Method for preserving fermentation broth of engineered bacteria producing D-allulose-3-epimerase

Abstract

To provide a method for preserving a fermentation broth of D-allulose-3-isomerase engineered bacteria.SOLUTION: A method for preserving a fermentation broth of D-allulose-3-isomerase engineered bacteria includes the steps for: adding a lysozyme into a fermentation broth of D-allulose-3-isomerase engineered bacteria, stirring to make the OD600 of the fermentation broth less than or equal to 1 to obtain an enzyme solution; and adjusting the enzyme solution to be weakly alkaline with an alkali solution, and storing the weakly alkaline enzyme solution at room temperature.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for preserving the fermentation broth of D-allose-3-isomerase engineered bacteria.

Background Art

[0002] D-allose is a rare sugar and an epimer of D-fructose. Its sweetness is equivalent to 70% of sucrose, but its calorie content is only 0.3% of sucrose, making it an excellent substitute for sucrose. According to research, D-allose can inhibit the absorption of fructose and glucose in the body, reduce the amount of fat accumulation, and reduce the risk of diseases such as type II diabetes and obesity. Currently, D-allose is expected to have a wide range of applications in food and healthcare products.

[0003] The industrial preparation method of D-allose is mainly based on the biocatalytic method, that is, using fructose as a substrate and D-allose-3-isomerase to convert fructose into D-allose. Most of the D-allose-3-isomerase is obtained by large-scale fermentation culture of engineered bacteria. The release of enzymes in the fermentation broth of D-allose-3-isomerase and the preservation of the enzyme solution are very important for the industrial production of D-allose.

[0004] The conventional treatment and preservation process of the fermentation broth of D-allose-3-isomerase involves centrifugation, redissolution, low-temperature homogenization, secondary centrifugation, and then preservation under low-temperature conditions (generally 4°C). However, as the preservation time lengthens, the inactivation of D-allose-3-isomerase becomes serious, and after 15 days of low-temperature preservation, the enzyme solution begins to emit an odor (the enzyme activity decreases), increasing the production cost of D-allose for enterprises.

[0005] Currently, there have also been reports related to improving the preservation effect of D-allulose-3-epimerase solution and ensuring enzyme activity. For example, in Patent Document 1 (Chinese Patent Application Publication No. 115074350), a method for suppressing the decrease in the enzyme activity of D-allulose 3-epimerase solution is disclosed. This method involves adding MgSO 4 or Na 2 CO 3 to the supernatant (i.e., the enzyme solution) after secondary centrifugation and storing it at a low temperature (i.e., 4°C). This method can extend the storage time of the enzyme solution to a certain extent, but the treatment steps of the fermentation broth are still very complex. During the storage process, the enzyme solution still becomes smelly (the enzyme activity decreases), and it is necessary to store the enzyme solution at a low temperature of 4°C. When the amount of the enzyme solution obtained by scale culture is large, enterprises not only need a large number of freezers to store the enzyme solution, but also the electricity energy consumption during the storage process is large.

[0006] From the above, how to solve the problems that the treatment steps of the fermentation broth of D-allulose-3-epimerase are complicated and the storage cost of the enzyme solution is high has great significance for the industrial production of D-allulose.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] In view of the above, the present invention provides a method for storing the fermentation broth of D-allulose-3-epimerase engineering bacteria. The treatment steps of this method are simple, not only ensuring the enzyme activity during the storage process, but also realizing the long-term storage of D-allulose-3-epimerase solution under room temperature conditions, reducing not only the storage cost of the enzyme solution but also the production cost of D-allulose.

Means for Solving the Problem

[0009] In order to achieve the above object, the present invention adopts the following technical solutions.

[0010] The present invention provides a method for storing a fermentation broth of D-allose-3-isomerase engineering bacteria. The storage method includes the steps of adding lysozyme to the fermentation broth of D-allose-3-isomerase engineering bacteria, stirring to obtain an enzyme solution such that the fermentation broth has an OD600 ≤ 1, and adjusting the enzyme solution to a weakly alkaline condition with an alkaline solution and storing the weakly alkaline enzyme solution at room temperature.

