阅读说明：本技术 一种连续生产1,3-二羟基丙酮和赤藓酮糖的制备方法 (Preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose ) 是由 严燕兵 俞丽燕 钱敏帆 华超 胡钦 吴自成 张利坤 杨卫华 汤必成 谈聪 于 2021-08-09 设计创作，主要内容包括：本发明公开了一种连续生产1,3-二羟基丙酮和赤藓酮糖的制备方法,利用1,3-二羟基丙酮的酵母菌反应体系的菌体废料通过硅藻土、聚乙烯亚胺、戊二醛进行包埋/交联获得固定化菌体,再建立可重复转化的赤藓酮糖生物催化反应体系,通过控制赤藓糖醇底物、补料时机和浓度,有效提高了赤藓酮糖的产量,且固定化菌体可用于连续化工业生产赤藓酮糖,并保持相当高度的酶活。(The invention discloses a preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose, which comprises the steps of embedding/crosslinking thallus waste of a yeast reaction system of the 1,3-dihydroxyacetone through diatomite, polyethyleneimine and glutaraldehyde to obtain immobilized thallus, then establishing an erythrulose biocatalysis reaction system capable of being repeatedly converted, effectively improving the yield of the erythrulose by controlling erythritol substrates, feeding time and concentration, and enabling the immobilized thallus to be used for continuously and industrially producing the erythrulose and keeping high enzyme activity.)

1. A preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose is characterized by comprising the following steps:

(1) performing biological fermentation by using a first reaction system containing glycerol and saccharomycetes, and obtaining bacterial liquid by putting the first reaction system in a tank when the concentration of the 1,3-dihydroxyacetone in the first reaction system is 180-220g/L, wherein the bacterial liquid is separated to obtain 1,3-dihydroxyacetone clear liquid and first thalli;

(2) adding glutaraldehyde, polyethyleneimine and diatomite to the first thallus to obtain immobilized thallus;

(3) establishing a second reaction system, adding the immobilized bacteria, erythritol with a first concentration and water, maintaining the pH at 5.6-6.0, aerating until the erythritol content in the second reaction system is reduced to a second concentration, starting erythritol feeding, and gradually increasing the erythritol content after feeding and maintaining the erythritol content at a third concentration; wherein the third concentration of erythritol is 15-25 g/L;

(4) when the content of the erythrulose in the second reaction system is 180g/L, the erythritol feeding amount is reduced; when the content of the erythrulose in the second reaction system is 190-210g/L, stopping the reaction;

(5) and separating bacteria liquid to obtain an erythrulose clear liquid and a second thallus, wherein the second thallus is repeatedly used for the conversion of a second system reaction, and the erythrulose clear liquid is concentrated and decolored to obtain the L-erythrulose.

2. The method according to claim 1, wherein the first reaction system comprises:

35-45g/L of glycerin,

5-10g/L of yeast extract,

0 to 0.06g/L of defoaming agent,

the balance of water;

the feed was glycerol and the pH was maintained at 5.6-5.8 by addition of NaOH.

3. The method according to claim 1 or 2, wherein the step (1) comprises:

(1A) culturing for 10-16 h at a proper temperature to obtain first-level seeds, and transferring the first-level seeds into a first-level seed tank;

(1B) culturing for 8-10h at a proper temperature to obtain secondary seeds, and transferring into a fermentation tank;

(1C) the inoculation amount is 8-10 percent, the ventilation amount is not higher than 1.5vvm, the pH is maintained between 5.6 and 5.8, when the content of the 1,3-dihydroxyacetone in the reaction system is 180-220g/L, the reaction system is placed in a tank, and the bacteria liquid is separated to obtain a first thallus and a clear 1,3-dihydroxyacetone liquid.

4. The method for continuously producing 1,3-dihydroxyacetone and erythrulose according to claim 3, wherein in step (2), the mass concentration of diatomite is 6-7 g/L, the volume fraction of polyethyleneimine is 2-4%, and the volume fraction of glutaraldehyde is 0.5-1%.

5. The method according to claim 1 or 4, wherein in the step (3), the second reaction system comprises immobilized bacteria, 30-50g/L erythritol and water, the pH is maintained at 5.6-6.0, the erythritol feeding is started after the aeration reaction is carried out until the erythritol content in the system is 8-12g/L, and the erythritol content gradually increases and is maintained at 16-22g/L after feeding.

