A pyruvate oxidase preparation and its industrial production method and application

Abstract

The invention discloses a pyruvate oxidase preparation and an industrial production method and application thereof. First, a strain containing pyruvate oxidase is expanded and cultured for fermentation, lactose is added for induction, and then vitamin B1 and vitamin B2 solutions are added, and fermentation is continued for 26 hours, and then magnesium sulfate solution is added and fermentation is continued until the bacterial liquid concentration is greater than 120g / L to prepare a fermentation liquid, and the fermentation liquid is washed and then a nuclease is added for homogenization treatment, and then a flocculant is added to the treated liquid, and the mixture is stirred and filtered, and the filtrate is concentrated to obtain a pyruvate oxidase preparation solution. In the present invention, vitamin B1 and vitamin B2 are added after lactose induction during the fermentation process, and the time for adding magnesium sulfate is changed to after 26 hours after fermentation, so that the enzyme activity of the prepared pyruvate oxidase preparation is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a pyruvate oxidase preparation and an industrial production method and application thereof. Background Art

[0002] Pyruvate oxidase is a thiaminepyrophosphate (TPP)-dependent oxidase. When inorganic phosphate and oxygen molecules are present, pyruvate oxidase can catalyze the oxidative decarboxylation of pyruvate to generate acetyl phosphate, CO2 and H2O2 with the participation of coenzymes TPP, FAD and Mg2+. Under the joint action of peroxidase, pyruvate oxidase can be used to determine alanine aminotransferase and aspartate aminotransferase in human blood. At the same time, it can also be used to determine the phosphate ion content in river water and wastewater. As an enzyme biocatalyst, pyruvate oxidase plays a very important role in biochemical tests, including clinical biochemical tests, environmental testing and biosensors, and is used for real-time online detection of pyruvate, an important intermediate product in fermentation production, and the pyruvate content in blood.

[0003] The industrial production of pyruvate oxidase enzyme preparations involves a multi-coenzyme and cofactor system. After the fermentation medium has been steam sterilized for a long time, the organic nutrients are lost significantly. This makes it impossible to determine whether the fermentation process can meet the nutritional requirements for coenzyme synthesis, causing the problem of unstable fermentation activity. When the fermentation tank is released and entered into the post-processing stage, the coenzymes and cofactors are easily lost, resulting in a low overall yield. Summary of the invention

[0004] In order to overcome the technical problem of low overall yield of pyruvate oxidase enzyme preparation in industrial production in the prior art, the present invention provides a pyruvate oxidase preparation and an industrial production method and application thereof. During the fermentation process, vitamin B1 and vitamin B2 are added after lactose induction, and the time for adding magnesium sulfate is changed to 26 hours after fermentation, so that the enzyme activity of the prepared pyruvate oxidase preparation is greatly improved.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0006] The present invention provides an industrial production method of a pyruvate oxidase preparation, comprising the following steps:

[0007] S1. Expand the culture of the bacteria containing pyruvate oxidase, add glucose when the bacterial solution concentration is 35-40g / L, ferment until the bacterial solution concentration is 45-55g / L, add lactose for induction after cooling, and then add vitamin B1 and vitamin B2 solutions;

[0008] S2, fermenting the bacterial solution in step S1 for 26 hours, adding a divalent metal ion solution and continuing the fermentation until the bacterial solution concentration is greater than 120 g / L, to prepare a fermentation solution;

[0009] S3, washing the fermentation liquid in step S3, adding nuclease to homogenize, adding nuclease again to the homogenized liquid, heating to 50±1°C, and heat preservation to obtain a treated liquid, then adding a flocculant to the treated liquid, stirring evenly, and filtering;

[0010] S4. Concentrating the filtrate obtained in step S3 to obtain a pyruvate oxidase preparation solution.

[0011] According to the present invention, after adding lactose for induction during bacterial liquid fermentation, adding vitamin B1 and vitamin B2 can improve the activity of pyruvate oxidase. The reason is that in the initial stage of induction, the bacterial concentration is not high and the expression level of pyruvate oxidase protein is high. At this time, adding auxiliary factors vitamin B1 and vitamin B2 can increase the binding amount of target protein and improve the activity of pyruvate oxidase.

[0012] Divalent metal ions are consumed in the process of microbial metabolism and are used as enzyme activators. In the present invention, they are added after the bacterial solution has been fermented for 26 hours to avoid adding them too early to affect the binding of divalent metal ions, and adding them too late to stop fermentation after the bacteria have aged in the late stage, resulting in insufficient binding time for divalent metal ions.

