Formate Dehydrogenase Mutant with Improved Enzyme Activity and Stability and Construction Method thereof

Abstract

The present invention discloses a formate dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, which belongs to the technical field of genetic engineering. The mutant of the present invention is obtained by mutating alanine at a 10.sup.th site to cysteine based on the amino acid shown in SEQ ID NO. 2. The specific enzyme activity of the mutant enzyme obtained by the present invention is improved by 1.3 times compared with that before the mutation, a half-life period (t.sub.1/2) at 60 C. is increased by 6.8 times compared with that in the mutation period, the copper ion tolerance is increased by 30 times compared with that before the mutation, and when pH is 4, the stability is improved by 2.0 times, and the catalytic efficiency is increased by 1.4 times. The present invention shows that an amino acid residue at a 10.sup.th site is mutated to the cysteine which forms a correct disulfide bond with a cysteine residue at a 30.sup.th site of the natural formate dehydrogenase, so that the stability and the catalytic efficiency of the enzyme are improved, and the industrial application potential of the enzyme is improved.

Claims

1. A formate dehydrogenase mutant, wherein an amino acid sequence of the mutant is set forth in SEQ ID NO. 1.

2. A nucleotide sequence encoding the mutant of claim 1.

3. The nucleotide sequence according to claim 2, wherein the nucleotide sequence is set forth in SEQ ID NO. 3.

4. A recombinant expression vector comprising the nucleotide sequence according to claim 2.

5. A genetically engineered bacterium expressing the formate dehydrogenase mutant of claim 1.

6. The genetically engineered bacterium according to claim 5, wherein the genetically engineered bacterium comprises a recombinant plasmid having the nucleotide sequence set forth in SEQ ID NO. 3 connected to an expression vector.

7. The genetically engineered bacterium according to claim 5, wherein the genetically engineered bacterium is a recombinant Escherichia coli genetically engineered bacterium.

8. (canceled)

9. A method, wherein the method comprises expressing a formate dehydrogenase mutant with an amino acid sequence set forth in SEQ ID NO. 1, and constructing a coenzyme NADH cycling system by comprising the mutant.

10. The method of claim 9, comprising using the system for NADH regeneration in a bioconversion production.

Description

DETAILED DESCRIPTION

[0024] TV fermentation medium: 8 g.Math.L.sup.1 of yeast powder, 12 g.Math.L.sup.1 of peptone, 4.02 g.Math.L.sup.1 of K.sub.3PO.sub.4, 3 g.Math.L.sup.1 of NaCl, 2 g.Math.L.sup.1 of citric acid, 0.3 g.Math.L.sup.1 of ammonium ferric citrate, 10 g.Math.L.sup.1 of glycerinum, 2.5 g.Math.L.sup.1 of (NH).sub.4SO.sub.4, and MgSO.sub.4.7H.sub.2O.sub.2. The pH was adjusted to 7.2.

[0025] Definition of enzyme activity: an enzyme amount required by reducing 1 mol of NAD.sup.+ to NADH in every one minute was defined as one enzyme activity unit U. The specific enzyme activity was defined as the enzyme activity U.Math.mg.sup.1 of per unit protein.

[0026] Method for determining enzyme activity of formate dehydrogenase: a reaction system was 10 mmol.Math.L.sup.1 of potassium phosphate buffer solution with the pH being 7.5 which contains 1.67 mmol.Math.L.sup.1 of NAD and 167 mmol.Math.L.sup.1 of sodium formate. An appropriate amount of enzyme liquid was added to initiate the reaction, the reaction was performed for 1.5 minutes at 30 C., an absorbance value at 340 nm was measured every 30 s, and the data was recorded. The concentration of NADH was calculated according to the increment of the absorbance value at 340 nm of the reaction solution, the enzyme activity was calculated, and a reaction formula was as follows: NaCOOH+NAD.sup.+=CO.sub.2+NADH+Na.sup.+.

EXAMPLE 1: CONSTRUCTION OF A RECOMBINANT VECTOR COMPRISING THE FORMATE DEHYDROGENASE MUTANT

[0027] (1) Acquisition of mutant A10C: the nucleotide sequence shown in SEQ ID NO. 4 was adopted as a template, and F primer (the sequence was shown in SEQ ID NO. 5) and R primer (the sequence was shown in SEQ ID NO. 6) were adopted as primers to perform PCR, thus obtaining the recombinant gene shown in SEQ ID NO. 3.

