CN103555797A - Rapid optimization method for realizing naringinase catalytic naringin based on DNS analysis - Google Patents
Rapid optimization method for realizing naringinase catalytic naringin based on DNS analysis Download PDFInfo
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Abstract
The invention discloses a rapid optimization method for realizing naringinase catalytic naringin based on DNS analysis. The method comprises the steps of preparation of a culture medium, solid fermentation, preparation of a crude enzyme solution, enzymic catalytic reaction and analysis of reducing sugar yield by a DNS method. The DNS method is that the reducing sugar yield of the enzyme catalytic reaction is analyzed by using 2,4-dinitrosalicylic acid (DNS) and specifically comprises the steps of mixing 0.2 mL of a reaction liquid and 0.6 mL of a DNS solution; shaking thoroughly; bathing for 15 min at a temperature of 100 DEG C; cooling with tap water instantly by taking out the solution; diluting to 5 mL by using distilled water; measuring a light absorption value under a wavelength of 520 nm; and calculating the yield of the reducing sugar in the reaction solution according to a standard curve. The method can achieve an object of rapid optimization the naringinase catalytic naringin and further optimize catalytic conditions, and conversion rate of naringin is greatly increased under the conditions.
Description
Technical field
The invention belongs to the technical field of biocatalysis, relate in particular to a kind of fast Optimization that realizes naringinase catalysis naringin of analyzing based on DNS.
Background technology
Naringin (4,5,7-trihydroxy flavanone-7-β-L-rhamnoglucoside-(1,2)-α-D-glucopyranoside) be a kind of flavanone glucoside compound, it is rutaceae, as the main bitter material of natsudaidai, sweet shaddock, bitter orange etc., there is antiulcer agent, anti-inflammatory, a series of biological activity such as anti-oxidant and antibacterial, in fields such as food, medicine and makeup, there is good application prospect.Yet, naringin solvability is at normal temperatures poor, so that it has been subject to restriction largely in industrial application, water-soluble or fat-soluble for improving it, or improve its physiologically active, increase new function etc., and then improve its bioavailability, main chemical method and the enzyme process of adopting carries out study on the modification to naringin both at home and abroad at present, than chemic modified method, it is strong that enzyme modification naringin has selectivity, reaction conditions is gentle, and product is easily separated and to advantages such as environmental hazard are little, thereby has better Research Significance and value.
The prozyme that naringinase is comprised of alpha-L-Rhamnosidase and β-D-Glucose glycosides enzyme, the hydrolysis reaction of its catalysis naringin mainly contains two processes: first, naringin resolves into rhamnosyl and Pu Luning under the effect of alpha-L-Rhamnosidase, secondly, Pu Luning resolves into glucose and naringenin again under the effect of β-D-Glucose glycosides enzyme.
In the prior art, conventionally adopt high performance liquid chromatography to detect the reduction of naringin or the growing amount of naringenin, analyze the hydrolytic process of naringin.In China Patent No., be 200510014880.9, measuring method > > and China Patent No. that name is called naringin content in < < gastrointestinal disease treating pill are 200910073856.0, name is called in the two pieces of patent documentations of method for measuring naringin content > > in < < preparation for reunion of bone, all by the method for high performance liquid chromatography, to measure the content of naringin, although, this method accuracy is high, but this method is comparatively loaded down with trivial details consuming time, extensive mensuration for naringin, for example, when fermentation condition optimization, need to consume a large amount of manpower and materials.
The process that generates naringenin in view of naringinase catalysis naringin is also the glycogenetic process of reduction simultaneously, and the generation of reducing sugar should have dependency with the hydrolysis of naringin and the generation of naringenin in this process.Therefore, the inventor studies and has designed a kind of fast Optimization that realizes naringinase catalysis naringin of analyzing based on DNS, and this case produces thus.
Summary of the invention
The object of this invention is to provide a kind of fast Optimization that realizes naringinase catalysis naringin of analyzing based on DNS, by DNS, analyze the rhamnosyl reducing sugar that produces in naringinase catalysis naringin reaction process and the content of Reduction of Glucose sugar, reach the object of rapid Optimum naringinase catalysis naringin, and further analyzed the impact of different technical parameters on naringin conversion process, and then for utilizing the modified product of naringinase modified pomelo peel glycosides acquisition particular organisms activity that Research foundation is provided.
