Process for producing trehalose under catalysis of compound enzyme

Abstract

The invention belongs to the technical field of trehalose, and particularly relates to a process for producing trehalose through compound enzyme catalysis. The production process comprises the following steps: (1) inoculating escherichia coli into an LB culture medium for culturing to obtain an escherichia coli seed solution; (2) inoculating the Escherichia coli seed solution into a TB culture medium for fermentation culture, and adding isopropyl-beta-D-thiogalactoside into a fermentation tank for induction when OD600 is measured to be 1.0-1.2, so as to obtain a fermentation solution; (3) adding colistin sulfate into the fermentation liquor, centrifuging and discarding supernate after culture is finished, then discarding supernate after centrifugation, washing with a phosphate buffer solution, and resuspending to obtain suspension liquid; and (4) mixing the suspension with a maltose aqueous solution, after the reaction is finished, cooling, centrifuging, and taking supernate to obtain a product containing trehalose. The production process provided by the invention can obviously improve the trehalose conversion rate, and has important significance for improving the economic benefits of enterprises.

Description

Technical Field

[0001] The present invention belongs to the technical field of trehalose, and particularly relates to a production process of trehalose catalyzed by a composite enzyme. Background Art

[0002] As a multifunctional natural disaccharide, trehalose exhibits irreplaceable important value in multiple fields. It not only plays a key role in medical treatment, pharmaceuticals, biochemistry, and molecular biology research, but also finds extensive applications in the food industry, biological products and pharmaceutical industries, cosmetics development, and agriculture. With the in-depth exploration of its functions, the application prospects of trehalose are becoming increasingly broad, and it is gradually becoming an important force driving the development of related industries.

[0003] Currently, the production methods of trehalose mainly include direct extraction method, enzyme synthesis method, microbial fermentation method, and genetic engineering method. Although the direct extraction method is simple to operate, due to the low yield during the extraction process, the production cost is too high. At the same time, this method may cause a certain degree of environmental pollution, so it is not suitable for large-scale industrial applications. Although the enzyme synthesis method can achieve a high conversion efficiency, the high price of enzyme reagents becomes a major obstacle. Although the production of trehalose by genetic engineering method has the advantages of high efficiency, environmental protection, and strong scalability, it also faces many challenges such as complex technology and high cost.

[0004] In recent years, with the development of biotechnology, the microbial fermentation method has gradually become a more feasible way to produce trehalose. This method accumulates trehalose under suitable conditions by utilizing the metabolic ability of specific microorganisms, showing significant advantages. However, there is still a problem of low trehalose conversion rate in current practical applications, which cannot meet the market demand and poses a great challenge to the input cost of enterprises.

[0005] Therefore, there is an urgent need for a production process of trehalose catalyzed by a composite enzyme. Summary of the Invention

[0006] The purpose of the present invention is to provide a production process of trehalose catalyzed by a composite enzyme.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A production process of trehalose catalyzed by a composite enzyme, comprising the following steps:

[0009] (1) Inoculate Escherichia coli into LB medium for cultivation to obtain an Escherichia coli seed solution;

[0010] (2) Inoculate the Escherichia coli seed solution into TB medium for fermentation cultivation. When OD is measured 600When it is 1.0 - 1.2, isopropyl - β - D - thiogalactoside is added to the fermenter for induction for 6 - 7 h to obtain a fermentation broth;

[0011] The TB medium comprises the following components: peptone 14 - 16 g / L, yeast extract 24 - 27 g / L, glycerol 4 - 5 mL / L, a first inducer 3.0 - 5.2 g / L, a second inducer 0.06 - 0.09 g / L, potassium dihydrogen phosphate 2.3 - 2.4 g / L, and dipotassium hydrogen phosphate 0.2 - 0.4 g / L, and the balance is made up with water;

[0012] (3) Colistin sulfate is added to the fermentation broth and cultivation is continued. After cultivation is completed, the supernatant is discarded by centrifugation. After centrifugation, the supernatant is discarded, washed with phosphate buffer solution, and resuspended to obtain a suspension;

[0013] (4) The suspension is mixed with a maltose aqueous solution and reacted at 25 - 27 °C and 220 - 240 r / min for 15 - 17 h. After cultivation is completed, centrifugation is carried out and the supernatant is taken to obtain a product containing trehalose.

[0014] Further, the first inducer comprises lactose and L - arabinose with a weight ratio of 1:(1.2 - 1.4).

