CN100513552C - Bile salt hydrolase and preparation method and special preparing strain thereof - Google Patents
Bile salt hydrolase and preparation method and special preparing strain thereof Download PDFInfo
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- CN100513552C CN100513552C CNB2006101126962A CN200610112696A CN100513552C CN 100513552 C CN100513552 C CN 100513552C CN B2006101126962 A CNB2006101126962 A CN B2006101126962A CN 200610112696 A CN200610112696 A CN 200610112696A CN 100513552 C CN100513552 C CN 100513552C
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Abstract
the invention discloses a lactic acid bacteria bile salt hydrolase and manufacturing method and specific manufacturing strain, which comprises the following steps: 1) fermenting Lactobacillus casei (KTx CGMCCNo..1739); gathering bacteria; 2) suspending bacterial through phosphoric buffer; grinding in the icy bath through supersonic wave; collecting the rough enzyme liquid; sedimenting protein through ammonia sulfate; centrifuging to obtain the product.
Description
Technical field
The present invention relates to a kind of bile salt hydrolase and production method thereof and special preparing strain.
Background technology
Probiotic bacterium is meant the microorganism useful to the humans and animals body.They are by improving the enteric microorganism eubiosis, and the control intestinal tract infections reduces serum cholesterol level, improve the utilization of lactose intolerance people to lactose, and can stimulate specificity or nonspecific immune reaction, have the effect of enhancing immunity.The viable bacteria metabolic stability of probiotic composition, activity is stronger, by large number of viable behind the digestive tube, brings into play probiotic action after entering enteron aisle.Has the ability of reducing cholesterol with regard to the sour milk that confirms lactobacillus ferment as far back as people in 1963.Many researchs have also shown content and the low density lipoprotein cholesterol content that some Bacterium lacticum can reducing total cholesterol.Absorb the content that the sour milk that contains certain Bacterium lacticum or bifidus bacillus can reduce serum cholesterol.
A large amount of discovers, the often edible fermented product that contains profitable probliotics (mainly being milk-acid bacteria) helps the reduction of serum cholesterol content.Mann and Spoerry find the people in the African Massai warrios clan, owing to drink the milk by the wild lactobacillus-fermented in locality for a long time in a large number, the content of serum cholesterol is obviously low than the general population.This phenomenon has caused people from all walks of life's such as trophology, medical science, microbiology attention, thereby has started the research boom of milk-acid bacteria reducing cholesterol effect.Up to now, in a large number discover and confirmed that different types of milk-acid bacteria has the effect of reducing cholesterol effect.
Mechanism about milk-acid bacteria reduction serum cholesterol does not have clear and definite answer at present as yet.Both at home and abroad investigator's viewpoint mainly concentrate on following some: (1) milk-acid bacteria somatic cells directly absorbs cholesterol; (2) milk-acid bacteria degraded combined cholate changes the free state cholate into, and latter's solubleness descends and cholesterol generation co-precipitation; Cholate after the degraded is free cholate, and it is in the solubleness of enteron aisle with to be absorbed ability all poor than the combined cholate, and form that can ight soil excretes, and has caused the metabolism of cholesterol to be quickened thus, reaches the effect that reduces serum cholesterol level; (3) other theories.
Bile salt hydrolase is that milk-acid bacteria is at the intravital a kind of meta-bolites of people, this enzyme energy hydrolysis combined cholate, make in conjunction with cholate and be degraded to the free state cholate, the latter's solubleness decreases than the former, therefore cholesterol is excreted with sedimentary form, its meta-bolites is amino acid, and is harmless.
The decreasing cholesterol that Klaver etc. utilize many strains Bacterium lacticum and bifidus bacillus to carry out studies confirm that: the bile salt hydrolase that milk-acid bacteria produces can make in conjunction with cholate and be decomposed into free cholate, free cholate and cholesterol form the mixture coprecipitation gets off, thereby causes the reduction of cholesterol level in the environment.
Verstrate thinks that high this feature of bile salt hydrolase vigor of milk-acid bacteria is playing an important role aspect the reducing cholesterol content.In addition, Ahn thinks that the bile salt hydrolase of milk-acid bacteria has vital role aspect reducing cholesterol.
