CN110093395A - A kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide - Google Patents
A kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide Download PDFInfo
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Abstract
The invention discloses a kind of compound proteases prepared suitable for albumen effectively hydrolyzing and small peptide, including strain, mold fermentation culture medium, bacterial fermentation culture medium, Tilapia mossambica intestinal protease, protease purification sample, protein hydrolyzate and compound protease, the strain includes for aspergillus oryzae, Penicillium citrinum, Mucor, Methylotrophic bacillus, bacillus licheniformis, bacillus subtilis and bafillus natto, the mold fermentation culture medium includes test tube slant culture medium and fermentation medium, the bacterial fermentation culture medium includes test tube slant culture medium, liquid seed culture medium and fermentation medium.The compound protease that this is suitable for albumen effectively hydrolyzing and prepared by small peptide, the multiple protease components complementary with peptide bond selection are obtained by multiplex screening, then compound protease is constructed by their compatibilities, the peptide bond selectivity for having widened single protease significantly in this way, to realize compound protease to the depth hydrolysis of a variety of substrate proteins.
Description
Technical field
It is specially a kind of to be answered suitable for prepared by albumen effectively hydrolyzing and small peptide the present invention relates to compound protease technical field
Hop protein enzyme.
Background technique
Protease be one kind can aminosal, the general designation of the hydrolase of peptide and amino acid is translated into, in day
The fields such as change, food processing, Feed Manufacturing and cultivation have a wide range of applications.Acid hydrolysis technique compared to early stage is come
It says, realizes that proteolysis is a kind of production technology of cleaning green with protease, have more potential for development.In recent years, with more
The physiology and healthcare function of peptide are constantly recognized by people, and polypeptide is as raw material in health food, clinical food and feed addition
Continuous expanded application in the industries such as agent, protease are gradually applied to the production of polypeptide.Protease is weight in different kind organism body
The digestive ferment wanted, it takes part in a series of physiological and biochemical procedures related with proteolysis in organism.So different kind organism
Body all has the ability of certain synthetic proteins enzyme including animal, plant and microorganism.Has a large amount of document report from difference
Separation obtains protease in organism: such as papain and bromelain from plant；From aspergillus oryzae point
The protease secreted, the protease of Mucor secretion, and the protease etc. from animal alimentary canal secretion.
However, the effect that protease is undertaken in different organisms is usually different: the object that it needs to hydrolyze is not
Together；The hydrolysis target for needing to realize is also different；As a result, under the driving of natural evolution, the protease from different organisms
It would generally show different catalysis characteristics, especially peptide bond selectivity.For example, pepsin from animal digestive system,
Trypsase；And there is significant difference in peptide bond selectivity between the papain and bromelain from plant；
With from bacillus subtilis neutral proteinase and bacillus licheniformis alkali protease there is also biggish peptide bond selectivity
Difference.It can thus be seen that the protease from different organisms typically exhibits certain peptide bond selective difference, each other
Between there are certain synergistic action effect, compound protease can be constructed by the protease of these separate sources and realize substrate protein
The efficient production of depth hydrolysis and polypeptide.
As protease preparation is widely used in the production of polypeptide, existing commercialization protease preparation is produced in polypeptide
Present in deficiency also it is increasingly prominent come out:
1, the peptide bond of single protease is selectively very limited, is not applied for the hydrolysis of all types peptide bond, therefore single egg
White enzyme is lower to the hydrolysis efficiency of animal/vegetable protein, can not achieve albumen to the Efficient Conversion of polypeptide；
2, existing commercial protease species are more single, and unification is more apparent, and the peptide bond between protease selects complementary
It is not significant, therefore, although the composite hydrolysis effect of two kinds of protease can be better than single protease, conversion of the albumen to polypeptide
Efficiency is still relatively low；
3, existing compound protease preparation is generally not to be produced as application target with polypeptide, it mainly passes through releasing and produces
Object polypeptide to maximize the hydrolysis ability for playing protease, and improves hydrolyzate to the feedback inhibition of endo type protease
Flavor.But it (is still limited to existing several since endo type protease type is more single in this compound protease preparation
Protease is commercialized), peptide bond selects face not wide, therefore can not achieve the depth hydrolysis of substrate protein and improve polypeptide conversion effect
Rate.
Summary of the invention
The purpose of the present invention is to provide a kind of compound proteases prepared suitable for albumen effectively hydrolyzing and small peptide, to solve
The existing commercial protease kind currently on the market that above-mentioned background technique proposes is more single, tends to homogeneity, Bu Nengshi
The depth hydrolysis of existing albumen, it is difficult to meet the hydrolysis process of different albumen to the diversified demand of protease, and compound protease
Compatibility is simpler, is usually compounded by endo type protease with circumscribed-type protease, the product of protein hydrolysate is with amino acid
Based on, be not suitable for the production of polypeptide, and the peptide bond for having commercialization protease selectively lacks concertedness, the effect of compounding is not
Significant problem.
To achieve the above object, the invention provides the following technical scheme: a kind of be suitable for albumen effectively hydrolyzing and small peptide preparation
Compound protease, including strain, mold fermentation culture medium, bacterial fermentation culture medium, Tilapia mossambica intestinal protease, protease is pure
Change sample, protein hydrolyzate and compound protease, the strain includes for aspergillus oryzae, Penicillium citrinum, Mucor, Methylotrophic gemma
Bacillus, bacillus licheniformis, bacillus subtilis and bafillus natto, the mold fermentation culture medium include test tube slant training
Base and fermentation medium are supported, the bacterial fermentation culture medium includes test tube slant culture medium, liquid seed culture medium and fermentation training
Support base.
Preferably, the test tube slant culture medium in the mold fermentation culture medium are as follows: test tube slant culture medium: potato 200g
Liquor, glucose 20g, 15~20g of agar, water 1000mL, natural pH121 DEG C of sterilizing 20min, in the mold fermentation culture medium
Fermentation medium are as follows: in every 250mL triangular flask fill 12 grams of wheat bran, 1.2 grams of peptone, 0.036 gram of dipotassium hydrogen phosphate, tween
80 0.024 grams, 1mol/L sodium hydroxide 1mL, water 8mL, 121 DEG C of sterilizing 20min.
Preferably, the test tube slant culture medium (%) in the bacterial fermentation culture medium are as follows: yeast powder 0.5, peptone 1,
NaCl0.5, agar 1.5 ~ 2, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min, the liquid seeds culture in the bacterial fermentation culture medium
Base (%) are as follows: yeast powder 0.5, peptone 1, NaCl0.5, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min, the bacterial fermentation culture medium
In fermentation medium (%): sucrose 4, peptone 2, wheat bran 3, potassium dihydrogen phosphate 0.3, calcium carbonate 0.3, Tween 80 0.1,
PH7.5,121 DEG C of sterilizing 20min.
Preferably, the preparation step of the Tilapia mossambica intestinal protease are as follows:
1) fresh Tilapia mossambica fish intestines, are taken, the NaCl solution that concentration is 0.15mol/L is added according to the ratio of 1:7, is smash with tissue
Broken machine breaks into pulpous state, is subsequently placed in and extracts 15min at room temperature；
2), leaching liquor is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, collects supernatant to get crude enzyme liquid is arrived；
3), above-mentioned enzyme solution is concentrated by ultrafiltration in the ultrafiltration membrane for being 30000Da with molecular cut off；
4) lactose, is added in enzyme solution after concentration, its concentration is made to reach 1%；Then enzyme solution is placed in freeze drier and is frozen
It is dry, obtain protease dry powder formulations.