[0013] In step S3, nuclease is added before homogenizing the cells to reduce the viscosity of the fermentation liquid and improve the subsequent homogenization efficiency. Nuclease is added after homogenization to decompose nucleic acids to reduce the viscosity of the material, which is conducive to flocculation. Flocculants are added to condense broken cell fragments and some small particles into larger particles, which is conducive to improving the effect of solid-liquid separation.

[0014] As an optional embodiment, in the industrial production method provided by the present invention, the amount of vitamin B1 added in step S1 is 0.03-0.07% of the volume of the fermentation broth, and the amount of vitamin B2 added is 0.01-0.04% of the volume of the fermentation broth, wherein the vitamin B1 solution and the vitamin B2 solution are prepared using sodium chloride solution.

[0015] As an optional embodiment, in the industrial production method provided by the present invention, the divalent metal ions in step S2 are magnesium ions, and the final concentration of the added divalent metal ions is 0.05-0.2%.

[0016] In the present invention, the concentration of divalent metal ions is limited within the above range, which can improve the enzyme activity of pyruvate oxidase. After the concentration continues to increase, the enzyme activity is not significantly improved. However, the salt concentration of the culture medium is already the optimal ratio at the beginning of the process. Increasing the concentration of divalent metal ions will change the conductivity value of the fermentation broth and increase the impurity content in the final pyruvate oxidase preparation.

[0017] As an optional embodiment, in the industrial production method provided by the present invention, the amount of nuclease added before homogenization in step S3 is 2-4 g per ton of fermentation broth, and the amount of nuclease added after homogenization is 4-6 g per ton of fermentation broth.

[0018] As an optional embodiment, in the industrial production method provided by the present invention, the pH is maintained at 6.0-6.2 during the heat preservation in step S3.

[0019] During the insulation process, the pH was maintained in the range of 6.0 to 6.2, and the enzymatic activity of pyruvate oxidase was most stable.

[0020] As an optional embodiment, in the industrial production method provided by the present invention, the flocculant in step S3 includes chitosan and sodium polyacrylate, the chitosan is added in an amount of 12-25% of the mass of the treated liquid, and the sodium polyacrylate is added in an amount of 8-16% of the mass of the treated liquid.

[0021] The present invention uses a composite flocculant, and compared with the single flocculant chitosan in the prior art, the time and pressure of the plate and frame filtration are reduced, and the yield and filtration efficiency of the pyruvate oxidase solution are improved.

[0022] As an optional embodiment, in the industrial production method provided by the present invention, the enzyme activity of the pyruvate oxidase preparation solution after concentration in step S4 is maintained at ≥200 U / mL.

[0023] As an optional embodiment, in the industrial production method provided by the present invention, the pH of the pyruvate oxidase preparation obtained in step S4 is adjusted to 7.6-7.8, filtered, and then added with 43-45% v / v glycerol and stored at -5--10°C.

[0024] In the present invention, when the pyruvate oxidase preparation is stored, after the enzyme liquid activity is concentrated to more than 200 U / mL, the glycerol concentration is adjusted from the existing 30% to the range of 43-45%, thereby increasing the low-temperature storage time of the enzyme liquid. After being placed for three months, the enzyme liquid is still clear, and the enzyme activity loss is basically no more than 10%, which will not affect the use of the pyruvate oxidase preparation in the later stage.

[0025] The second aspect of the present invention provides the use of the above industrial production method in the production of pyruvate oxidase preparations.

[0026] The third aspect of the present invention provides a pyruvate oxidase preparation, which is produced by the above-mentioned industrial production method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) In the industrial production method of the pyruvate oxidase preparation described in the present invention, vitamin B1 and vitamin B2 are added after lactose induction during the fermentation process, and the time for adding magnesium sulfate is changed to 26 hours after fermentation, so that the enzyme activity of the prepared pyruvate oxidase preparation is greatly improved.

[0029] (2) In the present invention, chitosan and sodium polyacrylate are used as flocculants in the flocculation stage, and the flocs have good formability, so that the plate and frame rapid filtration speed is fast, the solid-liquid separation is thorough, the supernatant obtained after separation is clear, and the yield of pyruvate oxidase preparation obtained from different batches is stable.