Domestic research about lactobacterium casei mainly is on the health-care effecies such as its reducing blood-fat, hypertension, treatment anaphylactic disease, and as the Chinese patent that Cao Xinggen applied for: (number of patent application is the antibacterial agent of novel anti-helicobacter pylori and the lactobacillus paracasei class cheese subspecies bacterial strain of intestinal bacteria 157:H7: CN01805665.2); Re Erwei Dalai Nur Co., Ltd is applied for: (number of patent application is: CN01810051.1) in the application of lactobacterium casei in immunostimulatory peptides; Shanghai Bright Dairy ﹠ Food Co., Ltd's application: (number of patent application is: CN03128995.9) for lactobacterium casei Bd-II bacterial strain and the application aspect blood fat reducing thereof; (number of patent application is: CN03129450.2) for lactobacterium casei LC2W bacterial strain and the application aspect hypertension thereof.Also has Jingyue Biological Science and Technology Co., Ltd. application in addition: new microbial strain lactobacillus paracasei GM-080 and treat the purposes of irritated relative disease (number of patent application is: CN200410038566.X).
Abroad the patent about lactobacterium casei also mainly is to concentrate on lactobacterium casei as on the preventive and therapeutic effect of probiotic bacterium to various diseases, mainly contain heredity anaphylactic disease, cancer, halitosis, metabolic syndrome, probiotic therapy etc., wider general than domestic its research.Lactobacterium casei can also suppress growth of pathogenic bacteria with it as bio protective agent.Glenn; Matthew etc. separate the research that the polypeptides matter that obtains is a special efficacy material that milk-acid bacteria is produced from milk-acid bacteria.And domestic no any milk-acid bacteria produces the article report and the patent report of bile salt hydrolase.
Summary of the invention
The purpose of this invention is to provide a kind of Conjugated bile salt hydrolase (Bile Salt Hydrolase, BSH) and production method and special preparing strain.
The special preparing strain of bile salt hydrolase provided by the present invention, be lactobacterium casei (Lactobacilluscasei) KTx, be preserved in Chinese microorganism strain preservation board of trustee reason person on 06 23rd, 2006 and understood common micro-organisms center (abbreviation CGMCC, the address is: the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City), preserving number is CGMCC № .1739.
Lactobacterium casei (Lactobacillus casei) KTx separates the Kai Feier from the northeast average family.On the MRS substratum, the bacterium colony of lactobacterium casei (Lactobacillus casei) KTx is oyster white or little band yellow, slightly projection.Individual morphology is irregular G
+Sporeless bacterium presents shaft-like.
Bile salt hydrolase provided by the present invention is the protein that fermentation lactobacterium casei KTx CGMCC № .1739 obtains.
The method of production bile salt hydrolase provided by the present invention comprises the steps:
1) fermentation lactobacterium casei KTx CGMCC № .1739 collects thalline;
2) broken thalline is collected albumen, obtains bile salt hydrolase.
In the described method, the carbon source and the nitrogenous source of the substratum of fermentation lactobacterium casei KTx CGMCC № .1739 are respectively glucose and soy peptone in the described step 1).Described substratum is preferably and contains glucose 2%～4% among the 1L, soy peptone 0.5%～3%, MnSO
40.025%, extractum carnis 1.00%, yeast extract paste 0.5%, tween 80 0.10%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.50%, ammonium citrate 0.20%, the substratum of sal epsom 0.058%; Described percentage composition is quality hundred content.
Described substratum is preferably and contains glucose addition 2%, soy peptone addition 3%, MnSO among the 1L
40.025%, the substratum of extractum carnis 1.00%, yeast extract paste 0.5%, tween 80 0.10%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.50%, ammonium citrate 0.20%, sal epsom 0.058%; Described percentage composition is the quality percentage composition.
In the described method, the temperature of fermentation lactobacterium casei KTx CGMCC № .1739 can be 30～40 ℃ in the described step 1), and fermentation time can be 10～12h, and the initial pH value of fermention medium can be 5.0～7.0.
The temperature of the lactobacterium casei of fermentation described in described step 1) KTx CGMCC № .1739 is preferably 37 ℃, and the initial pH value that fermentation time is preferably 12h, fermention medium is preferably 6.0.
