Antarctic krill-derived xylose isomerase, its mutant and application

Abstract

The present invention relates to a xylose isomerase derived from Antarctic krill and its mutants and applications, belonging to the field of enzymology, and the amino acid sequence of the xylose isomerase is shown in SEQ ID NO.1. The gene encoding the xylose isomerase is shown in SEQ ID NO.2. The present invention also provides a gene recombinant plasmid and a recombinant bacterium containing the xylose isomerase; the present invention also provides a xylose isomerase mutant, specifically EsXLI‑K253R and EsXLI‑D328A. The Antarctic krill xylose isomerase and its mutants obtained by the present invention can not only catalyze the isomerization of D‑xylose to generate D‑xylulose, but also have specificity for different substrates, and convert and produce many important raw materials and rare sugars in the pharmaceutical industry, and have very good application prospects. The xylose isomerase of the present invention has good adaptability to low temperatures and can be expressed in Escherichia coli cells.

Description

Technical Field

[0001] The invention belongs to the field of enzymology, and specifically relates to a xylose isomerase derived from Antarctic krill and a mutant and application thereof. Background Art

[0002] The conservative biomass of krill in the Antarctic waters can reach 62.6 million tons, and the annual catch can reach 60-100 million tons, and it will not affect the ecosystem of the Southern Ocean. Antarctic krill contains proteins, phospholipids, unsaturated fatty acids and other ingredients with extremely rich nutritional value. If this resource can be used by humans, it will greatly promote the development of marine resources. Numerous studies have shown that the adaptability of Antarctic krill to extreme environments is closely related to the enzymes in its body, which also provides additional possibilities for Antarctic krill-derived enzymes as industrial biocatalysts. At present, the development and utilization of Antarctic krill-derived enzymes is still in its early stages, so we need to conduct more and deeper research on Antarctic krill-derived enzymes.

[0003] Xylose isomerase (Ⅺ) (EC 5.3.1.5) can catalyze the conversion of pentose D-xylose into D-xylulose, and plays an important role in the sugar metabolism process in microorganisms; it can also catalyze the isomerization reaction of D-glucose to D-fructose in vitro, so it is also called glucose isomerase. After pretreatment and enzymatic hydrolysis, lignocellulosic biomass will produce a large amount of glucose and xylose. There are some microorganisms in nature that can naturally utilize xylose, including bacteria, yeast and fungi, but most of them cannot use xylose as a fermentation substrate, so the xylose resources in the hydrolyzate are not used as effectively as glucose. Xylose isomerase has extremely important industrial application value, and is called an important industrial enzyme together with protease and amylase. In recent years, scientists have found that under certain conditions, xylose isomerase can convert and produce many rare sugars that are important raw materials for the pharmaceutical industry, such as ribose, mannose, arabinose, lyxose, etc. These discoveries have injected new vitality into the research of xylose isomerase.

[0004] At present, there are few reports on heterologous expression of xylose isomerase. Therefore, discovering xylose isomerase genes with high activity and constructing genetically engineered bacteria through gene recombination to efficiently heterologously express xylose isomerase have important industrial application value and potential. Summary of the invention

[0005] The purpose of the present invention is to provide a xylose isomerase from Antarctic krill and its mutants and applications. The present invention mines the Antarctic krill XLI gene (EsXLI) based on genome analysis and heterologously expresses it in Escherichia coli PET28a, which has higher enzyme activity to meet industrial production needs.

[0006] The present invention is achieved through the following technical solutions:

[0007] A xylose isomerase derived from Antarctic krill, wherein the amino acid sequence of the xylose isomerase is shown as SEQ ID NO.1.

[0008] The gene encoding the xylose isomerase, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0009] A recombinant plasmid PET28a-EsXLI contains the nucleotide sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a first recombinant Escherichia coli engineered bacterium, wherein the first recombinant Escherichia coli engineered bacterium contains the recombinant plasmid PET28a-EsXLI.

[0011] A xylose isomerase mutant derived from Antarctic krill, wherein the mutant is characterized in that, based on the amino acid sequence shown in SEQ ID NO.1, the lysine at position 253 is mutated to arginine, i.e., EsXLI-K253R, or the aspartic acid at position 328 is mutated to alanine, i.e., EsXLI-D328A.

