A STRAIN FOR PRODUCTION OF GLUCOSAMINE BY MICROBIAL FERMENTATION, AND ITS METHODS

Abstract

This Invention relates to, for production of N-acetyl-D-glucosamine and D-glucosamine by microbial fermentation: A nongenetic recombinant strain, preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; NJ090259, a strain of Bacillus subtilis, Preservation No. CGMCC10257; and NJ091195, a strain of Bacillus licheniformis, Preservation No. CGMCC10258 and Preservation Date Dec. 29, 2014. The beneficial effects of this Invention are: Provide a new strain for production of N-acetyl-D-glucosamine and D-glucosamine, and its production methods. By this method, it may achieve stable production and supply of N-acetyl-D-glucosamine, and may also achieve non-animal-derived, safety production of N-acetyl-D-glucosamine and D-glucosamine; the method is of short production phase and low cost, and is more environmentally-friendly.

Claims

1. A strain for production of N-acetyl-D-glucosamine and D-glucosamine by microbial fermentation, which is characterized by: Being preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; and NJ090259, a strain of Bacillus subtilis, Preservation No. CGMCC10257 and Preservation Date Dec. 29, 2014.

2. A method for production of N-acetyl-D-glucosamine by microbial fermentation, which is characterized by production of N-acetyl-D-glucosamine by seed culture and optimized medium fermentation, using NJ090259 (a strain of Bacillus subtilis) based on claim 1 as initial strain, including the following steps: (1) Screening, Identification, and Cultivation of Strains Collect 50 soil samples, dilute and then smear on the plates for initial screening; using the medium for initial screening: Colloidal chitin 2.5 g/L, dipotassium hydrogen phosphate 0.7 g/L, potassium dihydrogen phosphate 0.3 g/L, magnesium sulfate 0.5 g/L, ferrous sulfate 0.01 g/L, and agar 20 g/L, pH 7.0; the culture temperature is 37 C. and the cultivation time is 72 h. Incubate to obtain individual colonies, separate colonies to obtain Bacillus subtilis, cultivate it by shake flask fermentation, measure chitinase activities in the fermented solution, and screen strains by chitinase activities in the fermented solution. (2) Cultivate by Fermentation Inoculate NJ090259 (a strain of Bacillus subtilis) activated in the plate medium into the seed medium, carry out a constant-temperature shaking-bed incubation, using the solution as seed solution; inoculate the solution into the fermentation medium, carry out a constant-temperature shaking-bed incubation, centrifuge and transfer the supernatant, and measure the content of N-acetyl-D-glucosamine; Plate medium: Colloidal chitin 30 g/L, ammonium sulfate 2 g/L, potassium dihydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L, sodium chloride 0.5 g/L, and agar 20 g/L, pH 6.5; Seed medium: Peptone 5.0 g/L, beef extract 5.0 g/L, and sodium chloride 5.0 g/L, pH 7.0-7.2; Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L; Fermentation conditions: Temperature 35 C., fermentation time 18 h, initial pH 6.5, inoculum dose 10%, and volume of loaded solution, 50 mL/250 mL; (3) Purification of N-acetyl-D-glucosamine Fermented Solution Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt, heat the salt-removed fermented solution under vacuum to concentrate to supersaturation. Cool the concentrated fermented solution, add 5 volumes of anhydrous alcohol, stir, and centrifuge to obtain a high-purity N-acetyl-glucosamine.