To achieve these goals, the technical solution adopted in the present invention is as follows:
Based on DNS, analyze a fast Optimization that realizes naringinase catalysis naringin, present method comprises the following steps:
The preparation of step 1, substratum: the preparation of described substratum comprises the preparation of slant medium and the preparation of solid medium, wherein: the preparation of described slant medium, by the component preparation of following mass body volume concentrations: MgSO
47H
2o 1.0 g/L, KH
2pO
41.0 g/L, (NH4)
2sO
41.5 g/L, KCl 0.5 g/L, KNO
31.5 g/L, anhydrous CaCl
20.1 g/L, yeast extract 2.0 g/L, naringin 2.5 g/L, agar 20 g/L, initial pH 6.0,1 * 105 Pa sterilizing 20 min; The preparation of described solid medium, prepares by the component of following weight: shaddock ped powder 160g, soybean cake powder 24g, wheat bran 1%, KNO
31%, solid water is than being 1:1.5,1 * 10
5pa sterilizing 20 min;
The preparation of step 3, crude enzyme liquid: with the solid-to-liquid ratio of 1:20, with phosphate buffered saline buffer, solid state fermentation thing is soaked to 1 h, stirs evenly rear suction filtration and collect filtrate, by described filtrate in 4 ° of C with centrifugal 10 min of 12000 * g, collect supernatant liquor and obtain crude enzyme liquid, under 0-4 ° of C, preservation is standby;
Step 4, enzymic catalytic reaction: described enzymic catalytic reaction is the free enzymic catalytic reaction of naringin and/or the enzymic catalytic reaction of immobilization naringin, wherein, the enzymic catalytic reaction of described free naringin is: in the reaction system of 50 mL, naringin concentration is 0.04-0.24g/100 mL, enzyme dosage is 4-12 U/mL, take concentration as 0.02 mol/L acetic acid-sodium acetate soln be damping fluid, reaction pH value is 3.0-7.0, be placed in constant temperature oscillation tank, temperature of reaction is 30-70oC, oscillatory reaction 4 h, vibration velocity is 0-160 time/min, from adding enzyme liquid to start timing, every 30 min extract reaction solution once, after boiling water deactivation, be cooled to room temperature, the enzymic catalytic reaction of described immobilization naringin is: in the reaction system of 50 mL, the resin that adds 1 g weight in wet base, naringin concentration is 0.08-0.24g/100 mL, enzyme dosage is 4-12 U/mL, take concentration as 0.02 mol/L acetic acid-sodium acetate soln be damping fluid, reaction pH value is 3.0-7.0, be placed in constant temperature oscillation tank, temperature of reaction is 30-70oC, oscillatory reaction 4 h, and vibration velocity is 0-160 time/min, during this time from adding enzyme liquid to start timing, extract reaction solution once at set intervals, and use immediately boiling water bath deactivation 20 min, be cooled to room temperature, in the situation that other conditions are constant, respectively described naringin concentration, enzyme dosage, pH value in reaction, temperature of reaction and vibration velocity are carried out to single factor experiment, to determine optimum reaction condition,
Step 5, DNS method analysis-reduction sugar growing amount: adopt 2, 4-dinitrosalicylic acid is the reducing sugar growing amount that DNS analyzes described enzymic catalytic reaction: get reaction solution described in 0.2 mL and mix with 0.6 mL DNS solution, shake up, 100 ° of C water-bath 15 min, take out with tap water immediately cooling, with distilled water, be settled to 5 mL again, under 520 nm wavelength, measure light absorption value, establishing criteria curve calculates the growing amount of reducing sugar in reaction solution, wherein, during drawing standard curve, the mixing solutions that the rhamnosyl of equimolar ratio and glucose forms of take is standardized solution, its concentration is 0-2 mg/mL.
As the optimal way of embodiment, in described step 4, in the enzymic catalytic reaction of described free naringin, naringin concentration is 0.2 g/100 mL, and enzyme dosage is 8 U/mL, and pH value is 5.0, and temperature of reaction is 50oC, enzyme digestion reaction 120 min.
As the optimal way of embodiment, in described step 4, in the enzymic catalytic reaction of described immobilization naringin, naringin concentration is 0.2 g/100 mL, and enzyme dosage is 8 U/mL, and pH value is 5.0, and temperature of reaction is 50oC, and vibration velocity is 80 times/min, enzyme digestion reaction 6 h.
Optimal way as embodiment, described method also comprises the purification procedures of enzymolysis product, after enzymic catalytic reaction finishes, 20 min of boiling water bath deactivation immediately, be cooled to room temperature, filter and collect resin, after dress post, first with distilled water, the flow velocity with 1 mL/min cleans, then with the ethanol of certain volume concentration, carry out wash-out, with substep collector Fractional Collections elutriant, adopt tlc to follow the tracks of detection to the composition in elutriant qualitative simultaneously, composition is identical, be that the elutriant that spot is identical merges, and elutriant is carried out to the concentrated enriched material that to obtain of underpressure distillation, reclaim ethanol simultaneously, and then by enriched material recrystallizing methanol, obtain enzymolysis product sterling, it is naringenin sterling.
Optimal way as embodiment, described method also comprises the qualitative analysis step of enzymolysis product, after collecting described naringenin sterling, take described naringenin sterling and naringenin standard substance, with after dissolve with methanol constant volume, adopt respectively uv-spectrophotometric, infrared spectra and high-efficient liquid phase chromatogram HPLC method to carry out qualitative analysis and purity testing to described naringenin sterling.
Optimal way as embodiment, described method also comprises the determination step of naringinase vigor, after collecting described crude enzyme liquid, at 1.9 mL, in pH 5.0 phosphate buffered saline buffer test tubes, add 0.1 mL crude enzyme liquid to mix, preheating 5 min in 50 ° of C water-baths, add again 2 mL, 300 mg/L naringin reference liquids, 50 ° of C, react after 5 min, in 100 ° of C heating in water bath 20 min, make enzyme deactivation, after taking-up, be cooled to rapidly room temperature, the sample that reaction is obtained moves to 1.5 mL centrifuge tubes, centrifugal 15 min of 12 000 * g, HPLC is to be measured, control group is for first will adding naringin reference liquid again after crude enzyme liquid deactivation, other operations are identical, under the condition at 50 ° of C, pH 5.0, per minute generates the required enzyme amount of 1 μ g naringenin and is defined as a naringinase unit of activity.
