PO 4And water, wherein the weight ratio of wheat bran and water is 1: 1.0～2.0, and the lactose addition is 0.05～0.1g/mL, and the peptone addition is 0.04～0.08g/mL, KH 2PO 4Addition is 0.05～0.15% of a solid medium gross weight, NaH 2PO 4Addition is 0.10～0.2% of a solid medium gross weight.
When above-mentioned solid medium was selected for use through the solid medium after 2～3 fermentation culture, born of the same parents produced the better effects if of enzyme outward, can improve 40～55% Sumylact L output.
Culture condition in the step (1) is: culture temperature is 30～50 ℃, incubation time is 60～80h, per 24～36h turns over Qu Yici in the culturing process, can improve the output of Sumylact L, by suitably turning over song material inoculation and temperature are kept evenly, help the utilization of raw material, turn over the bent time that also can postpone the spore appearance simultaneously, make mycelia secrete more enzyme.
(2) crude enzyme liquid after the cultivation adds the inorganic flocculating agent precipitation, filters then and obtains work in-process;
Inorganic flocculating agent in the step (2) is a kind of in alum, Tai-Ace S 150 or the magnesium chloride, and not only flocculating effect is good for it, and the rate of recovery of enzyme is also high.By using flocculation agent, make that the little suspended particle of various solids and pigment flocculating settling get off in the Sumylact L crude enzyme liquid, obtain clarifying enzyme liquid, reach the effect of preliminary purification Sumylact L crude enzyme liquid, reduce the influence that the Sumylact L enzyme is lived.
The measuring method of flocculating rate is:
Get 100mL Sumylact L crude enzyme liquid and place the 500mL beaker, under room temperature and the natural pH condition, add Tai-Ace S 150, mixing 1min fast, mixing 1min at a slow speed again behind 2～3min leaves standstill 60min, and blank is not added flocculation agent.Get upper strata liquid dilution certain multiple, sentencing distilled water at 620nm is that reference is measured absorbancy OD value.Flocculating rate (FR) is calculated as follows:
Figure GDA0000021899780000031
The measuring method of flocculation liquid filtering rate is:
Recording volume behind the filtration 5min is converted into the volume (mL/min) in the unit time, as the index of weighing filtering rate (Fv).
Filter method in the step (2) is: behind the fermented liquid flocculation treatment 60min, filter with 9cm conical hopper, 12cm filter paper, filtrate directly splashes in the graduated cylinder.
(3) work in-process through saltout and chromatography purification after obtain the Sumylact L finished product.
What saltouing in the step (3) used is the ammonium sulfate precipitation method, and chromatography purification comprises Sephadex G-100 gel chromatography and DEAE-Mierocrystalline cellulose-52 ion exchange chromatography purifying.
The process of ammonium sulfate precipitation method is:
Ammonium sulfate is dried porphyrize, get the 200mL supernatant liquor, reach 20%, 4 ℃ and leave standstill 2h to wherein slowly adding ammonium sulfate powder to its concentration, centrifugal (7000r/min, 15min, 4 ℃), precipitation is dissolved in the phosphate buffered saline buffer of 0.005M, pH6.28, surveys enzyme and lives.Supernatant liquor continue to add ammonium sulfate powder to concentration and reaches 40%, leaves standstill centrifugally, and precipitation is dissolved in damping fluid, surveys enzyme and lives.Get supernatant liquor adding ammonium sulfate and make its concentration reach 60%, 80% and 90% respectively, operate the same.
The process of Sephadex G-100 gel chromatography is:
1.Sephadex the pre-treatment of G-100
Get 2.5g Sephadex G-100, in beaker, add distilled water, stir gently, discard suspended particle, so repetitive scrubbing several times after, add the phosphate buffered saline buffer of excessive 0.005M, pH6.28, swelling 72h under the room temperature.
