CN113817790A - Method for preparing alanyl glutamine by immobilized enzyme - Google Patents
Method for preparing alanyl glutamine by immobilized enzyme Download PDFInfo
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- CN113817790A CN113817790A CN202111211456.9A CN202111211456A CN113817790A CN 113817790 A CN113817790 A CN 113817790A CN 202111211456 A CN202111211456 A CN 202111211456A CN 113817790 A CN113817790 A CN 113817790A
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 107
- 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 35
- 229960002648 alanylglutamine Drugs 0.000 title claims abstract description 33
- 108010044940 alanylglutamine Proteins 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000006243 chemical reaction Methods 0.000 claims abstract description 89
- 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 32
- 238000001914 filtration Methods 0.000 claims abstract description 21
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 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
- 238000003756 stirring Methods 0.000 claims abstract description 6
- 230000003213 activating effect Effects 0.000 claims abstract description 5
- 238000002386 leaching Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 94
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 239000008213 purified water Substances 0.000 claims description 22
- 239000008055 phosphate buffer solution Substances 0.000 claims description 20
- 239000012295 chemical reaction liquid Substances 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 10
- 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
- 238000005119 centrifugation Methods 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- -1 alpha-amino acid ester Chemical class 0.000 abstract description 6
- 235000008206 alpha-amino acids Nutrition 0.000 abstract description 6
- 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
- 239000012456 homogeneous solution Substances 0.000 description 14
- 230000008569 process Effects 0.000 description 10
- 238000001308 synthesis method Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 1
- 230000001925 catabolic effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000007547 defect 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
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification 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
- 230000009469 supplementation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 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 liquor of the alpha-amino acid ester acyltransferase, and collecting hypha; secondly, breaking the hypha wall, centrifuging, and collecting the centrifugal clear liquid; activating an immobilized enzyme carrier for later use; fourthly, preparing immobilized alpha-amino acid ester acyltransferase, and storing at low temperature; fifthly, preparing a glutamine solution to ensure that the pH value of the solution is 6.5-9.5 and the temperature is 5-40 ℃; sixthly, adding the alanine methyl ester solution into the glutamine solution, and stirring to ensure that the pH value of the solution is 6.5-9.5 and the temperature is 5-40 ℃; seventhly, adding the a-amino acid ester acyltransferase immobilized enzyme into the solution prepared by the sixth step, and performing timed conversion; eighthly, filtering after the conversion is finished, and leaching and mechanically applying the a-amino acid ester acyltransferase immobilized enzyme. The invention provides the method which can be continuously applied, improves the utilization rate of enzyme, reduces the protein amount and is suitable for preparing alanyl glutamine by 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 using immobilized enzyme.
Background
L-alanyl-L-glutamine (also known as glutamine dipeptide) with molecular formula C8H15N3O4White or off-white loose lumps. Alanylglutamine is a component of parenteral nutrition and is indicated 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, the synthesis method uses biological enzyme as a catalyst, L-alanyl glutamine can be quickly and efficiently synthesized, and compared with the traditional chemical synthesis method, the enzymatic synthesis method has the advantages of simple process and low cost, so that the current enzymatic synthesis method of alanyl glutamine is widely applied.
However, the bacterial sludge or the bacterial sludge homogenized enzyme liquid is directly used for reaction, (1) the enzyme utilization rate is low, and (2) the existence of a large amount of protein has large pressure on the extraction equipment of the L-alanyl glutamine in the later period and has large environmental protection pressure.
Disclosure of Invention
The invention solves the defects of the prior art and provides the method for preparing alanyl glutamine by immobilized enzyme, which can be continuously applied, improves the utilization rate of enzyme and reduces protein.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for preparing alanyl glutamine by immobilized enzyme comprises the following steps:
step one, centrifuging fermentation liquor of a-amino acid ester acyltransferase, and collecting hypha;
step two, hypha wall breaking treatment;
step three, carrying out centrifugal treatment on enzyme liquid after wall breaking, and collecting centrifugal clear liquid;
step four, taking an immobilized enzyme carrier, activating and packing the immobilized enzyme carrier into a column for later use;
taking an immobilized enzyme carrier after the centrifugal clear liquid is subjected to over-current activation;
step six, after the overflowing, the immobilized enzyme carrier is placed statically, free centrifugal clear liquid in the immobilized enzyme carrier is replaced by purified water, and the obtained product is filtered to obtain the a-amino acid ester acyltransferase immobilized enzyme which is stored at low temperature;
seventhly, preparing a glutamine solution, and adjusting the pH value of the glutamine solution to 6.5-9.5 by using an adjusting solution at the temperature of 5-40 ℃;
step eight, adding an alanine methyl ester solution into the glutamine solution, and stirring to ensure that the pH value of the solution is 6.5-9.5 and the temperature is 5-40 ℃;
step nine, adding the a-amino acid ester acyltransferase immobilized enzyme prepared in the step six into the solution prepared in the step eight, and performing timed conversion;
step ten, converting for 10-120min, finishing the reaction, filtering the reaction liquid, and leaching and mechanically applying the immobilized a-amino acid ester acyltransferase.
