CN105925650A - Method for degrading casein by utilizing recombinant bacillus subtilis proline aminopeptidase - Google Patents
Method for degrading casein by utilizing recombinant bacillus subtilis proline aminopeptidase Download PDFInfo
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- CN105925650A CN105925650A CN201610382986.2A CN201610382986A CN105925650A CN 105925650 A CN105925650 A CN 105925650A CN 201610382986 A CN201610382986 A CN 201610382986A CN 105925650 A CN105925650 A CN 105925650A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/14—Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
- C12Y304/14012—Xaa-Xaa-Pro tripeptidyl-peptidase (3.4.14.12), i.e. prolyltripeptidyl aminopeptidase
Abstract
The invention discloses a method for degrading casein by utilizing recombinant bacillus subtilis proline aminopeptidase, and belongs to the fields of enzyme engineering and food additives. The method for degrading the casein by utilizing the recombinant bacillus subtilis proline aminopeptidase hydrolyzes the casein by utilizing proline aminopeptidase and through cooperation with alkaline protease and leucine aminopeptidase; the content of free amino acids is 3.968mg/mL, and is 63 times of the content of free amino acids before hydrolysis; polypeptide above 2,000Da does not exist basically; the content of small peptide below 180Da reaches 36.66%; exposed N-terminal proline residues are sufficiently hydrolyzed; the content of free proline is 0.044mg/mL, and is 3.67 times of the content of free proline which is not hydrolyzed; a hydrolysis extent of the proline is improved. The method for degrading the casein by utilizing the recombinant bacillus subtilis proline aminopeptidase has quite good application prospects in fields of food, beverages, and processing and utilization of food protein resources.
Description
Technical field
The present invention relates to one and utilize the recombinant bacillus proline aminopeptidase caseic method of degraded, belong to enzyme preparation, food interpolation
Agent field.
Background technology
Enzyme preparation refers to the material with enzyme character extracted from biology, now has been supplied in medicine, chemical industry, food, brewages
Etc. each field, range of application is the most extensive.Food processing industry and the life close relation of people, enzyme is in food processing industry
Application is more and more, acts on more and more important, meat packing, the depth hydrolysis of protein and as food additive in have
Very gross appearance.
Enzyme preparation is more extensive along with its application of development of society, and aminopeptidase is also constantly opening up its application.Non-
The hydrolyzed protein of special aminopeptidase energy wide spectrum or the amino acid residue of polypeptide N-end, can be the most of the same race with hydrolyzed peptide or n-end of albumen
The amino acid residue of class, dissociate different types of aminoacid.Casein hydrolysate can apply to food additive, inscribe egg
Casein can be hydrolyzed by white enzyme, but the product after hydrolysis contains strong bitterness, thus limit it and be widely applied.
Further investigation revealed that these bitterness derive from the intermediate peptide containing proline that casein is formed after protease hydrolysis, cheese
In albumen, proline content is higher, particularly beta-casein, has 34 proline in its 209 aminoacid sequences.For non-spy
For the aminopeptidase of the opposite sex, they can hydrolyze most amino acid residue from peptide termini, but but can not cut N-terminal
Polypeptide for proline.Therefore, how caseic degree of hydrolysis, the content increasing little peptide and trip are improved by the Synergistic degradation of enzyme
From amino acid content, be also current problem demanding prompt solution.
Summary of the invention
In order to solve the problems referred to above, the present invention utilizes proline aminopeptidase to work in coordination with alkaline protease and leucine amino peptidase degraded cheese egg
In vain, result substantially increases caseic degree of hydrolysis, adds the content of little peptide and free amino acid content.This laboratory is raw
The proline aminopeptidase produced is a kind of aminopeptidase with strict substrate specificity, it can only the proline of single-minded hydrolyzing N end residual
Base, to Pro-Leu, Pro-Lys, Pro-Pro, Leu-Pro, Pro-Phe-Gly-Lys,
When 6 kinds of dipeptides such as Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys or many hydrolase polypeptides, show the different ends
Thing selectivity and hydrolysis rate.The present invention utilizes proline aminopeptidase can specifically excise the proline residue of N-terminal, permissible
Remove the highest aminopeptidase of some specificitys and hydrolyze the obstacle of Pro further, the protein raw materials kind that proline content is high can be risen
Preferably synergetic hydrolysis and de-hardship effect.