Optimal way as embodiment, described optimum reaction condition is that the maximum conversion rate with naringin draws, the method of calculating naringin transformation efficiency is: wherein, and HPLC method: the total amount (mg) of naringin before catalytic amount (mg) * 100/ enzymic catalytic reaction of naringin transformation efficiency (%)=naringin; DNS method: the growing amount (mg) of reducing sugar when growing amount (mg) * 100/ naringin of reducing sugar is by complete catalysis in naringin transformation efficiency (%)=enzymic catalytic reaction.
Adopt after aforesaid method, the present invention is by analyzing the rhamnosyl reducing sugar that produces in naringinase catalysis naringin reaction process and the content of Reduction of Glucose sugar by DNS, reach the object of rapid Optimum naringinase catalysis naringin, and further analyzed the impact of different technical parameters on naringin conversion process, determined optimum optimizing condition, the naringin transformation efficiency of gained is high, wherein, the transformation efficiency of the enzymic catalytic reaction naringin of free naringin has reached 85.1%, the transformation efficiency of the enzymic catalytic reaction naringin of immobilization naringin has reached 93.5%, and then for utilizing the modified product of naringinase modified pomelo peel glycosides acquisition particular organisms activity that Research foundation is provided.
accompanying drawing explanation
Fig. 1 is the relation of the naringin transformation efficiency that records of HPLC method of the present invention and DNS method;
Fig. 2 is in the free naringin process optimization of naringinase catalysis of the present invention, the optimization of enzyme dosage to the effect of naringin enzymolysis;
Fig. 3 is in the free naringin process optimization of naringinase catalysis of the present invention, the optimization of concentration of substrate to the effect of naringin enzymolysis;
Fig. 4 is in the free naringin process optimization of naringinase catalysis of the present invention, the optimization of pH value to the effect of naringin enzymolysis;
Fig. 5 is in the free naringin process optimization of naringinase catalysis of the present invention, the optimization of temperature to the effect of naringin enzymolysis;
Fig. 6 is that in the free naringin process optimization of naringinase catalysis of the present invention, oscillation rate is to naringinase majorization of solutions;
Fig. 7 is during the enzymolysis process of immobilization naringin of the present invention is optimized, the optimization of enzyme dosage to the effect of naringin enzymolysis;
Fig. 8 is during the enzymolysis process of immobilization naringin of the present invention is optimized, the optimization of concentration of substrate to the effect of naringin enzymolysis;
Fig. 9 is during the enzymolysis process of immobilization naringin of the present invention is optimized, the optimization of pH value to the effect of naringin enzymolysis;
Figure 10 is during the enzymolysis process of immobilization naringin of the present invention is optimized, the optimization of temperature to the effect of naringin enzymolysis;
Figure 11 is during the enzymolysis process of immobilization naringin of the present invention is optimized, the optimization of oscillation rate to the effect of naringin enzymolysis.
Embodiment
First the present invention adopts high performance liquid chromatography and DNS method to analyze the variation relation of naringin, Pu Luning, naringenin and reducing sugar content in naringin enzymolysis process, and explored on this basis and only by DNS method, analyzed the feasibility of naringin enzymolysis process, then utilize the method, analyzed the impact of different technical parameters on naringin conversion process, for the modified product that utilizes naringinase modified pomelo peel glycosides to obtain particular organisms activity provides Research foundation.
embodiment mono-materials and methods
Based on DNS, analyze a fast Optimization that realizes naringinase catalysis naringin, present method comprises the following steps:
The preparation of step 1, substratum: the preparation of described substratum comprises the preparation of slant medium and the preparation of solid medium, wherein: the preparation of described slant medium, by the component preparation of following mass body volume concentrations: MgSO
47H
2o 1.0 g/L, KH
2pO
41.0 g/L, (NH4)
2sO
41.5 g/L, KCl 0.5 g/L, KNO
31.5 g/L, anhydrous CaCl
20.1 g/L, yeast extract 2.0 g/L, naringin 2.5 g/L, agar 20 g/L, initial pH 6.0,1 * 105 Pa sterilizing 20 min; The preparation of described solid medium, prepares by the component of following weight: shaddock ped powder 160g, soybean cake powder 24g, wheat bran 1%, KNO
31%, solid water is than being 1:1.5,1 * 10
5pa sterilizing 20 min;
The preparation of step 3, crude enzyme liquid: with the solid-to-liquid ratio of 1:20, with phosphate buffered saline buffer, solid state fermentation thing is soaked to 1 h, stirs evenly rear suction filtration and collect filtrate, by described filtrate in 4 ° of C with centrifugal 10 min of 12000 * g, collect supernatant liquor and obtain crude enzyme liquid, under 0-4 ° of C, preservation is standby;
Step 5, DNS method analysis-reduction sugar growing amount: adopt 2, 4-dinitrosalicylic acid is the reducing sugar growing amount that DNS analyzes described enzymic catalytic reaction: get reaction solution described in 0.2 mL and mix with 0.6 mL DNS solution, shake up, 100 ° of C water-bath 15 min, take out with tap water immediately cooling, with distilled water, be settled to 5 mL again, under 520 nm wavelength, measure light absorption value, establishing criteria curve calculates the growing amount of reducing sugar in reaction solution, wherein, during drawing standard curve, the mixing solutions that the rhamnosyl of equimolar ratio and glucose forms of take is standardized solution, its concentration is 0-2 mg/mL.