In the described method, also comprising step 2) bile salt hydrolase that obtains adopts anion chromatography method purifying, obtains the bile salt hydrolase of purifying; Described separation condition is: with DEAE-Sepharose Fast Flow is gel filler, and 0.05mol/L NaCl collects 280nm absorption peak place sample as eluent.
Lactobacterium casei of the present invention (Lactobacillus casei) KTx, fermentation can produce highly active bile salt hydrolase, and it produces the bile salt hydrolase vigor can reach 8.85U/2 * 10
7Cfu, the ratio vigor behind the purifying reaches 1259.84U/mg.Lactobacterium casei of the present invention (Lactobacillus casei) KTx has reliable security to human body, makes active bacteria formulation, can save loaded down with trivial details technologies such as conventional fermentation, extraction.The bile salt hydrolase that lactobacterium casei of the present invention is produced can be used as additive and adds the purpose that reaches decreasing cholesterol in food or the medicine to, is new bio functional additive efficient, that have no side effect; This bile salt hydrolase can also be directly edible in the mode of zymin, but the content of same reducing cholesterol.By the method that lactobacterium casei of the present invention is produced bile salt hydrolase, low for equipment requirements, simple to operate, cost is lower, is suitable for suitability for industrialized production.
Description of drawings
The bile salt hydrolase vigor that Fig. 1 produces for lactobacterium casei KTx ferments is with the variation of pH value.
The bile salt hydrolase vigor that Fig. 2 produces for lactobacterium casei KTx ferments is with variation of temperature.
The bile salt hydrolase vigor that Fig. 3 produces for lactobacterium casei KTx ferments is with the variation of concentration of substrate.
Fig. 4 is the Km value of the bile salt hydrolase of lactobacterium casei KTx fermentation generation.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
1, the screening of bacterial strain
Kai Feier filtrate 5ml adds 45ml physiological saline (band granulated glass sphere) and makes bacteria suspension, then 10 times of gradient dilutions 10
6～10
8Doubly, getting 1ml is poured into flat board and (contains CaCO
3The MRS agar plate) cultivate 24h in 37 ℃, picking has the bacterium colony of dissolving circle to place 37 ℃ of MRS liquid lines to increase bacterium cultivation 12h, Grans dyeing, look its purity, drawing that enrichment liquid is inoculated in the cholesterol with the inoculum size of 2% (volume ratio) is the I substratum (substratum that contains salts solution I1L and cholesterol 1.0g of sole carbon source; Salts solution I (g/L): KH
2PO
40.25; MgSO
47H
2O 0.25; FeSO
45mg; NaCl 5mg; During preparation, cholesterol is dissolved in behind the salts solution I with supersonic cell disintegrating machine 500w concussion emulsification 20min; Adjust pH to 7.0,121 ℃ of sterilization 20min) cultivate 20h for 37 ℃ in, o-phthalaldehyde method is surveyed the cholesterol residual content, according to the reduction of cholesterol level in the I substratum, and the bacterial strain that primary dcreening operation obtains with decreasing cholesterol function.
With obtain behind the primary dcreening operation can decreasing cholesterol bacterial strain, be inoculated in the Oxford agar diffusion method and contain 0.3% ox sulphur cholate, 0.1% cholesterol, 0.2% CaCl
2The MRS nutrient agar in (contain extractum carnis 10g among the 1L, yeast extract 5g, peptone 10g, glucose 20g, dibasic ammonium citrate 2g, sodium acetate 5g, tween 80 1mL, K
2HPO
42g, MnSO
40.25g, MgSO
47H
2O 0.58g, agar 15g) MRS of adding 0.1ml cultivates bacterium liquid in the cup of Oxford, and whether observe the Oxford cup in 37 ℃ of anaerobic jars behind the cultivation 72h has opaque precipitation circle generation on every side.Not contain ox sulphur cholate, 0.1% cholesterol, 0.2% CaCl
2The MRS nutrient agar in contrast.White depositions around the cup of Oxford is scraped gently, be dissolved in the test tube of a small amount of distilled water, drip triketohydrindene hydrate colour developing liquid, observe whether the bluish voilet reaction is arranged, determine tentatively with this whether bacterial strain produces bile salt hydrolase.Obtain a strain by experiment and produce the bacterial strain of bile salt hydrolase, called after KTx.The streak inoculation of ninhydrin reaction male bacterial strain in the MRS slant tube, is cultivated the back and preserved standby.
