Method for preparing trehalose through microbial fermentation catalysis and application of trehalose

Abstract

The invention relates to a method for preparing trehalose through microbial fermentation catalysis and application of the trehalose, and the method comprises the following steps: inoculating an activated seed solution of escherichia coli genetically engineered bacteria for producing trehalose synthase into a fermentation culture medium for fermentation culture to obtain a fermentation solution; centrifuging the fermentation liquor to obtain wet thalli, suspending the wet thalli in a permeabilization reagent for permeabilization treatment, centrifuging and collecting supernate to obtain catalytic enzyme liquid; the permeabilization reagent is a solution containing colistin sulfate, Tween 80 and ethylenediamine tetraacetic acid; maltodextrin is used as a substrate and is mixed with a catalytic enzyme solution, and a catalytic reaction is performed to obtain the trehalose. The invention develops a brand new method for preparing trehalose, abandons a traditional mode of ultrasonically crushing fermentation liquor obtained by microbial fermentation, and adopts a permeabilization reagent with a specific formula to treat the fermentation liquor, so that the final enzyme activity can be greatly improved, and meanwhile, the conversion rate of trehalose is increased; the production efficiency is improved and the production cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of trehalose preparation, and relates to a method for preparing trehalose by microbial fermentation catalysis and its application. Background Art

[0002] In the field of biomedicine, trehalose has profound significance for the protection of life and is praised as the "sugar of life" in the 21st century. The special issue on trehalose published by the journal Nature in July 2000 pointed out that "for many living organisms, the presence or absence of trehalose means life or death", and other sugars such as sucrose and glucose in nature do not have this function. Exogenous trehalose also has good protective effects and can be used as a biological activity protectant. It plays an important role in the protection of biological tissues and organs, biological germ cells, liposomes, antibody drugs, vaccines, enzymes, etc. Trehalose has successfully replaced plasma proteins as a stabilizer for bioactive substances such as blood products, lymphocytes, and vaccines.

[0003] Secondly, studies have found that trehalose has the efficacy of treating Huntington's disease, atherosclerosis, Parkinson's disease and other diseases. In 2015, trehalose was included in the Chinese Pharmacopoeia as a pharmaceutical excipient and can be used as a tablet diluent and freeze-drying excipient for drugs. It is precisely the biological protection function of trehalose that is different from other carbohydrates that greatly expands the function of trehalose as a natural sweetener.

[0004] At present, the main production methods of trehalose include microbial extraction method, microbial fermentation method, gene recombination method, chemical synthesis method, enzyme synthesis method, etc. The production methods of trehalose have been continuously optimized and improved, and the cost has gradually decreased.

[0005] At present, the industrial production process route of trehalose is to first ferment by engineering bacteria, and after the cells are broken, the trehalose synthase system is separated and purified, and finally catalyzed to react with the substrate to generate trehalose. However, physical crushing is mostly used for cell crushing, which inactivates the enzyme when used on a large scale, resulting in generally low enzyme use efficiency. In addition, intracellular complex components will be introduced after cell crushing, increasing the separation and purification pressure and the industrial cost of separation. Moreover, the raw material cost of fermentation is relatively high, which further increases the production cost. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing trehalose by microbial fermentation catalysis and its application.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a method for preparing trehalose by microbial fermentation catalysis, and the method for preparing trehalose includes the following operation steps:

[0009] (1) Inoculate the activated seed liquid of the genetically engineered Escherichia coli strain producing trehalose synthase into a fermentation medium for fermentation culture to obtain a fermentation broth;

[0010] (2) Centrifuge the fermentation broth to obtain wet bacterial cells, suspend them in a permeabilization reagent for permeabilization treatment, and centrifuge to collect the supernatant to obtain a catalytic enzyme solution;

[0011] The permeabilization reagent is a solution containing polymyxin sulfate, Tween 80, and ethylenediaminetetraacetic acid;

[0012] (3) Use maltodextrin as a substrate, mix it with the catalytic enzyme solution, and carry out a catalytic reaction to obtain trehalose.

[0013] The genetically engineered Escherichia coli strain producing trehalose synthase used in the method of the present invention can be selected from all publicly known genetically engineered Escherichia coli strains with the function of producing trehalose synthase in the art or Escherichia coli prepared by conventional genetic engineering techniques.

