A high-temperature-resistant cellobiose epimerase mutant and its application

Abstract

The present invention discloses a thermostable cellobiose epimerase mutant and its application. The mutant is obtained by a single mutation at position 242 of the amino acid sequence shown in SEQ ID No. 1. The present invention molecularly modifies the cellobiose epimerase (DiCE) from Dictyoglomus thermophilum to screen for mutants with improved thermostability. The resulting mutants have significantly improved lactulose conversion efficiency. Mutant A242L, in particular, can tolerate temperatures of 80°C and has a lactulose conversion rate of 64.41%, showing promising prospects for industrial application.

Description

(1) Technical field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a high-temperature-resistant cellobiose epimerase mutant and application thereof. (2) Background technology

[0002] Lactulose (4-O-β-D-galactopyranosyl-D-glucose), also known as isomerized lactose, is a non-natural sugar that is only found in small amounts in heated dairy products. Lactulose has good prebiotic activity and can be used to treat constipation and hepatic encephalopathy, and has been widely used in the food and pharmaceutical industries. Currently, the industrial production method of lactulose is mainly based on chemical isomerization, that is, lactose isomerized through the Lobry-de Bruyn-Alberda van Ekenstein (LA) arrangement. However, this method has problems such as lengthy product separation and purification steps, low yield, and serious environmental pollution. Therefore, the development of a green, safe, and efficient enzymatic production process for lactulose is of great practical significance.

[0003] Lactulose can be produced enzymatically from D-fructose and lactose using β-galactosidase (EC 3.2.1.23) or β-glucosidase (EC 3.2.1.21). However, the production of lactulose in this reaction is often accompanied by the addition of high concentrations of D-fructose and the formation of byproducts, such as D-galactose and D-glucose produced by lactose hydrolysis, and galacto-oligosaccharides (GOS) produced by lactose polymerization. Consequently, the lactulose content is relatively low, and the separation and purification process is complex. Lactulose can also be produced enzymatically by isomerizing lactose to lactulose using cellobiose epimerase (EC 5.1.3.11). In 2011, Park et al. discovered that cellobiose epimerase (CsCE) from Caldicellulosiruptor saccharolyticus catalyzes the conformational transition at the C2 position of an aldose, converting glucose to mannose. It was subsequently confirmed by Kim et al. in 2012 that the enzyme has the ability to isomerize lactose to lactulose.

[0004] All reported cellobiose epimerases have high catalytic efficiencies. Among them, the cellobiose epimerase (DiCE) from Dictyoglomus thermophilum has a lactulose conversion efficiency of 50.7%. While this cellobiose epimerase has a high lactulose conversion efficiency, attempts to increase the solubility of the substrate lactose and the catalytic efficiency of lactose isomerization to lactulose by raising the reaction temperature of the biocatalytic process face the bottleneck of poor thermal stability, a common problem for cellobiose epimerases. (3) Summary of the invention

[0005] The present invention aims to provide a thermostable cellobiose epimerase mutant and its application. By mutating cellobiose epimerase (DiCE) from Dictyoglomus thermophilum, the thermal stability is improved and the efficiency of catalyzing lactose to produce lactulose is enhanced to meet the needs of future industrial production and solve the problem of poor thermal stability of existing cellobiose epimerase.

[0006] The technical solution adopted in the present invention is:

[0007] The present invention provides a high-temperature resistant cellobiose epimerase mutant, which is obtained by performing a single mutation on position 242 of the amino acid sequence shown in SEQ ID No. 1.

[0008] Furthermore, preferably, the mutant is a mutant in which the alanine at position 242 of the amino acid sequence shown in SEQ ID No. 1 is mutated to leucine, the amino acid sequence is shown in SEQ ID No. 4, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 3.