[0030] (3) The prepared pyruvate oxidase preparation solution was aseptically treated and then stored by adding 43-45% glycerol, which could meet the low-temperature storage requirements. After three months of storage, the enzyme solution did not become turbid, and the enzyme activity loss was basically no more than 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a pyruvate oxidase preparation production process flow chart;

[0032] Figure 2 The single tank capacity and fermentation tank activity results in Examples 1-8;

[0033] Figure 3 The results of comparing the activity of the samples before and after adding magnesium sulfate in Example 1-8;

[0034] Figure 4 The results are a comparison of the flocculation yield and the total extraction yield in Examples 1-8. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] Example 1

[0039] Industrial production method of pyruvate oxidase preparation, such as Figure 1 shown.

[0040] 1. Fermentation technology route

[0041] 1.1. Bacteria activation

[0042] Use 100mL of LB medium as the medium for strain activation, including 0.5g of yeast powder, 1g of peptone, 1g of sodium chloride, 3mg of kanamycin sulfate, and adjust the pH to 7.2 with NaOH. Take out the glycerol freeze-dried tube of Escherichia coli containing pyruvate oxidase stored at -80℃, and inoculate it into 100mL of LB medium (8 bottles in total) at a ratio of 1:100 to 1:200 after natural thawing. Put the shaking flask into the shaking incubator for culture at 37℃ and 220rpm. Cultivate for about 12h to obtain a qualified seed solution with an OD600 of 3-5.

[0043] 1.2 Seed cultivation

[0044] The seed culture medium formula is: yeast extract powder: 24g / L, peptone: 12g / L, glycerol: 5g / L, polyether defoamer: 0.75mL / L, prepare 350L. After mixing the ingredients according to the above formula, adjust the pH with liquid alkali so that the pH after sterilization is 7.0±0.2, and steam sterilize at 121℃ for 30min. The initial fermentation conditions are: temperature 37℃, tank pressure 0.03±0.01MPa, rotation speed 330rpm, air flow 20±2m 3 / h. Take qualified seed liquid (not less than 400mL) and inoculate it into the seed tank. When the dissolved oxygen is lower than 35%, gradually increase the stirring to the highest level, then gradually increase the air flow to the maximum, and finally increase the tank pressure to 0.05MPa to accelerate the growth of bacteria. After 4-6 hours of cultivation in the seed tank, take samples for bacterial inspection and measure the bacterial concentration. When the bacterial concentration is ≥15g / L and the bacterial inspection is normal, the seeds can be transplanted to the fermentation tank.

[0045] 1.3 Production of large tank secondary fermentation

[0046] 1.3.1 Preparation and sterilization of large tank fermentation medium

[0047] The fermentation tank is a modified fermentation formula with a batching volume of 9T, including 90Kg of glycerol, 22.5Kg of yeast extract powder, 36Kg of peptone, 135Kg of dipotassium hydrogen phosphate, 67.5Kg of potassium dihydrogen phosphate, 18Kg of citric acid, 90Kg of ammonium sulfate, 4.5Kg of magnesium sulfate, 9L of polyether defoamer, and 450mL of trace element solution (including CoCl2·2H2O, FeSO4·7H2O, CuSO4·5H2O, MnSO4·H2O, ZnCl2, Na2MoO4·2H2O, KI, H3BO3, and concentrated sulfuric acid (H2SO4) 98%). After the ingredients are prepared according to the formula, steam sterilize at 121±1℃ for 30min, and control the specified volume after sterilization to 8000L. After sterilization, adjust the pH with ammonia water to 7.0±0.2.

[0048] 1.3.2 Feed preparation and sterilization

[0049] Feed 1: Lactose is prepared at 12g / L in a small feed tank and sterilized at 121℃ steam for 20min. After sterilization, the concentration is controlled at about 350g / L and the temperature is controlled at 70±10℃ for standby use. Stirring should be turned on during the lactose sterilization process to prevent incomplete dissolution.

[0050] Feed 2: The volume of glucose solution was prepared according to 250L / T fermentation liquid, and the feed concentration was: glucose was 650g / L, MgSO4·7H2O was 10g / L, and trace element solution was 0.25mL / L. Steam sterilized at 121℃ for 20min, and after sterilization, the glucose temperature was controlled at 70±10℃ with hot water.

[0051] Feed 3: VB1 and VB2 were mixed at a ratio of 0.5‰ and 0.1‰ respectively, and 40L of solution was prepared using 0.9% NaCl solution in a feed cylinder. The solution was sterilized in a 115℃ steam boiler for 20 min and then used for later use.

