CN109504671A

CN109504671A - A method of extracting from moringa seeds has hydrolysing activity protease

Info

Publication number: CN109504671A
Application number: CN201910036097.4A
Authority: CN
Inventors: 王雪峰; 黄艾祥; 陈越; 施亚楠
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2019-01-15
Filing date: 2019-01-15
Publication date: 2019-03-22
Anticipated expiration: 2039-01-15
Also published as: CN109504671B

Abstract

The present invention provides a kind of to extract the method with hydrolysing activity protease from moringa seeds, belongs to neutral protease extractive technique field.The protease species and content of moringa seeds are improved by germination technology, are then parsed according to moringa seeds proteomics, the information of up-regulated expression and the protease with hydrolysing activity are obtained, for instructing subsequent extracted.Extracting method provided by the invention can not only be extracted to obtain a variety of protease with hydrolysing activity, and it is strong to extract obtained protease content height, enzymatic activity.The 8 kinds of moringa seeds protease extracted from germination moringa seeds compare the protease content extracted from dry moringa seeds and have correspondinglyd increase 10%~21%, wherein 2 kinds of protease, which accordingly hydrolyze vigor compared to dry moringa seeds protease, improves 8.0%~9.9%.Using method provided by the invention, the problem of avoiding the blindness, triviality and heavy workload of traditional protein separation method, have the characteristics that easy to operate reproducible.

Description

A method of extracting from moringa seeds has hydrolysing activity protease

Technical field

The invention belongs to neutral protease extractive technique fields, and in particular to one kind is extracted from moringa seeds, and there is hydrolysis to live The method of property protease.

Background technique

As enzyme engineering is more and more widely used in the food industry, the demand of protease resource is also more next It is bigger.Although protease can be obtained by means such as genetic engineering, artificial synthesized or microbial fermentations, to natural animal-plant Still demand is larger for protease resource, and more especially there is the protease of hydrolysing activity there are problems that critical shortage.

Moringa tree is the perennial torrid zone, subtropical zone deciduous tree, is belonged to by cervical orifice of uterus Moringaceae Moringa platymiscium, Classification system is Moringa oleifera Lam., English name Moringa, in addition also there are many alias, according to its beanpod shape (it is elongated, It is triangular in shape), referred to as drumstick tree (Drumstick tree) has acid, referred to as horseradish tree according to its root (Horseradish tree)；It can be extracted oil according to its seed, referred to as run quickly oil tree (Ben oil tree or Benzoil tree). Moringa is Yunnan special advantage industries, resourceful, nutritional ingredient rich in the seed and leaf of moringa seeds, but Moringa Seed itself is containing alkaloids, Moringa flavones, and hypertension, diabetic and pregnant woman etc. preferably consider after doctor indicates edible. Currently, the edible or medical value report in relation to Moringa and its accessory is more, however protease and its bioactivity are ground Study carefully and be but rarely reported, more without a kind of on how to extract high-content, high hydrolysing activity protease method.

Summary of the invention

The side with hydrolysing activity protease is extracted from moringa seeds in view of this, the purpose of the present invention is to provide a kind of Method, not only content is high for the protease extracted using the method, but also hydrolysing activity is strong.

A kind of method extracted from moringa seeds with hydrolysing activity protease provided by the invention, comprising the following steps:

1) protein non-marked relative quantitative assay is carried out respectively from germination moringa seeds and dry moringa seeds, screen dry Moringa Differential protein between seed total protein and germination moringa seeds total protein；A length of 10~the 15cm of bud of germination moringa seeds, germinate Moringa The quality of seed is 12.57~13.09g/15；The quality of dry moringa seeds is 4.31-4.53g/15；

2) statistic procedure 1) protein of up-regulated expression in the differential protein, by the protein of obtained up-regulated expression into Row GO functional annotation, obtains the protease with hydrolysing activity；

3) using salt method germination moringa seeds protease, moringa seeds protease salt extract is obtained；

4) obtain having the PI value of the protease of hydrolysing activity, set-up procedure 4 according to screening in step 2)) in moringa seeds egg The pH value of white enzyme salt extract is collected protease precipitate, is redissolved, desalination obtains moringa seeds albumen enzyme extract to the PI value；

5) molecular weight for obtaining having the protease of hydrolysing activity according to screening in step 2), selects ultrafiltration membrane to described peppery Albumen in the wooden seed albumen enzyme extract carries out ultrafiltration retention, collects the protease of target molecular weight, obtains with hydrolysing activity Protease.

Preferably, germinate described in step 1) moringa seeds cultural method by 4~10 DEG C of water infiltrations of moringa seeds, then 20 24~26h is impregnated in~25 DEG C of water, obtained immersion moringa seeds are cultivated in the environment of constant temperature and humidity；

It cultivates the 1st~3 day, cultivation temperature is 25~30 DEG C, humidity is 70%~75%；It cultivates the 4th~6 day, culture temperature Degree is 22~25 DEG C, humid control is 60%~65%.

Preferably, the protease in step 2) with hydrolysing activity includes 8 kinds of protease；The amino acid sequence of 8 kinds of protease In column respectively sequence table shown in No.1~8 SEQ ID；

The PI of protease shown in the SEQ ID No.1 is 10.10；Protease shown in the SEQ ID No.1 Molecular weight is 11789.27；

The PI of protease shown in the SEQ ID No.2 is 7.55；Point of protease shown in the SEQ ID No.2 Son amount is 18459.82；

The PI of protease shown in the SEQ ID No.3 is 7.15；Point of protease shown in the SEQ ID No.3 Son amount is 18811.2；

The PI of protease shown in the SEQ ID No.4 is 6.23；Point of protease shown in the SEQ ID No.4 Son amount is 52880.49；

The PI of protease shown in the SEQ ID No.5 is 10.63；Protease shown in the SEQ ID No.5 Molecular weight is 6802.67；

The PI of protease shown in the SEQ ID No.6 is 5.24；Point of protease shown in the SEQ ID No.6 Son amount is 31591.8；

The PI of protease shown in the SEQ ID No.7 is 10.36；Protease shown in the SEQ ID No.7 Molecular weight is 9427.98；

The PI of protease shown in the SEQ ID No.8 is 7.75；Point of protease shown in the SEQ ID No.8 Son amount is 18756.12.

Preferably, the method for salt method germination moringa seeds protease includes that the germination moringa seeds crush in step 2), Sieving, obtained screenings and sodium chloride solution mixing, extracts, is separated by solid-liquid separation, obtains moringa seeds protease salt extract.

Preferably, the sieve pore of the sieving is not less than 60 mesh；

The quality of the screenings and the volume of sodium chloride solution are 1g:5mL；The concentration of the sodium chloride solution is 0.25 ~0.30mol/L.

Preferably, the temperature of extraction is 30 DEG C；The time of extraction is 0.5~1h；The pH value of feed liquid is 7.15 when extraction.

