CN107502563A - One plant of kluyveromyces marxianus bacterium and its method for producing β glucuroides - Google Patents
One plant of kluyveromyces marxianus bacterium and its method for producing β glucuroides Download PDFInfo
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Abstract
The invention discloses one plant of kluyveromyces marxianus bacterium and its method for producing β glucuroides, kefir grains fermented ginseng slurry, is coated on improvement esculin medium and cultivates, and the bacterial strain C21 that black hydrolysis spot is produced in screening is productionβGlucuroide bacterium strain, it is kluyveromyces marxianus bacterium further to identify bacterial strain, and deposit number is CGMCC No.13907；Set out with the bacterial strain, with new fresh bean dreg culture medium fermenting and producingβGlucuroide, productionβGlucoside enzyme heat stability is good, and enzyme activity more than 80% in the range of pH4.0 6.5, acidproof alkaline stability is good, purification 12.8, and yield is that 1L zymotic fluids obtain the pure enzyme 80mg of single product；Strain high temperature resistant provided by the invention, growth are fast, using more cheap substrate or inducer production enzyme；ProductionβGlucuroide can be used for cellulose degradation, improve flavour of food products, production isoflavones etc..
Description
Technical field
The invention belongs to biotechnology and field of fermentation engineering, and in particular to one plant of kluyveromyces marxianus bacterium and its life
ProductionβThe method of-glucuroide.
Background technology
β- glucuroide, also referred to asβ- glucoside hydrolase（EC 3.2.1.21）, be cellulase system important set
Into part.ReportβThe isoelectric point of-glucuroide typically between 3.5 ~ 5.5, but optimum pH can more than 7.0, and
And degrees are strong, optimum temperature is between 40 ~ 110 DEG C.β- glucuroide most early in 1987 by Liebig and
Wohler has found that then research shows that it is widely present in many animals, plant and microorganism, and joins in semen armeniacae amarae first
With the glycometabolism to organism, the normal physiological function for maintaining organism is played a key effect.
β- glucuroide has a wide range of applications in food industry, health products, bioenergy, agricultural, medicine and other fields.
Particularly food industry and field of health care products,βThe fragrance precursor material that-glucuroide can be acted in fruit, beneficial to fruit
The de- bitter and fruit juice flavouring of juice, and can be used as food additives；The glucosides terpenes precursor substance in grape wine can be dramatically increased
Hydrolysis and Monoterpenes release, played an important role in Wine Aroma forming process；For soybean isoflavone glucoside
Hydrolysis for producing high-purity isoflavone genin, can effectively increase yield and reduce production cost, to meet the country
Demand of the outer market to isoflavones；Polydatin can be hydrolyzed to resveratrol, operating method is simple, reaction condition temperature
It is high with, purpose product yield, there is more considerable industrial applications prospect.β- glucuroide wide material sources, in plant, lactation
All be distributed in animal and microorganism, but its because source heterogeneity it is widely different.ProducingβThe microorganism of-glucuroide comes
In terms of source, most of researchs are concentrated mainly on bacterium, saccharomycete and mould etc., and at present, the country is in aspergillus nigerβ- grape
Glycosidase research is more, but growth that the inulinase-producing activity of Aspergillus niger strain can be over time and reduce, be unfavorable for storing, and
There is the hidden danger in terms of food safety and sanitation in bacterial strain, so the application in food is restricted.Selection one plant it is safe efficient,
The bacterial strain easily preserved is paid close attention to by more and more scholars.
Kluyveromyces marxianus bacterium（K.Marxianus）, it is a strain important in Kluyveromyces, Yin Qinai
High temperature, growth rate are fast, substrate composes many advantages such as wide and is increasingly being applied to industrial biotechnology field.The bacterial strain is not
The advantage saccharomycete being to be considered only as in Kefir grains Yoghourt and the Sudan acidified milk Rob, and it is a variety of to people containing having in the bacterial strain
The healthy and beneficial active material of body.Recent study shows that kluyveromyces marxianus can both secrete inulinase, beta galactose
A variety of widely used hydrolases such as glycosides enzyme；A variety of non-grains such as inulin class raw material, whey, molasses and xylose can be utilized again
Substrate produces ethanol.Bulletin through the issue of national health State Family Planning Commission on 3 kinds of new raw-food materials such as approval ampelopsis grossdentata leaves
（2013 No. 10）, approval kluyveromyces marxianus are new raw-food material.Application in terms of producing enzyme is it in modern work
One of most important part during industry biotechnology applications.K.MarxianusHave in terms of enzyme is produced excellent
Gesture, the high temperature resistant of removing itself, the features such as growth is fast, can also utilize relatively inexpensive substrate or inducer, as whey with
The fermenting and producing enzyme such as corn steep liquor；Meanwhile some thermostabilization enzymes such as lipase, glucuroide etc. can be fromK. MarxianusIncubation in obtain.