3. A method for production of D-glucosamine by microbial fermentation, which is characterized by production of D-glucosamine by seed culture and optimized medium fermentation, using NJ090259 (a strain of Bacillus subtilis) based on claim 1, including the following steps: (1) Screening, Identification, and Cultivation of Strains Collect 50 soil samples, dilute and then smear on the plates for initial screening; using the medium for initial screening: Colloidal chitin 2.5 g/L, dipotassium hydrogen phosphate 0.7 g/L, potassium dihydrogen phosphate 0.3 g/L, magnesium sulfate 0.5 g/L, ferrous sulfate 0.01 g/L, and agar 20 g/L, pH 7.0; the culture temperature is 37 C. and the cultivation time is 72 h. Incubate to obtain individual colonies, separate colonies to obtain Bacillus subtilis, cultivate it by shake flask fermentation, measure chitinase activities in the fermented solution, and screen strains by chitinase activities in the fermented solution. (2) Cultivate by Fermentation Inoculate NJ090259 (a strain of Bacillus subtilis) activated in the plate medium into the seed medium, carry out a constant-temperature shaking-bed incubation, using the solution as seed solution; inoculate the solution into the fermentation medium, carry out a constant-temperature shaking-bed incubation, centrifuge and transfer the supernatant, and measure the content of N-acetyl-D-glucosamine; Plate medium: Colloidal chitin 30 g/L, ammonium sulfate 2 g/L, potassium dihydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L, sodium chloride 0.5 g/L, and agar 20 g/L, pH 6.5; Seed medium: Peptone 5.0 g/L, beef extract 5.0 g/L, and sodium chloride 5.0 g/L, pH 7.0-7.2; Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L; Fermentation conditions: Temperature 35 C., fermentation time 18 h, initial pH 6.5, inoculum dose 10%, and volume of loaded solution, 50 mL/250 mL; (3) Purification of N-acetyl-D-glucosamine Fermented Solution Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt, heat the salt-removed fermented solution under vacuum to concentrate to supersaturation. Cool the concentrated fermented solution, add 5 volumes of anhydrous alcohol, stir, and centrifuge to obtain a high-purity N-acetyl-glucosamine. (4) Acidified Hydrolysis of N-acetyl-D-glucosamine Prepare a saturated solution with N-acetyl-D-glucosamine, add 37% concentrated hydrochloric acid to a final concentration of 12-16%, keep at 90 C. for 45-90 min, allow to cool overnight, filter to obtain crystals, wash with alcohol, vacuum-dry and measure, to obtain a high-purity D-glucosamine hydrochloride.

4. A strain for production of N-acetyl-D-glucosamine and D-glucosamine by microbial fermentation, which is characterized by: Being preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CCCCM); and NJ091195, a strain of Bacillus licheniformis, Preservation No. CGMCC10258 and Preservation Date Dec. 29, 2014.

5. A method for production of N-acetyl-D-glucosamine by microbial fermentation, which is characterized by production of N-acetyl-D-glucosamine by seed culture and optimized medium fermentation, using NJ091195 (a strain of Bacillus subtilis) based on claim 4 as initial strain, including the following steps: (1) Screening, Identification, and Cultivation of Strains Collect 50 soil samples, dilute and then smear on the plates for initial screening; using the medium for initial screening: Colloidal chitin 2.5 g/L, dipotassium hydrogen phosphate 0.7 g/L, potassium dihydrogen phosphate 0.3 g/L, magnesium sulfate 0.5 g/L, ferrous sulfate 0.01 g/L, and agar 20 g/L, pH 7.0; the culture temperature is 37 C. and the cultivation time is 72 h. Incubate to obtain individual colonies, separate colonies to obtain Bacillus licheniformis, cultivate it by shake flask fermentation, measure chitinase activities in the fermented solution, and screen strains by activities of chitinase in the fermented solution. (2) Cultivate by Fermentation Inoculate NJ091195 (a strain of Bacillus licheniformis) activated in the plate medium into the seed medium, carry out a constant-temperature shaking-bed incubation, using the solution as seed solution; inoculate the solution into the fermentation medium, carry out a constant-temperature shaking-bed incubation, centrifuge and transfer the supernatant, and measure the content of N-acetyl-D-glucosamine; Plate medium: Colloidal chitin 30 g/L, ammonium sulfate 2 g/L, potassium dihydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L, sodium chloride 0.5 g/L, and agar 20 g/L, pH 6.5; Seed medium: Peptone 5.0 g/L, beef extract 5.0 g/L, and sodium chloride 5.0 g/L, pH 7.0-7.2; Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L; Fermentation conditions: Temperature 35 C., fermentation time 18 h, initial pH 6.5, inoculum dose 10%, and volume of loaded solution, 50 mL/250 mL; (3) Purification of N-acetyl-D-glucosamine Fermented Solution Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt, heat the salt-removed fermented solution under vacuum to concentrate to supersaturation. Cool the concentrated fermented solution, add 5 volumes of anhydrous alcohol, stir, and centrifuge to obtain a high-purity N-acetyl-glucosamine.

