CN112011584A - Method for preparing acetyl chitosamine by enzymatic conversion method - Google Patents
Method for preparing acetyl chitosamine by enzymatic conversion method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 10
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 10
- -1 acetyl chitosamine Chemical compound 0.000 title claims abstract description 7
- 238000000855 fermentation Methods 0.000 claims abstract description 36
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 239000001963 growth medium Substances 0.000 claims abstract description 28
- 241000588724 Escherichia coli Species 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000011218 seed culture Methods 0.000 claims abstract description 20
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 238000003756 stirring Methods 0.000 claims abstract description 19
- 239000011259 mixed solution Substances 0.000 claims abstract description 18
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract description 16
- 229960004308 acetylcysteine Drugs 0.000 claims abstract description 16
- 230000000415 inactivating effect Effects 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000004806 packaging method and process Methods 0.000 claims abstract description 10
- 239000002244 precipitate Substances 0.000 claims abstract description 10
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- 238000007789 sealing Methods 0.000 claims abstract description 10
- 238000011081 inoculation Methods 0.000 claims abstract description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 34
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 22
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 18
- 239000008103 glucose Substances 0.000 claims description 18
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 17
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 17
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- 235000005822 corn Nutrition 0.000 claims description 12
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 11
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
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- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 9
- 239000000411 inducer Substances 0.000 claims description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- 238000001291 vacuum drying Methods 0.000 claims description 9
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 8
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
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- 239000011573 trace mineral Substances 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 5
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- 230000008569 process Effects 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000002904 solvent Substances 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 15
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 15
- 229960002442 glucosamine Drugs 0.000 description 15
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000012258 culturing Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 6
- 229950006780 n-acetylglucosamine Drugs 0.000 description 6
- 229920002101 Chitin Polymers 0.000 description 5
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 5
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- NDVRKEKNSBMTAX-MVNLRXSJSA-N (2s,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;phosphoric acid Chemical compound OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O NDVRKEKNSBMTAX-MVNLRXSJSA-N 0.000 description 2
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- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 108010092528 Phosphate Transport Proteins Proteins 0.000 description 2
- 102000016462 Phosphate Transport Proteins Human genes 0.000 description 2
- 108010076086 acetylglucosamine transporter Proteins 0.000 description 2
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- MTDHILKWIRSIHB-UHFFFAOYSA-N (5-azaniumyl-3,4,6-trihydroxyoxan-2-yl)methyl sulfate Chemical compound NC1C(O)OC(COS(O)(=O)=O)C(O)C1O MTDHILKWIRSIHB-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
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- 206010016946 Food allergy Diseases 0.000 description 1
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- 101150056133 GNPNAT1 gene Proteins 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 150000004263 amino monosaccharides Chemical class 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 150000002016 disaccharides Chemical group 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229960001911 glucosamine hydrochloride Drugs 0.000 description 1
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- 101150100121 gna1 gene Proteins 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- Bioinformatics & Cheminformatics (AREA)
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing acetyl chitosamine by an enzymatic conversion method, which comprises the following steps: the recombinant escherichia coli strain is dropped into a seed culture medium, aerobic culture is carried out for 13-18 h at the stirring speed of 200rpm and the temperature of 35-45 ℃, and strain seed liquid is obtained; inoculating the strain seed liquid into a fermentation tank containing a fermentation culture medium in an inoculation amount of 5-10% for fermentation; continuously inactivating the fermented mixed solution at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (4) drying and crushing the obtained precipitate in vacuum, sealing and packaging. The invention has the beneficial effects that: the yield is high, the purity of the obtained acetylcysteine can reach more than 98 percent, the preparation method is simple in process and low in cost, the used solvent can be recycled, and the method is environment-friendly, economical and suitable for large-scale popularization and application.
Description
Technical Field
The invention belongs to the field of preparation processes of acetyl chitosamine, and particularly relates to a method for preparing acetyl chitosamine by an enzymatic conversion method.
