A rare earth-dependent thermostable alcohol dehydrogenase mutant and application thereof

Abstract

The application discloses a rare earth-dependent heat-stable alcohol dehydrogenase mutant and application thereof, and relates to the technical field of enzyme engineering. The rare earth-dependent heat-stable alcohol dehydrogenase mutant constructed by the application has improved stability compared with a wild type (62.51 DEG C), wherein the stability of the mutant S155T / S216T / A375G / G419P (72.07 DEG C) is improved by 9.6 DEG C, and the enzyme activity (0.53 U / mg) of the mutant is slightly higher than that (0.5 U / mg) of the wild type. The application constructs a rare earth-dependent heat-stable alcohol dehydrogenase mutant, and uses the rare earth enzyme mutant catalyst with HMF activity and high stability to catalyze FDCA in the conversion of HMF. The application is expected to change the technical monopoly situation of single enzyme source in the process of catalyzing HMF to FDCA by a single enzyme, and has a promoting effect on realizing green manufacturing of FDCA.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a rare earth-dependent thermostable alcohol dehydrogenase mutant and its applications. Background Technology

[0002] 2,5-Furandicarboxylic acid (FDCA), as an important bio-based platform compound, has its most promising application as a substitute for terephthalic acid (PTA) in the synthesis of renewable polyethylene 2,5-furandicarboxylic acid (PEF), which is widely used in the production of various bio-based polymers (such as polyamides, polyesters, and polyurethanes). Currently, FDCA is mainly synthesized chemically, using noble metal catalysts to oxidize 5-hydroxymethylfurfural (HMF) under high temperature and pressure with O2. The process mainly involves two steps: first, the aldehyde group on the HMF side chain is oxidized to generate a carboxyl group, yielding 5-hydroxymethyl-2-furancarboxylic acid (HFCA); then, the hydroxymethyl group on the HFCA side chain is further oxidized to generate an aldehyde group (FFCA) and FDCA. However, the chemical production of FDCA requires organic solvents and high concentrations of alkali, resulting in high costs and significant pollution.

[0003] The bioenzymatic conversion of HMF to FDCA has attracted widespread attention due to its high selectivity and mild conditions. Currently, enzymes that catalyze the oxidation of HMF mainly include xanthine oxidase, laccase, and lipase. However, because enzymes have different selectivities for HMF intermediates, it is usually difficult to achieve the direct catalysis of HMF to FDCA with a single enzyme.

[0004] Rare earth-dependent alcohol dehydrogenases (Ln-ADHs) are a class of alcohol dehydrogenases that catalyze the conversion of alcohols or aldehydes to aldehydes or acids in the absence of oxygen. They are mainly distributed in methyl-metabolizing bacteria, but have also been found in non-methyl-metabolizing bacteria such as *Pseudomonas putida* KT2440. Ln-ADHs are selective for the substrates of oxidation, primarily targeting primary alcohols or aldehydes for dehydrogenation. The molecular weight of Ln-ADHs is typically around 130 KD. Ln-ADHs have binding sites for pyrroloquinoline quinone (PQQ) and rare earth ions (La-Lu, etc.), where the rare earth ions act as Lewis acids, and PQQ participates in the oxidation reaction as a cofactor. During the dehydrogenation reaction, the cofactor PQQ in Ln-ADH is reduced to PQQH2, and then a hydrogen proton is transferred to a hydrogen acceptor, completing the hydrogen transfer, i.e., the dehydrogenation process. Oxygen is not required in the dehydrogenation reaction, and no hydrogen peroxide is generated. Under certain conditions, alcohols can be directly converted to acids. Ln-ADH therefore has the potential to be applied to the single-enzyme conversion of HMF to FDCA.

