A maleate isomerase mutant and its application in the preparation of fumaric acid

Abstract

The present invention discloses a maleate isomerase mutant and its application in the preparation of fumaric acid, relating to the field of bioengineering. The nucleic acid molecule for preparing fumaric acid provided by the present invention encodes a maleate isomerase mutant, and the base sequences are as shown in SEQ ID NO.4 and SEQ ID NO.6; the recombinant engineered bacterium transformed with this nucleic acid molecule can be used as a catalyst to catalyze maleic acid substrate to form fumaric acid; the method for preparing fumaric acid using the maleate isomerase engineered bacterium provided by the present invention uses the engineered bacterium cells containing the maleate isomerase gene as a catalyst to catalyze the conversion of maleic acid to obtain fumaric acid, which has good stability and high catalytic activity and is environmentally friendly during the production process.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a mutant of maleate isomerase and its application in the preparation of fumaric acid. Background Art

[0002] Fumaric acid, also known as trans-butenedioic acid, is an important four-carbon dicarboxylic acid widely existing in nature and is a key intermediate metabolite in the tricarboxylic acid cycle (TCA cycle) of organisms. As a high-value-added chemical, fumaric acid has important applications in the fields of food, medicine, chemical industry, etc. For example, in the food industry, fumaric acid can be used as an acidulant and preservative; in the pharmaceutical field, its derivatives can be used to synthesize anti-tumor drugs (such as doxorubicin) and anti-inflammatory drugs; in the chemical industry, fumaric acid is an important raw material for the production of unsaturated polyester resins and biodegradable plastics (such as polyfumarate). With the increasing global demand for green chemistry and sustainable development, the production technology of bio-based fumaric acid has become a research hotspot.

[0003] The industrial production of traditional fumaric acid mainly relies on chemical synthesis methods. Using maleic acid (cis-butenedioic acid) as a raw material, fumaric acid is produced through a catalytic isomerization reaction at high temperature (>150°C). Commonly used catalysts include strong acids (such as sulfuric acid), metal oxides or thiourea compounds. However, this method has significant defects:

[0004] High energy consumption and environmental pollution: The reaction requires high temperature and high pressure conditions, resulting in high energy consumption; strong acid catalysts are prone to cause equipment corrosion, and the treatment of waste liquid is difficult, posing an environmental pollution risk.

[0005] Low product purity: By-products (such as maleic anhydride) are easily generated during the chemical isomerization process, and multiple purification processes are required, increasing costs.

[0006] Unsustainability: The raw material maleic acid is mostly prepared by catalytic oxidation of petroleum-based compounds (such as benzene or butane), relying on non-renewable resources.

[0007] To overcome the defects of the chemical method, biocatalysis has gradually become an alternative. Its core is to use microorganisms or enzymes to catalyze the isomerization of maleic acid into fumaric acid, which has the advantages of mild conditions, environmental friendliness, high substrate specificity, etc. The existing biocatalytic pathways are mainly divided into two categories:

[0008] (I) Microbial fermentation method

[0009] Some filamentous fungi (such as Rhizopus) can directly ferment sugars to produce fumaric acid. For example, Rhizopus oryzae can accumulate fumaric acid through the reductive TCA cycle pathway. However, this method has the following bottlenecks:

[0010] Low yield: The intracellular metabolic pathway of microorganisms is complex, and fumaric acid is easily further converted by downstream enzymes (such as fumarase), resulting in a low concentration of the end product (usually <100 g / L).

[0011] Difficult process control: Filamentous fungi are prone to form mycelial pellets, which affect the mass transfer efficiency, and the fermentation cycle is long (>72 h).

[0012] High substrate cost: It depends on carbon sources such as glucose, and the raw material cost accounts for more than 30% of the total production cost.