Preferably, the preparation step of the protease purification sample are as follows:
1), DEAE-Sepharose anion exchange
With 0.02M, the Tris-HCl buffer balance DEAE-Sepharose anion-exchange column of pH7.5 (foundation balanced:
Efflux pH7.5, stable conductivity), a certain amount of enzyme powder 0.02M is taken, the Tris-HCl buffer solution of pH7.5 is loaded onto
Anion-exchange column, with 0.02M, the Tris-HCl buffer of pH7.5 is sufficiently washed after column (about 300ml), with 0.5MNaCl solution
(being dissolved in 0.02M, the Tris-HCl buffer of pH7.5) carries out ladder isocratic elution, collects activated protein peak, uses ultra-filtration centrifuge tube
Collection liquid is concentrated in (molecular cut off 10000Da), and with 0.05M, and pH5.0 acetate buffer solution adjusts its pH to 5.0；
2), CM-Sepharose cation exchanges
With 0.05M, pH5.0 acetate buffer solution balances CM-Sepharose cation exchange column, the concentration enzyme for taking previous step to collect
Liquid is loaded cation exchange column, after sufficiently washing column with equilibration buffer, (is dissolved in 0.05M, pH5.0 vinegar with the NaCl solution of 0.5M
Acid buffer) gradient elution is carried out, activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration；
3), Phenyl-Sepharose hydrophobic chromatography
Phenyl-Sepharose hydrophobic chromatography is balanced with 1.2M ammonium sulfate (being dissolved in 0.05M, the PBS buffer solution of pH7.5)
Column, the concentration enzyme solution that previous step is collected and 2.4M ammonium sulfate (being dissolved in 0.1M, the PBS buffer solution of pH7.5) are mixed in equal volume
With rear sample-adding Phenyl-Sepharose hydrophobic chromatography column, drainage column is sufficiently rinsed with equilibration buffer, gradient is then carried out and washes
It is de-, activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration；
4), with SuperdexG75 gel permeation chromatography
With 0.02M, the PBS buffer solution of pH7.5 balances Sepharose6B gel column, and the concentration enzyme solution that sample-adding previous step is collected is used
The PBS buffer solution of 0.02M, pH7.5 elute, and collecting activated protein peak is to purify enzyme solution.
Preferably, the preparation step of the protein hydrolyzate are as follows:
1) soy bean proteinous soln that substrate protein concentration is 5%, is prepared with distilled water, 20min is kept the temperature in 80 DEG C of water-baths, after cooling
Its pH value is adjusted to pH8.0, liquid of protease is added according to the enzyme concentration of 4000U/g albumen, mixes well and is placed on 50 DEG C of water-baths
Middle heat preservation digests 5h；
2) enzymolysis liquid pH value, is adjusted to 5.0, is kept the temperature 10min in 100 DEG C of water-baths and is carried out enzyme deactivation；
3) it, is centrifuged 10min under conditions of 4000r/min, collects supernatant and obtains protein hydrolyzate.
Preferably, the step of compound protease are as follows:
1), using insulin oxidation of beta chain as substrate model, with the different protease hydrolytics substrate, using liquid phase and spectrometer analysis
Hydrolysis gained polypeptide fragment, determines restriction enzyme site of the various protease on insulin β chain, filters out and select complementation with peptide bond
The protease type of property；
2), using soybean protein as natural substrate, test is hydrolyzed using single protease and compounding protease, investigates different eggs
Synergistic action effect between white enzyme has carried out point hydrolyzate of soybean protein with Tricine-SDS-PAGE electrophoresis system
It is complementary further to analyze the selection of the peptide bond between each protease according to the electrophoretic band quantity of polypeptide and position for analysis, and thus
Determining, there are four kinds of protease P1, P4, P7 and P9 of preferable peptide bond selection complementary relationship to be combined the compound protease of building；
3), using soybean protein as substrate, different compound proteases is respectively adopted and carries out proteolysis assay, analyzes different composite albumen
Rational proportion and hydrolysis effect difference between enzyme component.
Preferably, the compound proportion of the compound protease is P1:P4:P7:P9=1:3:1:1, and compound protease is suitable
Suitable amount ranges are 3000~4000U/g albumen, and best catalytic temperature range is 40~50 DEG C of ranges, most suitable catalytic pH range
Are as follows: pH8~10, suitable enzymolysis time are 4h.
Compared with prior art, the beneficial effects of the present invention are: should be prepared suitable for albumen effectively hydrolyzing and small peptide compound
Protease obtains the multiple protease components complementary with peptide bond selection by multiplex screening, then by their compatibilities Lai
Compound protease is constructed, the peptide bond selectivity of single protease has been widened significantly in this way, to realize compound protease to substrate
The depth hydrolysis of albumen start with from the acquisition of protease, seek more protease types, and by their peptide bonds selectivity
Analysis come screen be suitble to building compound protease component type, so that it is guaranteed that building compound protease have it is broadest
Peptide bond selectivity, to different well adapting to property of protein substrate, has higher hydrolysis efficiency to substrate protein；
1, the building of compound protease
1.1 use microbial fermentation technology, and have prepared multiple protein enzyme preparation in conjunction with vacuum freeze-drying method；
1.2 purify to have obtained multiple protein enzyme purification sample using a variety of chromatography means；
1.3 using insulin oxidation of beta chain as substrate model, with the different protease hydrolytics substrate, using liquid phase and spectrometer analysis
Hydrolysis gained polypeptide fragment, determines restriction enzyme site of the various protease on insulin β chain, filters out and select complementation with peptide bond
The protease type of property；
1.4 using soybean protein as natural substrate, and test is hydrolyzed using single protease and compounding protease, investigates different eggs
Synergistic action effect between white enzyme.Hydrolyzate of soybean protein has been carried out point with Tricine-SDS-PAGE electrophoresis system
It is complementary further to analyze the selection of the peptide bond between each protease according to the electrophoretic band quantity of polypeptide and position for analysis, and thus
Determine that the component of compound protease is constituted；
1.5 using soybean protein as natural substrate, has investigated the compatibility relationship between each protease component, determines the master of compound protease
Enzyme component, and the proportion compatibility of apoenzyme and coenzyme has been investigated on this basis, the composition of compound protease has finally been determined；
2. the catalytic property of compound protease is analyzed
Using soybean protein as natural substrate, the catalysis characteristics of compound protease are investigated, it is determined that the compound protease is most preferably urged
Change condition are as follows: the Optimum of compound protease is 3000~4000U/g albumen, best catalytic temperature in 40~50 DEG C of ranges,
For most suitable catalytic pH in pH8~10, suitable enzymolysis time is 4h；
3. verifying the substrate adaptability of compound protease
Soyabean expeller, the semen sojae atricolor dregs of rice, Gluten, peanut meal, dephenolize cotton dregs, Symeplectoteuthis oualaniensis, leftovers of tilapia and spirulina is respectively adopted
Powder is protein raw materials, prepares polypeptide with composite protease hydrolysis, the results show that the compound protease is to a variety of common animals and plants eggs
Bai Jun shows higher hydrolysis efficiency, digests the polypeptide yield of substrate protein usually 83% or more, the degree of hydrolysis of polypeptide is reachable
To 14% or more, for the polypeptide product being prepared based on small peptide, the peptide chain length that is averaged is 100/14=7.14 amino acid,
Thus the average molecular weight of small peptide illustrates that the peptide key type of cutting may be selected in difference in the compound protease in 1000Da or so
There is higher coating ratio on the peptide chain of substrate protein, this further demonstrates the synergy of the compound protease.