2. dress post
Cut-off footpath 1.0cm, the chromatography column of long 50cm is vertically fixed on the iron stand, with the sealing screw means of chromatography column lower end, Xiang Zhuzhong adds the 0.005M of about 5～7cm, the phosphate buffered saline buffer of pH6.28, slowly adds the good gel of swelling then, note making not have bubble in the post, open sample output valve.Gel slowly sinks, and continues to add, and stirs gently with glass stick, makes boundlessness in the post.After treating that gel column installs, put one deck filter paper, destroy the glue face when avoiding sample in gel surface.With damping fluid balance chromatography column, and to open constant flow pump control flow velocity be 1mL/5min.
3. go up sample
The upper end of post is opened, and with the damping fluid sucking-off above the gel, exhaustion is not stayed the skim liquid level, in order to avoid air admission with suction pipe.Slowly add sample along tube wall, note not upsetting gel surface.Turn on the screw clip of lower end, sample is entered in the gel, when soon having advanced, add 1mL～2mL damping fluid flushing post jamb, use the elutriant wash-out of volume immediately.
4. wash-out, collection
After sample entered gel fully, with the phosphate buffered saline buffer elution samples of 0.005M, pH 6.28, elution speed was 1mL/5min, and collects.
The process of DEAE-Mierocrystalline cellulose-52 ion exchange chromatography purifying is:
The activation 1.DEAE-Mierocrystalline cellulose-52 expands
Take by weighing 3.0g DEAE-Mierocrystalline cellulose-52, be spread in about 45mL0.5mol/L NaOH solution, soak about 1h, stir frequently.Suction filtration (B adds two layers of filter paper), with distilled water wash, suction filtration till the nearly neutrality of filtrate, is soaked in Mierocrystalline cellulose 1h among the 0.5mol/LHCl more again, and same suction filtration liquid is near neutral.Mierocrystalline cellulose is dipped in the 0.5mol/L NaOH liquid, the same processing is washed till neutrality again.
2. balance
DEAE-Mierocrystalline cellulose-52 is put into 0.02mol/L pH7.4 phosphate buffered saline buffer (being initial damping fluid), static 1h, stir frequently, after treating that Mierocrystalline cellulose sinks, incline supernatant liquor or suction filtration removed washing lotion, so to inclining several times repeatedly the pH of phosphoric acid buffer of the pH of liquid and adding when close till.
3. adorn post, go up sample
Adorn post, go up the sample process with Sephadex G-100 gel dress post process.
4. wash-out, collection
With contain 0.15,0.25,0.35,0.45, the 0.02mol/L pH7.4 phosphate buffered saline buffer of 0.55mol/LNaCl carries out gradient elution, and collects.
The calibration method that enzyme is lived is:
1.ONP the preparation of typical curve
ONP (o-NP) solution with concentration known is done typical curve, according to the OD value of the concentration c correspondence of ONP, utilizes method of least squares to set up regression equation:
OD＝51648c+0.0079
The OD=420nm light absorption value
C:ONPG concentration μ mol/rnL
Coefficient R=0.9908; The measurement range of c is 0～0.15 μ mol/mL
O-nitrophenol-β-D-galactoside (ONPG) is leucocompound soluble in water, and beta-D-galactosidase catalysis ONPG hydrolysis generates o-NP (ONP), and it is yellow that ONP is in basic solution, at 420nm maximum absorption band arranged.
(1) gets the 1mL crude enzyme liquid in the EP pipe, the centrifugal 10min of 10000r/min.Get supernatant liquor 0.5mL, mix with the 0.5mL reaction buffer.
(2) add 0.2mL ONPG solution, reaction 10min.
(3) add 0.5mL saturated sodium carbonate solution termination reaction.Moisturizing is to 3.5mL.
(4) measure the OD value at the 420nm place with ultraviolet-visible pectrophotometer.
(5) enzyme activity is defined as: under optimum reaction conditions, per minute catalysis 1 μ mol substrate conversion is that the required enzyme amount of product is defined as enzyme unit alive.