[0052] Feed 4: Magnesium sulfate was prepared into 200L aqueous solution at a ratio of 1g / L in a small feed tank and steam sterilized at 121℃ for 30min.

[0053] 1.3.3 Fermentation control after transplanting

[0054] Before transplanting: fermentation temperature 37±1℃, air flow 80±10M 3 / h, rotation speed 35rpm, tank pressure 0.02MPa, under this state, the dissolved oxygen is calibrated as 100%.

[0055] Initial stage of fermentation: after transplanting, the dissolved oxygen value will gradually decrease. When the dissolved oxygen value drops to 30%, the speed will be gradually increased by 10rpm / time. When the speed reaches the maximum (130rpm), the air flow rate will be increased by 30M. 3 / h / times to the maximum air flow, and at the same time increase the tank pressure to 0.04-0.05MPa to control the dissolved oxygen to not less than 30%. During the process of increasing the speed, air flow and tank pressure, the dissolved oxygen should be controlled at 10%-30%, and wait for the dissolved oxygen to rebound after the increase; the pH value drops at this stage, and the pH can be controlled at 7.0±0.2 by adding ammonia water.

[0056] Feeding stage control: It is necessary to wait for the dissolved oxygen to rebound to 60% (reference bacterial concentration: 35-40g / L). During this initial adaptation stage, glucose is added at the slowest flow rate (reference value for the minimum amount of glucose added per 3T batching volume: 5-10L / h) to prevent excessive glucose addition. The initial adaptation stage is controlled within 1-2h. In the subsequent adjustment stage, the glucose addition rate is gradually increased to the maximum (reference value for the maximum amount of glucose added per 3T batching volume is 20-25L / h) based on the parameter requirement that the dissolved oxygen is not less than 30%. To prevent excessive addition of glucose, stop adding glucose and judge the glucose addition rate based on the dissolved oxygen rebound time and previous control experience. After entering the induced stabilization stage, fine-tune the glucose addition rate until the tank is released based on controlling the dissolved oxygen at 20%-40%.

[0057] Control during the induction phase: Start measuring the bacterial concentration after 4 hours of fermentation, and then take samples every hour to measure the bacterial concentration; when the bacterial concentration is close to 45g / L, cool it down to 25±1℃ with ice water (the time is controlled within 1 to 1.5 hours), and then add lactose solution for induction, and add it within 1 hour (Note: the bacterial concentration needs to be predicted based on the measured value. When it is greater than 55g / L, lactose can be added while cooling).

[0058] After lactose is added, sterilized VB1 and VB2 solutions are added at one time. Both VB1 and VB2 solutions are dissolved using 0.9% NaCl solution. The final concentration of VB1 is 0.05%, and the final concentration of VB2 is 0.01%. Here, the final concentrations of VB1 and VB2 refer to the masses of 0.0005kg and 0.0001kg of VB1 and VB2 added to every 1L of fermentation broth.

[0059] When the fermentation reaches 26 hours, a sterilized magnesium sulfate solution is added at one time, and the final concentration of magnesium sulfate is 0.1%. Here, the final concentration of magnesium sulfate refers to the mass of magnesium sulfate added to each 1L of fermentation liquid is 0.001kg.

[0060] Release criteria: fermentation cycle of more than 40 hours, bacterial concentration of more than 120g / L, activity> 45U / mL. If the activity of the fermentation liquid shows an upward trend in the later stage, the tank can be released in advance.

[0061] 2. Enzyme extraction technology route

[0062] 2.1 Microfiltration and Bacteria Washing

[0063] The fermentation liquid is microfiltered with a ceramic membrane, and after all the bacteria are enriched, they are washed with tap water. Ultimately, the conductivity of the bacterial suspension is controlled below 3000μS / cm, and the bacterial concentration is controlled at 250-400g / L. The concentrated liquid is pumped into the homogenizer circulation material tank.

[0064] 2.2 Homogenization

[0065] After washing, add 0.05 mol / L phosphate (i.e., add 1.14 kg of potassium dihydrogen phosphate to 100 L of material) to the concentrated liquid, adjust and control the pH to 6.5-7.0 with dilute phosphoric acid solution (i.e., 85% concentrated phosphoric acid solution diluted 20-30 times with water), add nuclease (2 g / T material, added after dissolution), and inject into the homogenizer. According to the operating procedures of the homogenizer, control the homogenization pressure to 800-900 bar, release more than 95% of the activity (at least homogenize twice), generally homogenize 2-3 times, and take samples to measure the activity.

