High-temperature-resistant cellobiose epimerase mutant and application thereof

Abstract

The invention discloses a high-temperature-resistant cellobiose epimerase mutant and application thereof. The mutant is obtained by performing single mutation on the 242nd site of an amino acid sequence as shown in SEQ ID No.1. The invention also discloses a preparation method of the high-temperature-resistant cellobiose epimerase mutant. According to the invention, molecular modification is carried out on cellobiose epimerase (DiCE) from Dictyoglomus thermophilum, a mutant with improved thermal stability is screened out, the lactulose conversion efficiency of the obtained mutant is obviously improved, the mutant A242L can tolerate the temperature of 80 DEG C, the lactulose conversion rate is 64.41%, and the industrial application prospect is good.

Description

(I) 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. (II) Background technology

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

[0003] The enzymatic production of lactulose can be completed by β-galactosidase (EC 3.2.1.23) or β-glucosidase (EC3.2.1.21) catalyzing D-fructose and lactose. However, the production of lactulose by this reaction is often accompanied by the addition of high concentrations of D-fructose and the generation of by-products. For example, D-galactose and D-glucose produced by lactose hydrolysis and galacto-oligosaccharides (GOS) produced after lactose polymerization, so the lactulose content is relatively low and the separation and purification process is complicated. The enzymatic production of lactulose can also be completed by cellobiose epimerase (EC 5.1.3.11) isomerizing lactose into lactulose. In 2011, Park et al. found that cellobiose epimerase (CsCE) from Caldicellulosiruptor saccharolyticus can catalyze the configuration change of the C2 position of aldose to convert glucose into mannose. It was subsequently confirmed by Kim et al. in 2012 that the enzyme has the ability to isomerize lactose into lactulose.

[0004] All the cellobiose epimerases reported so far have high catalytic efficiency. Among them, the cellobiose epimerase (DiCE) from Dictyoglomus thermophilum has a lactulose conversion rate of 50.7%. Although the cellobiose epimerase has a high conversion rate in producing lactulose, the attempt to increase the solubility of the substrate lactose and the catalytic efficiency of lactose isomerization to lactulose by increasing the reaction temperature of the biocatalytic process will face the bottleneck problem of poor thermal stability that is common in cellobiose epimerases. (III) Summary of the invention

[0005] The present invention aims to provide a high temperature resistant cellobiose epimerase mutant and its application, by mutating cellobiose epimerase (DiCE) from Dictyoglomus thermophilum, improving thermal stability, enhancing the efficiency of catalyzing lactose to prepare lactulose, so as 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 by the present invention is:

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

[0008] Furthermore, preferably, the mutant is a mutant in which the 242nd alanine in 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 coding gene for the thermostable cellobiose epimerase mutant, a recombinant expression vector constructed by the coding gene, and a recombinant genetic engineering bacterium. The recombinant expression vector uses pET28a(+) as a basic plasmid; and the recombinant genetic engineering 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 that: the present invention performs molecular modification on cellobiose epimerase (DiCE) from Dictyoglomus thermophilum, screens out mutants with improved thermal stability, and obtains mutants with significantly improved lactulose conversion efficiency, wherein mutant A242L tolerates a temperature of 80°C, and has a lactulose conversion rate of 64.41%, which has good industrial application prospects. (IV) Description of the drawings

[0016] Figure 1 Agarose gel electrophoresis 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 The SDS-PAGE electrophoresis diagram of DiCE and A242L pure enzyme solution. M is a marker; 1 is DiCE; 2 is A242L.

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

[0020] Figure 5 Thermal stability changes of A242L.

[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] Fig. 9HPLC chromatograms of the conversion products. 1. Lactose standard 40 mg / mL (article number: L103492-500g); 2. Lactulose standard 40 mg / mL (article number: jg-yy-S11049-5g); 3. Lactulose 40 mg / mL and lactose 40 mg / mL mixed standard; 4. Conversion product of the best reaction system of Example 5. (V) Specific implementation methods

[0025] The present invention is further described below in conjunction with 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 strains

[0028] The amino acid sequence of cellobiose epimerase from Dictyoglomus thermophilum (GeneBank No.: WP_012547288.1) (SEQ ID No. 1) was obtained from the NCBI database, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to perform codon optimization and gene synthesis (the nucleotide sequence is shown in SEQ ID No. 2) to obtain plasmid pET28a(+)-DiCE, which was introduced into Escherichia coli BL21(DE3) to obtain wild-type recombinant genetically engineered bacteria 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 / ), using the Gibbs free energy calculation tools in FireProt - FoldX and Rosttea, to obtain simulated mutants based on energy mutation and evolutionary mutation and their changes in Gibbs free energy. In the selection of simulated mutants, we selected mutants with a Gibbs free energy less than -1 kcal / mol calculated based on FoldX and a 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 tested by agarose gel electrophoresis. Figure 1 As shown, after the plasmid size is detected correctly, it is introduced into Escherichia coli BL21 (DE3), and after sequencing, it is confirmed to be a genetically engineered bacterium containing a mutant.

