Method for removing protein from fermentation liquor containing dicarboxylate

Abstract

The invention relates to a method for removing protein from dicarboxylate-containing fermentation liquor, which comprises the following steps of: in the acidification process of the dicarboxylate-containing fermentation liquor, respectively adding a compound protease composition I and a compound protease composition II when the pH value is 8-11 and the pH value is reduced to 8-6, and carrying out step-by-step combined protein hydrolysis, and finally, the trouble of filtering, washing and drying in the dicarboxylic acid primary product extraction process caused by the protein is eliminated. According to the invention, the compound protease is added by stages, so that the hydrolysis of protein in the dicarboxylate filtrate is more complete, the separation of dicarboxylic acid and organic nitrogen is more thorough in the subsequent primary product crystallization and filtering washing steps, the nitrogen content of the dicarboxylic acid primary product is lower, and the dicarboxylic acid filter cake is lighter in color due to good washing effect; the practical application value is higher.

Description

Technical Field

[0001] The invention relates to a dicarboxylic acid extraction technology, in particular to a deproteinization process in the process of extracting a primary dicarboxylic acid product. Background Art

[0002] Bio-based dicarboxylic acids are metabolites produced by microbial cells using carbon sources such as normal alkanes. Its molecular formula is C n H 2n-2 O 4 , where n is 8-18, dicarboxylic acid contains two terminal carboxyl groups and is a precursor raw material for synthesizing polymers such as engineering plastics and hot melt adhesives.

[0003] The growth process of microbial cells is divided into four periods: the retardation period, the logarithmic period, the stable period, and the decay period. Similarly, the dicarboxylic acid fermentation process also includes the above four periods, especially in the late fermentation period, the bacterial cells continue to decay, the cell membrane ruptures and autolyzes, and the cell membrane or intracellular proteins are released and dissolved in the fermentation liquid. In the process of extracting dicarboxylic acid primary products, the fermentation liquid in which the dicarboxylic acid disalt is dissolved is filtered through one or several combined equipment such as microfiltration, ultrafiltration, centrifugation or plate and frame to remove insoluble solids such as cells and proteins, but water-soluble proteins and some oil-soluble proteins pass through the filter medium to the dicarboxylate filtrate side. In the further acidification and heating of the dicarboxylate filtrate to convert it into the dicarboxylic acid precipitation process, the soluble protein is precipitated or denatured and precipitated under this condition, and the dicarboxylate is also precipitated in the acidification process, and then through the filtration step, the precipitated soluble protein is mixed with the dicarboxylic acid. Since the protein content in the filter cake of the primary dicarboxylic acid product is relatively high, the protein acts as a binder, making the dicarboxylic acid filter cake viscous, and the water has a large resistance and is difficult to wash thoroughly, making the primary product yellow and easy to agglomerate, and making the energy consumption of drying and dehydrating the primary dicarboxylic acid product higher. In the subsequent solvent-based dicarboxylic acid refining process, part of the protein precipitates under the action of the solvent, blocking the gap between the activated carbon pores and activated carbon particles used for decolorization after the solvent dissolves the primary dicarboxylic acid product, resulting in a decrease in the activated carbon adsorption decolorization effect and filtration speed. The remaining part of the protein dissolves in the solvent, and is adsorbed to the crystal surface as the dicarboxylic acid crystallizes and precipitates, ultimately resulting in a high protein content in the refined product, affecting the application of the product. Summary of the invention

[0004] In view of the above shortcomings, the present invention provides a method for primary deproteinization of dicarboxylic acid under mild conditions, which has a simple and safe operation process, can be operated for a long time, and is easy for industrial-scale production.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A method for removing protein from a fermentation broth containing dicarboxylate, comprising:

[0007] (1) removing solids from a fermentation broth containing dicarboxylates to obtain a dicarboxylate filtrate, adding a composite protease composition I at a pH of 8-11, and hydrolyzing at 20-70° C., wherein the composite protease composition I comprises the following proteases by weight based on 100% of the total weight of the protease:

[0008] Subtilisin 20%-50%

[0009] Bacillus licheniformis protease 20%-50%

[0010] Bromelain 10%-30%

[0011] Trypsin 5%-20%;

[0012] (2) The above solution is further acidified to convert the dicarboxylic acid disalt into the dicarboxylic acid monosalt. When the pH drops to 8-6, the composite protease composition II is added thereto, and hydrolyzed at 20-70° C., and the enzyme is filtered to obtain a clear dicarboxylate filtrate; the composite protease composition II comprises the following proteases by weight based on 100% of the total weight of the protease:

[0013] Papain 45%-80%

[0014] Ficin 10%-35%

[0015] Pepsin 10%-20%;

[0016] (3) Continue to add acid to the above solution until the pH is 2-4 to convert the dicarboxylic acid monosalt into dicarboxylic acid, heat, cool, filter, wash, and dry to obtain a dicarboxylic acid solution.

[0017] Further, the compound protease composition I and compound protease composition II are added according to the protein content in the dicarboxylate filtrate at 0.5u-50000u / g protein, preferably 5u-20000u / g protein, more preferably 50u-10000u / g protein, and most preferably 50u-1000u / g protein. Wherein, the protein content in the solution is determined according to the nitrogen content of the solution, and the nitrogen content is determined according to the method of GB5009.5-2016. According to the source of the solution of the present invention and the type of protein therein, the protein content is obtained by multiplying the nitrogen content by the conversion factor 6.25.

[0018] Furthermore, the composite protease composition I preferably comprises the following proteases by weight, based on the total weight of the proteases being 100%:

[0019] Subtilisin 30%-50%

[0020] Bacillus licheniformis protease 25%-45%

[0021] Bromelain 10%-20%

[0022] Trypsin 5%-20%.

[0023] Furthermore, the composite protease composition II comprises the following proteases by weight, based on the total weight of the proteases being 100%:

[0024] Papain 50%-70%

[0025] Ficin 20%-30%

[0026] Pepsin 10%-20%.

[0027] Furthermore, the hydrolysis temperature in step (1) and step (2) is preferably 40-65° C. The hydrolysis time in step (1) is 30-240 min. The hydrolysis time in step (2) is 30-60 min.

[0028] Furthermore, the dicarboxylate filtrate is obtained by solid-liquid separation of the fermentation broth, and the methods include but are not limited to flocculation sedimentation, microfiltration, ultrafiltration, centrifugation, pressure filtration and vacuum filtration to obtain a clarified dicarboxylate solution.

[0029] It should be understood by those skilled in the art that the dicarboxylate filtrate is usually alkaline, and the pH gradually decreases during the acidification process. The present invention adds corresponding proteases at different pH stages to perform step-by-step combined hydrolysis of proteins, ultimately eliminating the troubles caused by proteins in the filtration, washing and drying process of the dicarboxylic acid primary product extraction process.

[0030] Furthermore, the acidification in step (1) and step (2) is carried out by a method known in the prior art. Specifically, the acid used in the acidification is H 2 SO 4 , HNO 3 , HCl, H 3 PO 4 , formic acid, acetic acid, propionic acid and other inorganic or organic acids. Clean acids or recycled acids can be selected. Single acids, multiple acids, or acids mixed with inorganic and organic acids can be selected.

[0031] Furthermore, the filtration in step (2) is carried out by membrane filtration, using a ceramic membrane, ultrafiltration membrane or the like at the micron or nanometer level or a combination of filtration methods to remove the protease to obtain a clarified filtrate, and the enzyme concentrate can be recovered and used for protein hydrolysis in the next batch of dicarboxylic acid filtrate.

[0032] Furthermore, the heating in step (3) is to heat to 75-100° C., so that the dicarboxylic acid crystal particle size is larger, which is convenient for filtering and washing. The cooling is to cool to room temperature.