[0066] 2.3 Heating

[0067] The homogenate was diluted with tap water to a bacterial concentration of 220-270 g / L, and the pH was adjusted to 6.15-6.25 with a dilute phosphoric acid solution (i.e., 85% concentrated phosphoric acid solution was diluted 20-30 times with water). Nuclease (4-6 g / T material, added after dissolution) was added, and the temperature was raised to 50±1°C with hot water (65-75°C). The timing was started at 49°C and the temperature was kept for 40 minutes, during which the pH was maintained at 6.0-6.2 (adjusted with 1-1.5 mol / L sodium hydroxide solution). Samples were taken to measure activity.

[0068] 2.4 Flocculation Filtration

[0069] After heating, the feed liquid is cooled to 28-30°C, and potassium dihydrogen phosphate and 1.0-1.5 mol / L sodium hydroxide solution are used to adjust the feed liquid pH to 7.2±0.1 and the conductivity to 19-21 mS / cm. 12% chitosan solution (6‰) is added in turn according to the volume of the feed liquid, and 1-3% diatomaceous earth (used as a filter aid) is added after stirring evenly, and 8% sodium polyacrylate (1‰) is added after stirring evenly. The process controls the pH to be not less than 7.0. After adding the flocculant, stir evenly, take a sample to test the activity of the flocculated clear liquid, let it stand for 5-10 minutes, and filter it with a plate and frame. The plate and frame top wash uses 5%-6% glycerol water, and the last top wash liquid is used to top wash the ultrafiltration membrane device for material concentration.

[0070] 2.5 Concentration

[0071] Use 10KD spiral membrane ultrafiltration to concentrate, control the enzyme activity ≥ 200U / mL and take samples to measure activity, specific activity and impurities.

[0072] 2.6 Save

[0073] The concentrated enzyme was adjusted to pH 7.6-7.8 with potassium dihydrogen phosphate solution, and the enzyme solution was filtered through a filter element with a pore size of 0.2 μm. Then, (v / v) 43% glycerol was added to the clear solution, stirred evenly, divided into portions, and sealed and stored in a low temperature (-5--10° C.) freezer.

[0074] Example 2

[0075] The production method is the same as in Example 1, except that:

[0076] During the fermentation control process after transplanting, after lactose is added, sterilized VB1 and VB2 solutions are added at one time. Both VB1 and VB2 solutions are dissolved using 0.9% NaCl solution. The final concentration of VB1 is 0.07% and the final concentration of VB2 is 0.03%.

[0077] Example 3

[0078] The production method is the same as in Example 1, except that:

[0079] During the fermentation control process after transplanting, after lactose is added, sterilized VB1 and VB2 solutions are added at one time. Both VB1 and VB2 solutions are dissolved using 0.9% NaCl solution. The final concentration of VB1 is 0.06%, and the final concentration of VB2 is 0.02%.

[0080] Example 4

[0081] The production method is the same as in Example 1, except that:

[0082] When the fermentation reaches 26 hours, add the sterilized magnesium sulfate solution at one time, and the final concentration of magnesium sulfate is 0.05%.

[0083] Example 5

[0084] The production method is the same as in Example 1, except that:

[0085] When the fermentation reaches 26 hours, add the sterilized magnesium sulfate solution at one time, and the final concentration of magnesium sulfate is 0.2%.

[0086] Example 6

[0087] The production method is the same as in Example 1, except that:

[0088] When the fermentation reaches 26 hours, add the sterilized magnesium sulfate solution at one time, and the final concentration of magnesium sulfate is 0.15%.

[0089] Example 7

[0090] The production method is the same as in Example 2, except that:

[0091] During the flocculation and filtration process, 25% chitosan solution (6‰) was added in turn according to the volume of the feed liquid, and after stirring evenly, 1-3% diatomaceous earth was added, and after stirring evenly, 12% sodium polyacrylate (1‰) was added. The pH of the process was controlled to be not less than 7.0.

[0092] Example 8

[0093] The production method is the same as in Example 2, except that:

[0094] During the flocculation and filtration process, 20% chitosan solution (6‰) was added in turn according to the volume of the feed liquid, and after stirring evenly, 1-3% diatomaceous earth was added, and after stirring evenly, 16% sodium polyacrylate (1‰) was added. The pH of the process was controlled to be not less than 7.0.

