NADP < + >-dependent glucose dehydrogenase mutant and application thereof

Abstract

The invention discloses an NADP < + >-dependent glucose dehydrogenase mutant and application thereof. The mutant is obtained by replacing at least one amino acid in the 96th site, the 199th site and the 261th site of an amino acid sequence shown as SEQ ID No.1; glutamic acid Glu at the 96th site is changed into glutamine Gln, lysine Lys at the 199th site is mutated into methionine Met, and glycine Gly at the 261th site is mutated into arginine Arg. The glucose dehydrogenase mutant is obtained by modifying wild glucose dehydrogenase through a directed evolution technology, site-directed mutagenesis is carried out at key amino acid residues based on structural analysis of a wild enzyme activity pocket and an action site thereof, the mutant with significantly improved enzyme activity is obtained through screening, the reaction conditions of the mutant are optimized, and the yield of the mutant is improved. Therefore, the method has important value in the field of industrial application.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and genetic engineering, and particularly to an NADP... + Glucose dehydrogenase mutants dependent on glucose and their applications. Background Technology

[0002] Among the various types of glucose dehydrogenase (GDH), NADP + Dependent GDH (NADP) + GDH plays an irreplaceable core role in biomanufacturing due to its ability to efficiently regenerate the high-value reduced coenzyme NADPH. Unlike NADH, which is mainly used for energy metabolism, NADPH specializes in the biosynthesis of high-value-added products such as fatty acids and sterols, as well as cellular antioxidant defense. This functional specificity makes it irreplaceable by NADH. Furthermore, NADPH has a significantly higher market price than NADH, and its production cost is high while its stability is poor, further highlighting the urgency of developing efficient NADPH regeneration systems. Especially in high-end biocatalytic processes such as the synthesis of chiral drugs and high-value fine chemicals, many key enzymes (such as imine reductases and P450 monooxygenases) are strictly dependent on NADPH, making NADP, which can specifically and efficiently regenerate NADPH, a crucial technology. + -GDH becomes the key factor restricting the economic feasibility of the entire process. Therefore, developing NADP with high enzyme activity and high selectivity is crucial. + -GDH mutants have significant application value for reducing the cost of coenzyme use and promoting the upgrading of green biomanufacturing.

[0003] In the field of industrial biotechnology, NADP + The application of glucose-dependent dehydrogenases is crucial. They are widely designed into multi-enzyme coupling systems for the continuous regeneration of high-value NADPH, driving the catalytic cycles of key enzymes such as leucine dehydrogenase and P450 monooxygenase, thereby efficiently synthesizing high-value-added products such as chiral amino acids and pharmaceutical intermediates, significantly reducing coenzyme regeneration costs. Furthermore, this enzyme also shows clear application prospects in biosensing and in vitro diagnostics; its catalyzed NADPH generation reaction is easily detected by spectroscopic methods, providing a stable and sensitive detection basis for metabolite analysis. However, naturally derived or existing engineered enzymes still have significant limitations in practical scale-up and continuous applications, mainly manifested in insufficient catalytic efficiency (kcat, kcat / Km) and limited sensitivity to NADP. + The unsatisfactory selectivity, poor thermal stability and resistance to processing conditions, easy suppression of substrates or products, and poor interfacial compatibility with supports or electrodes limit the development of NADP. + The promotion and application of GDH in cofactor regeneration modules and industrial biocatalysis systems. Summary of the Invention

[0004] Purpose of the invention: The primary objective of this invention is to provide a catalyst with both high catalytic activity and high NADP content. + Selective NADP + A glucose-dependent dehydrogenase mutant; a second object of the present invention is to provide the NADP-dependent glucose dehydrogenase mutant. + The application of glucose dehydrogenase mutants that are dependent on glucose.

[0005] Technical solution: The NADP described in this invention + The mutant is a glucose dehydrogenase-dependent mutant, wherein at least one amino acid is replaced at positions 96, 199, and 261 of the amino acid sequence shown in SEQ ID No. 1; glutamic acid at position 96 (Glu) is replaced with glutamine (Gln), lysine at position 199 (Lys) is replaced with methionine (Met), and glycine at position 261 (Gly) is replaced with arginine (Arg).