[0014] The present invention has developed a completely new method for preparing trehalose, abandoning the traditional method of ultrasonic disruption of the fermentation broth obtained by microbial fermentation. Instead, a fermentation broth is treated with a permeabilization reagent with a specific formulation, which can greatly improve the final enzyme activity, simultaneously increase the conversion rate of trehalose, improve production efficiency, and reduce production costs. The permeabilization reagent used in the present invention contains three components: a solution of polymyxin sulfate, Tween 80, and ethylenediaminetetraacetic acid, without chemical reagents that are toxic to the human body, relatively safe, not easily causing environmental pollution, and these three components complement each other and cooperate synergistically in the permeabilization effect on Escherichia coli, synergistically enhancing the improvement of enzyme activity and the conversion rate of trehalose.

[0015] Preferably, the composition of the fermentation medium includes: glucose, fructose, peptone, yeast extract, sodium chloride, ammonium sulfate, MgSO4·7H2O, KH2PO4, K2HPO4.

[0016] The fermentation medium used in the method of the present invention uses glucose and fructose as two carbon sources, peptone and yeast extract as two nitrogen sources, and is combined with sodium chloride, ammonium sulfate, MgSO4·7H2O, KH2PO4, and K2HPO4. By optimizing the formula of the fermentation medium, the fermentation efficiency of Escherichia coli is greatly improved, the fermentation cycle of trehalose synthase is shortened, the enzyme activity is increased, and the production capacity is greatly improved.

[0017] Preferably, the composition of the fermentation medium includes, by concentration: glucose 5 - 15 g / L, fructose 5 - 10 g / L, peptone 5 - 15 g / L, yeast powder 1 - 10 g / L, sodium chloride 5 - 15 g / L, ammonium sulfate 20 - 30 g / L, MgSO4·7H2O 1 - 5 g / L, KH2PO4 2 - 6 g / L, K2HPO4 2 - 6 g / L.

[0018] The concentration of the glucose can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12 g / L, 13 g / L, 15 g / L, etc.; the concentration of the fructose can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc.; the concentration of the peptone can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12 g / L, 13 g / L, 15 g / L, etc.; the concentration of the yeast powder can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 10 g / L, etc.; the concentration of the sodium chloride can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12 g / L, 13 g / L, 15 g / L, etc.; the concentration of the ammonium sulfate can be 20 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, etc.; the concentration of MgSO4·7H2O can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc.; the concentration of KH2PO4 can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, etc.; the concentration of K2HPO4 can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, etc. Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0019] Preferably, the fermentation culture is carried out at 25 - 37 °C (such as 25 °C, 28 °C, 30 °C, 32 °C, 35 °C, 37 °C, etc.) for 20 - 30 h (such as 20 h, 24 h, 28 h, 30 h, etc.), and the pH of the fermentation system is controlled at 6.0 - 7.0 (such as 6.0, 6.2, 6.5, 6.8, 7.0, etc.). Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0020] Preferably, the pH value of the fermentation system is adjusted with ammonia water.

[0021] In the present invention, the activated seed liquid is prepared by the following method:

[0022] The genetically engineered Escherichia coli strain producing trehalose synthase was cultured on solid LB medium containing ampicillin by the streaking method to obtain single colonies of Escherichia coli; single colonies were picked and inoculated into liquid LB medium containing ampicillin and activated at 35-38 °C (such as 35 °C, 36 °C, 37 °C, 38 °C, etc.) for 12-24 h (such as 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, etc.).

[0023] Preferably, feeding is carried out during the fermentation culture process; the composition of the feedstock includes, by concentration: 200-300 g / L of glycerol, 400-500 g / L of glucose, 20-40 g / L of lactose, 30-50 g / L of arabinose, 100-120 g / L of yeast powder, and the pH is adjusted to 6.0-7.0 with ammonia water.

[0024] In the method of the present invention, feeding is carried out by monitoring the consumption of fermentation substrates during the fermentation culture process. The feedstock formula is specifically selected with glycerol and glucose as carbon sources, yeast powder as nitrogen source, and lactose and arabinose as inducers, which has a beneficial effect on improving enzyme activity.

