CN109504671B - Method for extracting protease with hydrolytic activity from moringa seeds - Google Patents
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Abstract
The invention provides a method for extracting protease with hydrolytic activity from moringa seeds, belonging to the technical field of natural protease extraction. The method comprises the steps of improving the types and the content of protease of moringa seeds by a germination technology, and then obtaining information of protease with up-regulated expression and hydrolytic activity according to the proteomics analysis of the moringa seeds for guiding subsequent extraction. The extraction method provided by the invention can be used for extracting various proteases with hydrolytic activity, and the extracted proteases have high content and strong enzymatic activity. Compared with the protease extracted from the dry moringa seeds, the content of the 8 moringa seed proteases extracted from the germinated moringa seeds is correspondingly improved by 10-21%, wherein the corresponding hydrolytic activity of the 2 proteases is improved by 8.0-9.9% compared with the dry moringa seed proteases. The method provided by the invention avoids the problems of blindness, complexity and large workload of the traditional protein separation method, and has the characteristics of simple operation and good repeatability.
Description
Technical Field
The invention belongs to the technical field of natural protease extraction, and particularly relates to a method for extracting protease with hydrolytic activity from moringa seeds.
Background
As enzyme engineering is becoming more and more widely used in the food industry, there is an increasing demand for protease resources. Although the protease can be obtained by means of genetic engineering, artificial synthesis or microbial fermentation, the natural animal and plant protease resources are still in great demand, and particularly, some proteases with hydrolytic activity have the problem of serious shortage.
Moringa tree is a perennial tropical and subtropical deciduous tree, belongs to Moringa plants of Moringaceae, has a Latin scientific name of Moringa oleifera lam, has an English name of Moringa, has various other names, is called Drumstick tree (Drumstick tree) according to the pod shape (slender and triangular), has pungent taste according to the root part, and is called horse radish tree (Horseradish tree); oil can be extracted from its seeds, called a Benoil tree (Benoil tree or Benzoil tree). Moringa oleifera is a characteristic and advantageous industry in Yunnan, has rich resources, and the seeds and leaves of the moringa oleifera seeds contain rich nutrient components, but the moringa oleifera seeds contain alkaloid and moringa oleifera flavone, and are preferably eaten by doctors in a proper amount after the instructions of the doctors. At present, the edible or medicinal value of the moringa oleifera and the auxiliary products thereof is reported more, however, the research on the protease and the biological activity thereof is rarely reported, and a method for extracting the protease with high content and high hydrolytic activity is not provided.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for extracting protease with hydrolytic activity from moringa seeds, wherein the protease extracted by the method has high content and strong hydrolytic activity.
The invention provides a method for extracting protease with hydrolytic activity from moringa seeds, which comprises the following steps:
1) respectively carrying out protein non-labeled relative quantitative analysis on the germinated moringa seeds and the dried moringa seeds, and screening differential proteins between the total protein of the dried moringa seeds and the total protein of the germinated moringa seeds; the bud length of the germinated moringa seeds is 10-15 cm, and the mass of the germinated moringa seeds is 12.57-13.09 g/15 grains; the mass of the dried moringa seeds is 4.31-4.53g per 15 grains;
2) counting the protein up-regulated and expressed in the differential protein in the step 1), and performing GO function annotation on the obtained protein up-regulated and expressed to obtain protease with hydrolytic activity;
3) extracting the germinated moringa seed protease by a salt method to obtain moringa seed protease salt extract;
4) obtaining a PI value of protease with hydrolytic activity by screening in the step 2), adjusting the pH value of the moringa seed protease salt extract in the step 4) to the PI value, collecting protease precipitate, redissolving and desalting to obtain a crude moringa seed protease extract;
5) and 2) screening according to the molecular weight of the protease with hydrolytic activity obtained in the step 2), selecting an ultrafiltration membrane to carry out ultrafiltration interception on the protein in the crude extract of the moringa seed protease, and collecting the protease with target molecular weight to obtain the protease with hydrolytic activity.
Preferably, the culture method of the germinated moringa seeds in the step 1) is characterized in that the moringa seeds are soaked in water at the temperature of 4-10 ℃, then soaked in water at the temperature of 20-25 ℃ for 24-26 hours, and the obtained soaked moringa seeds are cultured in a constant-temperature and constant-humidity environment;
culturing for 1-3 days at 25-30 deg.C and 70-75% humidity; culturing for 4-6 days at 22-25 deg.C and humidity of 60-65%.