Further, the centrifugation treatment in the first step is performed at a rotation speed of 9500-.
Further, in the step two, adding phosphate buffer solution into the hyphae for dissolving 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 centrifugation treatment in the third step is carried out at the rotating speed of 9000-.
And further, activating in the fourth step, soaking the immobilized enzyme carrier in a phosphate buffer solution for activation, wherein 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 that of the immobilized enzyme carrier.
Further, the flow rate of the centrifuged clear liquid in the fifth step is 5-10 times of the volume of the immobilized enzyme carrier.
Further, standing for 24 hours in the sixth step; the volume of purified water is 1 time of the volume of the immobilized enzyme carrier.
Further, the conditioning solution in the seventh step is one of ammonia water or sodium hydroxide solution.
Further, the mol ratio of the glutamine to the alanine methyl ester solution in the step eight is 1: 2-3.
Further, in the ninth step, the a-amino acid ester acyltransferase is 1-2% of glutamine by mass, the conversion temperature is 5-40 ℃, and the pH value is 6.5-9.5.
As the invention adopts the invention, compared with the prior art, the invention has the technical progress that:
the immobilized enzyme can be continuously applied, the utilization rate is improved, the macromolecular protein of the conversion solution after the conversion is finished and the enzyme is filtered is less, the environmental protection pressure and the cost investment of the post-purification of alanyl glutamine are reduced, the process operation is simple and easy to control, and the method can be used for large-scale production;
in conclusion, the invention provides the method which can be continuously applied, improves the utilization rate of enzyme, reduces the protein amount and is suitable for preparing alanyl glutamine by immobilized enzyme.
Detailed Description
The following description is given in conjunction with preferred embodiments of the present invention. It should be understood that the preferred embodiments described herein are 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 fermentation broth of alpha-amino acid ester acyltransferase, centrifuging at 10000r/min for 30min to obtain mycelium.
(2) And (3) adding 500g of centrifuged hyphae into a phosphate buffer solution with the pH value of 4.0 and the concentration of 0.015M to dissolve until the solid content is 2500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Collecting 2500ml of the homogenized solution, centrifuging at 10000r/min for 30min to obtain 1950ml of centrifugate.
(4) 100g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 200ml of 0.015M phosphate buffer with the pH value of 4.0, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to pass through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 9 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 100ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) 0.1mol of glutamine is added with 300ml of water and stirred to be dissolved, and the PH value of the solution is adjusted to 6.5 by ammonia water, and the temperature of the solution is 10 ℃.
(8) Adding 0.23mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 6.5 and the temperature of the solution to be 10 ℃ to obtain reaction liquid.
(9) 0.33g of immobilized enzyme is added into the reaction liquid, and the reaction liquid is converted at a timing, wherein the conversion temperature is controlled to be 10 ℃ and the conversion pH value is 6.5 in the conversion process.
(10) And (3) converting for 100min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 98.15%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Example 2
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of a-amino acid ester acyltransferase, centrifuging at 10000r/min for 30min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH value of 4.0 and the concentration of 0.015M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous solution for centrifugal treatment, rotating speed 10000r/min, and centrifuging for 30min to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.02M phosphate buffer with the pH value of 4.0, and packed into a column for standby.
(5) Taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 10 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) Adding 300ml of water into 0.1mol of glutamine, stirring for dissolving, and adjusting the pH value of the solution to 9.0 by using ammonia water, wherein the temperature of the solution is 30 ℃.
(8) Adding 0.25mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 9.0 and the temperature of the solution to be 30 ℃ to obtain reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 30 ℃ and the conversion pH value is 9.0 in the conversion process.
(10) And (3) converting for 100min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 98.25%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Example 3
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of a-amino acid ester acyltransferase, centrifuging at 10000r/min for 30min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH value of 4.0 and the concentration of 0.015M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous solution for centrifugal treatment, rotating speed 10000r/min, and centrifuging for 30min to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.02M phosphate buffer with the pH value of 4.0, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 8 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) 0.1mol of glutamine is added with 300ml of water and stirred to be dissolved, and the PH value of the solution is adjusted to 9.5 by ammonia water, and the temperature of the solution is 35 ℃.
(8) Adding 0.25mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 9.5 and the temperature of the solution to be 35 ℃ to obtain reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 35 ℃ and the conversion pH value is 9.5 in the conversion process.
