CN113981017A - Biosynthesis method of gluconate - Google Patents
Biosynthesis method of gluconate Download PDFInfo
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Abstract
The invention discloses a biosynthesis method of gluconate, which comprises the following steps: preparing a material glucose, and adding glucose oxidase and catalase required for glucose conversion into a buffer system; high-concentration glucose and hydrogen peroxide mother liquor are added into a buffer system, glucose oxidase catalyzes and synthesizes gluconic acid, catalase catalyzes hydrogen peroxide to decompose and generate oxygen, the dissolved oxygen concentration of the reaction system is improved, and the conversion efficiency of the glucose is improved; the method comprises the steps of adding an alkaline solution, neutralizing gluconic acid generated by reaction, and preparing gluconate, wherein a semi-circulation system for generating oxygen by catalyzing hydrogen peroxide to decompose through catalase is reversely constructed, the gluconate is synthesized by a biological catalysis method, the content of dissolved oxygen is improved by enzyme catalysis, and the glucose is promoted to be efficiently converted into the gluconate.
Description
Technical Field
The invention relates to the technical field of gluconate biosynthesis, in particular to a gluconate biosynthesis method.
Background
Gluconate is an important biochemical product as polyhydroxy carboxylate and is widely applied to the fields of medicines, foods, chemical industry, light industry and the like. The calcium gluconate can be used for treating osteoporosis, skin allergy, etc.; the zinc gluconate, calcium gluconate, zinc, magnesium and ferrous iron are used in food industry to supplement elements required by human body; the gluconate is used as a cleaning agent and a metal rust remover in the electroplating industry, and is used as a water reducing agent for concrete in the construction industry; it is used as assistant and tanning agent for leather.
The current production methods of gluconate mainly comprise: chemical oxidation, electrolytic oxidation, microbial fermentation, and enzymatic oxidation. Compared with the chemical oxidation method and the electrolytic oxidation method, the fermentation method has become the leading technology for producing the gluconic acid and the salt thereof due to the advantages of mild reaction conditions, simple and environment-friendly process route, low product cost and the like. But still has the problems of long fermentation time in the production process, further treatment of the fermented thalli and the like. The enzyme rule is that glucose is dehydrogenated into gluconolactone under the action of molecular oxygen by utilizing the specificity of glucose oxidase, the gluconolactone is hydrolyzed to generate gluconic acid, and simultaneously hydrogen peroxide is generated and is decomposed into oxygen and water under the action of catalase. The enzyme catalysis has the characteristics of high specificity, easy separation of products and the like, but the biological catalysis efficiency becomes the key for competing with a chemical method.
In China, the preparation of gluconate by enzyme catalysis is reported, and the improvement of the conversion efficiency of glucose is mainly realized by changing the immobilization mode. However, the level of dissolved oxygen during gluconate synthesis directly affects the activity of glucose oxidase. The content of dissolved oxygen in water is low, and the content of dissolved oxygen in water is obviously insufficient for the conversion of high concentration and glucose.
Therefore, it is necessary to develop a method for biosynthesis of gluconate to solve the above problems.
Disclosure of Invention
In order to overcome the above defects in the prior art, an embodiment of the present invention provides a method for biosynthesis of gluconate, including the following steps:
s1, preparing a material glucose, converting the glucose into required glucose oxidase and catalase, and adding the glucose oxidase and the catalase into a buffer system;
s2, adding high-concentration glucose and hydrogen peroxide mother liquor into a buffer system, catalyzing and synthesizing gluconic acid by using glucose oxidase, catalyzing hydrogen peroxide to decompose and generate oxygen by using catalase, and improving the concentration of dissolved oxygen of a reaction system so as to improve the conversion efficiency of glucose;
and S3, adding an alkaline solution into the mixture, and neutralizing the gluconic acid generated by the reaction to prepare the gluconate.
Preferably, the protein content of glucose oxidase in the reaction system in S2 is 2-20mg/L, and the protein content of catalase is 5-50 mg/L.
Preferably, the activity of the glucose oxidase is 50-500U/mg, and the activity of the catalase is 1000-5000U/mg.
Preferably, the conditions of the buffer system in S1 are as follows: the temperature is 30-50 ℃, and the pH value is 3.5-6.0.
