Recombinant engineering bacterium for producing recombinant lactate oxidase as well as construction method and application of recombinant engineering bacterium

Abstract

The invention discloses a recombinant engineering bacterium for producing recombinant lactate oxidase as well as a construction method and application of the recombinant engineering bacterium, and the construction method comprises the following steps: (1) introducing an HsoLOX gene for coding lactate oxidase into an expression plasmid to construct a recombinant expression vector containing the HsoLOX gene; the nucleotide sequence of the HsoLOX gene is as shown in SEQ ID NO.1 or a sequence of which the homology with the sequence as shown in SEQ ID NO.1 is greater than 90%; and (2) transforming the recombinant expression vector into host bacteria, screening positive clones by taking KanR as a screening marker, and culturing to obtain recombinant engineering bacteria. When the engineering strain is subjected to high-density fermentation for 46 hours, the relative enzyme activity of thalli is 1773 U / g, and the thalli density of a fermentation tank is 262.5 g / L. The invention further provides application of the recombinant lactate oxidase or the whole-cell catalyst and catalase in catalyzing L-sodium lactate together to generate sodium pyruvate, and the conversion rate can reach 99.5%. The operation method is simple and safe, low in production cost, mild in preparation condition, green and pollution-free, and has high economic value and social benefit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a recombinant engineering bacterium producing recombinant lactate oxidase, a construction method and application thereof, in particular, application in efficient conversion of L-sodium lactate into sodium pyruvate. BACKGROUND

[0002] Pyruvate is an important organic acid, which is widely used in the fields of pharmaceuticals, food additives and agricultural chemicals. At present, the production methods of pyruvate mainly include chemical synthesis, microbial fermentation and enzyme catalysis. The chemical synthesis method usually involves high temperature and high pressure and toxic catalysts, which causes great environmental pollution; the microbial fermentation method has problems of long cycle and many by-products.

[0003] Lactate oxidase (EC 1.1.3.15) belongs to the L-2-hydroxy acid oxidase family, is a flavoprotein enzyme, uses flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as a tightly bound coenzyme, and uses oxygen as an electron acceptor to catalyze the oxidation of L-lactic acid to produce pyruvate and hydrogen peroxide (H2O2). Since its catalytic process does not require additional free coenzyme, it completely solves the problem of coenzyme regeneration and cost, and is therefore considered as one of the most potential catalysts for the enzyme catalytic preparation of pyruvate. At present, the commonly used lactate oxidase AviLOX in research is derived from Aerococcus viridans, and its protein sequence (GenBank: WP_003142047.1) has been published. However, the lactate oxidase preparation expressed by the current lactate oxidase still has problems of high expression difficulty and low catalytic efficiency, which greatly restricts the industrial application of lactate oxidase.

[0004] Therefore, it is very important to explore lactate oxidase with higher catalytic activity and stability for promoting the enzyme production of pyruvate. SUMMARY

[0005] The present application aims to make up for the shortcomings of the prior art, and provides a recombinant engineering bacterium producing recombinant lactate oxidase, a construction method and application thereof, which are used to solve the problems of high expression difficulty and low catalytic efficiency of traditional lactate oxidase.

[0006] To solve the above problems, the technical scheme adopted by the present application is as follows:

[0007] The first object of the present application is to provide a construction method of a recombinant engineering bacterium producing recombinant lactate oxidase, comprising the following steps:

[0008] (1) introducing the HsoLOX gene encoding lactate oxidase into an expression plasmid to construct a recombinant expression vector containing the HsoLOX gene; the nucleotide sequence of the HsoLOX gene is shown in SEQ ID NO. 1 or a sequence having a homology of more than 90% with the sequence shown in SEQ ID NO. 1;

[0009] (2) transforming the recombinant expression vector obtained in step (1) into a host bacterium, using KanR as a screening marker, screening positive clones and culturing to obtain a recombinant engineering bacterium.

[0010] Preferably, the expression plasmid is pET-28a(+).

[0011] Preferably, the host bacterium is Escherichia coli BL21 (DE3).

[0012] The second object of the present application is to provide a recombinant engineering bacterium producing recombinant lactate oxidase, which is obtained by using the construction method of the lactate oxidase.