Detailed description of the invention
Fig. 1 is a kind of protease preparation list of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention
Schematic diagram；
Fig. 2 is a kind of peptide bond selectivity of the protease of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention
Analyze result schematic diagram；
Fig. 3 is a kind of different protease of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to soybean egg
The comparison schematic diagram of white hydrolysis effect；
Fig. 4 is the electrophoresis of polypeptide in a kind of hydrolyzate of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention
Analyze schematic diagram；
Fig. 5 is that a kind of protease P 1 of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention compounds ratio with P4
Example influences schematic diagram to soya-bean polypeptides yield；
Fig. 6 is that a kind of protease P 1 of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention compounds ratio with P7
Example influences schematic diagram to soya-bean polypeptides yield；
Fig. 7 is that a kind of protease P 1 of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention compounds ratio with P9
Example influences schematic diagram to soya-bean polypeptides yield；
Fig. 8 is that a kind of protease P 4 of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention compounds ratio with P7
Example influences schematic diagram to soya-bean polypeptides yield；
Fig. 9 is that a kind of protease P 4 of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention compounds ratio with P9
Example influences schematic diagram to soya-bean polypeptides yield；
Figure 10 is that a kind of protease P 7 of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention is compounded with P9
Ratio influences schematic diagram to soya-bean polypeptides yield；
Figure 11 is a kind of different composite protease pair of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention
The comparison schematic diagram of soybean protein hydrolysis efficiency；
Figure 12 is a kind of different composite protease pair of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention
The comparison schematic diagram of soybean protein hydrolysis efficiency；
Figure 13 is a kind of compound protease of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to soybean
The comparison schematic diagram of proteolysis effect；
Figure 14 is a kind of enzyme concentration of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to soybean protein
The influence schematic diagram of hydrolysis process；
Figure 15 is a kind of temperature of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to soybean protein water
Solve the influence schematic diagram of technique；
Figure 16 is a kind of pH of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to soybean protein hydrolysis
The influence schematic diagram of technique；
Figure 17 is a kind of enzymolysis time of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to soybean egg
The influence schematic diagram of white hydrolysis process；
Figure 18 is a kind of compound protease of the compound protease prepared suitable for albumen effectively hydrolyzing and small peptide of the present invention to difference
The hydrolysis effect schematic diagram of substrate protein.
Specific embodiment
Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
The present invention provides a kind of technical solution: a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide, packet
Include strain, mold fermentation culture medium, bacterial fermentation culture medium, Tilapia mossambica intestinal protease, protease purification sample, proteolysis
Liquid and compound protease, strain include for aspergillus oryzae, Penicillium citrinum, Mucor, Methylotrophic bacillus, bacillus licheniformis,
Bacillus subtilis and bafillus natto, mold fermentation culture medium include test tube slant culture medium and fermentation medium, bacterium
Fermentation medium includes test tube slant culture medium, liquid seed culture medium and fermentation medium.
Test tube slant culture medium in mold fermentation culture medium are as follows: test tube slant culture medium: potato 200g liquor, glucose
20g, 15~20g of agar, water 1000mL, natural pH121 DEG C of sterilizing 20min, the fermentation medium in mold fermentation culture medium are as follows:
12 grams of wheat bran of dress in every 250mL triangular flask, 1.2 grams of peptone, 0.036 gram of dipotassium hydrogen phosphate, 0.024 gram of Tween-80,
1mol/L sodium hydroxide 1mL, water 8mL, 121 DEG C of sterilizing 20min.
Test tube slant culture medium (%) in bacterial fermentation culture medium are as follows: yeast powder 0.5, peptone 1, NaCl0.5, agar
1.5 ~ 2, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min, the liquid seed culture medium (%) in bacterial fermentation culture medium are as follows: yeast powder
0.5, peptone 1, NaCl0.5, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min, the fermentation medium (%) in bacterial fermentation culture medium:
0.1, pH7.5,121 DEG C of sucrose 4, peptone 2, wheat bran 3, potassium dihydrogen phosphate 0.3, calcium carbonate 0.3, Tween 80 sterilizing 20min.
The preparation step of Tilapia mossambica intestinal protease are as follows:
1) fresh Tilapia mossambica fish intestines, are taken, the NaCl solution that concentration is 0.15mol/L is added according to the ratio of 1:7, is smash with tissue
Broken machine breaks into pulpous state, is subsequently placed in and extracts 15min at room temperature；
2), leaching liquor is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, collects supernatant to get crude enzyme liquid is arrived；
3), above-mentioned enzyme solution is concentrated by ultrafiltration in the ultrafiltration membrane for being 30000Da with molecular cut off；
4) lactose, is added in enzyme solution after concentration, its concentration is made to reach 1%；Then enzyme solution is placed in freeze drier and is frozen
It is dry, obtain protease dry powder formulations.
The preparation step of protease purification sample are as follows:
1), DEAE-Sepharose anion exchange
With 0.02M, the Tris-HCl buffer balance DEAE-Sepharose anion-exchange column of pH7.5 (foundation balanced:
Efflux pH7.5, stable conductivity), a certain amount of enzyme powder 0.02M is taken, the Tris-HCl buffer solution of pH7.5 is loaded onto
Anion-exchange column, with 0.02M, the Tris-HCl buffer of pH7.5 is sufficiently washed after column (about 300ml), with 0.5MNaCl solution
(being dissolved in 0.02M, the Tris-HCl buffer of pH7.5) carries out ladder isocratic elution, collects activated protein peak, uses ultra-filtration centrifuge tube
Collection liquid is concentrated in (molecular cut off 10000Da), and with 0.05M, and pH5.0 acetate buffer solution adjusts its pH to 5.0；
2), CM-Sepharose cation exchanges
With 0.05M, pH5.0 acetate buffer solution balances CM-Sepharose cation exchange column, the concentration enzyme for taking previous step to collect
Liquid is loaded cation exchange column, after sufficiently washing column with equilibration buffer, (is dissolved in 0.05M, pH5.0 vinegar with the NaCl solution of 0.5M
Acid buffer) gradient elution is carried out, activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration；
3), Phenyl-Sepharose hydrophobic chromatography
Phenyl-Sepharose hydrophobic chromatography is balanced with 1.2M ammonium sulfate (being dissolved in 0.05M, the PBS buffer solution of pH7.5)
Column, the concentration enzyme solution that previous step is collected and 2.4M ammonium sulfate (being dissolved in 0.1M, the PBS buffer solution of pH7.5) are mixed in equal volume
With rear sample-adding Phenyl-Sepharose hydrophobic chromatography column, drainage column is sufficiently rinsed with equilibration buffer, gradient is then carried out and washes
It is de-, activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration；
4), with SuperdexG75 gel permeation chromatography
With 0.02M, the PBS buffer solution of pH7.5 balances Sepharose6B gel column, and the concentration enzyme solution that sample-adding previous step is collected is used
The PBS buffer solution of 0.02M, pH7.5 elute, and collecting activated protein peak is to purify enzyme solution.