[0033] The implementation of the embodiments of the present invention has the following advantages:

[0034] (1) In the process of extracting the primary dicarboxylic acid product, the protein removal process is indispensable. Only the insoluble precipitated protein solids are removed by filtration, while the removal of dissolved protein is often not the focus of the research in the prior art. However, the inventors found that the presence of protein in the primary dicarboxylic acid product not only affects the effects of filtration, washing and drying, but also directly affects the subsequent refining operations and whether the product can meet the relevant requirements. The inventors found in the research that the use of a single component of protease, such as Bacillus subtilis or Bacillus licheniformis protease, cannot obtain a satisfactory protein removal effect. Through research, the inventors obtained a composite protease combination. For the protein in the dicarboxylate filtrate, the dicarboxylic acid and organic nitrogen are better separated by adding protease for hydrolysis, the washing and filtration speed of the dicarboxylic acid filter cake is faster, the nitrogen content of the primary dicarboxylic acid product is low, and the subsequent solvent recrystallization and refining can easily obtain a product that meets the polymerization requirements.

[0035] (2) The inventors of this case have found that, compared with adding protease directly to the dicarboxylate fermentation broth, adding protease to the filtrate of the dicarboxylate fermentation broth has a better hydrolysis effect and a lower amount of hydrolase. Further research has found that adding protease only in one pH range, such as pH 8-11 or pH 6-8, can only hydrolyze and remove part of the protein. The pH of the dicarboxylate filtrate decreases in a step-by-step manner during the acidification process. Through research, the inventors have combined two composite proteases and added them in two stages, pH 11-8 and pH 8-6, respectively, according to the optimal hydrolysis pH conditions of alkaline protease and neutral protease. Combined protein hydrolysis can achieve more satisfactory results, more complete protein hydrolysis in the dicarboxylate filtrate, more thorough separation of dicarboxylic acid and organic nitrogen in the subsequent primary product crystallization and filtration washing steps, lower nitrogen content in the dicarboxylic acid primary product, and lighter color of the dicarboxylic acid filter cake due to good washing effect, which has more practical application value.

[0036] (3) The inventors of this case further discovered that the method of grading with different proteases and deproteinizing by hydrolysis in different pH ranges can effectively reduce the amount of activated carbon used in subsequent processes and improve the decolorization and filtration efficiency.

[0037] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0038] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0039] The following examples provide the extraction process of the primary dicarboxylic acid product produced by fermentation, wherein the nitrogen content of the primary dicarboxylic acid product is detected using the GB5009.5-2016 method.

[0040] Example 1

[0041] (1) 100 liters of fermentation liquid containing decacarboxylic acid was filtered to remove solid impurities such as bacteria to obtain a clear filtrate with a pH of 9.0. The protein content was 0.47 g / L after detection. 25,000 u of the composite protease composition I was added to the filtrate and the mixture was hydrolyzed at 50° C. for 240 min.

[0042] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0043] Subtilisin 30%

[0044] Bacillus licheniformis protease 30%

[0045] Bromelain 20%

[0046] Trypsin 20%.

[0047] (2) Add sulfuric acid to the above dicarboxylate solution to acidify it. When the pH value drops to 6, add 10,000 u of the composite protease composition II and treat at 50° C. for 60 minutes. Filter by ultrafiltration to obtain a clear dicarboxylate filtrate;

[0048] The compound protease composition II is compounded with different neutral proteases, and based on 100% of the total weight of the protease, it is:

[0049] Papain 70%

[0050] Ficin 20%

[0051] Pepsin 10%.

[0052] (3) Sulfuric acid is continuously added to the above dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain decacarboxylic acid.

[0053] The nitrogen content of the obtained decacarboxylic acid product is shown in Table 1.

[0054] Example 2

[0055] (1) 100 liters of fermentation liquid containing undecanedicarboxylic acid was filtered to remove solid impurities such as bacteria to obtain a clear filtrate with a pH of 8.7. The protein content was 0.52 g / L after detection. 20,000 u of the composite protease composition I was added thereto and the mixture was hydrolyzed at 50° C. for 240 min.