[0005] Rare earth enzymes derived from methyl-metabolizing bacteria are difficult to express correctly in *E. coli*, limiting their in vitro catalytic applications. *Pseudomonas putida* KT2440 has similar characteristics to *E. coli*, therefore, rare earth enzymes derived from KT2440 can be normally expressed and purified in *E. coli* while maintaining high activity. However, rare earth enzymes derived from *Pseudomonas putida* KT2440 suffer from low activity and low stability, limiting their industrial applications. Currently, there are no known cases of using rare earth-dependent alcohol dehydrogenases to catalyze the conversion of HMF to FDCA using a single enzyme. Therefore, improving the stability of rare earth-dependent alcohol dehydrogenases is beneficial for promoting their industrial applications. Summary of the Invention

[0006] The purpose of this invention is to provide a rare-earth-dependent, thermostable alcohol dehydrogenase mutant with high activity and stability, and the application of this highly stable mutant in catalyzing the production of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural. Identifying the potential mutation sites for rare-earth-dependent alcohol dehydrogenases using computer-aided protein design methods, and then screening for mutants with improved stability and / or enhanced HMF activity, is of great significance.

[0007] The specific technical solution is as follows: Wild-type rare earth-dependent alcohol dehydrogenases are derived from *Pseudomonas putida* (… Pseudomonas putida KT2440, whose amino acid sequence is shown in SEQ ID NO.1, and whose encoding gene is shown in SEQ ID NO.2. The mutant was modified by site-directed mutagenesis to increase the thermal stability of the protein. The mutation site is one or more of the following: (1) position 155, (2) position 216, (3) position 375, (4) position 419, and the specific mutation is any one of the following: (1) The amino acid at position 155 is mutated from serine to threonine; (2) The amino acid at position 216 is mutated from serine to threonine; (3) The amino acid at position 375 is mutated from alanine to glycine; (4) The amino acid at position 419 is mutated from glycine to proline; (5) The amino acid at position 419 is mutated from glycine to methionine; (6) The amino acid at position 155 is mutated from serine to threonine, and the amino acid at position 419 is mutated from glycine to proline; (7) The amino acid at position 155 is mutated from serine to threonine, and the amino acid at position 216 is mutated from serine to threonine; (8) The 155th amino acid is mutated from serine to threonine, and the 375th amino acid is mutated from alanine to glycine; (9) The 216th amino acid is mutated from serine to threonine, and the 375th amino acid is mutated from alanine to glycine; (10) The amino acid at position 216 is mutated from serine to threonine, and the amino acid at position 419 is mutated from glycine to proline; (11) The amino acid at position 375 is mutated from alanine to glycine, and the amino acid at position 419 is mutated from glycine to proline; (12) The amino acid at position 155 was mutated from serine to threonine, the amino acid at position 216 was mutated from serine to threonine, and the amino acid at position 375 was mutated from alanine to glycine; (13) The amino acid at position 155 was mutated from serine to threonine, the amino acid at position 216 was mutated from serine to threonine, and the amino acid at position 419 was mutated from glycine to proline; (14) The amino acid at position 216 was mutated from serine to threonine, the amino acid at position 375 was mutated from alanine to glycine, and the amino acid at position 419 was mutated from glycine to proline; (15) The amino acid at position 155 was mutated from serine to threonine, the amino acid at position 216 was mutated from serine to threonine, the amino acid at position 375 was mutated from alanine to glycine, and the amino acid at position 419 was mutated from glycine to proline.

[0008] Among them, the mutants S155T, S216T, A375G, G419P, G419M, S155T / G419P, S155T / S216T, S155T / A375G, S216T / A375G, S216T / G419P, A375G / G419P, S155T / S216T / A375G, S155T / S216T / G419P, S155T / A375G / G419P, S216T / A375G / G419P, and S155T / S216T / A375G / G419P all showed higher stability than the wild-type WT. Among them, the mutants S155T / G419P, S155T / S216T / A375G, S216T, S155T, S155T / S216T / A375G / G419P, S216T / G419P, and S155T / S216T all showed higher enzyme activity and stability than the wild-type WT.

