L-arabinose isomerase, engineering bacteria and application

Abstract

The invention discloses L-arabinose isomerase, engineering bacteria and application, the amino acid sequence of the L-arabinose isomerase is shown as SEQ ID NO: 3, and the gene sequence for coding the L-arabinose isomerase is shown as SEQ ID NO: 2. On the basis of a genome of a Peribacillussp.S4 strain, a gene for coding the L-arabinose isomerase is excavated, and a stable prokaryotic expression system is constructed by performing codon optimization on the gene, so that the large-scale controllable production of the L-arabinose isomerase is realized, the yield of the enzyme is remarkably increased, the production cost of the enzyme is reduced, and the method is suitable for industrial production. A stable and economical enzyme source is provided for the production of D-tagatose; the L-arabinose isomerase expressed by the constructed engineering bacteria can significantly improve the yield of D-tagatose, and industrialization of D-tagatose is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, and in particular to an L-arabinose isomerase, an engineered bacterium and applications. BACKGROUND

[0002] D-tagatose is a new low-calorie sweetener (92% of sucrose, 1.5 kcal / g of heat), which is one of the low-calorie functional sweeteners that have attracted much attention in recent years. D-tagatose exists naturally in small amounts in foods such as cheese and cocoa beans, so D-tagatose is mainly obtained by chemical or biological conversion. Chemical conversion uses lactose as a raw material, hydrolyzes lactose into D-galactose, and then isomerizes D-galactose into D-tagatose using an alkali catalyst, but the chemical conversion method requires strong acid / high temperature conditions, resulting in many by-products and high separation costs.

[0003] Biological conversion includes enzyme method and genetic engineering technology, and the core of both methods is to rely on L-arabinose isomerase. L-arabinose isomerase (LAI, EC 5.3.1.4) is a key enzyme for catalyzing the isomerization of D-galactose into D-tagatose, and belongs to the aldose-ketose isomerase family. The enzyme widely exists in microorganisms such as Bacillus subtilis and thermophilic bacteria, and the active center contains highly conserved catalytic residues (such as E306, E333), which realizes efficient stereoselective conversion through an enediol intermediate mechanism. However, the traditional enzyme method is limited by low enzyme activity and substrate inhibition effect. Genetic engineering technology can significantly improve the catalytic efficiency by recombinant expression of L-arabinose isomerase, but the existing technology still has limitations in enzyme thermal stability and substrate affinity, and the conversion rate of D-tagatose is low. SUMMARY

[0004] Therefore, the present application provides an L-arabinose isomerase, an engineered bacterium and applications.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The L-arabinose isomerase provided by the present application has an amino acid sequence as shown in SEQ ID NO: 3.

[0006] The present application also provides a gene encoding L-arabinose isomerase, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 2; and the amino acid sequence of the L-arabinose isomerase is as shown in SEQ ID NO: 3.

[0007] The present application also provides a recombinant plasmid comprising a gene encoding L-arabinose isomerase, wherein the expression vector is PTIG, and the sequence of the gene encoding L-arabinose isomerase is as shown in SEQ ID NO: 2.

[0008] The application further constructs an engineered bacterium expressing L-arabinose isomerase, which is obtained by transforming a host cell with a recombinant plasmid E. coli The recombinant plasmid includes a gene encoding L-arabinose isomerase, and the nucleotide sequence of the gene is shown as SEQ ID NO: 2.

[0009] The application proposes an application of the engineered bacterium expressing L-arabinose isomerase in producing L-arabinose isomerase. Specifically, the engineered bacterium expressing L-arabinose isomerase is induced to culture, and L-arabinose isomerase is obtained. Peribacillus The application is based on the genome of the strain S4, mines the gene producing L-arabinose isomerase, optimizes the gene by codon, constructs a stable prokaryotic expression system, and then realizes the controllable production of L-arabinose isomerase, significantly improves the yield of the enzyme, reduces the production cost of the enzyme, and provides a stable and economical enzyme source for the commercial application of L-arabinose isomerase.