Preferably, the concentration of glucose in S2 is 40-60 (w/v)%, and the concentration of hydrogen peroxide is 15-30 (w/v)%.
Preferably, the feeding speed of the glucose in the S2 is 3-15g/h, and the feeding speed of the hydrogen peroxide is 1-5 g/h.
Preferably, the alkaline solution in S3 may be a strong alkali solution, a strong alkali weak acid solution or a suspension thereof.
Preferably, the gluconate is one of potassium salt, sodium salt, magnesium salt, zinc salt, calcium salt or ferrous salt.
The invention has the technical effects and advantages that:
through the experimental preparation process, a semi-circulation system for generating oxygen through hydrogen peroxide decomposition catalyzed by catalase is reversely constructed, and dissolved oxygen in the reaction process is improved, so that the enzyme catalysis efficiency is improved, the time-space yield of gluconate synthesis is improved, gluconate is synthesized by a biological catalysis method, the content of the dissolved oxygen is improved by enzyme catalysis, glucose is promoted to be efficiently converted into gluconate, and the method has the characteristics of higher time-space yield of gluconate synthesis, simple process and environmental protection.
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FIG. 1 is a schematic diagram showing the structure of the dissolved oxygen in different flow-acceleration reactors according to the present invention.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a biosynthesis method of gluconate, which comprises the following steps:
s1, preparing a material glucose, converting the glucose into required glucose oxidase and catalase, and adding the glucose oxidase and the catalase into a buffer system;
s2, adding high-concentration glucose and hydrogen peroxide mother liquor into a buffer system, catalyzing and synthesizing gluconic acid by using glucose oxidase, catalyzing hydrogen peroxide to decompose and generate oxygen by using catalase, and improving the concentration of dissolved oxygen of a reaction system so as to improve the conversion efficiency of glucose;
and S3, adding an alkaline solution into the mixture, and neutralizing the gluconic acid generated by the reaction to prepare the gluconate.
Further, in the technical scheme, the protein content of glucose oxidase in the reaction system in S2 is 2-20mg/L, and the protein content of catalase is 5-50 mg/L.
Further, in the technical scheme, the activity of the glucose oxidase is 50-500U/mg, and the activity of the catalase is 1000-5000U/mg.
Further, in the above technical solution, the conditions of the buffer system in S1 are as follows: the temperature is 30-50 ℃, and the pH value is 3.5-6.0.
Furthermore, in the above technical solution, the concentration of glucose in S2 is 40-60 (w/v)%, and the concentration of hydrogen peroxide is 15-30 (w/v)%.
Furthermore, in the technical scheme, the flow acceleration rate of glucose in S2 is 3-15g/h, and the flow acceleration rate of hydrogen peroxide is 1-5 g/h.
Further, in the above technical solution, the alkaline solution in S3 may be a strong alkali solution, a strong alkali weak acid solution, or a suspension thereof.
Further, in the above technical scheme, the gluconate is one of potassium salt, sodium salt, magnesium salt, zinc salt, calcium salt or ferrous salt.
Example 1:
adding 5mg of glucose oxidase, 5mg of catalase and 140ml of buffer solution with the pH value of 5.0 into a 0.6L reaction tank, stirring at the speed of 400rpm/min and the ventilation rate of 600ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose and 7% of hydrogen peroxide solution at a constant flow rate of 6ml/h for 10h, controlling the pH value in the reaction process through 300g/L of zinc oxide suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable, finishing the catalytic reaction, continuously adding the zinc oxide suspension to ensure that the pH value is 7.0, thus obtaining the corresponding zinc gluconate solution. The DO value in the reaction system is about 100 percent, and the conversion rate of the glucose is 95 percent.
Example 2:
adding 5mg of glucose oxidase, 10mg of catalase and 135ml of buffer solution with the pH value of 4.5 into a 0.6L reaction tank, stirring at the speed of 500rpm/min and the ventilation rate of 800ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose and 8% of hydrogen peroxide solution at a constant flow rate of 6ml/h for 10h, controlling the pH value in the reaction process through 280g/L of calcium carbonate suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable, finishing the catalytic reaction, continuously adding the calcium carbonate suspension to ensure that the pH value is 7.0 to obtain the corresponding calcium gluconate solution. The DO value in the reaction system is about 200%, and the conversion rate of glucose is 99%.
