CN113817790B - Method for preparing alanyl glutamine by immobilized enzyme - Google Patents
Method for preparing alanyl glutamine by immobilized enzyme Download PDFInfo
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 119
- 238000000034 method Methods 0.000 title claims abstract description 33
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 title claims abstract description 31
- 229960002648 alanylglutamine Drugs 0.000 title claims abstract description 29
- 108010044940 alanylglutamine Proteins 0.000 title claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 98
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 102000057234 Acyl transferases Human genes 0.000 claims abstract description 32
- 108700016155 Acyl transferases Proteins 0.000 claims abstract description 32
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000001914 filtration Methods 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 claims abstract description 6
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 238000002386 leaching Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 102
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 239000008055 phosphate buffer solution Substances 0.000 claims description 24
- 239000008213 purified water Substances 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 230000004913 activation Effects 0.000 claims description 11
- 238000001994 activation Methods 0.000 claims description 11
- 239000012295 chemical reaction liquid Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 9
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 3
- 239000000969 carrier Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 1
- -1 alpha-amino acid ester Chemical class 0.000 abstract description 9
- 235000018102 proteins Nutrition 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 235000008206 alpha-amino acids Nutrition 0.000 abstract 1
- 210000002421 cell wall Anatomy 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 9
- 235000004279 alanine Nutrition 0.000 description 8
- 238000004090 dissolution Methods 0.000 description 7
- 238000002791 soaking Methods 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 6
- 239000012456 homogeneous solution Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000001925 catabolic effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003166 hypermetabolic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000016236 parenteral nutrition Nutrition 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
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Abstract
The invention discloses a method for preparing alanyl glutamine by immobilized enzyme, which comprises the following steps: centrifuging the fermentation broth of the alpha-amino acid ester acyl transferase, and collecting hyphae; 2. breaking the cell wall of hypha, centrifuging, and collecting the centrifugated clear liquid; 3. activating an immobilized enzyme carrier for standby; 4. preparing an immobilized enzyme of the a-amino acid ester acyltransferase, and storing at a low temperature; 5. preparing glutamine solution, wherein the pH value of the solution is 6.5-9.5, and the temperature is 5-40 ℃; 6. adding alanine methyl ester solution into glutamine solution, stirring to make pH value of the solution be 6.5-9.5, and temperature be 5-40 ℃; 7. adding an immobilized enzyme of a-amino acid ester acyltransferase into the solution prepared in six steps, and carrying out timing conversion; 8. after the conversion is finished, filtering, leaching and applying the immobilized enzyme of the a-amino acid ester acyltransferase. The invention provides a method for preparing alanyl glutamine by using immobilized enzyme, which can be continuously used, improves the utilization rate of enzyme, reduces the protein amount and is suitable for preparing alanyl glutamine by using immobilized enzyme.
Description
Technical Field
The invention belongs to the field of alanyl glutamine production, and particularly relates to a method for preparing alanyl glutamine by immobilized enzyme.
Background
L-alanyl-L-glutamine also known as proglutide, with formula C 8 H 15 N 3 O 4 Is a white or white-like loose lump. Alanylglutamine is a component of parenteral nutrition and is suitable for patients in need of glutamine supplementation, such as patients in catabolic or hypermetabolic conditions.
At present, alanyl glutamine can be produced by adopting an enzymatic synthesis method, and the synthesis method uses biological enzyme as a catalyst, so that L-alanyl glutamine can be quickly and efficiently synthesized.
However, the use of bacterial sludge or bacterial sludge homogeneous enzyme solution directly participates in the reaction, (1) the enzyme utilization rate is low, and (2) the existence of a large amount of proteins has great pressure on the extraction equipment of L-alanyl glutamine in the later stage and environmental protection pressure.