[0012] A recombinant plasmid containing a gene encoding the xylose isomerase mutant.

[0013] The present invention also provides a second recombinant Escherichia coli engineered bacterium, wherein the second recombinant Escherichia coli engineered bacterium contains a gene of a xylose isomerase mutant.

[0014] The present invention also provides the xylose isomerase, the xylose isomerase mutant, the recombinant plasmid PET28a-EsXLI, the recombinant plasmid, the first recombinant Escherichia coli engineering bacteria and the second recombinant Escherichia coli engineering bacteria in isomerizing xylose.

[0015] The present invention also provides an enzyme preparation, which contains the xylose isomerase and / or the xylose isomerase mutant.

[0016] The beneficial effects of the present invention compared with the prior art are as follows:

[0017] The present invention adopts the bioinformatics method of Antarctic krill protein transcriptome to obtain the gene sequence of xylose isomerase, amplifies the target gene in vitro by polymerase chain reaction (PCR), and expresses soluble active gene in Escherichia coli, laying a theoretical foundation for the application of xylose isomerase in the preparation of rare ketose in the fields of food and medicine.

[0018] The present invention also obtains two mutants of Antarctic krill xylose isomerase, wherein the enzyme activity of the mutant EsXLI-K253R is increased by 1.25 times compared with the original enzyme, and the optimum enzyme activity temperature of the mutant EsXLI-D328A is reduced by 10°C.

[0019] The Antarctic krill xylose isomerase and its mutant obtained by the present invention can not only catalyze the isomerization of D-xylose to generate D-xylulose, the isomerization product of L-arabinose is D-tagatose, the isomerization product of D-ribose is D-ribulose, and the isomerization product of D-glucose is D-fructose, but also have specificity for different substrates and have very good application prospects. The xylose isomerase of the present invention has good adaptability to low temperature and can be expressed in Escherichia coli cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a line graph showing the effect of temperature on the activity of xylose isomerase;

[0021] Figure 2 is a line graph showing the effect of temperature on the stability of xylose isomerase;

[0022] Figure 3 It is a line graph showing the effect of pH on the activity of xylose isomerase;

[0023] Figure 4 is a line graph showing the effect of pH on the stability of xylose isomerase;

[0024] Figure 5 It is a bar graph showing the effect of metal ions on the activity of xylose isomerase;

[0025] Figure 6 It is a bar graph showing the effect of enzyme inhibitors on the activity of xylose isomerase;

[0026] Figure 7 The bar graph shows the isomerization ability of xylose isomerase to different substrates.

[0027] Figure 8 The hydrogen bonding diagram between the catalytic active center of xylose isomerase and the substrate;

[0028] Fig. 9 Figure 2 shows the substrate binding site map of the wild-type xylose isomerase and its two mutants. DETAILED DESCRIPTION

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0030] Example 1: A batch of xylose isomerase genomic sequences were discovered and analyzed through the previously discovered Antarctic krill genome database (unpublished). After the amino acid sequence of these sequences was aligned, the confidence and homology of xylose isomerases from other species were compared by using the Blast analysis tool in the NCBI database. A batch of unverified functional gene sequences that were predicted to be hypothetical xylose isomerases or have potential xylose isomerase activity in bioinformatics were selected, and the homology of the Antarctic krill amino acid sequence with the known source sequence was between 70-90%. After further classification and analysis of the domain and protein family, the candidate sequence was determined. Subsequently, the front and rear primers were designed for the candidate sequence, and the Antarctic krill cDNA was used as a template to verify it by PCR. After the PCR product was sequenced correctly, the candidate gene EsXLI was obtained.

[0031] After large-scale screening and in-depth research, a new xylose isomerase was identified and separated from Antarctic krill, and its amino acid and nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. It can not only catalyze the isomerization of D-xylose to generate D-xylulose, but also has specificity for different substrates, and has very good application prospects. The xylose isomerase of the present invention has good adaptability to low temperatures and can be expressed in Escherichia coli cells. At the same time, the present invention also optimizes the recombinant expression method of the xylose isomerase, so that it is efficiently expressed in host cells.