[0009] S155T indicates that the 155th amino acid is changed from serine to threonine; S155T / G419P indicates that the 155th amino acid is changed from serine to threonine, and the 419th amino acid is changed from glycine to proline; the others are similar.

[0010] The present invention also provides the application of the rare earth-dependent, heat-stable alcohol dehydrogenase mutant in the catalytic production of 5-hydroxymethylfurfural from 5-hydroxymethyl-2-furanoic acid.

[0011] The present invention also provides a gene encoding the rare earth-dependent, thermostable alcohol dehydrogenase mutant.

[0012] The present invention also provides the application of the gene in catalyzing the production of 5-hydroxymethylfurfural from 5-hydroxymethyl-2-furanoic acid. The present invention also provides an expression vector comprising the gene.

[0013] The present invention also provides a genetically engineered bacterium expressing the rare earth-dependent, thermostable alcohol dehydrogenase mutant.

[0014] The present invention also provides the application of the genetically engineered bacteria in catalyzing the production of 5-hydroxymethyl-2-furanoic acid from 5-hydroxymethylfurfural.

[0015] The present invention also provides a method for preparing 5-hydroxymethyl-2-furanic acid, wherein the rare earth-dependent heat-stable alcohol dehydrogenase mutant is used to catalyze the oxidation reaction of 5-hydroxymethylfurfural to prepare 5-hydroxymethyl-2-furanic acid.

[0016] Specifically, using wet cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the encoding gene of wild-type rare earth-dependent alcohol dehydrogenase or mutant, or the supernatant obtained by ultrasonic disruption of wet cells, and enzyme purified by nickel affinity chromatography as a catalyst, HMF as a substrate, PQQ as a coenzyme, rare earth ions as the active center, phenazine ethyl sulfate (PES) or phenazine methyl sulfate (PMS) as electron transporters, dichlorophenolindophenol (DCPIP) or N,N,N',N'-tetramethyl-p-phenylenediamine (WB) as hydrogen acceptors, and 25 mM imidazole as an activator, the reaction is carried out in Tris-HCl buffer at pH 8.0 at 30°C.

[0017] The beneficial effects of this invention are: The rare earth-dependent thermostable alcohol dehydrogenase mutant constructed in this invention has improved stability compared with the wild type (62.51℃). Among them, the stability of mutant S155T / S216T / A375G / G419P (72.07℃) is 9.6℃ higher than that of wild type, and its enzyme activity (0.53 U / mg) is also slightly higher than that of wild type WT (0.5 U / mg).

[0018] This invention constructs a rare-earth-dependent, thermostable alcohol dehydrogenase mutant, using a rare-earth enzyme mutant catalyst with HMF activity and high stability for the conversion of HMF to FDCA. This invention is expected to change the current technological monopoly of relying on a single enzyme source in the single-enzyme catalysis of HMF to FDCA, and will promote the green manufacturing of FDCA. Attached Figure Description

[0019] Figure 1 A three-dimensional structural model of the rare earth-dependent alcohol dehydrogenase PedH (6ZCW.pdb).

[0020] Figure 2 A schematic diagram of PedH catalysis for the production of FDCA from HMF.

[0021] Figure 3 For point mutations and their ΔT m Value graph. Detailed Implementation

[0022] Reagents used in upstream genetic engineering: those used in the embodiments of this invention E. coli BL21(DE3) and plasmid pET28a were purchased from Novagen; the gene synthesis, primer synthesis and sequence sequencing of PedH were completed by Qingke Biotechnology Co., Ltd.

