Chymosin and preparation method thereof

Abstract

The preparation method comprises the following steps: carrying out acidolysis and salt extraction on a raw material, carrying out ultrafiltration desalination by adopting an ultrafiltration membrane, carrying out column chromatography, and freeze-drying a chromatography filtrate to obtain the chymosin. According to the preparation method provided by the invention, a plurality of steps of acidolysis and salt extraction, ultrafiltration and desalination, column chromatography, freeze drying and the like are used for synergism, the chymosin with the product activity of more than 60U / mg and the product yield of more than 90% is obtained, and the method is simple to operate, low in process condition requirement, low in production cost, high in safety and suitable for industrial production. And the requirements of product safety, simple process flow and high curding activity can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of chymotrypsin, and particularly relates to a chymotrypsin and a preparation method thereof. Background Art

[0002] Chymosin is a key enzyme in the cheese production process, which can coagulate milk and plays an irreplaceable role in the cheese ripening process. The coagulation process of chymosin includes two stages: the first stage is to hydrolyze the peptide bond connected by Phe105-Met106 of κ-casein, exposing the internal α-casein and β-casein; the second stage is in the presence of Ca 2+ Under the action, chemical bonds are formed between casein molecules and aggregated to form a three-dimensional network gel.

[0003] Calf chymosin was the first to be applied to cheese making. However, with the increasing market demand for cheese, calf chymosin has gradually become in short supply, and people have to look for its substitutes. Animal-derived chymosins such as camel chymosin, lamb chymosin, and porcine pepsin all have good coagulation activity, with less proteolytic activity, less bitter taste in the produced cheese, and better flavor and quality. Among the substitutes for animal-derived chymosin, camel chymosin has a very small molecular structure difference from calf chymosin, and its coagulation activity is higher than that of calf chymosin, but its proteolytic activity is only 30% of that of calf chymosin. Lamb chymosin has higher pH stability, but its coagulation activity is lower than that of calf chymosin. The stability and proteolytic activity of buffalo rennet are not much different from those of calf chymosin, but its coagulation activity is lower. Therefore, there is currently no substitute with high coagulation activity, strong proteolytic activity, and wide access.

[0004] At the same time, the current preparation methods of chymotrypsin have many deficiencies. For example, the patent document with the publication number CN105368810A discloses a method for preparing chymotrypsin, in which acetone precipitation is used, and there is a risk of excessive organic solvent residues in the product, reducing its safety; and ammonium sulfate salting-out is used with low resolution, making it difficult to achieve high-precision separation. The patent document with the publication number CN102272299A discloses recombinant porcine chymotrypsin, which discloses a chymotrypsin obtained by a synthetic gene. However, the preparation process of this invention is cumbersome, requires high process conditions, and has high production costs. Zhang Fuxin et al. in the paper "Study on the Extraction, Isolation and Characteristics of Lamb Chymosin", prepared lamb chymosin through steps such as sample concentration, gel filtration (Sephacry MT 5200) chromatography, and DEAE cellulose (DE-32) ion exchange chromatography, and its highest coagulation activity was only 73.3 U / mg. Therefore, the existing preparation methods of chymotrypsin cannot meet the requirements of product safety, simple process flow, and high coagulation activity.

[0005] In view of this, this patent application is proposed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing preparation methods of chymosin cannot meet the multi-faceted requirements of product safety, simple process, high chymosin activity, and wide raw material sources. To solve the above technical problems, the present invention provides a preparation method of chymosin and provides the chymosin product.

[0007] The first object of the present invention is to provide a preparation method of chymosin. Using lamb abomasum mucosa as the raw material, after acid hydrolysis and salt extraction, ultrafiltration desalination is carried out using an ultrafiltration membrane, and then column chromatography and freeze-drying of the chromatography filtrate are carried out to obtain chymosin.