[0032] The inventors first optimized the codons according to the expression conditions to improve the expression efficiency and stability of the DNA fragment. The optimized sequence was inserted into the plasmid PET28a to obtain the recombinant plasmid PET28a-EsXLI, and then the recombinant plasmid PET28a-EsXLI was transformed into Escherichia coli BL1 (DE3) competent cells for heterologous expression.

[0033] Example 2: This example provides a construction process of a recombinant Escherichia coli engineering bacterium of a recombinant expression vector, as follows:

[0034] The EsXLI gene was synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). The synthesized EsXLI gene was used as a template to amplify EsXLI using forward primer 5'-ATGGCAGATCCAGCTGTCAA-3' (SEQ ID NO.3) and reverse primer 5'-CTAAACATAGTGGTTAAATACGGCC-3' (SEQ ID NO.4). The amplified product was purified and double-digested with EcoRI / NotI, and then ligated into the pET28a vector after double-digestion with EcoRI / NotI.

[0035] The plasmid was recovered using a plasmid extraction kit. After gene sequencing, it was transformed into Escherichia coli competent cells, and the transformants were transferred to LB medium for culture (the medium consisted of 10g / L peptone, 5g / L yeast extract, 10g / LNaCl, and 50 μg / mL carbomer). The temperature was 37°C, the shaking speed was 200 r / min, and the Escherichia coli bacterial solution was used as a template. T7-F (TAATACGACTCACTATAGGG, SEQ ID NO.5) and T7-R (GCTAGTTATTGCTCAGCGG, SEQ ID NO.6) were used as front and back primers. The annealing temperature was set to 48°C for PCR, and 20 μL of the PCR product was sent to Biotech for sequencing to verify the sequence. After the sequencing was correct, the bacterial solution was transferred to fresh LB medium and cultured at 37°C for shaking until OD 600 The value reached 0.6-0.8. Add 0.6M IPTG for induction culture for 20 h at 37°C and oscillation speed of 200 r / min, then centrifuge at 8000 r / min for 10 minutes to collect the cells, add the collected cells to the buffer and resuspend them three times. After resuspension, add PMSF with a final concentration of 1 mM, place on ice for ultrasonic disruption, and the supernatant of centrifugation is the crude enzyme solution.

[0036] Nickel column purification of recombinant enzyme: The recombinantly expressed EsXLI gene carries an 8×His tag at the N-terminus, which can bind to the Ni of the HisTrap HP column packing, and Ni can bind to imidazole. Therefore, elution with different concentrations of imidazole can achieve the purpose of purification, thereby obtaining a single protein. First, flush the A and B pumps and the system of the AKTA protein rapid purification instrument with ddH2O, and then flush the A and B pumps with equilibrium buffer respectively; secondly, adjust the flow rate to 1 mL / min, and adjust the flow rate to 2 mL / min after connecting the nickel column, and load the crude enzyme solution after passing through a 0.45 um filter membrane, with a loading flow rate of 1 mL / min; after the penetration peak runs flat, rinse with washing buffer to remove impurities; finally, after the baseline is stable, elute the target protein with elution buffer, sample the peak, and stop sampling until the peak runs flat.

[0037] The equilibration buffer is: 20 mM Tris-HCl buffer; 500 mM NaCl; pH 8;

[0038] The washing buffer is: 20 mM Tris-HCl buffer; 500 mM NaCl; 10 mM imidazole; pH 8;

[0039] The elution buffer was: 20 mM Tris-HCl buffer; 500 mM NaCl; 500 mM imidazole; pH 8.