Preferably, the method for collecting protease precipitate includes centrifugation；The revolving speed of the centrifugation is 4500-5000r/min；Institute The time for stating centrifugation is 20~30min.

Preferably, the method for redissolution is the protease precipitate that will be collected and water mixing, and it is molten that obtained mixed liquor carries out ultrasound Solution obtains protein enzyme solution；The quality of the protease precipitate and the volume ratio of water are 5g:5~10mL；

The power of the ultrasound is 200Hz；The time of the ultrasound is 2~5min.

It preferably, also successively include that pH value and pre-separation are adjusted to the protein enzyme solution before the desalination；

The pH value of protein enzyme solution is adjusted to 7.0；

The pre-separation includes dialysis；The molecular cut off of dialysis bag filter is 3500Da；The dialysis when Between be 50~56h.

Preferably, the protein non-marked relative quantitative assay includes TCA/ acetone precipitation and SDT cracking process, SDS- PAGE gel electrophoresis, FASP enzymatic hydrolysis, high performance liquid chromatography and the mass spectrometric mass spectral analysis of Q-Exactive, mass spectral analysis original number The analysis of checking storehouse identification and quantification is carried out according to and using MaxQuant software.

A kind of method extracted from moringa seeds with hydrolysing activity protease provided by the invention, passes through germination skill first The protease species and content of art raising moringa seeds；Then it is parsed according to moringa seeds proteomics, obtains up-regulated expression and tool There is the information of the protease of hydrolysing activity, to instruct later separation.Extracting method provided by the invention can not only be extracted To a variety of protease with hydrolysing activity, and the protease that extraction obtains has the characteristics that content is high, enzymatic activity is strong.Experiment Showing: the 8 kinds of moringa seeds protease extracted have correspondinglyd increase 10% compared to the protease content extracted in dry moringa seeds~ 21%, it filters out wherein 2 kinds of protease and accordingly hydrolyzes vigor compared to the protease in dry moringa seeds and improve 8.0%~9.9%.

Method provided by the invention simultaneously, is combined using proteome analysis and extraction means, avoids tradition point The problem of blindness, triviality and heavy workload from method, keep extracting method more simple and efficient and efficient.Using the present invention Method extract 2 kinds of protease not only hydrolytic enzyme activities with higher also simultaneously have both condenser water level, experiments have shown that: use 2 kinds of enzymes that method provided by the invention is extracted improve 8.3% than the protease extracted in dry moringa seeds in terms of condenser water level ~9.1%.Therefore, using the method separation and Extraction to moringa seeds protease all there is preferable proteolysis vigor, solidifying Newborn vigor is conducive to expansion scale and is produced in batches, is expected to realize the industrial applications of subsequent moringa seeds protease.

Detailed description of the invention

Fig. 1 is that moringa seeds protein relative molecular mass distribution figure is identified in the present invention；

Fig. 2 is the protein Venn figure that repetitive identified arrives three times in dry moringa seeds group in the present invention；

Fig. 3 is the protein Venn figure that repetitive identified arrives three times in germination moringa seeds group in the present invention；

Fig. 4 is the protein Venn figure that germination moringa seeds and dry moringa seeds sample group identify in the present invention；

Fig. 5 is germinate in the present invention moringa seeds and dry moringa seeds volcano illustrated example.

Specific embodiment

The present invention carries out protein non-marked relative quantitative assay respectively from germination moringa seeds and dry moringa seeds, and screening is dry Differential protein between moringa seeds total protein and germination moringa seeds total protein；A length of 10~the 15cm of bud of germination moringa seeds, germination The quality of moringa seeds is 12.57~13.09g/15；The quality of dry moringa seeds is 4.31-4.53g/15.

In the present invention, the cultural method of the germination moringa seeds is preferably by 4~10 DEG C of water infiltrations of moringa seeds, then 20 24~26h is impregnated in~25 DEG C of water, obtained immersion moringa seeds are cultivated in the environment of constant temperature and humidity.The culture the 1st~3 It, cultivation temperature is preferably 25~30 DEG C, and more preferably 28 DEG C.The humidity of culture is preferably 70%~75%, more preferably 73%.The culture the 4th~6 day, cultivation temperature is preferably 22~25 DEG C, and more preferably 23 DEG C.The culture humidity is preferably 60%~65%, more preferably 63%.The long bud of germination moringa seeds is preferably 12~14cm, more preferably 13cm.Germinate Moringa The quality of seed is preferably 12.57~13.09g/15；The quality of dry moringa seeds is preferably 4.48g/15.Using germination Moringa Seed is extracted as raw material, is conducive to the protease species, content and the enzymatic activity that improve moringa seeds.

In the present invention, the protein non-marked relative quantitative assay preferably includes TCA/ acetone precipitation and SDT cracking Method, PAGE gel electrophoresis, FASP enzymatic hydrolysis, high performance liquid chromatography and the mass spectrometric mass spectral analysis of Q-Exactive, mass spectrum point It analyses initial data and the analysis of checking storehouse identification and quantification is carried out using MaxQuant software.Protein non-marked relative quantitative assay Specific steps are shown in following bibliography: [1] Cox, J.and M.Mann (2008) " MaxQuant enables high peptide identification rates,individualized p.p.b.-range mass accuracies and proteome-wide protein quantification."Nat Biotechnol 26(12):1367-1372.

[2]Cox,J.,M.Y.Hein,et al.(2014)."Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ."Mol Cell Proteomics 13(9):2513-2526.

[3]Cox,J.,N.Neuhauser,et al.(2011)."Andromeda:a peptide search engine integrated into the MaxQuant environment."J Proteome Res 10(4):1794-1805.

Differential protein is obtained, the present invention counts the protein of up-regulated expression in the differential protein, the upper mileometer adjustment that will be obtained The protein reached carries out GO functional annotation, obtains the protease with hydrolysing activity.

In the present invention, the quantity of the differential protein is 43, and wherein the quantity of the differential protein of up-regulated expression is 26.The present invention is not particularly limited the method for the GO functional annotation, using the functional annotation side GO known in the art Case.

In the present invention, the protease with hydrolysing activity preferably includes 8 kinds of protease；The amino acid sequence of 8 kinds of protease In column respectively sequence table shown in No.1~8 SEQ ID；The PI of protease shown in the SEQ ID No.1 is 10.10；Institute The molecular weight for stating protease shown in SEQ ID No.1 is 11789.27；Name through protease shown in retrieval SEQ ID No.1 Referred to as Chitin-binding protein 2.

The PI of protease shown in the SEQ ID No.2 is 7.55；Point of protease shown in the SEQ ID No.2 Son amount is 18459.82；Entitled 2S albumin through protease shown in retrieval SEQ ID No.2.