[0023] Reagents used in the catalytic reaction: Tris and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; PQQ (pyrroloquinoline quinone) and PMS (phenazine methyl sulfate) were purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; neodymium chloride and HMF (5-hydroxymethylfurfural) were purchased from Aladdin Reagent (Shanghai) Co., Ltd.; DCPIP (2,6-dichlorophenolindophenol) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0024] Enzyme activity units (U) are defined using the generally accepted academic definition: the amount of enzyme required to consume 1 μM DCPIP per minute in the reaction system. The amount of DCPIP consumed in the reaction system was quantitatively analyzed using a microplate reader, and the specific enzyme activities of the original rare-earth-dependent alcohol dehydrogenase and the rare-earth-dependent thermostable alcohol dehydrogenase mutant to the substrate 5-hydroxymethylfurfural were calculated. The reaction system contained appropriate amounts of enzyme solution, 15 μM PQQ, 15 μM NdCl3, and 10 mM 5-hydroxymethylfurfural, with a total volume of 200 μL. The reaction medium was Tris HCl buffer (100 mM pH 8.0), containing 1 mM PMS and 200 μM DCPIP.

[0025] Figure 2 This is a schematic diagram of PedH catalyzing the production of FDCA from HMF. PedH can act in every step of the process.

[0026] Example 1 Construction of wild-type rare earth-dependent alcohol dehydrogenase recombinant bacteria.

[0027] The rare earth-dependent alcohol dehydrogenase PedH (amino acid sequence shown in SEQ ID NO. 1, three-dimensional structural model shown in [image missing]) derived from *Pseudomonas putida* KT2440 is shown in [image missing]. Figure 1 (As shown) is a template, based on E. coli Codon optimization was performed based on codon bias, and the nucleotide sequence of the enzyme was synthesized by Suzhou Genewiz using a whole-genome synthesis method, as shown in SEQ ID NO.2. A 6×his-tag coding sequence was added to the end of the PedH gene sequence and cloned into the restriction enzyme site of the vector pET28a(+). BamH I and Hind Between stages III, the recombinant expression plasmid pET28b-PedH was obtained. The recombinant plasmid was then transformed into [a specific target] using a heat shock method. E. coli After sequencing verification, wild-type rare earth-dependent alcohol dehydrogenase recombinant bacteria were obtained from BL21(DE3) competent cells.

[0028] Example 2 Design of rare earth-dependent alcohol dehydrogenase mutants.

[0029] High-performance mutants of PedH were predicted using the online tools Fireprot (https: / / loschmidt.chemi.muni.cz / fireprotweb / ) and PROSS (https: / / pross.weizmann.ac.il / step / pross-terms / ). The results from both online tools were combined, and four potential mutation sites were identified based on a set of single-point mutations.

[0030] Example 3 Construction of rare earth-dependent thermostable alcohol dehydrogenase mutants.

[0031] Primers were designed based on the mutant obtained in Example 2 to perform site-directed mutagenesis on PedH. The specific method is as follows: 1. Whole plasmid PCR Using pET28a-PedH plasmid as a template, upstream and downstream primers covering the mutation sites were designed (Table 1) for whole plasmid PCR to obtain recombinant genes. The recombinant genes are the nucleotide sequences shown in SEQ ID NO.2, where AGC is mutated to ACC (S155T), AGC is mutated to ACC (S216T), GCA is mutated to GGT (A375G), GGT is mutated to CCT (G419P), and GGT is mutated to ATG (G419M).

[0032] Table 1 Primers used for site-directed mutagenesis construction

[0033] PCR amplification system: 25 μL of DNA polymerase Upstream primer (10 μM) 1 μL, Downstream primer (10 μM) 1 μL, Template (5 ng / μL) 1μL, ddH2O 22μL.

[0034] PCR amplification conditions: 1) Pre-denaturation: 98℃ for 3 min; 2) Denaturation: 98℃ for 10s; Annealing: 60℃ for 15s; Extension: 72℃ for 1min; 33 cycles in total; 3) Post-extension: 72℃ for 5 minutes; 4) Store at 4℃.

[0035] 2. Conversion and Validation The above PCR products were directly transformed into [the target material] using a heat shock method. E. coli After sequencing verification, rare earth-dependent thermostable alcohol dehydrogenase mutant strains were obtained from BL21(DE3) competent cells and named mutant strains S155T, S216T, A375G, G419P and G419M.