[0006] The NADP + The glucose-dependent dehydrogenase is derived from Bacillus megaterium, with the amino acid sequence SEQ ID NO.1 and the gene sequence SEQ ID NO.2.

[0007] Preferably, the mutant is K199M, K199M / G261R, or K199M / G261R / E96Q.

[0008] The mutant K199M is formed by the mutation of lysine at position 199 (Lys) to methionine (Met).

[0009] The mutant K199M / G261R has a 199th lysine (Lys) mutated to methionine (Met) and a 261st glycine (Gly) mutated to arginine (Arg).

[0010] The mutant K199M / G261R / E96Q has a 199th position lysine (Lys) mutated to methionine (Met), a 261st position glycine (Gly) mutated to arginine (Arg), and a 96th position glutamate (Glu) mutated to glutamine (Gln).

[0011] The gene described in this invention is: encoding the NADP... + The gene for a glucose dehydrogenase mutant protein that is dependent on glucose.

[0012] The recombinant plasmid described in this invention is a recombinant plasmid containing the aforementioned gene.

[0013] Preferably, the expression vector of the recombinant plasmid is a PET series expression vector.

[0014] The recombinant bacteria described in this invention are recombinant bacteria carrying the gene of the mutant or the recombinant plasmid.

[0015] Preferably, the host of the mutant gene or recombinant plasmid is Escherichia coli.

[0016] The method for constructing the recombinant bacteria:

[0017] (1) Construction of recombinant plasmid pET22b-GDH: The glucose dehydrogenase gene was ligated with the enzyme-digested plasmid pET22b to obtain the recombinant expression vector pET22b-GDH;

[0018] (2) Construction of recombinant E.coli BL21(DE3) / pET22b-GDH: The constructed recombinant expression vector pET22b-GDH was heat-transformed into Escherichia coli BL21(DE3) competent cells, and the recombinant E.coli BL21(DE3) / pET22b-GDH was obtained by culture and screening.

[0019] The NADP described in this invention + The glucose-dependent dehydrogenase mutant, the recombinant plasmid, or the recombinant bacteria act as a catalyst in the catalytic dehydrogenation of glucose to gluconic acid, while simultaneously releasing the coenzyme NADP. + Reverting to its application in NADPH.

[0020] The application includes the following steps: using a glucose dehydrogenase mutant as a catalyst and glucose as a substrate, catalyzing the dehydrogenation of glucose to gluconic acid, while simultaneously introducing the coenzyme NADP. + Reduction to NADPH; the catalytic process is shown below:

[0021] .

[0022] Preferably, the pH of the catalytic reaction system is 6-9.

[0023] Mechanism of Invention: This invention employs a strategy combining protein engineering and directed evolution. By constructing diverse mutant libraries and utilizing high-throughput methods such as spectroscopy, electrochemistry, and automated micro-screening, iterative optimization is performed on cofactor binding sites, catalytic centers, structural stability domains, and surface interface residues. This strategy aims to obtain proteins with both high catalytic activity and high NADP. + A new type of NADP with selectivity, excellent thermal stability and workability + The GDH mutant is dependent on it, thereby meeting the needs of advanced applications such as industrial cofactor regeneration, continuous flow biocatalysis and high-performance biosensing.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) By improving the NADP +The active site of the glucose dehydrogenase mutant was mutated, resulting in a mutant that improved the ability to catalyze the dehydrogenation of glucose to gluconic acid while simultaneously releasing the coenzyme NADP. + (2) The catalytic efficiency of reduction to NADPH; (3) The catalytic activity of mutant E96Q / K199M / G261R is 2.5 times that of wild-type glucose dehydrogenase. Attached Figure Description

[0025] Figure 1 The relative activities of different mutants of glucose dehydrogenase;

[0026] Figure 2 The relative activity of mutant K199M / G261R / E96Q at different pH values. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the embodiments.

[0028] Example 1

[0029] Construction and preparation of wild-type glucose dehydrogenase:

[0030] NADP from Bacillus megaterium + The codon-optimized gene sequence of glucose-dependent dehydrogenase (BmGDH, amino acid sequence as shown in SEQ ID NO.1) (as shown in SEQ ID NO.2) was synthesized by Suzhou Genewiz and cloned into the pET22b vector (provided by Suzhou Genewiz) to obtain the recombinant plasmid pET22b-BmGDH.