[0015] Further, the second inducer is pantothenic acid.

[0016] In the TB medium of the present invention, the first inducer and the second inducer are added simultaneously, which can improve the trehalose conversion rate. It is analyzed that the addition of the first inducer and the second inducer can play a synergistic role, can activate the expression of the trehalose synthase gene in Escherichia coli, and promote the synthesis of related enzymes such as trehalose synthase. Pantothenic acid has an effect on the biosynthesis of the phospholipid bilayer interface in the cell membrane of Escherichia coli, which helps to improve the number, affinity, and effective utilization rate of the recognition sites related to the first inducer on the cell membrane surface.

[0017] Further, the inoculation amount of the Escherichia coli seed liquid in the step (2) is 0.8 - 1.2% of the volume percentage of the TB medium.

[0018] Further, the fermentation culture conditions in the step (2) are: 35 - 38 °C, 220 - 250 rpm, and the ventilation volume is 7.0 - 7.5 L / min.

[0019] Further, the LB medium in the step (1) comprises the following components: peptone 10 - 12 g / L, yeast powder 1 - 2 g / L, NaCl 10 - 13 g / L; the pH value is adjusted to 7.3 - 7.6, and the balance is made up with water.

[0020] In the production process of the present invention, the decline in trehalose conversion rate can be improved under specific cell concentrations and inductions.

[0021] Further, the culture conditions in step (1) are: culturing at 35 - 38 °C for 12 - 13 h.

[0022] Further, isopropyl-β-D-thiogalactoside is added to a concentration of 0.7 - 0.9 mmol / L.

[0023] Further, the concentration of polymyxin sulfate in step (3) is 1.3 - 1.4 g / L.

[0024] Further, the continued culture time in step (3) is 70 - 80 min.

[0025] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0026] 1. In the TB medium of the present invention, the simultaneous addition of the first inducer and the second inducer can improve the trehalose conversion rate.

[0027] 2. In the production process of the present invention, the decline in trehalose conversion rate can be improved under specific cell concentrations and inductions. Specific Embodiments

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] All raw materials used in the following embodiments of the present invention are commercially available products:

[0030] Escherichia coli, preservation number CGMCC 4.1174, from the China General Microbiological Culture Collection Center.

[0031] Isopropyl-β-D-thiogalactoside, Zhangjiajie Hengkang Biopharmaceutical Co., Ltd.

[0032] Polymyxin sulfate, Hunan Yunbang Biotechnology Co., Ltd.

[0033] Example 1

[0034] This example provides a trehalose production process catalyzed by a composite enzyme, including the following steps:

[0035] (1) Inoculate Escherichia coli into LB medium and culture it at 37 °C for 13 h to obtain an Escherichia coli seed solution; the LB medium in step (1) includes the following components: peptone 11 g / L, yeast extract 1.5 g / L, NaCl 12 g / L; adjust the pH value to 7.5 and make up the balance with water.

[0036] (2) Inoculate the Escherichia coli seed solution into TB medium for fermentation culture, and the inoculation amount of the Escherichia coli seed solution is 1% of the volume percentage of the TB medium; the conditions for fermentation culture are: 37 °C, 230 rpm, aeration volume 7.2 L / min. When the measured OD 600 is 1.1, add isopropyl-β-D-thiogalactoside to the fermenter to make the concentration of isopropyl-β-D-thiogalactoside in the fermenter 0.9 mmol / L, and perform induction for 7 h to obtain a fermentation broth;

[0037] The TB medium includes the following components: peptone 15 g / L, yeast extract 25 g / L, glycerol 4.5 mL / L, first inducer 4 g / L, second inducer 0.08 g / L, potassium dihydrogen phosphate 2.3 g / L, and dipotassium hydrogen phosphate 0.2 g / L, and make up the balance with water; the first inducer includes lactose and L-arabinose with a weight ratio of 1:1.3. The second inducer is pantothenic acid.

[0038] (3) Add colistin sulfate to the fermentation broth until the concentration of colistin sulfate in the fermentation broth reaches 1.4 g / L, continue to culture for 75 min. After the culture is completed, centrifuge at 6500 r / min for 10 min, discard the supernatant, wash twice with phosphate buffer, and resuspend with phosphate buffer at pH 8.0 to obtain a suspension with a solid concentration of 10 wt%.