6. The process according to claim 5, wherein when the content of erythrulose in the second reaction system is 150-170g/L, the erythritol feed is reduced; when the content of erythrulose in the second reaction system reached 195-205g/L, the reaction was stopped.

7. The method according to claim 1 or 4, wherein the second reaction system is stopped, and the immobilized bacteria and the erythrulose clear solution are separated by centrifugation or suction filtration.

8. The method for continuously producing 1,3-dihydroxyacetone and erythrulose according to claim 6, wherein the erythrulose supernatant of step (5) is concentrated at 32-50 ℃.

9. The method according to claim 1, wherein the step of continuously producing 1,3-dihydroxyacetone and erythrulose comprises: the transformation frequency of the second thallus repeatedly used in the second reaction system is not less than 20 times.

10. The method according to claim 9, wherein the step of continuously producing 1,3-dihydroxyacetone and erythrulose comprises: the second thallus is repeatedly used in a second reaction system, the enzyme activity is not less than 80 percent, and the repeated times are 20-30 times.

Technical Field

The invention relates to the field of microbial fermentation and biotransformation, in particular to a preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose.

Background

1,3-Dihydroxyacetone (DHA) is simple ketose, has a structural formula shown in formula I, is a white powdery crystal in a normal state, has cool and sweet taste, and is easy to absorb moisture and decompose; mainly uses the glycerol dehydrogenase generated in the growth process of microorganisms to oxidize and dehydrogenate the secondary hydroxyl of glycerol.

The preparation of 1,3-dihydroxyacetone is mainly a microbial conversion method, wherein the secondary hydroxyl of glycerol is subjected to oxidative dehydrogenation by using glycerol dehydrogenase generated in the growth process of microorganisms to generate the 1, 3-dihydroxyacetone. The biotransformation has the advantages of high reaction rate, mild transformation conditions, excellent chemical selectivity, environmental friendliness and the like, belongs to a green processing technology, and meets the requirements of low-carbon economy and sustainable development advocated by the state at present.

The structural formula of the L-erythrulose (DHB) is shown in a formula II, the L-erythrulose (DHB) is a light yellow liquid at normal temperature, the L-erythrulose exists in the nature but has a very small content, belongs to rare sugar, and the high-purity (78-85%) L-erythrulose is a yellow viscous liquid and has sweet taste.

1,3-dihydroxyacetone and L-erythrulose have been widely used in practice as important chemical raw materials, medical intermediates and functional additives abroad.

The traditional method for producing L-erythrulose is a chemical synthesis method using erythritol as a precursor, and the method is complicated, and the product is a racemate chiral compound, so that the resolution is difficult and the purification is not facilitated. The method for preparing the L-erythrulose by using the biotransformation method has the advantages of simple operation, mild reaction conditions, easy process control, high raw material utilization rate and product purity, environmental protection and the like, and has bright prospect.

Chinese patent CN109503340A (published as 2019, 03, 22) discloses a preparation process of 1,3-dihydroxyacetone, which comprises the steps of adding a glycerol aqueous solution and a catalyst into a high-pressure reaction kettle, completely sealing, evacuating with high-purity oxygen for three times at room temperature, then filling with 1.0MPa high-purity oxygen, stirring and heating to 70-90 ℃, and separating to obtain the 1, 3-dihydroxyacetone.

Chinese patent CN107141208B (published 2021, 02/09/h) discloses a method for preparing 1,3-dihydroxyacetone, which contains intermediate products such as 1, 3-dichloro-2-propanol and 1, 3-dichloro-2-propanone, and has complicated reaction and complicated steps.

Chinese patent CN103952334B (published 2014, 07, 30) discloses a technology for preparing L-erythrulose by biological fermentation of lactobacillus acetobacter and conventional nitrogen sources (such as corn steep liquor, urea, beef extract and the like), and 186g/L of L-erythrulose is obtained by secondary fermentation culture. The reaction system of the process contains more calcium carbonate and potassium phosphate, which is not beneficial to the purification of erythrulose and has low recycling rate.