Experimental example 3:
adding 5mg of glucose oxidase, 15mg of catalase and 150ml of buffer solution with the pH value of 4.8 into a 0.6L reaction tank, stirring at the speed of 350rpm/min and the ventilation rate of 600ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose and 5% of hydrogen peroxide solution at a constant flow rate of 6ml/h for 12h, controlling the pH value in the reaction process through 350g/L magnesium carbonate suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable and the catalytic reaction is finished, continuously adding the magnesium carbonate suspension to ensure that the pH value is 7.0 to obtain the corresponding magnesium gluconate solution. The DO value in the reaction system is about 70%, and the conversion rate of glucose is 93%.
Example 4:
adding 10mg of glucose oxidase, 15mg of catalase and 130ml of buffer solution with the pH value of 5.2 into a 0.6L reaction tank, stirring at the speed of 350rpm/min and the ventilation rate of 600ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose and 6% of hydrogen peroxide solution at a constant flow rate of 6ml/h for 12h, controlling the pH value in the reaction process through 260g/L of potassium hydroxide solution, and when the reaction system does not consume alkali any more and the pH value tends to be stable, finishing the catalytic reaction, continuously adding potassium hydroxide to ensure that the pH value is 7.0 to obtain the corresponding potassium gluconate solution. The DO value in the reaction system is about 120 percent, and the conversion rate of the glucose is 96 percent.
Example 5:
adding 10mg of glucose oxidase, 10mg of catalase and 155ml of buffer solution with the pH value of 5.4 into a 0.6L reaction tank, stirring at the speed of 300rpm/min and the ventilation rate of 400ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose and 6% of hydrogen peroxide solution at a constant flow rate of 6ml/h for 12h, controlling the pH value in the reaction process through 250g/L of sodium hydroxide solution, and when the reaction system does not consume alkali any more and the pH value tends to be stable, finishing the catalytic reaction, continuously adding sodium hydroxide to ensure that the pH value is 7.0 to obtain the corresponding sodium gluconate solution. The DO value in the reaction system is about 100 percent, and the conversion rate of the glucose is 98 percent.
Example 6:
adding 10mg of glucose oxidase, 10mg of catalase and 140ml of buffer solution with the pH value of 5.0 into a 0.6L reaction tank, stirring at the speed of 400rpm/min and the ventilation rate of 600ml/min, heating to 35 ℃, adding 50 (w/v)% of glucose and 5% of hydrogen peroxide solution at the constant speed of 14.4ml/h for 6h, controlling the pH value in the reaction process through 320g/L of zinc oxide suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable, and the catalytic reaction is finished, continuously adding the zinc oxide suspension to ensure that the pH value is 7.0 to obtain the corresponding zinc gluconate solution. The DO value in the reaction system is about 150%, and the conversion rate of glucose is 99%.
Example 7:
adding 10mg of glucose oxidase, 20mg of catalase and 150ml of buffer solution with the pH value of 5.0 into a 0.6L reaction tank, stirring at the speed of 400rpm/min and the ventilation rate of 600ml/min, heating to 35 ℃, adding 55 (w/v)% of glucose and 7% of hydrogen peroxide solution at the constant speed of 19.2ml/h for 4h, controlling the pH value in the reaction process through 330g/L of zinc oxide suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable, and the catalytic reaction is finished, continuously adding the zinc oxide suspension to ensure that the pH value is 7.0 to obtain the corresponding zinc gluconate solution. The DO value in the reaction system is about 300%, and the conversion rate of glucose is 90%.
Example 8:
adding 10mg of glucose oxidase, 30mg of catalase and 170ml of buffer solution with the pH value of 5.2 into a 0.6L reaction tank, stirring at the speed of 500rpm/min and the ventilation rate of 800ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose and 7% of hydrogen peroxide solution at a constant flow rate of 24ml/h for 3h, controlling the pH value in the reaction process through 350g/L of zinc oxide suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable, finishing the catalytic reaction, continuously adding the zinc oxide suspension to ensure that the pH value is 7.0, thus obtaining the corresponding zinc gluconate solution. The DO value in the reaction system is about 250%, and the conversion rate of glucose is 96%.
Comparative example 1:
in comparison with example 1, no hydrogen peroxide was fed during the reaction and the effect of hydrogen peroxide as an oxygen precursor was tested.