E = OD - 0.0079 5.1648 T × V × N
E is the enzyme activity of Sumylact L in the crude enzyme liquid, and T is the reaction times, and V is the reaction system volume, and N is an extension rate.Owing to be the solid state fermentation Sumylact L, last enzyme activity unit will become U/g song (Y)
Y = Ev m
E is a Sumylact L vigor in the crude enzyme liquid, and V is the cumulative volume of crude enzyme liquid, and m is the quality of wheat bran
(6) because OD value is directly proportional with enzyme work, promptly enzyme live high more, reaction many more, the OD value is also high more accordingly, so can come the work of indirect reaction enzyme with the OD value.
Embodiment
The plant and instrument that relates in the present embodiment comprises J-25 type high speed freezing centrifuge, DL201 type constant temperature automatic drier, BS-2F constant-temperature shaking culture case, the full temperature shaking culture of HZQ-F160 case, magnetic stirring apparatus, JA2003 electronic balance, DZKW-C type water-bath, steam sterilizer, Delta 320pH meter.
1. preparation spore suspension
(1) the graceful radiation of cultured activation Mucor bacterial strain bacterial classification is put into sterilisable chamber, lights spirit lamp, with 75% cotton ball soaked in alcohol wiping both hands and desktop, the sterilized water of getting 3～5mL joins in the viable bacteria culture dish, in flame region, use the spreader of the bacterium of going out to scrape spore, make it to be dissolved in the sterilized water.
(2) with the spore liquid in the liquid-transfering gun absorption culture dish, move in the triangular flask of the bacterium of going out.Get an amount of sterilized water dilution spore liquid.Put into the little granulated glass sphere of some bacterium of going out in triangular flask, fully vibration is scattered spore fully.
(3) with the spore count of blood counting chamber, utilize the sterilized water diluting effect to make the spore count of spore suspension 10 at microscopically metering spore suspension 7The order of magnitude
2. the preparation of solid medium
(1) wheat bran and distilled water mixing in 1: 1.5 are put into the 250mL triangular flask, wherein solid matter quantitatively is 10g, stirs, as basic medium.
(2) 0.1% of the peptone of the lactose of interpolation 0.06g/mL, interpolation 0.06g/mL, interpolation solid medium gross weight KH in basic medium 2PO 4, add 0.15% NaH of solid medium gross weight 2PO 4
(3) triangular flask is wrapped with newspaper put into high-pressure sterilizing pot sterilization, sterilising conditions: 121 ℃, 0.1Mpa, 17min.
(4) triangular flask is taken out, is cooled to normal temperature from high-pressure sterilizing pot.
3. bacterial classification inoculation
(1) enters sterilisable chamber, light spirit lamp, with 75% cotton ball soaked in alcohol wiping both hands and desktop.Take off the rope that is bundled in the Erlenmeyer flask bottleneck, keep gauze, bottleneck is from flame nearby.
(2) insert the bacteria suspension of solid medium gross weight 15% in the triangular flask with liquid-transfering gun, the went out glass stick of bacterium of usefulness stirs it then.
4. cultivate
The constant incubator that vaccinated triangular flask is put into 45 ℃ is cultivated, and cultivates 72h left and right sides best results.In culturing process, stir 1 time every 24h.
5. the preparation of crude enzyme liquid
The wheat bran that fermented with the flooding of 8 volumes also at the uniform velocity stirs 30min.After four layers of filtered through gauze, promptly get crude enzyme liquid.
6. the flocculation of crude enzyme liquid
Add Tai-Ace S 150 in the crude enzyme liquid and prepare the Sumylact L work in-process, the crude enzyme liquid addition is 0.002-0.003g/mL.Under 30 ℃, its flocculating effect the best.Under this condition, filtration velocity is 1.52mL/min, and flocculating rate is 88.27%, and the clarification rate is 91.46%, the enzyme rate of recovery 87.9%.
7. Sumylact L is half-finished saltouts and purifying
(1) the Sumylact L work in-process through the ammonium sulfate precipitation method saltout, Sephadex G-100 gel chromatography and DEAE-Mierocrystalline cellulose-52 ion exchange chromatography purifying.