Disclosure of Invention
The invention solves the defects in the prior art, and provides a method for preparing alanyl glutamine by immobilized enzyme, which can be continuously used, improves the utilization rate of enzyme and reduces protein.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for preparing alanyl glutamine by immobilized enzyme, comprising the following steps:
step one, centrifuging a-amino acid ester acyl transferase fermentation liquor, and collecting hyphae;
step two, performing hypha wall breaking treatment;
step three, enzyme solution is subjected to centrifugal treatment after wall breaking, and centrifugal clear liquid is collected;
step four, taking an immobilized enzyme carrier, activating and loading the immobilized enzyme carrier into a column for later use;
step five, taking immobilized enzyme carriers after the centrifugal clear liquid is subjected to overcurrent activation;
standing the immobilized enzyme carrier after the overflow, replacing the free centrifugal clear liquid in the immobilized enzyme carrier by using purified water, filtering to obtain the immobilized enzyme of the a-amino acid ester acyltransferase, and storing at a low temperature;
step seven, preparing a glutamine solution, and regulating the PH value of the glutamine solution to be 6.5-9.5 by using a regulating solution at the temperature of 5-40 ℃;
step eight, adding an alanine methyl ester solution into the glutamine solution, and stirring to enable the pH value of the solution to be 6.5-9.5 and the temperature to be 5-40 ℃;
step nine, adding the immobilized enzyme of the a-amino acid ester acyltransferase prepared in the step six into the solution prepared in the step eight, and carrying out timing conversion;
step ten, converting for 10-120min, ending the reaction, filtering the reaction liquid, leaching the immobilized enzyme of the a-amino acid ester acyltransferase, and applying the immobilized enzyme.
Further, in the first step, the centrifugal treatment is carried out at the rotating speed of 9500-10500r/min for 28-32min.
Further, in the second step, a phosphate buffer solution is added into the mycelium for dissolution until the solid content is 20%, wherein the pH value of the phosphate buffer solution is 3.0-9.0, and the concentration is 0.01-0.1M.
Further, the centrifugal treatment in the third step is performed at the rotating speed of 9000-11000r/min and the centrifugal treatment is performed for 26-34min.
Further, in the step four, a phosphate buffer solution is used for soaking the immobilized enzyme carrier for activation, the PH of the phosphate buffer solution is 3.0-9.0, the concentration of the phosphate buffer solution is 0.01-0.2M, and the volume of the phosphate buffer solution is 2 times of the volume of the immobilized enzyme carrier.
Further, the overflow of the centrifugate in the step five is 5-10 times of the volume of the immobilized enzyme carrier.
Further, standing still for 24 hours in the step six; the volume of purified water was 1 time the volume of the immobilized enzyme carrier.
Further, in the seventh step, the regulating solution is one of ammonia water or sodium hydroxide solution.
Further, in the step eight, the molar ratio of the glutamine to the alanine methyl ester solution is 1:2-3.
Further, in step nine, the a-amino acid ester acyltransferase is 1-2% of the mass of glutamine, the conversion temperature is 5-40 ℃, and the PH value is 6.5-9.5.
Compared with the prior art, the invention adopts the invention, and the technical progress is that:
the immobilized enzyme can be continuously used, the utilization rate is improved, the converted liquid after the filtering enzyme is converted has less macromolecular protein, the environmental protection pressure and cost input of the subsequent purification of alanyl glutamine are reduced, the process operation is simple and easy to control, and the immobilized enzyme can be used for large-scale production;
in conclusion, the invention provides a method which can be continuously used, improves the enzyme utilization rate, reduces the protein amount and is suitable for preparing alanyl glutamine by immobilized enzyme.
Detailed Description
The following description is made in connection with the preferred embodiments of the present invention. It should be understood that the preferred embodiments described herein are presented for purposes of illustration and explanation only and are not intended to limit the present invention.
Example 1
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 5L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 10000r/min for 30min to obtain hypha.
(2) Taking 500g of centrifuged mycelium, adding phosphate buffer with the pH value of 4.0 and the concentration of 0.015M, dissolving until the solid content is 2500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 2500ml of homogeneous solution, centrifuging at 10000r/min for 30min to obtain 1950ml of supernatant.
(4) 100g of immobilized enzyme carrier is taken, dissolved, activated and soaked for 12 hours by 200ml of phosphate buffer solution with the pH value of 4.0 and the concentration of 0.015M, and the mixture is filled into a column for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 9 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, replacing the free centrifugal supernatant with 100ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 6.5 with ammonia water, and the temperature of the solution was 10 ℃.
(8) Adding 0.23mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to be 6.5, and controlling the temperature of the solution to be 10 ℃ to obtain reaction solution.
(9) 0.33g of immobilized enzyme is taken and added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 10 ℃ and the conversion pH value is controlled to be 6.5 in the conversion process.
(10) The reaction is completed after 100min of conversion, the reaction solution is filtered, the conversion rate is 98.15%, and the immobilized enzyme is used after being leached by 10ml of purified water for the next batch conversion.