The content of the invention
The invention aims to improveβ- glucosidase production, and provide one plant of kluyveromyces marxianus bacterium and its
The method for producing beta-glucosidase.
One plant of kluyveromyces marxianus bacterium, its deposit number are CGMCC No.13907.
βThe production method of-glucuroide, it includes：
1）Kluyveromyces marxianus bacterium was activated into for 3 generations in basal medium, the strain that picking has activated is inoculated into basis
In culture medium, cultivated in shaking table, temperature is 25-35 DEG C, time 10-15h；Centrifugation, washing, then be resuspended, make bacteria suspension concentration
It is adjusted to 107Cfu/mL, it isβ- glucuroide leavening；
2）Step 1）Inβ5-15% is inoculated on fermentation medium-glucuroide leavening by mass percentage；In shaking table
Fermentation, temperature are 35-37 DEG C, rotating speed 100-130r/min；The zymotic fluid after fermentation is taken, refrigerated centrifuge 15-25min, is collected
Supernatant, obtainβ- glucuroide crude enzyme liquid；
3）Take step 2）Inβ- glucuroide crude enzyme liquid 25-35mL, ammonium sulfate is added, stood in 4 DEG C of refrigerators, 8500-
9500r/min centrifuges 15-30min, and precipitation is redissolved with buffer solution, dialyses；Enzyme liquid is splined on Sephadex G-75 pillars,
Elution, collection containβThe eluting peak mixing of-glucuroide；DEAE Cellulose52 weak anion exchange columns are splined on again,
Elution, collection containβThe component of-glucuroide, desalination again is carried out to purifying enzyme, vacuum freeze drying, obtainedβ- glucose
Glycosides enzyme enzyme powder；
Step 2）Described in fermentation medium be that bean dregs moisture is 80-99%；121 DEG C of sterilizing 20min.；
Step 3）Described in 35 DEG C of fermentation temperature, the r/min of rotating speed 120, centrifuge 20min；
Step 4）Described in centrifugal rotational speed be 9000 r/min, time 20min；
Step 1）Described in the component of basal medium be：Beef extract powder 8g/L, peptone 10g/L, yeast extract 4g/L, Portugal
Grape sugar 20g/L, dipotassium hydrogen phosphate 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.2g/L, manganese sulfate
0.04g/L, Tween 80 1g/L, deionized water 1000mL, pH5.7 ± 0.2.
The invention provides one plant of kluyveromyces marxianus bacterium and its method for producing beta-glucosidase.Kefir grains
Fermented ginseng is starched, and is coated on improvement esculin medium and is cultivated, and the bacterial strain C21 that black hydrolysis spot is produced in screening is productionβ- grape
Glycosidase bacterium strain, it is kluyveromyces marxianus bacterium further to identify bacterial strain, and deposit number is CGMCC No.13907；With this
Bacterial strain sets out, with new fresh bean dreg culture medium fermenting and producingβ- glucuroide, productionβ- glucoside enzyme heat stability is good,
Enzyme activity more than 80% in the range of pH4.0-6.5, acidproof alkaline stability are good；The final purification of beta-glucosidase is
12.8；Yield is that 1L zymotic fluids obtain the pure enzyme 80mg of single product.Strain high temperature resistant provided by the invention, growth are fast, using more honest and cleaner
Substrate or inducer the production enzyme of valency；Productionβ- glucuroide through clasmatosis without just can obtain, available for fiber
Element degraded, improve flavour of food products, production isoflavones and insect-pest etc..
Brief description of the drawings
Fig. 1 is producedβThe culture shape and morphological feature of-glucuroide bacteria strain；A. c21 bacterial strains are in improvement aesculin training
Support the form in base；B. form of the c21 bacterial strains in seed culture medium；C. c21 bacterial strains are in microscope（×100）Under shape
State；
Fig. 2 strains to be tested（C21）The PCR in 26S rDNA D1/D2 regions；
Phylogenetic trees of the Fig. 3 according to 26SrDNA sequences；
Fig. 4 various concentrations ammonium sulfate pairβThe influence of-glucuroide enzyme activity；
Fig. 5 fermentation temperatures, fermentation time, inoculum concentration pairβ- glucuroide effect of vigor；
Under Fig. 6 different temperaturesβThe enzyme activity value of-glucuroide；
Fig. 7βThe stability result of-glucuroide；
Under Fig. 8 condition of different pHβThe enzyme activity value of-glucuroide；
Fig. 9β- glucuroide at various ph values after 12h, 24h enzyme activity change；
Influence of Figure 10 metal ions to enzyme activity；
Figure 11 culture medium the selection results.