Chinese patent CN109251948B (published 2019, 01, 22) discloses a method for preparing D-erythrulose by an immobilized enzyme catalysis method, a reaction system of the technology contains a plurality of enzymes and coenzyme, the immobilization is carried out by using epoxy resin, ADP/ATP is eluted by anion exchange, and then the D-erythrulose is obtained by adsorbing phosphoric acid by column chromatography.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose, aiming at solving the problems of complex reaction system, complicated steps, high equipment requirement, inconvenience for industrial production and the like in the prior art.

The technical scheme is as follows: in order to achieve the above object, the method of the present invention comprises the steps of:

(1) performing biological fermentation by using a first reaction system containing glycerol and saccharomycetes, and obtaining bacterial liquid by putting the first reaction system in a tank when the concentration of the 1,3-dihydroxyacetone in the first reaction system is 180-220g/L, wherein the bacterial liquid is separated to obtain 1,3-dihydroxyacetone clear liquid and first thalli;

(2) adding glutaraldehyde, polyethyleneimine and diatomite to the first thallus to obtain immobilized thallus;

(3) establishing a second reaction system, adding the immobilized bacteria, erythritol with a first concentration and water, maintaining the pH at 5.6-6.0, aerating until the erythritol content in the second reaction system is reduced to a second concentration, starting erythritol feeding, and gradually increasing the erythritol content after feeding and maintaining the erythritol content at a third concentration; wherein the third concentration of erythritol is 15-25 g/L;

(4) when the content of the erythrulose in the second reaction system is 180g/L, the erythritol feeding amount is reduced; when the content of the erythrulose in the second reaction system is 190-210g/L, stopping the reaction;

(5) separating bacteria liquid to obtain an erythrulose clear liquid and a second thallus, repeatedly using the second thallus for the conversion of a second system reaction, and concentrating and decoloring the erythrulose clear liquid to obtain the L-erythrulose.

It is known that in the fields of cosmetics and the like, L-erythrulose and 1,3-dihydroxyacetone have similar tanning effects and are frequently used in combination, while the prior art has a report of combining the bio-enzyme catalysis process routes of L-erythrulose and 1,3-dihydroxyacetone, and the thallus transformed in the traditional 1,3-dihydroxyacetone process is directly treated as waste; in addition, even if the processes of 1,3-dihydroxyacetone and erythrulose are related, how to effectively improve the yield and ensure the continuous production of the reaction system is often a technical difficulty which is difficult to overcome by the technical personnel in the field.

According to the invention, yeast and glycerol in the first reaction system are utilized to obtain 1,3-dihydroxyacetone through biocatalysis, the separated first thallus is immobilized to obtain an immobilized thallus, and the immobilized thallus is utilized to catalyze the bio-enzyme catalysis system of erythrulose, so that the repeated utilization rate of the immobilized thallus is improved while the content of L-erythrulose is remarkably improved, and the industrial continuous production of 1,3-dihydroxyacetone and erythrulose is facilitated. In a preferred embodiment of the present invention, the first microbial cell is selected from methylotrophic yeasts, and more preferably, the first microbial cell is Pichia pastoris (Pichia pastoris).

The first reaction system is used for producing 1,3-dihydroxyacetone and comprises 35-45g/L of glycerol, 5-10g/L of yeast extract, 0-0.06g/L of defoaming agent and the balance of water; the feed was glycerol and the pH was maintained at 5.6-5.8 by addition of NaOH.

Further, the step (1) is to perform 2L shake flask seed culture (first-stage seed liquid), 500L seed tank culture (second-stage seed liquid) and 5000L fermentation tank culture step by step expansion at a proper temperature, the total time is 18-30 hours, the pH value in the fermentation process is not lower than 5.6, and the fermentation temperature is 29 +/-0.5 ℃.

Still further, the step (1) includes:

(1A) culturing for 10-16 h at a proper temperature to obtain first-level seeds, and transferring the first-level seeds into a first-level seed tank;

(1B) culturing for 8-10h at a proper temperature to obtain secondary seeds, and transferring into a fermentation tank;

(1C) the inoculation amount is 8-10 percent, the ventilation amount is not higher than 1.5vvm, the pH is maintained between 5.6 and 5.8, when the content of the 1,3-dihydroxyacetone in the reaction system is 180-220g/L, the reaction system is placed in a tank, and the bacteria liquid is separated to obtain a first thallus and a clear 1,3-dihydroxyacetone liquid. In order to maintain the activity of the first thallus, the first thallus is generally preserved at the temperature of-20 ℃ or prepared into immobilized thallus for preparing erythrulose for the first time.