[0010] Preferably, after the induction culture is completed, the L-arabinose isomerase crude enzyme solution is obtained by treating the bacterial liquid: the bacterial liquid is centrifuged at 4 DEG C, the supernatant is discarded, and the bacterial precipitate is collected; the bacterial precipitate is resuspended with pre-cooled Tris-HCl buffer, and after resuspension, centrifugation is performed, and the resuspension and washing are performed for multiple times to remove the residual culture medium; after washing, the bacterial precipitate is resuspended, pre-cooled at 0-4 DEG C, ultrasonically broken, centrifuged to collect the supernatant, and filtered to obtain the L-arabinose isomerase crude enzyme solution. The L-arabinose isomerase obtained by fermentation has good stability and activity, can promote the generation of D-tagatose, and improve the yield of D-tagatose.

[0011] More preferably, after the crude enzyme solution is obtained, purification treatment is performed on the crude enzyme solution, specifically including: column chromatography purification is adopted, and the buffer liquid containing 20 mM imidazole, 50 mM imidazole and 500 mM imidazole is sequentially eluted. Through experiments, the specific enzyme activity of the pure enzyme solution is 5.2 U / mg, the conversion rate of D-tagatose is more than 40%, and the applicable temperature is 4-60 DEG C. The results show that the L-arabinose isomerase has good catalytic activity, and can significantly improve the yield of D-tagatose, and has popularization value and application prospect.

[0012] The application further proposes an application of the L-arabinose isomerase in preparing D-tagatose. In the preparation of D-tagatose, D-galactose is used as a substrate, L-arabinose isomerase is used to catalyze the conversion of D-galactose into D-tagatose, and the substrate system contains Mn 2+ , and the concentration is 2 mmol / L.

[0013] Compared with the prior art, the application has the following advantages: The application is based onPeribacillus The present application is based on the genome of the strain S4, the gene producing L-arabinose isomerase is mined, the stable prokaryotic expression system is constructed by codon optimization of the gene, and then the controllable production of L-arabinose isomerase is realized, the yield of the enzyme is significantly improved, the production cost of the enzyme is reduced, a stable and economical enzyme source is provided for the commercial application of L-arabinose isomerase, and the industrialization of D-tagatose is facilitated.

[0014] Through experiments, the specific enzyme activity of the pure enzyme liquid of the present application is above 5.2 U / mg at 4-60 DEG C, that is, the enzyme shows high activity in a wide temperature range, and is not only suitable for low temperature conditions, but also suitable for medium and high temperature conditions; the conversion rate of D-tagatose is above 40%, and the conversion rate of D-tagatose in a low temperature environment (4-20 DEG C) is slightly higher than that in a high temperature environment. The results show that the L-arabinose isomerase of the present application has a wide temperature adaptation range, and also shows high catalytic activity, so that the conversion rate of D-tagatose is as high as above 45%, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the map of the recombinant plasmid of the present application.

[0016] Figure 2 is the plasmid double enzyme digestion verification diagram of the recombinant strain. Figure 2 Marker 1, recombinant plasmid 2.

[0017] Figure 3 is the electrophoresis diagram of the eluent in Example 2. Figure 3 M is a protein marker; 1 is the precipitate after centrifugal separation of the cell body broken in Example 2; 2 is the crude enzyme liquid in Example 2; 3 is the flow-through liquid of the crude enzyme liquid in the loading process; 4 is the washing liquid obtained by washing with 20 mM imidazole; 5 and 6 are the washing liquid in the washing process with 50 mM imidazole; 7-9 are the pure enzyme liquid obtained by elution with 500 mM imidazole. DETAILED DESCRIPTION

[0018] The present application will be described in detail below with reference to the accompanying drawings. It should be pointed out that the chemical reagents used in the present application are all conventional reagents commercially available, and the instruments used are all commonly used laboratory instruments.

[0019] It should be pointed out that the starting strain in the present application is Peribacillus sp. S4, the whole genome of which has been uploaded to National Center for Biotechnology Information (NCBI), and the genome number is PRJNA1193798; the host cell E. coliBL21(DE3) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; the vector PTIG was provided by the Henan Provincial Engineering Research Center for Applied Microbiology, Henan University. This application utilizes a homologous recombinase to integrate the gene expressing sulfur oxidase (TrxA) into PTIG to increase the expression level of the target protein (i.e., L-arabinose isomerase). Furthermore, specific enzyme activity in this invention is defined as the amount of enzyme required to catalyze the production of 1 μmol of D-tagatose per minute, expressed in U / mg.