Preferably, the protease having hydrolytic activity in step 2) includes 8 proteases; the amino acid sequences of the 8 proteinases are respectively shown as SEQ ID No. 1-8 in the sequence table;
the PI of the protease shown as SEQ ID No.1 is 10.10; the molecular weight of the protease shown in SEQ ID No.1 is 11789.27;
the PI of the protease shown as SEQ ID No.2 is 7.55; the molecular weight of the protease shown in SEQ ID No.2 is 18459.82;
the PI of the protease shown as SEQ ID No.3 is 7.15; the molecular weight of the protease shown in SEQ ID No.3 is 18811.2;
the PI of the protease shown as SEQ ID No.4 is 6.23; the molecular weight of the protease shown in SEQ ID No.4 is 52880.49;
the PI of the protease shown as SEQ ID No.5 is 10.63; the molecular weight of the protease shown in SEQ ID No.5 is 6802.67;
the PI of the protease shown as SEQ ID No.6 is 5.24; the molecular weight of the protease shown in SEQ ID No.6 is 31591.8;
the PI of the protease shown as SEQ ID No.7 is 10.36; the molecular weight of the protease shown in SEQ ID No.7 is 9427.98;
the PI of the protease shown as SEQ ID No.8 is 7.75; the molecular weight of the protease shown in SEQ ID No.8 is 18756.12.
Preferably, the method for extracting the germinated moringa seed protease by the salt method in the step 2) comprises the steps of crushing and sieving the germinated moringa seeds, mixing obtained undersize with a sodium chloride solution, extracting, and carrying out solid-liquid separation to obtain the moringa seed protease salt extract.
Preferably, the sieve mesh of the sieve is not less than 60 meshes;
the mass of the undersize product and the volume of the sodium chloride solution are 1g:5 mL; the concentration of the sodium chloride solution is 0.25-0.30 mol/L.
Preferably, the temperature of extraction is 30 ℃; the extraction time is 0.5-1 h; the pH of the feed liquid during extraction was 7.15.
Preferably, the method of collecting the protease precipitate comprises centrifugation; the rotating speed of the centrifugation is 4500-; the centrifugation time is 20-30 min.
Preferably, the redissolution method is to mix the collected protease precipitate with water, and the obtained mixed solution is subjected to ultrasonic dissolution to obtain a protease solution; the mass of the protease precipitate and the volume ratio of water are 5 g: 5-10 mL;
the power of the ultrasound is 200 Hz; the time of the ultrasonic treatment is 2-5 min.
Preferably, the steps of adjusting the pH value of the protease solution and pre-separating are sequentially included before desalting;
adjusting the pH value of the protease solution to 7.0;
the pre-separating comprises dialysis; the cut-off molecular weight of the dialysis bag for dialysis is 3500 Da; the dialysis time is 50-56 h.
Preferably, the non-labeled relative quantitative analysis of the protein comprises TCA/acetone precipitation and SDT cracking method, SDS-PAGE gel electrophoresis, FASP enzymolysis, high performance liquid chromatography and mass spectrum analysis of a Q-active mass spectrometer, and the mass spectrum analysis of raw data and library checking identification and quantitative analysis are carried out by adopting MaxQuant software.
The invention provides a method for extracting protease with hydrolytic activity from moringa seeds, which comprises the steps of firstly, improving the types and the contents of the protease of the moringa seeds by a germination technology; and then, obtaining information of protease with up-regulated expression and hydrolytic activity according to proteomics analysis of moringa seeds so as to guide subsequent separation. The extraction method provided by the invention can be used for extracting various proteases with hydrolytic activity, and the extracted proteases have the characteristics of high content and strong enzyme activity. Experiments show that: compared with the protease extracted from the dried moringa seeds, the content of the extracted 8 moringa seed proteases is correspondingly improved by 10-21%, and 2 of the extracted moringa seed proteases are correspondingly improved by 8.0-9.9% in hydrolysis activity compared with the protease in the dried moringa seeds.
Meanwhile, the method provided by the invention combines proteomics analysis and extraction means, so that the problems of blindness, complexity and large workload of the traditional separation method are avoided, and the extraction method is simpler, more convenient, faster and more efficient. The 2 proteases extracted by the method have high hydrolase activity and curd activity, and experiments prove that: compared with the protease extracted from the dry moringa seeds, the 2 enzymes extracted by the method provided by the invention have the advantage that the milk curd activity is improved by 8.3-9.1%. Therefore, the moringa seed protease separated and extracted by the method has better proteolytic activity and curd activity, is beneficial to large-scale batch production, and is expected to realize the industrial application of the subsequent moringa seed protease.
Drawings
FIG. 1 is a graph showing the relative molecular mass distribution of the proteins identified in Moringa seed according to the present invention;
FIG. 2 is a Venn diagram of the protein identified in triplicate within a group of dried moringa seeds according to the invention;
FIG. 3 is a Venn diagram of the protein identified in the seed group of the sprouted Moringa oleifera of the present invention in triplicate;
FIG. 4 is a Venn diagram of the proteins identified in the sample set of germinated moringa seeds and dried moringa seeds of the present invention;
FIG. 5 is a volcanic image of germinated and dried Moringa seeds according to the present invention.
