CN116445455B - Heat-resistant alkali-resistant xylanase mutant and application thereof - Google Patents
Heat-resistant alkali-resistant xylanase mutant and application thereof Download PDFInfo
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- CN116445455B CN116445455B CN202310420629.0A CN202310420629A CN116445455B CN 116445455 B CN116445455 B CN 116445455B CN 202310420629 A CN202310420629 A CN 202310420629A CN 116445455 B CN116445455 B CN 116445455B
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
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Abstract
The invention discloses a heat-resistant alkali-resistant xylanase mutant and application thereof, wherein the heat stability of xylanase is improved by utilizing high-pressure-based molecular dynamics simulation and combining various heat stability algorithm combination predictions, and further the surface charge of xylanase is calculated and designed by utilizing Rosetta supercharge based on a surface coupling method so as to improve the alkali resistance of enzyme, and a series of mutants are obtained by screening. The xylanase mutant has greatly improved heat resistance and alkali resistance, particularly remarkably prolonged half life of enzyme under high temperature and strong alkali conditions, and improved enzyme activity to a certain extent, and solves the limitations of short half life, poor stability and contradiction between protein storage and actual catalytic reaction conditions of xylanase in the current industrial environment.
Description
Technical Field
The invention relates to a heat-resistant alkali-resistant xylanase mutant and application thereof, belonging to the technical fields of enzyme engineering and genetic engineering.
Background
Xylan is a rich renewable resource with the content inferior to that of cellulose in nature, and xylanase can efficiently degrade xylan of a polymerization mixture to form xylose with lower polymerization degree, so that on one hand, the process of degrading polysaccharide with higher polymerization degree has high industrial application value in industry, and on the other hand, the xylose with lower polymerization degree has high commercial value. Therefore, xylanase has wide economic prospect in the fields of paper industry, food industry, feed industry, bioenergy and the like. Along with the progress of biotechnology, the xylanase can be subjected to molecular modification by utilizing a genetic engineering means so as to improve the heat resistance or enzyme activity and other performances of the xylanase and expand the condition range of the xylanase in industrial application.
The alkaline xylanase can degrade xylan into xylooligosaccharide, xylodisaccharide and other xylooligosaccharide, and very little xylose and arabinose under alkaline condition. The heat stability is related to the production, storage and application of the alkaline xylanase preparation, and the heat resistance and the decay rate are important parameters for representing the heat stability. The half-life of the enzyme refers to the time required for the activity to be reduced to the original general value under certain conditions, and the half-life depends on various factors such as temperature, pH and the like. In general, the shorter the half-life of an enzyme, the poorer the stability of the enzyme and vice versa.
In the existing alkaline xylanase stability modification, the half-life is generally within 60min, and a small number of the half-life reaches about 70min, for example, xylanase from Bacillus megaterium has an optimal temperature of 85 ℃, an optimal pH of 7.5, and specific enzyme activity of 25.6U/mg, but the half-life is only 7min (Ray et al, 1997); xylanase from Talaromyces purpureogenus, with optimal temperature of 60 ℃, optimal pH of 7, specific enzyme activity of 47.6U/mg, and half-life of 60min (ECHEVERRIA ET al.2019); xylanase from Alkalihalobacillus halodurans with optimum temperature of 80 ℃, optimum pH of 9, specific activity of 375U/mg and half-life of only 35min (Kumar et al, 2015); patent 201110451326.2 provides a xylanase mutant with a half-life of 73.6min at 65 ℃. In summary, the stability of the alkaline xylanase is to be further improved, and the present invention has been proposed.
Disclosure of Invention
In order to solve the problems, the invention provides a xylanase mutant with improved heat resistance and alkali resistance, the specific enzyme activity of the optimal mutant H136K is improved from 280.33U/mg of a wild type to 619.36U/mg, the half life of the xylanase mutant in the environment of 70 ℃ and pH 9.0 is improved from 6.50min to 129.68min, and the T m and the catalytic efficiency are also improved obviously.
The first object of the invention is to provide a heat-resistant alkali-resistant xylanase mutant, which is obtained by mutating xylanase with an amino acid sequence shown as SEQ ID NO. 1:
Histidine 136 was mutated to lysine (H136K), isoleucine 197 to lysine (I197K), glycine 213 to leucine (G213L), or arginine 247 to leucine (R247L).