[0040] Determination of enzyme activity: D-xylulose is converted into furfural substances under the action of concentrated sulfuric acid. Furfural reacts with cysteine-carbazole reagent to produce a color reaction. The product of the catalytic reaction, D-xylulose, can be quantitatively detected by spectrophotometry. The maximum absorption peak at a wavelength of 540 nm is used to characterize the enzyme activity of the recombinant xylose isomerase: dilute the enzyme solution appropriately with pH8 Tris-HCl buffer, then take 100 μL of the diluted enzyme solution, add 50 μL 10 mM MnCl2, 800 uL pH8 Tris-HCl buffer, add 50 uL 100 mM D-xylose solution (control plus inactivated enzyme), and react in a 50℃ water bath for 30 min. After the reaction is completed, take it out immediately, add 200 μL trichloroacetic acid immediately to terminate the reaction, take 200 μL of the reaction mixture to the wells of the ELISA plate, and measure the absorbance at a wavelength of 540 nm.

[0041] Enzyme properties (1) Optimum temperature determination: The enzyme activity of EsXLI was measured at different temperatures of 20°C, 30°C, 40°C, 50°C, 60°C, 70°C and 80°C. Three parallels were made for each temperature group, and the relative enzyme activity was measured, with the highest enzyme activity measured at 50°C being 100%.

[0042] (2) Determination of optimal pH: The effect of pH on enzyme activity was analyzed from the change of pH. Citrate buffer (pH 4.0-5.0), phosphate buffer (pH 5.0-7.0), Tris-HCl buffer (pH 7.0-9.0) and glycine sodium hydroxide buffer (pH 9.0-10.0) were prepared. The reaction system was placed in a 50℃ water bath for 30 min. Three parallels were made for each temperature group. The relative enzyme activity was measured, and the highest enzyme activity at pH 7 was set as 100%.

[0043] (3) Temperature stability determination: The purified enzyme solution was placed in a metal bath at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 240 min. The solution was taken out every 60 min and the enzyme activity was determined at pH 8.5. Three parallel experiments were performed for each time group. The relative enzyme activity was determined, with the highest enzyme activity at 30°C being 100%.

[0044] (4) pH stability determination: The purified enzyme solution was placed in a buffer solution at 4°C and pH 4-9 for 240 min. The enzyme solution was taken out every 60 min and the enzyme activity was determined at 60°C. Three parallel assays were performed for each pH group to determine the relative enzyme activity, with the highest enzyme activity at pH 8.5 being set as 100%.

[0045] (5) Effects of metal ions on the activity of recombinant EsXLI: Various metal ions Na2O were prepared at a concentration of 20 mM. + , K + Mg2+ , Ba 2+ , Ca 2+ , Cu 2+ 、Ni 2+ , Fe 3+ , Mn 2+ 、Co 2+ The final concentration of metal ions was configured to 1 mM and 10 mM. The purified enzyme solution was placed in a metal ion system with different concentrations at 4°C for 120 min, and then reacted in a water bath at pH = 7 and 50°C for 30 min. Three parallel reactions were performed for each temperature group, and the relative enzyme activity was determined, with the highest enzyme activity under ddH2O conditions being 100%.

[0046] (6) Effects of protease inhibitors on recombinant EsXLI enzyme activity

[0047] Use ddH2O to prepare 20 mM SDS, PMSF, β-mercaptoethanol, DTT and EDTA, and configure their final concentrations to 1 mM and 10 mM. Place the purified enzyme solution in a protease inhibitor system with different concentrations at 4°C for 120 min, then react in a water bath at pH = 7 and 50°C for 30 min. Make three parallels for each temperature group and measure the relative enzyme activity. The highest enzyme activity under standard buffer conditions is 100%.

[0048] (7) Isomerization ability of xylose isomerase on different substrates:

[0049] Six different polyhydroxyaldehyde monosaccharide substrates with a concentration of 10 mM were prepared, including D-xylose, D-ribose, D-glucose, D-mannose, D-galactose, and L-arabinose. 50 μL of each was added to 1 ml reaction system, and then reacted in a water bath at pH = 7 and 50°C for 30 min. Three parallels were made for each group of substrates, and the relative enzyme activity was determined. The highest enzyme activity under the condition of D-xylose as the substrate was 100%.