[0031] pET22b-BmGDH was first transformed into E. coli Top10 strain (purchased from Shanghai Sangon Biotech Co., Ltd.) for amplification. The resulting recombinant bacteria were inoculated into 5 mL LB medium containing 100 μg / mL ampicillin and cultured at 37℃ with shaking at 220 rpm for 12 h. After culture, the bacterial cells were collected, and the pET22b-BmGDH plasmid was extracted using a high-purity plasmid miniprep kit as a template for subsequent mutant construction.

[0032] The plasmid was further transformed into the expression host *E. coli* BL21(DE3) (purchased from Shanghai Sangon Biotech Co., Ltd.) to construct the recombinant expression strain *E. coli* BL21(DE3) / pET22b-BmGDH. The successfully transformed strain was plated on a solid plate containing 100 μg / mL ampicillin and incubated upside down at 37°C for 18 h. A single colony was then picked and inoculated into 5 mL of LB broth containing the appropriate antibiotic and pre-cultured at 37°C and 200 rpm for 18 h. Subsequently, the inoculum was transferred to 100 mL of TB broth (containing 100 μg / mL ampicillin) at a 1% (v / v) inoculation rate. When the OD600 reached approximately 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and expression was induced for 14 h at 18°C ​​and 220 rpm.

[0033] After induction, the bacterial suspension was centrifuged at 4℃ and 4000 rpm for 10 min to collect the bacterial cells. The cells were resuspended in an appropriate amount of 100 mM pH 7.5 phosphate buffer and the suspension was adjusted to OD600 = 3 before use for subsequent enzyme activity assays. Alternatively, the bacterial cells could be further disrupted by sonication in an ice bath (3 s operation time, 6 s interval, total operation time 5 min). The disrupted solution was centrifuged at 4℃ and 4000 rpm for 10 min, and the resulting supernatant was the crude enzyme solution, which was aliquoted and stored at -20℃ for later use.

[0034] Example 2

[0035] Construction and preparation of glucose dehydrogenase mutant K199M:

[0036] Gene mutation was detected using whole-plasmid PCR to obtain the target mutant gene. The primers are as follows:

[0037] K199M upstream primer: GATTAAACGCGGAAatgTTTGCCGATC;

[0038] K199M downstream primer: GATCGGCAAAcatTTCCGCGTTAATC.

[0039] The PCR system is shown in Table 1, and the reaction conditions are shown in Table 2.

[0040] Table 1 PCR reaction system

[0041]

[0042] Table 2 PCR reaction conditions

[0043]

[0044] After PCR amplification, the amplification product was detected by 0.9% agarose gel electrophoresis. The results showed that the amplification product was a single band, approximately 7000 bp in size. The amplification product was purified and recovered using a DNA recovery and purification kit.

[0045] The purified gene fragment was digested with DpnI enzyme to remove the template, and then recombined with recombinase. The recombinant product was transformed into Escherichia coli DH5α competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated at 37°C for 12 h.

[0046] After the culture was completed, single colonies were picked and cultured in LB liquid containing 100 μg / mL ampicillin. After 6 hours of culture, the culture was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to verify the correctness of the mutation site. After verification, a portion of the bacterial culture was stored at -80℃ for later use, and the recombinant plasmid pET22b-BmGDH-K199M was extracted from the other portion of the bacterial culture and stored at -20℃.

[0047] The successfully sequenced recombinant expression plasmid pET22b-BmGDH-K199M was transformed into E. coli BL21(DE3) to construct the recombinant mutant expression strain E. coli BL21(DE3) / pET22b-BmGDH-K199M.

[0048] The successfully constructed recombinant mutant expression strain E. coli BL21(DE3) / pET22b-BmGDH-K199M was plated onto agar plates containing ampicillin at a final concentration of 100 μg / mL and incubated at 37°C for 18 h.