The PI of protease shown in the SEQ ID No.3 is 7.15；Point of protease shown in the SEQ ID No.3 Son amount is 18811.2；Entitled 2S albumin through protease shown in retrieval SEQ ID No.3

The PI of protease shown in the SEQ ID No.4 is 6.23；Point of protease shown in the SEQ ID No.4 Son amount is 52880.49；Entitled Ribulose bisphosphate through protease shown in retrieval SEQ ID No.4 carboxylase large chain(RBCL)。

The PI of protease shown in the SEQ ID No.5 is 10.63；Protease shown in the SEQ ID No.5 Molecular weight is 6802.67；Entitled 2.1 protein through protease shown in retrieval SEQ ID No.5.

The PI of protease shown in the SEQ ID No.6 is 5.24；Point of protease shown in the SEQ ID No.6 Son amount is 31591.8；Entitled Actin through protease shown in retrieval SEQ ID No.6.

The PI of protease shown in the SEQ ID No.7 is 10.36；Protease shown in the SEQ ID No.7 Molecular weight is 9427.98；Entitled Photosystem I P700 through protease shown in retrieval SEQ ID No.7 apoprotein A1。

The PI of protease shown in the SEQ ID No.8 is 7.75；Point of protease shown in the SEQ ID No.8 Son amount is 18756.12.Entitled 2S albumin through protease shown in retrieval SEQ ID No.8.

The present invention obtains moringa seeds protease salt extract using salt method germination moringa seeds protease.

In the present invention, the method for the salt method germination moringa seeds protease preferably includes the germination moringa seeds powder Broken, sieving, obtained screenings and sodium chloride solution mixing extracts, is separated by solid-liquid separation, obtains moringa seeds protease salt extract.Institute The sieve pore for stating sieving is preferably not less than 60 mesh.The quality of the screenings and the volume of sodium chloride solution are preferably 1g:5mL；Institute The concentration for stating sodium chloride solution is preferably 0.25~0.30mol/L.The temperature of extraction is preferably 30 DEG C；The time of extraction is preferably 0.5~1h；The pH value of feed liquid is preferably 7.15 when extraction.

After obtaining moringa seeds protease salt extract, the present invention obtains having the PI of the protease of hydrolysing activity according to screening Value, set-up procedure 4) in moringa seeds protease salt extract pH value to the PI value, collect protease precipitate, redissolve, desalination obtains To moringa seeds albumen enzyme extract.

In the present invention, the method for the pH value of the adjustment moringa seeds protease salt extract preferably uses the hydrogen-oxygen of 0.1mol/L Change the hydrogen chloride solution of sodium solution and 0.1mol/L to instill in moringa seeds protease salt extract with the speed of 0.5~1.0mL/s, make The pH value of salt extract respectively reaches the PI for 8 kinds of protease that above-mentioned proteomic techniques screen.

In the present invention, the method for collecting protease precipitate preferably includes to be centrifuged；The revolving speed of the centrifugation is preferably 4500 ~5000r/min；The time of the centrifugation is preferably 20~30min.Precipitating is collected after centrifugation.

In the present invention, the method for redissolution preferably mixes the protease precipitate of collection and water, obtained mixed liquor into Row ultrasonic dissolution obtains protein enzyme solution.The quality of the protease precipitate and the volume ratio of water are preferably 5g:5~10mL.Institute The power for stating ultrasound is preferably 200Hz；The time of the ultrasound is preferably 2~5min.

It in the present invention, further preferably successively include that pH value and pre-separation are adjusted to the protein enzyme solution before the desalination. The pH value of protein enzyme solution is preferably adjusted to 7.0.The pre-separation preferably includes to dialyse；The retention point of the dialysis bag filter Son amount is preferably 3500Da；The time of the dialysis is preferably 50~56h.

The present invention obtains having the molecular weight of the protease of hydrolysing activity according to screening, selects ultrafiltration membrane to the moringa seeds Albumen in albumen enzyme extract carries out ultrafiltration retention, collects the protease of target molecular weight, obtains with hydrolysing activity albumen Enzyme.

In the present invention, it is further preferably placed in after -80 DEG C of quick-frozen 5h and is put into very including lower layer's trapped fluid that ultrafiltration retains Empty low-temperature freeze-drying machine frozen dried 48h, obtains protease powders.

The protease that the present invention extracts content with higher and bioactivity, wherein shown in SEQ ID No.6 Ribulose bisphosphate carboxylase large chain shown in Actin the and SEQ ID No.4 of protease (RBCL) there is hydrolytic enzyme activities and milk-clotting activity, the processing applied to cheese, beer, beverage/food.

Below with reference to embodiment to a kind of side extracted from moringa seeds with hydrolysing activity protease provided by the invention Method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.

Embodiment 1

The germination of the dry moringa seeds of 100g and the separation of specific proteases

1) dry moringa seeds warm water impregnates: the dry moringa seeds of 100g first being moistened with cold water sprays, then put it into 25 DEG C of warm water For 24 hours, soaking process need to be covered with strainer, guarantee that moringa seeds are completely immersed in warm water for middle immersion；

2) moringa seeds germinate: soaked moringa seeds being put into and spread in the seedlings nursing plate through water wetting filter paper that haves three layers every two Seedlings nursing plate, is then put into growth cabinet by moringa seeds interval 1cm, germination 1~3 day during the temperature inside the box control 25 DEG C, Humid control the temperature inside the box during 70%, germination 4~6 days is controlled in 22 DEG C, humid control 60%；

3) proteomics of dry moringa seeds and germination moringa seeds parses: it is long to choose bud for the moringa seeds of germination the 6th day 10cm, quality 12.57g/15 germination moringa seeds and quality 4.31g/15 dry moringa seeds carry out protein respectively Label-free relative quantitative assay, digested by TCA/ acetone precipitation and SDT cracking process, PAGE gel electrophoresis, FASP, High performance liquid chromatography and the mass spectrometric mass spectral analysis of Q-Exactive, mass spectral analysis initial data and using MaxQuant software into The identification of row checking storehouse and quantitative analysis (Fig. 1), the dry moringa seeds of comparative analysis (A-1 in Fig. 2) and germination moringa seeds (B-1 in Fig. 3) go out Existing 43 species diversity albumen, wherein the up-regulation of 26 kinds of protein contents occur in germination moringa seeds, GO functional annotation filters out 8 kinds of up-regulation eggs White matter is the protease with hydrolysing activity, the molecular weight ranges and iso-electric point of uniprot database displaying 8 kinds of protease Range is shown in Table 1.