[0008] The preparation method of chymosin provided by the present invention utilizes the combination and synergistic effect of multiple steps such as acid hydrolysis and salt extraction, ultrafiltration desalination, column chromatography, and freeze-drying to obtain chymosin with a product activity of more than 60 U / mg and a product yield of more than 90%. This method is simple to operate, has low process conditions requirements, low production costs, and high safety.

[0009] As a preferred design, the lamb abomasum mucosa is broken and added with water and hydrochloric acid to obtain a grinding slurry, and an extraction solution is added to the grinding slurry for acid hydrolysis and salt extraction.

[0010] As a preferred design, the extraction solution is a mixed extraction solution of hydrochloric acid and calcium chloride. The hydrochloric acid concentration in the mixed extraction solution is 5% - 10%, and the calcium chloride concentration is 0.01% - 1.0%.

[0011] As a preferred design, during acid hydrolysis and salt extraction, stirring extraction is carried out at 25°C - 35°C for 2h - 6h. After the acid hydrolysis is completed, the temperature is lowered to 10°C to obtain an acid hydrolysis solution.

[0012] As a preferred design, 8 - 10 times the volume of hydrochloric acid solution (pH 5.0 - 7.0) is added to the obtained acid hydrolysis solution, stirring extraction is carried out for 2 - 4h, the insoluble matter is filtered off, and the filtered clear liquid is collected;

[0013] Hydrochloric acid solution is added to the filter residue again, stirring extraction is carried out at 25 - 35°C for 1h - 3h, filtered, the filtered clear liquids from the two times are mixed, the pH is adjusted to 5.0 - 7.0 and then ultrafiltration desalination is carried out. The filtrate after ultrafiltration desalination is collected and adjusted to 5.0 - 7.0 and then subjected to column chromatography.

[0014] As a preferred design, during column chromatography, a phosphate buffer solution is used to rinse and balance the DEAE chromatography column, and a glycine salt buffer solution is used for elution.

[0015] As a preferred design, the concentration of the phosphate buffer solution is 0.01M to 0.3M, pH = 5.0 to 7.0, and the concentration of the glycinate buffer solution is 0.1M to 0.3M, pH = 5.0 to 7.0.

[0016] As a preferred design, after column chromatography, the pH of the collected eluate is adjusted to 5.0 to 7.0, allowed to stand for 30 minutes, and then the filtrate is collected and freeze-dried.

[0017] As a preferred design, the freeze-drying process is as follows: drying at -45°C for 5 hours, drying at -15°C for 8 hours, drying at -5°C for 12 hours, drying at 0°C for 4 hours, drying at 35°C for 12 hours, and drying at 70°C for 6 hours to obtain chymosin.

[0018] The second object of the present invention is to provide a chymosin prepared by using the method described in any one of the above.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention uses sheep abomasum mucosa as a material, and after acid hydrolysis and salt extraction, membrane separation is adopted. Through ultrafiltration membranes, biological macromolecules and inorganic salts are effectively removed to prepare chymosin. Finally, through column chromatography and freeze-drying, the obtained product has an activity of more than 160 U / mg and a product yield of more than 90%. The process used in the present invention is simple, fast, and mild in conditions, without the need for complex equipment and the risk of exceeding the standard of organic solvent residues. It can meet the requirements of product safety, simple process flow, and high chymosin activity, and can be put into industrial production, opening up a new way for the industrial preparation of chymosin.

[0021] 2. The present invention uses freeze-drying to avoid the use of organic solvents, enhancing the safety of the product; and sheep mucosa, as a livestock by-product, is rich in resources and low in cost. Detailed Embodiments

[0022] The following further describes in detail the embodiments of the present application in conjunction with the examples. The detailed descriptions of the following examples are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application, that is, the present application is not limited to the described embodiments.

[0023] The "ranges" disclosed in this application are defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The ranges defined in this way can include or exclude the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a specific parameter, ranges of 60 to 110 and 80 to 120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, then the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise specified, the numerical range "a to b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0 to 5" means that all real numbers between "0 to 5" are fully listed herein, and "0 to 5" is only an abbreviated representation of these numerical combinations. Additionally, when stating that a certain parameter is an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0024] If there is no special instruction, all embodiments and optional embodiments of this application can be combined with each other to form a new technical solution.