The preparation step of protein hydrolyzate are as follows:
1) soy bean proteinous soln that substrate protein concentration is 5%, is prepared with distilled water, 20min is kept the temperature in 80 DEG C of water-baths, after cooling
Its pH value is adjusted to pH8.0, liquid of protease is added according to the enzyme concentration of 4000U/g albumen, mixes well and is placed on 50 DEG C of water-baths
Middle heat preservation digests 5h；
2) enzymolysis liquid pH value, is adjusted to 5.0, is kept the temperature 10min in 100 DEG C of water-baths and is carried out enzyme deactivation；
3) it, is centrifuged 10min under conditions of 4000r/min, collects supernatant and obtains protein hydrolyzate.
The step of compound protease are as follows:
1), using insulin oxidation of beta chain as substrate model, with the different protease hydrolytics substrate, using liquid phase and spectrometer analysis
Hydrolysis gained polypeptide fragment, determines restriction enzyme site of the various protease on insulin β chain, filters out and select complementation with peptide bond
The protease type of property；
2), using soybean protein as natural substrate, test is hydrolyzed using single protease and compounding protease, investigates different eggs
Synergistic action effect between white enzyme has carried out point hydrolyzate of soybean protein with Tricine-SDS-PAGE electrophoresis system
It is complementary further to analyze the selection of the peptide bond between each protease according to the electrophoretic band quantity of polypeptide and position for analysis, and thus
Determining, there are four kinds of protease P1, P4, P7 and P9 of preferable peptide bond selection complementary relationship to be combined the compound protease of building；
3), using soybean protein as substrate, different compound proteases is respectively adopted and carries out proteolysis assay, analyzes different composite albumen
Rational proportion and hydrolysis effect difference between enzyme component.
The compound proportion of compound protease is P1:P4:P7:P9=1:3:1:1, and the Optimum range of compound protease
For 3000~4000U/g albumen, best catalytic temperature range is 40~50 DEG C of ranges, most suitable catalytic pH range are as follows: pH8~10,
Suitable enzymolysis time is 4h.
The preparation of embodiment 1, protease preparation
1.1 strains:
Aspergillus oryzae；Penicillium citrinum；Mucor；Methylotrophic bacillus；Bacillus licheniformis；Bacillus subtilis；Natto gemma
Bacillus；
The measurement of 1.2 proteinase activities
By national standard using the enzyme activity of Folin method measurement neutral proteinase, prolease activity is defined as, and lmL enzyme solution is at 40 DEG C
Under conditions of pH7.5, it is 1 enzyme activity unit that caseinhydrolysate per minute, which generates l μ g tyrosine, is indicated with U/mL；
The preparation of 1.3 mold fermentations and protease
1.3.1 culture medium
(1) test tube slant culture medium: potato 200g liquor, glucose 20g, 15~20g of agar, water 1000mL, natural pH, 121
DEG C sterilizing 20min.
(2) fermentation medium: 12 grams of wheat bran of dress in every 250mL triangular flask, 1.2 grams of peptone, dipotassium hydrogen phosphate 0.036
Gram, 0.024 gram of Tween-80,1mol/L sodium hydroxide 1mL, water 8mL, 121 DEG C of sterilizing 20min.
1.3.2 mold fermentation culture
(1) aspergillus oryzae ferments: being inoculated with 1 ring test tube slant strain in the triangular flask equipped with fermentation medium, is placed on after shaking up
Constant temperature stationary culture 72h in 22 DEG C of environment；
(2) Penicillium citrinum ferments: being inoculated with 1 ring test tube slant strain in the triangular flask equipped with fermentation medium, is placed on after shaking up
Constant temperature stationary culture is for 24 hours in 32 DEG C of environment；24 DEG C are cooled to continue to cultivate 72h；
(3) Mucor is fermented: being inoculated with 1 ring test tube slant strain in the triangular flask equipped with fermentation medium, is placed on 24 after shaking up
DEG C environment in constant temperature stationary culture 72h.
1.3.3 the extraction of protease
The NaCl solution of 10mL concentration 0.3mol/L is added according to every gram of wheat bran in the wheat bran fermented, stirs evenly and is placed on 40 DEG C
Heat preservation extraction 30min, is filtered with gauze and absorbent cotton respectively, filtrate is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, received
Collecting supernatant is enzyme solution.
1.3.4 the concentration and freeze-drying of enzyme solution
Above-mentioned enzyme solution is concentrated by ultrafiltration in the ultrafiltration membrane for being 30000Da with molecular cut off, and enzyme solution volume is made to be reduced to substance
Long-pending 1/10th.Lactose is added in enzyme solution after concentration, its concentration is made to reach 1%；Then enzyme solution is placed in freeze drier
Middle freeze-drying obtains protease dry powder formulations.
The preparation of 1.4 bacterial fermentations and protease
1.4.1 culture medium
(1) test tube slant culture medium (%): yeast powder 0.5, peptone 1, NaCl0.5, agar 1.5 ~ 2, pH7.0 ~ 7.2,121 DEG C
Sterilize 20min；
(2) liquid seed culture medium (%): yeast powder 0.5, peptone 1, NaCl0.5, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min；
(3) fermentation medium (%): sucrose 4, peptone 2, wheat bran 3, potassium dihydrogen phosphate 0.3, calcium carbonate 0.3, Tween 80 0.1,
PH7.5,121 DEG C of sterilizing 20min；
1.4.2 liquid seeds culture
Pick them separately 1 ring activation after test tube slant strain be inoculated in 50mL seed culture medium, by triangular flask be placed in 35 DEG C,
Shake culture is for 24 hours on the shaking table of 200r/min.
1.4.3 fermented and cultured
(1) Methylotrophic fermentation of bacillus culture: drawing the switching of 2mL seed culture fluid in 50mL fermentation medium, will
Triangular flask be placed in 32 DEG C, the shaking table shake culture of 200r/min for 24 hours；It cools to 28 DEG C and continues culture to 96h.
(2) fermentation of bacillus subtilis culture: the switching of 2mL seed culture fluid is drawn in 50mL fermentation medium, by three
Angle bottle is placed in the shaking table shake culture 46h of 36 DEG C, 200r/min.
(3) bacillus natto to ferment culture: the switching of 2mL seed culture fluid is drawn in 50mL fermentation medium, by three
Angle bottle is placed in the shaking table shake culture 72h of 32 DEG C, 200r/min.
(4) the lichen bacillus ferments culture: the switching of 2mL seed culture fluid is drawn in 50mL fermentation medium, by three
Angle bottle is placed in the shaking table shake culture 72h of 35 DEG C, 200r/min.
1.4.4 the processing of fermentation liquid and enzyme solution freeze-drying
Fermentation liquid is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, collecting supernatant is enzyme solution.According to fermentation liquid enzyme activity
Just, above-mentioned enzyme solution is concentrated by ultrafiltration in the ultrafiltration membrane for being 30000Da with molecular cut off；It is added in enzyme solution after concentration
Lactose makes its concentration reach 1%；Then enzyme solution is placed in freeze drier and is lyophilized, obtain protease dry powder formulations.
The preparation of 1.5 Tilapia mossambica intestinal proteases
Fresh Tilapia mossambica fish intestines are taken, the NaCl solution that concentration is 0.15mol/L is added according to the ratio of 1:7, uses tissue mashing
Machine breaks into pulpous state, is subsequently placed in and extracts 15min at room temperature；Leaching liquor is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, is received
Collect supernatant to get crude enzyme liquid is arrived.Above-mentioned enzyme solution is concentrated by ultrafiltration in the ultrafiltration membrane for being 30000Da with molecular cut off；?
