Method For Promoting Synthesis Of Sorbitol Dehydrogenase And Coenzyme Pyrroloquinoline Quinone From Gluconobacter Oxydans

Abstract

The present invention discloses a method for promoting the fermentation of Gluconobacter oxydans to produce D-sorbitol dehydrogenase and pyrroloquinoline quinone. The method comprises: Gluconobacter oxydans is inoculated to a fermentation culture medium, fermented and cultured under the conditions of 28-32 C. and 150-180 rpm for 6-24 hours, the fermented solution is centrifuged, and wet bacteria are collected, thus acquiring bacteria cells containing D-sorbitol dehydrogenase and pyrroloquinoline quinone. The method promotes the synthesis of coenzyme pQQ and the enzyme activity of per unit volume D-sorbitol dehydrogenase, Gluconobacter oxydans cultured and acquired using the method is biotransformed to synthesize miglitol precursor 6-deoxy-6-amino(N-hydroxyethyl)--L-furan sorbose (6NSL), the conversion progress of the product 6NSL is increased by 21-35%, and a biotransformation step cycle is reduced from 48 hours to 36 hours. In addition, under a same substrate concentration (60 g/L), the cumulative concentration of the product 6NSL is increased by 10 g/L or more.

Claims

1. A method for promoting the fermentation of Gluconobacter oxydans to produce D-sorbitol dehydrogenase and pyrroloquinoline quinone, the method comprising: Gluconobacter oxydans is inoculated to a fermentation culture medium, fermented and cultured under the conditions of 28-32 C. and 150-180 rpm for 6-24 hours, a fermented solution is centrifuged, and wet bacteria are collected, thus acquiring bacteria cells containing D-sorbitol dehydrogenase and pyrroloquinoline quinone; the fermentation culture medium comprises D-sorbitol 50-80 g/L, yeast extract 0.5-30 g/L, KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, amino acid 0.2-2 g/L, deionized water as solvent, pH 6.5.

2. The method according to claim 1, wherein the amino acid in the fermentation culture medium is sodium glutamate, leucine, tyrosine or proline.

3. The method according to claim 2, wherein a concentration of the amino acid in the fermentation culture medium is 0.5-1 g/L.

4. The method according to claim 2, wherein the amino acid in the fermentation culture medium is sodium glutamate.

5. The method according to claim 1, wherein a concentration of the yeast extract is 1-7 g/L.

6. The method according to claim 1, wherein the fermentation culture medium comprises D-sorbitol 50 g/L, yeast extract 5 g/L, KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, sodium glutamate 1 g/L, deionized water as solvent, pH 6.5.

7. The method according to claim 1, wherein Gluconobacter oxydans is subjected to bevel activated culture and seed culture before fermental cultivation, and then a seed solution at an inoculated dose of concentration of 5-15% by volume is inoculated to the fermentation culture medium to be fermented.

8. The method according to claim 7, wherein the bevel activated culture is to inoculate Gluconobacter oxydans to a slant medium, and cultivate in a constant temperature incubator at 28 C. for 3-5 days to obtain a slanted bacterium; the slant medium comprises yeast extract 2-10 g/L, calcium carbonate 5-20 g/L, glucose 10-60 g/L, agar 24 g/L, deionized water as solvent, the pH value is natural.

9. The method according to claim 8, wherein the seed culture is to inoculate the slanted bacterium to a seed culture medium, and then shaking bottle fermental cultivate under the conditions of 28 C. and 225-235 rpm for 24-48 hours to obtain a seed solution; the seed culture medium comprises D-sorbitol 30-60 g/L, yeast extract 10-30 g/L, KH.sub.2PO.sub.4 1-5 g/L, K.sub.2HPO.sub.4 0.1-1 g/L, deionized water as solvent, pH4.0-7.0.

10. The method according to claim 1, wherein Gluconobacter oxydans is Gluconobacter oxydans CCTCC No. M 208069.

Description

DESCRIPTION OF DRAWINGS

[0020] FIG. 1 is a synthetic route of miglitol by a chemical and biological group method.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS THEREOF

[0021] Hereafter, the present invention will be described specifically with reference to the examples. The examples are given only for illustration of the technical solution of the present invention. However, the high-activity D-sorbitol dehydrogenase and its coenzyme pQQ obtained from resting cells of Gluconobacter oxydans cultured in the fermentation culture medium provided by the present invention are not only used for the synthesis of miglitol precursor 6NSL, but also used for the selective oxidation catalysis process of other polyols.