[0036] Example 4 Strains culture and protein expression and purification.

[0037] Single colonies of the correctly sequenced recombinant bacteria (original rare-earth-dependent alcohol dehydrogenase recombinant bacteria and rare-earth-dependent thermostable alcohol dehydrogenase mutant strain) from Example 3 were incubated in 5 mL LB liquid medium (containing 50 μg / mL kanamycin) at 37°C with shaking for 12 h. The culture was then transferred at a 2% inoculum to 50 mL LB liquid medium containing 50 μg / mL kanamycin and 10 g / L α-lactose, and incubated at 30°C with shaking for 12 h.

[0038] After culturing, the bacterial culture was centrifuged at 4000×g at 4℃ for 15 min, the supernatant was discarded, and the bacterial cells were collected. The cells were washed with 100mM Tris-HCl buffer (pH 8.0), resuspended in Tris-HCl buffer, and sonicated in an ice-water bath until clear. The lysed culture was centrifuged at 8000×g at 4℃ for 20 min, and the supernatant was subjected to affinity chromatography using a nickel column. Impurities were washed away with washing buffer (100mM Tris-HCl buffer, 150mM NaCl, 50mM imidazole, pH 8.0), and the target protein was eluted with elution buffer (100mM Tris-HCl buffer, 150mM NaCl, 250mM imidazole, pH 8.0). The isolated target protein was placed in an ultrafiltration centrifuge tube for thorough desalting and concentration to obtain a pure target protein. The theoretical molecular weight of PedH (63.0 kDa) was determined by SDS-PAGE.

[0039] Example 5 Rare earth-dependent alcohol dehydrogenases T m Value determination.

[0040] The concentration of rare earth-dependent alcohol dehydrogenase and its mutant was uniformly adjusted to 0.5 mg / ml and added to a high-precision quartz glass capillary array. Protein stability was measured using a Prometheus NT.Plex protein analyzer. T m Values, measurement results are shown in Figure 3 ,in T m The single-point mutations that increased the value are S155T (increased by 3.03℃), S216T (increased by 0.66℃), A375G (increased by 4.71℃), G419P (increased by 1.77℃), and G419M (increased by 0.16℃).

[0041] Example 6 Construction of rare earth-dependent thermostable alcohol dehydrogenase combinatorial mutants.

[0042] To further improve the thermal stability modification effect, this invention further combines the single-point mutants to obtain combined mutants with more significant effects. Among the single-point mutants, the mutants obtained in Example 5—S155T mutant, S216T mutant, A375G mutant, and G419P mutant—showed significantly improved stability. Therefore, these four single-point mutants were further combined to obtain mutants with even better stability.

[0043] Using the same method as in Example 1, combined mutagenesis was performed to obtain 11 combined mutants with further improved stability compared to single-point mutants. The genes encoding the 11 combined mutants are as follows: AGC mutation to ACC and GGT mutation to CCT (S155T / G419P) in the nucleotide sequence shown in SEQ ID NO.1; AGC mutation to ACC and AGC mutation to ACC (S155T / S216T); AGC mutation to ACC and GCA mutation to GGT (S155T / A375G); AGC mutation to ACC and GCA mutation to GGT (S216T / A375G); AGC mutation to ACC and GGT mutation to CCT (S216T / G419P); GCA mutation to GGT and GGT mutation to CCT (A375G / G419P); AGC mutation to ACC; AGC mutation to ACC and GCA mutation to CCT (A375G / G419P); AGC mutation to ACC; AGC mutation to ACC and GCA mutation to CCT (S216T / G419P); AGC mutation to ACC; AGC mutation to ACC and GCA mutation to CCT (S375G ... It becomes GGT (S155T / S216T / A375G), AGC mutates to ACC, AGC mutates to ACC and GGT mutates to CCT (S155T / S216T / G419P), AGC mutates to ACC, GCA mutates to GGT and GGT mutates to CCT (S155T / A375G / G419P), AGC mutates to ACC, GCA mutates to GGT and GGT mutates to CCT (S216T / A375G / G419P), AGC mutates to ACC, AGC mutates to ACC, GCA mutates to GGT and GGT mutates to CCT (S155T / S216T / A375G / G419P).