Detailed Description
The invention provides a method for extracting protease with hydrolytic activity from moringa seeds, which comprises the following steps:
1) respectively carrying out protein non-labeled relative quantitative analysis on the germinated moringa seeds and the dried moringa seeds, and screening differential proteins between the total protein of the dried moringa seeds and the total protein of the germinated moringa seeds; the bud length of the germinated moringa seeds is 10-15 cm, and the mass of the germinated moringa seeds is 12.57-13.09 g/15 grains; the mass of the dried moringa seeds is 4.31-4.53g per 15 grains;
2) counting the protein up-regulated and expressed in the differential protein in the step 1), and performing GO function annotation on the obtained protein up-regulated and expressed to obtain protease with hydrolytic activity;
3) extracting the germinated moringa seed protease by a salt method to obtain moringa seed protease salt extract;
4) obtaining a PI value of protease with hydrolytic activity by screening in the step 2), adjusting the pH value of the moringa seed protease salt extract in the step 4) to the PI value, collecting protease precipitate, redissolving and desalting to obtain a crude moringa seed protease extract;
5) and 2) screening according to the molecular weight of the protease with hydrolytic activity obtained in the step 2), selecting an ultrafiltration membrane to carry out ultrafiltration interception on the protein in the crude extract of the moringa seed protease, and collecting the protease with target molecular weight to obtain the protease with hydrolytic activity.
The method comprises the steps of respectively carrying out protein non-labeled relative quantitative analysis on germinated moringa seeds and dried moringa seeds, and screening differential proteins between the total protein of the dried moringa seeds and the total protein of the germinated moringa seeds; the bud length of the germinated moringa seeds is 10-15 cm, and the mass of the germinated moringa seeds is 12.57-13.09 g/15 grains; the mass of the dried moringa seeds is 4.31-4.53g per 15 grains.
In the method for culturing the germinated moringa seeds, the moringa seeds are preferably soaked in water at the temperature of 4-10 ℃, then soaked in water at the temperature of 20-25 ℃ for 24-26 hours, and the obtained soaked moringa seeds are cultured in a constant-temperature and constant-humidity environment. The culture temperature is preferably 25-30 ℃ and more preferably 28 ℃ on days 1-3. The humidity of the culture is preferably 70% to 75%, more preferably 73%. The culture temperature is preferably 22-25 ℃ and more preferably 23 ℃ on the 4 th-6 th day of culture. The culture humidity is preferably 60% to 65%, more preferably 63%. The bud length of the germinated moringa seeds is preferably 12-14 cm, and more preferably 13 cm. The mass of the germinated moringa seeds is preferably 12.57-13.09 g/15 grains; the mass of the dried moringa seeds is preferably 4.48g/15 grains. The germinated moringa seeds are used as raw materials for extraction, and the protease variety, content and enzyme activity of the moringa seeds are improved.
In the invention, the non-labeled relative quantitative analysis of the protein preferably comprises TCA/acetone precipitation and SDT cracking method, SDS-PAGE gel electrophoresis, FASP enzymolysis, high performance liquid chromatography and mass spectrum analysis of Q-active mass spectrometer, and the mass spectrum analysis of the original data and database checking identification and quantitative analysis by adopting MaxQuant software. Specific procedures for unlabeled relative quantification of proteins are described in the following references: [1] cox, J.and M.Man (2008), "MaxQuant enzymes high peptide identification rates, induced p.p.b. -range of acids and polypeptides-protein identification," Nat Biotechnol 26(12): 1367-.
[2]Cox,J.,M.Y.Hein,et al.(2014)."Accurate proteome-wide label-freequantification 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 engineintegrated into the MaxQuant environment."J Proteome Res 10(4):1794-1805.
Obtaining differential protein, counting the protein up-regulated and expressed in the differential protein, and carrying out GO function annotation on the obtained up-regulated and expressed protein to obtain the protease with hydrolytic activity.
In the present invention, the number of the differential proteins is 43, wherein the number of the up-regulated expression of the differential proteins is 26. The GO function annotation method is not particularly limited in the present invention, and a GO function annotation scheme well known in the art may be adopted.
In the present invention, the protease having hydrolytic activity preferably includes 8 proteases; the amino acid sequences of the 8 proteinases are respectively shown as SEQ ID No. 1-8 in the sequence table; the PI of the protease shown as SEQ ID No.1 is 10.10; the molecular weight of the protease shown in SEQ ID No.1 is 11789.27; the name of the protease shown by SEQ ID No.1 is searched and is Chitin-binding protein 2.
The PI of the protease shown as SEQ ID No.2 is 7.55; the molecular weight of the protease shown in SEQ ID No.2 is 18459.82; the name of the protease shown by the searched SEQ ID No.2 is 2S albumin.
The PI of the protease shown as SEQ ID No.3 is 7.15; the molecular weight of the protease shown in SEQ ID No.3 is 18811.2; the name of the protease shown by the searched SEQ ID No.3 is 2S albumin
The PI of the protease shown as SEQ ID No.4 is 6.23; the molecular weight of the protease shown in SEQ ID No.4 is 52880.49; the name of the protease shown in SEQ ID No.4 was searched and named as Ribulose bisphosphaguard carboxylase large chain (RBCL).
The PI of the protease shown as SEQ ID No.5 is 10.63; the molecular weight of the protease shown in SEQ ID No.5 is 6802.67; the name of the protease shown by SEQ ID No.5 was searched for 2.1 protein.
The PI of the protease shown as SEQ ID No.6 is 5.24; the molecular weight of the protease shown in SEQ ID No.6 is 31591.8; the name of the protease shown by SEQ ID No.6 is Actin by searching.