Embodiment
Embodiment 1 is producedβThe screening of the bacterial strain of-glucuroide
Kefir grains are fermented at 28 DEG C after ginseng pulp 48h, and it is pressed into 10-3、10-4、10-5、10-6Four gradient dilutions, respectively take 50
μ L bacterium solutions are spread evenly across improvement esculin medium, 25 DEG C of 48 h of culture, and the hydrolysis spot that black occurs in periphery of bacterial colonies is then productionβ- glucuroide bacterial strain.Kefir grain fermented ginseng bacterium solutions be coated on improvement esculin medium on cultivate 36 h after, filter out
Produce the bacterial strain C21 of black hydrolysis spot（Figure 1A）, judge it for productionβ- glucuroide bacterium strain.The bacterial strain is accessed into solid seed
Observation finds that bacterium colony is creamy white, is smooth, be opaque after culture medium, and surface is smooth, and bacterium colony is easily provoked（Figure 1B）.Microscope（×
100）As shown in Figure 1 C, Preliminary Identification result is one plant of production saccharomycete to lower observation form result.
Described improvement esculin medium be 1 g aesculins, 0.5 g ironic citrates, 2 g yeast extracts, 0.5g peptones,
20 g agar, through 121 DEG C of min of high pressure steam sterilization 20；Seed culture based formulas is 10 g dusty yeasts, 20 g peptones, 20
G glucose, 20 g agar, 1000 mL distilled water, it is adjusted to pH 6.0, through 121 DEG C of min of high pressure steam sterilization 20.
Described ginseng pulp preparation method is to select the complete fresh ginseng of no disease and pests harm, cleans up, steams 20 min, press
According to ginseng water quality ratio（g/g）1：2nd, more crawls of 5000 rpm/min amount to 3 min mashing, 110 DEG C of sterilizing 20min.
Embodiment 2 is producedβThe Molecular Identification of the bacterial strain of-glucuroide
Method for identifying molecules：The sequence analysis in 26S rDNA D1/D2 areas.Strain to be tested DNA extractions use SK8257 kits
Operation；The use of primer is NL1（5'-GCATATCAATAAGCGGAGGAAAAG-3'）And NL4（5'-GGTCCGTGTTT
CAAGACGG-3'）, by Shanghai Sheng Gong limited companies synthetic primer and sequencing, obtained strains DNA sequence dna information exists
The upper Blast of NCBI compare analysis.PCR reaction systems（25 μL）, including genomic DNA（20-50 ng/μL）0.5 μ L, 10 ×
Buffer（Contain Mg2+）2.5 μ L, dNTP（Each 2.5 mM）1 μ L, μ L of enzyme 0.2, F（10 μM）0.5 μ L, R（10 μM）0.5
μ L, add distilled water to 25 μ L；PCR response procedures are 94 DEG C of min of pre-degeneration 4,94 DEG C of 45 s of extension, 30 circulations；55 ℃
Extend 45 s, 30 circulations；72 DEG C of 1 min of extension, 30 circulations；72 DEG C are repaired 1 min of extension；4 DEG C of terminating reactions.PCR
Amplified production is detected with 1% agarose gel electrophoresis, and deposition condition is 150 V, 100 mA, 20 min, is seen using gel imager
Examine result.
Using NL1 and NL4 pair of primers amplification strain to be tested 26S rDNA nearly 5 ' ends D1/D2 regions, amplification is shown in figure
2.As shown in Figure 2, amplified production through electrophoresis detection occur 578 bp fluorescent bands, it is consistent with expected results, clip size with
The fragment of report（500~600 bp）It is consistent.The D1/D2 regions of strain to be tested are sequenced, homology sequence is carried out in GenBank
Row search（BLAST Search）, strain to be tested is as a result shown compared with kluyveromyces marxianus sequence, and homology is up to 99%
（Fig. 3）, therefore further checking strain to be tested is kluyveromyces marxianus（Kluyveromyces marxianus）.The bacterium
Kind it is a very important strain in Kluyveromyces, because of its high temperature resistant, growth rate is fast and that security is high etc. is many excellent
Gesture and be applied to such as enzyme, ethanol industrial biotechnology field.Contain micro- lifes such as various lactobacillus and saccharomycete in kefir grains
Thing, but during kefir grains fermentation full constituent ginseng, find productionβThe microorganism of-glucuroide is kluyveromyces marxianus.