[0011] In the present invention, after the fermentation of the engineering bacteria is completed, lysozyme is added to obtain an enzyme solution, and then the enzyme solution is adjusted to a weakly alkaline condition, so that it can be stored at room temperature for a long time, and the storage time can be up to 60 days or more. This method is simple and reliable, and can simplify the processing steps of the fermentation broth of the engineering bacteria.

[0012] In the present invention, by adding lysozyme to the fermentation broth, the fermented engineering bacteria are lysed, and D-allose-3-isomerase in the engineering bacteria cells is released. On the other hand, during the storage process, lysozyme can avoid the degradation of D-allose-3-isomerase by protein enzymes secreted by surviving engineering bacteria, and can also avoid the degradation of D-allose-3-isomerase by protein enzymes secreted due to the growth of other microorganisms during the storage process.

[0013] In the present invention, by storing the obtained enzyme solution at room temperature under weakly alkaline conditions, the growth of microorganisms in the enzyme solution is suppressed, and during the storage process, situations such as the enzyme solution becoming smelly and the enzyme activity decreasing severely can be avoided as much as possible. On the other hand, room temperature storage eliminates the need for low-temperature storage equipment such as refrigerators, and not only reduces the number of refrigerators but also reduces the power consumption.

[0014] In a preferred embodiment of the present invention, the concentration of the lysozyme is ≧0.06 g / L. That is, at least 0.06 g of lysozyme is added to 1 L of the fermentation broth, and the engineered bacteria after fermentation can be dissolved as much as possible.

[0015] In a more preferred embodiment of the present invention, the concentration of the lysozyme is 0.06 - 0.2 g / L. That is, the amount of lysozyme used in 1 L of the fermentation broth is preferably 0.06 - 0.2 g. More preferably, the concentration of the lysozyme is 0.1 g / L.

[0016] In a preferred embodiment of the present invention, the alkaline solution for adjusting the enzyme solution to be weakly alkaline with the above alkaline solution is a sodium hydroxide solution, a potassium hydroxide solution, a disodium hydrogen phosphate solution or a dipotassium hydrogen phosphate solution, and the pH of the enzyme solution adjusted with the alkaline solution is 7.5 - 8.5. In the present invention, when adjusting the pH of the enzyme solution, not only a strong alkaline solution (for example, a sodium hydroxide solution generally used in experiments), but also a weak alkaline solution such as a disodium hydrogen phosphate solution or a dipotassium hydrogen phosphate solution can be used, and the alkaline solution can be flexibly selected according to the actual situation. Furthermore, in the present invention, by adjusting the pH of the enzyme solution to 7.5 - 8.5, the growth of microorganisms in the enzyme solution can be effectively suppressed, and during the storage process, situations such as the enzyme solution becoming smelly and the enzyme activity decreasing severely can be prevented. More preferably, the alkaline solution is a sodium hydroxide solution, and the pH of the enzyme solution adjusted with the alkaline solution is 8.0.

[0017] In a preferred embodiment of the present invention, when storing the weakly alkaline enzyme solution at room temperature, the room temperature is ≦30°C. The indoor temperature in spring and autumn is generally 20°C, the indoor temperature in winter is generally within 10°C, and the indoor temperature in summer is generally 30°C or lower. The storage method of the present invention can store the fermentation broth of D-allose-3-isomerase engineering bacteria at room temperature throughout the year, not only ensuring the enzyme activity of the enzyme solution and avoiding odors, but also reducing power consumption, reducing the industrial production cost of D-allose, and having great significance for the revitalization of D-allose production enterprises.

[0018] Compared with the prior art (i.e., centrifugation + homogenization + centrifugation + storage at 4°C), the present invention has the following advantages.

[0019] After the fermentation of the engineering bacteria is completed in the present invention, a certain proportion of lysozyme is added to obtain an enzyme solution, and then the enzyme solution is adjusted to be weakly alkaline and can be stored at room temperature for a long time. The steps such as the conventional two centrifugations and high-pressure homogenization can be completely omitted, making the treatment and storage of the fermentation broth simpler and more convenient. The enzyme solution treated by the present invention can be stored at room temperature for more than 60 days without emitting odors, and the reduction rate of enzyme activity after being stored at room temperature for 60 days is suppressed within 15% (when stored at room temperature in winter for 60 days, the enzyme activity of the enzyme solution basically does not decrease), which can meet the industrial production requirements of D-allose for enterprises.