It is a second object of the present invention to provide a gene encoding the xylanase mutant.
Further, the nucleotide sequence of the gene is shown as SEQ ID NO.2 (H136K).
Further, the nucleotide sequence of the gene is shown in SEQ ID NO.3 (I197K).
Further, the nucleotide sequence of the gene is shown as SEQ ID NO.4 (G213L).
Further, the nucleotide sequence of the gene is shown in SEQ ID NO.5 (R247L).
A third object of the present invention is to provide a recombinant plasmid carrying the gene.
Further, vectors of the recombinant plasmid include, but are not limited to, pET-28a, pET-28b, pET-20b, etc.
It is a fourth object of the present invention to provide host cells expressing the xylanase mutants.
Further, the host cell is a bacterial, fungal, plant cell or animal cell.
Further, the bacterium is E.coli, preferably E.coli BL21 (DE 3).
It is a fifth object of the present invention to provide the use of the xylanase mutant, gene, expression vector or host cell described above for hydrolyzing xylan.
Further, the application is to add the xylanase mutant to a system containing xylan for reaction.
It is a sixth object of the present invention to provide a method for hydrolyzing xylan using the xylanase mutant described above.
Further, the reaction temperature is not higher than 80 ℃.
Further, the reaction pH is 7.0-10.0.
The enzyme molecule reconstruction method provided by the invention comprises the following steps:
(1) Through molecular dynamics simulation, a key region is determined based on isothermal compression coefficient perturbation engineering strategy screening analysis, and the thermal stability of the improved xylanase is predicted and calculated by combining four thermal stability algorithms, namely FoldX, I-Mutant, dDFIRE and Rosetta Cartesian _ddG;
(2) On the premise of keeping the stability of the enzyme, reasonably designing and mutating the surface charge of the enzyme, and calculating and designing the surface charge of the enzyme by using Rosetta software to improve the alkali resistance of the enzyme;
(3) And taking intersection of the mutant and the mutant to determine a potential mutant, and detecting enzymatic properties of the mutant, so as to obtain the mutant with optimal heat resistance and alkali resistance.
The invention has the beneficial effects that:
According to the invention, the heat stability of xylanase is improved by utilizing high-pressure-based molecular dynamics simulation and combining FoldX, I-Mutant, dDFIRE and Rosetta Cartesian _ddG heat stability algorithm combination prediction calculation, the surface charge of xylanase is calculated and designed by utilizing Rosetta supercharge, the alkali resistance of enzyme is improved, and mutants with high industrial application potential are screened by the method for simultaneously improving the heat stability and the alkali resistance. The half life of the xylanase mutant obtained by the invention is improved by 18.95 times compared with that of a wild enzyme in the environment of 70 ℃ and pH 9.0, and the xylanase mutant is remarkably improved.
Drawings
FIG. 1 shows the crystal structure of wild-type xylanase.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
In the following examples, the xylanase gene from Bacillus halodurans S7 was protein expressed using an E.coli expression system, the amino acid sequence of the xylanase gene was shown in SEQ ID NO.1, and the plasmid selected was pET-28a, and the E.coli host was BL21 (DE 3).
The media and the required solutions involved in the examples below are as follows:
LB liquid medium: 10g/L peptone, 5g/L, naCl g/L yeast extract.
LB solid medium: 10g/L peptone, 5g/L, naCl g/L yeast extract, 2% (m/v) agar powder.
IPTG (1 mol/L): IPTG is isopropyl thio-beta-D-galactoside (molecular weight is 238.3), after 2.38gIPTG is dissolved in 8mL of distilled water, distilled water is used for fixing the volume to 10mL, a 0.22 mu m filter is used for filtering and sterilizing, and 1mL of small portions are split and stored at the temperature of minus 20 ℃ and mainly used for inducing the escherichia coli strain to express target proteins;
PBS buffer: 8g of NaCl, 0.2g of KCl and 0.24g of Na 2HPO4·12H2O 3.63g、KH2PO4 in 900mL of double distilled water, adjusting the pH to 7.4 by hydrochloric acid, adding water to a constant volume of 1L, and preserving the heat at normal temperature for later use.
