CN119020341B

CN119020341B - Antarctic krill-derived xylose isomerase, mutant and application thereof

Info

Publication number: CN119020341B
Application number: CN202411513932.6A
Authority: CN
Inventors: 徐甲坤; 谭佳豪; 王芳; 邹华英
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2024-10-29
Filing date: 2024-10-29
Publication date: 2025-01-24
Anticipated expiration: 2044-10-29
Also published as: CN119020341A

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

Antarctic krill-derived xylose isomerase, mutant and application thereof

Technical Field

The invention belongs to the field of enzymology, and particularly relates to antarctic krill-derived xylose isomerase, a mutant thereof and application thereof.

Background

The conserved biomass of krill in the antarctic sea area can reach 6260 ten thousand tons, the annual fishing gain can reach 0.6-1.0 hundred million tons, and the ecological system of the south ocean is not affected. Euphausia superba contains components such as proteins, phospholipids, unsaturated fatty acids, etc. which are extremely rich in nutritional value, and if this resource can be utilized by humans, the development of marine resources will be greatly promoted. Numerous studies have shown that the adaptation of euphausia superba to extreme environments is closely related to the enzymes in its body, which also provides additional possibilities for euphausia superba derived enzymes as industrial biocatalysts. The current development and utilization of euphausia superba source enzymes is still in the primary stage, so we need to explore euphausia superba source enzymes more and more.

Xylose isomerase (Xylose isomerase, XI) (EC 5.3.1.5) can catalyze the conversion of five-carbon sugar D-xylose into D-xylulose, plays an important role in the glucose metabolism process in microorganisms, and can catalyze the isomerization reaction from D-glucose to D-fructose in vitro, so that the xylose isomerase is also called glucose isomerase. The lignocellulose biomass can generate a large amount of glucose and xylose after pretreatment and enzymatic hydrolysis, and certain microorganisms capable of naturally utilizing xylose exist in nature, including bacteria, saccharomycetes and fungi, but most of the microorganisms cannot use xylose as a fermentation substrate, so that xylose resources in hydrolysate are not utilized equally effectively compared with glucose. Xylose isomerase has extremely important industrial application value, and proteases, amylases and the like are also called important industrial enzymes. In recent years, research by scientists has found that xylose isomerase can be converted to produce rare sugars, such as ribose, mannose, arabinose, lyxose and the like, which are important raw materials in the pharmaceutical industry under certain conditions, and the research of xylose isomerase is infused with new activity.

Currently, there are few reports of heterologous expression of xylose isomerase. Therefore, a xylose isomerase gene with higher activity is explored, and a genetic engineering bacterium is constructed by a genetic recombination method, so that the xylose isomerase is efficiently and heterologously expressed, and the xylose isomerase gene has important industrial application value and potential.

Disclosure of Invention

The invention aims to provide antarctic krill-derived xylose isomerase, a mutant thereof and application thereof. The invention digs the euphausia superba XLI gene (EsXLI) based on genome analysis and carries out heterologous expression in the escherichia coli PET28a, which has higher enzyme activity so as to meet the industrial production requirement.

The invention is realized by the following technical scheme:

a euphausia superba-derived xylose isomerase has an amino acid sequence shown in SEQ ID NO. 1.

The nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

A recombinant plasmid PET28a-EsXLI contains a nucleotide sequence shown as SEQ ID NO. 2.

The invention also provides a first recombinant escherichia coli engineering bacterium which contains the recombinant plasmid PET28a-EsXLI.

A mutant of antarctic krill-derived xylose isomerase, which is characterized in that on the basis of the sequence of amino acid shown as SEQ ID NO.1, lysine at 253 is mutated to arginine, namely EsXLI-K253R, or aspartic acid at 328 is mutated to alanine, namely EsXLI-D328A.

A recombinant plasmid comprising a gene encoding said xylose isomerase mutant.

The invention also provides a second recombinant escherichia coli engineering bacterium which contains the genes of the xylose isomerase mutant.

The invention also provides application of the xylose isomerase, xylose isomerase mutant, recombinant plasmid PET28a-EsXLI, the recombinant plasmid, the first recombinant escherichia coli engineering bacteria and the second recombinant escherichia coli engineering bacteria in isomerising xylose.