[0013] Preferably, the HsoLOX gene is codon-optimized, and the nucleotide sequence thereof is shown in SEQ ID NO. 1 or a sequence having a homology of more than 90% with the sequence shown in SEQ ID NO. 1.

[0014] Preferably, the amino acid sequence of the HsoLOX gene is shown in SEQ ID NO. 2.

[0015] The third object of the present application is to provide an application of the recombinant engineering bacterium producing recombinant lactate oxidase in the fermentation preparation of lactate oxidase.

[0016] The fourth object of the present application is to provide a preparation method of recombinant lactate oxidase, which comprises the step of using the genetic engineering bacterium to perform shake flask fermentation or high-density fermentation culture to express the lactate oxidase.

[0017] Preferably, the relative enzyme activity of the recombinant lactate oxidase reaches 1773 U / g at 46 h of fermentation, and the cell density in the fermenter is 262.5 g / L.

[0018] The fifth object of the present application is to provide a recombinant lactate oxidase, which is prepared by using the preparation method of the lactate oxidase.

[0019] The sixth object of the present application is to provide a preparation method of a whole-cell catalyst, which comprises:

[0020] Step 1, performing shake flask fermentation or high-density fermentation culture on the recombinant engineering bacterium to obtain a fermentation liquor;

[0021] Step 2, after the fermentation broth is centrifuged and washed for multiple times, the wet bacteria are collected, which are the whole-cell catalyst containing lactate oxidase.

[0022] A seventh object of the present application is to provide a whole-cell catalyst.

[0023] An eighth object of the present application is the use of a lactate oxidase or a whole-cell catalyst in the conversion of sodium L-lactate into sodium pyruvate.

[0024] Preferably, the use comprises:

[0025] S1, adding a substrate solution, catalase and the recombinant lactate oxidase of claim 6 or the whole-cell catalyst of claim 8 into a reaction container; wherein the initial concentration of sodium lactate in the substrate solution is 500-800 mM; the added amount of the lactate oxidase or the whole-cell catalyst is 8500-42500 U / L in terms of the lactate oxidase enzyme activity contained therein; and the enzyme activity ratio of the endogenous lactate oxidase in the whole-cell catalyst to the added catalase is controlled to be 1:25 to 1:300.

[0026] S2, controlling the reaction temperature to be 31-43°C, the reaction pH to be 6.5-8.5, and introducing oxygen or air into the reaction system, and reacting for 5-9 hours to obtain the target product after separation and purification.

[0027] Preferably, the enzyme activity added amount is 34000 U / L (corresponding to a whole-cell catalyst concentration of 20 g / L), the initial concentration of sodium lactate is 680 mM, the double-enzyme enzyme activity ratio is 1:100, the reaction temperature is 40°C, the reaction pH is 7.0, the reaction time is 9 hours, and the introduced gas is oxygen, so as to realize complete conversion of the substrate.

[0028] Compared with the prior art, the beneficial effects of the present application are as follows: after the HsoLOX gene screened in the present application is introduced into the E. coli strain BL21 (DE3), an E. coli engineering strain is screened, and high-activity whole-cell catalyst can be obtained by fermentation culture of the engineering strain. The conversion rate of 680 mM L-lactic acid sodium in 9 h can reach 99.5%, compared with the traditional fermentation method and the enzyme catalysis method of the rest lactate oxidases for producing pyruvic acid. The operation method provided by the present application is simple, safe, low in cost, green and efficient, the preparation conditions are mild, and the method has universality, thereby providing a reference for industrial production of pyruvic acid. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A map of the recombinant expression vector pET-28a-HsoLOX;

[0030] Figure 2SDS-PAGE result of recombinant lactic acid oxidase prepared for strain HsoLOX;

[0031] Figure 3 Standard curve of pyruvic acid in the present application;

[0032] Figure 4 HPLC spectrum of sodium pyruvate standard;

[0033] Figure 5 HPLC spectrum of sodium lactate standard. DETAILED DESCRIPTION

[0034] The present application can be implemented or applied in other different specific embodiments, and the details in the present specification can be modified or changed based on different views and applications without departing from the spirit of the present application.