[0020] Example 1 Construction and Identification of Engineered Bacteria This invention addresses data already uploaded to NCBI. Peribacillus Bioinformatics analysis was performed on the whole genome sequence of sp. S4, and the putative protein gene of L-arabinose isomerase was obtained through protein functional annotation, the nucleotide sequence of which is shown in SEQ ID NO:1. This invention constructs an engineered bacterium expressing L-arabinose isomerase based on SEQ ID NO:1, specifically including the following: The first step was to optimize the design of the putative protein gene of L-arabinose isomerase based on the codon preference of E. coli, resulting in the target gene shown in SEQ ID NO:2. The target gene (i.e., nucleotide sequence) shown in SEQ ID NO:2 was synthesized by Suzhou Junji Gene Technology Co., Ltd. The amino acid sequence of the target gene after transcription and translation is shown in SEQ ID NO:3. The second step is to synthesize the target gene from the first step. EcoR I / Xho I Double digestion was performed, followed by ligation with PTIG carrying the TrxA gene using T4 DNA ligase to construct the recombinant expression plasmid RALI. The plasmid map is shown below. Figure 1 ; The third step is to transform the recombinant expression plasmid RALI constructed in the second step. E. coli BL21(DE3), specifically: a. Add 3 μL (10 ng / μL) of the recombinant plasmid obtained in step 2 to the thawed plasmid on ice. E. coli In BL21(DE3) competent cells, after mixing, the mixture was placed in an ice-water mixture and allowed to stand for 10 min; then, it was placed in a 45°C metal bath for 90 s heat shock, then quickly removed and placed on ice for 5 min; finally, 600 μL of LB medium was added, and the cells were revived and cultured at 37°C and 200 rpm for 45 min. b. After the resuscitation culture is completed, 50 μL of the transformation product is evenly spread on an LB plate containing the resistance (Kana, final concentration 50 μg / mL) and cultured at 37℃ for 12 h. Then, single clones are picked and transferred to LB liquid medium containing the resistance (Kana, final concentration 50 μg / mL) and cultured at 37℃ and 200 rpm for 12 h. c. After culture, extract the plasmid using a plasmid extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and then extract the plasmid... EcoR I / XhoI Double enzyme digestion and electrophoresis results showed that the target band of the extracted plasmid was consistent with the target gene (see...). Figure 2 This indicates that the plasmid transformation was correct. After the plasmid digestion was verified to be correct, Shanghai Sangon Biotech Co., Ltd. was commissioned to perform sequencing and identification of the plasmid sequence (to ensure that the target gene recombination was correct (without any mutations)). Finally, sterile glycerol was added to the strain containing the correct recombinant plasmid (i.e., the strain expressing L-arabinose isomerase), and it was frozen and stored at -80℃.

[0021] Example 2: Fermentation culture and purification method of L-arabinose isomerase I. Fermentation culture of L-arabinose isomerase This invention proposes a method for producing L-arabinose isomerase, which involves fermenting the recombinant strain from Example 1 to obtain L-arabinose isomerase, specifically including the following: The first step is to thaw the frozen strain (i.e. the strain constructed in Example 1) at 37°C, pick the strain with a sterile inoculation loop, and streak it in three zones onto an LB solid medium plate containing kanamycin (final concentration 50 μg / mL). Invert the plate and incubate it in a 37°C incubator for 12-16 h until a single colony is formed. The second step involves picking a single colony from the first step and inoculating it into LB liquid medium containing kanamycin (final concentration 50 μg / mL) (volume ≤ 20% of shake flask volume), and incubating at 37℃ with shaking at 200 rpm. During the incubation process, the OD of the bacterial culture is monitored using a UV spectrophotometer. 600nm value; The third step is when the OD of the fermentation broth... 600nm When the value reaches 0.6-0.8 (mid-logarithmic growth phase), add isopropyl-β-D-thiogalactoside (IPTG) to the culture system to a final concentration of 0.5 mM, and then immediately transfer to a 16℃ low-temperature shaker, adjust the rotation speed to 200 rpm, and continue to induce culture for 20 h-24 h. Fourth step: Centrifuge the induced expression broth at 8000×g for 10 min at 4℃ (pre-cool the centrifuge rotor), discard the supernatant, and collect the cell pellet; resuspend the cells in pre-cooled Tris-HCl buffer (50 mM, pH = 7.0), and centrifuge again at 8000×g for 10 min at 4℃, discard the supernatant, and repeat this process several times to remove residual culture medium; After washing, the bacterial cells were gently resuspended in pre-cooled binding-wash buffer (containing 500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.5) and placed in an ice-water bath at 0-4℃ for 10 min. The ratio of bacterial cells to buffer was 1 g bacterial cells : 5 mL binding-wash buffer. After pre-cooling, the bacterial cells were sonicated using a titanium alloy probe sonicator under ice bath conditions. After sonication, the cells were centrifuged at 4℃, 12,000×g for 10 min. The supernatant was collected and filtered through a 0.22 μm PVDF aqueous filter membrane to obtain crude enzyme solution, which was aliquoted and stored at -80℃.