Background
Glucosamine (GlcN), also known as Glucosamine, Glucosamine or Glucosamine, is a compound obtained by substituting one hydroxyl group of glucose with an amino group, and is an important functional monosaccharide, and is also the first amino monosaccharide with a confirmed structure. Currently, GlcN-related compounds generally include: glucosamine hydrochloride, glucosamine sulfate, and acetylglucosamine (also known as acetylcysteine, N-acetylglucosamine, GlcNAc). Wherein the GlcN molecular formula is C6H13O5N, commonly called amino sugar. GlcN is present in almost all organisms, including bacteria, yeasts, filamentous fungi, plants and animals, and is a major constituent of glycoproteins and proteoglycans, and also a major constituent of chitosan and chitin, and can be produced intracellularly by amination of glucose-6-phosphate.
In the human body, acetylglucosamine is a synthetic precursor of disaccharide unit of glycosaminoglycan, which plays an important role in the repair and maintenance of the function of cartilage and joint tissues. Therefore, acetylglucosamine is widely used as a pharmaceutical and dietary supplement to treat and repair joint damage. In addition, acetylglucosamine has many applications in the cosmetic and pharmaceutical fields. At present, the acetyl glucosamine is mainly produced by acidolysis of chitin in shrimp shells or crab shells, the waste liquid produced by the method has serious environmental pollution, and the obtained product is easy to cause anaphylactic reaction and is not suitable for people with seafood allergy to take.
GlcN and its derivatives are widely used in medicine, food, cosmetics, etc. and the main production methods of GlcN include chitin hydrolysis, enzyme biotransformation and microbial fermentation. The chitin hydrolysis method has the defects of limited raw material sources, easy environmental pollution and anaphylactic reaction, and difficult realization of industrial production; compared with a chitin hydrolysis method, the enzyme biotransformation method has little environmental pollution, but has the problems of long reaction time and low efficiency; the microbial transformation method avoids the determination of the former two methods, has higher efficiency, becomes the main method for producing GlcN at present, but the existing microbial transformation method still has the defects of insufficient yield and purity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for preparing the acetyl chitosamine by an enzymatic conversion method, which comprises the following steps:
s1, the recombinant Escherichia coli strain is dropped into a seed culture medium, and aerobic culture is carried out for 13-18 h at the stirring speed of 200rpm and the temperature of 35-45 ℃ to obtain strain seed liquid;
s2, inoculating the strain seed liquid cultured in the step S1 into a fermentation tank containing a fermentation culture medium in an inoculation amount of 5-10%, adding an inducer xylose after inoculating for 2 hours, performing fermentation culture for 30-48 hours at a stirring speed of 180rpm and at a temperature of 30-35 ℃, and controlling the pH of the fermentation process to be 6-8 by using ammonia water;
s3, continuously inactivating the mixed liquor fermented in the step S2 at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
Further, the fermentation medium contains: 75-85 g/L of glucose, 30g/L of ammonium sulfate, 15g/L of corn steep liquor, 1g/L of monopotassium phosphate, 1g/L of magnesium sulfate, 30-35 g/L of calcium carbonate and 0.01-50 mg/L of trace elements.
Further, the fermentation medium contains: 40g/L glucose, 2g/L potassium dihydrogen phosphate, 17g/L ammonium sulfate, 4g/L yeast powder, 30g/L calcium carbonate, 1g/L magnesium sulfate and 0.2 g/L-threonine.
Further, the seed culture medium is an LB culture medium.
Further, the seed medium contains: 10-15 g/L of glucose, 1.25-2 g/L of urea, 15-25 g/L of corn steep liquor, 1g/L of monopotassium phosphate and 0.5g/L of magnesium sulfate.
The invention has the beneficial effects that: the yield is high, the purity of the obtained acetylcysteine can reach more than 98 percent, the preparation method is simple in process and low in cost, the used solvent can be recycled, and the method is environment-friendly, economical and suitable for large-scale popularization and application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that the following description is only illustrative of the present invention and is not to be construed as limiting the present invention.