[0044] The constructed E. coli vector of the combined mutant was used to express and purify the combined mutant protein according to the same method as in Example 4 to obtain the pure enzyme solution of the combined mutant.

[0045] The concentrations of rare-earth-dependent alcohol dehydrogenase, single-point mutants, and combined mutants were uniformly adjusted to 0.5 mg / ml and added to a high-precision quartz glass capillary array. Protein stability was measured using a Prometheus NT.Plex protein analyzer. T m The values ​​and results are shown in Table 2, for all mutants. T m The values ​​were all higher than those of wild-type WT.

[0046] Table 2

[0047] Example 7 Determination of the activity of rare earth-dependent alcohol dehydrogenases.

[0048] The amount of DCPIP consumed in the reaction system was quantitatively analyzed using a microplate reader. The specific enzyme activities of the original rare-earth-dependent alcohol dehydrogenase and the rare-earth-dependent thermostable alcohol dehydrogenase mutant to the substrate 5-hydroxymethylfurfural were calculated. The reaction system contained appropriate amounts of enzyme solution, 1.5 μM PQQ, 1.5 μM NdCl3, 10 mM substrate, and 25 mM imidazole, with a total volume of 200 μL. The reaction medium was Tris-HCl buffer (100 mM, pH 8.0) containing 1 mM PMS and 200 μM DCPIP. The results of enzyme activity assays are shown in Table 2. Among them, the mutants S155T / G419P, S155T / S216T / A375G, S216T, S155T, S155T / S216T / A375G / G419P, S216T / G419P, and S155T / S216T all showed higher activity than the wild-type WT.

Claims

1. A rare earth-dependent, thermostable alcohol dehydrogenase mutant, characterized in that, It is from Pseudomonas putida ( Pseudomonas putida It is obtained by mutating the wild-type rare earth-dependent alcohol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.1, and the specific mutation is any one of the following (1)-(7): (1) The amino acid at position 155 is mutated from serine to threonine; (2) The amino acid at position 155 is mutated from serine to threonine, and the amino acid at position 419 is mutated from glycine to proline; (3) The amino acid at position 155 is mutated from serine to threonine, and the amino acid at position 216 is mutated from serine to threonine; (4) The amino acid at position 155 is mutated from serine to threonine, and the amino acid at position 375 is mutated from alanine to glycine; (5) The amino acid at position 155 is mutated from serine to threonine, the amino acid at position 216 is mutated from serine to threonine, and the amino acid at position 375 is mutated from alanine to glycine; (6) The amino acid at position 155 is mutated from serine to threonine, the amino acid at position 216 is mutated from serine to threonine, and the amino acid at position 419 is mutated from glycine to proline; (7) The amino acid at position 155 was mutated from serine to threonine, the amino acid at position 216 was mutated from serine to threonine, the amino acid at position 375 was mutated from alanine to glycine, and the amino acid at position 419 was mutated from glycine to proline.

2. The application of the rare earth-dependent, heat-stable alcohol dehydrogenase mutant as described in claim 1 in catalyzing the oxidation of 5-hydroxymethylfurfural.

3. A gene encoding the rare earth-dependent, thermostable alcohol dehydrogenase mutant of claim 1.

4. The application of the gene as described in claim 3 in catalyzing the oxidation reaction of 5-hydroxymethylfurfural.

5. An expression vector comprising the gene of claim 3.

6. A genetically engineered bacterium expressing the rare earth-dependent, thermostable alcohol dehydrogenase mutant of claim 1.

7. The application of the genetically engineered bacteria as described in claim 6 in catalyzing the oxidation reaction of 5-hydroxymethylfurfural.

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