Example 2
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 10000r/min for 30min to obtain hypha.
(2) Taking 300g of centrifuged mycelium, adding phosphate buffer with the pH value of 4.0 and the concentration of 0.015M, dissolving until the solid content is 1500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 1500ml of homogeneous solution for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 10000r/min for 30 min.
(4) 60g of immobilized enzyme carrier is taken, 120ml of phosphate buffer solution with the pH value of 4.0 and the concentration of 0.02M is used for dissolution, activation and soaking for 12 hours, and the column is filled for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 10 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 9.0 with ammonia water, and the temperature of the solution was 30 ℃.
(8) Adding 0.25mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 9.0, and controlling the temperature of the solution to 30 ℃ to obtain reaction solution.
(9) 0.3g of immobilized enzyme is taken and added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 30 ℃ and the conversion pH value is controlled to be 9.0 in the conversion process.
(10) The reaction was completed after 100min of conversion, the reaction solution was filtered to 98.25% conversion, and the immobilized enzyme was used after washing with 10ml of purified water for the next batch conversion.
Example 3
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 10000r/min for 30min to obtain hypha.
(2) Taking 300g of centrifuged mycelium, adding phosphate buffer with the pH value of 4.0 and the concentration of 0.015M, dissolving until the solid content is 1500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 1500ml of homogeneous solution for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 10000r/min for 30 min.
(4) 60g of immobilized enzyme carrier is taken, 120ml of phosphate buffer solution with the pH value of 4.0 and the concentration of 0.02M is used for dissolution, activation and soaking for 12 hours, and the column is filled for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 8 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 9.5 with ammonia water, and the temperature of the solution was 35 ℃.
(8) Adding 0.25mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 9.5, and controlling the temperature of the solution to 35 ℃ to obtain reaction solution.
(9) 0.3g of immobilized enzyme is taken and added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 35 ℃ and the conversion pH value is controlled to be 9.5 in the conversion process.
(10) The reaction was completed after 80min of conversion, the reaction solution was filtered to a conversion of 97.34%, and the immobilized enzyme was used after rinsing with 10ml of purified water for the next batch conversion.
Example 4
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 5L of a-amino acid ester acyl transferase fermentation liquor, centrifuging at the rotating speed of 9500r/min for 32min to obtain hypha.
(2) Adding 500g of centrifuged mycelium into phosphate buffer with pH value of 3.0 and concentration of 0.01M to dissolve until the solid content is 2500ml, and homogenizing and breaking wall to obtain homogenized liquid.
(3) Taking 2500ml of homogenized liquid for centrifugal treatment, and obtaining 1950ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 9000r/min and centrifugal treatment for 34min.
(4) 100g of immobilized enzyme carrier is taken, dissolved, activated and soaked for 12 hours by 200ml of phosphate buffer solution with the pH value of 3.0 and the concentration of 0.01M, and the mixture is filled into a column for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 5 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, replacing the free centrifugal supernatant with 100ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 7.0 with ammonia water, and the temperature of the solution was 5 ℃.
(8) Adding 0.23mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 7.0 and the temperature of the solution to 5 ℃ to obtain reaction solution.
(9) 0.33g of immobilized enzyme is added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 5 ℃ and the conversion pH value is controlled to be 7.0 in the conversion process.
(10) The reaction was completed after 120min of conversion, the reaction mixture was filtered to obtain a conversion of 97.26%, and 10ml of immobilized enzyme was used
Purified water is used for the next batch conversion after being leached.
Example 5
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotational speed of 10500r/min for 28min to obtain hypha.
(2) Taking 300g of centrifuged mycelium, adding a phosphate buffer solution with the pH value of 5.0 and the concentration of 0.1M, dissolving until the solid content is 1500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 1500ml of homogenized liquid for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 9000r/min and centrifugal treatment for 34min.
(4) 60g of immobilized enzyme carrier is taken, 120ml of phosphate buffer solution with the pH value of 5.0 and the concentration of 0.1M is used for dissolution, activation and soaking for 12 hours, and the column is filled for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 10 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 9.5 with sodium hydroxide solution, and the temperature of the solution was 40 ℃.
(8) Adding 0.25mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 9.5, and controlling the temperature of the solution to 40 ℃ to obtain reaction solution.
(9) 0.3g of immobilized enzyme is taken and added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 40 ℃ and the conversion pH value is controlled to be 9.5 in the conversion process.