[0050] The above reaction results are shown in the figure. Figure 1 , 2It can be seen that the optimal conditions for enzyme activity are pH 7.0 and the optimal temperature is 40℃~50℃. The optimal reaction temperature of the enzyme is 50℃. As the reaction temperature increases from 20℃ to 45℃, the enzyme activity of EsXLI has been increasing. When the temperature is 50℃, the enzyme activity reaches the highest value. Then, as the temperature gradually increases, the catalytic activity of EsXLI decreases significantly. The temperature stability of EsXLI was tested and it was found that the enzyme was highly stable at 20℃ and could maintain more than 95% of the original activity after incubation for 4 hours. After incubation for 4 hours at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃, the enzyme activity was 95.22%, 91.64%, 82.34%, 70.27%, 5.21%, 4.29% and 4.02% of the original activity, respectively, indicating that the enzyme has a very low stability in an environment greater than 50℃, but can maintain a high stability at low temperatures. Compared with xylose isomerase from other sources, the optimum temperature is around 60-90°C, but xylose isomerase from Antarctic krill can reach its highest enzyme activity at 50°C.

[0051] like Figure 3 , 4 As shown in the figure, the optimum pH is 7. Between pH 4.0 and 7.0, the catalytic activity of the enzyme increases with the increase of pH and reaches the maximum at pH 7.0. As the pH further increases from 7.0 to 10.0, the catalytic activity of EsXLI decreases. Figure 4 The stability of EsXLI under different pH conditions was shown. After incubation for 4 h at pH 7.0, the enzyme activity could be maintained at 95% of its original activity. With the gradual increase of acidity or alkalinity, its stability gradually decreased. However, at pH 4.0, 5.0 and 10.0, the enzyme activity was less than 50% after 4 h of incubation.

[0052] like Figure 5 , Figure 6 The results showed that 1 mmol / L metal ions and enzyme inhibitors had no significant effect on EsXLI enzyme activity. 2+ , Mn 2+ and Co 2+ After the influence of plasma, the relative enzyme activities were 121.25%, 128.13% and 148.32%, respectively, while Na + , K + , Cu 2+ 、Al 3+ , Fe 3+The effects on enzyme activity were relatively small, namely 108.06%, 99.31%, 86.85%, 90.40% and 89.42%, respectively, while Ca 2+ , Ba 2+ 、Ni 2+ The effects on enzyme activity were slightly greater, at 75.32%, 67.54% and 81.28% respectively.

[0053] like Figure 6 As shown, under the influence of 10 mM PMSF and SDS, the enzyme activity retained 84.31% and 89.01%, while 10 mM EDTA and β-mercaptoethanol only retained 39.32% and 52.91% of EsXLI activity.

[0054] like Figure 7 As shown in the figure, among the six xylose isomerase substrates (working concentration 100 mM, the substrates are D-xylose, L-arabinose, D-ribose, D-glucose, D-mannose, and D-galactose), the recombinant xylose isomerase has the strongest effect on D-xylose, followed by L-arabinose (22.32%), D-ribose (13.47%), and D-glucose (3.09%). D-xylose is isomerized to D-xylulose, the isomerization product of L-arabinose is D-tagatose, the isomerization product of D-ribose is D-ribulose, and the isomerization product of D-glucose is D-fructose. There is no isomerization effect on the other two substrates, indicating that the Antarctic krill-derived xylose isomerase has strong substrate specificity.

[0055] According to substrate specificity, three kinds of strong D-xylose (100%), L-arabinose (22.32%), and D-ribose (13.47%) were respectively configured to substrate concentrations of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, and 225 mM. The kinetics of xylose isomerase under different substrate conditions were determined according to the above enzyme activity determination method. Through the kinetic characterization of the enzyme, the maximum reaction rate and Michaelis constant for D-xylose were 0.74 U / mg and 67.99 mM, respectively. The catalytic efficiency of L-arabinose was 0.38 U / mg and 195.53 mM, respectively. The catalytic efficiency of D-ribose was 0.34 U / mg and 202.36 mM, respectively.