As the optimal way of embodiment, in described step 4, in the enzymic catalytic reaction of described free naringin, naringin concentration is 0.2 g/100 mL, and enzyme dosage is 8 U/mL, and pH value is 5.0, and temperature of reaction is 50oC, enzyme digestion reaction 120 min.
As the optimal way of embodiment, in described step 4, in the enzymic catalytic reaction of described immobilization naringin, naringin concentration is 0.2 g/100 mL, and enzyme dosage is 8 U/mL, and pH value is 5.0, and temperature of reaction is 50oC, and vibration velocity is 80 times/min, enzyme digestion reaction 6 h.
Optimal way as embodiment, described method also comprises the purification procedures of enzymolysis product, after enzymic catalytic reaction finishes, 20 min of boiling water bath deactivation immediately, be cooled to room temperature, filter and collect resin, after dress post, first with distilled water, the flow velocity with 1 mL/min cleans, then with the ethanol of certain volume concentration, carry out wash-out, with substep collector Fractional Collections elutriant, adopt tlc to follow the tracks of detection to the composition in elutriant qualitative simultaneously, composition is identical, be that the elutriant that spot is identical merges, and elutriant is carried out to the concentrated enriched material that to obtain of underpressure distillation, reclaim ethanol simultaneously, and then by enriched material recrystallizing methanol, obtain enzymolysis product sterling, it is naringenin sterling.
Optimal way as embodiment, described method also comprises the qualitative analysis step of enzymolysis product, after collecting described naringenin sterling, take described naringenin sterling and naringenin standard substance, with after dissolve with methanol constant volume, adopt respectively uv-spectrophotometric, infrared spectra and high-efficient liquid phase chromatogram HPLC method to carry out qualitative analysis and purity testing to described naringenin sterling.
Optimal way as embodiment, described method also comprises the determination step of naringinase vigor, after collecting described crude enzyme liquid, at 1.9 mL, in pH 5.0 phosphate buffered saline buffer test tubes, add 0.1 mL crude enzyme liquid to mix, preheating 5 min in 50 ° of C water-baths, add again 2 mL, 300 mg/L naringin reference liquids, 50 ° of C, react after 5 min, in 100 ° of C heating in water bath 20 min, make enzyme deactivation, after taking-up, be cooled to rapidly room temperature, the sample that reaction is obtained moves to 1.5 mL centrifuge tubes, centrifugal 15 min of 12 000 * g, HPLC is to be measured, control group is for first will adding naringin reference liquid again after crude enzyme liquid deactivation, other operations are identical, under the condition at 50 ° of C, pH 5.0, per minute generates the required enzyme amount of 1 μ g naringenin and is defined as a naringinase unit of activity.
Optimal way as embodiment, described optimum reaction condition is that the maximum conversion rate with naringin draws, the method of calculating naringin transformation efficiency is: wherein, and HPLC method: the total amount (mg) of naringin before catalytic amount (mg) * 100/ enzymic catalytic reaction of naringin transformation efficiency (%)=naringin; DNS method: the growing amount (mg) of reducing sugar when growing amount (mg) * 100/ naringin of reducing sugar is by complete catalysis in naringin transformation efficiency (%)=enzymic catalytic reaction.
embodiment bis-DNS methods are analyzed the feasibility of naringin enzymolysis process
Adopt respectively HPLC method and DNS method to analyze the enzyme digestion reaction of naringin, the transformation efficiency of the naringin recording in both cases with the variation relation of enzymolysis time as shown in Figure 1.
As shown in Figure 1, the transformation efficiency of the naringin being recorded by DNS method and HPLC method overlaps substantially with the change curve of enzymolysis time, that is to say that the naringin transformation efficiency being recorded by DNS is consistent with the result of HPLC method mensuration, therefore adopt DNS method to analyze the enzymolysis process of naringin, its result accurately and reliably.
the enzyme-catalyzed reaction optimization of embodiment tri-free naringins
1. the optimization of enzyme concentration to naringin enzymolysis process
In 2 mL reaction systems, in the constant situation of other condition, naringinase crude enzyme liquid (enzyme work the is 100 U/mL) consumption of take is respectively 4,6,8,10,12 U/mL enzyme digestion reaction 210 min, and the transformation efficiency of mensuration naringin is situation over time, and its result as shown in Figure 2.