[0035] It should be noted that the process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art.

[0036] Furthermore, it should be understood that the one or more method steps mentioned in the present application do not exclude that other method steps can exist before and after the mentioned combination steps or other method steps can be inserted between the mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between the one or more devices mentioned in the present application does not exclude that other devices can exist before and after the mentioned combination devices or other devices can be inserted between the two mentioned devices, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool to identify each method step, and is not intended to limit the arrangement order of each method step or to limit the range of the present application, and the change or adjustment of the relative relationship without substantial change of the technical content is also considered as the range of the present application.

[0037] LB plate components: NaCl 10 g / L, peptone 10 g / L, yeast powder 5 g / L, agar 20 g / L, kanamycin 100 μg / ml.

[0038] LB liquid medium components: NaCl 10 g / L, peptone 10 g / L, yeast powder 5 g / L, kanamycin 100 μg / ml.

[0039] The liquid chromatography detection method in the following examples is as follows: after sampling the reaction solution, centrifuge at 12000 r / min for 5 min, then take the supernatant for detection. The liquid chromatography conditions are as follows: CAPCELL PAK ADME-HR S5 chromatographic column, 4.6*250 mm; mobile phase: 10 mM potassium dihydrogen phosphate aqueous solution (phosphoric acid is added to adjust pH to 2.5); flow rate: 1 ml / min; column temperature: 35℃; detection wavelength: 213 nm; injection volume: 10 μL.

[0040] Example 1, cloning and expression of recombinant lactate oxidase gene

[0041] In this example, DLKcat and CataPro calculation models are used to screen a large number of lactate oxidases in the UniProt database, predict and evaluate the catalytic activity of potential sequences, and finally select a candidate HsoLOX gene (GenBank: WP_028118949.1) with high predicted activity for encoding lactate oxidase. The above gene is synthesized by Beijing Qikong Biotechnology Co., Ltd. and cloned into pET-28a(+) vector to obtain recombinant expression vector pET-28a-HsoLOX, the map of which is shown in Figure 1 .

[0042] Take 1 μL of the above recombinant expression vector pET-28a-HsoLOX, and add it to 100 μL of E. coli competent BL21 (DE3) under ice bath conditions. Place in ice bath for 30 min, 42℃ water bath heat shock for 45 s, immediately place on ice for 2 min, add 900 μL of LB medium without antibiotics, and incubate at 37℃, 220 rpm for 60 min. Centrifuge at 4000 r / min for 5 min, discard 900 μL of supernatant, resuspend the bacterial pellet and evenly spread on LB plates containing 100 μg / mL kanamycin, and incubate in a 37℃ incubator overnight. Select positive monoclonal colonies for colony PCR and sequencing verification to obtain strain HsoLOX, which is a recombinant genetically engineered bacterium.

[0043] Colony PCR reaction system: 2*TransStart FastPfu Fly PCR SuperMix 10 μL; ddH2O 7 μL; primer T7-F 1 μL; primer T7-R 1 μL; bacterial liquid template 1 μL.

[0044] Reaction program: 95℃ 5min; 98℃ 10sec, 57℃ 15sec, 72℃ 10sec, run for 30 cycles; 72℃

[0045] 5min.

[0046] Primer T7-F sequence: 5'-CCGCGAAATTAATACGACTCAC-3';

[0047] Primer T7-R sequence: 5'-TCAAGACCCGTTTAGAGGCC-3'.

[0048] The correct recombinant genetically engineered bacteria were inoculated into 5 mL LB liquid medium containing kanamycin, and cultured at 37°C with 200 r / min shaking overnight. The seed liquid was transferred to fresh 50 mL LB liquid medium containing kanamycin at a 1:100 inoculation amount, and cultured at 37°C until the OD 600 was about 0.6-0.8. IPTG was added to a final concentration of 0.05 mM, and the culture temperature was reduced to 25°C, and the expression was induced for 20 h.