The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
Example 1
In this example, the recombinant E.coli strain was purchased from Rixing Biotech, Yangzhou Co., Ltd. Coli as a host is Escherichia coli W3110, nagE Escherichia coli W3110 in which the gene encoding the acetylglucosamine transporter has been deleted, manXYZ Escherichia coli W3110 in which the gene encoding the mannose phosphate transporter has been deleted, nagA Escherichia coli W3110 in which the gene encoding the deacetylase has been deleted, or nagB Escherichia coli W3110 in which the gene encoding the deaminase has been deleted. The glucosamine synthase encoding gene GlmS in the glucosamine synthesis pathway was cloned into the expression vector pTargetF and then transformed into E.coli.
The recombinant escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is an LB culture medium in the embodiment, and aerobic culture is carried out for 13 hours at the stirring speed of 200rpm and the temperature of 35 ℃ to obtain a strain seed solution;
the cultured strain seed solution was inoculated at an inoculum size of 5% into a fermenter containing a fermentation medium containing: 75g/L of glucose, 30g/L of ammonium sulfate, 15g/L of corn steep liquor, 1g/L of monopotassium phosphate, 1g/L of magnesium sulfate, 30g/L of calcium carbonate and 0.01mg/L of trace elements.
Inoculating for 2h, adding inducer xylose, fermenting and culturing at 30 deg.C and stirring speed of 180rpm for 30h, and controlling pH of fermentation process with ammonia water 6;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was determined by HPLC method, the yield was 83.5%, and the purity was 98.4%.
Example 2
In this example, the recombinant E.coli strain was purchased from Hiden Bio-technology Co., Ltd. Coli as a host is Escherichia coli W3110, nagE Escherichia coli W3110 in which the gene encoding the acetylglucosamine transporter has been deleted, manXYZ Escherichia coli W3110 in which the gene encoding the mannose phosphate transporter has been deleted, nagA Escherichia coli W3110 in which the gene encoding the deacetylase has been deleted, or nagB Escherichia coli W3110 in which the gene encoding the deaminase has been deleted. The glucosamine acetylase-encoding gene GNA1 in the glucosamine synthesis pathway was cloned into expression vector pTargetF and transformed into E.coli.
The recombinant Escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is 10g/L of glucose, 1.25g/L of urea, 15g/L of corn steep liquor, 1g/L of potassium dihydrogen phosphate and 0.5g/L of magnesium sulfate, and aerobic culture is carried out for 13h at the stirring speed of 200rpm and the temperature of 35 ℃ to obtain strain seed liquid;
the cultured strain seed solution was inoculated at an inoculum size of 5% into a fermenter containing a fermentation medium containing: 40g/L glucose, 2g/L potassium dihydrogen phosphate, 17g/L ammonium sulfate, 4g/L yeast powder, 30g/L calcium carbonate, 1g/L magnesium sulfate and 0.2 g/L-threonine.
Inoculating for 2h, adding inducer xylose, fermenting and culturing at 30 deg.C and stirring speed of 180rpm for 30h, and controlling pH of fermentation process with ammonia water 6;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was determined by HPLC method, the yield was 84.3%, and the purity was 98.2%.
Example 3
In this example, the recombinant E.coli strain was purchased from Rixing Biotech, Yangzhou Co., Ltd. The same as the recombinant E.coli strain of example 1.
The recombinant escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is an LB culture medium in the embodiment, and aerobic culture is carried out for 18h at the stirring speed of 200rpm and the temperature of 45 ℃ to obtain a strain seed solution;
the cultured strain seed solution was inoculated at an inoculum size of 10% into a fermenter containing a fermentation medium containing: 85g/L of glucose, 30g/L of ammonium sulfate, 15g/L of corn steep liquor, 1g/L of monopotassium phosphate, 1g/L of magnesium sulfate, 35g/L of calcium carbonate and 50mg/L of trace elements.
Inoculating for 2h, adding inducer xylose, fermenting and culturing at stirring speed of 180rpm and 35 deg.C for 48h, and controlling pH of fermentation process with ammonia water 8;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was determined by HPLC method, the yield was 85.5%, and the purity was 98.7%.
Example 4
In this example, the recombinant E.coli strain was purchased from Hiden Bio-technology Co., Ltd. The same as the recombinant E.coli strain of example 2.