The amino acid sequence (SEQ ID NO. 2) of the wild-type xylanase of the invention is as follows:
MITLFKKPFVAGLAISLLVGGGLGNVAAAQGGPPKSGVFGENQKRNDQPFAWQVASLSERYQEQFDIGAAVEPYQLEGRQAQILKHHYNSLVAENAMKPVSLQPREGEWNWEGADKIVEFARKHNMELRFHTLVWHSQVPEWFFIDENGNRMVDETDPEKRKANKQLLLERMENHIKTVVERYKDDVTSWDVVNEVIDDGGGLRESEWYQITGTDYIKVAFETARKYGGEEAKLYINDYNTEVPSKRDDLYNLVKDLLEQGVPIDGVGHQSHIQIGWPSIEDTRASFEKFTSLGLDNQVTELDMSLYGWPPTGAYTSYDDIPEELFQAQADRYDQLFELYEELSATISSVTFWGIADNHTWLDDRAREYNNGVGVDAPFVFDHNYRVKPAYWRIID
the nucleotide sequence of the xylanase mutant related to the invention is as follows:
SEQ ID NO.2(H136K):
ATGATTACACTTTTTAAAAAGCCTTTTGTTGCTGGACTAGCGATCTCTTTATTAGTTGGAGGGGGGCTAGGCAATGTAGCTGCTGCTCAAGGAGGACCACCAAAATCTGGAGTCTTTGGAGAAAATCAAAAAAGAAATGATCAGCCTTTTGCATGGCAAGTTGCTTCTCTTTCTGAGCGATATCAAGAGCAGTTTGATATTGGAGCTGCGGTTGAGCCCTATCAATTAGAAGGAAGACAAGCCCAAATTTTAAAGCATCATTATAACAGCCTTGTGGCGGAAAATGCAATGAAACCTGTATCACTCCAGCCAAGAGAAGGTGAGTGGAACTGGGAAGGCGCTGACAAAATTGTGGAGTTTGCCCGCAAACATAACATGGAGCTTCGCTTCCACACACTCGTTTGGAAAAGCCAAGTACCAGAATGGTTTTTCATCGATGAAAATGGCAATCGGATGGTTGATGAAACCGATCCAGAAAAACGTAAAGCGAATAAACAATTGTTATTGGAGCGAATGGAAAACCATATTAAAACGGTTGTTGAACGTTATAAAGATGATGTGACTTCATGGGATGTGGTGAATGAAGTTATTGATGATGGCGGGGGCCTCCGTGAATCAGAATGGTATCAAATAACAGGCACTGACTACATTAAGGTAGCTTTTGAAACTGCAAGAAAATATGGTGGTGAAGAGGCAAAGCTGTACATTAATGATTACAACACCGAAGTACCTTCTAAAAGAGATGACCTTTACAACCTGGTGAAAGACTTATTAGAGCAAGGAGTACCAATTGACGGGGTAGGACATCAGTCTCATATCCAAATCGGCTGGCCTTCCATTGAAGATACAAGAGCTTCTTTTGAAAAGTTTACGAGTTTAGGATTAGACAACCAAGTAACTGAACTAGACATGAGTCTTTATGGCTGGCCACCGACAGGGGCCTATACCTCTTATGACGACATTCCAGAAGAGCTTTTTCAAGCTCAAGCAGACCGTTATGATCAGCTATTTGAGTTATATGAAGAATTAAGCGCTACTATCAGTAGTGTAACCTTCTGGGGAATTGCTGATAACCATACATGGCTTGATGACCGCGCTAGAGAGTACAATAATGGAGTAGGGGTCGATGCACCATTTGTTTTTGATCACAACTATCGAGTGAAGCCTGCTTACTGGAGAATTATTGATTAA
SEQ ID NO.3(I197K):