18 kinds of protease information lists of table

Sequence number	Protease title	Molecular weight (kDa)	Isoelectric point
				SEQ ID No.1	Chitin-binding protein 2	11789.27	10.10
SEQ ID No.2	2S albumin	18459.82	7.55
				SEQ ID No.3	2S albumin	18811.2	7.15
SEQ ID No.4	Ribulose bisphosphate carboxylase large chain	52880.49	6.23
				SEQ ID No.5	2.1 protein	6802.67	10.63
SEQ ID No.6	Actin	31591.8	5.24
				SEQ ID No.7	Photosystem I P700 apoprotein A1	9427.98	10.36
SEQ ID No.8	2S albumin	18756.12	7.75

4) salt method of dry moringa seeds and germination moringa seeds protease: dry moringa seeds and germination moringa seeds are crushed with high speed Machine smashes 2min, crosses 60 meshes and removes most of moringa seeds shell, respectively according to dry moringa seeds screenings and sodium chloride solution according to The ratio of 1:10 (g/mL) mixes, and germination moringa seeds screenings and sodium chloride solution are mixed according to the mass volume ratio of 1:5g/mL, Moringa seeds extracting solution is obtained under conditions of 30 DEG C of Extracting temperature, pH7.15, extraction time 1.0h, extracting solution, which is put into, to be filtered in cup It is handled to obtain moringa seeds protease salt extract with 2 layers of middling speed qualitative filter paper；

5) separation of dry moringa seeds and specific proteases in germination moringa seeds: the sodium hydroxide solution of 0.1mol/L is used respectively It is instilled in moringa seeds protease salt extract with the hydrogen chloride solution of 0.1mol/L with the speed of 0.5mL/s, makes the pH value of salt extract PI5.24~10.63 for respectively reaching 8 kinds of protease that above-mentioned proteomic techniques screen, successively with 4500r/min Protein precipitation is obtained after centrifugation 20min, the protein precipitation obtained under different protease P I is surpassed with the deionized water of 5mL Sound dissolution 2min is completely dissolved precipitating, then is instilled with the sodium hydroxide solution of 0.1mol/L or the hydrogen chloride solution of 0.1mol/L Each protein solution pH value is set to be adjusted to 7.0, the bag filter that each protein solution is then respectively put into molecular cut off 3500Da is saturating It analyses 50h and carries out desalting processing, then each dialyzate is selected the different ultrafiltration membranes of molecular cut off 6kDa-60kDa to carry out ultrafiltration and cut It stays, lower layer's trapped fluid is put into vacuum and low temperature freeze drier frozen dried 48h after being placed in -80 DEG C of quick-frozen 5h, obtains germination Moringa 8 kinds of protease contents of 8 kinds of protease contents of seed and dry moringa seeds.It the results are shown in Table 2.

The germination 8 kinds of protease contents (mg) of 8 kinds of protease contents of moringa seeds and dry moringa seeds of table 2

Sequence table numbering	Germinate moringa seeds protease content	Dry moringa seeds protease content
			SEQ ID No.1	568	501
SEQ ID No.2	578	511
			SEQ ID No.3	608	523
SEQ ID No.4	830	703
			SEQ ID No.5	523	475
SEQ ID No.6	659	556
			SEQ ID No.7	544	489
SEQ ID No.8	604	513

It obtains germination 8 kinds of protease content split-phases of moringa seeds and has correspondinglyd increase 10.0% (SEQ ID than dry moringa seeds No.5), 11.2% (SEQ ID No.7), 13.4% (SEQ ID No.1), 15.3% (SEQ ID No.2), 16.2% (SEQ ID No.3), 17.7% (SEQ ID No.8), 18.1% (SEQ ID No.4), 18.5% (SEQ ID No.6)；

6) dry moringa seeds and the hydrolysing activity of germination moringa seeds specific proteases are analyzed: by 8 kinds of moringa seeds obtained above Protease freeze-dried powder is configured to the solution that mass concentration is 10mg/L with deionized water and carries out condenser water level and protein point respectively Vitality test is solved, 3g skimmed milk power is weighed first and is added to the redissolution cream that mass volume ratio 1:10g/mL is made in 30mL distilled water, It is heated to being put into 60 DEG C of thermostat water baths after standing cooling 5min after boiling, reaches 60 DEG C wait redissolve newborn central temperature, be added 10mL enzyme solution, continues water-bath after being sufficiently mixed, accurate recording is added to time (T) calculating that lactoprotein solidifies completely from enzyme solution and coagulates Newborn vigor=(3*2400)/(T*10) SU/mg, next takes 2mL, 1.5% casein solution in 60 DEG C of heat preservation 5min, 1mL is added The enzyme solution to be measured of preheating mixes reaction 30min, and 4mL, the termination reaction of 8% trichloroacetic acid is added, filtrate is measured after filtering and is existed Absorbance (the A of 280nm₂₈₀), first to keep addition 2mL casein after enzyme solution inactivation molten with trichloroacetic acid mixing the enzyme solution of preheating Liquid measures filtrate in the absorbance (A ' of 280nm after keeping the temperature 5min, filtering at 60 DEG C₂₈₀) it is blank control, calculate 1mL enzyme solution Proteolytic activity=(A₂₈₀-A′₂₈₀)/1 filters out 2 kinds of protease by condenser water level and Proteolytic activity measurement, respectively For protease shown in SEQ ID No.6 and SEQ ID No.4.It the results are shown in Table 3.

The condenser water level and Proteolytic activity of 32 kinds of protease of table

Sequence number	Condenser water level (SU/mg)	Hydrolysis vigor
			SEQ ID No.6	12.45	168
SEQ ID No.4	15.87	199

Germination 2 kinds of protease of moringa seeds compared to dry moringa seeds accordingly hydrolyze vigor improve 7.6% (SEQ ID No.6), 9.5% (SEQ ID No.4), condenser water level improve 7.3% (SEQ ID No.6), 8.1% (SEQ ID No.4).

Embodiment 2

The germination of the dry moringa seeds of 500g and the separation of specific proteases

Implementation process is substantially with embodiment 1, and only the following is different:

1) dry moringa seeds warm water impregnates: the dry moringa seeds of 500g first being moistened with cold water sprays, then put it into 22 DEG C of warm water Middle immersion is for 24 hours；

2) moringa seeds germinate: soaked moringa seeds being put into and spread in the seedlings nursing plate through water wetting filter paper that haves three layers every two Seedlings nursing plate, is then put into growth cabinet by moringa seeds interval 1.2cm, germination 1-3 days during the temperature inside the box control 28 DEG C, Humid control the temperature inside the box during 72%, germination 4~6 days is controlled in 23 DEG C, humid control 63%；

3) proteomics of dry moringa seeds and germination moringa seeds parses: it is long to choose bud for the moringa seeds of germination the 6th day 12cm, quality 12.76g/15 germination moringa seeds and quality 4.41g/15 dry moringa seeds carry out protein respectively Label-free relative quantitative assay searches library result and sees in Fig. 2 B-2 in A-2 and Fig. 3；

4) salt method of dry moringa seeds and germination moringa seeds protease: dry moringa seeds and germination moringa seeds are crushed with high speed Machine smashes 3min, crosses 60 meshes and removes most of moringa seeds shell, is added respectively according to the mass volume ratio of 1:10 and 1:5g/mL The sodium chloride solution of 0.25mol/L obtains moringa seeds under conditions of 30 DEG C of Extracting temperature, pH7.15, extraction time 1.2h and mentions Take liquid；