[0056] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0057] Subtilisin 40%

[0058] Bacillus licheniformis protease 30%

[0059] Bromelain 20%

[0060] Trypsin 10%.

[0061] (2) Sulfuric acid was added to the above dicarboxylate solution to acidify it. When the pH value dropped to 6.5, 10,000 u of the composite protease composition II was added and treated at 50° C. for 60 min. The solution was filtered through ultrafiltration to obtain a clear dicarboxylate filtrate.

[0062] The compound protease composition II is compounded with different neutral proteases, and the total weight of the proteases is 100%, which is:

[0063] Papain 60%

[0064] Ficin 30%

[0065] Pepsin 10%.

[0066] (3) Sulfuric acid is further added to the above dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain undecanedicarboxylic acid.

[0067] The nitrogen content of the obtained undecanedicarboxylic acid product is shown in Table 1.

[0068] Example 3

[0069] (1) 100 liters of fermentation liquid containing dodecanedicarboxylic acid was filtered to remove solid impurities such as bacteria to obtain a clear filtrate with a pH of 8.7. The protein content was 0.49 g / L after detection. 18,000 u of the composite protease composition I was added thereto and the mixture was hydrolyzed at 50° C. for 240 min.

[0070] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0071] Subtilisin 45%

[0072] Bacillus licheniformis protease 30%

[0073] Bromelain 18%

[0074] Trypsin 7%.

[0075] (2) Add sulfuric acid to the above dicarboxylate solution to continue acidification. When the pH value drops to 6.2, add 20,000 u of the composite protease composition II and treat at 50° C. for 60 minutes. Filter by ultrafiltration to obtain a clear dicarboxylate filtrate;

[0076] The compound protease composition II is compounded with different neutral proteases, and based on 100% of the total weight of the protease, it is:

[0077] Papain 65%

[0078] Ficin 20%

[0079] Pepsin 15%.

[0080] (3) Sulfuric acid is further added to the dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain dodecanedicarboxylic acid.

[0081] The nitrogen content of the obtained dodecanedicarboxylic acid product is shown in Table 1.

[0082] Example 4

[0083] (1) 100 liters of fermentation liquid containing tridecanedicarboxylic acid was filtered to remove solid impurities such as bacterial cells to obtain a clear filtrate with a pH of 8.5. The protein content was 0.55 g / L after testing. 17000 u of the composite protease composition I was added thereto and the mixture was hydrolyzed at 50° C. for 240 min.

[0084] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0085] Subtilisin 45%

[0086] Bacillus licheniformis protease 35%

[0087] Bromelain 15%

[0088] Trypsin 5%.

[0089] (2) Sulfuric acid was added to the above dicarboxylate solution. When the pH value dropped to 6.3, 21000 u of the composite protease composition II was added and treated at 50° C. for 60 min. The solution was filtered through ultrafiltration to obtain a clear dicarboxylate filtrate.

[0090] The compound protease composition II is compounded with different neutral proteases, and based on 100% of the total weight of the protease, it is:

[0091] Papain 60%

[0092] Ficin 25%

[0093] Pepsin 15%.

[0094] (3) Sulfuric acid is further added to the above dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain tridecanedicarboxylic acid.

[0095] The nitrogen content of the obtained tridecanedicarboxylic acid product is shown in Table 1.

[0096] Example 5

[0097] (1) 100 liters of fermentation liquid containing tetradecanedicarboxylic acid was filtered to remove solid impurities such as bacterial cells to obtain a clear filtrate with a pH of 8.3. The protein content was found to be 0.52 g / L by testing. 18,000 u of the composite protease composition I was added thereto and the mixture was hydrolyzed at 50° C. for 240 min.

[0098] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0099] Subtilisin 40%

[0100] Bacillus licheniformis protease 35%

[0101] Bromelain 15%

[0102] Trypsin 10%.