[0049] After cultivation, a single colony was picked and inoculated into a test tube containing 0.5 mL of LB medium with a final concentration of 100 μg / mL ampicillin. The culture was incubated at 37 °C and 200 rpm for 18 h. Then, a 1% inoculum was transferred to 2 mL of TB medium containing a final concentration of 100 μg / mL ampicillin. The culture was then incubated at OD... 600 When the concentration of β-D-thiogalactoside (IPTG) was 0.6, it was added to a final concentration of 0.5 mM and induced at 18 °C for 20 h.

[0050] After centrifuging at 4000 rpm for 10 min to obtain bacterial cells, the cells were resuspended in 100 mM pH 7.5 phosphate buffer to obtain OD. 600 =3 bacterial cells containing the glucose dehydrogenase mutant K199M were broken up and used for subsequent catalytic studies and enzyme activity determination.

[0051] Example 3

[0052] Construction and preparation of glucose dehydrogenase mutant K199M / G261R:

[0053] Based on the plasmid in Example 2, the K199M / G261R mutant was modified by adding primers in step 1, while keeping all other conditions unchanged. The primers are as follows:

[0054] G261R upstream primer: CAAGCGGGCCGCcggCTCGAGcaccac;

[0055] G261R downstream primer: gtggtgCTCGAGccgGCGGCCCGCTTG.

[0056] Example 4

[0057] Construction and preparation of glucose dehydrogenase mutant K199M / G261R / E96Q:

[0058] Based on the plasmid in Example 3, the K199M / G261R / E96Q mutant was modified by adding primers in step 1, while keeping all other conditions unchanged. The primers are as follows:

[0059] E96Q upstream primer: CGCGGGCGTGcagAACCCGGTGC;

[0060] E96Q downstream primer: GCACCGGGTTctgCACGCCCGCG.

[0061] Performance testing

[0062] 1. Performance testing of glucose dehydrogenase mutants

[0063] Construction of glucose dehydrogenase reaction:

[0064] Glucose dehydrogenase catalyzes the dehydrogenation of glucose to produce gluconic acid, while simultaneously releasing the coenzyme NADP. + The NADPH generated in the reaction is reduced to NADPH, and the change in absorbance of the NADPH generated in the reaction at 340 nm is detected to indirectly detect the enzyme activity of glucose dehydrogenase.

[0065] The reaction system consisted of 20 μL of the OD200 obtained from Examples 1-4 after resuspending. 600 Add the bacterial culture (=2) to a 96-well microplate, lyse, centrifuge, collect the supernatant, and add 10 mM glucose and 10 mM NADP to each well. + The absorbance at 340 nm was measured using an ELISA reader within 10 minutes, with data recorded every minute. The test results are shown in Table 3 and [Table data missing]. Figure 1 .

[0066] Table 3. Absorbance changes of samples catalyzed by wild-type enzyme and mutant K199M / G261R / E96Q to produce gluconic acid and NADPH from glucose and NADP⁺.

[0067]

[0068] According to the sample absorbance A detected in Table 3 340 The formula for calculating enzyme activity and relative yield is as follows:

[0069] Relative yield (%) = (A340) mut-10min - A340 mut-1min ) ÷ (A340) wt-10min - A340 wt-1min )×100

[0070] Among them, the relative yield of wild-type glucose dehydrogenase was 100%, A340 mut-10min A340 mut-1min The A values ​​of the mutant at 10 min and 1 min were respectively. 340 Value, A340 wt-10min A340 wt-1min The A values ​​of wild-type enzyme at 10 min and 1 min are respectively. 340 value.

[0071] The relative yields are shown in Table 4, and the statistical results are as follows: Figure 1 As shown.

[0072] Table 4. Relative activities of wild-type and mutant enzymes in catalyzing the production of gluconic acid and NADPH from glucose and NADP⁺.

[0073]

[0074] Table 4 shows that mutants K199M, K199M / G261R, and K199M / G261R / E96Q catalyze glucose and NADP. + The relative activities for generating gluconic acid and NADPH were improved compared to WT, with K199M / G261R / E96Q increasing the yield by 250%.

[0075] 2. Optimization of pH conditions for glucose dehydrogenase

[0076] Engineered bacteria capable of expressing glucose dehydrogenase mutant K199M / G261R / E96Q and their mutants were constructed and cultured according to Examples 2 and 3, and the whole cells or pure enzyme solutions obtained were used as catalysts.