5) separation of dry moringa seeds and specific proteases in germination moringa seeds: the sodium hydroxide solution of 0.1mol/L is used respectively It is instilled in moringa seeds protease salt extract with the hydrogen chloride solution of 0.1mol/L with the speed of 0.8mL/s, makes the pH value of salt extract PI5.24~10.63 for respectively reaching 8 kinds of protease that above-mentioned proteomic techniques screen, successively with 4800r/min Protein precipitation is obtained after centrifugation 25min, the protein precipitation obtained under different protease P I is surpassed with the deionized water of 8mL Sound dissolution 3min is completely dissolved precipitating, then is instilled with the sodium hydroxide solution of 0.1mol/L or the hydrogen chloride solution of 0.1mol/L Each protein solution pH value is set to be adjusted to 7.0, the bag filter that each protein solution is then respectively put into molecular cut off 3500Da is saturating It analyses 52h and carries out desalting processing, then each dialyzate is selected the different ultrafiltration membranes of molecular cut off 6kDa~60kDa to carry out ultrafiltration and cut It stays, lower layer's trapped fluid is put into vacuum and low temperature freeze drier frozen dried 60h after being placed in -80 DEG C of quick-frozen 5.5h, obtains germinateing peppery 8 kinds of protease contents of 8 kinds of protease contents of the wooden seed and dry moringa seeds.It the results are shown in Table 4.

The germination 8 kinds of protease contents (mg) of 8 kinds of protease contents of moringa seeds and dry moringa seeds of table 4

Sequence table numbering	Germinate moringa seeds protease content	Dry moringa seeds protease content
			SEQ ID No.1	2839	2497
SEQ ID No.2	2958	2561
			SEQ ID No.3	3048	2625
SEQ ID No.4	4183	3536
			SEQ ID No.5	2650	2381
SEQ ID No.6	3349	2803
			SEQ ID No.7	2721	2443
SEQ ID No.8	2998	2554

As shown in Table 4, germination 8 kinds of protease contents of moringa seeds have correspondinglyd increase 11.3% (SEQ ID compared to dry moringa seeds No.5), 11.4% (SEQ ID No.7), 13.7% (SEQ ID No.1), 15.5% (SEQ ID No.2), 16.1% (SEQ ID No.3), 17.4% (SEQ ID No.8), 18.3% (SEQ ID No.4), 19.5% (SEQ ID No.6).

6) dry moringa seeds and the hydrolysing activity of germination moringa seeds specific proteases are analyzed:

2 kinds of protease, respectively SEQ ID No.6 and SEQ are filtered out by condenser water level and Proteolytic activity measurement Protease shown in ID No.4.Using the enzymatic activity analysis method in embodiment 1, the hydrolysis vigor and curdled milk of protease are obtained Vigor the results are shown in Table 5.

The condenser water level and Proteolytic activity of 52 kinds of protease of table

Sequence number	Condenser water level (SU/mg)	Hydrolysis vigor
			SEQ ID No.6	12.38	171
SEQ ID No.4	15.79	195

As shown in Table 5, germination 2 kinds of protease of moringa seeds accordingly hydrolyze vigor compared to dry moringa seeds and improve 8.0% (SEQ ID No.6), 9.9% (SEQ ID No.4), condenser water level improves 8.3% (SEQ ID No.6), 9.1% (SEQ ID No.4)。

Embodiment 3

The germination of the dry moringa seeds of 1000g and the separation of specific proteases

1) dry moringa seeds warm water impregnates: the dry moringa seeds of 1000g first being moistened with cold water sprays, then put it into 20 DEG C of warm water Middle immersion is for 24 hours；

2) moringa seeds germinate: soaked moringa seeds being put into and spread in the seedlings nursing plate through water wetting filter paper that haves three layers every two Seedlings nursing plate, is then put into growth cabinet by moringa seeds interval 1.5cm, and the temperature inside the box control is 30 during germination 1~3 day DEG C, humid control 75%, the temperature inside the box control is in 23 DEG C, humid control 65% during germination 4~6 days；

3) proteomics of dry moringa seeds and germination moringa seeds parses: it is long to choose bud for the moringa seeds of germination the 6th day 15cm, quality 13.09g/15 germination moringa seeds and quality 4.53g/15 dry moringa seeds carry out protein respectively Label-free relative quantitative assay searches library result and sees in Fig. 2 B-3 in A-3 and Fig. 3；

4) salt method of dry moringa seeds and germination moringa seeds protease: dry moringa seeds and germination moringa seeds are crushed with high speed Machine smashes 5min, crosses 60 meshes and removes most of moringa seeds shell, is added respectively according to the mass volume ratio of 1:10 and 1:5g/mL The sodium chloride solution of 0.30mol/L obtains moringa seeds under conditions of 30 DEG C of Extracting temperature, pH7.15, extraction time 1.5h and mentions Take liquid；

5) separation of dry moringa seeds and specific proteases in germination moringa seeds: the sodium hydroxide solution of 0.1mol/L is used respectively It is instilled in moringa seeds protease salt extract with the hydrogen chloride solution of 0.1mol/L with the speed of 1.0mL/s, makes the pH value of salt extract PI5.24~10.63 for respectively reaching 8 kinds of protease that above-mentioned proteomic techniques screen, successively with 5000r/min Protein precipitation is obtained after centrifugation 30min, the protein precipitation obtained under different protease P I is carried out with the deionized water of 10mL Ultrasonic dissolution 3min is completely dissolved precipitating, then is dripped with the sodium hydroxide solution of 0.1mol/L or the hydrogen chloride solution of 0.1mol/L Enter to make each protein solution pH value to be adjusted to 7.0, each protein solution is then respectively put into the bag filter of molecular cut off 3500Da The 56h that dialyses carries out desalting processing, and then each dialyzate selects the different ultrafiltration membranes of molecular cut off 6kDa~60kDa to carry out ultrafiltration Retention, lower layer's trapped fluid are put into vacuum and low temperature freeze drier frozen dried 72h after being placed in -80 DEG C of quick-frozen 6h, obtain germinateing peppery 8 kinds of protease contents of 8 kinds of protease contents of the wooden seed and dry moringa seeds.It the results are shown in Table 6.

The germination 8 kinds of protease contents (mg) of 8 kinds of protease contents of moringa seeds and dry moringa seeds of table 6

Sequence table numbering	Germinate moringa seeds protease content	Dry moringa seeds protease content
			SEQ ID No.1	5663	5003
SEQ ID No.2	5909	5112
			SEQ ID No.3	6045	5198
SEQ ID No.4	8503	7127
			SEQ ID No.5	5387	4801
SEQ ID No.6	6789	5611
			SEQ ID No.7	5415	4788
SEQ ID No.8	6062	5142

As shown in Table 6, germination 8 kinds of protease contents of moringa seeds have correspondinglyd increase 12.2% (SEQ ID compared to dry moringa seeds No.5), 13.1% (SEQ ID No.7), 13.2% (SEQ ID No.1), 15.6% (SEQ ID No.2), 16.3% (SEQ ID No.3), 17.9% (SEQ ID No.8), 19.3% (SEQ ID No.4), 21.0% (SEQ ID No.6).