[0013] (2) Enzymatic catalysis method

[0014] Directly using maleate isomerase (MIase) to catalyze the isomerization of maleic acid is another research direction. MIase can efficiently catalyze the reversible conversion of maleic acid and fumaric acid (EC 5.2.1.1), and its catalytic mechanism depends on metal ion cofactors (such as Fe 2+ )). Existing studies have isolated MIase from strains such as Pseudomonas and Achromobacter, but the industrial application of natural enzymes faces severe challenges:

[0015] Poor enzyme stability: Free enzymes are easily inactivated in the reaction system, and the reusability is low.

[0016] Low expression efficiency: The expression level of the natural MIase gene is insufficient in common hosts such as Escherichia coli, resulting in low enzyme activity.

[0017] Limited reaction conditions: The optimal pH of most MIases is acidic (pH 5.0 - 6.0), which does not match the high solubility conditions (pH>7.0) of the substrate maleic acid in the chemical method, resulting in limited substrate feeding concentration.

[0018] In recent years, genetic engineering technology has provided new ideas for the modification of MIase. For example, the expression level of MIase in engineered bacteria can be improved by codon optimization, promoter enhancement or fusion tags (such as His-tag); or the catalytic stability can be enhanced by using immobilized cell technology. However, existing technologies still have problems such as insufficient catalytic efficiency, poor host adaptability and low process integration.

[0019] Therefore, there is an urgent need for maleate isomerase with good stability and high catalytic activity at present. Summary of the invention

[0020] The purpose of the present invention is to provide a maleate isomerase mutant for the preparation of fumaric acid, and a nucleic acid molecule encoding the enzyme mutant. The recombinant engineered bacteria transformed with the nucleic acid molecule can be used as a catalyst to catalyze maleic acid as a substrate to form fumaric acid, which has good stability and catalytic activity compared with the known existing maleate isomerase.

[0021] The specific technical solution of the present invention is as follows:

[0022] The present invention provides a maleate isomerase PbFu, and its amino acid sequence is shown in SEQ ID NO.1.

[0023] The present invention also provides two maleate isomerase mutants Fu-a and Fu-b, which are obtained by mutating the wild-type maleate isomerase PbFu. The amino acid sequence of the wild-type maleate isomerase PbFu is shown in SEQ ID NO.1, and the specific mutations are as follows:

[0024] (1) Fu-a: A115I_G139L_S250Y;

[0025] (2) Fu-b: A114I_G139L_S250Y;

[0026] Wherein A115I_G139L_S250Y means that the amino acid at position 115 is mutated from alanine to isoleucine, the amino acid at position 139 is mutated from glycine to leucine, and the amino acid at position 250 is mutated from serine to tyrosine; A114I_G139L_S250Y means that the amino acid at position 114 is mutated from alanine to isoleucine, the amino acid at position 139 is mutated from glycine to leucine, and the amino acid at position 250 is mutated from serine to tyrosine.

[0027] On the other hand, the present invention also provides a nucleic acid molecule encoding the maleate isomerase mutant.

[0028] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4 or SEQ ID NO.6.

[0029] On yet another aspect, the present invention also provides a vector containing the nucleic acid molecule.

[0030] Furthermore, in some embodiments of the present invention, the above vector is a cloning vector or an expression vector; more preferably, it is a recombinant expression vector for expression in bacteria, such as Escherichia coli ( E.coli ). Preferably, in some embodiments of the present invention, the expression vector is pET28a.

[0031] On still another aspect, the present invention also provides a host cell containing the vector.

[0032] Preferably, the host cell is a host cell for expressing the maleate isomerase from the vector, preferably a bacterial cell, such as Escherichia coli ( E.coli ). Preferably, in some embodiments of the present invention, the above Escherichia coli isE. coli BL21(DE3).

[0033] Experiments show that, in terms of the conversion of the substrate maleic acid to fumaric acid, the maleic acid isomerase mutant provided by the present invention has the use of catalyzing the isomerization of maleic acid to fumaric acid with higher catalytic efficiency.