As a preferred embodiment, said step (1)The method comprises the following steps: culturing at 29 ℃ and 200rpm for 10-16 h to obtain the OD of the first-stage seed liquid₅₆₀(2% hydrochloric acid is diluted by 10 times) and is moved into a first-class seed tank when the dilution is more than or equal to 5; culturing at 29-30 deg.C and 200rpm under 1.3vvm for 8-10 hr, and determining the OD of the secondary seed liquid₅₆₀(2% hydrochloric acid is diluted by 10 times) and is moved into a fermentation tank when the dilution is more than or equal to 3; the inoculation amount is 8% -10%, and the initial ventilation amount is 0.8vvm (20 m)³H) gradually increased to 1.3vvm (30 m) with decreasing dissolved oxygen³H); and (3) tank pressure: at the beginning, 0.03MPa is increased to 0.05MPa gradually along with the decrease of dissolved oxygen. Stirring speed: at the beginning 150rpm (30Hz), gradually increased to 250rpm (50Hz) with decreasing dissolved oxygen. The pH value is 6.0-6.2 before digestion and 5.8-6.0 after digestion. The pH slowly dropped to 5.6 during the fermentation, and was maintained at 5.6-5.8 by addition of NaOH. The fermentation temperature is 29 plus or minus 0.5 ℃.

In the early stage of feeding, the concentration of the anhydrous glycerol fed each time is about 20-30g/L and can be maintained for about 2 hours, when the logarithmic phase is entered, the growth rate of the 1,3-dihydroxyacetone can reach 15 g/L.h, and then the growth rate is gradually reduced, and when the content of the 1,3-dihydroxyacetone initially reaches 140g/L of 100-. The specific supplement amount is determined according to the content of residual anhydrous glycerin. According to the current production experience, the glycerol content is maintained at 20-30g/L by adopting a mode of feeding substrates in a variable speed manner, so that the production speed of the 1,3-dihydroxyacetone can be greatly improved.

When the content of the 1,3-dihydroxyacetone in the reaction system is increased to 220g/L of 180-.

Concentrating the clear 1,3-dihydroxyacetone obtained in the step (1) at 30-50 ℃, and adding absolute ethyl alcohol in equal proportion to obtain 1, 3-dihydroxyacetone; and (3) preparing the obtained first thallus into an immobilized thallus through the step (2), and using the immobilized thallus for biotransformation of erythrulose by using a second reaction system. In particular, to maintain the activity of the first cells, it is generally necessary to preserve the cells at-20 ℃ or prepare immobilized cells for the first time to prepare erythrulose.

And (3) adding diatomite and polyethyleneimine for adsorption for a period of time, and then adding glutaraldehyde for crosslinking reaction to obtain the immobilized bacteria. As a preferred embodiment, the step (2) specifically includes: the mass concentration of the diatomite is 6-7 g/L, the volume fraction of the polyethyleneimine is 2-4%, and after adsorption for 1h, glutaraldehyde with the volume fraction of 0.5-1% is added for crosslinking for 1h, so that the immobilized bacteria is obtained. The mode of combining adsorption and crosslinking fixation is adopted in the step (2), so that the enzyme recovery activity and the enzyme reuse rate are favorably improved, and especially the bacterial residue after the production of 1,3-dihydroxyacetone is unexpectedly improved in the erythrulose conversion rate.

Further, in the step (3), a second reaction system is established, the immobilized bacteria, erythritol with the first concentration and water are added, the pH is maintained at 5.6-6.0, the erythritol is fed after the erythritol content in the second reaction system is reduced to the second concentration by aeration, and the erythritol content gradually rises and is maintained at a third concentration after feeding; wherein the third concentration of erythritol is 15-25 g/L. In the second reaction system biocatalysis process, the control on the erythritol concentration is particularly important, the concentration of the substrate erythrulose is a main factor determining the enzyme catalysis reaction rate, and when the concentration of the substrate is too high, the enzyme reaction rate is reduced due to the inhibition effect of the substrate; at very low substrate concentrations, the enzyme is not fully saturated with substrate and the efficiency of the catalytic reaction is compromised.