[0039] (4) Mix the suspension with a volume ratio of 3:10 and 30% (W / V) maltose aqueous solution, react at 26 °C and 230 r / min for 16 h. After the reaction is completed, terminate the reaction by heating in a boiling water bath for 10 min. After cooling, centrifuge at 5300 r / min for 10 min, and take the supernatant to obtain a product containing trehalose.

[0040] Example 2

[0041] This example provides a trehalose production process catalyzed by a composite enzyme, including the following steps:

[0042] (1) Inoculate Escherichia coli into LB medium and culture it at 38 °C for 12 h to obtain an Escherichia coli seed solution; the LB medium in step (1) includes the following components: peptone 12 g / L, yeast extract 1 g / L, NaCl 13 g / L; adjust the pH value to 7.3 and make up the balance with water.

[0043] (2) Inoculate the Escherichia coli seed liquid into the TB medium for fermentation culture. The inoculation amount of the Escherichia coli seed liquid is 1% of the volume percentage of the TB medium; the conditions for fermentation culture are: 38 °C, 250 rpm, aeration rate 7.0 L / min. When the measured OD 600 reaches 1.0, add isopropyl-β-D-thiogalactoside to the fermenter to make the concentration of isopropyl-β-D-thiogalactoside 0.9 mmol / L, and perform induction for 7 h to obtain the fermentation broth;

[0044] The TB medium comprises the following components: peptone 16 g / L, yeast extract 27 g / L, glycerol 4 mL / L, first inducer 5.2 g / L, second inducer 0.06 g / L, potassium dihydrogen phosphate 2.4 g / L, and dipotassium hydrogen phosphate 0.2 g / L. Make up the balance with water; the first inducer comprises lactose and L-arabinose with a weight ratio of 1:1.2. The second inducer is pantothenic acid.

[0045] (3) Add colistin sulfate to the fermentation broth until the concentration of colistin sulfate in the fermentation broth reaches 1.4 g / L, continue culturing for 70 min. After the culturing is completed, centrifuge at 6500 r / min for 10 min, discard the supernatant, wash twice with phosphate buffer solution, and resuspend with phosphate buffer solution at pH 8.0 to obtain a suspension with a solid concentration of 10 wt%;

[0046] (4) Mix the suspension with a volume ratio of 3:10 and 30% (W / V) maltose aqueous solution, react at 27 °C and 220 r / min for 17 h. After the reaction is completed, terminate the reaction by heating in a boiling water bath for 10 min. After cooling, centrifuge at 5300 r / min for 10 min, and take the supernatant to obtain a product containing trehalose.

[0047] Comparative Example 1

[0048] The difference between this comparative example and Example 1 is that: the first inducer and the second inducer are not added to the TB medium.

[0049] Comparative Example 2

[0050] The difference between this comparative example and Example 1 is that: the first inducer is lactose.

[0051] Comparative Example 3

[0052] The difference between this comparative example and Example 1 is that: the second inducer is not added to the TB medium.

[0053] Comparative Example 4

[0054] The difference between this comparative example and Example 1 is that the TB medium comprises the following components: peptone 20 g / L, yeast extract 20 g / L, glycerol 4.5 mL / L, a first inducer 2.5 g / L, a second inducer 0.12 g / L, potassium dihydrogen phosphate 2.3 g / L, and dipotassium hydrogen phosphate 0.2 g / L, and the balance is made up with water; the first inducer comprises lactose and L-arabinose in a weight ratio of 1:1.3. The second inducer is pantothenic acid.

[0055] Comparative Example 5

[0056] The difference between this comparative example and Example 1 is that when the OD 600 is measured to be 0.8, isopropyl-β-D-thiogalactoside is added to the fermenter to make the concentration of isopropyl-β-D-thiogalactoside 0.9 mmol / L, induction is carried out for 7 h, and a fermentation broth is obtained.

[0057] Comparative Example 6

[0058] The difference between this comparative example and Example 1 is that when the OD 600 is measured to be 1.3, isopropyl-β-D-thiogalactoside is added to the fermenter to make the concentration of isopropyl-β-D-thiogalactoside 0.9 mmol / L, induction is carried out for 7 h, and a fermentation broth is obtained.

[0059] Comparative Example 7

[0060] The difference between this comparative example and Example 1 is that the induction time is 5 h.

[0061] Comparative Example 8

[0062] The difference between this comparative example and Example 1 is that the induction time is 8 h.