[0009] The present invention also provides a gene encoding the thermostable cellobiose epimerase mutant, a recombinant expression vector constructed from the gene, and a recombinant genetically engineered bacterium. The recombinant expression vector uses pET28a(+) as a base plasmid, and the recombinant genetically engineered bacterium uses Escherichia coli BL21(DE3) as a host bacterium.

[0010] The present invention also provides an application of the high-temperature-resistant cellobiose epimerase mutant in catalyzing the production of lactulose from lactose.

[0011] Furthermore, the application is: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing the gene encoding the high-temperature-resistant cellobiose epimerase mutant as a catalyst, lactose as a substrate, and a pH 6-8 buffer as a reaction medium to form a reaction system, and performing a conversion reaction at 70-90°C and 150-200rpm to obtain lactulose.

[0012] Furthermore, in the reaction system, the concentration of the substrate lactose is 100-400 g / L (preferably 200 g / L), and the amount of wet bacteria is 30-70 g / L (preferably 50 g / L).

[0013] Furthermore, the reaction time is 2-6 hours, preferably 4 hours.

[0014] Furthermore, the buffer was 50 mM, pH 7.0 phosphate buffer.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention screens out mutants with improved thermal stability by molecularly modifying the cellobiose epimerase (DiCE) from Dictyoglomus thermophilum, and the obtained mutants have significantly improved lactulose conversion efficiency. Among them, the mutant A242L can tolerate a temperature of 80°C and has a lactulose conversion rate of 64.41%, which has good industrial application prospects. (IV) Description of the accompanying drawings

[0016] Figure 1 Agarose gel electrophoresis diagram of the recombinant plasmid of the mutant: M is Marker; 1 is N30W; 2 is T145L; 3 is N211F; 4 is A242L; 5 is N264D; 6 is S326D; 7 is N341K; 8 is S335L; 9 is N355W; 10 is N355Y; 11 is C361F; 12 is E368I.

[0017] Figure 2 This is the SDS-PAGE electrophoresis of DiCE and mutant crude enzyme solutions: M is Marker; 1 is DiCE; 2 is N30W; 3 is T145L; 4 is N211F; 5 is A242L; 6 is N264D; 7 is S326D; 8 is N341K; 9 is S335L; 10 is N355W; 11 is N355Y; 12 is C361F; 13 is E368I.

[0018] Figure 3 Figure 2 is an SDS-PAGE electrophoresis diagram of pure DiCE and A242L enzyme solutions. M is a marker; 1 is DiCE; 2 is A242L.

[0019] Figure 4 The thermal stability of DiCE changes.

[0020] Figure 5 The thermal stability of A242L changes.

[0021] Figure 6 Effect of reaction time on lactulose production.

[0022] Figure 7 Effect of substrate concentration on lactulose production.

[0023] Figure 8 The effect of bacterial dosage on lactulose production.

[0024] Figure 9HPLC chromatograms of conversion products. 1. Lactose standard 40 mg / mL (Cat. No. L103492-500g); 2. Lactulose standard 40 mg / mL (Cat. No. jg-yy-S11049-5g); 3. Mixed standard of 40 mg / mL lactulose and 40 mg / mL lactose; 4. Conversion product from the optimal reaction system of Example 5. (V) Specific implementation methods

[0025] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0026] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, solvent is water.

[0027] Example 1: Construction of wild-type strain

[0028] The amino acid sequence of cellobiose epimerase from Dictyoglomus thermophilum (GeneBank No.: WP_012547288.1) was obtained from the NCBI database (SEQ ID No. 1). Beijing Qingke Biotechnology Co., Ltd. was commissioned to perform codon optimization and gene synthesis (the nucleotide sequence is shown in SEQ ID No. 2). The plasmid pET28a(+)-DiCE was obtained, which was introduced into Escherichia coli BL21(DE3) to obtain the wild-type recombinant genetically engineered bacterium E. coli BL21(DE3)-pET28a(+)-DiCE containing the DiCE gene.