As can be seen from the figure,, under different enzyme dosages, the transformation efficiency that reacts naringin is at the end basically identical.Along with the increase of enzyme dosage, enzyme reaction reaches the needed time of balance and shortens gradually.This is due in the situation that concentration of substrate is certain, and along with the increase of enzyme dosage, enzyme molecule contacts substrate molecule chance in unit volume, in the unit time increases, thus the quickening of the speed of enzyme Journal of Molecular Catalysis substrate conversion, and enzyme reaction reaches the time shorten of balance.Experimental result demonstration, under 8 U/mL enzyme dosages, while reacting to 120 min, the transformation efficiency of naringin has reached 85.1%.And when enzyme dosage is over after 8 U/mL, though enzyme reaction reaches the required time of balance and shortens to some extent, rangeability is little.From economic angle, consider, select 8 U/mL as the suitableeest enzyme dosage.
2. the optimization of concentration of substrate to the effect of naringin enzymolysis
On the basis of best enzyme dosage, investigate the impact of concentration of substrate on the effect of naringin enzymolysis, the changing conditions of naringin transformation efficiency is as shown in Figure 3.
As can be seen from Figure 3, when other condition is fixedly time, the enzyme digestion reaction of naringin reaches balance required time and extends along with the increase of naringin concentration, and the transformation efficiency of naringin declines thereupon.When the concentration of naringin is lower, substrate is easy to by enzyme institute saturated, and enzyme reaction can reach balance soon, and when naringin concentration increases, the chance of enzyme-to-substrate contact also increases thereupon, and enzyme reaction just speed is accelerated relatively, but reaction reach the time of balance can be elongated.When concentration of substrate is 0.04 g/100 mL, during molecular balance, naringin transformation efficiency reaches 94.5%, and along with the increase of concentration of substrate, the transformation efficiency of naringin presents continuous decrease trend.Naringinase catalysis naringin is a reversing process, the have certain restraining effect of product to reaction, therefore, along with substrate naringin concentration increases, the production concentration that reaction obtains also increases, and restraining effect strengthens, thereby causes the transformation efficiency of naringin to increase and decline with concentration of substrate.For acquisition enzymolysis product naringenin as much as possible, therefore the peak concentration 0.2 g/100 mL that selects naringin can reach under this experiment condition carries out follow-up enzyme digestion reaction as concentration of substrate.
3. the optimization of pH value to the effect of naringin enzymolysis
It is larger that the activity of biological enzyme is affected by environment pH, and its maximum vigor only just can show under its suitableeest pH condition, and peracid or excessively alkali all can make its vigor reduce, and even lose.Therefore it is very necessary on the impact of naringin enzymolysis effect to investigate pH value.When the pH of system value is respectively 3.0,4.0,5.0,6.0,7.0, in enzyme digestion reaction system, curve is as shown in Figure 4 over time for the transformation efficiency of reducing sugar growing amount and naringin.
As shown in Figure 4, pH value all has significant impact to the transformation efficiency of the initial rate of enzyme reaction and naringin.When pH value is 5.0, the initial rate of enzyme reaction and while reaching balance the transformation efficiency of naringin be maximum.And when pH value increases or reduces, all can there is reducing in various degree in the initial reaction rate of enzyme reaction and the transformation efficiency of naringin.
4. the optimization of temperature to the effect of naringin enzymolysis
Each enzyme has its optimal reactive temperature under certain condition, in lower than optimum temperature range, enzyme reaction speed increases with the rising of temperature, but because most of enzymes are all protein, when temperature surpasses after its optimum temperuture, along with the rising of temperature, zymoprotein is sex change and inactivation gradually, thereby causes that enzyme reaction speed declines.When other condition is constant, under differing temps, the transformation efficiency of reducing sugar content and naringin dynamic change situation in time in mensuration enzymolysis solution, its result is as shown in Figure 5.
As can be seen from Figure 5, when temperature increases to 50oC by 30oC, along with the rising of temperature, enzyme reaction initial rate is accelerated thereupon, reach the required time of balance to shorten, and the transformation efficiency of naringin also increases thereupon.Though initial enzyme reaction speed during 60oC is a little more than 50oC, along with the prolongation in reaction times, enzyme reaction speed significantly declines, and naringin transformation efficiency also declines thereupon.This is due to the initial period in enzyme reaction, the sex change of zymoprotein not yet shows, speed of reaction increases with the rising of temperature, but along with the carrying out of enzyme digestion reaction, zymoprotein sex change is outstanding gradually, the effect that speed of reaction raises with temperature is offset by zymoprotein denaturing effect gradually, speed of reaction declines rapidly, and the sex change of zymoprotein is prolongation in time and cumulative, thereby causes enzyme reaction to arrive the time lengthening of balance, naringin transformation efficiency reduces, and naringenin growing amount reduces.Therefore, the optimum temperuture that this naringinase transforms naringin is 50oC, and with this understanding, naringin transformation efficiency reaches 85.1%.
5. the optimization of oscillation rate to the effect of naringin enzymolysis
Generally speaking, make, in the environment of reaction system in vibration, may increase the touch opportunity between reactant, thereby add the carrying out of fast response.Investigate under different oscillation rate naringin transformation efficiency situation over time, its result as shown in Figure 6.