[0095] Comparative Example 1

[0096] The production method is the same as in Example 1, except that:

[0097] During the fermentation control process after transplanting, VB1 and VB2 solutions are not added after lactose is added.

[0098] Comparative Example 2

[0099] The production method is the same as in Example 1, except that:

[0100] During the fermentation control process after transplanting, after lactose is supplemented, VB1 and VB2 solutions are not added, magnesium sulfate solution is added to the culture medium, and magnesium sulfate solution is not added when the fermentation reaches 26 hours.

[0101] Comparative Example 3

[0102] The production method is the same as in Example 1, except that:

[0103] During flocculation filtration, 20% chitosan solution (6‰) was added according to the volume of the feed liquid.

[0104] Comparative Example 4

[0105] The production method is the same as in Example 1, except that:

[0106] When storing, the concentrated enzyme is adjusted to pH 7.6-7.8 with potassium dihydrogen phosphate solution, the enzyme solution is filtered through a filter element with a pore size of 0.2 μm, and then (v / v) 30% glycerol is added to the clear solution, stirred evenly, divided into portions, and sealed and stored in a low temperature (-5 to -10°C) freezer.

[0107] 2. Performance Testing

[0108] 1. Pyruvate oxidase activity detection method

[0109] (1) The principle of enzyme activity detection reaction is as follows:

[0110]

[0111] Note: POX is pyruvate oxidase; POD is horseradish peroxidase.

[0112] (2) Instruments and equipment

[0113] Electronic balance, common glass instruments, UV-visible spectrophotometer, constant temperature water bath, etc.

[0114] (3) Preparation of reagents

[0115] 5% sodium pyruvate solution: Dissolve 5 g of sodium pyruvate in 80 mL of deionized water, preheat to 25°C, adjust the pH to 6.7 with 85% concentrated phosphoric acid, and add deionized water to make up to 100 mL.

[0116] 0.21 mM o-dianisidine solution: 6.66 mg of o-dianisidine hydrochloride was dissolved in 0.3 M phosphate buffer at pH 6.7 and the volume was adjusted to 100 mL.

[0117] (4) Detection method

[0118] Sample pretreatment (mixed solution): The sample was first ball-milled and then diluted with deionized water to an enzyme activity of 0.4-0.8u / mL as the crude enzyme solution.

[0119] Detection: In a 1 cm quartz cuvette, add 2.4 mL of 0.21 mM o-dianisidine solution preheated to 25°C, 0.5 mL of 5% sodium pyruvate solution, and 100 uL of horseradish peroxidase in sequence, mix well and zero at a wavelength of 500 nm, then add 100 uL of crude enzyme solution, mix quickly for a while, measure the time, record the absorbance A500 every 10 seconds, record the absorbance A500 of 18 measuring points, establish a curve of measurement time (min)-absorbance A500, and calculate the slope.

[0120] Enzyme activity unit: Under certain reaction conditions, the amount of enzyme that produces 1 μmol of o-Dianisidine (oxidized) per minute is one unit.

[0121] 2. Performance comparison

[0122] The fermentation tank activities, i.e., the single tank production capacity, of Examples 1-8 were compared. The results are as follows: Figure 2 As shown, 01-08 represent Examples 1-8 respectively, all of which are fermented in a 15T fermentation tank, and the single batch tank volume is between 11-12T. Figure 2 It can be seen that the fermentation activity exceeds the process requirements (>40U / mL), the total enzyme extraction yield is about 50%, and the single tank production capacity is >300Mu. In terms of fermentation activity and single tank production capacity, it is proved that the production process route is feasible.

[0123] In the production process of the pyruvate oxidase preparation of Example 1-8, the enzyme activity of the samples was tested before and after the addition of magnesium sulfate. The test time was 24h sample and 27h sample respectively. The test results are as follows Figure 3 As shown, it is shown that the addition of magnesium sulfate has a significant improvement on the fermentation activity, and the highest increase is the sample in Example 4, which increases by 67%.

[0124] The enzyme activity yield in the homogenization process is generally greater than 100%, and the enzyme activity loss is mainly in flocculation. In Example 1-8, after the flocculation process using chitosan + sodium polyacrylate two flocculants combined, the flocculation process yield can be stabilized at more than 80%. The results are as follows Figure 4 As shown, the formed flocs are better, the plate and frame filtration speed is fast, and the yield is high.