The invention provides a kind of caseic method for hydrolysis, be successively with alkaline protease, leucine amino peptidase, proline ammonia
Peptidase is hydrolyzed.
Described method, be preparation mass concentration be the casein solution of 1-3%,
(1) add appropriate alkaline protease, at 40-50 DEG C, react 2.5-3.5h, boiling water bath 10-20min；
(2) appropriate leucine amino peptidase is continuously added after cooling, 45-50 DEG C of reaction 2.5-3.5h, boiling water bath 10-20min；
(3) appropriate proline aminopeptidase is added after cooling, 45-50 DEG C of reaction 2.5-3.5h, boiling water bath 10-20min.
Described step (1) neutral and alkali protease addition is 30000-34000U/g casein.
In described step (2), leucine amino peptidase addition is 800-1000U/g casein.
In described step (3), proline aminopeptidase addition is 400-500U/g casein.
In one embodiment of the invention, described casein solution is to use PBS preparation.
In one embodiment of the invention, described PBS concentration 50mM, pH7.5.
In one embodiment of the invention, the nucleotide sequence of described proline aminopeptidase is as shown in SEQ ID NO.1.
In one embodiment of the invention, described proline aminopeptidase utilizes recombinant bacterium to produce, purification obtains.Described
Recombinant bacterium is on the basis of early stage builds the recombined bacillus subtilis of (Chinese patent 201510497845.0), at coding dried meat
The upstream of propylhomoserin aminopeptidase gene adds the gene of encoding histidine label, and obtains.
In one embodiment of the invention, described purification, is by centrifugal for the fermentation liquor of recombinant bacterium, ultrafiltration, dialysis, nickel
The electrophoretically pure proline aminopeptidase that affinity chromatograph, dialysis obtain.
In one embodiment of the invention, described method, specifically: with the PBS preparation of 50mM pH7.5
The casein solution of 2%；Then,
(1) appropriate basic protein enzyme powder is added, 50 DEG C of reactions 3h, boiling water bath 15min；
(2) appropriate leucine amino peptidase powder is continuously added after cooling, 50 DEG C of reactions 3h, boiling water bath 15min；
(3) appropriate proline aminopeptidase enzyme liquid is added after cooling, 50 DEG C of reactions 3h, boiling water bath 15min.
After described step (3) reaction, by gained hydrolyzed solution centrifugal 15min under conditions of 10000rpm after cooling, take supernatant
Liquid；Being diluted with 10% trichloroacetic acid equal-volume by supernatant, place 1h, then with syringe filter, filtrate is in 10000
Rpm is centrifuged 10min, again takes supernatant.Carry out polypeptide molecular weight distribution, free amino acid analysis the most respectively.
The present invention is hydrolyzed with different small molecule substrates, and hydrolysis rate is as index, it is judged that the substrate selective of this enzyme；With
Casein is substrate, this enzyme and alkaline protease, leucine amino peptidase synergetic hydrolysis, with little peptide and free aminoacid content for depending on
According to, optimize proline aminopeptidase effect in casein degradation.
The present invention also provide for described method in Food & Drink, utilize the application in field in food protein resources processing.
The present invention also provides for a kind of method of proline aminopeptidase described in purification, is by recombinant bacterium fermentation liquid centrifuging and taking supernatant, obtains slightly
Enzyme liquid, the ultra-filtration centrifuge tube of recycling molecular cut off 30kDa is concentrated by ultrafiltration, and the ultrafiltration concentration liquid obtained is in buffer A
Middle dialysed overnight, takes dialysis solution loading nickel affinity chromatography post, and flow velocity keeps 1mL/min, first by the buffer A of 5 column volumes
Eluting, then with the buffer B eluting of 5 column volumes, change according to UV-detector reading, collect eluent in batches, will
The eluent arrived, dialysed overnight in Tris-HCl pH7.5 buffer, i.e. obtain proline aminopeptidase pure enzyme liquid.