[0025] If there is no special instruction, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0026] If there is no special instruction, all steps of this application can be carried out sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out sequentially, or can also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b), and (c), or can also include steps (a), (c), and (b), or can also include steps (c), (a), and (b), etc.

[0027] If there is no special instruction, the "including" and "comprising" mentioned in this application mean open-ended or can also be closed-ended. For example, the "including" and "comprising" can mean that other components not listed can also be included or comprised, or can also mean that only the listed components are included or comprised.

[0028] The present invention uses sheep mucosa as the material, adopts the membrane separation technology, and effectively removes biological macromolecules and inorganic salts through an ultrafiltration membrane to obtain chymotrypsin. The product activity reaches more than 160 U / mg. The process is simple, there is no risk of exceeding the standard of organic solvent residues, the safety is high, and the raw material source is wide. The specific preparation process is as follows:

[0029] A preparation method of chymotrypsin is carried out according to the following steps:

[0030] Step 1: Take the abomasal mucosa of a lamb, break it, and add an appropriate amount of purified water and hydrochloric acid to obtain a ground slurry;

[0031] Step 2: Acid hydrolysis and salt extraction: Add 300 mL - 500 mL of a mixed extraction solution (hydrochloric acid concentration is 5% - 10%, calcium chloride concentration is 0.01% - 1.0%) to the ground slurry, stir and extract at 25°C - 35°C for 2 h - 6 h. After the acid hydrolysis is completed, cool down to 10°C to obtain an acid hydrolysis solution;

[0032] Step 3: Add 8 - 10 times the volume of hydrochloric acid solution (pH is 5.0 - 7.0) to the above acid hydrolysis solution, stir and extract for 2 - 4 h, filter to remove insoluble substances, and collect the filtered and clarified solution; Add 100 ml of hydrochloric acid solution to the filter residue again, stir and extract at 25°C - 35°C for 1 h - 3 h, filter, mix the two filtrates, and adjust the pH to 5.0 - 7.0.

[0033] Step 4: Ultrafiltration for impurity removal: Ultrafilter and desalt the filtrate in Step 3 using a 2000 Da ultrafiltration membrane, collect the filtrate, and adjust the pH to 5.0 - 7.0.

[0034] Step 5: Column chromatography: Rinse and balance the DEAE chromatography column with a phosphate buffer solution (0.01 M - 0.3 M, pH = 5.0 - 7.0), load the filtrate with adjusted pH in Step 4 onto the column, elute with a glycine salt buffer solution (0.1 M - 0.3 M, PH = 5.0 - 7.0), and collect the eluate.

[0035] Step 6: Adjust the pH of the eluate collected in Step 5 to 5.0 - 7.0, let it stand for 30 minutes, and collect the filtrate;

[0036] Step 7: Lyophilize the filtrate obtained in Step 6. The drying program is: dry at -45°C for 5 hours, dry at -15°C for 8 hours, dry at -5°C for 12 hours, dry at 0°C for 4 hours, dry at 35°C for 12 hours, and dry at 70°C for 6 hours to obtain chymotrypsin.

[0037] The following is a detailed description through specific examples.

[0038] Example 1:

[0039] Step 1: Take 1 kg of lamb abomasum mucosa, break it up, and add an appropriate amount of purified water and hydrochloric acid to obtain a ground slurry;

[0040] Step 2: Acidolysis and salt extraction: Add 500 mL of a mixed extraction solution (hydrochloric acid concentration is 5%, calcium chloride concentration is 0.01%) to the ground slurry, stir and extract at 25 °C for 4 h. After acidolysis is completed, cool down to 10 °C to obtain an acidolysis solution;

[0041] Step 3: Add 8 times the volume of hydrochloric acid solution (pH = 5.0) to the above acidolysis solution, stir and extract for 2 h, filter to remove insoluble substances, and collect the filtered clear liquid; Add 100 ml of hydrochloric acid solution to the filter residue again, stir and extract at 25 °C for 2 h, filter, mix the two filtrates, and adjust the pH to 5.0.

