A tagatose-4-epimerase mutant and its application

Abstract

The present invention belongs to the field of biopharmaceuticals and biotransformation, and specifically relates to a tagatose-4-epimerase mutant and its application. The tagatose-4-epimerase mutant of the present invention is obtained by single mutation or combined mutation of the amino acid residues at specific positions of the amino acid sequence shown in SEQ ID NO.1. The mutant has improved catalytic activity when converting and preparing D-tagatose compared to the wild-type tagatose-4-epimerase. The tagatose-4-epimerase mutant obtained by the present invention has the advantages of mild reaction conditions, green environmental protection, single product, and simple separation and purification, which greatly reduces the production cost.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals and biotransformation, and specifically relates to a tagatose-4-epimerase mutant, a mutant encoding gene, a recombinant vector containing the mutant encoding gene, a recombinant genetic engineering bacterium containing the mutant encoding gene, and application of the tagatose-4-epimerase mutant in the preparation of D-tagatose. Background Art

[0002] Tagatose is a diastereomer of fructose, in which five carbon molecules and one oxygen molecule form a ring structure. It is a natural six-carbon ketose that exists in nature but is relatively rare. Its sweetness is similar to that of sucrose, but the calories it produces are only one-third of sucrose. Tagatose has the advantages of low energy, lowering blood sugar, improving intestinal flora, and resisting dental caries, so it has broad application prospects in the fields of medicine and health products.

[0003] Tagatose is present in very low concentrations in nature and is currently mainly synthesized by chemical or biological methods. The chemical method uses D-galactose as a raw material and prepares D-tagatose through a two-step reaction. However, the chemical method has the disadvantages of severe reaction conditions that are difficult to control, easy to cause pollution, complex process, and a large number of by-products that are not conducive to separation and purification. The biological method for preparing D-tagatose has the advantages of being green and environmentally friendly, having a single product, and being simple to separate and purify, so it has become the main method for synthesizing D-tagatose. At present, the dual enzyme method is mainly used, that is, the synthesis of D-tagatose from lactose is catalyzed by β-galactosidase and L-arabinose isomerase. This method requires two-step reactions to synthesize D-tagatose: first, β-galactosidase hydrolyzes lactose to generate the intermediate product D-galactose, and then L-arabinose isomerase catalyzes the synthesis of D-tagatose. However, the cost of the substrate lactose of this method is high, and the two-step reaction is required, resulting in a complicated process; at the same time, the low utilization rate of the substrate is limited by the hydrolysis of lactose in the first step, which further increases the cost of the method. Therefore, finding other efficient ways to synthesize D-tagatose is of great research significance and application value.

[0004] Tagatose-4-epimerase can synthesize D-tagatose through epimerization reaction using cheap D-fructose as substrate. It is not only environmentally friendly, simple to separate and purify, and has mild reaction conditions, but also has the advantages of single reaction and low cost, and has good application prospects. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art D-tagatose synthesis process, such as complicated steps, complex separation and purification, severe reaction conditions, difficulty in control and high preparation cost. The present invention provides a tagatose-4-epimerase mutant, a mutant encoding gene, a recombinant vector containing the mutant encoding gene, and a recombinant genetic engineering bacterium containing the mutant encoding gene. The tagatose-4-epimerase mutant is applied to the process for preparing D-tagatose.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0007] A tagatose-4-epimerase mutant is obtained by site-directed mutagenesis of the amino acids in the sequence shown in SEQ ID NO. 1, wherein the mutation site is one or more of the following: (1) position 75, (2) position 140, (3) position 165, and (4) position 361.

[0008] Preferably, a tagatose-4-epimerase mutant is derived from the amino acid sequence shown in SEQ ID NO.1 by site-directed mutagenesis, wherein the mutation site is one or more of the following: (1) position 75, (2) position 140, (3) position 165, (4) position 361, (5) position 69, and (6) position 70.

[0009] Preferably, the tagatose-4-epimerase mutant is derived from the amino acid sequence shown in SEQ ID NO.1 by site-directed mutagenesis, and the mutation sites are one or two of the following: (1) position 75, (4) position 361.