Lactose is added in enzyme solution after concentration, its concentration is made to reach 1%；Then enzyme solution is placed in freeze drier and is lyophilized, obtain albumen
Enzyme dry powder formulations.
1.6 protease preparation enzyme activity determination results
The enzyme activity that each protease preparation is measured using Folin- phenol method, as a result as shown in figure 1 shown in table 1.
1 protease preparation list of table
The peptide bond selectively analysis of embodiment 2, different protease
The preparation of 2.1 protease purification samples
2.1.1DEAE-Sepharose anion exchange
With 0.02M, the Tris-HCl buffer balance DEAE-Sepharose anion-exchange column of pH7.5 (foundation balanced:
Efflux pH7.5, stable conductivity), a certain amount of enzyme powder 0.02M is taken, the Tris-HCl buffer solution of pH7.5 is loaded onto
Anion-exchange column.With 0.02M, the Tris-HCl buffer of pH7.5 is sufficiently washed after column (about 300ml), with 0.5MNaCl solution
(being dissolved in 0.02M, the Tris-HCl buffer of pH7.5) carries out ladder isocratic elution, collects activated protein peak, uses ultra-filtration centrifuge tube
Collection liquid is concentrated in (molecular cut off 10000Da), and with 0.05M, and pH5.0 acetate buffer solution adjusts its pH to 5.0.
2.1.2CM-Sepharose cation exchange
With 0.05M, pH5.0 acetate buffer solution balances CM-Sepharose cation exchange column, the concentration enzyme for taking previous step to collect
Liquid is loaded cation exchange column.After sufficiently washing column with equilibration buffer, (0.05M, pH5.0 vinegar are dissolved in the NaCl solution of 0.5M
Acid buffer) carry out gradient elution.Activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration.
2.1.3Phenyl-Sepharose hydrophobic chromatography
Phenyl-Sepharose hydrophobic chromatography is balanced with 1.2M ammonium sulfate (being dissolved in 0.05M, the PBS buffer solution of pH7.5)
Column.The concentration enzyme solution that previous step is collected and 2.4M ammonium sulfate (being dissolved in 0.1M, the PBS buffer solution of pH7.5) are mixed in equal volume
With rear sample-adding Phenyl-Sepharose hydrophobic chromatography column, drainage column is sufficiently rinsed with equilibration buffer, gradient is then carried out and washes
It is de-.Activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration.
2.1.4 with SuperdexG75 gel permeation chromatography
With 0.02M, the PBS buffer solution of pH7.5 balances Sepharose6B gel column, and the concentration enzyme solution that sample-adding previous step is collected is used
The PBS buffer solution of 0.02M, pH7.5 elute,.Collecting activated protein peak is to purify enzyme solution.
The peptide bond of 2.2 protease is selectively analyzed
Using bovine insulin oxidation of beta chain as model substrates, respectively with above-mentioned protease hydrolytic bovine insulin oxidation of beta chain after purification,
Then the enzyme-digest fragment of insulin oxidation of beta chain is analyzed using liquid phase and mass spectrum, determines main small peptide in hydrolysate
Segment, and then determine restriction enzyme site of the protease on bovine insulin oxidation of beta chain.
2.2.1 the enzymatic hydrolysis of insulin oxidation of beta chain
Take 100 μ g(Sigma company reagent of bovine insulin oxidation of beta chain), with the Tris-HCl buffer solution of 0.2mLpH8.0, add
Enter protease 5U after purification, mixing is placed on 40 DEG C of water enzyme digestion 2h, and then boiling water bath 5min is inactivated.
2.2.2 the mass spectral analysis of enzymatic fragment
The enzymolysis liquid of bovine insulin oxidation of beta chain is loaded to liquid chromatogram and is separated: using NUCLEOSIL C18 chromatographic column
(250 × 10mm), 10 μ L of loading；Trifluoroacetic acid (TFA) solution of eluent A:0.1%；Eluent B is 60% acetonitrile+0.1%
Trifluoroacetic acid (TFA) solution；Elution flow rate 1ml/min；Gradient is 0-50%B, time 50min.The polypeptide that liquid phase separation goes out
Segment imports tandem mass spectrum and carries out molecular weight detection.The polypeptide fragment molecular weight results and insulin oxidation of beta chain that measurement is obtained
The molecular weight of random fragment is compared, and thus determines the amino acid sequence of each polypeptide fragment, and position insulin oxidation of beta chain
On cleavage site.
The peptide bond of 2 protease of table selectively analysis result
It can be seen that by comparing the above protease such as 14 kinds of separate sources in Fig. 2 table 2 in addition to P13 and P14 has complete phase
Same restriction enzyme site, the restriction enzyme site of other several protease have certain difference.On bovine insulin oxidation of beta chain, P1 tool
There are most restriction enzyme sites, followed by P7, and the restriction enzyme site of P9 is minimum；P1 contains most of other albuminoid enzymes (as)
Restriction enzyme site；P9 has the restriction enzyme site entirely different with other protease, in addition, the restriction enzyme site of P10 and other protease
Between also have biggish difference；Have the selection of preferable peptide bond complementary between tetra- kinds of protease of P1, P4, P7 and P9, by them
The compound protease of composition contains above 14 kinds of protease restriction enzyme site all on bovine insulin oxidation of beta chain substantially.
The analysis of synergistic action effect between 2.3 different protease
2.3.1 hydrolysate of soybean protein technique
The soy bean proteinous soln that substrate protein concentration is 5% is prepared with distilled water, 20min is kept the temperature in 80 DEG C of water-baths, is adjusted after cooling
Its pH value is added liquid of protease according to the enzyme concentration of 4000U/g albumen, mixes well to be placed in 50 DEG C of water-baths and protect to pH8.0
Temperature enzymatic hydrolysis 5h；Enzymolysis liquid pH value is adjusted to 5.0,10min is kept the temperature in 100 DEG C of water-baths and carries out enzyme deactivation；In the condition of 4000r/min
Lower centrifugation 10min collects supernatant and obtains protein hydrolyzate.
2.3.2 the detection of content of peptides and yield calculate in hydrolyzate
The measurement of content of peptides uses national standard GB/T22492-2008 method in hydrolyzate: taking protein hydrolyzate 5mL, concentration is added
The trichloroacetic acid 5mL of 2mol/L, stands 10min after mixing well；Then it is centrifuged 10min in 8000r/min, collects supernatant；
Using nitrogen content in Kjeldahl nitrogen determination supernatant.Polypeptide yield calculates in hydrolyzate:
Total nitrogen content in peptide nitrogen content/original enzymolysis liquid in polypeptide yield (%)=hydrolyzate
2.3.3 the hydrolysis effect of different protease and concertedness variance analysis
Using soybean protein as raw material, test is hydrolyzed with different protease respectively, further investigates the water of different protease
The synergistic effect between effect and protease is solved, as a result as shown in Fig. 3 table 3.