Beneficial effects of the present invention:
Proline aminopeptidase of the present invention passes through ni-sepharose purification, simplifies extraction process, and the selectivity of small molecule substrates, for proline
The application of aminopeptidase provides reference, after this enzyme works in coordination with alkaline protease and leucine amino peptidase caseinhydrolysate, and free amino acid
Content is 3.968mg/mL, and for 63 times of the free aminoacid content before not hydrolyzing, the polypeptide of more than 2000Da is the most
Do not exist, and the little peptide content of below 180Da reached 36.66%, be fully hydrolyzed the N end proline residue exposed,
Free proline content is 0.044mg/mL, is do not hydrolyze free proline content 3.67 times, improves casein hydrolysis
The degree of depth, has good application prospect in Food & Drink.
Accompanying drawing explanation
Fig. 1: SDS-PAGE result (M: low molecular weight protein (LMWP) standard, 1: crude enzyme liquid, 2: nickel affinity chromatography eluent).
Detailed description of the invention
Buffer A: 40mmol/L imidazoles, 50mM Tris-HCl (pH7.5) and 0.5mol/L NaCl.
Buffer B: 0.5mol/L imidazoles, 50mM Tris-HCl (pH7.5) and 0.5mol/L NaCl.
Proline aminopeptidase enzyme activity determination method: (the substrate stock solution Tris-HCl 7.5 with L-PROLINE-paranitroanilinum as substrate
Preparation, concentration is 4.25mM), reactant mixture includes the enzyme liquid after 1mL dilution, 2mL Tris-HCl 7.5 buffer and 1
ML substrate stock solution, 50 DEG C of water-bath 10min, at 405nm, measure light absorption value.
Needed for enzyme unit (U) alive is defined as 50 DEG C of decomposition L-PROLINEs per minute-paranitroanilinum 1 μM of paranitroanilinum of generation
Enzyme amount.
Polypeptide molecular weight distribution detection: Waters600 high performance liquid chromatograph (joins 2487 UV-detector and Empower work
Stand).
The mensuration of free amino acid in hydrolyzed solution: use aminoacids solution chromatography (Ag1100) to be measured.
Embodiment 1: the preparation of recombinant bacillus proline aminopeptidase
Take the recombined bacillus subtilis WB600 fermentation liquid that 40mL cultivates through 24h, 10000rpm, under the conditions of 4 DEG C from
Heart 10min, takes supernatant, abandons precipitation, obtains crude enzyme liquid, then is concentrated by ultrafiltration, used for molecular cut off 30kDa ultrafiltration
Centrifuge tube, ultra-filtration conditions is 5000rpm, 4 DEG C of centrifugal 20min, and cycles of concentration controls at 3-4 times, be concentrated by ultrafiltration liquid 4 DEG C,
Dialysed overnight in 1L buffer A, takes 5mL dialysis solution loading nickel affinity chromatography post, and flow velocity keeps 1mL/min, first with 5
The buffer A eluting of column volume, then with the buffer B eluting of 5 column volumes, change according to UV-detector reading, in batches
Collecting eluent, the eluent that will obtain, in Tris-HCl pH7.5 buffer, 4 DEG C of dialysed overnight, dialysis solution is carried out
SDS-PAGE。
Embodiment 2: recombinant bacillus proline aminopeptidase small molecule substrates selectivity
With PBS pH 7.5 buffer of 0.05M configure the dipeptides of 1mM synthesis and polypeptide: Pro-Leu, Pro-Lys, Pro-Pro,
Leu-Pro、Pro-Phe-Gly-Lys、Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys。
Take dipeptides and the polypeptide solution of 0.9mL respectively, be subsequently adding 0.1mL and dilute through PBS pH 7.5 buffer of 0.05M
Enzyme liquid (enzyme liquid is that method described in embodiment 1 obtains, and enzyme is lived as 15.4U/mL).
After reacting 10min at 50 DEG C, addition 2.5mL acetic acid and 2.5mL ninhydrin solution immediately, boiling water bath 30min,
Surveying light absorption value at 480nm, blank is that PBS pH 7.5 buffer of 0.1mL0.05M replaces dilution enzyme liquid, with hydrolysis
Ability soprano is 100%.Result shows: the relative activity of hydrolysis Pro-Leu is 69.5%, relative activity to Pro-Lys
Be 74.4%, to Pro-Pro relative activity be 44.1%, to the relative activity of Leu-Pro be 0, to Pro-Phe-Gly-Lys's
Relative activity is 100%, relative activity to Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys is 17.4%.
The hydrolysis ability of different small molecule substrates be there are differences by restructuring proline aminopeptidase, and small molecule substrates hydrolysis is had selectivity.