[0020] By adding lysozyme to the fermentation broth in the present invention, the fermented engineering bacteria are lysed, and the D-allose-3-isomerase in the engineering bacteria cells is released. During the storage process, lysozyme can avoid the decomposition of D-allose-3-isomerase by the protein enzymes secreted by the surviving engineering bacteria, and can suppress the decomposition of D-allose-3-isomerase by the protein enzymes secreted by the growth of other microorganisms.

[0021] By storing the obtained enzyme solution at room temperature under weakly alkaline conditions, the growth of microorganisms in the enzyme solution can be suppressed, and during the storage process, situations such as the enzyme solution becoming smelly and the enzyme activity significantly decreasing can be avoided as much as possible. On the other hand, room temperature storage eliminates the need for low-temperature storage equipment such as refrigerators, not only reducing the number of refrigerators but also reducing power consumption.

[0022] Taking the storage of 4000L of enzyme solution as an example, under the conventional 4°C storage condition, when storing 4000L of enzyme solution, 20 refrigerators with a capacity of 300L are required. The daily power consumption of a refrigerator is 1kWh, and the total power consumption for 60 days of low-temperature storage is 1200kWh. When operating for 300 days a year, the annual power consumption is 36,000kWh (i.e., 36,000 degrees). The present invention can be stored at room temperature throughout the year, completely eliminating the need for refrigerators, saving at least the equipment investment of 20 refrigerators, with a remarkable energy-saving effect (able to save at least 36,000 degrees of electricity annually), reducing the production cost of enterprises, and having great significance for wide popularization.

Embodiments for Carrying out the Invention

[0023] Hereinafter, the present invention will be described in detail with reference to specific examples. Among them, unless otherwise specified, all the equipment used in the present invention is ordinary experimental equipment, and the reagents used are ordinary reagents.

[0024] In addition, the enzyme activity in each embodiment of the present invention is measured by the following method: a fructose solution with a mass fraction of 1% (the fructose solution is prepared using a potassium phosphate buffer with pH = 8.0, and the potassium phosphate buffer contains 1mM of Mn 2+Using (containing) it as a substrate, an appropriate proportion of enzyme solution is added and reacted at 60°C for 10 minutes. After the reaction is completed, it is treated in a boiling water bath for 5 minutes, and the content of the product D-allose is measured using HPLC. Based on the measured content of D-allose and the reaction time (i.e., 10 minutes), the enzyme activity of the D-allose-3-isomerase solution is calculated. The enzyme activity in the present invention is defined as 1 unit (U) of enzyme activity when 1 μmol of D-allose is produced per minute at pH 8.0 and 60°C.

[0025] In addition, the D-allose-3-isomerase engineering bacteria mentioned in each example of the present invention are all recombinant Bacillus subtilis engineering bacteria B-3-1 (this strain is disclosed in Chinese Invention Patent CN202010496928.9, and the invention name of this patent is "Allose 3-isomerase mutant, engineering bacteria expressing the mutant, and application"). In addition, the fermentation broth of the D-allose-3-isomerase engineering bacteria used in each example of the present invention is fermented by the following steps.

[0026] Step S1: Preparation of the medium (including the seed medium, fermentation medium, and fed-batch medium). Seed medium: 10 g / L of peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, sterilized at 121°C for 20 minutes, and then kanamycin is added to 50 mg / L under aseptic conditions. Fermentation medium: 10 g / L of peptone, 5 g / L of yeast powder, 2.5 g / L of potassium dihydrogen phosphate, 15 g / L of dipotassium hydrogen phosphate, 0.1 g / L of manganese chloride tetrahydrate, 6 g / L of glucose, sterilized at 121°C for 20 minutes. Fed-batch medium: 50% by mass of glucose, sterilized at 121°C for 20 minutes.