6. A method for production of D-glucosamine by microbial fermentation, which is characterized by production of N-acetyl-D-glucosamine by seed culture and optimized medium fermentation, using NJ091195 (a strain of Bacillus subtilis) based on claim 4 as initial strain, including the following steps: (1) Screening, Identification, and Cultivation of Strains Collect 50 soil samples, dilute and then smear on the plates for initial screening; using the medium for initial screening: Colloidal chitin 2.5 g/L, dipotassium hydrogen phosphate 0.7 g/L, potassium dihydrogen phosphate 0.3 g/L, magnesium sulfate 0.5 g/L, ferrous sulfate 0.01 g/L, and agar 20 g/L, pH 7.0; the culture temperature is 37 C. and the cultivation time is 72 h. Incubate to obtain individual colonies, separate colonies to obtain Bacillus licheniformis, cultivate it by shake flask fermentation, measure chitinase activities in the fermented solution, and screen strains by activities of chitinase in the fermented solution. (2) Cultivate by Fermentation Inoculate NJ091195 (a strain of Bacillus licheniformis) activated in the plate medium into the seed medium, carry out a constant-temperature shaking-bed incubation, using the solution as seed solution; inoculate the solution into the fermentation medium, carry out a constant-temperature shaking-bed incubation, centrifuge and transfer the supernatant, and measure the content of N-acetyl-D-glucosamine; Plate medium: Colloidal chitin 30 g/L, ammonium sulfate 2 g/L, potassium dihydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L, sodium chloride 0.5 g/L, and agar 20 g/L, pH 6.5; Seed medium: Peptone 5.0 g/L, beef extract 5.0 g/L, and sodium chloride 5.0 g/L, pH 7.0-7.2; Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L; Fermentation conditions: Temperature 35 C., fermentation time 18 h, initial pH 6.5, inoculum dose 10%, and volume of loaded solution, 50 mL/250 mL; (3) Purification of N-acetyl-D-glucosamine Fermented Solution Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt, heat the salt-removed fermented solution under vacuum to concentrate to supersaturation. Cool the concentrated fermented solution, add 5 volumes of anhydrous alcohol, stir, and centrifuge to obtain a high-purity N-acetyl-glucosamine. (4) Acidified Hydrolysis of N-acetyl-D-glucosamine Prepare a saturated solution with N-acetyl-D-glucosamine, add 37% concentrated hydrochloric acid to a final concentration of 12-16%, keep at 90 C. for 45-90 min, allow to cool overnight, filter to obtain crystals, wash with alcohol, vacuum-dry and measure, to obtain a high-purity D-glucosamine hydrochloride.

7. A method for production of N-acetyl-D-glucosamine by microbial fermentation, based on claim 2, which is characterized by that the carbon and nitrogen sources of the said medium provided below: The said carbon source consists of one or more of glucose, aspergillus niger dregs, trichoderma dregs, leftovers from production of black fungus, leftovers from production of glossocardia bidens, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses; the said nitrogen source consists of one or more of ammonia water, bean flour, malt, corn steep, cotton seed meal, yeast extract, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, and urea.

8. A method for production of D-glucosamine by microbial fermentation, based on claim 3, which is characterized by that the carbon and nitrogen sources of the said medium provided below: The said carbon source consists of one or more of glucose, aspergillus niger dregs, trichoderma dregs, leftovers from production of black fungus, leftovers from production of glossocardia bidens, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses; the said nitrogen source consists of one or more of ammonia water, bean flour, malt, corn steep, cotton seed meal, yeast extract, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, and urea.

9. A method for production of N-acetyl-D-glucosamine by microbial fermentation, based on claim 5, which is characterized by that the carbon and nitrogen sources of the said medium provided below: The said carbon source consists of one or more of glucose, aspergillus niger dregs, trichoderma dregs, leftovers from production of black fungus, leftovers from production of glossocardia bidens, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses; the said nitrogen source consists of one or more of ammonia water, bean flour, malt, corn steep, cotton seed meal, yeast extract, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, and urea.

10. A method for production of D-glucosamine by microbial fermentation, based on claim 6, which is characterized by that the carbon and nitrogen sources of the said medium provided below: The said carbon source consists of one or more of glucose, aspergillus niger dregs, trichoderma dregs, leftovers from production of black fungus, leftovers from production of glossocardia bidens, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses; the said nitrogen source consists of one or more of ammonia water, bean flour, malt, corn steep, cotton seed meal, yeast extract, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, and urea.