(10) The reaction was completed after 90min of conversion, the reaction solution was filtered to a conversion of 98.10%, and the immobilized enzyme was used after rinsing with 10ml of purified water for the next batch conversion.
Example 6
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 9500r/min for 32min to obtain hypha.
(2) Taking 300g of centrifuged mycelium, adding a phosphate buffer solution with the pH value of 7.0 and the concentration of 0.1M, dissolving until the solid content is 1500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 1500ml of homogenized liquid for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 11000r/min for 26 minutes.
(4) 60g of immobilized enzyme carrier is taken, 120ml of phosphate buffer solution with the pH value of 7.0 and the concentration of 0.2M is used for dissolution, activation and soaking for 12 hours, and the column is filled for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 8 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 8.0 with ammonia water, and the temperature of the solution was 30 ℃.
(8) 0.25mol of alanine methyl ester solution is taken and added into glutamine solution, the PH of the solution is controlled to be 8.0, the temperature of the solution is 30 ℃, and reaction solution is obtained.
(9) 0.3g of immobilized enzyme is taken and added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 30 ℃ and the conversion pH value is controlled to be 8.0 in the conversion process.
(10) After 70 minutes of conversion, the reaction was completed, the reaction solution was filtered to a conversion of 97.17%, and the immobilized enzyme was used after washing with 10ml of purified water for the next batch conversion.
Example 7
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 10000r/min for 30min to obtain hypha.
(2) And (3) adding 300g of centrifuged mycelium into a phosphate buffer with the pH of 8.0 and the concentration of 0.05M to dissolve until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogenized solution.
(3) Taking 1500ml of homogenized liquid for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 11000r/min and centrifugal treatment for 26 min.
(4) 60g of immobilized enzyme carrier is taken, 120ml of phosphate buffer solution with the pH value of 8.0 and the concentration of 0.05M is used for dissolution, activation and soaking for 12 hours, and the column is filled for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 7 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, and the pH was adjusted to 8.5 with sodium hydroxide solution at 20 ℃.
(8) Adding 0.25mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 8.5, and controlling the temperature of the solution to 20 ℃ to obtain reaction solution.
(9) 0.3g of immobilized enzyme is added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 20 ℃ and the conversion pH value is controlled to be 8.5 in the conversion process.
(10) The reaction was completed after 60min of conversion, the reaction solution was filtered to a conversion of 96.73%, and the immobilized enzyme was used after washing with 10ml of purified water for the next batch conversion.
Example 8
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 10000r/min for 30min to obtain hypha.
(2) Taking 300g of centrifuged mycelium, adding phosphate buffer with the pH value of 9.0 and the concentration of 0.08M, dissolving until the solid content is 1500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 1500ml of homogeneous solution for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 10000r/min for 30 min.
(4) 60g of immobilized enzyme carrier is taken, and is soaked for 12 hours by 120ml of phosphate buffer solution with the pH of 9.0 and the concentration of 0.08M for washing, activation and column loading for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 6 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 7.5 with ammonia water, and the temperature of the solution was 25 ℃.
(8) Adding 0.2mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 7.5 and the temperature of the solution to 25 ℃ to obtain reaction solution.
(9) 0.3g of immobilized enzyme is added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 25 ℃ and the conversion pH value is controlled to be 7.5 in the conversion process.
(10) The reaction was completed after 10min of conversion, the reaction solution was filtered to a conversion of 93.42%, and the immobilized enzyme was used after rinsing with 10ml of purified water for the next batch conversion.
Example 9
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of a-amino acid ester acyltransferase fermentation liquor, centrifuging at the rotating speed of 10000r/min for 30min to obtain hypha.
(2) Taking 300g of centrifuged mycelium, adding a phosphate buffer solution with the pH value of 7.5 and the concentration of 0.01M, dissolving until the solid content is 1500ml, and carrying out homogenizing wall breaking to obtain a homogenized solution.
(3) Taking 1500ml of homogeneous solution for centrifugal treatment, and obtaining 1200ml of centrifugal clear liquid after centrifugal treatment at the rotating speed of 10000r/min for 30 min.
(4) 60g of immobilized enzyme carrier is taken, 120ml of phosphate buffer solution with the pH value of 7.5 and the concentration of 0.02M is used for dissolution, activation and soaking for 12 hours, and the column is filled for later use.