As shown in Figure 6, when the oscillation rate of tank increases to 160 times/min by 0 time/min, the speed of enzyme digestion reaction, enzyme digestion reaction reach the time of balance and the transformation efficiency of naringin does not all change substantially.
the enzymolysis process optimization of embodiment tetra-immobilization naringins
1. the optimization of enzyme dosage to immobilization naringin enzymolysis process
In 50 mL reaction systems, in the constant situation of other condition, the naringinase crude enzyme liquid (enzyme work is 100 U/mL) of take is catalyzer, at its consumption, be respectively 4,6,8,10, during 12 U/mL, catalysis naringin enzymolysis reacts 8 h, the transformation efficiency of measuring naringin in enzymolysis solution is situation over time, and its result respectively as shown in Figure 7.
As can be seen from Figure 7,, along with the increase of enzyme dosage, enzyme reaction reaches the needed time of balance and shortens gradually.This is due in the situation that concentration of substrate is certain, and along with the increase of enzyme dosage, enzyme molecule contacts substrate molecule chance in unit volume, in the unit time increases, thus the quickening of the speed of enzyme Journal of Molecular Catalysis substrate conversion, and enzyme reaction reaches the time shorten of balance.In this simultaneously, the transformation efficiency of naringin also presents synchronous variation tendency.But under different enzyme dosages, reacting the at the end transformation efficiency of naringin is consistent substantially.Experimental result demonstration, under the enzyme dosage of 8 U/mL, while reacting 6 h, the transformation efficiency of naringin can be up to 90.3%.And when enzyme dosage is over after 8 U/mL, though enzyme reaction reaches the required time of balance and shortens to some extent, rangeability is little.From economic angle, consider, select 8 U/mL as the suitableeest enzyme dosage.Yet, to compare with the enzymolysis conversion reaction of free naringin, the enzyme digestion reaction of immobilization naringin arrives balance needs the time of more growing, and this may be that existence due to resin carrier has hindered due to the contact of naringin enzyme-to-substrate naringin.
2. the optimization of concentration of substrate to the effect of immobilization naringin enzymolysis
In best enzyme dosage and other condition, certain in the situation that, the changing conditions of the naringin transformation efficiency of different concentration of substrate as shown in Figure 8.
As can be seen from Figure 8, the enzyme digestion reaction of naringin reaches balance required time and extends along with the increase of naringin concentration, but the transformation efficiency of naringin presents downtrending.When the concentration of naringin is lower, substrate is easy to by enzyme institute saturated, and enzyme reaction can reach balance soon, and when naringin concentration increases, the chance of enzyme-to-substrate contact also increases thereupon, and enzyme reaction just speed is accelerated relatively, but reaction reach the time of balance can be elongated.Known in figure, when concentration of substrate is 0.08 g/100 mL, during molecular balance, naringin transformation efficiency reaches 98.3%, but along with the increase of concentration of substrate, the transformation efficiency of naringin presents continuous decrease trend.For acquisition enzymolysis product naringenin as much as possible, therefore select the peak concentration that naringin can reach under this experiment condition, be that 0.2 g/100 mL carries out follow-up enzyme digestion reaction as concentration of substrate.
3. the optimization of pH value to the effect of immobilization naringin enzymolysis
Biological enzyme is as a kind of protein, and it is larger that its activity is affected by the pH of environment, and its maximum vigor only just can show under its suitableeest pH condition, and peracid or excessively alkali all can make its vigor reduce, and even lose.Therefore investigate pH value, the impact of naringin enzymolysis effect is seemed to very necessary.When the pH of system value is respectively 3.0,4.0,5.0,6.0,7.0, in enzyme digestion reaction system, curve is as shown in Figure 9 over time for the transformation efficiency of naringin.
As shown in Figure 9, pH value all has significant impact to the transformation efficiency of the initial rate of enzyme reaction and naringin.And along with the variation of system pH, the variation tendency of naringin transformation efficiency is consistent.When the pH of system value is 5.0, the initial rate of enzyme reaction and the transformation efficiency of naringin are maximum.And when pH value increases or reduces, all can there is reducing or reducing in various degree in the initial reaction rate of enzyme reaction and the transformation efficiency of naringin.
4. the optimization of temperature to the effect of immobilization naringin enzymolysis
In chemical reaction, the speed of most chemical reactions, all relevant with temperature, enzymic catalytic reaction is certainly no exception.Every kind of enzyme has its optimum temperuture under certain condition, and when below or above its optimum temperature range, the vigor of enzyme all can occur decline in various degree, even devitalization.When other condition is constant, under differing temps, the transformation efficiency of naringin dynamic change situation in time in mensuration enzymolysis solution, its result is as shown in figure 10.
As can be seen from Figure 10, when temperature increases to 50oC by 30oC, along with the rising of temperature, enzyme reaction initial rate is accelerated thereupon, reach the required time of balance to shorten, and the transformation efficiency of naringin also increases thereupon.Though initial enzyme reaction speed during 60oC is a little more than 50oC, along with the prolongation in reaction times, enzyme reaction speed significantly declines, and naringin transformation efficiency also declines thereupon.This is due to the initial period in enzyme reaction, the sex change of zymoprotein not yet shows, speed of reaction increases with the rising of temperature, but along with the carrying out of enzyme digestion reaction, zymoprotein sex change is outstanding gradually, the effect that speed of reaction raises with temperature is offset by zymoprotein denaturing effect gradually, speed of reaction declines rapidly, and the sex change of zymoprotein is prolongation in time and cumulative, thereby causes enzyme reaction to arrive the time lengthening of balance, naringin transformation efficiency reduces, and naringenin growing amount reduces.Therefore, the optimum temperuture that this naringinase transforms naringin is 50oC, and with this understanding, naringin transformation efficiency reaches 93.0%.