Description

EXAMPLE 1

[0043] Based on the other aspect of this Invention, the method for production of N-acetyl-D-glucosamine and D-glucosamine by fermentation of the above strains, which are used as initial strain for production N-acetyl-D-glucosamine and D-glucosamine by seed cultivation and optimized medium fermentation, including the following steps:

[0044] (1) Screening, Identification, and Cultivation of Strains

[0045] Collect 50 soil samples, dilute by 10-3 folds and then smear on the plates for initial screening; using the medium for initial screening: Colloidal chitin 2.5 g/L, dipotassium hydrogen phosphate 0.7 g/L, potassium dihydrogen phosphate 0.3 g/L, magnesium sulfate 0.5 g/L, ferrous sulfate 0.01 g/L, and agar 20 g/L, pH 7.0; the culture temperature is 37 C. and the cultivation time is 72 h. Incubate to obtain single colonies, separate such colonies, obtain 11 strains with good growth and large & bright transparent zones of colonies. The obtained strains are identified according to the morphological characteristics of such colonies, and by grain staining and physiological & biochemical tests, to obtain 3 strains of Bacillus subtilis, and 2 strains of Bacillus licheniformis.

[0046] Activated slant strains are inoculated into a 250-mL shake flask containing 50 mL of the seed medium and incubated. The incubated liquid strains are inoculated by 10% inoculum into a 500-mL shake flask containing 100 mL of the medium and incubate. The strains are screened, according to the activities of chitinase contained in the fermented solution.

[0047] Seed medium: Peptone 10 g/L, beef extract 3 g/L, and sodium chloride 5 g/L;

[0048] Culture conditions: pH 7.4; culture temperature 37 C., shake bed rotate speed 200 rpm, and culture time 8 h;

[0049] Fermentation medium: Chitin fine powder 10 g/L, corn powder 5 g/L, starch 3 g/L, sodium nitrate 3 g/L, dipotassium hydrogen phosphate 1.05 g/L, potassium dihydrogen phosphate 0.45 g/L, sodium chloride 0.1 g/L, magnesium sulfate 0.5 g/L, and ferrous sulfate 0.03 g/L;

[0050] Culture conditions: pH 7.0; culture temperature 37 C., shake bed rotate speed 220 rpm, and culture time 72 h;

[0051] Measurement of chitinase activities in the fermented solution: Weigh 10 g of chitin fine powder, prepare with phosphate buffer to produce a 10% suspension, add a centrifuged fermented solution in the ratio of 1:1 (V/V), and incubate at 45 C. for 4 h. Then centrifuge the enzymatic hydrolysate at 3000 rpm for 10 min, transfer the supernatant, add 2 volumes of anhydrous alcohol, allow to stand overnight, and centrifuge to remove any precipitate. Concentrate the supernatant under reduced pressure to 1% reducing sugar, and measure the content of N-acetyl-D-glucosamine by HPLC.

[0052] HPLC Measurement Conditions:

[0053] Apparatuses and Equipment: Shimadzu LC-15C type High Performance Liquid Chromatograph, RID-10A Differential Refraction Detector, Chromatographic Column: Aminex Hpx-87H Column (3007.8 mm); Mobile Phase: 5 mmol/L Sulfuric Acid Solution; flow rate: 0.6 mL/min; column temperature: 40 C.; injection volume 20 L; measurement: RI;

[0054] Definition of enzyme activity unit: Under the enzymatic reaction conditions, the enzyme amount required by the reducing sugar equivalent to 1 mol N-acetyl-D-glucosamine per minute is defined as one enzyme activity unit (IU).

[0055] Test Results: See Table 1 for the measured results of enzyme-producing activities from 3 strains of Bacillus subtilis, and 2 strains of Bacillus licheniformis.

TABLE-US-00001 TABLE 1 Chitinase Activities of 3 Strains of Bacillus subtilis and 2 Strains of Bacillus licheniformis Strain and No. Chitinase Activity (IU/mL) No. 1 Bacillus subtilis 0.31 No. 2 Bacillus subtilis 2.44 No. 3 Bacillus subtilis 1.34 No. 1 Bacillus lincheniformis 1.75 No. 2 Bacillus lincheniformis 0.41

[0056] The No. 2 Bacillus subtilis with the highest enzyme-producing activity is named as Bacillus subtilis NJ090259, and the No. 1 Bacillus Licheniformis with the highest enzyme-producing activity is named as Bacillus licheniformis NJ091195.