The PI of the protease shown as SEQ ID No.7 is 10.36; the molecular weight of the protease shown in SEQ ID No.7 is 9427.98; the name of the protease shown by SEQ ID No.7 was searched and found to be Photosystem I P700apoprotein A1.
The PI of the protease shown as SEQ ID No.8 is 7.75; the molecular weight of the protease shown in SEQ ID No.8 is 18756.12. The name of the protease shown by the searched SEQ ID No.8 is 2S albumin.
The invention adopts a salt method to extract the sprouted moringa seed protease to obtain the moringa seed protease salt extract.
In the invention, the method for extracting the germinated moringa seed protease by the salt method preferably comprises the steps of crushing and sieving the germinated moringa seeds, mixing the obtained undersize with a sodium chloride solution, extracting, and carrying out solid-liquid separation to obtain the moringa seed protease salt extract. The mesh size of the sieve is preferably not less than 60 mesh. The mass of the undersize product and the volume of the sodium chloride solution are preferably 1g:5 mL; the concentration of the sodium chloride solution is preferably 0.25-0.30 mol/L. The temperature of extraction is preferably 30 ℃; the extraction time is preferably 0.5-1 h; the pH of the feed solution during extraction is preferably 7.15.
After obtaining the moringa seed protease salt extract, the invention obtains the PI value of protease with hydrolytic activity according to the screening, adjusts the pH value of the moringa seed protease salt extract in the step 4) to the PI value, collects the protease precipitate, redissolves and desalts to obtain the moringa seed protease crude extract.
In the invention, the method for adjusting the pH value of the moringa seed protease salt extract is preferably to drop 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrogen chloride solution into the moringa seed protease salt extract at the speed of 0.5-1.0 mL/s, so that the pH values of the salt extracts respectively reach the PI of 8 proteases obtained by the screening of the proteomics technology.
In the present invention, the method of collecting the protease precipitate preferably comprises centrifugation; the rotation speed of the centrifugation is preferably 4500-5000 r/min; the time for centrifugation is preferably 20-30 min. And centrifuging and collecting precipitate.
In the present invention, the method of reconstitution is preferably a method in which the collected protease precipitate is mixed with water, and the resulting mixture is subjected to ultrasonic dissolution to obtain a protease solution. The ratio of the mass of the protease precipitate to the volume of water is preferably 5 g: 5-10 mL. The power of the ultrasound is preferably 200 Hz; the time of the ultrasonic treatment is preferably 2-5 min.
In the present invention, it is preferable that the desalting further comprises adjusting the pH of the protease solution and pre-separating in this order. The pH of the protease solution is preferably adjusted to 7.0. The pre-separation preferably comprises dialysis; the molecular weight cut-off of the dialysis bag for dialysis is preferably 3500 Da; the dialysis time is preferably 50-56 h.
According to the molecular weight of the protease with hydrolytic activity obtained by screening, an ultrafiltration membrane is selected to carry out ultrafiltration interception on the protein in the crude extract of the moringa seed protease, and the protease with the target molecular weight is collected to obtain the protease with hydrolytic activity.
In the invention, the lower layer trapped fluid obtained by ultrafiltration trapping is preferably put into a vacuum low-temperature freeze dryer for freeze-drying treatment for 48 hours after being quickly frozen at-80 ℃ for 5 hours, so as to obtain the protease powder.
The protease extracted by the invention has higher content and biological activity, wherein the Actin of the protease shown in SEQ ID No.6 and the Ribulose bisphosphate carboxylase large chain (RBCL) shown in SEQ ID No.4 have hydrolase activity and curd activity, and the protease is applied to processing cheese, beer and beverage food.
The method for extracting protease with hydrolytic activity from moringa seeds provided by the invention is described in detail by the following examples, but the method is not to be construed as limiting the scope of the invention.
Example 1
Germination of 100g dried Moringa seed and isolation of specific protease
1) Soaking dry moringa seeds in warm water: spraying cold water on 100g of dried moringa seeds for wetting, and then soaking the seeds in warm water at 25 ℃ for 24 hours, wherein a filter screen is required to cover the seeds in the soaking process, so that the moringa seeds are completely soaked in the warm water;
2) germination of moringa seeds: putting the soaked moringa seeds into a seedling tray paved with 3 layers of filter paper wetted by water, wherein the interval between every two moringa seeds is 1cm, then putting the seedling tray into an artificial climate box, and controlling the temperature and the humidity in the box to be 25 ℃ and 70% during germination for 1-3 days, 22 ℃ and 60% during germination for 4-6 days;
3) proteomics analysis of dried moringa seeds and germinated moringa seeds: selecting germinated Moringa seeds with bud length of 10cm and mass of 12.57g/15 seeds and dried Moringa seeds with mass of 4.31g/15 seeds for performing protein Label-free relative quantitative analysis, performing mass spectrometry on original data by using TCA/acetone precipitation and SDT cracking method, SDS-PAGE gel electrophoresis, FASP enzymolysis, high performance liquid chromatography and Q-active mass spectrometer, performing library checking identification and quantitative analysis by using MaxQuant software (figure 1), and comparing and analyzing 43 different proteins of dried moringa seeds (A-1 in figure 2) and germinated moringa seeds (B-1 in figure 3), wherein the germinated moringa seeds have the content of 26 proteins, 8 up-regulated proteins are screened out by GO function annotation and are proteases with hydrolytic activity, and the uniprot database shows that the molecular weight range and the isoelectric point PI range of the 8 proteases are shown in Table 1.