[0027] - Step S2: Obtaining a seed solution of D-allose-3-isomerase engineering bacteria using the seed medium. Recombinant Bacillus subtilis engineering bacteria B-3-1 are inoculated into the seed medium and cultured at 37°C and 200 rpm for 14 hours to obtain a seed solution of D-allose-3-isomerase engineering bacteria.

[0028] Step S3: Ferment and culture the inoculum to obtain a fermentation broth of D-allose-3-isomerase engineering bacteria. The inoculum is inoculated into the fermentation medium at a volume ratio of 0.1% (that is, 0.1% ml of the inoculum is inoculated into 1 ml of the fermentation medium), and fermentation culture is carried out. The fermentation culture conditions are: fermentation temperature 37 °C, initial stirring speed 200 rpm, aeration rate 1 vvm. During the fermentation process, the stirring speed is adjusted so that the dissolved oxygen is always 10% or more according to the dissolved oxygen. During the fermentation process, a fed-batch medium is supplied, sampled, and the OD600 value is detected. When the OD600 value stops increasing, the fermentation ends, and a fermentation broth of D-allose-3-isomerase engineering bacteria is obtained.

Example

[0029] 〔Example 1〕 After the fermentation of D-allose-3-isomerase engineering bacteria is completed (that is, after the OD600 value of the fermentation broth stops increasing), aeration is stopped, and lysozyme is added at a ratio of 0.1 g / L (that is, the concentration of lysozyme in the fermentation broth is 0.1 g / L), and stirring is carried out until the OD600 value of the fermentation broth ≤ 1, lysing the recombinant Bacillus subtilis engineering bacteria B-3-1 to obtain an enzyme solution of D-allose-3-isomerase. Next, the pH of the enzyme solution is adjusted to 8.0 with NaOH solution, stored at 20 °C (room temperature in spring and autumn), sampled every 10 days, and the enzyme activity is detected.

[0030] 〔Example 2〕 After the fermentation of D-allose-3-isomerase engineering bacteria is completed (that is, when the OD600 value of the fermentation broth stops increasing), aeration is stopped, and lysozyme is added at a ratio of 0.1 g / L (that is, the concentration of lysozyme in the fermentation broth is 0.1 g / L), and stirring is carried out until the OD600 value of the fermentation broth ≤ 1, lysing the recombinant Bacillus subtilis engineering bacteria B-3-1 to obtain an enzyme solution of D-allose-3-isomerase. Next, the pH of the enzyme solution is adjusted to 8.0 with NaOH solution, stored at 30 °C (room temperature in summer), sampled every 10 days, and the enzyme activity is detected.

[0031] 〔Example 3〕 After the fermentation of the engineered bacteria expressing D-allulose-3-epimerase is completed (i.e., the OD600 value of the fermentation broth no longer increases), aeration is stopped, and lysozyme is added at a rate of 0.1 g / L (i.e., the concentration of lysozyme in the fermentation broth is 0.1 g / L). Stir the fermentation broth until the OD600 value of the fermentation broth ≤ 1 to lyse the recombinant Bacillus subtilis engineered bacteria B-3-1 to obtain an enzyme solution of D-allulose-3-epimerase. Next, adjust the pH of the enzyme solution to 8.0 with NaOH solution and store it at 10°C (room temperature in winter), and sample every 10 days to detect the enzyme activity.

[0032] [Example 4] After the fermentation of the engineered bacteria expressing D-allulose-3-epimerase is completed (i.e., the OD600 value of the fermentation broth no longer increases), aeration is stopped, and lysozyme is added at a rate of 0.06 g / L (i.e., the concentration of lysozyme in the fermentation broth is 0.06 g / L). Stir the fermentation broth until the OD600 value of the fermentation broth ≤ 1 to lyse the recombinant Bacillus subtilis engineered bacteria B-3-1 to obtain an enzyme solution of D-allulose-3-epimerase. Next, adjust the pH of the enzyme solution to 8.0 with NaOH solution and store it at 30°C (room temperature in summer), and sample every 10 days to detect the enzyme activity.