[0057] (2) Cultivate by Fermentation

[0058] Bacillus subtilis NJ090259, Bacillus licheniformis NJ091195, and the standard strain Bacillus licheniformis ACCC02569 are activated in the plate medium, inoculated into the seed medium, respectively, and incubated in a constant-temperature (30 C.) shake bed for 18 h, as seed solution. For inoculation, inoculate at the 1:10 ratio into the fermentation medium, incubate in a shake bed at a constant temperature of 30 C. for 72 h, centrifuge at 12000 rpm for 5 min, then transfer the supernatant, and measure the content of N-acetyl-D-glucosamine by HPLC. See Table 2 for the measured results.

[0059] Plate medium: Colloidal chitin 30 g/L, ammonium sulfate 2.0 g/L, potassium dihydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L, sodium chloride 0.5 g/L, and agar 20 g/L, pH 6.5;

[0060] Seed medium: Peptone 5.0 g/L, beef extract 5.0 g/L, and sodium chloride 5.0 g/L, pH 7.0-7.2;

[0061] Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L;

[0062] Fermentation conditions: Temperature 35 C., fermentation time 18 h, initial pH 6.5, inoculum dose 10%, and volume of loaded solution, 50 mL/250 mL.

TABLE-US-00002 TABLE 2 N-acetyl-D-glucosamine Level in the Supernatant of the Fermentation Incubation Strain N-acetyl-D-glucosamine (g/L) Bacillus licheniformi ACCC02569 1.13 g/L Bacillus subtilis NJ090259 1.55 g/L Bacillus licheniformis NJ091195 1.11 g/L

[0063] Purification of N-acetyl-D-glucosamine Fermented Solution

[0064] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.01 mol/L; flow rate of the fermented solution in the dilute chamber: 40 L/h; flow rate of the fermented solution in the concentrated chamber: 40 L/h. The voltage of the single membrane pair is 0.5V. The fermented solution removed of salt is heated at 50 C. under vacuum conditions (0.095 MPa) and concentrated for 8 h until oversaturation. The concentrated fermented solution is cooled to 25 C. in water at 25 C., then cooled for 1 h to 0 C. in water at 0 C. Add 5 volumes of anhydrous alcohol, and stir for 15 min. Centrifuge at 700 rpm for 15 min, add an equal volume of anhydrous alcohol and stir at 10 rpm for 0.5 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 90%.

[0065] Acidified Hydrolysis of N-acetyl-D-glucosamine

[0066] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add 37% concentrated hydrochloric acid to the final concentration of 12%, keep at 90 C. for 45 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 97.5% D-glucosamine hydrochloride, white, in a total yield of 82%.

[0067] In another implementation example, the purification step of the N-acetyl-D-glucosamine fermented solution at Step (3) is provided below:

[0068] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.03 mol/L; flow rate of the fermented solution in the dilute chamber: 60 L/h; flow rate of the fermented solution in the concentrated chamber: 60 L/h. The voltage of the single membrane pair is 0.5-1.4 V. The fermented solution removed of salt is heated at 65 C. under vacuum conditions (0.095 MPa) and concentrated for 11 h until oversaturation. The concentrated fermented solution is cooled to 25 C. in water at 30 C., then cooled for 2 h to 0 C. in water at 5 C. Add 5 volumes of anhydrous alcohol, and stir for 37 min. Centrifuge at 1050 rpm for 37 min, add an equal volume of anhydrous alcohol and stir at 55 rpm for 1.2 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 93%.

[0069] The acid hydrolysis step of N-acetyl-D-glucosamine at Step (4) is provided below:

[0070] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add 37% concentrated hydrochloric acid to the final concentration of 14%, keep at 90 C. for 67 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 98.0% D-glucosamine hydrochloride, white, in a total yield of 84%.

[0071] In another implementation example, the purification step of the N-acetyl-D-glucosamine fermented solution at Step (3) is provided below:

[0072] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.05 mol/L; flow rate of the fermented solution in the dilute chamber: 80 L/h; flow rate of the fermented solution in the concentrated chamber: 80 L/h. The voltage of the single membrane pair is 1.4V. The fermented solution removed of salt is heated at 80 C. under vacuum conditions (0.095 MPa) and concentrated for 15 h until oversaturation. The concentrated fermented solution is cooled to 25 C. in water at 35 C., then cooled for 3 h to 0 C. in water at 10 C. Add 5 volumes of anhydrous alcohol, and stir for 1 h. Centrifuge at 1500 rpm for 60 min, add an equal volume of anhydrous alcohol and stir at 100 rpm for 2 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 95%.