Example 1

[0022] Gluconobacter oxydans (CCTCC No. M 208069, Chinese Patent Publication No. CN101591681A) is inoculated to a slant medium, and cultivated in a constant temperature incubator at 28 C. for 3-5 days to obtain a slanted bacterium. The slant medium comprise: yeast Extract 5 g/L, calcium carbonate 5 g/L, glucose 50 g/L, agar 24 g/L, deionized water as solvent, the pH value is natural.

[0023] The slanted bacterium are inoculated to a seed culture medium, and shaking cultivated at 28 C., 225 rpm for 48 hours to obtain a seed solution of Gluconobacter oxydans CCTCC No. M 208069; the seed culture medium comprises: D-sorbitol 50 g/L, yeast extract 20 g/L, KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, and deionized water as solvent, and the pH value is 6.5.

Example 2: Effect of Amino Acid Species in Fermentation Culture Medium on Enzyme Activity of Gluconobacter oxydans and SLDH

[0024] Preparing a fermentation culture medium with different components is to investigate effects of different amino acids on fermental cultivation of Gluconobacter oxydans to synthesize of sorbitol dehydrogenase. The fermentation culture medium formulation with different amino acids comprise: D-sorbitol 50 g/L, yeast extract 20 g/L, KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, 1 g/L (sodium glutamate, leucine, tyrosine, proline), deionized water as solvent, pH6.5. No amino acids are added as a blank control group under the same conditions.

[0025] A seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 is inoculated to a fermentation culture medium at a volume concentration of 2%, fermented at 28 C., 150 rpm for 24 h, and centrifuge a fermentation broth, and obtain bacterial cells for detecting a relative activity of sorbitol dehydrogenation SLDH.

[0026] Definition of enzyme activity units is referred to as an amount of enzyme to catalyze d-sorbitol and produce 1.0 moL L-sorbitose per minute, in a phosphate buffer system at 30 C., pH6.0 under 20 g/L sorbitol concentration. Concentrations of D-sorbitol and L-sorbose are determined by the High Performance Liquid Chromatography (HPLC).

[0027] Operation Procedure of Enzyme Activity Detection of SLDH:

[0028] Take 20 mL of a fermentation broth and centrifuge it at 10000 rpm for 10 min, discard a supernatant, wash it once, and then discard the supernatant after centrifuging again. Prepare a buffer 100 mM PBS. And afterwards dissolve 20 g/L sorbitol and 5 g/L magnesium sulfate heptahydrate to the buffer. Adjust pH to 6.0 again to establish a 10 mL reaction system at 28 C. for 30 min Take 1 mL sample to centrifuge for liquid phase detecting and analyzing conversion rate of sorbitol and yield of sorbitol product. Conditions of liquid phase detection are as follows: a mobile phase is 5 mM H.sub.2SO.sub.4 solution; a flow rate is 0.6 mL/min; an injection volume is 20 L; a column temperature is 60 C., a time is 12 min.

[0029] The determination results are shown in Table 1.

TABLE-US-00001 TABLE 1 Effects of different amino acids on producing SLDH by adding Gluconobacter oxydans Relative enzyme activity of Amino acid species SLDH (%) Blank control 100 Sodium glutamate 121 Leucine 112 Tyrosine 101 Proline 102

[0030] Analysis of Results:

[0031] As shown in Table 1, in comparison with without adding amino acids (the blank control group), its relative enzyme activities of Sorbitol dehydrogenase per unit volume have different degree changes after adding 1 g/L amino acid with different types to a culture medium and shaking bottle fermental cultivation for 24 hours. In particular, the enzyme activity of SLDH is almost unchanged after adding tyrosine or proline, compared with the blank control group. The enzyme activity of SLDH is respectively increased to 21% and 12% after adding sodium glutamate or leucine, compared with the blank control group, and the increase in sodium glutamate is greater. It shows that the addition of 1 g/L sodium glutamate can increase the relative activity of sorbitol dehydrogenase per unit volume by more than 20%, compared with a blank control group.