Adding 5mg of glucose oxidase, 5mg of catalase and 140ml of buffer solution with the pH value of 5.0 into a 0.6L reaction tank, stirring at the speed of 400rpm/min and the ventilation rate of 600ml/min, heating to 35 ℃, adding 60 (w/v)% of glucose solution at the flow speed of 6ml/h at a constant speed for 10h, controlling the pH value in the reaction process through 300g/L of zinc oxide suspension, and when the reaction system does not consume alkali any more and the pH value tends to be stable and the catalytic reaction is finished, continuously adding the zinc oxide suspension to ensure that the pH value is 7.0 to obtain the corresponding zinc gluconate solution. The DO value in the reaction system is about 15%, and the conversion rate of glucose is 61%.
From the examples and comparative examples, it can be seen that the gluconate space-time yield is influenced primarily by the amounts of glucose oxidase and catalase added, the rate of hydrogen peroxide flow, and the like. The more glucose oxidase and catalase are used, the higher the conversion efficiency. The fed-batch of hydrogen peroxide can effectively improve the DO and gluconate synthesis efficiency of the reaction system. The method has the advantages of simple process, high glucose conversion rate, convenient operation, and environmental friendliness.
Finally, it should be noted that: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (8)
1. A method for the biosynthesis of gluconate, comprising: the method comprises the following steps:
s1, preparing a material glucose, converting the glucose into required glucose oxidase and catalase, and adding the glucose oxidase and the catalase into a buffer system;
s2, adding high-concentration glucose and hydrogen peroxide mother liquor into a buffer system, catalyzing and synthesizing gluconic acid by using glucose oxidase, catalyzing hydrogen peroxide to decompose and generate oxygen by using catalase, and improving the concentration of dissolved oxygen of a reaction system so as to improve the conversion efficiency of glucose;
and S3, adding an alkaline solution into the mixture, and neutralizing the gluconic acid generated by the reaction to prepare the gluconate.
2. The method for the biosynthesis of gluconate according to claim 1, wherein: the protein content of glucose oxidase in the reaction system in S2 is 2-20mg/L, and the protein content of catalase is 5-50 mg/L.
3. The method for the biosynthesis of gluconate according to claim 1, wherein: the activity of the glucose oxidase is 50-500U/mg, and the activity of the catalase is 1000-5000U/mg.
4. The method for the biosynthesis of gluconate according to claim 1, wherein: the conditions of the buffer system in the S1 are as follows: the temperature is 30-50 ℃, and the pH value is 3.5-6.0.
5. The method for the biosynthesis of gluconate according to claim 1, wherein: the concentration of glucose in the S2 is 40-60 (w/v)%, and the concentration of hydrogen peroxide is 15-30 (w/v)%.
6. The method for the biosynthesis of gluconate according to claim 1, wherein: the feeding speed of glucose in the S2 is 3-15g/h, and the feeding speed of hydrogen peroxide is 1-5 g/h.
7. The method for the biosynthesis of gluconate according to claim 1, wherein: the alkaline solution in S3 may be a strong alkali solution, a strong alkali weak acid solution or a suspension thereof.
8. The method for the biosynthesis of gluconate according to claim 1, wherein: the gluconate is one of potassium salt, sodium salt, magnesium salt, zinc salt, calcium salt or ferrous salt.
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| CN114774482A (en) * | 2022-05-07 | 2022-07-22 | 山东欣宏药业有限公司 | Production process and production line of zinc gluconate solution |
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| CN110904164A (en) * | 2019-12-02 | 2020-03-24 | 武汉新华扬生物股份有限公司 | Biocatalysis method for preparing gluconate |
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| CN110904164A (en) * | 2019-12-02 | 2020-03-24 | 武汉新华扬生物股份有限公司 | Biocatalysis method for preparing gluconate |
Non-Patent Citations (1)
| Title |
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| M. ROSENBERG ET AL: "Gluconic acid production by Aspergillus niger with oxygen supply by hydrogen peroxide", 《BIOPROCESS ENGINEERING》, vol. 7, pages 309 - 313, XP002011042, DOI: 10.1007/BF00705160 * |
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| CN114774482A (en) * | 2022-05-07 | 2022-07-22 | 山东欣宏药业有限公司 | Production process and production line of zinc gluconate solution |
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