[0042] Step 4: Ultrafiltration for impurity removal: Ultrafilter and desalt the filtrate in Step 3 using a 2000 Da ultrafiltration membrane, collect the filtrate, and adjust the pH to 5.0.

[0043] Step 5: Column chromatography: Rinse and balance the DEAE chromatography column with phosphate buffer solution (0.01 M, pH = 5.0), load the filtrate in Step 4 onto the column, elute with glycine salt buffer solution (0.1 M, pH = 5.0), and collect the eluate.

[0044] Step 6: Adjust the pH of the eluate collected in Step 5 to 5.0, let it stand for 30 minutes, and collect the filtrate;

[0045] Step 7: Freeze-dry the filtrate. The drying procedure is: dry at -45 °C for 5 hours, dry at -15 °C for 8 hours, dry at -5 °C for 12 hours, dry at 0 °C for 4 hours, dry at 35 °C for 12 hours, and dry at 70 °C for 6 hours.

[0046] Example 2:

[0047] The difference between this example and Example 1 is that in the mixed extraction solution in Step 2, the calcium chloride concentration is 0.05%, and the rest are the same as in Example 1.

[0048] Example 3:

[0049] Step 1: Take 1 kg of lamb abomasum mucosa, break it up, and add an appropriate amount of purified water and hydrochloric acid;

[0050] Step 2: Acidolysis and salt extraction: Add 500 mL of a mixed extraction solution (hydrochloric acid concentration is 5%, calcium chloride concentration is 0.05%) to the ground slurry, stir and extract at 25 °C for 4 h. After acidolysis is completed, cool down to 10 °C to obtain an acidolysis solution;

[0051] Step 3: Add hydrochloric acid solution (pH = 5.5) with a volume 8 times that of the acid hydrolysate to the above acid hydrolysate, stir and extract for 2 h, filter to remove insoluble substances, and collect the filtered clear liquid; add 100 ml of hydrochloric acid solution to the filter residue again, stir and extract at 25 °C for 2 h, filter, mix the two filtrates, and adjust the pH to 5.5.

[0052] Step 4: Ultrafiltration for impurity removal: Ultrafilter and desalt the filtrate in Step 3 using a 2000 Da ultrafiltration membrane, collect the filtrate, and adjust the pH to 5.5.

[0053] Step 5: Column chromatography: Rinse and equilibrate the DEAE chromatography column with phosphate buffer (0.01 M, pH = 5.5), load the filtrate in Step 4 onto the column, elute with glycine buffer (0.1 M, pH = 5.5), and collect the eluate.

[0054] Step 6: Adjust the pH of the eluate collected in Step 5 to 5.5, let it stand for 30 minutes, and collect the filtrate;

[0055] Step 7: Freeze-dry the filtrate. The drying program is as follows: dry at -45 °C for 5 h, dry at -15 °C for 8 h, dry at -5 °C for 12 h, dry at 0 °C for 4 h, dry at 35 °C for 12 h, and dry at 70 °C for 6 h.

[0056] Example 4:

[0057] Step 1: Take 1 kg of lamb abomasum mucosa, break it up, and add an appropriate amount of purified water and hydrochloric acid;

[0058] Step 2: Acid hydrolysis and salt extraction: Add 500 mL of mixed extraction solution (hydrochloric acid concentration is 5%, calcium chloride concentration is 0.05%) to the ground slurry, stir and extract at 30 °C for 4 h. After the acid hydrolysis is completed, cool down to 10 °C to obtain the acid hydrolysate;

[0059] Step 3: Add hydrochloric acid solution (pH = 5.5) with a volume 8 times that of the above acid hydrolysate, stir and extract for 2 h, filter to remove insoluble substances, and collect the filtered clear liquid; add 100 ml of hydrochloric acid solution to the filter residue again, stir and extract at 30 °C for 2 h, filter, mix the two filtrates, and adjust the pH to 5.5.