6) dry moringa seeds and the hydrolysing activity of germination moringa seeds specific proteases are analyzed: passing through condenser water level and protein breakdown Vitality test filters out 2 kinds of protease, protease shown in respectively SEQ ID No.6 and SEQ ID No.4.Using embodiment Enzymatic activity analysis method in 1 obtains the hydrolysis vigor and condenser water level of protease, the results are shown in Table 7.

The condenser water level and Proteolytic activity of 72 kinds of protease of table

Sequence number	Condenser water level (SU/mg)	Hydrolysis vigor
			SEQ ID No.6	12.41	165
SEQ ID No.4	15.84	203

As shown in Table 7, germination 2 kinds of protease of moringa seeds accordingly hydrolyze vigor compared to dry moringa seeds and improve 8.2% (SEQ ID No.6), 10.1% (SEQ ID No.4), condenser water level improves 8.5% (SEQ ID No.6), 9.7% (SEQ ID No.4)。

Embodiment 4

It the protein of germination moringa seeds group extraction and the protein of dry moringa seeds extraction will compare, draw in Examples 1 to 3 (Fig. 4, wherein A indicates that the protein that germination moringa seeds seed group is extracted, B indicate the albumen that dry moringa seeds extract to Venn figure processed Matter).As shown in Figure 4, the protein of two groups of co-expressions has 650, and the germination distinctive protein of moringa seeds group has 119 kinds, does The distinctive protein of moringa seeds group has 110 kinds.

Using germination two groups of samples of moringa seeds and dry moringa seeds between protein expression fold differences (Fold change) and Two factors of P-value that T is examined draw volcano figure (Fig. 5) jointly, and the conspicuousness for showing two groups of sample datas is poor It is different.Abscissa indicate fold differences (with 2 for bottom logarithmic transformation), ordinate indicate difference conspicuousness P-value (be with 10 The logarithmic transformation at bottom).Volcano diagram form shows the data for being suitable for obtaining using T check algorithm as shown in Figure 5, illustrates two groups of samples This having differences property of protein expression.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Yunnan Prov Agriculture University

<120>a kind of that the method with hydrolysing activity protease is extracted from moringa seeds

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 104

<212> PRT

<213> Moringa oleifera Lam.

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Gln Pro Asp Phe Gln Arg Cys Pro Ser Leu Arg Cys Cys Gln Gln Leu

1 5 10 15

Arg Gln Ala Val Gln Leu Thr His Gln Gln Gln Gly Gln Val Gly Pro

20 25 30

Gln Gln Val Arg Gln Gln Phe Gln Thr His Gln Arg Ile Pro Ala Ile

35 40 45

Cys Asn Leu Gln Pro Met Arg Gln Ala Val Gln Leu Ala His Gln Gln

50 55 60

Gln Gln Gly Gln Val Gly Pro Gln Gln Val Arg Cys Cys Gln Gln Leu

65 70 75 80

Arg Gln Ala Val Gln Ser Gln Ala Ala Ala Ala Gly Gln Val Gly Pro

85 90 95

Gln Gln Val Gly His Met Tyr Arg

100

<210> 2

<211> 160

<212> PRT

<213> Moringa oleifera Lam.

<400> 2

Met Ala Lys Phe Thr Leu Leu Leu Ala Ile Phe Ala Leu Phe Leu Ile

1 5 10 15

Leu Ala Asn Ala Asn Val Tyr Arg Thr Thr Val Glu Leu Asp Glu Glu

20 25 30

Pro Asp Asp Asn Gln Gln Gly Gln Gln Gln Gln Gln Cys Arg Gln Gln

35 40 45

Phe Leu Thr His Gln Arg Leu Arg Ala Cys Gln Arg Phe Ile Arg Arg

50 55 60

Gln Thr Gln Gly Gly Gly Ala Leu Glu Asp Val Glu Asp Asp Val Glu

65 70 75 80

Glu Ile Glu Glu Val Val Glu Pro Asp Gln Ala Arg Arg Pro Ala Ile

85 90 95

Gln Arg Cys Cys Gln Gln Leu Arg Asn Ile Gln Pro Arg Cys Arg Cys

100 105 110

Pro Ser Leu Arg Gln Ala Val Gln Leu Ala His Gln Gln Gln Gly Gln

115 120 125

Val Gly Pro Gln Gln Val Arg Gln Met Tyr Arg Leu Ala Ser Asn Ile

130 135 140

Pro Ala Ile Cys Asn Leu Arg Pro Met Ser Cys Pro Phe Gly Gln Gln

145 150 155 160

<210> 3

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<213> Moringa oleifera Lam.

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Met Ala Lys Ile Thr Leu Leu Leu Ala Thr Phe Gly Leu Leu Leu Leu

1 5 10 15

Leu Thr Asn Ala Ser Ile Tyr Arg Thr Thr Val Glu Leu Asp Glu Glu

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Ala Asp Glu Asn Gln Gln Gln Arg Cys Arg Gln Gln Phe Gln Thr His

35 40 45

Gln Arg Leu Arg Ala Cys Gln Arg Phe Ile Arg Arg Arg Thr Gln Gly

50 55 60

Gly Gly Pro Leu Asp Glu Val Glu Asp Glu Val Asp Glu Ile Glu Glu

65 70 75 80

Val Val Glu Pro Asp Gln Gly Pro Gly Arg Gln Pro Ala Phe Gln Arg

85 90 95

Cys Cys Gln Gln Leu Arg Asn Ile Ser Pro Pro Cys Arg Cys Pro Ser

100 105 110

Leu Arg Gln Ala Val Gln Leu Thr His Gln Gln Gln Gly Gln Val Gly

115 120 125

Pro Gln Gln Val Arg Gln Met Tyr Arg Val Ala Ser Asn Ile Pro Ser

130 135 140

Met Cys Asn Leu Gln Pro Met Ser Cys Leu Phe Arg Gln Gln Gln Ser

145 150 155 160

Ser Trp Leu

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<213> Moringa oleifera Lam.