The recombinant Escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is 15g/L glucose, 2g/L urea, 25g/L corn steep liquor, 1g/L potassium dihydrogen phosphate and 0.5g/L magnesium sulfate, and aerobic culture is carried out for 18h at the stirring speed of 200rpm and the temperature of 45 ℃ to obtain strain seed liquid;
the cultured strain seed solution was inoculated at an inoculum size of 10% into a fermenter containing a fermentation medium containing: 40g/L glucose, 2g/L potassium dihydrogen phosphate, 17g/L ammonium sulfate, 4g/L yeast powder, 30g/L calcium carbonate, 1g/L magnesium sulfate and 0.2 g/L-threonine.
Inoculating for 2h, adding inducer xylose, fermenting and culturing at stirring speed of 180rpm and 35 deg.C for 48h, and controlling pH of fermentation process with ammonia water 8;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was determined by HPLC method, the yield was 84.7%, and the purity was 98.3%.
Example 5
In this example, the recombinant E.coli strain was purchased from Rixing Biotech, Yangzhou Co., Ltd. The same as the recombinant E.coli strain of example 1.
The recombinant escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is an LB culture medium in the embodiment, and aerobic culture is carried out for 15 hours at the stirring speed of 200rpm and the temperature of 40 ℃ to obtain a strain seed solution;
inoculating the cultured strain seed solution into a fermentation tank containing a fermentation medium with an inoculation amount of 8-9%, wherein the fermentation medium contains: 78-81 g/L of glucose, 27g/L of ammonium sulfate, 11-13 g/L of corn steep liquor, 1-2 g/L of monopotassium phosphate, 1-2 g/L of magnesium sulfate, 30g/L of calcium carbonate and 10mg/L of trace elements.
Inoculating for 2h, adding inducer xylose, fermenting and culturing at 32 deg.C and stirring speed of 180rpm for 40h, and controlling pH of fermentation process with ammonia water 7;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.3 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was measured by HPLC method, and the yield was 86.1% and the purity was 99.1%.
Example 6
In this example, the recombinant E.coli strain was purchased from Rixing Biotech, Yangzhou Co., Ltd. The same as the recombinant E.coli strain of example 1.
The recombinant Escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is 15g/L glucose, 2g/L urea, 25g/L corn steep liquor, 1g/L potassium dihydrogen phosphate and 0.5g/L magnesium sulfate, and aerobic culture is carried out for 15-18 h at the stirring speed of 200rpm and the temperature of 40-42 ℃ to obtain strain seed liquid;
inoculating the cultured strain seed solution into a fermentation tank containing a fermentation medium with an inoculation amount of 7-9%, wherein the fermentation medium contains: 60-75 g/L of glucose, 28-31 g/L of ammonium sulfate, 12-16 g/L of corn steep liquor, 2g/L of monopotassium phosphate, 1-2 g/L of magnesium sulfate, 30g/L of calcium carbonate and 20mg/L of trace elements.
Inoculating for 2h, adding an inducer xylose, fermenting and culturing for 37-41 h at the stirring speed of 180rpm and the temperature of 32-34 ℃, and controlling the pH value of the fermentation process to 6-7 by using ammonia water;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.5-1 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was measured by HPLC method, and the yield was 85.1% and the purity was 99.3%.
Example 7
In this example, the recombinant E.coli strain was purchased from Rixing Biotech, Yangzhou Co., Ltd. The same as the recombinant E.coli strain of example 1.
The recombinant Escherichia coli strain is dropped into a seed culture medium, wherein the seed culture medium is 15-18 g/L of glucose, 2-3 g/L of urea, 25-30 g/L of corn steep liquor, 1-1.3 g/L of potassium dihydrogen phosphate and 0.5-0.6 g/L of magnesium sulfate, and aerobic culture is carried out at the stirring speed of 200rpm and the temperature of 38-43 ℃ for 15-18 h to obtain a strain seed solution;
inoculating the cultured strain seed solution into a fermentation tank containing a fermentation medium with an inoculation amount of 7-8%, wherein the fermentation medium contains: 40-42 g/L glucose, 2-3 g/L potassium dihydrogen phosphate, 17-18 g/L ammonium sulfate, 4-6 g/L yeast powder, 30-32 g/L calcium carbonate, 1-3 g/L magnesium sulfate and 0.2-0.5 g/L-threonine.