2, the evaluation of bacterial strain KTx
The bacterial strain KTx of the product bile salt hydrolase that step 1 is obtained is inoculated in 37 ℃ of cultivations in the MRS liquid nutrient medium, carries out following evaluation.
The glycitols fermentation test, catalase (H
2O
2Enzyme) test, nitrate reduction test, methyl red test, V-P test, salt tolerant test (7.5% NaCl), differing temps test (10 ℃, 30 ℃, 40 ℃).
Qualification result is as shown in table 1.The result shows that bacterial strain KTx is a lactobacillus.Further adopt Biolog full automatic microorganism assessing instrument Analysis and Identification, show that bacterial strain KTx is Lactobacillus casei, i.e. lactobacterium casei.This bacterial strain has been preserved in Chinese microorganism strain preservation board of trustee reason person to be understood the common micro-organisms center (be called for short CGMCC, the address is: the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City), preserving number is CGMCC № .1739 on 06 23rd, 2006.On the MRS substratum, the bacterium colony of lactobacterium casei (Lactobacillus casei) KTx is oyster white or little band yellow, slightly projection.Individual morphology is irregular G
+Sporeless bacterium presents shaft-like.
Identification of indicator and qualification result that table 1. bacterial strain KTx belongs to
| Certified variety | Test-results | Certified variety | Test-results |
| Gramstaining | + | Glucose | + |
| Nitrate reduction | - | Rhamnosyl | - |
| The D-semi-lactosi | + | Turanose | + |
| D-N.F,USP MANNITOL | + | Fructose | + |
| Raffinose | - | Melibiose | + |
| Erythrose | + | Wood sugar | - |
| Sorbyl alcohol | - | 7.5％NaCl | - |
| Sucrose | + | 30℃ | + |
| Melizitose | + | 40℃ | + |
| Vitamin C2 | + | Methyl red | - |
| Catalase | - | V-P | - |
Annotate: "+" expression reacting positive, "-" expression reaction negative.
The detection of the bile salt hydrolase that the Optimizing Conditions of Fermentation of embodiment 2, lactobacterium casei (Lactobacillus casei) KTx and fermentation obtain
One, the Optimizing Conditions of Fermentation of lactobacterium casei (Lactobacillus casei) KTx
1, the selection of minimum medium: the inoculum size that lactobacterium casei KTx CGMCC № .1739 is adopted 2% (V/V), be seeded in the MRS liquid nutrient medium respectively, MRS liquid culture medium (it is that 3～4 ‰ corn steep liquors and mass content are 0.3～0.4 ‰ cysteine hydrochloride that the MRS substratum adds mass content), MRS broth culture (MRS-broth liquid nutrient medium) (Tryptones 5g, extractum carnis 10g, yeast extract 5g, glucose 10g, Triammonium citrate 1g, sodium acetate 2.5g, tween 80 0.5mL, Na
2HPO
41g, MnSO
40.025g, MgSO
47H
2O0.05g, distilled water 500mL), after cultivating 12h under 37 ℃, the amount of surveying its increment and producing bile salt hydrolase is produced the amount of bile salt hydrolase and is measured according to the extraction and the detection method of following bile salt hydrolase.
The extraction of bile salt hydrolase and detection method: with the fermented liquid of lactobacterium casei KTx CGMCC № .1739 at 4 ℃ of following frozen centrifugation collecting precipitations; Wash bacterial sediment with stroke-physiological saline solution, 10000rpm (10750g) is centrifugal, collects bacterial sediment; Join 0.1mol/L pH6.5 phosphoric acid buffer, bacterial concentration is transferred to A
600nmBe 1; Thallus suspension liquid carries out ultrasonic disruption in ice bath, 500w, and 3min/5ml collects crude enzyme liquid.With the 0.1ml crude enzyme liquid add the sodium phosphate buffer (pH6.0) of 1.8ml and 0.1ml in conjunction with cholate (6mmol/L Taurocholic acid sodium salt); vibration 1min mixing; mixture is cultivated 30min at 37 ℃ and is added trichoroacetic acid(TCA) stopped reaction (trichoroacetic acid(TCA) of 0.5ml reaction solution/0.5ml); 4 ℃ then, the centrifugal 10min of 10000rpm.Supernatant liquor 1ml is answered in negate, adds 1ml pH5.4, and 2mol/L acetate buffer solution and 1ml triketohydrindene hydrate colour developing liquid heat 15min behind the mixing in 100 ℃ of boiling water baths, and cooling after room temperature is placed 5min, adds 3ml 60% alcohol dilution, shakes up the back and measures A
570nm, the color of generation is stable in 60min.The amino acid that per minute generates 1mmol is defined as an enzyme activity unit.