(5) Taking the immobilized enzyme carrier of the centrifugal clear liquid in an overflow way, wherein the volume of the centrifugal clear liquid is 5 times of that of the immobilized enzyme carrier.
(6) Standing for 24 hr after overflow, displacing free centrifugal supernatant with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, i.e. immobilized enzyme, and refrigerating in refrigerator.
(7) 0.1mol of glutamine was dissolved in 300ml of water with stirring, the pH of the solution was adjusted to 7.5 with ammonia water, and the temperature of the solution was 15 ℃.
(8) Adding 0.3mol of methyl alanine solution into glutamine solution, controlling the pH value of the solution to 7.5, and controlling the temperature of the solution to 15 ℃ to obtain reaction solution.
(9) 0.3g of immobilized enzyme is added into the reaction liquid, and the conversion is timed, wherein the conversion temperature is controlled to be 15 ℃ and the conversion pH value is controlled to be 7.5 in the conversion process.
(10) After conversion for 30min, the reaction was completed, the reaction solution was filtered to a conversion of 95.68%, and the immobilized enzyme was used after rinsing with 10ml of purified water for the next batch conversion.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (6)
1. A method for preparing alanyl glutamine by immobilized enzyme is characterized in that: the method comprises the following steps:
step one, centrifuging a-amino acid ester acyl transferase fermentation liquor at the rotation speed of 9500-10500r/min for 28-32min, and collecting hyphae;
step two, hypha wall breaking treatment, namely adding a phosphate buffer solution into hypha to dissolve until the solid content is 20%, wherein the pH value of the phosphate buffer solution is 3.0-9.0, and the concentration is 0.01-0.1M;
step three, enzyme solution centrifugal treatment is carried out after wall breaking, the centrifugal treatment is carried out at the rotating speed of 9000-11000r/min, the centrifugation is carried out for 26-34min, and centrifugal clear liquid is collected;
step four, taking an immobilized enzyme carrier, activating and loading the immobilized enzyme carrier into a column for later use;
step five, taking immobilized enzyme carriers after the centrifugal clear liquid is subjected to overcurrent activation;
standing the immobilized enzyme carrier after the overflow, replacing the free centrifugal clear liquid in the immobilized enzyme carrier by using purified water, filtering to obtain the immobilized enzyme of the a-amino acid ester acyltransferase, and storing at a low temperature;
step seven, preparing a glutamine solution, wherein the pH value of the glutamine solution is regulated to be 6.5-9.5 by using a regulating solution, the regulating solution is one of ammonia water or sodium hydroxide solution, and the temperature is 5-40 ℃;
step eight, adding an alanine methyl ester solution into the glutamine solution, and stirring to enable the pH value of the solution to be 6.5-9.5 and the temperature to be 5-40 ℃;
step nine, adding the immobilized enzyme of the a-amino acid ester acyltransferase prepared in the step six into the solution prepared in the step eight, and carrying out timing conversion;
step ten, converting for 10-120min, ending the reaction, filtering the reaction liquid, leaching the immobilized enzyme of the a-amino acid ester acyltransferase, and applying the immobilized enzyme.
2. The method for preparing alanyl-glutamine by immobilized enzyme according to claim 1, wherein: in the fourth step, the immobilized enzyme carrier is soaked in a phosphate buffer solution for activation, wherein the pH value of the phosphate buffer solution is 3.0-9.0, the concentration of the phosphate buffer solution is 0.01-0.2M, and the volume of the phosphate buffer solution is 2 times that of the immobilized enzyme carrier.
3. The method for preparing alanyl-glutamine by immobilized enzyme according to claim 1, wherein: and step five, the overflow of the centrifugated clear liquid is 5-10 times of the volume of the immobilized enzyme carrier.
4. The method for preparing alanyl-glutamine by immobilized enzyme according to claim 1, wherein: standing still for 24 hours in the sixth step; the volume of purified water was 1 time the volume of the immobilized enzyme carrier.
5. The method for preparing alanyl-glutamine by immobilized enzyme according to claim 1, wherein: in the eighth step, the molar ratio of the glutamine to the alanine methyl ester solution is 1:2-3.
6. The method for preparing alanyl-glutamine by immobilized enzyme according to claim 1, wherein: in the step nine, the a-amino acid ester acyltransferase accounts for 1-2% of the mass of glutamine, the conversion temperature is 5-40 ℃, and the pH value is 6.5-9.5.
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