[0035] Table 1 Site-directed mutagenesis primers

[0036]

[0037] Example 3: Screening of mutants

[0038] 1. Inducible 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 at 37°C for 12 h. The colonies were picked and inoculated into LB liquid medium and cultured at 37°C and 200 rpm until OD 6 00 reached 0.6-0.8, IPTG was added to the culture medium with a final concentration of 0.1 mM, and the culture was 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 the induced bacterial solution and centrifuge at 8000 r / min, 4°C for 10 min, remove the supernatant, and wash twice with phosphate buffer (pH7.5, 50 mM) to obtain a bacterial precipitate. Add 5 mL of phosphate buffer (pH7.5, 50 mM) to the bacterial precipitate and resuspend it. Place it on ice and ultrasonically disrupt it at 360 W for 15 min (3s intervals for 3s after disruption). After disruption, centrifuge the mixed solution at 8000 r / min, 4°C for 10 min, and collect the supernatant as the crude enzyme solution. The SDS-PAGE electrophoresis of DiCE and mutant crude enzyme solutions is shown in Figure 2 shown.

[0043] (2) Initial enzyme activity

[0044] 0.9 mL of 68.4 g / L lactose solution (prepared with 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 with a 0.22 μm filter membrane, and the supernatant was used to detect the lactulose and fructose peak areas by HPLC, and 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 refractometer, 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 uses the external standard method to prepare lactulose and lactose standard solutions with a concentration gradient of 40, 20, 10, 5, and 2.5 mg / mL. After injection, the peak area and retention time are determined, and the lactulose and lactose standard curves are drawn based on the relationship between the 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 is completed, M lactose is the total lactose concentration before the reaction.

[0050] (3) Residual enzyme activity

[0051] Before the residual enzyme activity of DiCE and its mutants was detected, 0.1 mL of the 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 100% for the initial enzyme activity of DiCE, 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. 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 enzyme solution of DiCE and A242L: The preparation method of crude enzyme solution is as shown in Example 3. The obtained crude enzyme solution is passed through a 0.22 μm water filter membrane to remove cell debris. 5 mL of the filtrate is loaded onto a nickel-NTA agarose gel 6FF affinity chromatography column (column volume 3 mL, first equilibrated with 20 mM sodium phosphate buffer containing 20 mM imidazole and 0.5 M NaCl for 30 min), allowed to stand for binding for 60 min, and then the impurities are eluted with 90 mL of 20 mM sodium phosphate buffer containing 50 mM imidazole and 0.5 M NaCl, and then the target protein is eluted with 30 mL of 20 mM sodium phosphate buffer containing 200 mM imidazole and 0.5 M NaCl, and the remaining protein is 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 ultrafiltered through a 30 kDa ultrafiltration tube at 4 °C, 3000 g, 30 min to obtain pure enzyme solution. The SDS-PAGE electrophoresis of DiCE and A242L pure enzyme solution is shown in Figure 3 shown.

[0057] DiCE and A242L thermal stability test: After the pure enzyme solution with an enzyme concentration of 1 mg / mL was placed in 75℃, 80℃, and 85℃ for different time periods, the enzymatic reaction was carried out at pH 7.5 and 70℃, and 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 the incubation condition of 80°C, the half-life of A242L was increased by 11.8 min compared with DiCE, and the thermal stability was improved compared with 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 preparing lactulose by mutant A242L

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

[0062] 2. Response time

[0063] Reaction system: 200 g / L lactose and 50 g / L wet bacteria, 50 mM, pH 7.0 phosphate buffer solution constitute 50 mL reaction system. After reacting at 80°C, 150 rpm for 2-6 h, sample was taken and the reaction was terminated by ice bath for 15 min. 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 6 As shown, the optimal reaction time is 4h.

[0064] 3. Substrate concentration

[0065] Reaction system: 100-400 g / L lactose and 50 g / L wet bacteria, 50 mM, pH 7.0 phosphate buffer solution constitute 50 mL reaction system. After reacting at 80°C, 150 rpm for 4 h, sampling was performed, and the reaction was terminated by ice bath for 15 min. 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 7 As shown, the optimal substrate concentration is 200 g / L.

[0066] 4. Bacteria volume

[0067] Reaction system: 200 g / L lactose and 30-70 g / L wet bacteria, 50 mM, pH 7.0 phosphate buffer solution constitute 50 mL reaction system. After reacting at 80°C, 150 rpm for 4 h, sampling was performed, and the reaction was terminated by ice bath for 15 min. 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 8 As shown, the optimal bacterial cell quantity is 50g / L.

[0068] 5. Optimal conditions for transformation

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

[0070] L103492-500g), lactulose standard 40mg / mL (article number: jg-yy-S11049-5g), lactulose 40mg / mL and lactose 40mg / mL mixed standard, the results are shown in Fig. 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 as above by the embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can 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 performing a single mutation on position 242 of the amino acid sequence shown in SEQ ID No.

1.

2. The thermostable cellobiose epimerase mutant according to claim 1, 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.

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

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

5. The use according to claim 4, characterized in that 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.

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

7. The use according to claim 5, characterized in that The reaction time is 2-6h.

8. The use according to claim 5, characterized in that The buffer was 50 mM, pH 7.0 phosphate buffer.

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