(10) And (4) converting for 80min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 97.34%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Example 4
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 5L of fermentation broth of alpha-amino acid ester acyltransferase, centrifuging at 9500r/min for 32min to obtain mycelium.
(2) And (3) adding 500g of centrifuged hyphae into a phosphate buffer solution with the pH value of 3.0 and the concentration of 0.01M to dissolve until the solid content is 2500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Collecting 2500ml of the homogenized solution, centrifuging at 9000r/min for 34min to obtain 1950ml of centrifugate.
(4) 100g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 200ml of 0.01M phosphate buffer with the pH value of 3.0, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 5 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 100ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) 0.1mol of glutamine is added with 300ml of water and stirred to be dissolved, and the solution PH is adjusted to 7.0 by ammonia water, and the solution temperature is 5 ℃.
(8) Adding 0.23mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 7.0 and the temperature of the solution to be 5 ℃ to obtain reaction liquid.
(9) Adding 0.33g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 5 ℃ and the conversion pH value is 7.0 in the conversion process.
(10) Converting for 120min, finishing reaction, filtering reaction solution, converting rate is 97.26%, immobilized enzyme is used for 10ml
The purified water is used for conversion of the next batch after leaching.
Example 5
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of alpha-amino acid ester acyltransferase, centrifuging at 10500r/min for 28min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH value of 5.0 and the concentration of 0.1M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous solution for centrifugal treatment, rotating at 9000r/min, and centrifuging for 34min to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.1M phosphate buffer with the pH value of 5.0, and packed into a column for standby.
(5) Taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 10 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) Adding 300ml of water into 0.1mol of glutamine, stirring for dissolving, and adjusting the pH value of the solution to 9.5 by using sodium hydroxide solution, wherein the temperature of the solution is 40 ℃.
(8) Adding 0.25mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 9.5 and the temperature of the solution to be 40 ℃ to obtain reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 40 ℃ and the conversion pH value is 9.5 in the conversion process.
(10) And (3) converting for 90min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 98.10%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Example 6
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of alpha-amino acid ester acyltransferase, centrifuging at 9500r/min for 32min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH value of 7.0 and the concentration of 0.1M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous liquid for centrifugal treatment, rotating speed of 11000r/min, and centrifuging for 26 minutes to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.2M phosphate buffer with the pH value of 7.0, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 8 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) Adding 300ml of water into 0.1mol of glutamine, stirring for dissolving, and adjusting the pH value of the solution to 8.0 by using ammonia water, wherein the temperature of the solution is 30 ℃.
(8) Adding 0.25mol of alanine methyl ester solution into glutamine solution, controlling the pH of the solution to be 8.0 and the temperature of the solution to be 30 ℃ to obtain reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 30 ℃ and the conversion pH value is 8.0 in the conversion process.
(10) And (3) converting for 70 minutes, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 97.17%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Example 7
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of a-amino acid ester acyltransferase, centrifuging at 10000r/min for 30min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH of 8.0 and the concentration of 0.05M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous liquid for centrifugal treatment, rotating speed of 11000r/min, and centrifuging for 26min to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.05M phosphate buffer solution with the pH value of 8.0, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 7 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) 0.1mol glutamine is added with 300ml water and stirred to be dissolved, and the PH value of sodium hydroxide solution is adjusted to 8.5, and the solution temperature is 20 ℃.
(8) Adding 0.25mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 8.5 and the temperature of the solution to be 20 ℃, and obtaining reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 20 ℃ and the conversion pH value is 8.5 in the conversion process.
(10) And (3) converting for 60min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 96.73%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Example 8
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of a-amino acid ester acyltransferase, centrifuging at 10000r/min for 30min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH value of 9.0 and the concentration of 0.08M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous solution for centrifugal treatment, rotating speed 10000r/min, and centrifuging for 30min to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.08M phosphate buffer with the pH of 9.0, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to pass through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 6 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) 0.1mol of glutamine is added with 300ml of water and stirred to be dissolved, and the PH value of the solution is adjusted to 7.5 by ammonia water, and the temperature of the solution is 25 ℃.
(8) Adding 0.2mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 7.5 and the temperature of the solution to be 25 ℃, and obtaining reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 25 ℃ and the conversion pH value is 7.5 in the conversion process.
(10) And (3) converting for 10min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 93.42%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next batch of conversion.
Example 9
Method for preparing alanyl glutamine by immobilized enzyme
(1) Taking 3L of fermentation broth of a-amino acid ester acyltransferase, centrifuging at 10000r/min for 30min to obtain mycelium.