[0028] (2) The recombinant gene and pET28a were bi-digested by utilizing EcoRI and XhoI and were ligated overnight at 16 C. by utilizing T4 DNA ligase after being purified. A ligation product was converted into E. coli BL21 competent cells in a chemical method. A conversion solution was smeared on an LB flat panel containing kanamycin (50 mg.Math.L.sup.1), plasmids were extracted, and the constructed recombinant plasmids were verified by virtue of double-restriction enzyme digestion and were named as pET28a-A10C. Sequencing work was carried out by Shanghai Sangon Biotech.

EXAMPLE 2: CONSTRUCTION OF RECOMBINANT Escherichia coli ENGINEERED BACTERIA PRODUCING THE FORMATE DEHYDROGENASE MUTANT

[0029] A strain comprising correct recombinant plasmids pET28a-A10C obtained in the embodiment 1 was the recombinant gene engineered bacteria pET28a-A10C/E. coli BL21 of the present invention.

EXAMPLE 3: EXPRESS FORMATE DEHYDROGENASE WITH RECOMBINANT BACTERIA pET28A-A10C/E. Coli BL21 AND ENZYME ACTIVITY DETERMINATION

[0030] The recombinant bacteria pET28a-A10C/E. coli BL21 constructed in the embodiment 2 and a reference strain pET28a-FDH/E. coli BL21 expressing non-mutated wild enzyme CboFDH (amino acid sequence was shown in SEQ ID NO.2) were separately inoculated into 10 mL of LB culture medium containing kanamycin, were oscillated at 37 C. and cultured overnight to be transferred into a TY fermentation medium at a subsequent day in an amount of 4% of the inoculation amount, and were induced for 16 hours at 24 C. with the addition of 0.5 mM of IPTG after being cultured at 37 C. for 4 h. Cells were collected by centrifuge and disrupted, and the cell supernatant (crude enzyme liquid) was used for determining the enzyme activity.

[0031] The obtained crude enzyme liquid was purified to obtain the formate dehydrogenase mutant A10C, kinetic parameters of the purified recombinant formate dehydrogenase mutant A1OC were analyzed, as shown in table 1, the affinity K.sub.m of the mutant enzyme A10C to the substrate NAD.sup.+ was decreased by 1.4 times compared with that before the mutation, while the affinity K.sub.m of the mutant enzyme A10C to the substrate formic acid was not significantly decreased compared with that before the mutation, the catalytic efficiency (k.sub.cat/K.sub.m).sup.NAD+ was increased by 1.4 times, and the specific enzyme activity was improved by 1.3 times co pared with that before the mutation.

TABLE-US-00001 TABLE 1 A10C reaction kinetic parameters Specific Km (kcat/Km) enzyme Km NAD formate kcat NAD activity Enzyme (M) (mM) (s.sup.1) (M.sup.1 s.sup.1) (U mg.sup.1) CboFDH 53.6 3.4 7.3 0.6 3.3 0.3 0.06 5.6 0.4 A10C 74.2 2.6 8.2 0.4 6.2 0.5 0.08 7.4 0.5

[0032] The copper ion tolerance of the obtained pure enzyme was analyzed, the enzyme activities of the wild-type CboFDH and the mutant enzyme A10C under the condition of different concentrations of copperions were detected, where the required concentration of the copper ions was 15 mM when the residual enzyme activity of the mutant enzyme was 50%, and the required concentration of the copper ions was smaller than 0.5 mM when the residual enzyme activity of the wild-type enzyme CboFDH was 50%. This showed that the tolerance of the mutant enzyme A10C to the copper ions was increased by more than 30 times.

[0033] Thermal stability analysis was carried out on the obtained pure enzyme, and the pure enzyme was hatched for different times at the temperature of 60 C., the half-life period of the mutant enzyme was 21.6 min and was increased by 6.8 times compared with the wild-type CboFDH (3.2 min).

[0034] PH stability analysis was carried out on the obtained pure enzyme, and the obtained pure enzyme was hatched for 1 h under the condition of different pH, it was discovered that the residual enzyme activity of the mutant enzyme A10C under the acid addition was higher than the wild-type enzyme CboFDH, where when the pH was 4, the residual enzyme activity of the mutant enzyme A10C was 89.2%, while the residual enzyme activity of the wild-type enzyme CboFDH was only 45.3%.