[0073] The acid hydrolysis step of N-acetyl-D-glucosamine at Step (4) is provided below:

[0074] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add 37% concentrated hydrochloric acid to the final concentration of 16%, keep at 90 C. for 90 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 98.5% D-glucosamine hydrochloride, white, in a total yield of 86%.

EXAMPLE 2

[0075] (1) Induce Mutation by Ultraviolet Radiation

[0076] Transfer 10 mL of 110.sup.7 cells/mL Bacillus subtilis NJ090259 Suspension to a 9-cm culture dish, prewarm by an ultraviolet lamp for 20 min, and place the culture dish into a magnetic stirrer, approximately 30 cm vertically from the 10-W ultraviolet lamp; start the magnetic stirrer, and irradiate for 150, 200, 250, and 300 s. After induced mutation, the bacterial suspension is allowed to stand for 1-2 h in a refrigerator, protected from light. Pick up the strain after induced mutation, and the initial strains, inoculate into the chitin medium plate, pick up the largest mutants with rapid growth rate, of which the ratio of the chitin hydrolyzation circle to the diameter of the colony is more than that of the initial strain by 10%, and measure enzyme activities.

[0077] Chitin medium: Colloidal chitin 30 g/L, ammonium sulfate 2.0 g/L, magnesium sulfate 0.5 g/L, potassium dihydrogen phosphate 1.0 g/L, and sodium chloride 0.5 g/L.

[0078] Culture conditions: pH 6.5; culture temperature 32 C. and culture time 5 days.

[0079] (2) Production of N-acetyl-D-glucosamine

[0080] By the fermentation culture method as described in the Implementation Example 1, inoculate Bacillus subtilis NJ090259 mutant modified by ultraviolet radiation, carry out a fermentation incubation test, and analyze the supernatant of the culture solution by HPLC. The results show: The culture solution obtained from the medium contains 5.5 g/L N-acetyl-D-glucosamine.

[0081] (3) Purification of N-acetyl-D-glucosamine Fermented Solution

[0082] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.01-0.05 mol/L; flow rate of the fermented solution in the dilute chamber: 40-80 L/h; flow rate of the fermented solution in the concentrated chamber: 40-80 L/h. The voltage of the single membrane pair is 0.5-1.4 V. The fermented solution removed of salt is heated at 50-80 C. under vacuum conditions (0.095 MPa) and concentrated for 8-15 h until oversaturation. The concentrated fermented solution is cooled to 25-35 C. in water at 25 C., then cooled for 1-3 h to 0-10 C. in water at 0 C. Add 5 volumes of anhydrous alcohol, and stir for 15 min-1 h. Centrifuge at 700-1500 rpm for 15-60 min, add an equal volume of anhydrous alcohol and stir at 10-100 rpm for 0.5-2 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 94%.

[0083] (4) Acidified Hydrolysis of N-acetyl-D-glucosamine

[0084] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add (37%) concentrated hydrochloric acid to the final concentration of 12%-16%, keep at 90 C. for 45-90 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 98.0% D-glucosamine hydrochloride, white, in a total yield of 85%.

EXAMPLE 3

[0085] (1) Fed-Batch Fermentation of Ultraviolet-Induced Mutant of Bacillus subtilis NJ090259

[0086] The largest, ultraviolet induced mutant of Bacillus subtilis NJ090259 is activated in the plate medium, inoculated into the seed medium, and incubated in a constant-temperature (30 C.) shake bed for 18 h, as seed solution. For inoculation, inoculate in the 1:10 ratio into a 250-mL baffle conical flask containing 50 mL fermentation medium, and add 2.5 mL of the fed-batch medium at 24, 36, 48, and 60 h, respectively.

[0087] Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L;

[0088] Fed-batch Medium: Colloidal chitin 100 g/L and glucose 100 g/L, pH 6.0.

[0089] Culture conditions: pH 6.5; culture temperature 35 C. and culture time 72 days. Carry out a constant-temperature shake-bed incubation.

[0090] After completion of fermentation, centrifuge at 12000 rpm for 5 min, transfer the supernatant, and measure the content of N-acetyl-D-glucosamine by HPLC.