[0060] Step 4: Ultrafiltration for impurity removal: Ultrafilter and desalt the filtrate in Step 3 using a 2000 Da ultrafiltration membrane, collect the filtrate, and adjust the pH to 5.5.

[0061] Step 5: Column chromatography: Rinse and equilibrate the DEAE chromatography column with phosphate buffer (0.01 M, pH = 5.5), load the filtrate in Step 4 onto the column, elute with glycine buffer (0.1 M, pH = 5.5), and collect the eluate.

[0062] Step 6: Adjust the pH of the eluate collected in Step 5 to 5.5, let it stand for 30 minutes, and collect the filtrate.

[0063] Step 7: Freeze-dry the filtrate. The drying procedure is as follows: dry at -45°C for 5 hours, dry at -15°C for 8 hours, dry at -5°C for 12 hours, dry at 0°C for 4 hours, dry at 35°C for 12 hours, and dry at 70°C for 6 hours.

[0064] Comparative Example 1:

[0065] The difference between this comparative example and Example 3 is that in Step 2, hydrochloric acid acid extraction is deleted, that is, the extraction solution used is calcium chloride with a concentration of 0.05%. The rest is the same as in Example 2.

[0066] Comparative Example 2:

[0067] The difference between this comparative example and Example 3 is that the step of ultrafiltration and impurity removal by the ultrafiltration membrane is removed, and the rest of the process is the same as in Example 3.

[0068] Comparative Example 3:

[0069] The difference between this comparative example and Example 3 is that during the process of column chromatography, an acetate buffer solution (0.01M, pH = 5.5) is used to rinse and balance the DEAE chromatography column, and an acetate buffer solution (0.1M, pH = 5.5) is also used for elution. The rest is the same as in Example 3.

[0070] Comparative Example 4:

[0071] The difference between this comparative example and Example 3 is that a phosphate buffer solution (0.01M, pH = 5.5) is used to rinse and balance the DEAE chromatography column, and a phosphate buffer solution (0.1M, pH = 5.5) is used for elution. The rest is the same as in Example 3.

[0072] Comparative Example 5:

[0073] The difference between this comparative example and Example 3 is that a glycine buffer solution (0.01M, pH = 5.5) is used to rinse and balance the DEAE chromatography column, and a glycine buffer solution (0.1M, pH = 5.5) is used for elution. The rest is the same as in Example 3.

[0074] The activity and yield results of chymosin obtained in each example and comparative example are shown in Table 1.

[0075] Table 1

[0076] Category Activity (U / mg) Yield (%) Example 1 163 91 Example 2 171 95 Example 3 178 97 Example 4 160 90 Comparative Example 1 134 73 Comparative Example 2 128 71 Comparative Example 3 107 60 Comparative Example 4 121 67 Comparative Example 5 115 64

[0077] As can be seen from the table, by using the preparation method of the present invention, the activity values of the chymotrypsin obtained in each example are all at a relatively high level, all above 160 U / mg, and the yields are also all above 90%.

[0078] From the comparison between Example 1 and Example 2, it can be seen that calcium chloride has a certain effect on the activity of chymotrypsin, and the best effect is obtained when the concentration of calcium chloride is 0.05%.

[0079] From the comparison between Example 2 and Example 3, it can be seen that when pH = 5.5, the activity of chymotrypsin is higher.

[0080] By comparing Example 3 and Example 4, it can be seen that the extraction temperature has a certain effect on the activity of chymotrypsin, and the best effect is obtained when the temperature is 25 °C.