8. enzyme calibrating alive
The Sumylact L finished product obtains single band by the check of polyacrylate hydrogel acid amides electrophoresis, reaches electrophoresis purity, and finally it is than vigor 512.68 ± 0.25U/mg, and the Sumylact L yield is 44.38%, and the purification multiple is 35.82.
The Sumylact L finished product is that the zymologic property of substrate is with the o-NP: the suitableeest action pH is 4.5～5.5.Enzyme is lived and is significantly reduced when pH surpasses 6.0; The optimum temperuture of its reaction is 45～60 ℃.At 30～60 ℃ higher tolerance is arranged, pure enzyme liquid has satisfactory stability under 4 ℃ of refrigerated conditions.

Claims (8)

1. the preparation of an extracellular lactase and extraction process is characterized in that: may further comprise the steps:
(1) radiation of the grace after will activating mucormycosis is seeded in the solid medium and cultivates;
(2) crude enzyme liquid after the cultivation adds the inorganic flocculating agent precipitation, filters then and obtains work in-process;
(3) work in-process through saltout and chromatography purification after obtain the Sumylact L finished product.
2. the preparation of extracellular lactase according to claim 1 and extraction process is characterized in that: the grace radiation Mucor bacterial strain inoculum size after the activation described in the step (1) is 10～15% of a solid medium gross weight.
3. the preparation of extracellular lactase according to claim 1 and 2 and extraction process, it is characterized in that: described solid medium comprises:
(1) in wheat bran, analysis for soybean powder or the Semen Maydis powder a kind of, two or three;
(2) a kind of in maltose, sucrose or the lactose;
(3) a kind of in extractum carnis, peptone or the yeast extract paste;
(4) MnSO 4, KH 2PO 4, NaH 2PO 4Or CaCl 2In one or both;
(5) water.
4. according to the preparation and the extraction process of the described extracellular lactase of claim 1, it is characterized in that: solid medium is wheat bran, lactose, peptone, KH described in the step (1) 2PO 4, NaH 2PO 4And water, wherein the weight ratio of wheat bran and water is 1: 1.0～2.0, and the lactose addition is 0.05～0.1g/mL, and the peptone addition is 0.04～0.08g/mL, KH 2PO 4Addition is 0.05～0.15% of a solid medium gross weight, NaH 2PO 4Addition is 0.10～0.2% of a solid medium gross weight.
5. the preparation of extracellular lactase according to claim 1 and extraction process is characterized in that: described solid medium is for through the solid medium after 2～3 fermentation culture.
6. the preparation of extracellular lactase according to claim 1 and extraction process, it is characterized in that: culture condition is described in the step (1): culture temperature is 30～50 ℃, and incubation time is 60～80h, and per 24～36h turns over Qu Yici in the culturing process.
7. the preparation of extracellular lactase according to claim 1 and extraction process is characterized in that: the inorganic flocculating agent described in the step (2) is a kind of in alum, Tai-Ace S 150 or the magnesium chloride.
8. the preparation of extracellular lactase according to claim 1 and extraction process, it is characterized in that: what saltouing described in the step (3) used is the ammonium sulfate precipitation method, and chromatography purification comprises Sephadex G-100 gel chromatography and DEAE-Mierocrystalline cellulose-52 ion exchange chromatography purifying.
CN 201010187278 2010-05-31 2010-05-31 Preparation and extraction process of extracellular lactase Pending CN101812435A (en)

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WO2011150754A1 (en) * 2010-05-31 2011-12-08 鼎正动物药业（天津）有限公司 Method for preparing and extracting extracellular lactase
CN102676443A (en) * 2012-05-25 2012-09-19 鼎正动物药业（天津）有限公司 Synergist and medium for fermentation culture of hairy mould
CN102676443B (en) * 2012-05-25 2013-06-26 鼎正动物药业（天津）有限公司 Synergist and medium for fermentation culture of hairy mould

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