[0025] The concentration of the glycerol can be selected as 200 g / L, 220 g / L, 250 g / L, 280 g / L, 300 g / L, etc.; the concentration of the glucose can be selected as 400 g / L, 420 g / L, 450 g / L, 480 g / L, 500 g / L, etc.; the concentration of the lactose can be selected as 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, etc.; the concentration of the arabinose can be selected as 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, etc.; the concentration of the yeast powder can be selected as 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L, etc.; the pH can be selected as 6.0, 6.2, 6.5, 6.8, 7.0, etc. Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0026] Preferably, the feeding rate is 3-10 g / L·h, such as 3 g / L·h, 4 g / L·h, 5 g / L·h, 6 g / L·h, 7 g / L·h, 8 g / L·h, 9 g / L·h, 10 g / L·h, etc.

[0027] Preferably, the fermentation broth is centrifuged at 2 - 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 8°C, etc.) and 4000 - 8000 rpm (such as 4000 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 7000 rpm, 8000 rpm, etc.) for 5 - 20 min (such as 5 min, 10 min, 15 min, 20 min, etc.). Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0028] Preferably, the permeabilization treatment solution is centrifuged at 2 - 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 8°C, etc.) and 9000 - 15000 rpm (such as 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 15000 rpm, etc.) for 5 - 20 min (such as 5 min, 10 min, 15 min, 20 min, etc.). Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0029] Preferably, the permeabilization reagent is an aqueous solution or PBS solution containing 0.2 - 0.6 g / L polymyxin sulfate (such as 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, etc.), 0.1 - 0.3 g / L Tween 80 (such as 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, etc.) and 0.1 - 0.3 g / L ethylenediaminetetraacetic acid (such as 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, etc.). Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0030] Based on the potential synergistic effects of polymyxin sulfate, Tween 80 and ethylenediaminetetraacetic acid in improving the permeabilization effect of cells, enzyme activity and trehalose conversion rate, better effects can be achieved when the three components meet the above specific mass ratio relationship.

[0031] Preferably, the permeabilization treatment is carried out at 30 - 40°C (such as 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc.) for 50 - 90 min (such as 50 min, 60 min, 70 min, 80 min, 90 min, etc.).

[0032] Preferably, the substrate is an aqueous solution or PBS solution of 10 - 40% (such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, etc.) maltodextrin.

[0033] Preferably, the mixing volume ratio of the substrate to the catalytic enzyme solution is (3 - 5):1, such as 3:1, 7:2, 4:1, 9:2, 5:1, etc.

[0034] Preferably, the catalytic reaction is carried out at 20 - 25°C (such as 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, etc.) for 15 - 24 h (such as 15 h, 18 h, 20 h, 22 h, 23 h, 24 h, etc.).

[0035] Preferably, after the catalytic reaction, separation and purification treatment is also carried out.

[0036] In the second aspect, the present invention provides the application of the method according to the first aspect in the production of trehalose.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention develops a brand-new method for preparing trehalose, abandoning the traditional way of ultrasonic crushing of the fermentation broth obtained by microbial fermentation, but using a permeabilizing reagent with a specific formula to treat the fermentation broth, without ultrasonic crushing, so that the final enzyme activity can be greatly improved, while the conversion rate of trehalose is increased, and the intracellular complex components are not introduced, simplifying the separation and purification operation, improving the production efficiency, and reducing the production cost. The permeabilizing reagent adopted by the present invention comprises three components: a solution of polymyxin sulfate, Tween 80, and ethylenediaminetetraacetic acid, does not contain chemical reagents toxic to the human body, is relatively safe for the human body, is not likely to cause environmental pollution, and these three components complement each other and cooperate synergistically in the permeabilizing effect on Escherichia coli, synergistically enhancing the improvement of enzyme activity and the improvement of trehalose conversion rate.

[0039] Furthermore, by optimizing the formula of the strain fermentation medium and the formula of fed-batch feeding, the fermentation efficiency is greatly improved, further enhancing the enzyme activity and the trehalose conversion rate. Specific Embodiments

[0040] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solution of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0041] The construction method of the Escherichia coli genetic engineering bacteria used in the following examples or comparative examples adopts the methods disclosed in Examples 1 - 3 of Patent CN201510015409.5.