[0034] The present invention also provides the application of the maleic acid isomerase PbFu, the maleic acid isomerase mutants Fu-a and Fu-b, the nucleic acid molecule, the vector or the host cell in catalyzing the isomerization of maleic acid to fumaric acid. In the said application, the maleic acid isomerase PbFu, the maleic acid isomerase mutant or the maleic acid isomerase or maleic acid isomerase mutant obtained from the gene sequence, the vector or the host cell can be in the free form or immobilized form of the enzyme.

[0035] The present invention also provides a method for preparing fumaric acid, using the said host cell as a catalyst and maleic acid as a substrate to transform and prepare fumaric acid.

[0036] Preferably, the conditions for the said transformation and preparation are: using a buffer solution with a pH of 5.0 - 8.0 as the reaction medium and carrying out the transformation reaction at a temperature of 20 - 40°C.

[0037] More preferably, before the transformation and preparation, the said host cell is induced to culture, and the conditions for the said induction culture are: adding IPTG with a final concentration of 0.1 - 0.2 mM, and inducing the culture at 27 - 29°C and 180 - 200 rpm for 9 - 11 h.

[0038] Furthermore, in some embodiments of the invention, the concentration of the maleic acid isomerase engineering bacteria cells in the reaction system is 5 - 40 g / L.

[0039] Preferably, in some embodiments of the invention, the concentration of the maleic acid isomerase engineering bacteria cells in the reaction system is 30 g / L.

[0040] Furthermore, in some embodiments of the invention, the said buffer solution is PBS buffer solution with a pH of 7.4 - 7.6.

[0041] Preferably, in some embodiments of the invention, the said buffer solution is PBS buffer solution with a pH of 7.5.

[0042] Preferably, in some embodiments of the invention, the temperature of the transformation reaction is 40°C.

[0043] Furthermore, in some embodiments of the invention, the said maleic acid isomerase engineering bacteria are prepared by the following method:

[0044] The engineered bacteria containing the above-mentioned maleate isomerase gene are inoculated into an LB liquid medium containing kanamycin at a final concentration of 40 - 60 mg / L, and cultured at 36 - 38 °C and 180 - 200 rpm for 8 - 10 h to obtain a seed culture solution;

[0045] The seed culture solution is inoculated into an LB liquid medium containing kanamycin at a final concentration of 40 - 60 mg / L at an inoculation amount of 1% (v / v), and cultured at 36 - 38 °C and 180 - 200 rpm until the OD600 of the culture solution reaches 0.6 - 0.8. IPTG with a final concentration of 0.1 - 0.2 mM is added to the culture solution, and induced culture is carried out at 27 - 29 °C and 180 - 200 rpm for 9 - 11 h. The culture solution is centrifuged, and the precipitate is rinsed with PBS buffer to obtain the engineered bacteria cells of maleate isomerase as a catalyst.

[0046] Furthermore, in some embodiments of the invention, the LB liquid medium comprises: 8 - 12 g / L peptone, 4.5 - 5.5 g / L yeast extract, 9 - 11 g / L sodium chloride, and the pH value is 6.8 - 7.2.

[0047] The nucleic acid molecule for preparing fumaric acid provided by the present invention encodes maleate isomerase, and the base sequence is as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6; the recombinant engineered bacteria transformed with this nucleic acid molecule can be used as a catalyst to catalyze maleic acid substrate to form fumaric acid;

[0048] The present invention has the following beneficial effects:

[0049] The application of the wild-type maleate isomerase PbFu provided by the present invention and two maleate isomerase mutants obtained by mutating the wild-type maleate isomerase PbFu in catalyzing maleic acid substrate to form fumaric acid all have good activity and catalytic efficiency. Among them, the activities of the mutants with the two mutation methods of A115I_G139L_S250Y and A114I_G139L_S250Y are increased by 1.8 times and 2.4 times respectively compared with the wild-type maleate isomerase PbFu, and the catalytic efficiencies are increased by 29% and 39% respectively. Specific Embodiments

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0051] The features and properties of the present invention will be further described in detail below with reference to the embodiments.