As a preferred embodiment, the first concentration of the erythritol at the initial stage of the reaction is 30-50g/L, more preferably 35-45 g/L; when the erythritol starts to be fed, the second concentration of the erythritol in the second reaction system is 8-12 g/L; finally feeding to a third concentration and maintaining the reaction at the third concentration, and more preferably controlling the reaction at 16-22 g/L. In the second reaction system, the control of the erythritol concentration can obviously improve the erythrulose content and the finished product yield.

Step (4), when the content of the erythrulose in the second reaction system is up to 140-180g/L, more preferably up to 150-170g/L, the erythritol feeding amount is reduced; when the content of the erythrulose in the second reaction system reaches 190-210g/L, preferably 195-205g/L, the reaction is stopped.

And (5) after the bacterial liquid is separated, concentrating the obtained erythrulose clear liquid at the temperature of 32-50 ℃, and decoloring by using activated carbon to obtain an erythrulose (L-erythrulose) finished product. The second thallus is the immobilized thallus which is catalyzed once in the second reaction system and can be used for subsequent repeated reaction. According to production records, after the second thallus is recycled for 20-30 times, the enzyme activity is not lower than 80%, and preferably can reach more than 85%.

Has the advantages that: (1) the invention provides a preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose based on a glycerol and yeast fermentation system. (2) The immobilized bacteria obtained by the invention can be directly used for biotransformation of erythrulose in a reaction kettle, and after the transformation is finished, the immobilized bacteria and the transformation liquid are separated by centrifugation or suction filtration, so that the obtained immobilized bacteria can be used for biotransformation of erythrulose for more than 20 times. And the immobilized bacteria is directly used for biotransformation, so that fermentation impurities brought by the traditional process are reduced, and the centrifugal clear liquid can be directly concentrated at 30-50 ℃, packaged and stored at low temperature. The centrifugal separation reduces the loss caused by membrane filtration in the traditional process, improves the final yield of the erythrulose, and the theoretical yield can reach 100%. (3) After the traditional 1,3-dihydroxyacetone reaction is ended, the first thallus is waste, but the invention can be used for converting erythrulose after preparing the immobilized thallus.

Drawings

FIG. 1 is a flow chart for the continuous production of 1,3-dihydroxyacetone and erythrulose according to the present invention;

FIG. 2 is a high performance liquid chromatography spectrum of 1,3-dihydroxyacetone prepared in example 1 of the present invention;

FIG. 3 is a high performance liquid chromatography spectrum of erythrulose prepared in example 3 of the present invention;

FIG. 4 is a graph showing the number of reusability and relative enzyme activity in example 3 of the present invention.

Detailed Description

The invention is further described with reference to the following figures and specific examples.

Example 1

As shown in FIG. 1, in this example, a first reaction system containing glycerol and yeast is used for biological fermentation, when the concentration of 1,3-dihydroxyacetone in the first reaction system is 180-220g/L, the first reaction system is placed in a tank to obtain a bacterial liquid, and the bacterial liquid is separated to obtain a 1,3-dihydroxyacetone clear liquid and a first thallus. The specific method comprises the following steps:

firstly, using inoculating loop to pick out a loop of activated yeast from inclined plane, inoculating the yeast into 2L shake flask, making liquid content be 500mL, 29 deg.C, 200rpm, shake-culturing for 12h, measuring OD₅₆₀When the dilution of 2% hydrochloric acid is 10 times or more than 5, the first-class seed liquid is obtained.