[0056] like Figure 8 , Fig. 9As shown, molecular simulation of the catalytic active center of EsXLI and its substrate binding was performed based on sequence alignment and homology modeling. The mutation results showed that the enzyme activity of the EsXLI-K253R mutant (5.05 U / mg) was 1.25 times higher than that of the wild type (4.05 U / mg), because the sugar molecules in the reaction solution usually first bind to the ε-amino group (NH) of the lysine (Lys) residue in the catalytic domain. 3+ ) undergoes Schiff base reaction. This non-enzymatic glycopolymerization should be an important reason affecting the thermal stability of the enzyme. The mutation of lysine to arginine avoids the non-enzymatic glycopolymerization reaction, making xylose isomerase more stable when combined with the substrate at the optimum temperature (50°C) and having higher catalytic efficiency. The optimum temperature of the D328A mutant drops from 50°C to 40°C. The difference between the conservative residues of thermophilic xylose isomerase and its corresponding normal temperature enzyme is analyzed. After the mutation of aspartic acid to alanine, the interaction between the subunits in the conservative residue region of xylose isomerase is reduced, thereby improving the activity at low temperature.

[0057] An Antarctic krill xylose isomerase, the amino acid sequence of which is as follows (SEQ ID NO.1):

[0058] MADPAVKRPKMSDEIENGSGEFFPGIGVIPFKPDARPDETMVFKHYNAQDVVMGKTMEDWCRFSVVYWHTFRGTGLDPFGSGTINRHWDDGSETVDNAKRRLRAAFEFFTKLGN KYWTFHDRDVAPEGRNLAETNANLDALADLAADLQHRTGVKCLWATSNLFSHPRFMHGAATSSDSHVTAYAGAQVKKCMEVAHRLGAENFVFWGGREGYHSLLNTDVRKELDNF AAFFRMVIAYKEKIGFRGQLLIEPKPKEPTRHQYDYDAQTVMAFLHSYGLQDHFKLNIEPNHTTLAGHPYEHDIVMASAFKMLGSIDANTGSPDLGWDTDQFPMDVRNCTAIMK VVLEQGGLKPGGLNFDAKVRRESTDLKDLFIAHIAAMDTLARGLKNAAKIISDGILKKSLQKRYLSWDSGVGAKIAAGQCTLEECEKIIHEQGNPNPPSAHQEHFEAVFNHYV.

[0059] An Antarctic krill xylose isomerase, the nucleotide sequence of the enzyme is as follows (SEQ ID NO.2):

[0060]

Claims

1. A xylose isomerase derived from Antarctic krill, characterized in that: The amino acid sequence of the xylose isomerase is shown in SEQ ID NO.

1.

2. The gene encoding the xylose isomerase according to claim 1, characterized in that: The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A recombinant plasmid PET28a-EsXLI, characterized in that: The recombinant plasmid PET28a-EsXLI contains the nucleotide sequence shown in SEQID NO.

2.

4. The first recombinant Escherichia coli engineered bacterium is characterized in that: The first recombinant Escherichia coli engineered bacteria contains the recombinant plasmid PET28a-EsXLI described in claim 3.

5. A xylose isomerase mutant derived from Antarctic krill, characterized in that: The mutant is based on the amino acid sequence shown in SEQ ID NO.1, and the lysine at position 253 is mutated to arginine, namely EsXLI-K253R, or the aspartic acid at position 328 is mutated to alanine, namely EsXLI-D328A.

6. A recombinant plasmid, characterized in that: The recombinant plasmid contains a gene encoding the xylose isomerase mutant according to claim 5.

7. The second recombinant Escherichia coli engineered bacterium is characterized in that: The second recombinant Escherichia coli engineered bacteria contains the gene of the xylose isomerase mutant according to claim 5.

8. Use of the xylose isomerase according to claim 1, the recombinant plasmid PET28a-EsXLI according to claim 3 or the first recombinant Escherichia coli engineered bacterium according to claim 4 in isomerizing D-xylose, L-arabinose, D-ribose and D-glucose.

9. Use of the xylose isomerase mutant according to claim 5, the recombinant plasmid according to claim 6 or the second recombinant Escherichia coli engineered bacterium according to claim 7 in isomerizing D-xylose.

10. An enzyme preparation, characterized in that The enzyme preparation contains the xylose isomerase according to claim 1 and / or the xylose isomerase mutant according to claim 5.

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