[0077] The reaction system consisted of: crude enzyme solution with OD=2, 10 mM glucose, and 10 mM NADP. +The reaction buffers were different pH values: 50 mM disodium hydrogen phosphate-citric acid buffer (pH=5), 50 mM disodium hydrogen phosphate-citric acid buffer (pH=5.5), dipotassium hydrogen phosphate-dipotassium hydrogen phosphate buffer (pH=6), 50 mM dipotassium hydrogen phosphate-dipotassium hydrogen phosphate buffer (pH=6.5), 50 mM dipotassium hydrogen phosphate-dipotassium hydrogen phosphate buffer (pH=7.5), 50 mM Tris-HCl buffer (pH=8), and 50 mM Tris-HCl buffer (pH=9). The absorbance at 340 nm was measured using a microplate reader over 10 minutes, recorded every minute. The test results are shown below. Figure 2 .

[0078] Depend on Figure 2 It can be seen that the optimal pH for enzyme activity of mutant K199M / G261R / E96Q is 7. The activity is low under weakly acidic conditions (pH 5.0-6.5), but it can maintain extremely high activity in neutral to weakly alkaline environments (pH 7.0-9.0).

[0079] 3. Determination of glucose dehydrogenase kinetic parameters

[0080] Determination of glucose dehydrogenase in different pairs of NADP + The specific activity at different concentrations was determined, and the kinetic parameters were calculated by plotting a double reciprocal curve based on the specific activity and the reciprocal of the substrate concentration. The test results are shown in Table 5.

[0081] Table 5 Kinetic parameters of mutant E96Q / K199M / G261R

[0082]

[0083] Table 5 shows that the wild-type glucose dehydrogenase (WT) on NADP was measured. + The dynamic parameters are K m It is 0.18mM, K cat 695.43 S -1 K cat / K m The value was 3956.44; the mutant E96Q / K199M / G261R was resistant to NADP. + The dynamic parameters are K m It is 0.37 mM, K cat 43077.50 S -1 K cat / K m The result was 115830.77. The results indicate that the activity of the glucose dehydrogenase mutant E96Q / K199M / G261R was significantly enhanced.

Claims

1. A type of NADP + A glucose dehydrogenase-dependent mutant, characterized by, The mutant is formed by replacing at least one amino acid at positions 96, 199, and 261 of the amino acid sequence shown in SEQ ID No. 1; glutamic acid at position 96 (Glu) is replaced with glutamine (Gln), lysine at position 199 (Lys) is replaced with methionine (Met), and glycine at position 261 (Gly) is replaced with arginine (Arg).

2. The NADP according to claim 1 + A glucose dehydrogenase-dependent mutant, characterized by, The mutants are K199M, K199M / G261R, or K199M / G261R / E96Q.

3. A method for encoding the NADP as described in claim 1 or 2 + The gene for a glucose dehydrogenase mutant protein that is dependent on glucose.

4. A recombinant plasmid containing the gene of claim 3.

5. The recombinant plasmid according to claim 4, characterized in that, The expression vector for the recombinant plasmid is a PET series expression vector.

6. A recombinant bacterium carrying the gene of the mutant of claim 3 or the recombinant plasmid of claim 4.

7. The recombinant bacteria according to claim 6, characterized in that, The host of the mutant's gene or recombinant plasmid is Escherichia coli.

8. A NADP as described in claim 1 or 2 + The glucose-dependent dehydrogenase mutant, or the recombinant plasmid of claim 4, or the recombinant bacteria of claim 6, acts as a catalyst to catalyze the dehydrogenation of glucose to gluconic acid while simultaneously releasing the coenzyme NADP. + Reverting to its application in NADPH.

9. The application according to claim 8, characterized in that, The application includes the following steps: using a glucose dehydrogenase mutant as a catalyst and glucose as a substrate, catalyzing the dehydrogenation of glucose to gluconic acid while simultaneously introducing the coenzyme NADP. + Restored to NADPH.

10. The application according to claim 9, characterized in that, The pH of the catalytic reaction system is 6-9.

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