Kluyveromyces marxianus strong adaptability, it can speculate that kluyveromyces marxianus become advantage in kefir grain fermented ginsengs
Strain, it is suppressed that the growth of lactic acid bacteria.By the bacterial strain（Kluyveromyces marxianus）It is stored in Chinese microorganism strain
Preservation administration committee common micro-organisms center（CGMCC）, deposit number is CGMCC No.13907, and preservation date is 2017
March 21, preservation address are Institute of Microorganism, Academia Sinica, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Embodiment 3βThe preparation of-glucuroide leavening
Kluyveromyces marxianus bacterium was activated into for 3 generations in basal medium, the strain that picking has activated is inoculated into basic training
Support in base, cultivated in the shaking table that rotating speed is 120r/min, temperature is 30 DEG C, time 12h.Bacterium solution is in high speed freezing centrifuge
5min is centrifuged, temperature is 4 DEG C, rotating speed 7500r/min.Thalline after centrifugation is washed twice with sterilized water, then is resuspended, hangs bacterium
Liquid concentration is adjusted to 107Cfu/mL, as leavening.
The component of basal medium is：Beef extract powder 8g/L, peptone 10g/L, yeast extract 4g/L, glucose 20g/L,
Dipotassium hydrogen phosphate 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, Tween 80
1g/L, deionized water 1000mL, pH5.7 ± 0.2.
Embodiment 4βThe preparation of-glucuroide
1. the preparation of fermentation medium
Soybean and water 1 at room temperature:5 immersion 13h, water is drained, and the soybean after immersion and 100 DEG C of water are with cooking machine
Fast 3min carries out defibrination, filtering, obtains new fresh bean dreg, adjustment bean dregs moisture is about 85%.By pretreated fresh bean dreg
Liquid culture medium is configured to water different proportion.
2. βThe preparation of-glucuroide crude enzyme liquid
Fermentation medium uses moist hear heat test, is put into semi-automatic high-pressure steam sterilizing pan, 121 DEG C of sterilizing 20min.Will cooling
The leavening described in fermentation medium inoculation embodiment 3 afterwards（10%(v/w) ）.Then in 35 DEG C, 120r/min shaking table
Fermented, shaking table shaken cultivation to required time.Take the 9000r/min refrigerated centrifuges at 4 DEG C of the zymotic fluid after fermentation
20min, supernatant is collected, is obtainedβ- glucuroide crude enzyme liquid, refrigeration are standby.
3. βThe measure of-glucuroide crude enzyme liquid activity
1）P-nitrophenol standard curve
Take the p-nitrophenyl phenol solution of 1mmol/L standards, be added into volumetric flask, be respectively configured 0.1,0.2,0.3,0.4,
0.5mmol/L p-nitrophenyl phenol solution, with distilled water constant volume and is mixed.The solution 0.4mL of various concentrations is taken respectively, is added
Na2CO3Solution 0.4mL is fully mixed, and using distilled water as control group, light absorption value (OD) is determined at 405nm.
2）β- glucuroide enzyme activity determination
The accurate enzyme liquid drawn 0.16ml and moderately diluted, preheats 5min in 60 DEG C of waters bath with thermostatic control, adds and have been warmed up 10min's
1.6mmol/L p-NPG solution, accurate clock reaction 30min, immediately addition 0.4mL 1mol/L Na2CO3Solution is whole
Only react, place to room temperature, light absorption value (OD) is determined at 405nm.Enzyme unit (U)：Under these conditions, interior catalysis per minute
The enzyme amount generated needed for 1umol p-nitrophenols is 1 enzyme activity unit.
Calculation formula is：U=（C/(T*V)）*N.
In formula：U is enzyme activity, U/mL；C is content of p-nitrophenol, umol；V is enzyme liquid reaction volume, mL；T is reaction
Time；N is original enzyme liquid extension rate.