[0063] Performance test

[0064] The trehalose conversion rates of the fermentation processes of Examples 1-2 and Comparative Examples 1-8 are measured, and the test method is carried out with reference to the method for measuring the trehalose conversion rate in "Optimization of Fermentation Conditions for the Production of Trehalose Synthase by Escherichia coli" by Yang Meiyu et al.

[0065] The results are shown in Table 1.

[0066] Table 1 Test results of trehalose conversion rate

[0067] Trehalose conversion rate % Example 1 77.58 Example 2 76.91 Comparative Example 1 70.68 Comparative Example 2 72.27 Comparative Example 3 74.05 Comparative Example 4 73.46 Comparative Example 5 68.13 Comparative Example 6 75.89 Comparative Example 7 69.34 Comparative Example 8 75.72

[0068] As can be seen from Table 1, the fermentation processes of Examples 1-2 have high trehalose conversion rates.

[0069] In Comparative Example 1, the first inducer and the second inducer were not added. In Comparative Example 2, the first inducer was only lactose. In Comparative Example 3, the second inducer was not added to the TB medium. All of these led to a decrease in the trehalose conversion rate to varying degrees. It was analyzed that the addition of the first inducer and the second inducer could play a synergistic role, activate the expression of target genes in Escherichia coli, and promote the synthesis of related enzymes such as trehalose synthase, thereby synergistically promoting the increase in the trehalose conversion rate.

[0070] In Comparative Example 4, the concentrations of the components in the TB medium were different, resulting in a decrease in the trehalose conversion rate.

[0071] In Comparative Examples 5 and 6, the OD 600 was different, and the trehalose conversion rate decreased, indicating that at the cell concentration of the present invention, the conversion of trehalose could be better achieved.

[0072] In Comparative Examples 7 and 8, the induction time was different, and the trehalose conversion rate decreased, indicating that at the induction time of the present invention, the conversion of trehalose could be better achieved.

[0073] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composite enzyme-catalyzed trehalose production process, characterized in that: The following steps are involved: (1) inoculating Escherichia coli into LB medium and culturing it to obtain an Escherichia coli seed liquid; (2) Inoculate the E. coli seed solution into TB medium for fermentation. When the OD 600 When the pH value is 1.0-1.2, isopropyl-β-D-thiogalactoside is added to the fermentation tank for induction for 6-7 hours to obtain a fermentation broth; The TB culture medium comprises the following components: 14-16 g / L of peptone, 24-27 g / L of yeast extract, 4-5 mL / L of glycerol, 3.0-5.2 g / L of a first inducer, 0.06-0.09 g / L of a second inducer, 2.3-2.4 g / L of potassium dihydrogen phosphate and 0.2-0.4 g / L of potassium hydrogen phosphate, and the balance is supplemented with water; (3) adding colistin sulfate to the fermentation broth, continuing the culture, centrifuging and discarding the supernatant after the culture is completed, discarding the supernatant after centrifugation, washing with phosphate buffer, and resuspending to obtain a suspension; (4) The suspension is mixed with a maltose aqueous solution, and cultured at 25-27° C. and 220-240 r / min for 15-17 h. After the culture is completed, the mixture is centrifuged and the supernatant is collected to obtain a product containing trehalose.

2. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: The first inducer comprises lactose and L-arabinose in a weight ratio of 1:(1.2-1.4).

3. The process for producing trehalose catalyzed by complex enzyme according to claim 2, characterized in that: The second inducer is pantothenic acid.

4. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: In the step (2), the inoculation amount of the E. coli seed solution is 0.8-1.2% of the volume percentage of the TB culture medium.

5. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: The fermentation culture conditions in step (2) are: 35-38°C, 220-250rpm, and aeration volume of 7.0-7.5L / min.

6. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: In step (1), the LB culture medium comprises the following components: 10-12 g / L peptone, 1-2 g / L yeast powder, and 10-13 g / L NaCl; the pH value is adjusted to 7.3-7.6, and the balance is supplemented with water.

7. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: The culture conditions in step (1) are: culture at 35-38° C. for 12-13 hours.

8. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: Isopropyl-β-D-thiogalactoside was added to a concentration of 0.7-0.9 mmoL / L.

9. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: The concentration of colistin sulfate in step (3) is 1.3-1.4 g / L.

10. The process for producing trehalose catalyzed by complex enzyme according to claim 1, characterized in that: The culture time in step (3) is continued for 70-80 minutes.