Inoculating for 2h, adding an inducer xylose, fermenting and culturing for 40h at the stirring speed of 180rpm and the temperature of 32-35 ℃, and controlling the pH of the fermentation process to 7 by using ammonia water;
continuously inactivating the fermented mixed solution at 100 ℃ for 0.3-0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
The content was determined by HPLC method, the yield was 85.1% and the purity was 98.3%.
Claims (5)
1. A method for preparing acetyl chitosamine by enzymatic conversion is characterized in that: the method comprises the following steps:
s1, the recombinant Escherichia coli strain is dropped into a seed culture medium, and aerobic culture is carried out for 13-18 h at the stirring speed of 200rpm and the temperature of 35-45 ℃ to obtain strain seed liquid;
s2, inoculating the strain seed liquid cultured in the step S1 into a fermentation tank containing a fermentation culture medium in an inoculation amount of 5-10%, adding an inducer xylose after inoculating for 2 hours, performing fermentation culture for 30-48 hours at a stirring speed of 180rpm and at a temperature of 30-35 ℃, and controlling the pH of the fermentation process to be 6-8 by using ammonia water;
s3, continuously inactivating the mixed liquor fermented in the step S2 at 100 ℃ for 0.5 h; centrifuging the inactivated mixed solution, extracting supernatant, and precipitating the supernatant with ethanol solution; and (3) carrying out vacuum drying and crushing on the obtained precipitate at the temperature of 60 ℃, and then sealing and packaging to obtain the finished product of the acetylcysteine.
2. The method for preparing acetylcysteine by enzymatic conversion according to claim 1, wherein: the fermentation medium contains: 75-85 g/L of glucose, 30g/L of ammonium sulfate, 15g/L of corn steep liquor, 1g/L of monopotassium phosphate, 1g/L of magnesium sulfate, 30-35 g/L of calcium carbonate and 0.01-50 mg/L of trace elements.
3. The method for preparing acetylcysteine by enzymatic conversion according to claim 1, wherein: the fermentation medium contains: 40g/L glucose, 2g/L potassium dihydrogen phosphate, 17g/L ammonium sulfate, 4g/L yeast powder, 30g/L calcium carbonate, 1g/L magnesium sulfate and 0.2 g/L-threonine.
4. The method for preparing acetylcysteine by enzymatic conversion according to claim 1, wherein: the seed culture medium is LB culture medium.
5. The method for preparing acetylcysteine by enzymatic conversion as claimed in claim 4, wherein: the seed culture medium contains: 10-15 g/L of glucose, 1.25-2 g/L of urea, 15-25 g/L of corn steep liquor, 1g/L of monopotassium phosphate and 0.5g/L of magnesium sulfate.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101365785A (en) * | 2002-07-01 | 2009-02-11 | 阿基昂生命科学公司,以生物技术资源部的名义经营 | Process and materials for production of glucosamine and n-acetylglucosamine |
| CN107267577A (en) * | 2016-04-05 | 2017-10-20 | 孙镧 | The method that microbial fermentation produces N acetyl D Glucosamines and/or D glucosamine salts |
| CN107354188A (en) * | 2017-08-12 | 2017-11-17 | 河南巨龙生物工程股份有限公司 | The technique of ETEC JL GlcN fermenting and producing N acetylglucosamines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101365785A (en) * | 2002-07-01 | 2009-02-11 | 阿基昂生命科学公司,以生物技术资源部的名义经营 | Process and materials for production of glucosamine and n-acetylglucosamine |
| CN107267577A (en) * | 2016-04-05 | 2017-10-20 | 孙镧 | The method that microbial fermentation produces N acetyl D Glucosamines and/or D glucosamine salts |
| CN107354188A (en) * | 2017-08-12 | 2017-11-17 | 河南巨龙生物工程股份有限公司 | The technique of ETEC JL GlcN fermenting and producing N acetylglucosamines |
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