[0049] The induced bacterial liquid was then centrifuged at 4°C and 8000 r / min for 10 min, the bacterial bodies were collected, washed twice with pre-cooled PBS buffer (pH 7.4), weighed, resuspended with an equal volume of pre-cooled PBS buffer (pH 7.4), and the bacterial suspension was ultrasonically broken in an ice water bath (working parameters: power 300 W, ultrasonic 3 s, intermittent 7 s, total time 10-15 min). The broken bacterial liquid was centrifuged at 4°C and 12000 r / min for 5 min, and the supernatant was collected, which was the crude enzyme liquid containing lactate oxidase, and was analyzed by SDS-PAGE, and the results are shown in Figure 2 .

[0050] Example 2, activity determination of recombinant lactate oxidase

[0051] The activity of lactate oxidase is defined as the yield of pyruvate detected as the product of the oxidation of L-lactic acid to pyruvate by lactate oxidase.

[0052] Detection sample: 100 μL of 100 mM L-lactic acid sodium solution + 150 μL of enzyme liquid diluted by a certain multiple, mixed uniformly, and incubated at 37°C in a metal bath for 1 min; 250 μL of 1 mM 2,4-dinitrophenylhydrazine solution was added, and incubated for 20 min; 500 μL of 1 M sodium hydroxide solution was added, and the colorimetric reading value A1 was read at 520 nm.

[0053] Control sample: 100 μL of 100 mM L-lactic acid sodium solution + 150 μL of inactivated enzyme liquid diluted by a certain multiple, mixed uniformly, and incubated at 37°C in a metal bath for 1 min; 250 μL of 1 mM 2,4-dinitrophenylhydrazine solution was added, and incubated for 20 min; 500 μL of 1 M sodium hydroxide solution was added, and the colorimetric reading value A2 was read at 520 nm.

[0054] (A1-A2) is the actual reading value, and the control pyruvate standard curve (such asFigure 3 The enzyme activity was calculated.

[0055] Enzyme activity unit definition: the amount of enzyme required to produce 1 μmol pyruvic acid per minute, expressed in U;

[0056] After large-scale screening and determination of enzyme activity, it was found that the relative enzyme activity of strain HsoLOX was relatively high in shake flask fermentation, and the crude enzyme solution enzyme activity reached 18.43 U / ml, while the cell density could reach 262.5 g / L after high-density fermentation, and the cell enzyme activity could reach 1773 U / g. Therefore, the lactic acid oxidase or whole cell catalyst thereof was used for subsequent experiments.

[0057] Compared with the currently more studied AviLOX from Aerococcus urinae: strain HsoLOX is easy to express, while AviLOX is difficult to express and is prone to inclusion bodies; and the relative enzyme activity of the crude enzyme solution after fermentation HsoLOX is higher than that of AviLOX, and the relative enzyme activity of the crude enzyme solution of AviLOX in shake flask fermentation can only reach 15.6 U / mL.

[0058] Table 1, enzyme activity determination of lactic acid oxidase prepared by strain HsoLOX Strain name Shake flask fermentation relative enzyme activity (U / mL) HsoLOX 18.43 .

[0059] Example 3, high-density fermentation of engineering strain HsoLOX

[0060] The glycerol bacteria of the frozen recombinant genetically engineered bacteria HsoLOX were inoculated into 30 mL of LB liquid medium containing kanamycin at a ratio of 1:500 (60 μL), and cultured at 37°C and 220 r / min for 5 h to measure OD 600 to about 1.6, which was used as a seed solution.

[0061] 30 mL of seed solution was inoculated into a 3 L YT-M9 liquid medium in a fermenter at an inoculation amount of 1%, and the initial parameters were: stirring speed 200 r / min, air flow rate 1 vvm (3 L / min), 37°C, pH 7.0, tank pressure 0.03 Mpa. During the fermentation process, 2 mol / L HCl and 25% ammonia were used to control the pH.

[0062] The specific growth rate was controlled at 0.121 / h, and the DO was controlled above 30%; after the DO jumped, the feeding of the medium was started. When the OD 600 was 50, the feeding was stopped, 0.5 mmol / L (1.5 mL of 1 mol / L mother liquor) of IPTG was added for induction expression, and about 10 min later the feeding was started; after the induction started: the specific growth rate was controlled at 0.05 / h, and the induction temperature was 30°C. The DO was controlled above 30%; until the specific growth rate approached 0, the fermentation was terminated.