Embodiment 3: recombinant bacillus proline aminopeptidase effect in casein degradation
Prepare the casein solution of 2% with the PBS of 50mM pH7.5, add the caseic basic protein of 32000U/g
Enzyme powder, 50 DEG C of reactions 3h, boiling water bath 15min, continuously add 900U/g caseic leucine amino peptidase powder after cooling,
50 DEG C of reactions 3h, boiling water bath 15min, add 450U/g caseic restructuring proline aminopeptidase enzyme liquid and (implement after cooling
Example 1 method gained), 50 DEG C of reactions 3h, boiling water bath 15min, cooling, the polypeptide of more than 2000Da in casein hydrolyzate
Not existing, and the little peptide content of below 180Da has reached 36.66%, free proline content is 0.044
Mg/mL, is do not hydrolyze free proline content 3.67 times.
Additionally, inventor also compares the different enzyme solution impact on casein hydrolysis effect, result is as shown in table 1.Result
Display, when alkaline protease individually hydrolyzes, the little peptide content of below 180Da is 23.28%, during leucine amino peptidase single enzymolysis
The little peptide content of below 180Da is 6.17%, the little peptide of below 180Da when alkaline protease and leucine amino peptidase double-enzyme hydrolysis
Content is 34.25%, and when alkaline protease and proline aminopeptidase double-enzyme hydrolysis, the little peptide content of below 180Da is 22.41%.
The different enzyme solution impact on casein hydrolysis effect of table 1
Note: 1: casein solution, 2: alkaline protease single enzymolysis liquid, 3: leucine amino peptidase single enzymolysis liquid, 4: alkaline protease
With leucine amino peptidase double-enzyme hydrolysis liquid, 5: proline aminopeptidase, alkaline protease and leucine amino peptidase synergetic hydrolysis liquid, 6: alkalescence egg
White enzyme and proline aminopeptidase double-enzyme hydrolysis liquid.
It addition, the free aminoacid content that the inventive method obtains is 3.968mg/mL, for unhydrolysed free aminoacid content
63 times, wherein proline content is unhydrolysed 3.67 times.And alkaline protease when individually hydrolyzing free aminoacid content be 1.449
Mg/mL, during leucine amino peptidase single enzymolysis, free aminoacid content is 0.767mg/mL, alkaline protease and leucine aminopeptidase peptide
During enzyme double-enzyme hydrolysis, free aminoacid content is 3.361mg/mL, free when alkaline protease and proline aminopeptidase double-enzyme hydrolysis
Amino acid content is 2.193mg/mL and proline content is unhydrolysed 2.8 times.
The present invention by with the casein of proline rich as substrate, proline aminopeptidase and alkaline protease, leucine amino peptidase
In the hydrolysate that synergetic hydrolysis obtains, content and the little peptide content of free amino acid are all significantly increased, and caseic degree of hydrolysis is the biggest
Big raising, the hydrophobic amino acid of the least peptide end is hydrolyzed and also can significantly eliminate bitterness.
Embodiment 4: casein degradation method
The casein hydrolysis method of the present invention, be preparation mass concentration be the casein solution of 1.5%,
(1) add appropriate alkaline protease, at 40 DEG C, react 3.5h, boiling water bath 10min；
(2) appropriate leucine amino peptidase is continuously added after cooling, 45 DEG C of reactions 3.5h, boiling water bath 10min；
(3) appropriate proline aminopeptidase is added after cooling, 45 DEG C of reactions 3.5h, boiling water bath 10min；
Wherein alkaline protease addition is 34000U/g casein；Leucine amino peptidase addition is 800U/g casein；Dried meat
Propylhomoserin aminopeptidase addition is 400U/g casein.
In the casein hydrolyzate obtained, the polypeptide of more than 2000Da does not exists, and the little peptide of below 180Da
Content has reached 36.25%, and free aminoacid content is 3.849mg/mL, and free proline content is unhydrolysed 3.46 times.