TABLE 18 protease information List
| Sequence numbering | Name of protease | Molecular weight (kDa) | Isoelectric point |
| SEQ ID No.1 | Chitin-binding |
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 extraction of dry moringa seeds and germinated moringa seed protease: crushing dried moringa seeds and germinated moringa seeds for 2min by using a high-speed crusher, sieving by using a 60-mesh sieve to remove most of moringa seed shells, respectively mixing dried moringa seed undersize products and a sodium chloride solution according to a ratio of 1:10(g/mL), mixing the germinated moringa seed undersize products and the sodium chloride solution according to a mass-to-volume ratio of 1:5g/mL, obtaining a moringa seed extracting solution under the conditions of an extraction temperature of 30 ℃, a pH value of 7.15 and an extraction time of 1.0h, and placing the extracting solution into a filter cup to be treated by using 2 layers of medium-speed qualitative filter paper to obtain a moringa seed protease salt extracting solution;
5) separation of specific proteases in dried moringa seeds and germinated moringa seeds: dripping 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrogen chloride solution into Moringa seed protease salt extract at a speed of 0.5mL/s respectively to make the pH value of the salt extract reach PI 5.24-10.63 of 8 proteases obtained by the proteomics technology screening, centrifuging at 4500r/min for 20min in sequence to obtain protein precipitates, ultrasonically dissolving the protein precipitates obtained under different proteases PI with 5mL deionized water for 2min to completely dissolve the precipitates, dripping 0.1mol/L sodium hydroxide solution or 0.1mol/L hydrogen chloride solution to adjust the pH value of each protein solution to 7.0, putting each protein solution into a dialysis bag with a molecular weight cutoff of 3500Da for 50h for desalination treatment, and performing ultrafiltration on each dialysate by using different ultrafiltration membranes with molecular weight cutoff of 6-60 kDa, and quickly freezing the lower trapped fluid at-80 ℃ for 5h, and freeze-drying in a vacuum low-temperature freeze dryer for 48h to obtain the 8 protease content of the germinated moringa seeds and the 8 protease content of the dried moringa seeds. The results are shown in Table 2.
TABLE 2 germinating Moringa seed 8 protease content and dried Moringa seed 8 protease content (mg)
| Sequence Listing numbering | Content of protease in germinated moringa seeds | Content of protease in dried Moringa seed |
| 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 |
The obtained sprouted moringa seed 8 protease content is correspondingly increased by 10.0% (SEQ ID 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) and 18.5% (SEQ ID No.6) compared with the dry moringa seed;
6) analysis of hydrolytic Activity of specific proteases of dried Moringa seeds and germinated Moringa seeds: preparing the obtained 8 moringa seed protease freeze-dried powders into solutions with the mass concentration of 10mg/L by using deionized water, respectively measuring the milk curding activity and the protein decomposition activity, firstly weighing 3g of skimmed milk powder, adding 30mL of distilled water to prepare reconstituted milk with the mass-volume ratio of 1:10g/mL, heating to boil, standing, cooling for 5min, putting into a 60-DEG C constant-temperature water bath kettle, after the central temperature of the re-dissolved milk reaches 60 ℃, 10mL of enzyme solution is added, the water bath is continued after the enzyme solution is fully mixed, the time (T) from the addition of the enzyme solution to the complete coagulation of the milk protein is accurately recorded, the milk curd activity is (3 × 2400)/(T × 10) SU/mg, and then taking 2mL of 1.5% casein solution, preserving the heat at 60 ℃ for 5min, adding 1mL of preheated enzyme solution to be detected, uniformly mixing, reacting for 30min, adding 4mL of 8% trichloroacetic acid to stop the reaction, and measuring the absorbance (A) of the filtrate at 280nm after filtering.280) Mixing preheated enzyme solution with trichloroacetic acid to inactivate enzyme solution, adding 2mL casein solution, keeping at 60 deg.C for 5min, filtering, and measuring absorbance (A ') of the filtrate at 280 nm'280) As a blank control, the proteolytic activity of 1mL of the enzyme solution was calculated (A)280-A′280) And/1, screening out 2 proteases respectively shown as SEQ ID No.6 and SEQ ID No.4 by measuring the milk curd activity and the protein decomposition activity. The results are shown in Table 3.
TABLE 32 curd and proteolytic Activities of the proteases
| Sequence numbering | Milk curd vitality (SU/mg) | Hydrolytic activity |
| SEQ ID No.6 | 12.45 | 168 |
| SEQ ID No.4 | 15.87 | 199 |
Compared with the corresponding hydrolytic activity of the dry moringa seeds, the germination moringa seed 2 protease is improved by 7.6 percent (SEQ ID No.6) and 9.5 percent (SEQ ID No.4), and the curd activity is improved by 7.3 percent (SEQ ID No.6) and 8.1 percent (SEQ ID No. 4).