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Met Ser Phe Arg Glu Gly Leu Met Ser Pro Gln Thr Glu Thr Lys Ala

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Ser Val Gly Phe Lys Ala Gly Val Lys Asp Tyr Lys Leu Thr Tyr Tyr

20 25 30

Thr Pro Asp Tyr Glu Thr Lys Asp Thr Asp Ile Leu Ala Ala Phe Arg

35 40 45

Val Thr Pro Gln Pro Gly Val Pro Pro Glu Glu Ala Gly Ala Ala Val

50 55 60

Ala Ala Glu Ser Ser Thr Gly Thr Trp Thr Thr Val Trp Thr Asp Gly

65 70 75 80

Leu Thr Ser Leu Asp Arg Tyr Lys Gly Arg Cys Tyr His Ile Glu Pro

85 90 95

Ile Ala Gly Glu Glu Asn Gln Phe Ile Ala Tyr Val Ala Tyr Pro Leu

100 105 110

Asp Leu Phe Glu Glu Gly Ser Val Thr Asn Met Phe Thr Ser Ile Val

115 120 125

Gly Asn Val Phe Gly Phe Lys Ala Leu Arg Ala Leu Arg Leu Glu Asp

130 135 140

Leu Arg Ile Pro Pro Ala Tyr Ser Lys Thr Phe Gln Gly Pro Pro His

145 150 155 160

Gly Ile Gln Val Glu Arg Asp Lys Leu Asn Lys Tyr Gly Arg Pro Leu

165 170 175

Leu Gly Cys Thr Ile Lys Pro Lys Leu Gly Leu Ser Ala Lys Asn Tyr

180 185 190

Gly Arg Ala Val Tyr Glu Cys Leu Arg Gly Gly Leu Asp Phe Thr Lys

195 200 205

Asp Asp Glu Asn Val Asn Ser Gln Pro Phe Met Arg Trp Arg Asp Arg

210 215 220

Phe Leu Phe Cys Ala Glu Ala Ile Tyr Lys Ala Gln Ala Glu Thr Gly

225 230 235 240

Glu Ile Lys Gly His Tyr Leu Asn Ala Thr Ala Gly Thr Cys Glu Glu

245 250 255

Met Ile Lys Arg Ala Val Phe Ala Arg Glu Leu Gly Ala Pro Ile Ile

260 265 270

Met His Asp Tyr Leu Thr Gly Gly Phe Thr Ala Asn Thr Ser Leu Ala

275 280 285

His Tyr Cys Arg Asp Asn Gly Leu Leu Leu His Ile His Arg Ala Met

290 295 300

His Ala Val Ile Asp Arg Gln Lys Asn His Gly Met His Phe Arg Val

305 310 315 320

Leu Ala Lys Ala Leu Arg Leu Ser Gly Gly Asp His Ile His Ala Gly

325 330 335

Thr Val Val Gly Lys Leu Glu Gly Glu Arg Glu Ile Thr Leu Gly Phe

340 345 350

Val Asp Leu Leu Arg Asp Asp Phe Val Glu Lys Asp Arg Ser Arg Gly

355 360 365

Ile Phe Phe Thr Gln Asp Trp Val Ser Leu Pro Gly Val Leu Pro Val

370 375 380

Ala Ser Gly Gly Ile His Val Trp His Met Pro Ala Leu Thr Glu Ile

385 390 395 400

Phe Gly Asp Asp Ser Val Leu Gln Phe Gly Gly Gly Thr Leu Gly His

405 410 415

Pro Trp Gly Asn Ala Pro Gly Ala Val Ala Asn Arg Val Ala Leu Glu

420 425 430

Ala Cys Val Gln Ala Arg Asn Glu Gly Arg Asp Leu Ala Arg Glu Gly

435 440 445

Asn Glu Ile Ile Arg Glu Ala Ser Lys Trp Ser Pro Glu Leu Ala Ala

450 455 460

Ala Cys Glu Val Trp Lys Glu Ile Lys Phe Glu Phe Pro

465 470 475

<210> 5

<211> 160

<212> PRT

<213> Moringa oleifera Lam.

<400> 5

Met Ala Lys Phe Thr Leu Leu Leu Ala Ile Phe Ala Leu Phe Leu Ile

1 5 10 15

Leu Ala Asn Ala Asn Val Tyr Arg Thr Thr Val Glu Leu Asp Glu Glu

20 25 30

Pro Asp Asp Asn Gln Gln Gly Gln Gln Gln Gln Gln Cys Arg Gln Gln

35 40 45

Phe Leu Thr His Gln Arg Leu Arg Ala Cys Gln Arg Phe Ile Arg Arg

50 55 60

Gln Thr Gln Gly Gly Gly Ala Leu Glu Asp Val Glu Asp Asp Val Glu

65 70 75 80

Glu Ile Glu Glu Val Val Glu Pro Asp Gln Ala Arg Arg Pro Ala Ile

85 90 95

Gln Arg Cys Cys Gln Gln Leu Arg Asn Ile Gln Pro Arg Cys Arg Cys

100 105 110

Pro Ser Leu Arg Gln Ala Val Gln Leu Ala His Gln Gln Gln Gly Gln

115 120 125

Val Gly Pro Gln Gln Val Arg Gln Met Tyr Arg Leu Ala Ser Asn Ile

130 135 140

Pro Ala Ile Cys Asn Leu Arg Pro Met Ser Cys Pro Phe Gly Gln Gln

145 150 155 160

<210> 6

<211> 283

<212> PRT

<213> Moringa oleifera Lam.

<400> 6

Val Phe Pro Ser Ile Val Gly Arg Pro Arg His Thr Gly Val Met Val

1 5 10 15

Gly Met Gly Gln Lys Asp Ala Tyr Val Gly Asp Glu Ala Gln Ser Lys

20 25 30

Arg Gly Ile Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Ser

35 40 45

Asn Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu

50 55 60

Leu Arg Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro

65 70 75 80

Leu Asn Pro Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu

85 90 95

Thr Phe Asn Val Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser

100 105 110

Leu Tyr Ala Ser Gly Arg Thr Thr Gly Ile Val Leu Asp Ser Gly Asp

115 120 125

Gly Val Ser His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His

130 135 140

Ala Ile Leu Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Ala Leu

145 150 155 160

Met Lys Ile Leu Thr Glu Arg Gly Tyr Met Phe Thr Thr Thr Ala Glu

165 170 175

Arg Glu Ile Val Arg Asp Met Lys Glu Lys Leu Ala Tyr Val Ala Leu

180 185 190

Asp Tyr Glu Gln Glu Leu Glu Thr Ala Lys Ser Ser Ser Ser Val Glu

195 200 205

Lys Asn Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Ala Glu

210 215 220

Arg Phe Arg Cys Pro Glu Val Leu Phe Gln Pro Ser Leu Ile Gly Met

225 230 235 240

Glu Ala Ala Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys

245 250 255

Asp Val Asp Ile Arg Lys Asp Leu Tyr Gly Asn Ile Val Leu Ser Gly

260 265 270

Gly Ser Thr Met Phe Pro Gly Ile Ala Asp Arg

275 280

<210> 7

<211> 85

<212> PRT

<213> Moringa oleifera Lam.