[0103] (2) Sulfuric acid was added to the above dicarboxylate solution to acidify it. When the pH value dropped to 6.2, 16000u of the composite protease composition II was added and treated at 50°C for 60 minutes. The solution was filtered through ultrafiltration to obtain a clear dicarboxylate filtrate.

[0104] The compound protease composition II is compounded with different neutral proteases, and based on 100% of the total weight of the protease, it is:

[0105] Papain 55%

[0106] Ficin 25%

[0107] Pepsin 20%.

[0108] (3) Sulfuric acid is further added to the above dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain tetradecanedicarboxylic acid.

[0109] The nitrogen content of the obtained tetradecanedicarboxylic acid product is shown in Table 1.

[0110] Example 6

[0111] (1) 100 liters of fermentation liquid containing 15-carbon dicarboxylic acid was filtered to remove solid impurities such as bacterial cells, and a clear filtrate was obtained with a pH of 9.2. The protein content was 0.48 g / L after detection. 12000 u of composite protease composition I was added thereto and treated at 50° C. for 240 min.

[0112] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0113] Subtilisin 40%

[0114] Bacillus licheniformis protease 45%

[0115] Bromelain 10%

[0116] Trypsin 5%.

[0117] (2) Sulfuric acid was added to the above dicarboxylate solution to acidify it, and when the pH value was adjusted to 6.2, 24000u of the composite protease composition II was added and treated at 50°C for 60 minutes. The solution was filtered through ultrafiltration to obtain a clear dicarboxylate filtrate.

[0118] The compound protease composition II is compounded with different neutral proteases, and based on 100% of the total weight of the protease, it is:

[0119] Papain 65%

[0120] Ficin 25%

[0121] Pepsin 10%.

[0122] (3) Sulfuric acid is further added to the above dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain pentadecanedicarboxylic acid.

[0123] The nitrogen content of the obtained 15-carbon dicarboxylic acid product is shown in Table 1.

[0124] Example 7

[0125] (1) 100 liters of fermentation liquid containing hexadecanedicarboxylic acid was filtered to remove solid impurities such as bacterial cells to obtain a clear filtrate with a pH of 9.6. The protein content was found to be 0.45 g / L by testing. 13,000 u of the composite protease composition I was added thereto and the mixture was hydrolyzed at 50° C. for 240 min.

[0126] The compound protease composition I is compounded with different alkaline proteases, and based on 100% of the total weight of the protease, is:

[0127] Subtilisin 50%

[0128] Bacillus licheniformis protease 25%

[0129] Bromelain 10%

[0130] Trypsin 15%.

[0131] (2) Sulfuric acid was added to the above dicarboxylate solution to acidify it. When the pH value dropped to 6.2, 22000u of the composite protease composition II was added and treated at 50°C for 60 minutes. The solution was filtered through ultrafiltration to obtain a clear dicarboxylate filtrate.

[0132] The compound protease composition II is compounded with different neutral proteases, and based on 100% of the total weight of the protease, it is:

[0133] Papain 45%

[0134] Ficin 35%

[0135] Pepsin 20%.

[0136] (3) Sulfuric acid is further added to the above dicarboxylate filtrate to adjust the pH value to 4.0, and the mixture is heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain hexadecanedicarboxylic acid.

[0137] The nitrogen content of the obtained hexadecanedicarboxylic acid product is shown in Table 1.

[0138] Comparative Example 1

[0139] I. Filter 100 liters of fermentation liquid containing dodecanedicarboxylic acid to remove solid impurities such as bacteria to obtain a clarified filtrate.

[0140] II. Add sulfuric acid to the dicarboxylate decolorizing solution, adjust the pH to 4.0, heat to 95°C, then cool to room temperature, filter, wash and dry to obtain dodecanedicarboxylic acid.

[0141] The nitrogen content of the obtained dodecanedicarboxylic acid product is shown in Table 1.