[0042] Example 1

[0043] This example provides a method for preparing trehalose, and the operation steps are as follows:

[0044] (1) The genetically engineered Escherichia coli was cultured on solid LB medium supplemented with ampicillin using the streaking method to obtain single colonies of Escherichia coli. The formula of the solid LB medium supplemented with ampicillin is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L, agar 20 g / L.

[0045] (2) Single colonies were picked and inoculated into liquid LB medium supplemented with ampicillin to activate the strains. The activation conditions were 37 °C, 200 rpm, and the culture time was 20 h. The formula of the liquid LB medium supplemented with ampicillin is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L.

[0046] (3) The activated seed liquid was inoculated into the fermentation medium at 5%. The formula of the fermentation medium is as follows: glucose 10 g / L, fructose 5 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ammonium sulfate 25 g / L, KH2PO4 4 g / L, K2HPO4 4 g / L, MgSO4·7H2O 3 g / L. The fermentation temperature was 35 °C, the fermentation time was 28 h, and the fermentation pH was controlled at 6.0 - 7.0, adjusted with ammonia water.

[0047] During the fermentation process, the carbon source concentration was monitored. When the carbon source concentration dropped to 50%, feeding was carried out. The feeding formula is as follows: glycerol 250 g / L, glucose 450 g / L, lactose 30 g / L, arabinose 40 g / L, yeast extract 120 g / L, and the pH was adjusted to 6.0 - 7.0 with ammonia water; the feeding rate linearly increased from 3 g / L·h to 10 g / L·h.

[0048] (4) The fermentation broth was centrifuged at 4 °C and 6000 rpm for 10 min to obtain wet bacterial cells.

[0049] (5) The wet bacterial cells were suspended in the permeabilization treatment reagent and treated at 35 °C for 72 min. The formula of the permeabilization treatment reagent is as follows: polymyxin sulfate 0.4 g / L, Tween 80 0.2 g / L, ethylenediaminetetraacetic acid 0.2 g / L, and the solvent is water.

[0050] (6) The permeabilized bacterial liquid was centrifuged at 4 °C and 12000 rpm for 10 min. After removing the precipitate, the catalytic enzyme solution was obtained.

[0051] (7) Using a 20% maltodextrin aqueous solution as the substrate, 25% of the enzyme solution was added, and the catalytic reaction was carried out at 25 °C for 18 h to obtain a mixed solution rich in trehalose.

[0052] Example 2

[0053] This example provides a method for preparing trehalose, and the operation steps are as follows:

[0054] (1) The genetically engineered Escherichia coli was cultured on solid LB medium supplemented with ampicillin using the streaking method to obtain single colonies of Escherichia coli. The formula of the solid LB medium supplemented with ampicillin is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L, agar 20 g / L.

[0055] (2) The above single colonies were picked and inoculated into liquid LB medium supplemented with ampicillin to activate the strains. The activation conditions were 37 °C, 200 rpm, and the culture time was 20 h. The formula of the liquid LB medium supplemented with ampicillin is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L.

[0056] (3) The activated seed liquid was inoculated into the fermentation medium at 5%. The formula of the fermentation medium is as follows: glucose 5 g / L, fructose 10 g / L, peptone 5 g / L, yeast extract 10 g / L, sodium chloride 15 g / L, ammonium sulfate 20 g / L, KH2PO4 2 g / L, K2HPO4 6 g / L, MgSO4·7H2O 2 g / L. The fermentation temperature was 37 °C, the fermentation time was 25 h, and the fermentation pH was controlled at 6.0 - 7.0, adjusted with ammonia water.

[0057] During the fermentation process, the carbon source concentration was monitored. When the carbon source concentration dropped to 50%, feeding was carried out. The feeding formula is as follows: glycerol 200 g / L, glucose 500 g / L, lactose 20 g / L, arabinose 50 g / L, yeast extract 100 g / L, and the pH was adjusted to 6.0 - 7.0 with ammonia water; the feeding rate linearly increased from 3 g / L·h to 10 g / L·h.

[0058] (4) The fermentation broth was centrifuged at 4 °C and 5000 rpm for 15 min to obtain wet bacterial cells.