The invention also provides an enzyme preparation containing the xylose isomerase and/or xylose isomerase mutant.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts a antarctic krill protein transcriptome bioinformatics method to obtain the gene sequence of xylose isomerase, and the target gene is amplified in vitro by Polymerase Chain Reaction (PCR), and soluble active expression is carried out in escherichia coli, thus laying a theoretical foundation for the application of xylose isomerase in preparing rare ketose in the fields of food and medicine.

The invention also obtains two mutants of antarctic krill xylose isomerase, the enzyme activity of the mutant EsXLI-K253R is improved by 1.25 times compared with that of the original enzyme, and the optimal enzyme activity temperature of the mutant EsXLI-D328A is reduced by 10 ℃.

The euphausia superba xylose isomerase and the mutant thereof obtained by the invention can catalyze D-xylose to isomerize to generate D-xylulose, the L-arabinose isomerized product is D-tagatose, the D-ribose isomerized product is D-ribulose, and the D-glucose isomerized product is D-fructose, and also has specificity to different substrates and good application prospect. The xylose isomerase has good adaptability to low temperature and can be expressed in escherichia coli cells.

Drawings

FIG. 1 is a line graph of the effect of temperature on xylose isomerase enzyme activity;

FIG. 2 is a line graph of the effect of temperature on xylose isomerase stability;

FIG. 3 is a line graph of the effect of pH on xylose isomerase enzyme activity;

FIG. 4 is a line graph of the effect of pH on xylose isomerase stability;

FIG. 5 is a bar graph showing the effect of metal ions on xylose isomerase enzyme activity;

FIG. 6 is a bar graph showing the effect of enzyme inhibitors on xylose isomerase enzyme activity;

FIG. 7 is a bar graph of the isomerisation ability of xylose isomerase to different substrates

FIG. 8 is a graph of hydrogen bonding of xylose isomerase catalytic active centers to a substrate;

FIG. 9 is a plot of xylose isomerase wild type and two mutants binding sites for substrate.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Example 1A batch of xylose isomerase genomic sequences was developed and analyzed from a previously ascertained antarctic krill genome database (not disclosed), and after amino acid sequence alignment of this batch, a batch of unverified functional gene sequences that were bioinformatically predicted to be putative xylose isomerase or to have potential xylose isomerase activity was selected and the antarctic krill amino acid sequence was between 70-90% homologous to the known source sequences, and after further domain and protein family classification analysis, candidate sequences were determined by using Blast analysis tools in the NCBI database to compare confidence and homology to other species-derived xylose isomerases. And designing a front primer and a rear primer for the candidate sequence, taking the euphausia superba cDNA as a template, verifying the primers by using a PCR method, and sequencing a PCR product to obtain the candidate gene EsXLI.

Through large-scale screening and intensive research, a novel xylose isomerase is identified and separated from antarctic krill, the amino acid and nucleotide sequences of the xylose isomerase are shown as SEQ ID NO.1 and SEQ ID NO.2, the xylose isomerase can catalyze D-xylose to isomerize to D-xylulose, and the xylose isomerase has specificity to different substrates and has very good application prospect. The xylose isomerase has good adaptability to low temperature and can be expressed in escherichia coli cells. Meanwhile, the invention optimizes the recombinant expression method of the xylose isomerase, thereby realizing high-efficiency expression in host cells.

The present inventors first performed codon optimization according to the expression conditions to improve the expression efficiency and stability of the DNA fragment. The optimized sequence is inserted into a plasmid PET28a to obtain a recombinant plasmid PET28a-EsXLI, and then the recombinant plasmid PET28a-EsXLI is transformed into competent cells of escherichia coli BL1 (DE 3) for heterologous expression.

Example 2 this example provides a recombinant expression vector recombinant E.coli engineering bacteria construction process, specifically as follows:

The EsXLI gene was synthesized by the institute of biotechnology, inc. (Shanghai, china). EsXLI was amplified using the synthesized EsXLI gene as template and 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 digested with EcoRI/NotI, and then ligated into the EcoRI/NotI digested PET28a vector.