[0142] Comparative Example 2

[0143] The same as step (1) of Example 3, after enzymatic hydrolysis, ultrafiltration was performed to obtain a clear dicarboxylate filtrate.

[0144] Sulfuric acid is added to the above dicarboxylate filtrate to adjust the pH value to 4.0, heated to 95° C., then cooled to room temperature, filtered, washed and dried to obtain dodecanedicarboxylic acid.

[0145] The nitrogen content of the obtained dodecanedicarboxylic acid product is shown in Table 1.

[0146] Comparative Example 3

[0147] Compared with Example 1, the enzymes used in step (1) are only subtilisin and Bacillus licheniformis protease, and the weight ratio of the two is 1:1. The added amount is the same as the total amount of the composite protease composition I in Example 1; the enzyme used in step (2) is only papain, and the added amount is the same as the total amount of the composite protease composition II in Example 1, and the other steps are the same as Example 1. The nitrogen content of the obtained dicarboxylic acid product is shown in Table 1

[0148] Table 1.

[0149]

[0150]

Claims

1. A method for removing protein from a fermentation broth containing dicarboxylate, comprising: (1) removing solids from a fermentation broth containing dicarboxylates to obtain a dicarboxylate filtrate, adding a composite protease composition I at a pH of 8-11, and hydrolyzing at 20-70° C., wherein the composite protease composition I comprises the following proteases by weight based on 100% of the total weight of the protease: Subtilisin 20%-50% Bacillus licheniformis protease 20%-50% Bromelain 10%-30% Trypsin 5%-20%; (2) The above solution is further acidified to convert the dicarboxylic acid disalt into the dicarboxylic acid monosalt. When the pH drops to 8-6, the composite protease composition II is added thereto, and hydrolyzed at 20-70° C., and the enzyme is filtered to obtain a clear dicarboxylate filtrate; the composite protease composition II comprises the following proteases by weight based on 100% of the total weight of the protease: Papain 45%-80% Ficin 10%-35% Pepsin 10%-20%; (3) Continue to add acid to the above solution until the pH is 2-4 to convert the dicarboxylic acid monosalt into dicarboxylic acid, heat, cool, filter, wash, and dry to obtain a dicarboxylic acid solution.

2. The method according to claim 1, characterized in that The compound protease composition I and compound protease composition II are added at 0.5u-50000u / g protein based on the protein content in the dicarboxylate filtrate.

3. The method according to claim 2, characterized in that The compound protease composition I and compound protease composition II are added at 5u-20000u / g protein based on the protein content in the dicarboxylate filtrate.

4. The method according to claim 1, characterized in that The composite protease composition I comprises the following proteases by weight, based on the total weight of the proteases being 100%: Subtilisin 30%-50% Bacillus licheniformis protease 25%-45% Bromelain 10%-20% Trypsin 5%-20%.

5. The method according to claim 1, characterized in that The composite protease composition II comprises the following proteases by weight, based on the total weight of the proteases being 100%: Papain 50%-70% Ficin 20%-30% Pepsin 10%-20%.

6. The method according to claim 1, characterized in that The hydrolysis temperature in step (1) and step (2) is 40-65°C.

7. The method according to claim 1, characterized in that The hydrolysis time in step (1) is 30-240 min.

8. The method according to claim 1, characterized in that The hydrolysis time in step (2) is 30-60 min.

9. The method according to claim 1, characterized in that: The filtration in step (2) is carried out by membrane filtration, using a ceramic membrane, an ultrafiltration membrane or the like at the micron or nanometer level or a combination of filtration methods to remove the protease and obtain a clarified filtrate.

10. The method according to claim 1, characterized in that The dicarboxylate filtrate in step (1) is obtained by solid-liquid separation of the fermentation broth, and the method is selected from one or a combination of flocculation sedimentation, microfiltration, ultrafiltration, centrifugation, pressure filtration and vacuum filtration.

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