Example 3: Effect of Adding Sodium Glutamate on Fermental Cultivation of Gluconobacter oxydans to Synthesize Coenzyme pQQ and SLDH

[0032] Preparing a fermentation culture medium with different components is to investigate effects of sodium glutamate partially replacing yeast extract on fermental cultivation of Gluconobacter oxydans to synthesize sorbitol dehydrogenase and coenzyme pQQ. A fermentation culture medium formulation with different nitrogen sources comprises: D-sorbitol 50 g/L, yeast extract (30 g/L, 20 g/L, 15 g/L, 10 g/L, 5 g/L), KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, sodium glutamate 1 g/L, deionized water as solvent, pH6.5. No sodium glutamate is added as a blank control group under the same conditions.

[0033] The seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 is inoculated to the fermentation culture medium at a volume concentration of 2%, fermented at 28 C., 150 rpm for 24 h, and then centrifuge a fermentation broth to obtain a supernatant. The supernatant is used for detecting the relative cumulative concentration of pQQ. The bacteric cells are used for detecting the relative activity of SLDH. Wherein the former is determined by the method of glucose dehydrogenase recombinase, and the latter is determined by the method of High Performance Liquid Chromatography (HPLC). The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Effects of addition of sodium glutamate on synthesis of pQQ and activity of SLDH under different yeast extract concentrations Relative cumulative Relative enzyme concentration of pQQ (%) activity of SLDH (%) Concentration Adding Adding of Yeast sodium sodium Extract No sodium glutamate No sodium glutamate (g/L) glutamate (1 g/L) glutamate (1 g/L) 30 100 132 100 115 20 108 142 105 125 15 105 142 95 123 10 95 135 86 115 5 85 130 81 113

[0034] Analysis of Results:

[0035] As shown in Table 2, different conditions of adding 1 g/L sodium glutamate to the fermentation culture medium with different concentration yeast extracts are compared, after shaking bottle fermental cultivation for 24 hours. The concentration of pQQ and the coenzyme activity of sorbitol dehydrogenase per unit volume in the supernatant are significantly improved compared with the blank control group after fermental cultivating Gluconobacter oxydans for 24 hours. When the concentration of yeast extract is reduced to 5 g/L, adding 1 g/L sodium glutamate could ensure the content of coenzyme pQQ of the supernatant fermented is increased by more than 20%, compared with conventional culture medium. And the coenzyme activity of sorbitol dehydrogenase per unit volume is increased by more than 10%, compared with conventional culture medium. Consequently it effectively reduces the specific proportion of the yeast extract in the fermentation culture medium, and also reduces cost of fermentation.

Example 4: Effect of Adding Sodium Glutamate on Fermental Cultivation of Gluconobacter oxydans to Synthesize Coenzyme pQQ and SLDH in Different Time Periods

[0036] Preparing a fermentation culture medium with different components is to investigate effects of adding sodium glutamate on fermental cultivation of Gluconobacter oxydans to synthesize coenzyme pQQ and activity of SLDH in different time periods. The fermentation culture medium formulation comprises: D-sorbitol 50 g/L, yeast extract 5 g/L, KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, sodium glutamate 1 g/L, deionized water as solvent, the pH is natural. No sodium glutamate is added as a blank control group under the same conditions.

[0037] The seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 is inoculated to the fermentation culture medium at a volume concentration of 2%, fermented at 28 C., 150 rpm for 24 h. Wherein Samples are taken every 6 hours, centrifuge, and detect the relative content of pQQ and the relative enzyme activity of SLDH. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Accumulation of COENZYME PQQ and SLDH with culture time in fermentation culture medium Relative cumulative Relative enzyme concentration of pQQ (%) activity of SLDH(%) adding adding Fermentation sodium sodium time no sodium glutamate no sodium glutamate (H) glutamate (1 g/L) glutamate (1 g/L) 6 100 133 100 113 12 266 402 125 152 18 330 471 160 201 24 351 534 137 177

[0038] Analysis of Results:

[0039] As shown in Table 3, comparing different condition of adding 1 g/L sodium glutamate to the fermentation culture medium in different time periods after shaking bottle fermental cultivation for 24 hours. The content of coenzyme pQQ in the fermentation supernatant and the enzyme activity of sorbitol dehydrogenase per unit volume have significantly improved compared with the blank control group. When the concentration of yeast extract is 5 g/L, adding 1 g/L sodium glutamate could increase a synthetic amount of coenzyme PQQ by more than 30% in different time periods compared with the blank control group, in order to ensure the enzyme activity of sorbitol dehydrogenase per unit volume increased by more than 10% in different time periods compared with the blank control group.