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Ala Tyr Gly Leu Phe Phe Leu Gly Ala His Phe Val Trp Ala Phe Ser

1 5 10 15

Leu Met Phe Leu Phe Ser Gly Arg Gly Tyr Trp Gln Glu Leu Ile Glu

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Ser Ile Val Trp Ala His Asn Lys Leu Lys Val Ala Pro Ala Thr Gln

35 40 45

Pro Arg Ala Leu Ser Ile Val Gln Gly Arg Ala Val Gly Val Thr His

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Tyr Leu Leu Gly Gly Ile Ala Thr Thr Trp Ala Phe Phe Leu Ala Arg

65 70 75 80

Ile Ile Ala Val Gly

85

<210> 8

<211> 163

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<213> Moringa oleifera Lam.

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Met Ala Lys Leu Thr Leu Leu Leu Ala Thr Leu Ala Leu Leu Val Leu

1 5 10 15

Leu Ala Asn Ala Ser Ile Tyr Arg Thr Thr Val Glu Leu Asp Glu Glu

20 25 30

Pro Asp Asp Asn Gln Gln Gln Arg Cys Arg His Gln Phe Gln Thr Gln

35 40 45

Gln Arg Leu Arg Ala Cys Gln Arg Val Ile Arg Arg Trp Ser Gln Gly

50 55 60

Gly Gly Pro Met Glu Asp Val Glu Asp Glu Ile Asp Glu Thr Asp Glu

65 70 75 80

Ile Glu Glu Val Val Glu Pro Asp Gln Ala Arg Arg Pro Pro Thr Leu

85 90 95

Gln Arg Cys Cys Arg Gln Leu Arg Asn Val Ser Pro Phe Cys Arg Cys

100 105 110

Pro Ser Leu Arg Gln Ala Val Gln Ser Ala Gln Gln Gln Gln Gly Gln

115 120 125

Val Gly Pro Gln Gln Val Gly His Met Tyr Arg Val Ala Ser Arg Ile

130 135 140

Pro Ala Ile Cys Asn Leu Gln Pro Met Arg Cys Pro Phe Arg Gln Gln

145 150 155 160

Gln Ser Ser

Claims

1. a kind of extract the method with hydrolysing activity protease from moringa seeds, which comprises the following steps:

1) protein non-marked relative quantitative assay is carried out respectively from germination moringa seeds and dry moringa seeds, it is total to screen dry moringa seeds Differential protein between albumen and germination moringa seeds total protein；A length of 10~the 15cm of bud of germination moringa seeds, germinate moringa seeds Quality is 12.57~13.09g/15；The quality of dry moringa seeds is 4.31~4.53g/15；

2) statistic procedure 1) protein of up-regulated expression in the differential protein, the protein of obtained up-regulated expression is subjected to GO Functional annotation obtains the protease with hydrolysing activity；

4) obtain having the PI value of the protease of hydrolysing activity, set-up procedure 4 according to screening in step 2)) in moringa seeds protease The pH value of salt extract is collected protease precipitate, is redissolved, desalination obtains moringa seeds albumen enzyme extract to the PI value；

5) molecular weight for obtaining having the protease of hydrolysing activity according to screening in step 2), selects ultrafiltration membrane to the moringa seeds Albumen in albumen enzyme extract carries out ultrafiltration retention, collects the protease of target molecular weight, obtains with hydrolysing activity albumen Enzyme.

2. the method according to claim 1, wherein the cultural method for the moringa seeds that germinate described in step 1) will be peppery 4~10 DEG C of water infiltrations of the wooden seed, then 24~26h is impregnated in 20~25 DEG C of water, obtained immersion moringa seeds are in constant temperature and humidity Environment in cultivate；

It cultivates the 1st~3 day, cultivation temperature is 25~30 DEG C, humidity is 70%~75%；It cultivates the 4th~6 day, cultivation temperature is 22~25 DEG C, humid control is 60%~65%.

3. the method according to claim 1, wherein the protease in step 2) with hydrolysing activity includes 8 kinds Protease；The amino acid sequence of 8 kinds of protease is respectively in sequence table shown in No.1~8 SEQ ID；

The PI of protease shown in the SEQ ID No.1 is 10.10；The molecule of protease shown in the SEQ ID No.1 Amount is 11789.27；

The PI of protease shown in the SEQ ID No.2 is 7.55；The molecular weight of protease shown in the SEQ ID No.2 It is 18459.82；

The PI of protease shown in the SEQ ID No.3 is 7.15；The molecular weight of protease shown in the SEQ ID No.3 It is 18811.2；

The PI of protease shown in the SEQ ID No.4 is 6.23；The molecular weight of protease shown in the SEQ ID No.4 It is 52880.49；

The PI of protease shown in the SEQ ID No.5 is 10.63；The molecule of protease shown in the SEQ ID No.5 Amount is 6802.67；

The PI of protease shown in the SEQ ID No.6 is 5.24；The molecular weight of protease shown in the SEQ ID No.6 It is 31591.8；

The PI of protease shown in the SEQ ID No.7 is 10.36；The molecule of protease shown in the SEQ ID No.7 Amount is 9427.98；

The PI of protease shown in the SEQ ID No.8 is 7.75；The molecular weight of protease shown in the SEQ ID No.8 It is 18756.12.

4. the method according to claim 1, wherein in step 2) salt method germination moringa seeds protease side Method includes that the germination moringa seeds crush, sieving, and obtained screenings and sodium chloride solution mixing extracts, is separated by solid-liquid separation, obtains Moringa seeds protease salt extract.

5. according to the method described in claim 4, it is characterized in that, the sieve pore of the sieving is not less than 60 mesh；

The quality of the screenings and the volume of sodium chloride solution are 1g:5mL；The concentration of the sodium chloride solution be 0.25~ 0.30mol/L。

6. according to the method described in claim 4, it is characterized in that, the temperature extracted is 30 DEG C；The time of extraction be 0.5~ 1h；The pH value of feed liquid is 7.15 when extraction.

7. the method according to claim 1, wherein the method for collecting protease precipitate includes centrifugation；It is described from The revolving speed of the heart is 4500~5000r/min；The time of the centrifugation is 20~30min.

8. the method according to claim 1, wherein the method redissolved is to mix the protease precipitate of collection and water It closes, obtained mixed liquor carries out ultrasonic dissolution and obtains protein enzyme solution；The quality of the protease precipitate and the volume ratio of water are 5g:5~10mL；

The power of the ultrasound is 200Hz；The time of the ultrasound is 2~5min.

9. according to the method described in claim 8, it is characterized in that, also successively including to the protein enzyme solution before the desalination Adjust pH value and pre-separation；

The pH value of protein enzyme solution is adjusted to 7.0；

The pre-separation includes dialysis；The molecular cut off of dialysis bag filter is 3500Da；The time of the dialysis is 50~56h.

10. method described in any one according to claim 1~9, which is characterized in that the Protein L abel-free is opposite Quantitative analysis include TCA/ acetone precipitation and SDT cracking process, PAGE gel electrophoresis, FASP enzymatic hydrolysis, high performance liquid chromatography and The mass spectrometric mass spectral analysis of Q-Exactive, mass spectral analysis initial data simultaneously carry out checking storehouse identification using MaxQuant software and determine Amount analysis.