5. the optimization of oscillation rate to the effect of immobilization naringin enzymolysis
Generally speaking, the environment of vibration can promote the carrying out of enzymatic reaction.In the situation that other condition is certain, investigate under different oscillation rate naringin transformation efficiency situation over time, its result is as shown in figure 11.
As shown in Figure 11, when the oscillation rate of tank increases to 160 times/min by 0 time/min, the initial rate of enzyme digestion reaction is basically identical, but under each oscillation rate enzyme reaction to arrive time of molecular balance slightly different.In general, along with increasing progressively of oscillation rate, the time that enzyme digestion reaction arrives balance shortens gradually, but when the oscillation rate of tank increases to 160 times/min by 80 times/min, in enzyme reaction process, the time of the transformation efficiency of naringin and enzyme reaction arrival balance is basic identical.From the angle of save energy, consider, 80 times/min of this experimental selection is as the oscillation rate of tank.
Adopt after aforesaid method, the present invention is by analyzing the rhamnosyl reducing sugar that produces in naringinase catalysis naringin reaction process and the content of Reduction of Glucose sugar by DNS, reach the object of rapid Optimum naringinase catalysis naringin, and further analyzed the impact of different technical parameters on naringin conversion process, determined optimum optimizing condition, the naringin transformation efficiency of gained is high, wherein, the transformation efficiency of the enzymic catalytic reaction naringin of free naringin has reached 85.1%, the transformation efficiency of the enzymic catalytic reaction naringin of immobilization naringin has reached 93.5%, and then for utilizing the modified product of naringinase modified pomelo peel glycosides acquisition particular organisms activity that Research foundation is provided.
Above are only the specific embodiment of the present invention, but design concept of the present invention is not limited to this, allly utilizes this design to carry out the change of unsubstantiality to the present invention, all should belong to the behavior of invading protection domain of the present invention.
Claims (7)
1. based on DNS, analyze a fast Optimization that realizes naringinase catalysis naringin, it is characterized in that: present method comprises the following steps:
The preparation of step 1, substratum: the preparation of described substratum comprises the preparation of slant medium and the preparation of solid medium, wherein: the preparation of described slant medium, by the component preparation of following mass body volume concentrations: MgSO
47H
2o 1.0 g/L, KH
2pO
41.0 g/L, (NH4)
2sO
41.5 g/L, KCl 0.5 g/L, KNO
31.5 g/L, anhydrous CaCl
20.1 g/L, yeast extract 2.0 g/L, naringin 2.5 g/L, agar 20 g/L, initial pH 6.0,1 * 105 Pa sterilizing 20 min; The preparation of described solid medium, prepares by the component of following weight: shaddock ped powder 160g, soybean cake powder 24g, wheat bran 1%, KNO
31%, solid water is than being 1:1.5,1 * 10
5pa sterilizing 20 min;
Step 2, solid state fermentation: microorganism Aspergillus aculeatus bacterial classification is inoculated on described slant medium, 28 ° of C cultivate 3-4d, make to cover with spore on described slant medium, by stroke-physiological saline solution, wash lower spore, and adjust bacteria suspension OD value to 0.2 by stroke-physiological saline solution, 1 mL monospore bacteria suspension is accessed in the triangular flask of 250 mL that described solid medium is housed, every bottle of sample-loading amount is 5 g, under 28 ° of C, cultivates 7-8 d;
The preparation of step 3, crude enzyme liquid: with the solid-to-liquid ratio of 1:20, with phosphate buffered saline buffer, solid state fermentation thing is soaked to 1 h, stirs evenly rear suction filtration and collect filtrate, by described filtrate in 4 ° of C with centrifugal 10 min of 12000 * g, collect supernatant liquor and obtain crude enzyme liquid, under 0-4 ° of C, preservation is standby;
Step 4, enzymic catalytic reaction: described enzymic catalytic reaction is the free enzymic catalytic reaction of naringin and/or the enzymic catalytic reaction of immobilization naringin, wherein, the enzymic catalytic reaction of described free naringin is: in the reaction system of 50 mL, naringin concentration is 0.04-0.24g/100 mL, enzyme dosage is 4-12 U/mL, take concentration as 0.02 mol/L acetic acid-sodium acetate soln be damping fluid, reaction pH value is 3.0-7.0, be placed in constant temperature oscillation tank, temperature of reaction is 30-70oC, oscillatory reaction 4 h, vibration velocity is 0-160 time/min, from adding enzyme liquid to start timing, every 30 min extract reaction solution once, after boiling water deactivation, be cooled to room temperature, the enzymic catalytic reaction of described immobilization naringin is: in the reaction system of 50 mL, the resin that adds 1 g weight in wet base, naringin concentration is 0.08-0.24g/100 mL, enzyme dosage is 4-12 U/mL, take concentration as 0.02 mol/L acetic acid-sodium acetate soln be damping fluid, reaction pH value is 3.0-7.0, be placed in constant temperature oscillation tank, temperature of reaction is 30-70oC, oscillatory reaction 4 h, and vibration velocity is 0-160 time/min, during this time from adding enzyme liquid to start timing, extract reaction solution once at set intervals, and use immediately boiling water bath deactivation 20 min, be cooled to room temperature, in the situation that other conditions are constant, respectively described naringin concentration, enzyme dosage, pH value in reaction, temperature of reaction and vibration velocity are carried out to single factor experiment, to determine optimum reaction condition,
Step 5, DNS method analysis-reduction sugar growing amount: adopt 2, 4-dinitrosalicylic acid is the reducing sugar growing amount that DNS analyzes described enzymic catalytic reaction: get reaction solution described in 0.2 mL and mix with 0.6 mL DNS solution, shake up, 100 ° of C water-bath 15 min, take out with tap water immediately cooling, with distilled water, be settled to 5 mL again, under 520 nm wavelength, measure light absorption value, establishing criteria curve calculates the growing amount of reducing sugar in reaction solution, wherein, during drawing standard curve, the mixing solutions that the rhamnosyl of equimolar ratio and glucose forms of take is standardized solution, its concentration is 0-2 mg/mL.