The result shows, the lactobacterium casei KTx that cultivates in the MRS broth culture, and its increment maximum, the amount maximum of producing bile salt hydrolase shows the minimum medium of MRS broth culture (MRS-broth liquid nutrient medium) for optimum production bile salt hydrolase.
2, the pH value is preferred: with the MRS broth culture is minimum medium, is 1.0,2.0,3.0,4.0,5.0,6.0 or 7.0 to be initial pH value with the pH value respectively, according to inoculum size 2% (1.0 * 10
5The cfu/ml substratum) amount inoculation lactobacterium casei KTx, after cultivating 12 hours under 37 ℃, extract bile salt hydrolase according to the described method of step 1, and the vigor of detection bile salt hydrolase, the result shows that when initial pH is 6.0 the vigor of the bile salt hydrolase that cultivation lactobacterium casei KTx obtains is the highest.
3, temperature condition is preferred: with the MRS broth culture is minimum medium, and initial pH value is 6.0, and inoculum size is 2.0% (1.0 * 10
5The cfu/ml substratum) is 42 ℃, 37 ℃, 35 ℃, 30 ℃ or 25 ℃ with leavening temperature respectively and cultivates lactobacterium casei KTx, cultivate after 12 hours, extract bile salt hydrolase according to the described method of step 1, and the vigor of detection bile salt hydrolase, the result shows that when leavening temperature is 37 ℃ the vigor of the bile salt hydrolase that cultivation lactobacterium casei KTx obtains is the highest.
4, inoculum size is preferred: with the MRS broth culture is minimum medium, and initial pH value is 6.0, and inoculum size is respectively 0.5% (2.5 * 10
4The cfu/ml fermented liquid), 1.0% (5.0 * 10
4The cfu/ml fermented liquid), 2.0% (1.0 * 10
5The cfu/ml fermented liquid), 3.0% (1.5 * 10
5The cfu/ml fermented liquid) or 4.0% (2.0 * 10
5The cfu/ml fermented liquid), cultivate lactobacterium casei KTx at 37 ℃, after 12 hours, extract bile salt hydrolase according to the described method of step 1, and detect the vigor of bile salt hydrolase, the result shows that inoculum size is at 2% o'clock, the vigor of bile salt hydrolase is the highest, illustrates that 2% is the suitableeest inoculum size.
By the test of above-mentioned steps 1,2 and 3, determined the initial pH value 6.0 and the inoculum size 2.0% of 37 ℃ of best culture temperature, substratum.
5, medium component determines
Contrast different types of carbon source (glucose, sucrose, maltose, lactose, semi-lactosi, melibiose, fructose, cellobiose, raffinose and sorbyl alcohol), nitrogenous source (soy peptone, common peptone, polyprotein peptone, Tryptones, casein peptone), tensio-active agent (tween 80, polysorbas20 and polyoxyethylene glycol) respectively to the influence of bile salt hydrolase vigor, determine the optimal medium composition.Experimental result shows that best carbon source, nitrogenous source, tensio-active agent are respectively glucose, soy peptone, tween 80.
Result according to above-mentioned medium component selection, with glucose addition, soy peptone addition, culture temperature and inoculum size is factor, design four orthogonal experiments of quaternary (table 2), the conclusion setting that other factor (culture condition) obtains according to above-mentioned experiment.Fermented liquid according to the factor experiment of table 2 is fermented and obtained detects according to the described method extraction of step 1 bile salt hydrolase crude enzyme liquid according to the described method of step 1, detects the vigor of bile salt hydrolase.