[0063] OD at 23h 600 To 50, start inducing expression, specific growth rate decreased to 0 at 46h, cell density was 262.5g / L and stopped increasing, terminate fermentation. Centrifuge the fermentation broth at 10000r / min, 4℃, 10min, get the cell and determine the enzyme activity, the cell enzyme activity can reach 1773U / g.

[0064] YT-M9 medium components are as follows: yeast powder 12.0g / L, peptone 20.0g / L, NaCl 1.0g / L, (NH4)2SO4 6.0g / L, MgSO4·7H2O (sterilized separately) 2.0g / L, glycerol 7.5g / L, trace elements 1ml / L, KH2PO4 3g / L, Na2HPO4·12H2O 8.2g / L. MgSO4·7H2O and trace elements are separately sterilized and then added to the fermenter by flame protection, the initial volume of YT-M9 medium per tank is 3L.

[0065] Trace element solution: FeCl3·4H2O 22.87g / L, ZnCl2 1.31g / L, CoCl2·6H2O 2.0g / L, CaCl2 0.76g / L, CuCl2·2H2O 1.25g / l, H3BO3 0.5g / L, MnSO4·H2O 2.0g / L, concentrated HCl 100mL / L.

[0066] Feed medium: yeast powder 60.0g / L, peptone 100.0g / L, MgSO4·7H2O (sterilized separately) 10g / L, glucose (sterilized separately) 500.0g / L.

[0067] Example 4, Effect of different enzyme activity addition on the strain HsoLOX prepared whole cell catalyst catalyzing the synthesis of sodium pyruvate from sodium L-lactate

[0068] This example studies the effect of enzyme activity addition on the rate of whole cell catalyst catalyzing the synthesis of sodium pyruvate.

[0069] Within the range of 8500U / L~42500U / L enzyme activity addition, the effect of enzyme activity addition on the rate of whole cell catalyst catalyzing the synthesis of sodium pyruvate from strain HsoLOX was studied. Reaction system: 150ml 500mM L-lactic acid sodium aqueous solution was added to a three-necked flask, after preheating in a 37℃ metal bath, different enzyme activity addition corresponding whole cell catalyst was added and mixed well, the pH was controlled at 7.0, the oxygen flow rate was 0.3L / min, and the reaction was carried out at 37℃ for 5h. The mixture at 0h of reaction was used as the initial control, and HPLC was used to detect the conversion rate of L-lactic acid sodium.

[0070] The results are shown in Table 2. With the increase of enzyme activity, the conversion rate of L-sodium lactate at 5h also increased. When the enzyme activity reached 34000U / L, the conversion rate of L-sodium lactate at 5h reached 98.63%. With the continuous increase of enzyme activity, the conversion rate of L-sodium lactate per hour increased slowly. Therefore, the optimal enzyme activity was 34000U / L.

[0071] Table 2, Effect of different enzyme activities on the conversion rate of L-sodium lactate .

[0072] Example 5, Effect of different temperatures on the strain HsoLOX prepared whole cell catalyst catalyzing L-sodium lactate to synthesize sodium pyruvate

[0073] This example studies the effect of temperature on the rate of whole cell catalyst catalyzing the synthesis of sodium pyruvate. According to the experimental method of Example 5, the enzyme activity is 34000U / L, the temperature in this example is 31℃-43℃, and the rest of the experimental steps are the same as Example 3, which will not be repeated here.

[0074] The results are shown in Table 3. When the reaction temperature reached 40℃, the conversion rate of L-sodium lactate at 5h reached the highest, which was 99.5%. After that, with the increase of temperature, the conversion rate decreased. Therefore, the optimal reaction temperature was 40℃.

[0075] Table 3, Effect of different temperatures on the conversion rate of L-sodium lactate .

[0076] Example 6, Effect of different pH on the strain HsoLOX prepared whole cell catalyst catalyzing L-sodium lactate to synthesize sodium pyruvate

[0077] This example studies the effect of pH on the rate of whole cell catalyst catalyzing the synthesis of sodium pyruvate. According to the experimental method of Example 6, the temperature is 40℃, the pH in this example is 6.5-8.5, and the rest of the experimental steps are the same as Example 4, which will not be repeated here.