4. β- glucuroide isolates and purifies
Ammonium sulfate precipitation and separation method carries out preliminary purification, takes crude enzyme liquid 30mL, adds ammonium sulfate, respectively reaches its saturation degree
30%th, 40%, 50%, 60%, 70%, 80% and 90%, 12h is stood in 4 DEG C of refrigerators, 9000 r/min centrifuge 20 min, and precipitation is used
Buffer solution redissolves, and determines respectively in supernatant and precipitationβThe content of-glucoside enzyme activity and protein, to determine ammonium sulfate
The saturation point of fractional precipitation.By the crude enzyme liquid after ammonium sulfate preliminary purification, desalination of dialysing.Then enzyme liquid is splined on Sephadex
G-75 pillars (45.0cm × Φ 3.5cm), eluent be pH5.0 acetic acid-sodium acetate buffer solution, gradient 0.5mL/
Min, collection containβThe eluting peak mixing of-glucuroide, then it is splined on DEAE Cellulose52 weak anion exchange columns
(28.0cm × Φ 3.0cm), eluent are 0.5mol/L NaCl solutions, gradient 0.5mL/min, and collection containsβ- Portugal
The component of polyglycoside enzyme carries out enzyme activity determination and SDS-PAGE, and desalination again and vacuum freeze drying are carried out to prepare to purifying enzymeβ-
Glucuroide enzyme powder is standby, places and is preserved in 4 DEG C, for zymologic property research.
As shown in figure 4,βIn 20~60 % of ammonium sulfate concentrations, its enzyme activity significantly rises-glucuroide（P
＜ 0.05）；And during ammonium sulfate concentrations 60~80%, enzyme activity is remarkably decreased（P ＜ 0.05）, select ammonium sulfate saturation degree for
Saltoutd when 60%.Enzyme activity has purified 10.5 times after dialysis.Crude enzyme liquid coagulates through ammonium sulfate precipitation, Sephadex G-100
Glue-line analysis, the ion-exchange chromatographies of DEAE Cellulose 52 have obtained pureβ- glucuroide, molecular mass are about
66kD.As it can be seen from table 1 it is in this step of ammonium sulfate precipitation that vigor, which loses larger, it may be possible to because in ammonium sulfate precipitation
During local salt loading spend height and cause zymoprotein to inactivate, or in distilled water desalination overlong time reason.β-grape
The final purification of glycosidase is 12.8.
Beta-glucosidase production of enzyme is that 1L zymotic fluids obtain the pure enzyme 80mg of single product.
Embodiment 5β-The fermentation technology optimization of glucuroide
Investigate fermentation temperature, fermentation time, inoculum concentration, solid-liquid ratio pairβThe influence of-glucosidase activity.Withβ- glucoside
Enzyme enzyme activity is performance assessment criteria, determines the suitable fermentation condition of each single factor test, for productionβThe condition optimizing of-glucuroide provides ginseng
Scope is examined, each factor setting 3 of experimental design is parallel.According to Box-Behnken center combination design principles, in single factor test
In experimental basis, using three fermentation temperature, fermentation time and inoculum concentration factors as independent variable,β- glucosidase activity is response
Value, make the experiment of Three factors-levels response surface analysis, totally 17 testing sites.Wherein 12 are factorial, test and use centered on 5
With evaluated error.Test factor and level are shown in Table 2.Experimental design and it the results are shown in Table 3.
From the most important several factors for influenceing ferment effect, fermentation temperature, time and inoculum concentration are set out, and have studied mark
This kluyveromyces strains liquid fermentation fresh bean dreg is producedβ- glucoside enzyme effect（See Fig. 2）.As a result show, with fermentation temperature
Rise, accelerate the production metabolism of kluyveromyces marxianus bacterium, promoteβThe biosynthesis of-glucuroide, so thatβ- grape
Glycosidase enzyme activity is in rising trend, but works as temperature more than 35 DEG C, gradually uses up nutriment because temperature is too high, bacterium
The metabolism of body slows down；Protein easily loses activity at high temperature, so thatβThe yield of-glucuroide is reduced,β- grape
Glycosidase enzyme activity just reduces.Therefore 35 DEG C of selection is used as optimal reactive temperature.Fermentation time pairβ- glucoside enzyme activity
Power influences notable.When between when fermenting more than 96h,β- glucuroide enzyme activity reduces, and this is probably due to nutrients
The reason for matter exhausted too early, thalline aging, and harmful substance is gradually accumulated.When inoculum concentration is 10%,β- glucuroide enzyme is with respect to enzyme
Vigor reaches highest.More than 10%, enzyme activity reduces inoculum concentration on the contrary, and analysis reason is probably due to being less than in inoculum concentration
In the case of 10%, inoculum concentration is too small, and product accumulation lacks, and inoculum concentration is too small, unfavorable producing enzyme；But inoculum concentration is excessive to draw
It is too fast to play thalli growth, zymotic fluid viscosity increases and causes dissolved oxygen insufficient, have impact on the contraryβ- glucuroide enzyme activity
Reduce.To sum up, selection fermentation optimum temperature be 35 DEG C, fermentation optimum time 96h, the level that inoculum concentration 10% is response surface optimization
Central point.