Example 5: Comparison of the Activity of 6NSL Catalyzed and Synthesized by Resting Cells of Gluconobacter oxydans Obtained by Adding Sodium Glutamate to the Culture Medium

[0040] Preparing a fermentation culture medium with different components is to investigate effects of sodium glutamate partially replacing yeast extract on fermental cultivation of Gluconobacter oxydans to synthesize sorbitol dehydrogenase and coenzyme pQQ. A fermentation culture medium formulation with different nitrogen sources comprises: D-sorbitol 50 g/L, yeast extract (30 g/L, 20 g/L, 15 g/L, 10 g/L, 5 g/L), KH.sub.2PO.sub.4 5 g/L, K.sub.2HPO.sub.4 5 g/L, sodium glutamate 1 g/L, deionized water as solvent, the pH is natural. No sodium glutamate is added as a blank control group under the same conditions.

[0041] The seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 is inoculated to a fermentation culture medium at a volume concentration of 2%, fermented at 28 C., 150 rpm for 24 h. Centrifuge a fermentation broth at 10000 rpm for 10 min, discard a supernatant, and then collect resting cells of Gluconobacter oxydans, and afterwards wash bacteria cells with equal volume of clean water, re-centrifuge at 10000 rpm for 10 min, discard a supernatant, collect cells to catalyze and synthesize 6NSL.

[0042] Conversion System (Total Volume: 50 mL):

[0043] Set a baffle shake bottle 50 mL/500 mL, and then add a concentration of 80 g/L N-hydroxyethyl glucosamine, a concentration of 5 g/L MgSO.sub.4. 7H.sub.2O, 75 g/L wet bacteria, deionized water as a reaction medium to establish a catalytic reaction system. The specific reaction conditions are as follows: a temperature 15 C.; a rotation speed 220 rpm. Adjust an alkaline with 2M NaOH to maintain pH=4.5-5.0 of the conversion liquid. After transformation for 24 hours, the transformation is stopped. Take a transformation solution and then centrifuge at 10,000 rpm for 10 min. And afterwards take a supernatant and detect it by the HPLC method. The cumulative concentration of 6NSL is shown in Table 4.

TABLE-US-00004 TABLE 4 Comparison of cumulative concentrations of 6NSL catalyzed and synthesized by resting cells of Gluconobacter oxydans obtained from fermentation with addition of sodium glutamate Cumulative concentration Concentration of 6NSL (g/L) of Yeast Add sodium Extract No sodium glutamate (g/L) glutamate (1 g/L) 30 45.8 55.4 20 48.1 58.6 15 43.5 55.9 10 41.2 54.0 5 37.5 53.6

[0044] Analysis of Results:

[0045] As shown in Table 4, adding 1 g/L of sodium glutamate to the fermentation culture medium with different yeast extracts is compared. After fermental cultivating Gluconobacter oxydans for 24 hours, a cumulative concentration of miglitol precursor 6NSL synthesized by Gluconobacter oxydans resting cells resting cells catalyzing a substrate N-hydroxyethyl glucosamine has different degrees of improvement, comparing with the blank control group. Under a unit volume, when a concentration of yeast extract is reduced to 5 g/L, the cumulative concentration of 6NSL catalyzed and synthesized by Gluconobacter oxydans resting cells fermented by adding a culture medium of 1 g/L sodium glutamate is increased by more than 10%, comparing with cumulative concentrations of 6NSL without adding sodium glutamate, wherein, the concentration of yeast extract is 5-30 g/L. And at the same time, the cumulative concentration having 53.6 g/L of 6NSL under additions of a concentration 5 g/L of yeast extract is higher than that of the accumulation concentration having 45.8 g/L under additions of a concentration 30 g/L of yeast extract. The results suggest that adding 1 g/L of cheap sodium glutamate can effectively replace yeast extracts in the fermentation culture medium, and effectively reduces fermentation cost under the premise of ensuring the efficient transformation ability of the bacteria.