Embodiment 5: casein degradation method
The casein hydrolysis method of the present invention, be preparation mass concentration be the casein solution of 2.5%,
(1) add appropriate alkaline protease, at 45 DEG C, react 2.5h, boiling water bath 20min；
(2) appropriate leucine amino peptidase is continuously added after cooling, 50 DEG C of reactions 2.5h, boiling water bath 20min；
(3) appropriate proline aminopeptidase is added after cooling, 50 DEG C of reactions 2.5h, boiling water bath 20min；
Wherein alkaline protease addition is 30000U/g casein；Leucine amino peptidase addition is 1000U/g casein；
Proline aminopeptidase addition is 500U/g casein.
In the casein hydrolyzate obtained, the polypeptide of more than 2000Da does not exists, and the little peptide of below 180Da
Content has reached 37.82%, and free aminoacid content is 4.142mg/mL.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be with
What claims were defined is as the criterion.
Claims (10)
1. a caseic method for hydrolysis, it is characterised in that described method be successively with alkaline protease, leucine amino peptidase,
Proline aminopeptidase is hydrolyzed.
Method the most according to claim 1, it is characterised in that described method, be preparation mass concentration be the cheese of 1-3%
Protein solution,
(1) add appropriate alkaline protease, at 40-50 DEG C, react 2.5-3.5h, boiling water bath 10-20min；
(2) appropriate leucine amino peptidase is continuously added after cooling, 45-50 DEG C of reaction 2.5-3.5h, boiling water bath 10-20min；
(3) appropriate proline aminopeptidase is added after cooling, 45-50 DEG C of reaction 2.5-3.5h, boiling water bath 10-20min.
Method the most according to claim 1, it is characterised in that the nucleotide sequence of described proline aminopeptidase such as SEQ ID
Shown in NO.1.
Method the most according to claim 2, it is characterised in that described step (1) neutral and alkali protease addition is
30000-34000U/g casein.
Method the most according to claim 2, it is characterised in that in described step (2), leucine amino peptidase addition is
800-1000U/g casein.
Method the most according to claim 2, it is characterised in that in described step (3), proline aminopeptidase addition is
400-500U/g casein.
Method the most according to claim 2, it is characterised in that described method, specifically: with 50mM pH7.5's
The casein solution of PBS preparation 2%；Then,
(1) appropriate basic protein enzyme powder is added, 50 DEG C of reactions 3h, boiling water bath 15min；
(2) appropriate leucine amino peptidase powder is continuously added after cooling, 50 DEG C of reactions 3h, boiling water bath 15min；
(3) appropriate proline aminopeptidase enzyme liquid is added after cooling, 50 DEG C of reactions 3h, boiling water bath 15min.
8. the arbitrary described method of claim 1-7 is in the application of field of food.
9. the method for a purification proline aminopeptidase, it is characterised in that described method is by recombinant bacterium fermentation liquid centrifuging and taking supernatant,
Obtaining crude enzyme liquid, the ultra-filtration centrifuge tube of recycling molecular cut off 30kDa is concentrated by ultrafiltration, and the ultrafiltration concentration liquid obtained is in buffering
Dialysed overnight in liquid A, takes dialysis solution loading nickel affinity chromatography post, and flow velocity keeps 1mL/min, first by the buffering of 5 column volumes
Liquid A eluting, then with the buffer B eluting of 5 column volumes, change according to UV-detector reading, collect eluent in batches,
The eluent that will obtain, dialysed overnight in Tris-HCl pH7.5 buffer, i.e. obtain proline aminopeptidase pure enzyme liquid.
Method the most according to claim 9, it is characterised in that described recombinant bacterium is at Chinese patent 201510497845.0
Recombined bacillus subtilis on the basis of, add the base of encoding histidine label in the upstream of coding proline aminopeptidase gene
Because of, and obtain.
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| CN106967773A (en) * | 2017-04-20 | 2017-07-21 | 江南大学 | A kind of application restructuring aminopeptidase takes off bitter method to rice peptide |
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| CN105018407A (en) * | 2015-08-13 | 2015-11-04 | 江南大学 | Bacillus subtilis of secretory expression proline aminopeptidase and application thereof |
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| CN105018407A (en) * | 2015-08-13 | 2015-11-04 | 江南大学 | Bacillus subtilis of secretory expression proline aminopeptidase and application thereof |
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| 须瑛敏: "枯草芽孢杆菌氨肽酶的研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
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| CN106967773A (en) * | 2017-04-20 | 2017-07-21 | 江南大学 | A kind of application restructuring aminopeptidase takes off bitter method to rice peptide |
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