2. a kind of fast Optimization realize naringinase catalysis naringin of analyzing based on DNS as claimed in claim 1, it is characterized in that: in described step 4, in the enzymic catalytic reaction of described free naringin, naringin concentration is 0.2 g/100 mL, enzyme dosage is 8 U/mL, pH value is 5.0, and temperature of reaction is 50oC, enzyme digestion reaction 120 min.
3. a kind of fast Optimization realize naringinase enzymolysis of analyzing based on DNS as claimed in claim 1, is characterized in that:
In described step 4, in the enzymic catalytic reaction of described immobilization naringin, naringin concentration is 0.2 g/100 mL, and enzyme dosage is 8 U/mL, and pH value is 5.0, and temperature of reaction is 50oC, and vibration velocity is 80 times/min, enzyme digestion reaction 6 h.
4. a kind of fast Optimization realize naringinase catalysis naringin of analyzing based on DNS as claimed in claim 1, it is characterized in that: described method also comprises the purification procedures of enzymolysis product, after enzymic catalytic reaction finishes, 20 min of boiling water bath deactivation immediately, be cooled to room temperature, filter and collect resin, after dress post, first with distilled water, the flow velocity with 1 mL/min cleans, then with the ethanol of certain volume concentration, carry out wash-out, with substep collector Fractional Collections elutriant, adopt tlc to follow the tracks of detection to the composition in elutriant qualitative simultaneously, composition is identical, be that the elutriant that spot is identical merges, and elutriant is carried out to the concentrated enriched material that to obtain of underpressure distillation, reclaim ethanol simultaneously, and then by enriched material recrystallizing methanol, obtain enzymolysis product sterling, it is naringenin sterling.
5. a kind of fast Optimization realize naringinase catalysis naringin of analyzing based on DNS as claimed in claim 4, it is characterized in that: described method also comprises the qualitative analysis step of enzymolysis product, after collecting described naringenin sterling, take described naringenin sterling and naringenin standard substance, with after dissolve with methanol constant volume, adopt respectively uv-spectrophotometric, infrared spectra and high-efficient liquid phase chromatogram HPLC method to carry out qualitative analysis and purity testing to described naringenin sterling.
6. a kind of fast Optimization realize naringinase catalysis naringin of analyzing based on DNS as claimed in claim 1, it is characterized in that: described method also comprises the determination step of naringinase vigor, after collecting described crude enzyme liquid, at 1.9 mL, in pH 5.0 phosphate buffered saline buffer test tubes, add 0.1 mL crude enzyme liquid to mix, preheating 5 min in 50 ° of C water-baths, add again 2 mL, 300 mg/L naringin reference liquids, 50 ° of C, react after 5 min, in 100 ° of C heating in water bath 20 min, make enzyme deactivation, after taking-up, be cooled to rapidly room temperature, the sample that reaction is obtained moves to 1.5 mL centrifuge tubes, centrifugal 15 min of 12 000 * g, HPLC is to be measured, control group is for first will adding naringin reference liquid again after crude enzyme liquid deactivation, other operations are identical, under the condition at 50 ° of C, pH 5.0, per minute generates the required enzyme amount of 1 μ g naringenin and is defined as a naringinase unit of activity.
7. a kind of fast Optimization realize naringinase catalysis naringin of analyzing based on DNS as described in claim 1 or 4, it is characterized in that: in described step 4, described optimum reaction condition is that the maximum conversion rate with naringin draws, the method of calculating naringin transformation efficiency is: wherein, and HPLC method: the total amount (mg) of naringin before catalytic amount (mg) * 100/ enzymic catalytic reaction of naringin transformation efficiency (%)=naringin; DNS method: the growing amount (mg) of reducing sugar when growing amount (mg) * 100/ naringin of reducing sugar is by complete catalysis in naringin transformation efficiency (%)=enzymic catalytic reaction.
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