[0022] II. Purification of crude enzyme solution of L-arabinose isomerase In this invention, the crude enzyme solution is purified using a Ni-NTA column, with sequential elution to obtain the purified enzyme solution, specifically including: First, rinse the Ni-NTA column with 3-5 column volumes of ultrapure water to remove ethanol from the packing material. Then, add 3-5 column volumes of binding-wash buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.5) to equilibrate the column. After equilibration, add the obtained crude enzyme solution to the Ni-NTA column and incubate on ice for at least 30 minutes, shaking it from side to side during incubation to ensure that the target protein fully contacts and binds to the packing material. In the second step, after settling, first elute the non-bound proteins with the Ni-NTA column packing material with 3-5 column volumes of binding-wash buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.5) and collect the flow-through. Then elute the weakly bound proteins with the Ni-NTA column packing material with 3-5 column volumes of washing buffer (500 mM NaCl, 50 mM Tris, 50 mM imidazole, pH 7.5) and collect the washings. Finally, elute the target protein on the Ni-NTA column with 3-5 column volumes of elution buffer (500 mM NaCl, 50 mM Tris, 500 mM imidazole, pH 7.5) and collect the eluent. Electrophoretic analysis showed that the purified L-arabinose isomerase had a molecular weight of approximately 53 kDa, consistent with the theoretical molecular weight, and exhibited a single band, indicating high purity. See details below. Figure 3 ; The third step involves combining the eluents containing the target protein and using a 10 kDa ultrafiltration tube to concentrate and desalt the collected eluents, thereby obtaining a pure enzyme solution of L-arabinose isomerase.

[0023] Example 3: Preparation of D-tagatose based on the method in Example 2 This invention proposes a method for preparing D-tagatose, which uses the crude enzyme solution obtained in Example 2 as a catalyst to catalyze the conversion of D-galactose into D-tagatose, specifically including the following: Add 1 mL of substrate system (containing 100 g / L D-galactose, 2 mmol / L Mn) 2+ Add 200 μL of crude enzyme solution to 50 mmol / L Tris-HCl buffer (pH 7.5), react at 55℃ for 90 min, and immediately stop the reaction by boiling water bath. After inactivation, the reaction system was centrifuged and the supernatant was collected. The supernatant was diluted 10 times with ultrapure water. The mixture was filtered through a 0.22 μm filter membrane and D-tagatose was detected by high performance liquid chromatography (Agilent 1260 Infinity II system). The liquid chromatography conditions were as follows: ZORBAX Carbohydrate Analysis column (4.6×250 mm, 5 μm), mobile phase acetonitrile / ultrapure water (v / v 80:20), flow rate 0.8 mL / min, column temperature 30℃, injection volume 10 μL, and differential refractive index detector (RID) at 35℃ for 20 min. Before testing, the D-tagatose standard was diluted to different concentration gradients and analyzed by high performance liquid chromatography. A standard curve of D-tagatose was plotted based on the external standard method. The concentration of D-tagatose in the sample to be tested was calculated based on the standard curve. The amount of D-tagatose generated was determined based on the concentration and the volume of the supernatant after the reaction. The conversion rate of D-tagatose was calculated based on the yield of D-tagatose and the amount of D-galactose in the raw material. The conversion rate (%) of D-tagatose was calculated as: (D-tagatose yield / mass of D-galactose in the reaction system before reaction) × 100. The results showed that using the crude enzyme solution as a catalyst, the conversion rate of D-tagatose was 25.44%, and the enzyme activity of the crude enzyme solution was 7.845 U / mL. Therefore, the crude enzyme solution of this invention exhibits high enzyme activity and catalytic activity.