Secondly, the primary seed solution is inoculated into a 500L fermentation tank with the liquid loading capacity of 200L according to the inoculation amount of 8 percent, cultured for 10 hours at the temperature of 29-30 ℃ and at 200rpm under 1.3vvm, and then the OD is measured₅₆₀When the dilution of 2% hydrochloric acid is 10 times or more than 3, the second-level seed liquid is obtained;

finally, the secondary seed liquid is inoculated into a 5000L fermentation tank with a liquid loading capacity of 3000L according to the inoculation amount of 10 percent, the fermentation temperature is 29 +/-0.5 ℃, and the initial ventilation volume is 0.8vvm (20 m)³H) gradually increased to 1.3vvm (30 m) along with the decrease of dissolved oxygen in the fermentation process³H); the initial tank pressure is 0.03MPa, and gradually increases to 0.05MPa along with the decrease of dissolved oxygen in the fermentation process; the initial stirring speed was 150rpm (30Hz) and increased gradually to 250rpm (50Hz) as the dissolved oxygen decreased during the fermentation. NaOH is supplemented in the fermentation process to maintain the pH at 5.6-5.8, the glycerol content in the fermentation tank system is maintained at 25g/L by adopting a mode of adding substrates in a flow-variable manner, and the fermentation tank can be placed when the content of the converted 1,3-dihydroxyacetone is 200 g/L. Separating the bacterial liquid with a membrane (the material with the diameter of 0.1-1 micron is intercepted by a microfiltration membrane) to obtain 1,3-dihydroxyacetone clear liquid and first thallus, concentrating the 1,3-dihydroxyacetone clear liquid at 45 ℃, and adding absolute ethyl alcohol according to equal proportion to obtain the 1, 3-dihydroxyacetone. 1,3-dihydroxyacetone was measured by high performance liquid chromatography: chromatographic conditions are as follows: the instrument comprises the following steps: an Aglient 1260 high performance liquid chromatograph and an ultraviolet detector; a chromatographic column: lichrospher C18, 250X 4.5mm, 5 μm; flow rate: 1 mL/min; detection wavelength: 200 nm; mobile phase: 5% aqueous methanol (0.05% phosphoric acid adjusted to pH 3.0); sample introduction amount: 10 mu L of the solution; its high performance liquid chromatography spectrum is shown in FIG. 2.

Example 2

The first thallus obtained by separating the bacteria liquid in example 1 is wet thallus of yeast, 500g of the first thallus is added into 5L of water (100g/L) to prepare a bacterial suspension, stirring is started, the rotating speed is 140rpm (25Hz), 30g of diatomite (6g/L) and polyethyleneimine with the final volume fraction of 3% are added into the bacterial suspension, the pH value is adjusted to 8.0 by using a phosphoric acid solution, after 60min of cross-linking adsorption, glutaraldehyde with the final volume fraction of 1% is added and cross-linked for 60min, and solid obtained by suction filtration or centrifugal separation is the immobilized thallus. The temperature was controlled at 28 ℃ throughout the reaction at 150 rpm.

Example 3

300mL system: 8g of erythritol (40g/L) is added into the initial 200mL of water, 12g of the immobilized bacteria obtained in examples 1 and 2 (4% of the final volume) is added, air is introduced, the temperature of the water bath is 30 ℃, and the pH is controlled to be 5.6-6.0 by using 20% sodium hydroxide solution in the reaction process. After reacting for 3 hours (at the moment, the erythritol concentration in the system is 8-12g/L), feeding 50% erythritol solution at a speed of 4.8mL/h, continuously feeding 22 hours, reducing the feeding speed to 2.4mL/h, feeding about 6 hours (namely, maintaining the erythritol feeding concentration in the system to be 16-22g/L), and stopping the reaction, wherein the volume of the reaction solution is about 320mL, and the erythrulose content reaches about 200 g/L. And (3) carrying out suction filtration or centrifugal separation on the immobilized bacteria and the reaction liquid, directly concentrating the reaction liquid at 45 ℃ until the solid content is about 40, adding 3% of activated carbon for decoloring for 1h, and concentrating again at 40 ℃ until the solid content is about 68% to obtain about 90mL of an erythrulose finished product. The separated immobilized bacteria have the weight reused for the conversion of erythritol, the erythrulose content can still reach 183g/L after the reaction is repeated to the 22 th time, and the enzyme activity still reaches more than 85 percent.

Example 4

According to the method of example 1 and example 2, the immobilized cells were prepared by collecting the first cells after 1,3-dihydroxyacetone fermentation in a 500L reaction system, adding diatomaceous earth and polyethyleneimine for cross-linking adsorption, and then adding glutaraldehyde for immobilization.