[0091] (2) Production of N-acetyl-D-glucosamine

[0092] By the fermentation culture method as described in the Implementation Example 1, inoculate Bacillus subtilis NJ090259 mutant modified by ultraviolet radiation, carry out a fed-batch fermentation test, and analyze the supernatant of the culture solution by HPLC. The results show: The culture solution obtained from the medium contains 24.0 g/L N-acetyl-D-glucosamine.

[0093] (3) Purification of N-acetyl-D-glucosamine Fermented Solution

[0094] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.01-0.05 mol/L; flow rate of the fermented solution in the dilute chamber: 40-80 L/h; flow rate of the fermented solution in the concentrated chamber: 40-80 L/h. The voltage of the single membrane pair is 0.5-1.4 V. The fermented solution removed of salt is heated at 50-80 C. under vacuum conditions (0.095 MPa) and concentrated for 8-15 h until oversaturation. The concentrated fermented solution is cooled to 25-35 C. in water at 25 C., then cooled for 1-3 h to 0-10 C. in water at 0 C. Add 5 volumes of anhydrous alcohol, and stir for 15 min-1 h. Centrifuge at 700-1500 rpm for 15-60 min, add an equal volume of anhydrous alcohol and stir at 10-100 rpm for 0.5-2 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 96%.

[0095] (4) Acidified Hydrolysis of N-acetyl-D-glucosamine

[0096] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add (37%) concentrated hydrochloric acid to the final concentration of 12%-16%, keep at 90 C. for 45-90 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 98.7% D-glucosamine hydrochloride, white, in a total yield of 86.5%.

EXAMPLE 4

[0097] (1) Induce Mutation by a Mutagenic Agent

[0098] Bacillus licheniformis NJ091195 is activated and incubate to a culture solution in the log phase. Centrifuge and transfer the supernatant, and prepare an approximately 10.sup.8 cells/mL bacterial suspension. Transfer 0.5 mL of 400, 600, 800, and 1000 g/mL N-methyl-N-nitro-N-nitrosoguanidine to test tubes, and then transfer 0.5 mL each of the prepared bacterial suspension to the above test tubes. Mix well, then incubate in a water bath at 30 C. for 30 min (the treatment concentration are 200, 300, 400, and 500 g/mL), stop the reaction by the dilution method, dilute in a dark place and smear on the chitin medium plate, and incubate at 37 C. for 5 days. Pick up the largest mutants with rapid growth rate, of which the ratio of the chitin hydrolyzation circle to the diameter of the colony is more than that of the initial strain by 10%, and measure enzyme activities.

[0099] (2) Production of N-acetyl-D-glucosamine

[0100] By the fermentation culture method as described in the Implementation Example 1, inoculate Bacillus licheniformis NJ091195 mutant induced by a mutagenic agent, carry out a fermentation incubation test, and analyze the supernatant of the culture solution by HPLC. The results show: The culture solution obtained from the medium contains 3.0 g/L N-acetyl-D-glucosamine.

[0101] (3) Purification of N-acetyl-D-glucosamine Fermented Solution

[0102] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.01-0.05 mol/L; flow rate of the fermented solution in the dilute chamber: 40-80 L/h; flow rate of the fermented solution in the concentrated chamber: 40-80 L/h. The voltage of the single membrane pair is 0.5-1.4 V. The fermented solution removed of salt is heated at 50-80 C. under vacuum conditions (0.095 MPa) and concentrated for 8-15 h until oversaturation. The concentrated fermented solution is cooled to 25-35 C. in water at 25 C., then cooled for 1-3 h to 0-10 C. in water at 0 C. Add 5 volumes of anhydrous alcohol, and stir for 15 min-1 h. Centrifuge at 700-1500 rpm for 15-60 min, add an equal volume of anhydrous alcohol and stir at 10-100 rpm for 0.5-2 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 97%.

[0103] (4) Acidified Hydrolysis of N-acetyl-D-glucosamine

[0104] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add (37%) concentrated hydrochloric acid to the final concentration of 12%-16%, keep at 90 C. for 45-90 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 98.6% D-glucosamine hydrochloride, white, in a total yield of 86.6%.

EXAMPLE 5

[0105] (1) Fed-Batch Fermentation Test of Bacillus licheniformis NJ091195 Mutant Induced by a Mutagenic Agent

[0106] The largest mutant of Bacillus licheniformis NJ091195, induced by a mutagenic agent as described in Implementation Example 4, is activated in the plate medium, inoculated into the seed medium, and incubated in a constant-temperature (30 C.) shake bed for 18 h, as seed solution. For inoculation, inoculate in the 1:10 ratio into a 250-mL baffle conical flask containing 50 mL of the fermentation medium, incubate, add 2.5 mL of the fed-batch medium at 24, 36, 48, and 60 h. After completion of fermentation, centrifuge at 12000 rpm for 5 min, transfer the supernatant, and measure the content of N-acetyl-D-glucosamine by HPLC.