[0024] Example 4: Method for preparing D-tagatose based on the pure enzyme solution in Example 2 Add 1 mL of substrate system (containing 100 g / L D-galactose, 2 mmol / L Mn) 2+ The purified enzyme solution from Example 2 was added to 50 mmol / L Tris-HCl buffer (pH 7.5) to bring the final concentration of the enzyme solution to 0.5 mg / mL. The reaction was carried out at 55°C for 90 min, and the reaction was immediately terminated by boiling in a water bath. After centrifugation (same as Example 3), the yield of D-tagatose was determined by liquid chromatography (same as Example 3). In this example, the conversion rate of D-tagatose was greater than 45%.

[0025] Example 5: Effect of temperature on the L-arabinose isomerase of the present invention Prepare a substrate system containing 100 g / L D-galactose and 2 mmol / L Mn. 2+ 50 mmol / L Tris-HCl buffer, pH 7.5; take multiple 1 mL aliquots of substrate system, add an equal volume of pure enzyme solution (final concentration 0.5 mg / mL), and react at 4, 6, 8, 10, 20, 30, 40, 50, and 60 °C for 90 min respectively. After the reaction, immediately stop the reaction by boiling water bath, and calculate the conversion rate of D-tagatose. See Table 1 for details.

[0026] Table 1. Specific enzyme activity and catalytic properties of the L-arabinose isomerase of the present invention at different temperatures. As shown in Table 1, the L-arabinose isomerase of this invention exhibits high specific activity (over 5 U / mg) at temperatures ranging from 4 to 60°C. It also demonstrates high catalytic activity at these temperatures, achieving a D-tagatose conversion rate of over 40%. Furthermore, the D-tagatose conversion rate is slightly higher at lower temperatures (4-20°C) than at higher temperatures. These results indicate that the L-arabinose isomerase of this invention has a wide temperature adaptability range and exhibits high catalytic activity, resulting in a D-tagatose conversion rate exceeding 45%, demonstrating promising application prospects.

Claims

1. An L-arabinose isomerase, characterized in that: The amino acid sequence is shown in SEQ ID NO:

3.

2. A gene encoding the L-arabinose isomerase as described in claim 1, wherein, The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A recombinant plasmid comprising the gene of claim 2, wherein, The expression vector is PTIG.

4. An engineered bacterium expressing L-arabinose isomerase, characterized in that: Including the recombinant plasmid as described in claim 1, the host cell is E. coli BL21(DE3).

5. The use of the engineered bacteria according to claim 4 in the production of L-arabinose isomerase.

6. A method for producing L-arabinose isomerase, characterized in that: Seed culture was obtained by inoculating and culturing the engineered bacteria described in claim 4, and then inducing culture was performed to obtain L-arabinose isomerase.

7. The method for producing L-arabinose isomerase according to claim 6, characterized in that: After the induction culture was completed, the bacterial culture was treated to obtain the crude enzyme solution of L-arabinose isomerase: the fermentation culture was centrifuged and the cell precipitate was collected; the cells were then resuspended in pre-cooled Tris-HCl buffer, centrifuged again, and this process of resuspension and washing was repeated several times to remove residual culture medium; after washing, the cells were resuspended, pre-cooled at 0-4℃, sonicated and disrupted, the supernatant was collected by centrifugation, and filtered to obtain the crude enzyme solution of L-arabinose isomerase.

8. The method for producing L-arabinose isomerase according to claim 7, characterized in that: The method also includes the purification of the crude enzyme solution, specifically including: purification by column chromatography, eluting sequentially with buffers containing 20 mM imidazole, 50 mM imidazole and 500 mM imidazole.

9. The use of the L-arabinose isomerase produced according to any one of claims 6-8 in the preparation of D-tagatose.

10. The application according to claim 9, characterized in that: In the preparation of D-tagatose, D-galactose is used as a substrate, and L-arabinose isomerase is used to catalyze the conversion of D-galactose into D-tagatose.

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