[0078] The results are shown in Table 4. When the reaction pH was in the range of 6.5-7.5, the conversion rate of L-sodium lactate at 5h was relatively high, which could reach more than 98%. When the reaction pH was higher than 7.5, the conversion rate decreased rapidly. Therefore, the optimal pH was 7.0.

[0079] Table 4, Effect of different pH on the conversion rate of L-sodium lactate .

[0080] Example 7, Effect of different enzyme activity ratio on the strain HsoLOX prepared whole cell catalyst catalyzed synthesis of sodium pyruvate from sodium L-lactate

[0081] This example studies the effect of different enzyme activity ratio on the rate of whole cell catalyst catalyzed synthesis of sodium pyruvate. According to the experimental method of Example 6, the pH is 7.0, the enzyme activity ratio in this example is 1:25~1:300, and the rest of the experimental steps are the same as Example 5, which will not be repeated here.

[0082] The results are shown in Table 5, the conversion rate of sodium L-lactate increases with the increase of enzyme activity ratio, when the enzyme activity ratio exceeds 1:100, the sodium L-lactate is basically converted completely in 5h, which can reach more than 98%. Considering the cost factor, the enzyme activity ratio of 1:100 is selected as the optimal reaction condition.

[0083] Table 5, Effect of different enzyme activity ratio on the conversion rate of sodium L-lactate .

[0084] Example 8, Application of strain HsoLOX prepared whole cell catalyst catalyzed synthesis of sodium pyruvate from sodium L-lactate under optimal conditions

[0085] This example studies the application of HsoLOX whole cell catalyst catalyzed synthesis of sodium pyruvate under optimal conditions.

[0086] The results are shown in Table 6, using a 500ml reaction system, when the enzyme activity addition amount is 37000U / L (corresponding to the whole cell catalyst concentration of 20g / L), the enzyme activity ratio is 1:100, the reaction temperature is 40°C, the reaction pH is 7.0, and the oxygen is passed, 680mM sodium L-lactate is basically converted completely in 9 hours, which is at a high level in the current domestic and foreign research.

[0087] Table 6, Application of HsoLOX whole cell catalyst catalyzed synthesis of sodium pyruvate under optimal conditions Time 0h 1h 2h 3h 4h 5h 6h 7h 8h 9h Substrate concentration (mM) 680.0 567.15 469.48 406.92 345.47 282.26 226.61 168.83 85.08 0 Product concentration (mM) 0 110.72 183.25 263.30 324.43 373.59 446.86 498.78 557.70 652.4 .

[0088] The application obtains a gene HsoLOX gene coding lactic acid oxidase through computer model screening, carries out gene synthesis after codon optimization of Escherichia coli, constructs pET-28a-HsoLOX expression vector, transforms into Escherichia coli BL21 (DE3) for expression, and the recombinant lactic acid oxidase or the whole cell catalyst containing the recombinant lactic acid oxidase and catalase are used to catalyze L-lactic acid sodium to generate pyruvic acid sodium, and the conversion rate can reach 99.5%. The operation method provided by the application is simple and safe, the production cost is low, the preparation condition is mild, green and pollution-free, and the application has high economic value and social benefits.

[0089] Example 9, reaction liquid concentration cooling alcohol precipitation process for preparing pyruvic acid sodium

[0090] After the reaction, the bacteria were collected by primary centrifugation, the supernatant was subjected to microfiltration and ultrafiltration to remove cell debris and protein impurities, and then concentrated under reduced pressure at 60 DEG C. After the concentration of pyruvic acid sodium was close to saturation, 10 times of anhydrous ethanol was slowly added, the temperature was reduced to 0-4 DEG C, and the mixture was statically placed for 1 h. Pyruvic acid sodium was precipitated in large amounts, and after suction filtration and drying, pyruvic acid sodium powder was obtained. The purity of pyruvic acid sodium produced by this method was 94.1%, and the one-time yield was 75.3%.