[0033] [Example 5] After the fermentation of the engineered bacteria expressing D-allulose-3-epimerase is completed (i.e., the OD600 value of the fermentation broth no longer increases), aeration is stopped, and lysozyme is added at a rate of 0.15 g / L (i.e., the concentration of lysozyme in the fermentation broth is 0.15 g / L). Stir the fermentation broth until the OD600 value of the fermentation broth ≤ 1 to lyse the recombinant Bacillus subtilis engineered bacteria B-3-1 to obtain an enzyme solution of D-allulose-3-epimerase. Next, adjust the pH of the enzyme solution to 8.0 with NaOH solution and store it at 30°C (room temperature in summer), and sample every 10 days to detect the enzyme activity.

[0034] [Example 6] After the fermentation of the engineered bacteria of D-allulose-3-isomerase is completed (i.e., when the OD600 value of the fermentation broth no longer increases), aeration is stopped, and lysozyme is added at a ratio of 0.1 g / L (i.e., the lysozyme concentration in the fermentation broth is 0.1 g / L). Stir until the OD600 value of the fermentation broth ≤ 1 to lyse the recombinant Bacillus subtilis engineered bacteria B-3-1 to obtain an enzyme solution of D-allulose-3-isomerase. Next, adjust the pH of the enzyme solution to 7.5 with NaOH solution, store it at 30°C (room temperature in summer), sample it every 10 days, and detect the enzyme activity.

[0035] Example 7 After the fermentation of the engineered bacteria of D-allulose-3-isomerase is completed (i.e., when the OD600 value of the fermentation broth no longer increases), aeration is stopped, and lysozyme is added at a ratio of 0.1 g / L (i.e., the lysozyme concentration in the fermentation broth is 0.1 g / L). Stir until the OD600 value of the fermentation broth ≤ 1 to lyse the recombinant Bacillus subtilis engineered bacteria B-3-1 to obtain an enzyme solution of D-allulose-3-isomerase. Next, adjust the pH of the enzyme solution to 8.5 with NaOH solution, store it at 30°C (room temperature in summer), sample it every 10 days, and detect the enzyme activity.

[0036] Comparative Example 1 After the fermentation of the engineered bacteria of D-allulose-3-isomerase is completed (i.e., when the OD600 value of the fermentation broth no longer increases), aeration is stopped, and lysozyme is added at a ratio of 0.1 g / L (i.e., the concentration of lysozyme in the fermentation broth is 0.1 g / L). Stir until the OD600 value of the fermentation broth ≤ 1 to lyse the recombinant Bacillus subtilis engineered bacteria B-3-1 to obtain an enzyme solution of D-allulose-3-isomerase. Next, adjust the pH of the enzyme solution to 8.0 with NaOH solution, store it at 4°C, sample it for 10 days, and detect the enzyme activity.

[0037] Comparative Example 2 Centrifugation + Redissolution + Homogenization + Refrigeration at 4°C The fermentation broth of D-allulose-3-epimerase engineered bacteria was centrifuged at 5000 rpm for 30 minutes, and the supernatant was removed to obtain the fermented bacterial cells. The fermented bacterial cells were redissolved in an equal volume of potassium phosphate buffer at pH = 8.0 (i.e., the volume after redissolution was equal to the volume of the original fermentation broth of D-allulose-3-epimerase), and then homogenized with a homogenizer to release the intracellular bacterial cells. The homogenizer operated at a temperature of 4°C and an operating pressure of 800 bar. After homogenization, it was centrifuged at 10000 rpm for 20 minutes, and the obtained supernatant was stored at 4°C (low-temperature refrigeration), and sampled every 10 days to detect the enzyme activity.

[0038] Comparative Example 3: In this comparative example, the pH of the enzyme solution was not adjusted. After the fermentation of D-allulose-3-epimerase engineered bacteria was completed (i.e., when the OD600 value of the fermentation broth no longer increased), aeration was stopped, and lysozyme was added at a ratio of 0.1 g / L (i.e., the concentration of lysozyme in the fermentation broth was 0.1 g / L). The mixture was stirred until the OD600 value of the fermentation broth ≤ 1, and the recombinant Bacillus subtilis engineered bacteria B-3-1 were lysed to obtain the enzyme solution of D-allulose-3-epimerase. Then, it was stored at 30°C and sampled for 10 days to detect the enzyme activity.