Comparison of the different protease of table 3 to soybean protein hydrolysis effect
From the results shown in Table 3, peptide bond selectivity and protease are between the hydrolysis efficiency and polypeptide yield of substrate protein
There is direct relationship, peptide bond is selectively wide in range, and high to the hydrolysis efficiency of substrate protein, polypeptide yield is high；In addition, protease
The distribution of all types of peptide bonds also has direct relationship in hydrolysis efficiency and soybean protein, for example, the peptide bond selectivity of trypsase
It is relatively narrow, but stronger hydrolysis ability is but shown to soybean protein, this should be with the lysine in soybean protein containing about 13%
And the peptide bond of arginine composition has substantial connection；Although and papain has more digestion position on insulin oxidation of beta chain
Point, but other protease are but significantly lower than to the hydrolysis efficiency of soybean protein, and do not have association substantially with other protease
Same-action effect.Concertedness analysis is the results show that between P1 and P4, P1 and P7, P1 and P9, P4 and P7, P4 and P9, P7 and P9
Obvious synergistic action effect is shown, the protease after compounding has higher hydrolysis efficiency and polypeptide to obtain soybean protein
Rate.
2.3.4 in hydrolyzate polypeptide electrophoretic analysis
Using Tricine-SDS-PAGE electrophoresis system, 16.5% separation gel (glycerol of 10% (V/V) of addition), 10% squeegee, 4%
Glue is concentrated.3min is boiled after hydrolyzate is mixed with electrophoresis sample-loading buffer, respectively takes 6uL point sample to loading hole, is then run in 30v
1h, to be instructed dose of forward position reach on separation gel along when, voltage is adjusted to 90v, constant pressure to electrophoresis terminates.Film after taking electrophoresis is used
Coomassie brilliant blue staining liquid dyes 1h, then decolourizes.Observe the distribution situation of polypeptide band on each swimming lane of electrophoresis film.As a result such as
Shown in Fig. 1.
As seen from Figure 4, the polypeptide fragment that tetra- kinds of protease P 1, P4, P7 and P9 albumen soy proteins obtain
Difference, thus illustrate their restriction enzyme sites on soybean protein peptide chain be it is different, there are certain complementations for restriction enzyme site
Effect；The compound protease to be formed is compounded with P9 through P1, P4, P7, stronger hydrolysis ability is shown to soybean protein, and produce
For the band of object polypeptide also different from single protease, this further confirms there is synergistic effect between these four protease, this
As a result consistent with the peptide bond selectivity result that table 1 is shown.Thus explanation has peptide bond choosing using the screening of above two method
Selecting complementary protease has feasibility.
(in Fig. 4: swimming lane 1: Ultra-low molecular weight marker；Swimming lane 2: soybean protein solution；Swimming lane 3:P4 protease hydrolytic liquid；
Swimming lane 4:P7 protease hydrolytic liquid；Swimming lane 5:P9 protease hydrolytic liquid；Swimming lane 6:P1 protease hydrolytic liquid；Swimming lane 7: compound protein
Enzyme (P1:P4:P7:P9=1:1:1:1) hydrolyzate)
The building of embodiment 3, compound protease
3.1 hydrolysate of soybean protein techniques
The soy bean proteinous soln that substrate protein concentration is 5% is prepared with distilled water, 20min is kept the temperature in 80 DEG C of water-baths, is adjusted after cooling
Its pH value is added liquid of protease according to the enzyme concentration of 4000U/g albumen, mixes well to be placed in 50 DEG C of water-baths and protect to pH8.0
Temperature enzymatic hydrolysis 5h；Enzymolysis liquid pH value is adjusted to 5.0,10min is kept the temperature in 100 DEG C of water-baths and carries out enzyme deactivation；In the condition of 4000r/min
Lower centrifugation 10min collects supernatant and obtains protein hydrolyzate.
The detection of content of peptides and yield calculate in 3.2 hydrolyzates
The measurement of content of peptides uses national standard GB/T22492-2008 method in hydrolyzate: taking protein hydrolyzate 5mL, concentration is added
The trichloroacetic acid 5mL of 2mol/L, stands 10min after mixing well；Then it is centrifuged 10min in 8000r/min, collects supernatant；
Using nitrogen content in Kjeldahl nitrogen determination supernatant.Polypeptide yield calculates in hydrolyzate:
Total nitrogen content in peptide nitrogen content/original enzymolysis liquid in polypeptide yield (%)=hydrolyzate
The determination of 3.3 binary compound proteases
Four kinds of protease P1, P4, P7 and P9 each other with preferable peptide bond selection complementary relationship are selected to carry out combination of two structure
Build the compound protease of binary.Using soybean protein as substrate, different compound proteases is respectively adopted and carries out proteolysis assay, analysis
Rational proportion and hydrolysis effect difference between different composite protease component.As a result as shown in Fig. 8 ~ 10.
Certain synergistic effect is shown between can be seen that compound two component of protease of binary from above-mentioned test result
The concertedness of effect, especially P1 and P7 is the most significant；The compound egg of binary constructed by protease P 1 and P7 according to the ratio of 1:3
White enzyme has strongest hydrolysis ability.
The compatibility of 3.4 compound proteases optimizes
Based on the binary compound protease that P1 and P7 is constructed according to the ratio of 1:3, protease P 4 and P9 pairs are further investigated
The auxiliaring effect of the binary compound protease.As a result as shown in figure 11.
(note: Tu11Zhong: the A0 compound protease compounded for P1 and P7 in 1:3 ratio；A1 is A0 and P4 according to 1:1 ratio
The compound protease of compounding；A2 is the compound protease that A0 and P4 is compounded according to 2:1 ratio；A3 is A0 and P4 according to 3:1 ratio
The compound protease of compounding；A4 is the compound protease that A0 and P4 is compounded according to 4:1 ratio；A5 is A0 and P4 according to 5:1 ratio
The compound protease of compounding；A6 is the compound protease that A0 and P4 is compounded according to 6:1 ratio；A7 is A0 and P4 according to 7:1 ratio
The compound protease of compounding).
(note: Tu12Zhong: the A0 compound protease compounded for P1 and P7 in 1:3 ratio；B1 is A0 and P9 according to 1:1 ratio
The compound protease of compounding；B2 is the compound protease that A0 and P9 is compounded according to 2:1 ratio；B3 is A0 and P9 according to 3:1 ratio
The compound protease of compounding；B4 is the compound protease that A0 and P9 is compounded according to 4:1 ratio；B5 is A0 and P9 according to 5:1 ratio
The compound protease of compounding；B6 is the compound protease that A0 and P9 is compounded according to 6:1 ratio；B7 is A0 and P9 according to 7:1 ratio
The compound protease of compounding).
By the result of Figure 11 and Figure 13 it can be seen that P4 and P9 to binary compound protease A0(by P1 and P7 in 1:3 ratio
The compound protease of compounding) have the effect of certain synergistic effect, especially P9 more significant, compound according to a certain percentage and
At the hydrolysis efficiency of tri compound protease be apparently higher than binary compound protease；According to above-mentioned test result determined A0 with
The proper ratio of P9 is 4:1, i.e., the compound scheme of tri compound protease is P1:P7:P9=1:3:1.
In view of all there is certain synergistic action effect, and the peptide bond selectivity of P4 between protease P 4 and A0 and P9
There is certain complementarity really between P1, P7 and P9, the synergy of P9 has further been investigated on the basis of above-mentioned ternary built
Effect, as a result as shown in figure 13.