It can be seen that from the results of analysis of variance, contrast influencesβThree solid state fermentation conditionses factors of-glucoside enzyme activity
For inoculum concentration（C）＞ fermentation temperatures（A）＞ fermentation times（B）.By the Treatment Analysis of data, the maximum for trying to achieve response surface R is pre-
Measured value is 6.79U/g, and fermentation temperature now is 35 DEG C, fermentation time 98h, inoculum concentration 10%（V/w）.For the standard of testing model
True property, using fermentation test is carried out under optimal conditions of fermentation after optimization, test three timesβ- glucuroide enzyme activity average value is
(6.60±0.01)U/g.Predicted value approaches with actual value, and the cooperation technique for illustrating regression model optimization is effective.By ringing
The face method of answering optimizes what is obtainedβThe technique of-glucuroide can be used to refer to actual production.
Embodiment 6βThe zymologic property experiment of-glucuroide
1. the optimum temperature of enzymatic reaction
By the enzyme liquid suitably diluted after ammonium sulfate preliminary purification respectively 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C,
After 70 DEG C, 80 DEG C are incubated 10min, enzymatic reaction, measure are carried outβThe vigor of-glucuroide, highest enzyme activity is defined as
100%, calculate respectivelyβThe enzyme activity of-glucuroide at different temperatures（Residual enzyme i.e. under condition of different temperatures
Vigor accounts for the percentage of the wherein peak of enzyme activity）, it is determined thatβThe optimum temperature of-glucuroide enzymatic reaction.Such as Fig. 3 institutes
Show,βIn 20~60 DEG C of temperature, its enzyme activity significantly rises-glucuroide（P ＜ 0.05）；And 60~80 DEG C of temperature
When, enzyme activity is remarkably decreased（P ＜ 0.05）, it is determined thatβThe optimum temperature of-glucuroide enzymatic reaction is 60 DEG C.From folding
The steep of the trend of line chart can be seen thatβInfluence of-the glucuroide to temperature is very big, so the enzyme is to the quick of temperature
Perception is good.
2. βThe heat endurance of-glucuroide
By the enzyme liquid suitably diluted after ammonium sulfate preliminary purification respectively in 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C of temperature
After lower incubation 1h, 2h, 3h, 4h and 5h, enzymatic reaction, measure are carried outβThe vigor of-glucuroide.By the enzyme liquid without incubation
Enzyme activity be defined as 100%, calculate respectivelyβThe enzyme activity of-glucuroide at different temperatures（I.e. in different temperatures
Under the conditions of remaining enzyme activity account for hundred ratios of the wherein peak of enzyme activity）.Shown in Fig. 4βThe stability of-glucuroide, by
Knowable to figure,βWhen-glucuroide is below 55 DEG C, its enzyme activity is kept approximately constant in 5h；At 60 DEG C,β- Portugal
Polyglycoside half life of enzyme is 4h, and after being incubated 5h, its enzyme activity is more stable, still there is 41% enzyme activity；And when temperature surpasses
When crossing 65 DEG C, after being incubated 1h, enzyme activity disappears substantially.β- glucuroide has preferable heat-resistant stability.
3. the optimum pH of enzymatic reaction
By the enzyme liquid suitably diluted after ammonium sulfate preliminary purification respectively in different pH value（3.0、3.5、4.0、4.5、5.0、5.5、
6.0、6.5、7.0、7.5、8.0）Cushioning liquid in, carry out enzymatic reaction, measureβThe vigor of-glucuroide, by highest enzyme
Vigor is defined as 100%, calculates respectivelyβThe enzyme activity of-glucuroide at various ph values, it is determined thatβ- glucuroide
The optimum pH of enzymatic reaction.The different cushioning liquid applied are：Citrate buffer solution（PH value 3.0-3.5）；Sodium acetate
Buffer solution（PH value 4.0-7.0）；Phosphate buffer（PH value 7.5-8.0）.As shown in figure 5,β- glucuroide exists in pH value
During 3.0-5.0, its enzyme activity significantly rises（P ＜ 0.05）；And during pH value 5.0-8.0, enzyme activity is remarkably decreased（P
＜ 0.05）, it is determined thatβThe optimum pH of-glucuroide enzymatic reaction is 5.0.