(2) And (3) adding 300g of the centrifuged hyphae into a phosphate buffer solution with the pH value of 7.5 and the concentration of 0.01M to dissolve the hyphae until the solid content is 1500ml, and homogenizing and breaking the wall to obtain a homogeneous solution.
(3) Taking 1500ml of the homogeneous solution for centrifugal treatment, rotating speed 10000r/min, and centrifuging for 30min to obtain 1200ml of centrifugal clear liquid.
(4) 60g of immobilized enzyme carrier is taken, washed, activated and soaked for 12h by 120ml of 0.02M phosphate buffer with the pH value of 7.5, and packed into a column for standby.
(5) And (3) taking the centrifuged clear liquid to flow through the immobilized enzyme carrier, wherein the volume of the centrifuged clear liquid is 5 times of that of the immobilized enzyme carrier.
(6) Standing for 24h after overflowing, replacing free centrifugate with 60ml purified water, filtering to obtain immobilized enzyme of a-amino acid ester acyltransferase, and refrigerating in refrigerator.
(7) 0.1mol of glutamine is added with 300ml of water and stirred to be dissolved, and ammonia water is used for adjusting the pH value of the solution to 7.5, and the temperature of the solution is 15 ℃.
(8) Adding 0.3mol of alanine methyl ester solution into glutamine solution, controlling the pH value of the solution to be 7.5 and the temperature of the solution to be 15 ℃ to obtain reaction liquid.
(9) Adding 0.3g of immobilized enzyme into the reaction solution, and carrying out timed conversion, wherein the conversion temperature is controlled to be 15 ℃ and the conversion pH value is 7.5 in the conversion process.
(10) And (3) converting for 30min, finishing the reaction, filtering the reaction solution, wherein the conversion rate is 95.68%, and the immobilized enzyme is rinsed by 10ml of purified water and then is reused for the next conversion.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. A method for preparing alanyl glutamine by immobilized enzyme is characterized in that: the method comprises the following steps:
step one, centrifuging fermentation liquor of a-amino acid ester acyltransferase, and collecting hypha;
step two, hypha wall breaking treatment;
step three, carrying out centrifugal treatment on enzyme liquid after wall breaking, and collecting centrifugal clear liquid;
step four, taking an immobilized enzyme carrier, activating and packing the immobilized enzyme carrier into a column for later use;
taking an immobilized enzyme carrier after the centrifugal clear liquid is subjected to over-current activation;
step six, after the overflowing, the immobilized enzyme carrier is placed statically, free centrifugal clear liquid in the immobilized enzyme carrier is replaced by purified water, and the obtained product is filtered to obtain the a-amino acid ester acyltransferase immobilized enzyme which is stored at low temperature;
seventhly, preparing a glutamine solution, and adjusting the pH value of the glutamine solution to 6.5-9.5 by using an adjusting solution at the temperature of 5-40 ℃;
step eight, adding an alanine methyl ester solution into the glutamine solution, and stirring to ensure that the pH value of the solution is 6.5-9.5 and the temperature is 5-40 ℃;
step nine, adding the a-amino acid ester acyltransferase immobilized enzyme prepared in the step six into the solution prepared in the step eight, and performing timed conversion;
step ten, converting for 10-120min, finishing the reaction, filtering the reaction liquid, and leaching and mechanically applying the immobilized a-amino acid ester acyltransferase.
2. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: in the first step, the centrifugal treatment is carried out at the rotating speed of 9500- & gt 10500r/min and the centrifugation is carried out for 28-32 min.
3. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: and in the second step, hypha wall breaking treatment is carried out, phosphate buffer is added into the hypha to be dissolved until the solid content is 20%, the pH value of the phosphate buffer is 3.0-9.0, and the concentration is 0.01-0.1M.
4. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: the centrifugation treatment in the third step is carried out at the rotating speed of 9000-11000r/min and the centrifugation time of 26-34 min.
5. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: and step four, activating, namely soaking the immobilized enzyme carrier 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 of that of the immobilized enzyme carrier.
6. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: and in the fifth step, the flow rate of the centrifuged clear liquid is 5-10 times of the volume of the immobilized enzyme carrier.
7. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: standing for 24 hours in the sixth step; the volume of purified water is 1 time of the volume of the immobilized enzyme carrier.
8. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: and seventhly, adjusting liquid is one of ammonia water or sodium hydroxide solution.
9. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: in the step eight, the molar ratio of the glutamine to the alanine methyl ester solution is 1: 2-3.
10. The method for producing alanyl-glutamine by using immobilized enzyme according to claim 1, wherein: in the ninth step, the a-amino acid ester acyltransferase accounts for 1-2% of the glutamine, the conversion temperature is 5-40 ℃, and the pH value is 6.5-9.5.
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