[0039] The enzyme activities of the above Examples 1 to 7 and Comparative Examples 1 to 3 are shown in Table 1.

[0040]

Table 1

[0041] As can be seen from Table 1, after treating the fermentation broth using the conventional centrifugation + high-pressure homogenization method (i.e., Comparative Example 2), even when stored at a low temperature of 4°C, with the extension of the storage time, the decrease in enzyme activity was serious. The enzyme activity on the 60th day was 330, and the decrease in enzyme activity was more than 22%. The decrease in enzyme activity was large. From the 15th day, the enzyme solution began to emit a pungent odor, which affected the use of this enzyme solution. Also, in Comparative Example 3, the enzyme activity on the 60th day was 341, and the decrease in enzyme activity was as high as 20%. It smelled sour from the 20th day.

[0042] As can be seen by comparing Examples 1 to 7 of the present invention with Comparative Example 1, when compared with refrigeration at 4°C, although the enzyme activity of Comparative Example 1 is relatively high, refrigeration in a freezer is still required. In contrast, in Examples 1 to 7 of the present invention, there is no sour odor even after 60 days of storage at room temperature. Even when the storage temperature is 30°C, the enzyme solution on the 60th day still has very good enzyme activity (91.0% of the initial enzyme activity), the decrease in enzyme activity is small, the enzyme solution has no sour odor, and when the room temperature is 10°C, its enzyme activity is basically the same as that under the condition of refrigeration at 4°C. That is, the storage method of the present invention does not require refrigeration in a freezer, can completely achieve the storage of the enzyme solution at room temperature, and has a remarkable energy-saving effect.

[0043] Since none of Examples 1 to 7 of the present invention showed a pungent odor during the storage process, the storage method of the present invention can avoid microorganisms that appear in the enzyme solution even under room temperature conditions, and can ensure the enzyme activity and quality of the enzyme solution. Also, as can be seen from Examples 1 to 7, the enzyme activity after 60 days of storage by the storage method of the present invention is 89.4% to 95.3% of the initial enzyme activity. The present invention can achieve the storage of D-allose-3-isomerase solution at room temperature, can not only avoid the generation of sour odor due to the growth of microorganisms during the storage process, but also ensure the enzyme activity.

[0044] The above examples are only preferred embodiments of the present invention, and the embodiments of the present invention are not limited by the above examples. Any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be included in the protection scope of the present invention as equivalent substitutions.

Industrial Applicability

[0045] The present invention relates to the field of biotechnology, and in particular, can be used in a method for storing a fermentation broth of D-allose-3-isomerase engineering bacteria.

Claims

1. Adding lysozyme to the fermentation broth of D-allulose-3-epimerase engineered bacteria at a concentration of 0.06 to 0.15 g / L, stirring the fermentation broth so that OD600 ≤ 1 to obtain an enzyme solution, adjusting the enzyme solution to a weakly alkaline pH of 7.5 to 8.5 with an alkaline solution, and storing the weakly alkaline enzyme solution under room temperature conditions in the presence of lysozyme. A method for storing the fermentation broth of D-allulose-3-epimerase engineered bacteria, characterized by comprising the above steps.

2. The method for storing the fermentation broth of D-allulose-3-epimerase engineered bacteria according to Claim 1, wherein the concentration of the lysozyme is 0.1 g / L.

3. The alkaline solution for adjusting the enzyme solution to a weakly alkaline state with the above alkaline solution is a sodium hydroxide solution, a potassium hydroxide solution, a disodium hydrogen phosphate solution, or a dipotassium hydrogen phosphate solution. A method for storing the fermentation broth of D-allulose-3-epimerase engineered bacteria according to Claim 1, characterized by this.

4. The alkaline solution is a sodium hydroxide solution, and the pH of the enzyme solution adjusted with the alkaline solution is 8.

0. A method for storing the fermentation broth of D-allulose-3-epimerase engineered bacteria according to Claim 3, characterized by this.

5. When storing the above weakly alkaline enzyme solution under room temperature conditions, the room temperature is ≤ 30°C. A method for storing the fermentation broth of D-allulose-3-epimerase engineered bacteria according to Claim 1, characterized by this.

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