ATGATTACACTTTTTAAAAAGCCTTTTGTTGCTGGACTAGCGATCTCTTTATTAGTTGGAGGGGGGCTAGGCAATGTAGCTGCTGCTCAAGGAGGACCACCAAAATCTGGAGTCTTTGGAGAAAATCAAAAAAGAAATGATCAGCCTTTTGCATGGCAAGTTGCTTCTCTTTCTGAGCGATATCAAGAGCAGTTTGATATTGGAGCTGCGGTTGAGCCCTATCAATTAGAAGGAAGACAAGCCCAAATTTTAAAGCATCATTATAACAGCCTTGTGGCGGAAAATGCAATGAAACCTGTATCACTCCAGCCAAGAGAAGGTGAGTGGAACTGGGAAGGCGCTGACAAAATTGTGGAGTTTGCCCGCAAACATAACATGGAGCTTCGCTTCCACACACTCGTTTGGCATAGCCAAGTACCAGAATGGTTTTTCATCGATGAAAATGGCAATCGGATGGTTGATGAAACCGATCCAGAAAAACGTAAAGCGAATAAACAATTGTTATTGGAGCGAATGGAAAACCATATTAAAACGGTTGTTGAACGTTATAAAGATGATGTGACTTCATGGGATGTGGTGAATGAAGTTAAAGATGATGGCGGGGGCCTCCGTGAATCAGAATGGTATCAAATAACAGGCACTGACTACATTAAGGTAGCTTTTGAAACTGCAAGAAAATATGGTGGTGAAGAGGCAAAGCTGTACATTAATGATTACAACACCGAAGTACCTTCTAAAAGAGATGACCTTTACAACCTGGTGAAAGACTTATTAGAGCAAGGAGTACCAATTGACGGGGTAGGACATCAGTCTCATATCCAAATCGGCTGGCCTTCCATTGAAGATACAAGAGCTTCTTTTGAAAAGTTTACGAGTTTAGGATTAGACAACCAAGTAACTGAACTAGACATGAGTCTTTATGGCTGGCCACCGACAGGGGCCTATACCTCTTATGACGACATTCCAGAAGAGCTTTTTCAAGCTCAAGCAGACCGTTATGATCAGCTATTTGAGTTATATGAAGAATTAAGCGCTACTATCAGTAGTGTAACCTTCTGGGGAATTGCTGATAACCATACATGGCTTGATGACCGCGCTAGAGAGTACAATAATGGAGTAGGGGTCGATGCACCATTTGTTTTTGATCACAACTATCGAGTGAAGCCTGCTTACTGGAGAATTATTGATTAA
SEQ ID NO.4(G213L):
ATGATTACACTTTTTAAAAAGCCTTTTGTTGCTGGACTAGCGATCTCTTTATTAGTTGGAGGGGGGCTAGGCAATGTAGCTGCTGCTCAAGGAGGACCACCAAAATCTGGAGTCTTTGGAGAAAATCAAAAAAGAAATGATCAGCCTTTTGCATGGCAAGTTGCTTCTCTTTCTGAGCGATATCAAGAGCAGTTTGATATTGGAGCTGCGGTTGAGCCCTATCAATTAGAAGGAAGACAAGCCCAAATTTTAAAGCATCATTATAACAGCCTTGTGGCGGAAAATGCAATGAAACCTGTATCACTCCAGCCAAGAGAAGGTGAGTGGAACTGGGAAGGCGCTGACAAAATTGTGGAGTTTGCCCGCAAACATAACATGGAGCTTCGCTTCCACACACTCGTTTGGCATAGCCAAGTACCAGAATGGTTTTTCATCGATGAAAATGGCAATCGGATGGTTGATGAAACCGATCCAGAAAAACGTAAAGCGAATAAACAATTGTTATTGGAGCGAATGGAAAACCATATTAAAACGGTTGTTGAACGTTATAAAGATGATGTGACTTCATGGGATGTGGTGAATGAAGTTATTGATGATGGCGGGGGCCTCCGTGAATCAGAATGGTATCAAATAACACTGACTGACTACATTAAGGTAGCTTTTGAAACTGCAAGAAAATATGGTGGTGAAGAGGCAAAGCTGTACATTAATGATTACAACACCGAAGTACCTTCTAAAAGAGATGACCTTTACAACCTGGTGAAAGACTTATTAGAGCAAGGAGTACCAATTGACGGGGTAGGACATCAGTCTCATATCCAAATCGGCTGGCCTTCCATTGAAGATACAAGAGCTTCTTTTGAAAAGTTTACGAGTTTAGGATTAGACAACCAAGTAACTGAACTAGACATGAGTCTTTATGGCTGGCCACCGACAGGGGCCTATACCTCTTATGACGACATTCCAGAAGAGCTTTTTCAAGCTCAAGCAGACCGTTATGATCAGCTATTTGAGTTATATGAAGAATTAAGCGCTACTATCAGTAGTGTAACCTTCTGGGGAATTGCTGATAACCATACATGGCTTGATGACCGCGCTAGAGAGTACAATAATGGAGTAGGGGTCGATGCACCATTTGTTTTTGATCACAACTATCGAGTGAAGCCTGCTTACTGGAGAATTATTGATTAA