(note: Tu13Zhong: the C0 compound protease compounded for P1:P7:P9=1:3:1；C1 is P1:P7:P9:P4=3:1:1:1
The compound protease of the compound protease of compounding；C2 is the compound protease of P1:P7:P9:P4=3:1:1:2 compounding；C3 is P1:
The compound protease of P7:P9:P4=3:1:1:3 compounding；C4 is the compound protease of P1:P7:P9:P4=3:1:1:4 compounding；C5 is
The compound protease of P1:P7:P9:P4=3:1:1:5 compounding；C6 is the compound protease of P1:P7:P9:P4=3:1:1:2 compounding；
C7 is the compound protease of P1:P7:P9:P4=3:1:1:7 compounding).
By the result of Figure 13 it is found that further auxiliary addition 4 pairs of improvement of protease P are compound on the basis of former tri compound
The hydrolysis efficiency of protease is not significant, and weaker facilitation is only shown in low adding proportion, and as P4 adds ratio
The increase of example, the hydrolysis efficiency of compound protease is sharp fall instead.In view of protease P 4 peptide bond selectivity with P1,
It is implicitly present in certain complementarity between P7 and P9, selects face and to different substrate proteins to widen the peptide bond of compound protease
Adaptability determines the P4 protease component that low ratio is assisted in compound protease.Thus quaternary compound protein has finally been determined
The compound proportion of enzyme is P1:P4:P7:P9=1:3:1:1.
The technique that embodiment 4, composite protease hydrolysis soybean protein prepare polypeptide
4.1 basis enzymatic hydrolysis process conditions
The soy bean proteinous soln that substrate protein concentration is 50g/L is prepared with distilled water, 20min is kept the temperature in 80 DEG C of water-baths, after cooling
Its pH value is adjusted to pH8.0, compound protein enzyme solution is added according to the enzyme concentration of 4000U/g albumen, mixes well and is placed on 50 DEG C
Heat preservation enzymatic hydrolysis 5h in water-bath；Enzymolysis liquid pH value is adjusted to 4.5,10min is kept the temperature in 100 DEG C of water-baths and carries out enzyme deactivation；In 4000r/
It is centrifuged 10min under conditions of min, measures peptide concentration after collecting supernatant constant volume.
4.2 compound protein enzyme dosages
Using compound protease, respectively with 500U/g albumen, 1000U/g albumen, 2000U/g albumen, 3000U/g albumen,
4000U/g albumen, 5000U/g albumen, 6000U/g albumen, 7000U/g albumen additive amount, digested according to 4.1 method
Test measures the polypeptide yield of beans hydrolyzate, thereby determines that the Optimum of compound protease.
As shown in Figure 14, enzyme concentration has a significant impact enzymolysis process: when protein concentration is 50g/L, with enzyme concentration
Increase, the trend constantly risen is presented in the polypeptide yield of enzymatic hydrolysis system；When enzyme concentration reaches 3000U/g or more, polypeptide yield
Growth is slower, illustrates that enzyme amount at this time has tended to be saturated relative to substrate protein.It can determine compound protein according to test result
The Optimum of enzyme is in 3000~4000U/g albumen.
The optimum temperature of 4.3 compound proteases
Using compound protease, according to 4.1 method respectively at 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C
Under conditions of carry out proteolysis assay, measure the polypeptide yield of hydrolyzate, investigate the suitable catalyst temperature of compound protease.
As shown in Figure 15, the optimum temperature of the compound protease is in 40~50 DEG C of ranges.
The most suitable action pH of 4.4 compound proteases
Using compound protease, according to 4.1 method respectively in pH5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0
Under the conditions of carry out proteolysis assay, measure the polypeptide yield of hydrolyzate, thereby determine that the most suitable action pH of compound protease.
As shown in Figure 16, which has check and correction preferable catalytic activity in the range of pH7 to 12, in pH8
There is maximum catalytic efficiency under conditions of~10.
The appropriate effect time of 4.5 compound proteases
Using compound protease, carry out proteolysis assay according to 4.1 the methods, respectively enzymatic hydrolysis 0.5h, 1h, 2h, 3h, 4h, 5h,
The polypeptide yield of hydrolyzate is measured by sampling when 6h, 7h, thereby determines that the appropriate effect time range of compound protease.
As shown in Figure 17, at the initial stage of enzymatic hydrolysis (0~3h), proteolysis fast speed, polypeptide yield is increased rapidly；In enzyme
The mid-term (3~4h) of solution, proteolysis speed slow down gradually；After enzymatic hydrolysis is to 4h, the polypeptide yield of system tends towards stability, explanation
Proteolysis has reached reaction balance.Thus can determine, reach under the hydrolysising condition set enzymatic hydrolysis balance needed for time as
4h。
According to above-mentioned test result, it may be determined that the Optimum of the compound protease is 3000~4000U/g albumen, most preferably
Catalytic temperature is in 40~50 DEG C of ranges, and for most suitable catalytic pH in pH8~10, suitable enzymolysis time is 4h.
The comparative analysis of embodiment 5, compound protease to different substrate protein hydrolysis effects
The measurement of 5.1 degree of hydrolysis
(albumen) total nitrogen content in Kjeldahl nitrogen determination original hydrolyzate；
Using amino-acid nitrogen content in formol titration measurement hydrolyzate；
Degree of hydrolysis (DH) definition: the peptide bond number being cleaved in protein hydrolytic process accounts for the percentage of the total peptide bond number of given protein
Number.
Degree of hydrolysis (%)=(amino-acid nitrogen content/hydrolyzate Central Plains albumen total nitrogen content in hydrolyzate) × 100
The preparation of 5.2 hydrolyzates
Respectively with soyabean expeller, the semen sojae atricolor dregs of rice, Gluten, peanut meal, dephenolize cotton dregs, Symeplectoteuthis oualaniensis, leftovers of tilapia and spirulina powder
For raw material, preparation protein concentration is 50g/L, the compound protease of 3500U is added by every gram of albumen, in pH9.0,45 DEG C of condition
Lower enzymatic hydrolysis 4h.Enzymolysis liquid pH value is adjusted to 4.5,10min is kept the temperature in 100 DEG C of water-baths and carries out enzyme deactivation；In the condition of 4000r/min
Lower centrifugation 10min measures peptide concentration and amino-acid nitrogen content, and the polypeptide yield and water of counting system after collecting supernatant constant volume
Xie Du, as a result as shown in Figure 18 table 4.
Hydrolysis effect of 4 compound protease of table to different substrate proteins
Compound protease constructed by the present invention has preferable substrate adaptability it can be seen from Figure 18 table 4, to a variety of normal
See that animal/vegetable protein shows higher hydrolysis efficiency: under the most suitable catalytic condition of the compound protease, digesting substrate protein
White polypeptide yield 83% or more, be significantly higher than existing document announcement as a result, thus illustrating that this compound protease is optional
The peptide key class for selecting cutting has higher coating ratio on the peptide chain of a variety of substrate proteins, this sufficiently shows the compound egg
The advantage of white enzyme synergy.In addition, the degree of hydrolysis testing result to polypeptide is shown, the composite protease hydrolysis difference substrate protein
The degree of hydrolysis of white gained polypeptide has significant difference, this should be formed from the peptide chain of different substrate proteins direct relation.It is overall
For, the degree of hydrolysis for preparing gained polypeptide through composite protease hydrolysis is higher, can generally reach 14% or more, individually very
To can achieve 30% or so, this absolutely proves that compound protease can realize the depth hydrolysis of a variety of substrate proteins, is prepared
For polypeptide product based on small peptide, average peptide length is 100/14=7.14 amino acid, and the average molecular weight of small peptide exists
1000Da or so.