[0010] Preferably, the mutant is obtained by mutation of one or more of the following amino acids in the sequence as shown in SEQ ID NO.1: (1) glutamic acid at position 75 is mutated to alanine (E75A), (2) proline at position 140 is mutated to leucine (P140L), (3) arginine at position 165 is mutated to alanine (R165A), (4) phenylalanine at position 361 is mutated to histidine (F361H).

[0011] Preferably, the mutant is obtained by mutation of one or more of the following amino acids in the sequence as shown in SEQ ID NO.1: (1) glutamic acid at position 75 mutates to alanine (E75A), (2) proline at position 140 mutates to leucine (P140L), (3) arginine at position 165 mutates to alanine (R165A), (4) phenylalanine at position 361 mutates to histidine (F361H), (5) arginine at position 69 mutates to alanine (R69A), (6) asparagine at position 70 mutates to alanine (N70A).

[0012] As a further preference, the mutant is obtained by mutating the amino acids in the sequence shown in SEQ ID NO.1 by mutating the 75th glutamic acid to alanine (E75A) and the 361st phenylalanine to histidine (F361H), and its amino acid sequence is preferably as shown in SEQ ID NO.3.

[0013] A gene encoding a tagatose 4-epimerase mutant as described above, wherein the gene encoding a tagatose 4-epimerase mutant is preferably as shown in SEQ ID NO.4.

[0014] A recombinant vector constructed by the encoding gene as described above.

[0015] A recombinant genetically engineered bacterium transformed by the recombinant vector as described above.

[0016] The tagatose-4-epimerase mutant of the present invention is prepared by mutating multiple amino acids of the wild-type tagatose-4-epimerase, thereby improving its relative enzyme activity. First, the wild-type tagatose-4-epimerase encoding gene (sequence as shown in SEQ ID NO.2) is connected to the expression vector pET28a (+) to construct a recombinant expression plasmid. Then the constructed recombinant expression plasmid is transformed into E. coli BL21 (DE3). The recombinant expression plasmid containing the tagatose-4-epimerase encoding gene is used as a template, and genetic modification is performed by site-directed mutagenesis technology, and then the recombinant expression plasmid is transformed into E. coli BL21 (DE3) to obtain E. coli BL21 (DE3) recombinant genetic engineering bacteria containing the tagatose-4-epimerase mutant encoding gene. The obtained recombinant genetically engineered bacteria are induced to culture, and the culture fluid is separated to obtain bacterial cells containing the recombinant tagatose-4-epimerase mutant, and the culture fluid and bacterial cells are separated to obtain a crude enzyme solution of the tagatose-4-epimerase mutant. The relative activities of the mutant tagatose-4-epimerase and the wild-type tagatose-4-epimerase are compared to screen out a tagatose-4-epimerase mutant with higher activity.

[0017] Use of the tagatose-4-epimerase mutant as described above in the preparation of D-tagatose.

[0018] Preferably, the application is: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing a gene encoding a mutant of tagatose-4-epimerase, or crude enzyme extracted from wet bacteria after ultrasonic crushing, or purified pure enzyme as a catalyst, using D-fructose as a substrate, and reacting with Ni 2+ In the presence of , the reaction is carried out at 70-80°C in a sodium phosphate buffer solution with a pH value of 7-9. After the reaction is complete, the reaction solution is separated and purified to obtain D-tagatose.

[0019] As a further preferred application, the application is: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing a gene encoding a mutant of tagatose-4-epimerase, or crude enzyme extracted from wet bacteria after ultrasonic crushing, or purified pure enzyme as a catalyst, using D-fructose as a substrate, and using a sodium phosphate buffer solution with a pH value of 8 as a reaction medium to form a reaction system, at 75°C, 1 mM Ni 2+ , react at 1000 rpm for 30 min, separate and purify the reaction solution to obtain D-tagatose; wherein the amount of the catalyst is 40 g / L based on the weight of the wet bacteria, and the initial concentration of the substrate is 50 g / L.