[0081] By comparing Comparative Example 1 and Example 3, it is obtained that the activity of chymotrypsin is higher when acidolysis and salt extraction are used. In Comparative Example 1, hydrochloric acid acid extraction was cancelled, resulting in too high a pH value, and too high a pH value would significantly inhibit the activity of chymotrypsinogen. Adding an acidifying agent to lower the pH value can activate the proenzyme, thereby promoting protein decomposition and removing some protein impurities.

[0082] By comparing Comparative Example 2 and Example 3, it can be obtained that the activity of chymotrypsin prepared by adding an ultrafiltration membrane impurity removal step is higher. Because the ultrafiltration membrane can effectively remove a large number of small molecule impurities such as nucleic acids and endotoxins released by cell rupture, and can also filter out unwanted small molecules (such as inorganic salts, sugars), realizing the concentration of chymotrypsin in the solution.

[0083] By comparing Comparative Example 3, Comparative Example 4, Comparative Example 5 and Example 3, it is found that when passing through the chromatography column, compared with a single acetate buffer, phosphate buffer, and glycine buffer, the chymotrypsin prepared by using a combination of phosphate and glycine buffer has higher activity. This is because the combination of phosphate and glycine buffer can better maintain the pH value stability of the system and prevent enzyme inactivation caused by pH changes in the extraction solution system.

[0084] The above specific embodiments have further detailed the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing chymosin, characterized in that: The raw material is lamb abomasal mucosa, which is subjected to acid hydrolysis and salt extraction, ultrafiltration desalination with an ultrafiltration membrane, and then column chromatography and freeze-drying of the chromatography filtrate to obtain chymosin.

2. The method for preparing chymosin according to claim 1, characterized in that: The lamb abomasal mucosa is crushed, and water and hydrochloric acid are added to obtain a grinding solution, to which the extract is added for acid hydrolysis and salt extraction.

3. The method for preparing chymosin according to claim 2, characterized in that: The extract is a mixed extract of hydrochloric acid and calcium chloride, wherein the concentration of hydrochloric acid in the mixed extract is 5% to 10%, and the concentration of calcium chloride is 0.01% to 1.0%.

4. The method for preparing chymosin according to any one of claims 1 to 3, characterized in that: During acid hydrolysis and salt extraction, stir and extract at 25℃~35℃ for 2h~6h. After the acid hydrolysis is completed, cool to 10℃ to obtain the acid hydrolyzate.

5. The method for preparing chymosin according to claim 4, characterized in that: Add 8 to 10 times the volume of hydrochloric acid solution with a pH of 5.0 to 7.0 to the obtained acid hydrolysis solution, stir and extract for 2 to 4 hours, filter to remove insoluble matter, and collect the filtered clear liquid; add hydrochloric acid solution to the filter residue again, stir and extract at 25 to 35° C. for 1 hour to 3 hours, filter, mix the two filtered clear liquids, adjust the pH to 5.0 to 7.0, and then perform ultrafiltration desalination, collect the filtrate after ultrafiltration desalination, adjust the pH to 5.0 to 7.0, and then perform column chromatography.

6. The method for preparing chymosin according to claim 1, characterized in that: During column chromatography, the DEAE column was washed and equilibrated with phosphate buffer and eluted with glycine buffer.

7. The method for preparing chymosin according to claim 6, characterized in that: The concentration of the phosphate buffer is 0.01M-0.3M, and the pH is 5.0-7.

0. The concentration of the glycine buffer is 0.1M-0.3M, and the pH is 5.0-7.

0.

8. The method for preparing chymosin according to claim 6, characterized in that: After column chromatography, the pH of the collected eluate was adjusted to 5.0-7.0, and the filtrate was collected after standing for 30 minutes, and the collected filtrate was freeze-dried.

9. The method for preparing chymosin according to claim 1, characterized in that: The freeze-drying process is: drying at -45°C for 5 hours, drying at -15°C for 8 hours, drying at -5°C for 12 hours, drying at 0°C for 4 hours, drying at 35°C for 12 hours, and drying at 70°C for 6 hours to obtain chymosin.

10. A chymosin, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 9.