The plasmids were recovered using the plasmid extraction kit. After gene sequencing, the transformants were transformed into E.coli competent cells, and the transformants were transferred into LB medium (the medium consisted of 10g/L peptone, 5g/L yeast extract, 10g/LNaCl, 50. Mu.g/mL carbapenem). The temperature is 37 ℃, the oscillation speed is 200R/min, the escherichia coli bacterial liquid is used as a template, T7-F (TAATACGACTCACTATAGGG, SEQ ID No. 5) and T7-R (GCTAGTTATTGCTCAGCGG, SEQ ID No. 6) are used as front and rear primers, the annealing temperature is set to 48 ℃, PCR is carried out, and 20 mu L of PCR products are taken to send a sequencing verification sequence for the production. After sequencing was correct, the bacterial solution was transferred to fresh LB medium for shaking culture at 37℃until the OD ₆₀₀ reached 0.6-0.8. Adding 0.6M IPTG to perform induction culture for 20 h at 37deg.C with shaking speed of 200 r/min, centrifuging at 8000 r/min for 10 min to collect thallus, and adding buffer solution to the collected thallus to resuspend three times. And after the resuspension is finished, adding PMSF with the final concentration of 1mM, placing the mixture on ice for ultrasonic crushing, and obtaining a centrifugal supernatant as crude enzyme liquid.

The nickel column purification of the recombinant enzyme, wherein the recombinant expressed EsXLI gene carries an 8 XHis tag at the N end, can be combined with Ni of HIS TRAP HP column packing, and Ni can be combined with imidazole, so that the aim of purification can be achieved by eluting with imidazole with different concentrations, and single protein can be obtained. Washing A, B pump and system of AKTA protein rapid purifier with ddH ₂ O, washing A, B pump with balance buffer solution, regulating flow rate to 1 mL/min, connecting nickel column, regulating flow rate to 2 mL/min, loading crude enzyme solution to 0.45 um filter membrane, loading sample at 1 mL/min, washing with washing buffer solution after penetrating peak is flattened to remove impurity protein, eluting target protein with eluting buffer solution after base line is stabilized, taking out peak, collecting sample until peak is flattened, and stopping collecting sample.

The balance buffer is 20 mM Tris-HCl buffer, 500 mM NaCl, pH8;

The washing buffer solution is 20 mM Tris-HCl buffer solution, 500 mM NaCl;10 mM imidazole and pH8;

the elution buffer is 20mM Tris-HCl buffer, 500 mM NaCl;500 mM imidazole and pH8.

The method comprises the steps of converting D-xylulose into furfural substances under the action of concentrated sulfuric acid, generating color reaction between the furfural and a cysteine-carbazole reagent, detecting the maximum absorption peak of the product D-xylulose of the catalytic reaction at a wavelength of 540 nm by using a spectrophotometry method to characterize the enzyme activity of the recombinant xylose isomerase, properly diluting enzyme solution by using a buffer solution of pH8 Tris-HCl, taking 100 mu L of the diluted enzyme solution, adding 50 mu L of 10 mM MnCl ₂, 800 mu L of the buffer solution of pH8 Tris-HCl, adding 50 mu L of 100 mM D-xylose solution (contrast and inactivating enzyme), and carrying out water bath reaction at 50 ℃ for 30 min. Immediately after the completion of the reaction, 200. Mu.L of trichloroacetic acid was immediately added to terminate the reaction, 200. Mu.L of the reaction mixture was taken into the wells of the enzyme-labeled plate, and the absorbance was measured at 540 nm wavelength.

Enzymatic Properties (1) optimal temperature measurement the enzymatic activity of EsXLI was measured at different temperatures of 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, respectively. The relative enzyme activities were determined in 3 replicates per set of temperatures, with the highest enzyme activity measured at 50 ℃ being 100%.

(2) Determination of optimum pH from pH change analysis of the effect of pH on enzyme activity, citric acid buffer (pH 4.0-5.0), phosphoric acid 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 reaction at 30 min,3 replicates per set of temperatures, and relative enzyme activity was determined, taking the highest enzyme activity at pH 7 as 100%.

(3) Temperature stability determination the purified enzyme solution was placed in a metal bath at 30 ℃,40 ℃, 50 ℃, 60 ℃ and 70 ℃ 80 ℃ at 240 min intervals during which time the enzyme activity was determined at ph=8.5 every 60 min, 3 replicates were made for each set of time, and the relative enzyme activity was determined to be 100% of the highest enzyme activity at 30 ℃.

(4) And (3) measuring pH stability, namely placing the purified enzyme solution at the temperature of 4 ℃ under the buffer solution condition of pH4-9 for 240 min, taking out every 60: 60 min, measuring the enzyme activity at the temperature of 60 ℃, and measuring the relative enzyme activity by 3 parallels of each group of pH, wherein the highest enzyme activity at the pH of 8.5 is 100%.