[0020] The wet bacterial cells obtained by fermentation culture of the recombinant genetic engineering bacteria containing the tagatose-4-epimerase mutant encoding gene of the present invention are prepared as follows: the recombinant genetic engineering bacteria containing the tagatose-4-epimerase mutant encoding gene are inoculated into an LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C and 180 rpm for 10 hours, then inoculated into a fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 2% inoculum, cultured at 37°C and 180 rpm until the bacterial OD600 reaches 0.6-0.8, IPTG with a final concentration of 0.1 mM is added, and after induction culture at 28°C for 12 hours, the culture is centrifuged at 4°C and 8000 rpm for 10 minutes, the supernatant is discarded, and the wet bacterial cells are collected.

[0021] Preparation of the crude mutant tagatose-4-epimerase enzyme of the present invention: 0.2 g of wet cells of the recombinant genetically engineered bacteria containing the coding gene of the mutant tagatose-4-epimerase are added with 9.8 mL of 50 mM sodium phosphate buffer solution at pH 7.0 to resuspend the wet cells, and ultrasonically disrupt them under ice bath conditions (200 W power, 1 second, 2 seconds interval, continuous disruption for 15 minutes) to obtain a cell disruption solution. The cell disruption solution obtained after ultrasonic disruption is centrifuged at 8000 rpm and 4°C for 10 minutes, and the obtained supernatant is the desired crude enzyme solution.

[0022] Therefore, the present invention has the following beneficial effects:

[0023] The present invention prepares a tagatose-4-epimerase mutant with high catalytic activity and high stereoselectivity for D-tagatose, and the obtained mutant has the advantages of mild reaction conditions, green environmental protection, single product, and simple separation and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE picture of tagatose-4-epimerase.

[0025] Figure 2Schematic diagram of the enzymatic synthesis of D-tagatose.

[0026] Figure 3 Schematic diagram of HPLC detection of substrate D-fructose and product D-tagatose.

[0027] Figure 4 It is a bar graph showing the relative enzyme activities of wild-type tagatose-4-epimerase and tagatose-4-epimerase mutants. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0029] Example 1: Wild-type tagatose-4-epimerase genetically engineered bacteria E. coli Construction of BL21(DE3) / DTE

[0030] The gene sequence of tagatose-4-epimerase from Thermotoga petrophila in the gene library was codon optimized and the pET28a(+)-DTE plasmid was obtained by total gene synthesis. The plasmid was transformed into E. coli BL21(DE3) to obtain the wild-type E. coli BL21(DE3) / DTE, which was recorded as DTE.

[0031] Example 2: Construction of E. coli BL21(DE3) / DTE-muts by site-directed mutagenesis

[0032] Site-directed mutagenesis was introduced into tagatose-4-epimerase DTE by site-directed mutagenesis. The primers were designed as follows:

[0033] R69A:

[0034] Upstream Primer 1: 5'-CTTTTTCTTTTGCGAGgcgAACCATGAAAATC-3'

[0035] Downstream primer 2: 5'-CTCGCAAAAGAAAAAGCTCACGCCTTCC-3'

[0036] N70A:

[0037] Upstream Primer 1: 5'-CTTTTTCTTTTGCGAGCGCgcgCATGAAAATC-3'

[0038] Downstream primer 2: 5'-CTCGCAAAAGAAAAAGCTCACGCCTTCC-3

[0039] E75A:

[0040] Upstream primer 1: CCATGAAAATCTGgcgGTGCTGCGCAAATAT

[0041] Downstream primer 2: 5'- CAGATTTTCATGGTTGCGCTCGCAAAAG-3'

[0042] P140L:

[0043] Upstream primer 3: 5'- TACCTGGCGCGATGTTctgGATGATGCGAC-3'

[0044] Downstream primer 4: 5'-AACATCGCGCCAGGTACGACCAGTACGTTC-3'

[0045] R165A:

[0046] Upstream primer 5: 5'- GCGGATCATGTGAAAgcgCCGGAAGATTTGG-3'

[0047] Downstream primer 6: 5'-TTTCACATGATCCGCATCCGCGCCAAAGC-3'

[0048] F361H:

[0049] Upstream primer 7: 5'-GTGCGACTGGTGGTCTGcatCTGGTGAAAAC-3'

[0050] Downstream primer 8: 5'-CAGACCACCAGTCGCACTCGCAAACGC -3'

[0051] The primers for site-directed mutagenesis are as described above, and the mutation sites are marked with lowercase letters. Similarly, the plasmid DNA containing the DTE gene was used as a template to introduce mutations by PCR, and the PCR reaction program was as follows: 95℃ for 5min; 95℃ for 30s, 55℃ for 10s, 72℃ for 3min 30s, repeated for 35 cycles; 72℃ for further extension for 10min. The PCR product was treated with DpnI at 37℃ for 3h, inactivated at 80℃ for 10min, and then transformed into E. coli BL21 (DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg / L kanamycin resistance, and cultured at 37℃ for 12h. Single colonies were randomly picked for sequencing analysis, and DTE mutants DTE-R69A, DTE-N70A, DTE-E75A, DTE-P140L, DTE-R165A, DTE-F361H, and DTE-E75A / F361H were obtained.

[0052] Example 3: Preparation of wet cells of recombinant tagatose-4-epimerase mutant

[0053] The recombinant E. coli BL21 (DE3) / DTE-muts containing the gene expressing the recombinant tagatose-4-epimerase mutant obtained in Example 2 was inoculated into a LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 180 rpm for 10 hours, and then inoculated into a fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a 2% inoculum (v / v), and cultured at 37°C and 180 rpm until the bacterial OD600 reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and after induction culture at 28°C for 12 hours, the culture was centrifuged at 4°C and 8000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was collected to obtain the wet bacterial cell of the recombinant tagatose-4-epimerase mutant. The wet bacterial cell can be directly used as a biocatalyst or for protein purification.

[0054] Example 4: Isolation and purification of tagatose-4-epimerase mutants

[0055] Preparation of pure enzyme solution of mutant tagatose-4-epimerase described in the present invention: add 9.8mL of 100mM, pH8.0 sodium phosphate buffer solution to 0.2g of wet cells of recombinant genetically engineered bacteria containing mutant tagatose-4-epimerase encoding genes and resuspend the wet cells, perform ultrasonic disruption under ice bath conditions (200W power, lasting 1s, resting 2s, continuous disruption for 15min), and obtain cell disruption solution. The cell disruption solution obtained after ultrasonic disruption is centrifuged at 8000rpm and 4°C for 10min, and the supernatant obtained is the desired crude enzyme solution. The crude enzyme solution can be further purified to obtain a pure enzyme solution. The SDS-PAGE picture of tagatose-4-epimerase is shown below. Figure 1 As shown. Among them, Figure 1 Lane 1 from the left is a protein molecular weight marker, lane 2 is a crude enzyme of tagatose-4-epimerase, and lane 3 is a pure enzyme of a mutant tagatose-4-epimerase.

[0056] Example 5: Determination of catalytic activity of tagatose-4-epimerase mutants

[0057] The wild-type crude tagatose-4-epimerase and the recombinant crude tagatose-4-epimerase mutant enzymes DTE-R69A, DTE-N70A, DTE-E75A, DTE-P140L, DTE-R165A, DTE-F361H, and DTE-E75A / F361H separated and purified according to the method of Example 4 were used to catalyze the substrate (D-fructose).

[0058] The composition and catalytic conditions of the enzyme catalysis system are as follows: wet bacteria were resuspended and diluted with phosphate buffer (100 mM, pH 8.0), and D-fructose with a final concentration of 50 g / L and Ni with a final concentration of 1 mM were added. 2+ , phosphate buffer (50mM, pH8.0) was used to form a 1mL reaction system, and the reaction was carried out at 75°C and 1000rpm for 30min, and then the reaction was terminated by boiling in boiling water for 10min. After centrifugation at 13000rpm for 10min, the supernatant was diluted to a corresponding multiple and then passed through a membrane, and the conversion rate was detected by high performance liquid chromatography (HPLC). The schematic diagram of the enzymatic synthesis of D-tagatose is shown in Figure 2 shown.