4. βStability of-the glucuroide under different pH
By the enzyme liquid suitably diluted after ammonium sulfate preliminary purification and different pH value（3.0、3.5、4.0、4.5、5.0、5.5、
6.0、6.5、7.0、7.5、8.0）Cushioning liquid 1:After 9 is well mixed, after incubating 12h and 24h at 4 DEG C, enzyme is carried out
Promote reaction, measureβThe vigor of-glucuroide, the enzyme liquid vigor without incubation is defined as 100%, calculated respectivelyβ- grape
The enzyme activity of glycosidase at different temperatures.Shown in Fig. 6β- glucuroide relative enzyme after 12h, 24h at various ph values
The change of vigor, as seen from the figure, after being incubated 12h at 4 DEG C, when between pH 4.0-6.5,βThe relative enzyme of-glucuroide
Vigor is maintained at more than 80%；Between pH 3.0-3.5 under acid condition,βThe enzyme activity of-glucuroide is maintained at
More than 60%；Between pH7.0-7.5 under neutrallty condition,βThe enzyme activity of-glucuroide is maintained at more than 70%；
Under pH8.0 alkalescence condition,βThe enzyme activity of-glucuroide is maintained at more than 46%.After being incubated 24h at 4 DEG C, relatively
Enzyme activity largely loses compared with having for 12h, in optimal pH 5.0, enzyme activity loss 24%, and between pH3.5-7.0,β-
The enzyme activity of glucuroide is maintained at more than 60%, in summary,βThe acidproof alkaline stability of-glucuroide is preferable.
5. influence of the metal ion to enzyme activity
In the crude enzyme liquid that will suitably be diluted after ammonium sulfate preliminary purification, different metal ions is added：K+、Ca2+、Na+、Mg2+、
Mn2+、Ba2+、Fe3+、Fe2+、Zn2+、Ag+、Cu2+, make its final concentration of 10mmol/L, delayed with being not added with the sodium acetate of metal ion
Fliud flushing pH5.0 is blank control.Carry out enzymatic reaction, measureβThe vigor of-glucuroide, to be not added with the solution of metal ion
The enzyme activity of measure is 100%, determines influence of the metal ion to enzyme activity.As shown in fig. 7, different metal ions is to enzyme activity
What power influenceed.As a result show, Mg2+、K+、Na+、Ca2+, have certain activation, wherein Mg to enzyme2+、K+Enzyme activity is influenceed bright
It is aobvious, enzyme activity is added about 20%； Ba2+、Fe3+、Cu2+、Zn2+、Ag+There is certain inhibitory action, wherein Cu to enzyme2+、Ag+、Fe3+It is obvious to the inhibitory action of enzyme, the vigor of enzyme is largely lost, basic inactivation；Mn2+It is little to the influence of enzyme.
6. influence of the organic solvent to enzyme activity
In the crude enzyme liquid that will suitably be diluted after ammonium sulfate preliminary purification, different organic solvents is added：Respectively 10% methanol,
Ethanol, isopropanol, n-butanol and isoamyl alcohol, and（10-50%）Dimethyl sulfoxide (DMSO)（DMSO）, make its final concentration of 10mmol/
L, to be not added with the sodium acetate buffer pH5.0 of organic solvent as blank control.Carry out enzymatic reaction, measureβ- glucuroide
Vigor, using be not added with organic solvent solution determine enzyme activity as 100%, determine influence of the organic solvent to enzyme activity.Pass through
Measure studies alcohol pair different DMSO and 10% in the presence of various concentrationβ- glucoside enzyme hydrolysis pNPG influence.As a result
As shown in table 4, various concentrations（10%-50%）DMSO pairsβ- glucuroide enzyme activity is almost without influence.With prolonging for alcohol chain
Length makes the enzyme activity and increased.
The screening of the fermentation medium of embodiment 7
According toβAesculin in culture medium can be hydrolyzed to aesculetin by-glucuroide, its can and Fe3+Show after effect
The principle of brownish black, primary dcreening operation is carried out to culture medium using sxemiquantitative colorimetric method.By the bacterial strain activated access using aesculin as
Cultivate in the esculin medium of substrate, bean dregs esculin medium, yeast extract esculin medium, after cultivating 3d, select
Produce culture medium raw material of the big culture medium of black hydrolysis spot activity as bacterial strain.As shown in figure 8, A be esculin medium,
B is bean dregs esculin medium, C is yeast extract esculin medium, with the extension of time, black hydrolysis spot gradually becomes more,
Esculin medium does not occur before 48h, just occurs in 67h, the fast and area that bean dregs esculin medium occurs
Greatly, yeast extract medium is slow compared with B groups and area is small.So raw material of the selection bean dregs as culture medium.