(5) Metal ion Effect on recombinant EsXLI enzyme Activity the ddH ₂ O was used to formulate metal ions Na⁺、K⁺、Mg²⁺、Ba²⁺、Ca²⁺、Cu²⁺、Ni²⁺、Fe³⁺、Mn²⁺、Co²⁺, at a concentration of 20. 20 mM and the final concentrations of the metal ions were formulated to be 1 mM and 10 mM, the purified enzyme solution was placed in a metal ion system of different concentrations at 4℃for 120: 120 min, then reacted in a water bath at pH=7 at 50℃for 30: 30 min, 3 replicates were run per set of temperatures, and the relative enzyme activities were determined to give a maximum enzyme activity of 100% placed under ddH ₂ O.

(6) Protease inhibitors affecting recombinant EsXLI enzyme activity

The purified enzyme solutions were placed in protease inhibitor systems of different concentrations at 4 ℃ for 120 min, then reacted in water baths of ph=7, 50 ℃ for 30min at 3 parallel per set of temperatures, the relative enzyme activities were determined to give the highest enzyme activity at 100% placed under standard buffer conditions, and the final concentrations were set to 1mM and 10 mM using ddH ₂ O at 20 mM SDS, PMSF, β -mercaptoethanol, DTT and EDTA.

(7) Ability of xylose isomerase to isomerise different substrates:

6 different substrates of polyhydroxy aldehyde monosaccharides with the concentration of 10 mM are respectively prepared, specifically, D-xylose, D-ribose, D-glucose, D-mannose, D-galactose and L-arabinose, 50 mu L of each substrate is added into a1 ml reaction system, then the substrates react in a water bath with the pH value of 7 and 50 ℃ for 30 min, 3 parallel substrates are made, the relative enzyme activities are measured, and the highest enzyme activity under the condition that the D-xylose is taken as the substrate is 100%.

As shown in the above reaction results, as can be seen from FIGS. 1 and 2, the preferred condition of the enzyme activity is pH 7.0, and the preferred temperature is 40-50 ℃. The optimal reaction temperature of the enzyme is 50 ℃, the enzyme activity of EsXLI is always increasing along with the increase of the reaction temperature from 20 ℃ to 45 ℃, the enzyme activity reaches the highest value when the temperature is 50 ℃, and then the catalytic activity of EsXLI is obviously reduced along with the gradual increase of the temperature. The detection of EsXLI temperature stability shows that the enzyme is highly stable at 20 ℃, can keep more than 95% of original activity after incubating for 4h, and can keep relatively low stability at an environment of more than 50 ℃ and relatively high stability at a low temperature after incubating for 4h at 20 ℃,30 ℃,40 ℃,50 ℃, 60 ℃, 70 ℃ and 80 ℃ and the enzyme activities are respectively 95.22%, 91.64%, 82.34%, 70.27%, 5.21%, 4.29% and 4.02% of the original activity. Compared with xylose isomerase from other sources, the optimal temperature is about 60-90 ℃, but the highest enzyme activity can be achieved when the xylose isomerase from antarctic krill is at 50 ℃.

As shown in fig. 3 and 4, the optimum pH is 7, the pH is between 4.0 and 7.0, the catalytic activity of the enzyme increases with increasing pH, the enzyme activity reaches the maximum at pH 7.0, and the catalytic activity of EsXLI decreases with further increase in pH 7.0 to 10.0. Figure 4 shows the stability of EsXLI at various pH conditions, after incubation at pH 7.0 for 4 h, the enzyme activity can remain at 95% of its original activity, with a gradual decrease in stability with increasing acidity or basicity, but at pH 4.0, 5.0 and 10.0, the incubation at 4 h enzyme activity has been less than 50%.

As shown in fig. 5 and 6, the results showed that the effect of 1 mmol/L of metal ion and enzyme inhibitor on EsXLI enzyme activity was not significant. After incubation with 10 mmol/L of metal ions, the relative enzyme activities were 121.25%, 128.13% and 148.32% respectively, while the effects of Na ⁺、K⁺、Cu²⁺、Al³⁺、Fe³⁺ on the enzyme activities were 108.06%, 99.31%, 86.85%, 90.40% and 89.42% respectively, and the effects of Ca ²⁺、Ba²⁺、Ni²⁺ on the enzyme activities were slightly greater, 75.32%, 67.54% and 81.28% respectively, after being influenced by Mg ²⁺、Mn²⁺ and Co ²⁺ plasmas.