[0059] The HPLC detection method is as follows: Thermo High Performance Liquid Chromatography (HPLC) system (Milford, USA) and differential detector detection. The analytical column is Sugar-park (300×6.5mm). The mobile phase is 0.5mg / mL EDTA-disodium calcium, and the solvent is ultrapure water. The column temperature was set to 80°C, the flow rate was maintained at 0.4mL / min, and the injection volume was 10μL. The external standard method was used to determine the conversion rate of D-fructose and the yield of D-tagatose based on the retention time and peak area of ​​the peak. The schematic diagram of HPLC detection of substrate D-fructose and product D-tagatose is shown in the figure Figure 3 The relative enzyme activity bar graph of wild-type tagatose-4-epimerase and tagatose-4-epimerase mutants is shown in Figure 4 shown.

[0060] The relative activity results of wild-type DTE and tagatose-4-epimerase mutants are shown in Table 1 below:

[0061] Table 1: Relative activity results of wild-type DTE and tagatose-4-epimerase mutants

[0062]

[0063] Example 6: Application of recombinant tagatose-4-epimerase wild-type DTE in the preparation of D-tagatose

[0064] The wet cells of recombinant E. coli BL21 (DE3) / DTE containing the expression recombinant plasmid obtained in Example 4 were used as biocatalysts, and D-fructose was used as substrate to carry out catalytic reaction to prepare D-tagatose. Example 7 was used as a control.

[0065] The composition and catalytic conditions of the enzyme catalysis system are as follows: the crude enzyme was diluted with phosphate buffer (100 mM, pH 8.0), and D-fructose with a final concentration of 50 g / L and Ni with a final concentration of 1 mM were added. 2+ , phosphate buffer (50mM, pH 8.0) was used to form a 1mL reaction system, and the reaction was carried out at 75°C and 1000rpm for 30min, and then boiled in boiling water for 10min to terminate the reaction. After centrifugation at 13000rpm for 10min, the supernatant was diluted to a corresponding multiple and then passed through a membrane for detection by high performance liquid chromatography (HPLC).

[0066] Example 7: Application of the recombinant tagatose-4-epimerase mutant E75A / F361H in the preparation of D-tagatose

[0067] The crude enzyme solution of recombinant Escherichia coli BL21 (DE3) / DTEE75A / F361H containing the expression recombinant plasmid obtained in Example 4 was used as a biocatalyst and D-fructose was used as a substrate to enzymatically synthesize D-tagatose.

[0068] The catalytic reaction system and catalytic conditions are as follows: the final concentration of tagatose-4-epimerase is 40 g / L, the final concentration of D-fructose is 50 g / L, and the final concentration of Ni is 1 mM. 2+ The transformation system was composed of 1 mL of sodium phosphate buffer solution (pH 8.0) as the reaction medium, 75°C, 1000 rpm, and analyzed by HPLC. The catalytic results showed that its relative enzyme activity was 1.26 times higher than that of the original strain.

[0069] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A tagatose-4-epimerase mutant, characterized in that: The amino acid sequence is derived from SEQ ID NO.1 by site-directed mutagenesis, wherein the mutation sites are the following two: (1) glutamic acid at position 75 is mutated to alanine, and (2) phenylalanine at position 361 is mutated to histidine.

2. A gene encoding the tagatose-4-epimerase mutant according to claim 1.

3. A recombinant vector constructed from the encoding gene according to claim 2.

4. A recombinant genetically engineered bacterium transformed by the recombinant vector according to claim 3.

5. Use of the tagatose-4-epimerase mutant according to claim 1 in the preparation of D-tagatose.

6. The use according to claim 5, characterized in that: The application is: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing a gene encoding a mutant of tagatose-4-epimerase, or crude enzyme extracted from wet bacteria after ultrasonic crushing, or purified pure enzyme as a catalyst, using D-fructose as a substrate, and reacting with Ni 2+ In the presence of , the reaction is carried out at 70-80°C in a sodium phosphate buffer solution with a pH value of 7-9. After the reaction is complete, the reaction solution is separated and purified to obtain D-tagatose.

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