[0052] The sources of the main experimental materials used in the following examples are as follows:

[0053] Escherichia coli host strain E. coli BL21(DE3) was purchased from Invitrogen, the expression vector pET-28a(+) was purchased from Novagen, and the restriction endonucleases Nco I and Xho I were purchased from Fermentas. T4 DNA ligase and kanamycin were both purchased from Dalian Taosheng Biotechnology Co., Ltd.; isopropyl-β-D-thiogalactopyranoside (IPTG) was a product of Promega. DNA Marker and the staining agent Gold View were purchased from TakaRa; the Axygen DNA gel extraction kit, plasmid extraction kit, and PCR purification kit were purchased from Axygen Biotechnology Co., Ltd.

[0054] Example 1

[0055] Synthesis of the maleate isomerase gene

[0056] The maleate isomerase gene sequences shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6 were synthesized by total synthesis through conventional genetic engineering operations PbFu, Fu-a, Fu-b , and the amino acid sequences of the maleate isomerase encoded by this gene are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5.

[0057] Example 2

[0058] Construction of maleate isomerase gene expression vectors and their recombinant transformants

[0059] The synthesized maleate isomerase gene and pET28a were each digested with Nco I and Xho I. After digestion for about 5 h, the digestion products were recovered and ligated with T4 ligase at 16 °C for 16 h to obtain the recombinant expression plasmids pET28a-PbFu, pET28a-Fu-a, and pET28a-Fu-b.

[0060] The expression vectors pET28a-PbFu, pET28a-Fu-a, and pET28a-Fu-b were respectively transformed into E. coli BL21(DE3) recipient bacteria, spread on LB agar plates containing kanamycin (final concentration 50 mg / L), and cultured overnight at 37 °C. Colonies grew on the plates.

[0061] Single colonies were randomly selected, cultured, and plasmids were extracted for sequencing. The sequencing results showed that positive clones were obtainedE. coli BL21(DE3) / pET28b-PbFu, E. coli BL21(DE3) / pET28b-Fu-a, E. coli BL21(DE3) / pET28b-Fu-b.

[0062] Example 3

[0063] Expression of maleate isomerase.

[0064] The recombinant transformants obtained in Example 2 E. coli BL21(DE3) / pET28b-PbFu, E. coli BL21(DE3) / pET28b-Fu-a, E. coli BL21(DE3) / pET28b-Fu-b were respectively inoculated into LB liquid medium and cultured at 37 °C and 150 rpm for 10 - 12 h;

[0065] The LB liquid medium included: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the pH value was 7.0.

[0066] Then, they were inoculated into LB liquid medium containing kanamycin (final concentration 50 mg / L) at an inoculation amount of 2% by volume for scale-up culture. They were cultured at 37 °C and 150 rpm until the OD600 of the culture broth was between 0.6 - 0.8. IPTG was added to a final concentration of 0.1 mM, and they were induced to culture at 28 °C and 180 rpm for 10 h. The culture broth was centrifuged to collect the cells, and the wet cells were obtained after washing twice with physiological saline.

[0067] Example 4

[0068] Verification of the activity of maleate isomerase.

[0069] The wet cells obtained in Example 3 were subjected to a resting cell catalysis reaction to verify the activity of maleate isomerase towards maleic acid.

[0070] Experimental method: Weigh 0.4 g of wet cells (final concentration 40 g / L) and suspend them in 10 ml of 30 g / L maleic acid solution. React at 40 °C and 200 rpm for 10 min.

[0071] After the reaction was completed, 1 ml of the reaction solution was taken, 1 ml of 2 mol HCl was added to terminate the reaction, and then the supernatant was taken by centrifugation, diluted, and filtered through a 0.22 μm inorganic membrane.