As shown in FIG. 6, under the influence of 10 mM PMSF, SDS, the enzyme activities were retained 84.31% and 89.01%, whereas 10 mM EDTA and β -mercaptoethanol retained only EsXLI% of the activities 39.32% and 52.91%.

As shown in FIG. 7, among 6 xylose isomerase substrates (working concentration 100 mM, D-xylose, L-arabinose, D-ribose, D-glucose, D-mannose and D-galactose respectively), the recombinant xylose isomerase had the strongest effect on D-xylose, and then L-arabinose (22.32%), D-ribose (13.47%), D-glucose (3.09%), D-xylose was isomerized to D-xylulose, the L-arabinose isomerized product was D-tagatose, the D-ribose isomerized product was D-ribulose, the D-glucose isomerized product was D-fructose, and there was no isomerization on the other 2 substrates, indicating that the euphausia superba xylose isomerase had a stronger substrate specificity.

According to the substrate specificity, 3 types of D-xylose (100%), L-arabinose (22.32%) and D-ribose (13.47%) with strong activity are respectively configured into substrate concentrations of 25 mM, 50mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM and 225 mM, the kinetics of xylose isomerase under different substrate conditions are measured according to the enzyme activity measuring method, and the maximum reaction speed and the Mi constant on D-xylose are respectively 0.74U/mg and 67.99 mM by the dynamic characterization of the enzyme. The catalytic efficiency of L-arabinose was 0.38U/mg and 195.53 mM, respectively. The catalytic efficiency of D-ribose was 0.34U/mg and 202.36 mM, respectively.

As shown in fig. 8 and 9, molecular modeling of EsXLI catalytic active centers in combination with substrates was performed based on sequence alignment and homology modeling. The mutation result shows that the enzyme activity of EsXLI-K253R mutant (5.05U/mg) is 1.25 times higher than that of wild (4.05U/mg), and as sugar molecules in the reaction solution firstly usually react with epsilon-amino groups (NH ³⁺) of lysine (Lys) residues in a catalytic structural domain, schiff base reaction is carried out, the non-enzymatic glycosylation is an important reason for influencing the thermal stability of the enzyme, and the mutation of the lysine into arginine avoids the non-enzymatic glycosylation reaction, so that xylose isomerase is more stable in combination with a substrate at the optimal temperature (50 ℃) and has higher catalytic efficiency. The optimal temperature of the D328A mutant is reduced from 50 ℃ to 40 ℃, the optimal temperature of the D328A mutant is reduced, the difference between thermophilic xylose isomerase and the conserved residues of the corresponding normal-temperature enzymes is analyzed, and after aspartic acid is mutated into alanine, the interaction between subunits in the conserved residue region of the xylose isomerase is reduced, so that the activity at low temperature is improved.

A euphausia superba xylose isomerase has an amino acid sequence as follows (SEQ ID NO. 1):

MADPAVKRPKMSDEIENGSGEFFPGIGVIPFKPDARPDETMVFKHYNAQDVVMGKTMEDWCRFSVVYWHTFRGTGLDPFGSGTINRHWDDGSETVDNAKRRLRAAFEFFTKLGNKYWTFHDRDVAPEGRNLAETNANLDALADLAADLQHRTGVKCLWATSNLFSHPRFMHGAATSSDSHVTAYAGAQVKKCMEVAHRLGAENFVFWGGREGYHSLLNTDVRKELDNFAAFFRMVIAYKEKIGFRGQLLIEPKPKEPTRHQYDYDAQTVMAFLHSYGLQDHFKLNIEPNHTTLAGHPYEHDIVMASAFKMLGSIDANTGSPDLGWDTDQFPMDVRNCTAIMKVVLEQGGLKPGGLNFDAKVRRESTDLKDLFIAHIAAMDTLARGLKNAAKIISDGILKKSLQKRYLSWDSGVGAKIAAGQCTLEECEKIIHEQGNPNPPSAHQEHFEAVFNHYV.

a euphausia superba xylose isomerase having the nucleotide sequence as set forth in SEQ ID No. 2:

ATGGCAGATCCAGCTGTCAAGCGCCCCAAGATGTCAGATGAAATTGAAAATGGATCCGGAGAATTTTTTCCAGGTATTGGAGTGATCCCATTTAAGCCAGATGCTCGCCCAGACGAGACGATGGTGTTCAAGCACTACAATGCCCAGGATGTGGTCATGGGAAAGACCATGGAGGACTGGTGTCGCTTCTCTGTAGTGTACTGGCACACCTTCAGGGGAACTGGTTTGGACCCTTTTGGTTCTGGTACAATTAACAGACATTGGGATGATGGTAGTGAGACAGTGGACAATGCAAAACGTCGACTAAGAGCAGCTTTTGAATTTTTCACTAAACTTGGTAACAAGTACTGGACGTTTCATGACCGTGACGTAGCTCCTGAAGGGAGAAACTTGGCTGAGACTAACGCCAACCTTGATGCATTAGCAGATTTGGCTGCTGATCTTCAACATCGCACTGGTGTTAAGTGTTTATGGGCTACATCTAACCTCTTCTCACATCCTAGATTTATGCATGGAGCTGCAACAAGCAGTGATTCACATGTAACAGCATATGCTGGTGCACAGGTCAAGAAGTGTATGGAAGTTGCACATCGTTTGGGAGCTGAGAACTTTGTGTTCTGGGGAGGTCGAGAAGGCTATCATTCATTGCTTAACACAGATGTACGCAAGGAGCTAGACAATTTTGCTGCATTCTTCAGAATGGTTATCGCATACAAAGAGAAAATAGGTTTCCGAGGTCAGCTGCTTATTGAGCCCAAACCAAAGGAACCAACTCGTCACCAGTATGATTACGATGCCCAGACTGTGATGGCATTTTTGCACTCTTATGGTCTTCAGGATCATTTCAAACTTAATATTGAACCTAATCATACCACTTTGGCTGGCCATCCTTATGAACATGATATTGTCATGGCATCAGCTTTCAAGATGCTTGGTAGCATTGATGCTAATACAGGCTCCCCTGATCTTGGATGGGACACAGACCAGTTCCCAATGGATGTTCGCAATTGTACTGCTATCATGAAGGTTGTCTTAGAACAGGGTGGCCTAAAGCCAGGTGGGCTTAACTTTGATGCCAAGGTCCGCCGTGAGTCAACAGATCTTAAGGATTTGTTCATTGCTCACATTGCTGCTATGGACACCTTGGCCAGAGGCCTCAAGAATGCTGCAAAGATTATTTCCGATGGAATACTTAAGAAGAGTCTACAGAAACGTTACCTGTCTTGGGATTCTGGTGTTGGTGCAAAAATTGCAGCTGGGCAATGCACACTCGAGGAGTGTGAGAAAATAATCCACGAGCAGGGAAACCCTAATCCACCGTCTGCCCACCAGGAACATTTTGAGGCCGTATTTAACCACTATGTTTAG.

Claims

1. The antarctic krill-derived xylose isomerase is characterized in that the amino acid sequence of the xylose isomerase is shown as SEQ ID NO. 1.

2. A gene encoding the xylose isomerase as claimed in claim 1, characterized in that the nucleotide sequence of said gene is shown in SEQ ID No. 2.

3. The recombinant plasmid PET28a-EsXLI is characterized in that the recombinant plasmid PET28a-EsXLI contains a nucleotide sequence shown as SEQ ID NO. 2.

4. A first recombinant escherichia coli engineering bacterium, which is characterized by comprising the recombinant plasmid PET28a-EsXLI of claim 3.

5. A mutant of antarctic krill-derived xylose isomerase is characterized in that the mutant is characterized in that on the basis of the sequence of amino acid shown as SEQ ID NO.1, lysine at 253 is mutated into arginine, namely EsXLI-K253R, or aspartic acid at 328 is mutated into alanine, namely EsXLI-D328A.

6. A recombinant plasmid comprising a gene encoding the xylose isomerase mutant of claim 5.

7. A second recombinant escherichia coli engineering bacterium, which is characterized by comprising the xylose isomerase mutant gene of claim 5.

8. Use of the xylose isomerase of claim 1, the recombinant plasmid PET28a-EsXLI of claim 3 or the first recombinant escherichia coli engineering bacterium of claim 4 for isomerising D-xylose, L-arabinose, D-ribose and D-glucose.

9. Use of the xylose isomerase mutant of claim 5, the recombinant plasmid of claim 6or the second recombinant E.coli engineering bacterium of claim 7 in isomerising D-xylose.

10. An enzyme preparation comprising the xylose isomerase as claimed in claim 1 and/or the xylose isomerase mutant as claimed in claim 5.