[0107] Fermentation medium: Colloidal chitin 10 g/L, glucose 10 g/L, yeast extract 3.0 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, KH.sub.2PO.sub.4 0.4 g/L, K.sub.2HPO.sub.4 0.6 g/L, and ZnSO.sub.4 0.001 g/L;

[0108] Fed-batch Medium: Colloidal chitin 100 g/L and glucose 100 g/L, pH 6.0.

[0109] Culture conditions: pH 6.5; culture temperature 35 C. and culture time 72 days. Carry out a constant-temperature shake-bed incubation.

[0110] (2) Production of N-acetyl-D-glucosamine

[0111] By the fermentation culture method as described in the Implementation Example 1, inoculate Bacillus licheniformis NJ091195 mutant modified by ultraviolet radiation, carry out a fed-batch fermentation test, and analyze the supernatant of the culture solution by HPLC. The results show: The culture solution obtained from the medium contains 19.0 g/L N-acetyl-D-glucosamine.

[0112] (3) Purification of N-acetyl-D-glucosamine Fermented Solution

[0113] Transfer the supernatant obtained by centrifugation of the medium, carry an electrodialysis to remove salt; initial salt concentration of the fermented solution in the concentrated chamber tank: 0.01-0.05 mol/L; flow rate of the fermented solution in the dilute chamber: 40-80 L/h; flow rate of the fermented solution in the concentrated chamber: 40-80 L/h. The voltage of the single membrane pair is 0.5-1.4 V. The fermented solution removed of salt is heated at 50-80 C. under vacuum conditions (0.095 MPa) and concentrated for 8-15 h until oversaturation. The concentrated fermented solution is cooled to 25-35 C. in water at 25 C., then cooled for 1-3 h to 0-10 C. in water at 0 C. Add 5 volumes of anhydrous alcohol, and stir for 15 min-1 h. Centrifuge at 700-1500 rpm for 15-60 min, add an equal volume of anhydrous alcohol and stir at 10-100 rpm for 0.5-2 h, to obtain N-acetyl-D-glucosamine crystals in a purity of 97.9%.

[0114] (4) Acidified Hydrolysis of N-acetyl-D-glucosamine

[0115] Place the crude crystals of N-acetyl-D-glucosamine into a glass container, dissolve in water to prepare a saturated solution, add (37%) concentrated hydrochloric acid to the final concentration of 12%-16%, keep at 90 C. for 45-90 min, allow to cool to 4 C. and stand overnight, filter to obtain crystals, wash with alcohol, and vacuum-dry and measure, to obtain 98.8% D-glucosamine hydrochloride, white, in a total yield of 87%.

[0116] HPLC Measurement Conditions of the Content of D-glucosamine Hydrochloride:

[0117] Apparatuses and Equipment: Shimadzu LC-15C type High Performance Liquid Chromatograph; Detector: Variable wavelength ultraviolet detector. Chromatographic column: NH.sub.2 chromatographic column (4.6 mm15 cm, 5 m); mobile phase: Acetonitrile-phosphate buffer (60:40); flow rate: 1.5 mL/min; detection wavelength: 195 nm; column temperature: 35 C.; injection volume: 10 L.

[0118] As the bacteriological of common bacteria are extremely variable and unstable, therefore Bacillus subtilis NJ090259 and Bacillus licheniformis NJ091195 for production of N-acetyl-D-glucosamine and D-glucosamine are mutagenized. The metagenic methods to induce mutations of bacterial strains include known spontaneous mutation method or common artificial mutation method, such as ultraviolet radiation, X-ray radiation, or mutagenic agents (such as N-methyl-N-nitro-N-nitrosoguanidine). The results show that, all strains of the above bacteria, being able to produce N-acetyl-D-glucosamine, apply to this Invention, including their spontaneous mutants and artificial mutants.

[0119] The above described examples are just good implementation examples of this Invention, and are not intended to restrict this Invention. Any modification, equivalent replacement, or improvement, made within the spirits and principles of this Invention, falls into the scope of protection of this Invention.