[0059] (5) The wet bacterial cells were suspended in the permeabilization treatment reagent and treated at 30 °C for 80 min. The formula of the permeabilization treatment reagent is as follows: polymyxin sulfate 0.2 g / L, Tween 80 0.3 g / L, ethylenediaminetetraacetic acid 0.3 g / L, and the solvent is water.

[0060] (6) The permeabilized bacterial solution was centrifuged at 4 °C and 13000 rpm for 15 min. After removing the precipitate, the catalytic enzyme solution was obtained.

[0061] (7) Using 20% maltodextrin aqueous solution as the substrate, 25% of the enzyme solution was added, and the catalytic reaction was carried out at 25 °C for 18 h to obtain a mixed solution rich in trehalose.

[0062] Example 3

[0063] This embodiment provides a method for preparing trehalose, and the operation steps are as follows:

[0064] (1) The genetically engineered Escherichia coli was cultured on a solid LB medium supplemented with ampicillin by the streaking method to obtain Escherichia coli single colonies; the formula of the solid LB medium supplemented with ampicillin is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L, agar 20 g / L.

[0065] (2) The above single colonies were picked and inoculated into a liquid LB medium supplemented with ampicillin to activate the strains. The activation conditions were 37 °C, 200 rpm, and the culture time was 20 h. The formula of the liquid LB medium supplemented with ampicillin is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L.

[0066] (3) The activated seed liquid was inoculated into the fermentation medium at 5%. The formula of the fermentation medium is: glucose 8 g / L, fructose 7 g / L, peptone 8 g / L, yeast extract 7 g / L, sodium chloride 5 g / L, ammonium sulfate 30 g / L, KH2PO4 6 g / L, K2HPO4 2 g / L, MgSO4·7H2O 5 g / L. The fermentation temperature was 34 °C, the fermentation time was 30 h, and the fermentation pH was controlled at 6.0 - 7.0, adjusted with ammonia water.

[0067] During the fermentation process, the carbon source concentration was monitored. When the carbon source concentration dropped to 50%, feeding was carried out. The feeding formula is: glycerol 300 g / L, glucose 400 g / L, lactose 40 g / L, arabinose 30 g / L, yeast extract 100 g / L, and the pH was adjusted to 6.0 - 7.0 with ammonia water; the feeding rate linearly increased from 3 g / L·h to 10 g / L·h.

[0068] (4) The fermentation broth was centrifuged at 4 °C and 7000 rpm for 10 min to obtain wet bacterial cells;

[0069] (5) The wet bacterial cells were suspended in the permeabilization treatment reagent and treated at 37 °C for 65 min. The formula of the permeabilization treatment reagent is: polymyxin sulfate 0.6 g / L, Tween 80 0.1 g / L, ethylenediaminetetraacetic acid 0.1 g / L, and the solvent is water;

[0070] (6) The permeabilized bacterial liquid was centrifuged at 4 °C and 11000 rpm for 10 min. After removing the precipitate, the catalytic enzyme solution was obtained;

[0071] (7) Using a 20% maltodextrin aqueous solution as the substrate, 25% of the enzyme solution was added, and the catalytic reaction was carried out at 25 °C for 18 h to obtain a mixed solution rich in trehalose.

[0072] Example 4

[0073] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the carbon source of the fermentation medium is replaced from "10 g / L of glucose and 5 g / L of fructose" to "15 g / L of glucose", and other conditions remain unchanged.

[0074] Embodiment 5

[0075] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the carbon source of the fermentation medium is replaced from "10 g / L of glucose and 5 g / L of fructose" to "15 g / L of fructose", and other conditions remain unchanged.

[0076] Embodiment 6

[0077] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the carbon source of the fed-batch is replaced from "250 g / L of glycerol and 450 g / L of glucose" to "700 g / L of glycerol", and other conditions remain unchanged.

[0078] Embodiment 7

[0079] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the carbon source of the fed-batch is replaced from "250 g / L of glycerol and 450 g / L of glucose" to "700 g / L of glucose", and other conditions remain unchanged.

[0080] Embodiment 8

[0081] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the carbon source of the fed-batch is replaced from "250 g / L of glycerol and 450 g / L of glucose" to "250 g / L of fructose and 450 g / L of glucose", and other conditions remain unchanged.