[0091] SEQ ID NO: 1:

[0092]

[0093] SEQ ID NO: 2:

[0094] MTQDKYLAPSEVKPLRIINTIELEDMASEIVPHGGFNYMAGGSGDEFTLRRNVESFNFKGILPRMAADVEFPETDTKIFDHHLKVPFIMAPIAAHGLAHESKEAGTARGIHEFGGSLMSISAYSGASFEEISAGLQDTPRWFQIYMSKDDDFNRNILDEAKADGASAIILTADATISGNRDRDDKNEFVYPFGMPIVSRYLTGTGANMSLNNIYSQSKQKINLDDIRFIKEYSGLPVFLKGVQSPEDALAAIGAGADGIWVSNHGGRQLDGAPGSFEVLEEIAQAVAGQVPIVFDSGIRRGEHIFKALASGADIVALGRPVLFSLALGGWQGVQSVFEYFERDLKRVMQLAGTQTIEDVKKARLKDLWR.

[0095] Although the present application has been disclosed in its preferred embodiments as above, it is not intended to limit the present application thereto, and it will be apparent to those skilled in the art that various modifications and changes can be made thereto without departing from the spirit and scope of the present application, and it is intended that the scope of the present application be governed only by the appended claims.

Claims

1. A method for constructing a recombinant engineered bacterium producing a recombinant lactic acid oxidase, characterized by, The method comprises the following steps: (1) introducing a HsoLOX gene encoding lactic acid oxidase into an expression plasmid to construct a recombinant expression vector containing the HsoLOX gene; the nucleotide sequence of the HsoLOX gene is shown in SEQ ID NO. 1 or a sequence having a homology of more than 90% with the sequence shown in SEQ ID NO. 1; (2) transforming the recombinant expression vector obtained in step (1) into a host bacterium, using KanR as a screening marker, screening positive clones and culturing to obtain a recombinant engineering bacterium.

2. The construction method of claim 1, wherein, The expression plasmid is pET-28a(+), and the host bacterium is Escherichia coli BL21 (DE3).

3. A recombinant engineering bacteria producing recombinant lactic acid oxidase, characterized in that, The method is obtained by the construction method of claim 1 or 2.

4. Application of the recombinant engineering bacterium producing the recombinant lactic acid oxidase of claim 3 in the preparation of lactic acid oxidase by fermentation.

5. A method for producing a recombinant lactic acid oxidase, characterized by, The preparation method comprises the following steps: using the genetically engineered bacterium of claim 3 to perform shake flask fermentation or high-density fermentation culture to express the recombinant lactic acid oxidase.

6. A recombinant lactic acid oxidase, characterized in that, The method is obtained by the preparation method of claim 5.

7. A process for the preparation of a whole cell catalyst, characterized by, The method comprises the following steps: Step 1: performing shake flask fermentation or high-density fermentation culture on the recombinant engineering bacterium obtained by the construction method of claim 1 or 2 or the recombinant engineering bacterium of claim 3 to obtain a fermentation broth; Step 2: collecting wet bacterial cells after multiple centrifugation and washing treatments of the fermentation broth, which are whole-cell catalysts containing lactic acid oxidase.

8. A whole cell catalyst characterized by: The method is obtained by the preparation method of claim 7.

9. Application of the whole-cell catalyst obtained by the preparation method of claim 7 or the whole-cell catalyst of claim 8 in catalyzing the generation of sodium pyruvate from sodium L-lactate.

10. Use according to claim 9, characterized in that, The application comprises the following steps: S1: adding a substrate solution, catalase and the recombinant lactic acid oxidase of claim 6 or the whole-cell catalyst of claim 8 into a reaction container; wherein the initial concentration of sodium lactate in the substrate solution is 500-800 mM; the addition amount of the lactic acid oxidase or the whole-cell catalyst is 8500-42500 U / L in terms of the lactic acid oxidase enzyme activity contained therein; and the enzyme activity ratio of the endogenous lactic acid oxidase to catalase in the whole-cell catalyst is controlled to be 1:25 to 1:300; S2: controlling the reaction temperature to be 31-43°C, the reaction pH to be 6.5-8.5, and introducing oxygen or air into the reaction system, and collecting the reaction product after reaction for 5-9 hours.

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