Four orthogonal design level of factor of table 2. quaternary table
The result shows that it is to contain glucose addition 2%, soy peptone addition 3%, MnSO among the 1L that lactobacterium casei (Lactobacillus casei) KTx is inoculated in substratum
40.025%, extractum carnis 1.00%, yeast extract paste 0.5%, tween 80 0.10%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.50%, ammonium citrate 0.20%, sal epsom 0.058%, 37 ℃ of culture temperature, inoculum size 2%, initial pH value is 6.0, cultivates the fermented liquid (10 under 12 hours the condition
9The cfu/ml fermented liquid) enzyme is alive the highest, and the enzyme activity of bile salt hydrolase is 8.85U/2 * 10
7Cfu is that (the MRS substratum was cultivated inoculum size 2% (1.0 * 10 12 hours for 37 ℃ before optimizing
5The cfu/ml substratum), cultivate and finish, the bile salt hydrolase vigor reaches 0.66U/2 * 10
7Cfu) 13.4 times.
The lactobacterium casei that will obtain under above-mentioned optimal culture conditions (Lactobacillus casei) KTx fermented liquid extracts according to the method for step 1 and to obtain crude enzyme liquid, will add solid ammonium sulfate in the crude enzyme liquid that obtain, make its saturation ratio reach 10% respectively, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, whenever after adding ammonium sulfate, crude enzyme liquid is placed 24h in refrigerator, then in 10000rpm, centrifugal 20min, with the centrifugal collection respectively of each precipitation, with the 50mmol/L of about 2 times of volumes, after the dissolving of the sodium phosphate buffer of pH6.5, pack in the dialysis tubing, in identical damping fluid, dialyse to dialyzate through BaCl
2Till the nothing precipitation (it is complete to dialysis to change a damping fluid every 12h), measure enzyme activity and protein concentration respectively, calculating is than vigor (U/mg), and the result shows, 70% ammonium sulfate precipitation separation best results, protein content in the supernatant liquor is minimum, at utmost protein precipitation of 70% saturated ammonium sulphate is described, merges the part higher, and draw moisture in the dialysis band with Macrogol 2000 0 than vigor, reach 1～2 times of sample concentration, do being further purified; Adopt the anion chromatography post to carry out purifying then, the separation condition of described gel chromatography is: with DEAE-Sepharose Fast Flow is gel filler, application of sample 0.5mL, 0.05mol/L NaCl is as eluent, flow velocity wash-out with 1mL/min, collect 280nm absorption peak place sample, obtain the pure product of the bile salt hydrolase of lactobacterium casei (Lactobacillus casei) KTx, adopt SDS-PAGE electrophoresis detection purity.The electrophoretogram of bile salt hydrolase is a band after testing, and molecular weight is 30kDa.Can reach 1259.84 (U/mg) than vigor, purity is 17 times of former crude enzyme liquid.
Two, bile salt hydrolase characteristic research
(1) the optimum response pH of enzyme
Survey the enzyme of the crude enzyme liquid that the step 1 purifying obtains according to the method for the step 1 in the step 1 and live, wherein, after the crude enzyme liquid dilution, get in the damping fluid that 1ml is added in different pH (pH4.0～8.0) 37 ℃ of insulation 30min respectively.The result as shown in Figure 1, the optimal pH that shows BSH is 6.0, reaction conditions is a slant acidity.The optimal reaction pH value of enzyme has been described and has produced the optimum pH of enzyme consistent.
(2) mensuration of the optimal reactive temperature of enzyme
Live according to the enzyme of crude enzyme liquid under the differential responses temperature that method determination step one purifying of the step 1 in the step 1 obtains.Wherein, with after the crude enzyme liquid dilution, get 1ml respectively and join and place 10 ℃ in advance, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, the phosphoric acid buffer of preheating 10min in 70 ℃ the water-bath, in conjunction with in the cholate mixed solution, insulation 30min is even enzyme reaction temperature is respectively 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, measure enzyme and live.The result as shown in Figure 2, the optimal reactive temperature that shows enzyme is 30～40 ℃.
(3) mensuration of suitable concentration of substrate
Survey the enzyme of the crude enzyme liquid of step 1 purifying acquisition lives according to the method for the step 1 in the step 1.The substrate Taurocholic acid sodium salt is mixed with concentration is respectively 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, the solution of 8mmol/L, every then pipe add the crude enzyme liquid of the step 1 purifying acquisition of 1ml after dilution, 37 ℃ of insulation 30min, reaction finishes back survey enzyme and lives.The result as shown in Figure 3, the result shows that the suitableeest concentration of substrate of the bile salt hydrolase that lactobacterium casei KTx fermentation produces is 6mmol/L.