300L system: adding 8kg of erythritol (40g/L) and 12kg of the immobilized bacteria into initial 200L of water, introducing air, controlling the water bath temperature to be 30 ℃, and controlling the pH value to be 5.6-6.0 by using 20% sodium hydroxide solution in the reaction process. After reacting for 3.5h, feeding 50% erythritol solution at a speed of 4.8L/h, continuously supplementing for 22h, reducing the supplementing speed to 2.4L/h, supplementing for about 6h, and terminating the reaction, wherein the volume of the reaction solution is about 330L, and the erythrulose content reaches about 200 g/L. And (3) carrying out suction filtration or centrifugal separation on the immobilized bacteria and the reaction liquid, directly concentrating the reaction liquid at 45 ℃ until the solid content is about 40, adding 3% of activated carbon for decoloring for 1h, and concentrating again at 40 ℃ until the solid content is about 68% to obtain about 90L of an erythrulose finished product. The separated immobilized bacteria have the weight reused for the conversion of erythritol, the content of erythrulose can still reach 189g/L when the reaction is repeated to the 20 th time, and the enzyme activity still reaches more than 85 percent.

Example 5

3000L system: adding 80kg of erythritol (40g/L) into initial 2000L of water, fermenting 1,3-dihydroxyacetone in a 5 ton tank, collecting 120kg of immobilized bacteria prepared from bacterial sludge (the final volume is 4%), introducing air, controlling the water bath temperature to be 30 ℃, and controlling the pH to be 5.6-6.0 by using 20% sodium hydroxide solution in the reaction process. After reacting for 3h, feeding 50% erythritol solution at a speed of 48L/h, continuously supplementing for 21.5h, reducing the supplementing speed to 24L/h, supplementing for about 7h, and terminating the reaction, wherein the volume of the reaction solution is about 3250L, and the content of erythrulose reaches about 200 g/L. And (3) carrying out suction filtration or centrifugal separation on the immobilized bacteria and the reaction liquid, directly concentrating the reaction liquid at 45 ℃ until the solid content is about 40, adding 3% of activated carbon for decoloring for 1h, and concentrating again at 40 ℃ until the solid content is about 68 to obtain 980L of a finished product of erythrulose. Erythrulose was measured by high performance liquid chromatography: chromatographic conditions are as follows: lichrospher NH₂Analytical column, 250X 4.6mm, 5 μm; flow rate: 1 mL/min; detection wavelength: 277 nm; mobile phase: acetonitrile-water (volume ratio 9: 1); sample introduction amount: 10 mu L of the solution; its high performance liquid chromatography spectrum is shown in FIG. 2. The separated immobilized strain has the advantages that the weight is reused for the conversion of erythritol, the erythrulose content can still reach 174g/L after the 24 th reaction is repeated, and the enzyme activity still reaches more than 80 percent (as shown in figure 4).

Test example 1 Effect of free enzyme and different immobilization methods on Erythroside production

The test compares the influence of the free enzyme and the commonly used immobilization method on the yield of erythrulose and the recovery rate of enzyme activity, and shows that the method of immobilizing by glutaraldehyde, diatomite and polyethyleneimine is particularly suitable for industrial production, the conversion times are greatly repeated, and high enzyme activity is maintained.

TABLE 1 comparison of the effectiveness of the free enzymes and different immobilization methods

Test example 2 influence of erythritol initial concentration and timing of feeding on erythrulose yield

According to the invention, the yield of erythrulose is further optimized by exploring the dehumidification concentration of erythritol and the feeding time. The method used in this test example was substantially the same as in example 3, and the initial erythritol concentration, the timing of feeding, and the maintenance erythritol concentration were subjected to gradient design, and as shown in table 2, the influence of different level parameters on the difference in the yield of erythrulose was seen from the table.

TABLE 2 Effect of erythritol on the conversion Effect of erythrulose

The substrate erythritol solution is controlled within a certain range, and the reaction effect is influenced by lower or higher erythritol solution, so that the yield of erythrulose is reduced; the initial concentration, the feeding time and the maintaining concentration of the substrate erythritol have the optimal concentration, and the optimal concentration is controlled by an automatic fed-batch system.