The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field, although
Present invention has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, still can be right
Technical solution documented by foregoing embodiments is modified or equivalent replacement of some of the technical features, it is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in protection of the invention
Within the scope of.
Claims (8)
1. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide, including strain, mold fermentation culture medium, bacterium
Fermentation medium, Tilapia mossambica intestinal protease, protease purification sample, protein hydrolyzate and compound protease, it is characterised in that:
The strain includes for aspergillus oryzae, Penicillium citrinum, Mucor, Methylotrophic bacillus, bacillus licheniformis, bacillus subtilis
And bafillus natto, the mold fermentation culture medium include test tube slant culture medium and fermentation medium, the bacterial fermentation
Culture medium includes test tube slant culture medium, liquid seed culture medium and fermentation medium.
2. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 1, feature exist
In: the test tube slant culture medium in the mold fermentation culture medium are as follows: test tube slant culture medium: potato 200g liquor, glucose
20g, 15~20g of agar, water 1000mL, natural pH121 DEG C of sterilizing 20min, the fermented and cultured in the mold fermentation culture medium
Base are as follows: 12 grams of wheat bran of dress in every 250mL triangular flask, 1.2 grams of peptone, 0.036 gram of dipotassium hydrogen phosphate, Tween-80 0.024
Gram, 1mol/L sodium hydroxide 1mL, water 8mL, 121 DEG C of sterilizing 20min.
3. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 1, feature exist
In: the test tube slant culture medium (%) in the bacterial fermentation culture medium are as follows: yeast powder 0.5, peptone 1, NaCl0.5, agar
1.5 ~ 2, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min, the liquid seed culture medium (%) in the bacterial fermentation culture medium are as follows: yeast
Powder 0.5, peptone 1, NaCl0.5, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min, the fermented and cultured in the bacterial fermentation culture medium
Base (%): sucrose 4, peptone 2, wheat bran 3, potassium dihydrogen phosphate 0.3, calcium carbonate 0.3,0.1, pH7.5,121 DEG C of Tween 80 sterilizings
20min。
4. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 1, feature exist
In: the preparation step of the Tilapia mossambica intestinal protease are as follows:
1) fresh Tilapia mossambica fish intestines, are taken, the NaCl solution that concentration is 0.15mol/L is added according to the ratio of 1:7, is smash with tissue
Broken machine breaks into pulpous state, is subsequently placed in and extracts 15min at room temperature；
2), leaching liquor is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, collects supernatant to get crude enzyme liquid is arrived；
3), above-mentioned enzyme solution is concentrated by ultrafiltration in the ultrafiltration membrane for being 30000Da with molecular cut off；
4) lactose, is added in enzyme solution after concentration, its concentration is made to reach 1%；Then enzyme solution is placed in freeze drier and is frozen
It is dry, obtain protease dry powder formulations.
5. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 1, feature exist
In: the preparation step of the protease purification sample are as follows:
1), DEAE-Sepharose anion exchange
With 0.02M, the Tris-HCl buffer balance DEAE-Sepharose anion-exchange column of pH7.5 (foundation balanced:
Efflux pH7.5, stable conductivity), a certain amount of enzyme powder 0.02M is taken, the Tris-HCl buffer solution of pH7.5 is loaded onto
Anion-exchange column, with 0.02M, the Tris-HCl buffer of pH7.5 is sufficiently washed after column (about 300ml), with 0.5M NaCl solution
(being dissolved in 0.02M, the Tris-HCl buffer of pH7.5) carries out ladder isocratic elution, collects activated protein peak, uses ultra-filtration centrifuge tube
Collection liquid is concentrated in (molecular cut off 10000Da), and with 0.05M, and pH5.0 acetate buffer solution adjusts its pH to 5.0；
2), CM-Sepharose cation exchanges
With 0.05M, pH5.0 acetate buffer solution balances CM-Sepharose cation exchange column, the concentration enzyme for taking previous step to collect
Liquid is loaded cation exchange column, after sufficiently washing column with equilibration buffer, (is dissolved in 0.05M, pH5.0 vinegar with the NaCl solution of 0.5M
Acid buffer) gradient elution is carried out, activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration；
3), Phenyl-Sepharose hydrophobic chromatography
Phenyl-Sepharose hydrophobic chromatography is balanced with 1.2M ammonium sulfate (being dissolved in 0.05M, the PBS buffer solution of pH7.5)
Column, the concentration enzyme solution that previous step is collected and 2.4M ammonium sulfate (being dissolved in 0.1M, the PBS buffer solution of pH7.5) are mixed in equal volume
With rear sample-adding Phenyl-Sepharose hydrophobic chromatography column, drainage column is sufficiently rinsed with equilibration buffer, gradient is then carried out and washes
It is de-, activated protein peak is collected, the enzyme solution collected with ultra-filtration centrifuge tube concentration；
4), with SuperdexG75 gel permeation chromatography
With 0.02M, the PBS buffer solution of pH7.5 balances Sepharose6B gel column, and the concentration enzyme solution that sample-adding previous step is collected is used
The PBS buffer solution of 0.02M, pH7.5 elute, and collecting activated protein peak is to purify enzyme solution.
6. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 1, feature exist
In: the preparation step of the protein hydrolyzate are as follows:
1) soy bean proteinous soln that substrate protein concentration is 5%, is prepared with distilled water, 20min is kept the temperature in 80 DEG C of water-baths, after cooling
Its pH value is adjusted to pH8.0, liquid of protease is added according to the enzyme concentration of 4000U/g albumen, mixes well and is placed on 50 DEG C of water-baths
Middle heat preservation digests 5h；
2) enzymolysis liquid pH value, is adjusted to 5.0, is kept the temperature 10min in 100 DEG C of water-baths and is carried out enzyme deactivation；
3) it, is centrifuged 10min under conditions of 4000r/min, collects supernatant and obtains protein hydrolyzate.
7. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 1, feature exist
In: the compound protease the step of are as follows:
1), using insulin oxidation of beta chain as substrate model, with the different protease hydrolytics substrate, using liquid phase and spectrometer analysis
Hydrolysis gained polypeptide fragment, determines restriction enzyme site of the various protease on insulin β chain, filters out and select complementation with peptide bond
The protease type of property；
2), using soybean protein as natural substrate, test is hydrolyzed using single protease and compounding protease, investigates different eggs
Synergistic action effect between white enzyme has carried out point hydrolyzate of soybean protein with Tricine-SDS-PAGE electrophoresis system
It is complementary further to analyze the selection of the peptide bond between each protease according to the electrophoretic band quantity of polypeptide and position for analysis, and thus
Determining, there are four kinds of protease P1, P4, P7 and P9 of preferable peptide bond selection complementary relationship to be combined the compound protease of building；
3), using soybean protein as substrate, different compound proteases is respectively adopted and carries out proteolysis assay, analyzes different composite albumen
Rational proportion and hydrolysis effect difference between enzyme component.
8. a kind of compound protease prepared suitable for albumen effectively hydrolyzing and small peptide according to claim 7, feature exist
In: the compound proportion of the compound protease is P1:P4:P7:P9=1:3:1:1, and the Optimum range of compound protease is
3000~4000U/g albumen, best catalytic temperature range are 40~50 DEG C of ranges, most suitable catalytic pH range are as follows: pH8~10 are fitted
Suitable enzymolysis time is 4h.
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