Example 2
Germination of 500g dried Moringa seed and isolation of specific protease
The process was carried out essentially as in example 1, with the following differences:
1) soaking dry moringa seeds in warm water: spraying 500g of dry moringa seeds with cold water for wetting, and then soaking the seeds in warm water at the temperature of 22 ℃ for 24 hours;
2) germination of moringa seeds: putting the soaked moringa seeds into a seedling tray paved with 3 layers of filter paper wetted by water, wherein the interval between every two moringa seeds is 1.2cm, then putting the seedling tray into an artificial climate box, and controlling the temperature and the humidity in the box to be 28 ℃ and 72% during 1-3 days of germination, 23 ℃ and 63% during 4-6 days of germination;
3) proteomics analysis of dried moringa seeds and germinated moringa seeds: selecting germinated Moringa seeds with bud length of 12cm and mass of 12.76g/15 seeds and dried Moringa seeds with mass of 4.41g/15 seeds for protein Label-free relative quantitative analysis, wherein the library search result is shown as A-2 in FIG. 2 and B-2 in FIG. 3;
4) salt extraction of dry moringa seeds and germinated moringa seed protease: crushing the dried moringa seeds and the germinated moringa seeds for 3min by using a high-speed crusher, sieving by using a 60-mesh sieve to remove most of moringa seed shells, respectively adding 0.25mol/L sodium chloride solution according to the mass-volume ratio of 1:10 and 1:5g/mL, and obtaining a moringa seed extracting solution under the conditions of extraction temperature of 30 ℃, pH of 7.15 and extraction time of 1.2 h;
5) separation of specific proteases in dried moringa seeds and germinated moringa seeds: dripping 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrogen chloride solution into moringa seed protease salt extract at a speed of 0.8mL/s respectively to enable the pH value of the salt extract to reach 8 protease PI 5.24-10.63 obtained by the proteomics technology screening respectively, centrifuging at 4800r/min for 25min sequentially to obtain protein precipitates, ultrasonically dissolving the protein precipitates obtained under different proteases PI for 3min to completely dissolve the precipitates, dripping 0.1mol/L sodium hydroxide solution or 0.1mol/L hydrogen chloride solution to adjust the pH value of each protein solution to 7.0, putting each protein solution into a dialysis bag with a molecular weight cutoff of 3500Da for 52h to perform desalination treatment, putting different dialysate with a molecular weight cutoff of 6-60 kDa into an ultrafiltration membrane to perform ultrafiltration interception, putting the lower layer solution into a vacuum freeze-drying machine for freeze-drying at a temperature of-80 ℃ for 5.5h, and putting the lower layer into a vacuum freeze-drying machine for 60 low-temperature freeze-drying h, obtaining the content of 8 proteases in the germinated moringa seeds and the content of 8 proteases in the dried moringa seeds. The results are shown in Table 4.
TABLE 4 germinating Moringa seed 8 protease content and dried Moringa seed 8 protease content (mg)
| Sequence Listing numbering | Content of protease in germinated moringa seeds | Content of protease in dried Moringa seed |
| 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 can be seen from Table 4, the protease content of the germinated moringa seeds 8 was increased by 11.3% (SEQ ID 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) as compared to the dry moringa seeds.
6) Analysis of hydrolytic Activity of specific proteases of dried Moringa seeds and germinated Moringa seeds:
2 proteases shown as SEQ ID No.6 and SEQ ID No.4 are screened out through measuring the milk curd activity and the protein decomposition activity. The hydrolysis activity and curd activity of the protease obtained by the enzyme activity analysis method of example 1 are shown in Table 5.
TABLE 52 curd and proteolytic Activities of the proteases
| Sequence numbering | Milk curd vitality (SU/mg) | Hydrolytic activity |
| SEQ ID No.6 | 12.38 | 171 |
| SEQ ID No.4 | 15.79 | 195 |
As can be seen from Table 5, the corresponding hydrolytic activity of the germinated moringa seed 2 protease is improved by 8.0% (SEQ ID No.6) and 9.9% (SEQ ID No.4) compared with that of the dried moringa seed, and the curd activity is improved by 8.3% (SEQ ID No.6) and 9.1% (SEQ ID No. 4).