[0029] Example 2: Molecular modification of DiCE

[0030] 1. Selection of mutants

[0031] The three-dimensional structure model of DiCE was obtained from the UniProt database and entered into

[0032] Computational design of thermostable multiple-point mutants in FireProt (https: / / loschmidt.chemi.muni.cz / fireprot / ) was used. The Gibbs free energy calculation tools in FireProt, FoldX and Rosttea, were used to obtain simulated mutants based on energy mutation and evolutionary mutation and their Gibbs free energy changes. In the selection of simulated mutants, we selected mutants with Gibbs free energy less than -1 kcal / mol calculated based on FoldX and Gibbs free energy less than -2 kcal / mol calculated based on Rosttea (see Xia, Y. et al. Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol. Appl Microbiol Biotechnol 105, 7309-7319 (2021).) as potential target mutants for enhancing thermal stability, namely N30W, T145L, N211F, A242L, N264D, S326D, N341K, S335L, N355W, N355Y, C361F, and E368I.

[0033] 2. Construction of mutants

[0034] Site-directed mutagenesis primers (Table 1) were designed based on the DiCE nucleotide sequence (shown in SEQ ID No. 2) and the mutation was performed using plasmid pET28a(+)-DiCE as a template. The modified linear plasmid obtained by mutation amplification was subjected to agarose gel electrophoresis. Figure 1 As shown, after the plasmid size is detected to be correct, it is introduced into Escherichia coli BL21 (DE3), and after sequencing, it is a genetically engineered bacterium containing the mutant.

[0035] Table 1 Site-directed mutagenesis primers

[0036]

[0037] Example 3: Screening of mutants

[0038] 1. Induced expression of wild-type strains and mutants

[0039] The wild-type strain of Example 1 and the mutant strain constructed in Example 2 were inoculated into LB solid medium and cultured for activation at 37°C for 12 h. Colonies were picked and inoculated into LB liquid medium and cultured at 37°C and 200 rpm until the OD600 reached 0.6-0.8. IPTG was added to the culture solution at a final concentration of 0.1 mM and induced at 25°C and 200 rpm for 12 h.

[0040] 2. Enzyme activity determination:

[0041] (1) Crude enzyme solution

[0042] Take 100 mL of induced bacterial solution and centrifuge at 8000 r / min and 4°C for 10 minutes, remove the supernatant, and wash twice with phosphate buffer (pH 7.5, 50 mM) to obtain bacterial precipitate. Add 5 mL of phosphate buffer (pH 7.5, 50 mM) to the bacterial precipitate and resuspend it. Place it on ice and ultrasonically disrupt it at 360W for 15 minutes (3 seconds between disruptions). After disruption, centrifuge the mixed solution at 8000 r / min and 4°C for 10 minutes, and collect the supernatant as the crude enzyme solution. SDS-PAGE electrophoresis of DiCE and mutant crude enzyme solution is shown in Figure 2 shown.

[0043] (2) Initial enzyme activity

[0044] 0.9 mL of 68.4 g / L lactose solution (prepared in 50 mM phosphate buffer, pH 7.5) was mixed evenly with 0.1 mL of crude enzyme solution, reacted at 70°C and 150 rpm for 2 h, and terminated by ice bath for 15 min. The reaction solution was filtered through a 0.22 μm filter membrane, and the supernatant was collected and the lactulose and fructose peak areas were detected by HPLC. The lactulose and fructose contents were obtained according to the standard curve.

[0045] Enzyme activity is defined as: the amount of enzyme required to isomerize lactose to produce 1 μmol lactulose per minute at 70°C and pH 7.5 is defined as one enzyme activity unit (U).

[0046] HPLC conditions: Waters 1525 high performance liquid chromatograph, Waters 2414 differential refractive index detector, Shodex VG-50-4E chromatographic column, mobile phase was a mixture of 75% (v / v) acetonitrile, 20% (v / v) methanol and 5% (v / v) ultrapure water, column temperature was set at 40 °C, flow rate was 1 mL / min, injection volume was 10 μL.