[0072] The sample was analyzed by high performance liquid chromatography (C8 column). The analysis conditions of high performance liquid chromatography were as follows: the mobile phase was methanol∶0.1% phosphoric acid = 9∶1, the column temperature was 30 °C, the flow rate was 0.8 ml / min, and the detection wavelength was 214 nm. One enzyme activity unit was defined as the amount of enzyme required to catalyze 1 μmol of maleic acid per minute under the analysis conditions.

[0073] Table 1 shows the verification results of the catalytic activity of maleate isomerase measured using recombinant Escherichia coli BL21 / pET28b-PbFu, BL21 / pET28b-Fu-a, and BL21 / pET28b-Fu-b as enzyme sources.

[0074] Table 1

[0075]

[0076] It can be seen from Table 1 that Escherichia coli BL21 / pET28a-PbFu, BL21 / pET28b-Fu-a, and BL21 / pET28b-Fu-b after induced expression had enzyme activity.

[0077] Example 5

[0078] The method for preparing fumaric acid using the maleate isomerase engineering bacteria provided in this example is as follows:

[0079] Each group weighed 0.4 g of the wet bacterial cells obtained in Example 3 (final concentration 40 g / L) and suspended them in 10 ml of 30 g / L maleic acid solution, and reacted at 40 °C and 200 rpm for 1 h.

[0080] After the reaction was completed, 1 ml of the reaction solution was taken, 1 ml of 2 mol HCl was added to terminate the reaction, and then the supernatant was taken by centrifugation, diluted, and filtered through a 0.22 μm inorganic membrane.

[0081] The sample was analyzed by high performance liquid chromatography (C8 column). The analysis conditions of high performance liquid chromatography were as follows: the mobile phase was methanol∶0.1% phosphoric acid = 9∶1, the column temperature was 30 °C, the flow rate was 0.8 ml / min, and the detection wavelength was 214 nm.

[0082] Results: Maleate isomerase PbFu catalyzed maleic acid to produce fumaric acid. After 1 h of reaction, the remaining maleic acid was 17.1 g / L, and the catalytic efficiency was 43%. Maleate isomerase Fu-a catalyzed maleic acid to produce fumaric acid. After 1 h of reaction, the remaining maleic acid was 8.4 g / L, and the catalytic efficiency was 72%. Maleate isomerase Fu-b catalyzed maleic acid to produce fumaric acid. After 1 h of reaction, the remaining maleic acid was 5.3 g / L, and the catalytic efficiency was 82%.

Claims

1. A maleate isomerase mutant, characterized in that The maleate isomerase mutant is obtained by mutation of the wild-type maleate isomerase, and the amino acid sequence of the wild-type maleate isomerase is shown in SEQ ID NO.1; the mutation method is any one of the following: (1) A114I, G139L and S250Y; (2) A115I, G139L and S250Y.

2. A nucleic acid molecule encoding the maleate isomerase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.4 or SEQ ID NO.

6.

4. A vector comprising the nucleic acid molecule according to claim 2 or 3.

5. A host cell comprising the vector according to claim 4.

6. Use of the wild-type maleate isomerase according to claim 1, the maleate isomerase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the vector according to claim 4 or the host cell according to claim 5 in catalyzing the isomerization of maleate to produce fumarate.

7. A method for preparing fumaric acid, characterized in that: The host cell according to claim 5 is used as a catalyst and maleic acid is used as a substrate to convert and prepare fumaric acid.

8. The method for preparing fumaric acid according to claim 7, characterized in that: The conditions for the conversion preparation are: using a buffer solution with a pH of 5.0-8.0 as a reaction medium and carrying out the conversion reaction at a temperature of 20-40°C.

9. The method for preparing fumaric acid according to claim 8, characterized in that: The host cells described above are transformed and cultured under the conditions of induction: adding IPTG with a final concentration of 0.1-0.2 mM, and inducing culture at 27-29° C. and 180-200 rpm for 9-11 h.

10. The method for preparing fumaric acid according to claim 8, characterized in that: The concentration of the host cells in the reaction system is 5-40 g / L.

Images