Example 3
Germination of 1000g dried Moringa seed and isolation of specific protease
The process was carried out essentially as in example 1, with the following differences:
1) soaking dry moringa seeds in warm water: spraying 1000g of dry moringa seeds with cold water for wetting, and then soaking the seeds in warm water at the temperature of 20 ℃ for 24 hours;
2) germination of moringa seeds: putting the soaked moringa seeds into a seedling tray paved with 3 layers of filter paper wetted by water, wherein the interval between every two moringa seeds is 1.5cm, then putting the seedling tray into an artificial climate box, and controlling the temperature and the humidity in the box to be 30 ℃ and 75% during 1-3 days of germination, 23 ℃ and 65% during 4-6 days of germination;
3) proteomics analysis of dried moringa seeds and germinated moringa seeds: selecting germinated Moringa seeds with bud length of 15cm and mass of 13.09g/15 grains and dried Moringa seeds with mass of 4.53g/15 grains for performing protein Label-free relative quantitative analysis, wherein the library search result is shown as A-3 in FIG. 2 and B-3 in FIG. 3;
4) salt extraction of dry moringa seeds and germinated moringa seed protease: crushing the dried moringa seeds and the germinated moringa seeds for 5min by using a high-speed crusher, sieving by using a 60-mesh sieve to remove most of moringa seed shells, respectively adding 0.30mol/L sodium chloride solution according to the mass-volume ratio of 1:10 and 1:5g/mL, and obtaining a moringa seed extracting solution under the conditions of extraction temperature of 30 ℃, pH of 7.15 and extraction time of 1.5 h;
5) separation of specific proteases in dried moringa seeds and germinated moringa seeds: dripping 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrogen chloride solution into moringa seed protease salt extract at the speed of 1.0mL/s respectively to enable the pH value of the salt extract to reach 8 protease PI 5.24-10.63 obtained by the proteomics technology screening, centrifuging at 5000r/min for 30min in sequence to obtain protein precipitates, ultrasonically dissolving the protein precipitates obtained under different proteases PI for 3min to completely dissolve the precipitates, dripping 0.1mol/L sodium hydroxide solution or 0.1mol/L hydrogen chloride solution to adjust the pH value of each protein solution to 7.0, respectively putting each protein solution into a dialysis bag with the molecular weight cutoff of 3500Da for dialysis for 56h to carry out desalination treatment, then putting each dialysate into a vacuum low-temperature freeze-drying machine for freeze drying treatment after quickly freezing the lower layer solution at the temperature of-80 ℃ for 6h, and putting into a vacuum low-temperature freeze-drying machine for freeze drying treatment for 72 kDa h, obtaining the content of 8 proteases in the germinated moringa seeds and the content of 8 proteases in the dried moringa seeds. The results are shown in Table 6.
TABLE 6 germinating Moringa seed 8 protease content and dried Moringa seed 8 protease content (mg)
| Sequence Listing numbering | Content of protease in germinated moringa seeds | Content of protease in dried Moringa seed |
| 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 can be seen from Table 6, the protease content of the germinated moringa seeds 8 was increased by 12.2% (SEQ ID 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) as compared to the dry moringa seeds.
6) Analysis of hydrolytic Activity of specific proteases of dried Moringa seeds and germinated Moringa seeds: 2 proteases shown in SEQ ID No.6 and SEQ ID No.4 are screened out through measuring the milk curd activity and the protein decomposition activity. The hydrolysis activity and curd activity of the protease obtained by the enzyme activity analysis method in example 1 are shown in Table 7.
TABLE 72 curd and proteolytic Activity of the proteases
| Sequence numbering | Milk curd vitality (SU/mg) | Hydrolytic activity |
| SEQ ID No.6 | 12.41 | 165 |
| SEQ ID No.4 | 15.84 | 203 |
As can be seen from Table 7, the corresponding hydrolytic activity of the germinated moringa seed 2 protease is improved by 8.2% (SEQ ID No.6) and 10.1% (SEQ ID No.4) compared with that of the dried moringa seed, and the curd activity is improved by 8.5% (SEQ ID No.6) and 9.7% (SEQ ID No. 4).
Example 4
The proteins extracted from the germinated moringa seeds of examples 1-3 were compared with the proteins extracted from the dried moringa seeds to generate a Venn diagram (FIG. 4, wherein A represents the protein extracted from the germinated moringa seeds and B represents the protein extracted from the dried moringa seeds). As can be seen from FIG. 4, there were 650 proteins co-expressed in both groups, 119 proteins specific to the germinated moringa seeds, and 110 proteins specific to the dried moringa seeds.
A volcanic chart is drawn by using two factors, namely the Fold difference of protein expression (Fold change) between two groups of samples of germinated moringa seeds and dried moringa seeds and P-value obtained by T test (figure 5) together, and is used for displaying the significant difference of the two groups of sample data. The abscissa represents the multiple of the difference (base 2 logarithmic transformation) and the ordinate represents the significance P-value of the difference (base 10 logarithmic transformation). The data obtained using the T-test algorithm, shown in the volcano plot format in figure 5, is applicable to demonstrate the differences in protein expression between the two sets of samples.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A method for extracting protease with hydrolytic activity from moringa seeds is characterized by comprising the following steps:
1) respectively carrying out protein non-labeled relative quantitative analysis on the germinated moringa seeds and the dried moringa seeds, and screening differential proteins between the total protein of the dried moringa seeds and the total protein of the germinated moringa seeds; the bud length of the germinated moringa seeds is 10-15 cm, and the mass of the germinated moringa seeds is 12.57-13.09 g/15 grains; the mass of the dried moringa seeds is 4.31-4.53g/15 grains;
2) counting the protein up-regulated and expressed in the differential protein in the step 1), and performing GO function annotation on the obtained protein up-regulated and expressed to obtain protease with hydrolytic activity;
3) extracting the germinated moringa seed protease by a salt method to obtain moringa seed protease salt extract;
4) obtaining a PI value of protease with hydrolytic activity by screening in the step 2), adjusting the pH value of the moringa seed protease salt extract in the step 4) to the PI value, collecting protease precipitate, redissolving and desalting to obtain a crude moringa seed protease extract;
5) and 2) screening according to the molecular weight of the protease with hydrolytic activity obtained in the step 2), selecting an ultrafiltration membrane to carry out ultrafiltration interception on the protein in the crude extract of the moringa seed protease, and collecting the protease with target molecular weight to obtain the protease with hydrolytic activity.