[0125] The enzyme activity of the pyruvate oxidase preparation solutions prepared in Examples 1-8 and Comparative Examples 1-2 was measured. The results of the enzyme activity test are shown in Table 1.

[0126] After adding flocculants to the pyruvate oxidase preparation solutions prepared in Examples 1-8 and Comparative Examples 3-4, the test was performed. The test results are shown in Table 1.

[0127] Table 1: Flocculation yield test and preservation test

[0128]

[0129]

[0130] Comparative Example 2 is that after lactose is added, VB1 and VB2 solutions are not added, magnesium sulfate solution is added to the culture medium, and when fermented to 26 hours, magnesium sulfate solution is not added, and the detection result shows that the enzyme activity is low. Comparative Example 1 is that during the fermentation control process after transplanting, VB1 and VB2 solutions are not added after lactose is added, but magnesium sulfate solution is added immediately after lactose is added during the fermentation process. The samples prepared in Examples 1-8 have a total enzyme activity of the finished liquid enzyme much greater than that of Comparative Examples 1 and 2, indicating that the addition of magnesium sulfate and VB1 and VB2 can improve the enzyme activity to a certain extent.

[0131] In comparative example 3, where chitosan was used alone as a flocculant, the flocculation yield was relatively low; however, after chitosan and polyacrylamide were used in combination, the flocculation yield increased, showing a certain improvement. After the finished enzyme solution was concentrated and filtered, the enzyme activity loss rate showed a downward trend after the glycerol ratio was increased, while in comparative example 4, where the glycerol ratio was not increased, the enzyme activity loss was relatively large.

[0132] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. An industrial production method of a pyruvate oxidase preparation, characterized in that: The following steps are involved: S1. Expanding and culturing a strain containing pyruvate oxidase, adding glucose when the bacterial solution concentration is 35-40 g / L, fermenting until the bacterial solution concentration is 45-55 g / L, adding lactose for induction after cooling, and then adding vitamin B1 and vitamin B2 solutions; the strain is Escherichia coli; S2, fermenting the bacterial solution in step S1 for 26 hours, adding a divalent metal ion solution and continuing the fermentation until the bacterial solution concentration is greater than 120 g / L, to prepare a fermentation solution, wherein the divalent metal ion is a magnesium ion; S3, washing the fermentation liquid in step S2, adding nuclease to homogenize, adding nuclease again to the homogenized liquid, heating to 50±1°C, and heat preservation to obtain a treated liquid, then adding a flocculant to the treated liquid, stirring evenly, and filtering; S4. Concentrating the filtrate obtained in step S3 to obtain a pyruvate oxidase preparation solution.

2. The industrial production method according to claim 1, characterized in that: In step S1, the amount of vitamin B1 added is 0.03-0.07% of the volume of the fermentation broth, and the amount of vitamin B2 added is 0.01-0.04% of the volume of the fermentation broth, wherein the vitamin B1 solution and the vitamin B2 solution are prepared using sodium chloride solution.

3. The industrial production method according to claim 1, characterized in that: The final concentration of the divalent metal ions added in step S2 is 0.05-0.2%.

4. The industrial production method according to claim 1, characterized in that: In step S3, the amount of nuclease added before homogenization is 2-4 g per ton of fermentation broth, and the amount of nuclease added after homogenization is 4-6 g per ton of fermentation broth.

5. The industrial production method according to claim 1, characterized in that: During the heat preservation in step S3, the pH is maintained at 6.0-6.

2.

6. The industrial production method according to claim 1, characterized in that: In step S3, the flocculant includes chitosan and sodium polyacrylate, the chitosan is added in an amount of 12-25% of the mass of the treated liquid, and the sodium polyacrylate is added in an amount of 8-16% of the mass of the treated liquid.

7. The industrial production method according to claim 1, characterized in that: The enzyme activity of the concentrated pyruvate oxidase preparation solution in step S4 is maintained at ≥ 200 U / mL.

8. The industrial production method according to claim 1, characterized in that: The pH of the pyruvate oxidase preparation obtained in step S4 is adjusted to 7.6-7.8, filtered, and then added with 43-45% v / v glycerol and stored at -5 to -10°C.

9. Use of the industrial production method according to any one of claims 1 to 8 in the production of a pyruvate oxidase preparation.

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