[0047] Determination of Lactulose and Lactose Concentrations: HPLC using the external standard method, prepare lactulose and lactose standard solutions at concentration gradients of 40, 20, 10, 5, and 2.5 mg / mL. After injection, determine the peak area and retention time. Lactulose and lactose standard curves are constructed based on the relationship between peak area and concentration.

[0048] Calculation of lactulose conversion rate: Y = M lactulose / M lactose *100%

[0049] Where: M lactulose is the lactulose concentration calculated after the reaction, M lactose is the total lactose concentration before the reaction.

[0050] (3) Residual enzyme activity

[0051] Before the residual enzyme activity test of DiCE and its mutants, 0.1 mL of crude enzyme solution was placed in an 80°C water bath and incubated for 20 minutes. The remaining steps were consistent with the initial enzyme activity determination method. The residual enzyme activity (%) was based on the initial enzyme activity of DiCE as 100%, and the residual enzyme activity of the remaining mutants after incubation was compared with it. The initial enzyme activity and residual enzyme activity of DiCE and its mutants are shown in Table 2. Among them, the initial enzyme activity of mutant A242L and mutant C361F increased by 1.29 times and 1.05 times respectively compared with that of DiCE, and the residual enzyme activity retained 74.2% and 80.4% respectively compared with the initial enzyme activity of DiCE. Therefore, after comprehensive consideration, mutant A242L (amino acid sequence as shown in SEQ ID No.3, nucleotide sequence as shown in SEQ ID No.4) was selected as the subsequent research object.

[0052] Table 2 Initial and residual enzyme activities of DiCE and its mutants

[0053]

[0054] ND: indicates no activity detected;

[0055] Example 4. Thermal stability of mutant A242L

[0056] Preparation of pure DiCE and A242L enzyme solutions: The crude enzyme solution was prepared as described in Example 3. The crude enzyme solution was filtered through a 0.22 μm aqueous filter to remove cell debris. 5 mL of the filtrate was loaded onto a nickel-NTA sepharose 6FF affinity chromatography column (column volume 3 mL, equilibrated for 30 min with 20 mM sodium phosphate buffer containing 20 mM imidazole and 0.5 M NaCl). The column was allowed to bind for 60 min, and then the impurities were eluted with 90 mL of 20 mM sodium phosphate buffer containing 50 mM imidazole and 0.5 M NaCl. The target protein was then eluted with 30 mL of 20 mM sodium phosphate buffer containing 200 mM imidazole and 0.5 M NaCl, and the remaining protein was eluted with 30 mL of 20 mM sodium phosphate buffer containing 500 mM imidazole and 0.5 M NaCl. The target protein eluted in 20 mM sodium phosphate buffer containing 200 mM imidazole and 0.5 M NaCl was collected and concentrated by ultrafiltration through a 30 kDa ultrafiltration tube at 4 °C, 3000 g, and 30 min to obtain pure enzyme solution. The SDS-PAGE electrophoresis of DiCE and A242L pure enzyme solution is shown in the figure. Figure 3 shown.

[0057] DiCE and A242L thermal stability test: After the pure enzyme solution with an enzyme concentration of 1 mg / mL was incubated at 75°C, 80°C, and 85°C for different time periods, the enzymatic reaction was carried out at pH 7.5 and 70°C. The untreated pure enzyme solution was used as a control to measure the change in enzyme activity. Where Ln (residual enzyme activity / initial enzyme activity) = K d , T 1 / 2 =ln2 / K d Thermal stability constant K d , half-life T 1 / 2 As shown in Table 3, under incubation conditions of 80°C, the half-life of A242L was increased by 11.8 min compared to DiCE, and its thermal stability was improved compared to DiCE. Figure 4 、 Figure 5 shown.