2. The method according to claim 1, wherein the cultivation method of the germinated moringa seeds in the step 1) is characterized in that the moringa seeds are soaked in water at the temperature of 4-10 ℃ and then soaked in water at the temperature of 20-25 ℃ for 24-26 hours, and the obtained soaked moringa seeds are cultivated in a constant-temperature and constant-humidity environment;
culturing for 1-3 days at 25-30 deg.C and 70-75% humidity; culturing for 4-6 days at 22-25 deg.C and humidity of 60-65%.
3. The method according to claim 1, wherein the protease having hydrolytic activity in step 2) comprises 8 proteases; the amino acid sequences of the 8 proteinases are respectively shown as SEQ ID No. 1-8 in the sequence table;
the PI of the protease shown as SEQ ID No.1 is 10.10; the molecular weight of the protease shown in SEQ ID No.1 is 11789.27;
the PI of the protease shown as SEQ ID No.2 is 7.55; the molecular weight of the protease shown in SEQ ID No.2 is 18459.82;
the PI of the protease shown as SEQ ID No.3 is 7.15; the molecular weight of the protease shown in SEQ ID No.3 is 18811.2;
the PI of the protease shown as SEQ ID No.4 is 6.23; the molecular weight of the protease shown in SEQ ID No.4 is 52880.49;
the PI of the protease shown as SEQ ID No.5 is 10.63; the molecular weight of the protease shown in SEQ ID No.5 is 6802.67;
the PI of the protease shown as SEQ ID No.6 is 5.24; the molecular weight of the protease shown in SEQ ID No.6 is 31591.8;
the PI of the protease shown as SEQ ID No.7 is 10.36; the molecular weight of the protease shown in SEQ ID No.7 is 9427.98;
the PI of the protease shown as SEQ ID No.8 is 7.75; the molecular weight of the protease shown in SEQ ID No.8 is 18756.12.
4. The method as claimed in claim 1, wherein the method for extracting the germinated moringa seed protease by the salt method in the step 3) comprises the steps of crushing the germinated moringa seeds, sieving, mixing the obtained undersize with a sodium chloride solution, extracting, and carrying out solid-liquid separation to obtain the moringa seed protease salt extract.
5. The method of claim 4, wherein the screened mesh is no less than 60 mesh;
the mass of the undersize product and the volume of the sodium chloride solution are 1g:5 mL; the concentration of the sodium chloride solution is 0.25-0.30 mol/L.
6. The method of claim 4, wherein the temperature of extraction is 30 ℃; the extraction time is 0.5-1 h; the pH of the feed liquid during extraction was 7.15.
7. The method of claim 1, wherein the method of collecting the protease precipitate comprises centrifugation; the rotating speed of the centrifugation is 4500-5000 r/min; the centrifugation time is 20-30 min.
8. The method according to claim 1, wherein the redissolution method comprises mixing the collected protease precipitate with water, and subjecting the obtained mixture to ultrasonic dissolution to obtain a protease solution; the mass of the protease precipitate and the volume ratio of water are 5 g: 5-10 mL;
the power of the ultrasound is 200 Hz; the time of the ultrasonic treatment is 2-5 min.
9. The method according to claim 8, wherein the desalting comprises sequentially adjusting the pH value of the protease solution and pre-separating;
adjusting the pH value of the protease solution to 7.0;
the pre-separating comprises dialysis; the cut-off molecular weight of the dialysis bag for dialysis is 3500 Da; the dialysis time is 50-56 h.
10. The method according to any one of claims 1 to 9, wherein the protein non-labeled relative quantitative analysis comprises TCA/acetone precipitation and SDT fragmentation, SDS-PAGE gel electrophoresis, FASP enzymolysis, high performance liquid chromatography and mass spectrometry of Q-active mass spectrometer, mass spectrometry of raw data and database identification and quantitative analysis by MaxQuant software.
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| CN107058438A (en) * | 2017-05-16 | 2017-08-18 | 华南理工大学 | A kind of method that moringa seeds protein peptides are extracted from moringa seeds |
| CN107582581A (en) * | 2017-09-25 | 2018-01-16 | 华南理工大学 | A kind of extracting method of moringa oleifera leaf extractive |
| CN108192718A (en) * | 2018-01-05 | 2018-06-22 | 贵州省香瑞莱香料有限公司 | A kind of method that aqueous enzymatic method extracts seed oil of Moringa oleigera and protein hydrolysate simultaneously |
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