[0082] Embodiment 9

[0083] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the inducer of the fed-batch is replaced from "30 g / L of lactose and 40 g / L of arabinose" to "70 g / L of lactose", and other conditions remain unchanged.

[0084] Embodiment 10

[0085] This embodiment provides a method for preparing trehalose. The difference in the operation steps from those of Embodiment 1 is only that: in step (3), the inducer of the fed-batch is replaced from "30 g / L of lactose and 40 g / L of arabinose" to "70 g / L of arabinose", and other conditions remain unchanged.

[0086] Comparative Example 1

[0087] This comparative example provides a method for preparing trehalose. The difference in the operation steps from Example 1 is only that: in step (5), the formulation of the permeabilization treatment reagent is: polymyxin sulfate 0.55 g / L, ethylenediaminetetraacetic acid 0.25 g / L, and the solvent is water; other conditions remain unchanged.

[0088] Comparative Example 2

[0089] This comparative example provides a method for preparing trehalose. The difference in the operation steps from Example 1 is only that: in step (5), the formulation of the permeabilization treatment reagent is: polymyxin sulfate 0.55 g / L, Tween 80 0.25 g / L, and the solvent is water; other conditions remain unchanged.

[0090] Comparative Example 3

[0091] This comparative example provides a method for preparing trehalose. The difference in the operation steps from Example 1 is only that: in step (5), the formulation of the permeabilization treatment reagent is: Tween 80 0.4 g / L, ethylenediaminetetraacetic acid 0.4 g / L, and the solvent is water; other conditions remain unchanged.

[0092] Comparative Example 4

[0093] This comparative example provides a method for preparing trehalose. The difference in the operation steps from Example 1 is only in (5)-(6): The wet bacterial cells are resuspended in neutral PBS solution, subjected to ultrasonic disruption treatment, and the bacterial solution after ultrasonic disruption treatment is centrifuged at 4 °C and 6000 rpm for 10 min to obtain the catalytic enzyme solution; other conditions remain unchanged.

[0094] Test Example

[0095] Determination of trehalose conversion rate:

[0096] The concentration of trehalose in the products obtained in Examples 1-10 and Comparative Examples 1-4 was determined by high performance liquid chromatography, and the conversion rate of trehalose was calculated. The detection method for trehalose content was carried out according to the high performance liquid chromatography method in GB / T23529 - 2009, the national standard for trehalose. The conversion rate is the ratio of the concentration of the final product trehalose to the concentration of the initial substrate maltodextrin. The results are shown in Table 1.

[0097] Table 1

[0098] Group Trehalose conversion rate Example 1 84％ Example 2 81％ Example 3 82％ Example 4 79％ Example 5 75％ Example 6 71％ Example 7 76％ Example 8 70％ Example 9 70％ Example 10 77％ Comparative Example 1 66％ Comparative Example 2 65％ Comparative Example 3 62％ Comparative Example 4 64％

[0099] As can be seen from the data in Table 1, the method for catalytic preparation of trehalose by microbial fermentation according to the present invention can efficiently prepare trehalose with simple operation steps. By comparing the data results of Example 1 and Comparative Example 4, it can be known that the present invention abandons the traditional method of ultrasonic disruption of the fermentation broth obtained by microbial fermentation, but uses a permeabilizing reagent with a specific formula to treat the fermentation broth without ultrasonic disruption, which can greatly improve the final enzyme activity, increase the conversion rate of trehalose, and does not introduce complex intracellular components. By comparing the data results of Example 1 and Comparative Examples 1-3, it can be known that the three components of polymyxin sulfate, Tween 80 and ethylenediaminetetraacetic acid in the permeabilizing reagent used in the present invention complement each other and cooperate synergistically in the permeabilizing effect on Escherichia coli, and have a synergistic effect on the improvement of enzyme activity and the conversion rate of trehalose. By comparing the data results of Example 1 and Examples 4-10, it can be known that the medium formula and feeding formula used in strain fermentation also affect the conversion rate of trehalose to a certain extent.