(4) chemical inhibitor is to the influence of enzymic activity
Survey the enzyme of the bile salt hydrolase crude enzyme liquid of step 1 purifying acquisition lives according to the method for the step 1 in the step 1.Add 0.5%E D T A, 1%SDS, 4mol/L urea in substrate respectively, every pipe adds the crude enzyme liquid 1ml through dilution, is contrast not add the chemical reagents reaction system, in 37 ℃ of water bath heat preservation 30min, measures enzyme and lives.The result is as shown in table 3, and the influence of EDTA and the SDS bile salt hydrolase that fermentation produces to lactobacterium casei KTx is little, and the adding of urea has suppressed the enzyme of the bile salt hydrolase of lactobacterium casei KTx fermentation generation fully and lived.
The influence that the different inhibitor of table 3. are lived to enzyme
| Sample | Concentration/% | Enzyme (U) alive | Suppress percentage ratio (%) |
| |
0 | 0.15 | 0 |
| |
1 | 0.13 | 11.07 |
| EDTA | 0.5 | 0.14 | 6.34 |
| Urea | 4(mol/L) | 0.0 | 45.21 |
(5) metal ion is to enzyme influence alive
Survey the enzyme of the bile salt hydrolase crude enzyme liquid of step 1 purifying acquisition lives according to the method for the step 1 in the step 1.With Cu
2+, Mg
2+, Ca
2+, Fe
3+, AL
3+, Zn
2+, Mn
2+All add in the reaction system (crude enzyme liquid that contains the 1ml dilution) with the concentration of 30mmol/L, other reaction conditionss are constant, are contrast with the reaction solution that does not add metal ion, survey enzyme and live.The result is as shown in table 4.
The influence that the different activator of table 4. are lived to enzyme
| Sample | Concentration (mmol/L) | Enzyme (U) alive | Activate percentage ratio (%) |
| |
0 | 14.82 | 0 |
| Cu 2+ | 30 | 15.43 | 4.12 |
| Mg 2+ | 30 | 15.79 | 6.55 |
| Ca 2+ | 30 | 14.98 | 1.08 |
| Fe 3+ | 30 | 15.87 | 7.09 |
| AL 3+ | 30 | 17.55 | 18.42 |
| Zn 2+ | 30 | 14.82 | 0 |
| Mn 2+ | 30 | 15.87 | 7.09 |
(6) mensuration of kinetic constant
Survey the enzyme of the bile salt hydrolase crude enzyme liquid of step 1 purifying acquisition lives according to the method for the step 1 in the step 1.In the reaction system of different concentration of substrate (shown in the table 5), 37 ℃ of reaction 5min measure A570nm, to calculate the initial velocity of enzyme reaction, 1/V is to 1/[S] (1/V is the longitudinal axis) make the Lineweaver-Burk double reciprocal plot, is extrapolated to transverse axis to intersect, and the transverse axis intercept (X) is the 1/Km value.Can determine the quantitative relationship between enzyme reaction rate and the concentration of substrate with Michaelie-Menten equation (Michaelis-Menton equation), and satisfy hyp feature [Wang Jiazheng, Fan Ming chief editor.The protein technical manual.Science Press.200.4]。
The data that record are as shown in table 5, according to table 5 mapping, the result as shown in Figure 4, in conjunction with Michaelis-Menton equation, calculating the kinetic constant Km of bile salt hydrolase under this test conditions is 4.57mmol/L by Fig. 4.
The mensuration of table 5.Km value
| Concentration of substrate (mmol/L) | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Initial velocity of reaction (μ mol/min) | 0.0022 | 0.0022 | 0.0022 | 0.0022 | 0.0022 | 0.0022 | 0.0022 |
| 1/[s] | 0.5 | 0.333 | 0.25 | 0.2 | 0.167 | 0.143 | 0.125 |
| 1/v | 454.545 | 400 | 322.581 | 294.118 | 256.41 | 227.273 | 208.333 |
Claims (9)
1, lactobacterium casei (Lactobacillus casei) KTx CGMCC № .1739.