[0058] Table 3. Thermal stability constants K of DiCE and A242L d , half-life T 1 / 2

[0059]

[0060] Example 5: Optimization of conditions for producing lactulose from mutant A242L

[0061] 1. Wet cells: The E. coli BL21(DE3)-pET28a(+)-DiCE-A242L strain containing mutant A242L was induced and cultured according to the method of Example 3, and the wet cells were collected.

[0062] 2. Reaction time

[0063] Reaction system: 200 g / L lactose and 50 g / L wet cells, 50 mM, pH 7.0 phosphate buffer solution constitute a 50 mL reaction system. After reacting at 80°C, 150 rpm for 2-6 hours, samples were taken and the reaction was terminated by ice bath for 15 minutes. The lactulose content was determined by the HPLC method described in Example 3, and the lactulose conversion rate was calculated. The results are shown in Figure 6 As shown, the optimal reaction time is 4 h.

[0064] 3. Substrate concentration

[0065] Reaction system: 100-400 g / L lactose and 50 g / L wet cells, 50 mM, pH 7.0 phosphate buffer constitute a 50 mL reaction system. After reacting at 80°C, 150 rpm for 4 hours, sampling was performed and the reaction was terminated by ice bath for 15 minutes. The lactulose content was determined by the HPLC method described in Example 3, and the lactulose conversion rate was calculated. The results are shown in Figure 3. Figure 7 As shown, the optimal substrate concentration is 200 g / L.

[0066] 4. Bacteria volume

[0067] Reaction system: 200g / L lactose and 30-70g / L wet cells, 50mM, pH 7.0 phosphate buffer to form a 50mL reaction system. After reacting at 80℃, 150rpm for 4h, sampling was performed and the reaction was terminated by ice bath for 15min. The lactulose content was determined by HPLC method described in Example 3, and the lactulose conversion rate was calculated. The results are shown in Figure 8 As shown, the optimal bacterial cell volume is 50g / L.

[0068] 5. Optimal Conditions for Transformation

[0069] Reaction system: 200 g / L lactose and 50 g / L wet cells, 50 mM, pH 7.0 phosphate buffer constitute a 100 mL reaction system. After reacting at 80°C, 150 rpm for 4 hours, sampling was performed and the reaction was terminated by ice bath for 15 minutes. The lactulose content of the reaction solution was determined by HPLC method described in Example 3. At the same time, a lactose standard 40 mg / mL (Cat. No.:

[0070] L103492-500g), lactulose standard 40 mg / mL (catalog number: jg-yy-S11049-5g), mixed standard of lactulose 40 mg / mL and lactose 40 mg / mL, the results are shown in Figure 9 shown.

[0071] After catalysis for 4 h in the above optimal reaction system, the lactulose conversion rate of mutant A242L was as high as 64.41%.

[0072] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A thermostable cellobiose epimerase mutant, characterized in that: The mutant is obtained by mutating the 242nd alanine in the amino acid sequence shown in SEQ ID No. 1 to leucine.

2. A recombinant genetically engineered bacterium constructed with the gene encoding the thermostable cellobiose epimerase mutant according to claim 1.

3. Use of the thermostable cellobiose epimerase mutant according to claim 1 in catalyzing the production of lactulose from lactose.

4. The use according to claim 3, characterized in that The application is as follows: using wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing the gene encoding the thermostable cellobiose epimerase mutant as a catalyst, lactose as a substrate, and a pH 6-8 buffer as a reaction medium to form a reaction system, and performing a conversion reaction at 70-90°C and 150-200 rpm to obtain lactulose.

5. The use according to claim 4, characterized in that In the reaction system, the concentration of the substrate lactose is 100-400 g / L, and the amount of wet bacteria is 30-70 g / L.

6. The use according to claim 4, characterized in that The reaction time is 2-6h.

7. The use according to claim 4, characterized in that The buffer was 50 mM phosphate buffer, pH 7.0.

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