[0100] The applicant declares that the present invention uses the above-mentioned examples to illustrate the technical solution of the present invention, but the present invention is not limited to the above-mentioned examples, that is, it does not mean that the present invention must rely on the above-mentioned examples to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0101] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0102] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for preparing trehalose by microbial fermentation catalysis, characterized in that: The method for preparing trehalose comprises the following steps: (1) inoculating an activated seed solution of a genetically engineered Escherichia coli producing trehalose synthase into a fermentation medium for fermentation to obtain a fermentation solution; (2) centrifuging the fermentation broth to obtain wet bacterial cells, suspending the wet bacterial cells in a permeabilization reagent for permeabilization, and centrifuging and collecting the supernatant to obtain a catalytic enzyme solution; The permeabilization reagent is a solution containing colistin sulfate, Tween 80 and ethylenediaminetetraacetic acid; (3) Using maltodextrin as a substrate, mixing it with a catalytic enzyme solution, and carrying out a catalytic reaction to obtain trehalose.

2. The method for preparing trehalose by microbial fermentation catalysis according to claim 1, characterized in that: The fermentation medium comprises: glucose, fructose, peptone, yeast powder, sodium chloride, ammonium sulfate, MgSO4·7H2O, KH2PO4, K2HPO4; Preferably, the components of the fermentation medium include, by concentration, glucose 5-15 g / L, fructose 5-10 g / L, peptone 5-15 g / L, yeast powder 1-10 g / L, sodium chloride 5-15 g / L, ammonium sulfate 20-30 g / L, MgSO4·7H2O 1-5 g / L, KH2PO4 2-6 g / L, and K2HPO4 2-6 g / L.

3. The method for preparing trehalose by microbial fermentation catalysis according to claim 1 or 2, characterized in that: The fermentation culture is carried out at 25-37° C. for 20-30 hours, and the pH of the fermentation system is controlled at 6.0-7.0; Preferably, the pH value of the fermentation system is adjusted by aqueous ammonia.

4. The method for preparing trehalose by microbial fermentation catalysis according to any one of claims 1 to 3, characterized in that: The activated seed solution is prepared by the following method: The genetically engineered Escherichia coli producing trehalose synthase is cultured on a solid LB medium containing ampicillin by a streak method to obtain a single colony of the Escherichia coli; a single colony is picked and inoculated into a liquid LB medium containing ampicillin and the strain is activated at 35-38° C. for 12-24 hours to obtain the obtained strain.

5. The method for preparing trehalose by microbial fermentation catalysis according to any one of claims 1 to 4, characterized in that: Feed is added during the fermentation culture process; the composition of the feed includes, in terms of concentration, 200-300 g / L glycerol, 400-500 g / L glucose, 20-40 g / L lactose, 30-50 g / L arabinose, 100-120 g / L yeast powder, and ammonia water is used to adjust the pH to 6.0-7.0; Preferably, the flow acceleration of the feed is 3-10 g / L·h.

6. The method for preparing trehalose by microbial fermentation catalysis according to any one of claims 1 to 5, characterized in that: The fermentation broth was centrifuged at 2-8°C and 4000-8000 rpm for 5-20 min; Preferably, the permeabilization solution is centrifuged at 2-8°C and 9000-15000 rpm for 5-20 min.

7. The method for preparing trehalose by microbial fermentation catalysis according to any one of claims 1 to 6, characterized in that: The permeabilization reagent is an aqueous solution or PBS solution containing 0.2-0.6 g / L colistin sulfate, 0.1-0.3 g / L Tween 80 and 0.1-0.3 g / L ethylenediaminetetraacetic acid; Preferably, the permeabilization treatment is carried out at 30-40° C. for 50-90 min.

8. The method for preparing trehalose by microbial fermentation catalysis according to any one of claims 1 to 7, characterized in that: The substrate is a 10-40% maltodextrin aqueous solution or a PBS solution; Preferably, the mixing volume ratio of the substrate to the catalytic enzyme solution is (3-5):

1.

9. The method for preparing trehalose by microbial fermentation catalysis according to any one of claims 1 to 8, characterized in that: The catalytic reaction is carried out at 20-25° C. for 15-24 hours; Preferably, separation and purification treatment is also performed after the catalytic reaction is completed.

10. Use of the method according to any one of claims 1 to 9 in producing trehalose.