2, a kind of bile salt hydrolase is the product that the fermentation described lactobacterium casei of claim 1 (Lactobacilluscasei) KTx CGMCC № .1739 obtains.
3, a kind of method of producing bile salt hydrolase comprises the steps:
1) fermentation lactobacterium casei (Lactobacillus casei) KTx CGMCC № .1739 collects thalline;
2) broken thalline is collected albumen, obtains bile salt hydrolase.
4, method according to claim 3 is characterized in that: the carbon source and the nitrogenous source of the substratum of the lactobacterium casei that ferments in the described step 1) (Lactobacillus casei) KTx CGMCC № .1739 are respectively glucose and soy peptone.
5, method according to claim 4 is characterized in that: described substratum is to contain glucose 2%～4%, soy peptone 0.5%～3%, MnSO among the 1L
40.025%, extractum carnis 1.00%, yeast extract paste 0.5%, tween 80 0.10%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.50%, ammonium citrate 0.20%, the substratum of sal epsom 0.058%; Described percentage composition is the quality percentage composition.
6, method according to claim 5 is characterized in that: described substratum is to contain glucose 2%, soy peptone 3%, MnSO among the 1L
40.025%, the substratum of extractum carnis 1.00%, yeast extract paste 0.5%, tween 80 0.10%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.50%, ammonium citrate 0.20%, sal epsom 0.058%; Described percentage composition is the quality percentage composition.
7, method according to claim 6, it is characterized in that: the leavening temperature of lactobacterium casei (Lactobacilluscasei) the KTx CGMCC № .1739 that ferments in the described step 1) is 30～37 ℃, fermentation time is 10～12h, and the initial pH value of fermention medium is 5.0～7.0.
8, method according to claim 7 is characterized in that: described leavening temperature is 37 ℃, and fermentation time is that the initial pH value of 12h, fermention medium is 6.0.
9, method according to claim 8 is characterized in that: in the described method, also comprise step 2) bile salt hydrolase that obtains adopts anion chromatography method purifying, obtains the bile salt hydrolase of purifying; Described purification condition is: with DEAE-Sepharose Fast Flow is gel filler, and 0.05mol/L NaCl with the flow velocity wash-out of 1mL/min, collects 280nm absorption peak place sample as eluent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101331900B (en) * | 2007-06-28 | 2010-12-01 | 北京农学院 | Four kinds of lactobacillus for generating bilesalt hydrolase and extracellular polysaccharide and functionality yoghourt production technique thereof |
| CN101333518B (en) * | 2007-06-28 | 2010-12-22 | 北京农学院 | Process for extracting two kinds of yeast bilesalt hydrolase and production technology of functional posset |
| CN101642274B (en) * | 2009-08-21 | 2012-08-01 | 北京农学院 | Method for preparing cholesterol reducing egg milk fermenting drink by utilizing lactobacillus and microzyme which produce bile salt hydrolase |
| CN101948857B (en) * | 2010-08-16 | 2013-02-13 | 中国农业大学 | Recombinant lactobacillus casei engineering strain and preparation method thereof |
| CN101942411A (en) * | 2010-09-08 | 2011-01-12 | 大连工业大学 | Method for improving lactobacillus cholate hydrolase activity and culture medium |
| CN102220276B (en) * | 2011-05-10 | 2013-07-10 | 江南大学 | Genetic engineering bacteria generating bile salt hydrolase as well as construction method and application thereof |
| CN106591272A (en) * | 2016-12-14 | 2017-04-26 | 曹书华 | Bile salt hydrolase mutant with improved enzyme activity |
| CN109971844A (en) * | 2019-05-15 | 2019-07-05 | 四川剑南春（集团）有限责任公司 | A kind of method of Rapid identification wine brewing anaerobe |
| CN114015740B (en) * | 2021-11-18 | 2023-07-21 | 常德云港生物科技股份有限公司 | Production method of pig bile salt |
-
2006
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Non-Patent Citations (2)
| Title |
|---|
| 乳酸菌降胆固醇作用研究现状. 韩俊华等.中国乳品工业，第30卷第3期. 2002 |
| 乳酸菌降胆固醇作用研究现状. 韩俊华等.中国乳品工业，第30卷第3期. 2002 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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