Described esculin medium is 0.1% aesculin, 0.1% ironic citrate, 0.25% peptone, 2% agar, 0.1% phosphorus
Acid dihydride potassium, 0.05% magnesium sulfate, 1000mL water, under the conditions of pH=5.0,115 DEG C of sterilizing 30min.
Described bean dregs esculin medium is 1% bean dregs, 0.1% aesculin, 0.1% ironic citrate, 0.25% peptone, 2%
Agar, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 1000mL water, under the conditions of pH=5.0,115 DEG C of sterilizing 30min.
Described yeast extract esculin medium is 1% yeast extract, 0.1% aesculin, 0.1% ironic citrate, 0.25% albumen
Peptone, 2% agar, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 1000mL water, under the conditions of pH=5.0,115 DEG C of sterilizing 30min.
Claims (7)
1. one plant of kluyveromyces marxianus bacterium, its deposit number is CGMCC No.13907.
2.βThe production method of-glucuroide, it includes：
1）By kluyveromyces marxianus bacterium, activated for 3 generations in basal medium, the strain that picking has activated is inoculated into basis
In culture medium, cultivated in shaking table, temperature is 25-35 DEG C, time 10-15h；Centrifugation, washing, then be resuspended, make bacteria suspension concentration
It is adjusted to 107Cfu/mL, it isβ- glucuroide leavening；
2）Step 1）Inβ5-15% is inoculated on fermentation medium-glucuroide leavening by mass percentage；In shaking table
Fermentation, temperature are 35-37 DEG C, rotating speed 100-130r/min；The zymotic fluid after fermentation is taken, refrigerated centrifuge 15-25min, is collected
Supernatant, obtainβ- glucuroide crude enzyme liquid；
3）Take step 2）Inβ- glucuroide crude enzyme liquid 25-35mL, ammonium sulfate is added, stood in 4 DEG C of refrigerators, 8500-
9500r/min centrifuges 15-30min, and precipitation is redissolved with buffer solution, dialyses；Enzyme liquid is splined on Sephadex G-75 pillars,
Elution, collection containβThe eluting peak mixing of-glucuroide；DEAE Cellulose52 weak anion exchange columns are splined on again,
Elution, collection containβThe component of-glucuroide, desalination again is carried out to purifying enzyme, vacuum freeze drying, obtainedβ- glucose
Glycosides enzyme enzyme powder.
It is 3. according to claim 2βThe production method of-glucuroide, it is characterised in that：Described Marx's Crewe
Saccharomycete is tieed up, its deposit number is CGMCC No.13907.
4. according to Claims 2 or 3βThe production method of-glucuroide, it is characterised in that：Step 2）Described in
Fermentation medium is that bean dregs moisture is 80-99%；121 DEG C of sterilizing 20min.
It is 5. according to claim 4βThe production method of-glucuroide, it is characterised in that：Step 1）Described in base
The component of basal culture medium is：Beef extract powder 8g/L, peptone 10g/L, yeast extract 4g/L, glucose 20g/L, dipotassium hydrogen phosphate
2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, Tween 80 1g/L, go from
Sub- water 1000mL, pH5.7 ± 0.2.
It is 6. according to claim 5βThe production method of-glucuroide, it is characterised in that：Step 3）Described in hair
35 DEG C of ferment temperature, the r/min of rotating speed 120, centrifuge 20min.
It is 7. according to claim 6βThe production method of-glucuroide, it is characterised in that：Step 4）Described in centrifugation
Rotating speed is 9000 r/min, time 20min.
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| CN109295040A (en) * | 2018-11-29 | 2019-02-01 | 石河子开发区天易特色果蔬加工生产力促进中心（有限责任公司） | A kind of preparation method of Pichia pastoris beta-glucosidase enzyme preparation |
| CN110343686A (en) * | 2019-08-07 | 2019-10-18 | 张文霞 | A kind of production of the enzyme preparation that grape wine taste and quality can be improved and application method |
| CN112980913A (en) * | 2021-04-14 | 2021-06-18 | 北京欣康研医药科技有限公司 | A method for preparing baicalein from baicalin by fermentation |
| CN113583884A (en) * | 2021-08-21 | 2021-11-02 | 吉林农业大学 | Fermentation medium for producing gamma-aminobutyric acid and production method |
| CN117384799A (en) * | 2023-11-29 | 2024-01-12 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Lu Gesi bacillus A78.1, microbial inoculum, method for producing beta-glucosidase and application |
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