SEQ ID NO.5(R247L):
ATGATTACACTTTTTAAAAAGCCTTTTGTTGCTGGACTAGCGATCTCTTTATTAGTTGGAGGGGGGCTAGGCAATGTAGCTGCTGCTCAAGGAGGACCACCAAAATCTGGAGTCTTTGGAGAAAATCAAAAAAGAAATGATCAGCCTTTTGCATGGCAAGTTGCTTCTCTTTCTGAGCGATATCAAGAGCAGTTTGATATTGGAGCTGCGGTTGAGCCCTATCAATTAGAAGGAAGACAAGCCCAAATTTTAAAGCATCATTATAACAGCCTTGTGGCGGAAAATGCAATGAAACCTGTATCACTCCAGCCAAGAGAAGGTGAGTGGAACTGGGAAGGCGCTGACAAAATTGTGGAGTTTGCCCGCAAACATAACATGGAGCTTCGCTTCCACACACTCGTTTGGCATAGCCAAGTACCAGAATGGTTTTTCATCGATGAAAATGGCAATCGGATGGTTGATGAAACCGATCCAGAAAAACGTAAAGCGAATAAACAATTGTTATTGGAGCGAATGGAAAACCATATTAAAACGGTTGTTGAACGTTATAAAGATGATGTGACTTCATGGGATGTGGTGAATGAAGTTATTGATGATGGCGGGGGCCTCCGTGAATCAGAATGGTATCAAATAACAGGCACTGACTACATTAAGGTAGCTTTTGAAACTGCAAGAAAATATGGTGGTGAAGAGGCAAAGCTGTACATTAATGATTACAACACCGAAGTACCTTCTAAACTGGATGACCTTTACAACCTGGTGAAAGACTTATTAGAGCAAGGAGTACCAATTGACGGGGTAGGACATCAGTCTCATATCCAAATCGGCTGGCCTTCCATTGAAGATACAAGAGCTTCTTTTGAAAAGTTTACGAGTTTAGGATTAGACAACCAAGTAACTGAACTAGACATGAGTCTTTATGGCTGGCCACCGACAGGGGCCTATACCTCTTATGACGACATTCCAGAAGAGCTTTTTCAAGCTCAAGCAGACCGTTATGATCAGCTATTTGAGTTATATGAAGAATTAAGCGCTACTATCAGTAGTGTAACCTTCTGGGGAATTGCTGATAACCATACATGGCTTGATGACCGCGCTAGAGAGTACAATAATGGAGTAGGGGTCGATGCACCATTTGTTTTTGATCACAACTATCGAGTGAAGCCTGCTTACTGGAGAATTATTGATTAA
EXAMPLE 1 construction of xylanase recombinant plasmid pET-28a-XY
BamH I and EcoR I restriction sites were added to the 5 'and 3' ends of the xylanase gene, respectively. The synthesized xylanase full-length gene and pET-28a vector were double digested with BamH I and EcoR I, respectively, in a reaction system of 50. Mu.L: 20. Mu.L of xylanase gene (or pET-28a plasmid); 10xQ Buffer 5. Mu.L; bamH I and EcoR I are 2. Mu.L each; dd H 2 O21. Mu.L. Enzyme cutting conditions: 37℃for 2h. After the enzyme digestion is finished, performing nucleic acid gel electrophoresis verification on the enzyme digestion product, cutting gel according to the size of a target strip, and performing gel recovery treatment on the xylanase gene and the double enzyme digestion product of the pET-28a plasmid by using a DNA gel recovery kit. The xylanase gene and the vector pET-28a are subjected to ligation reaction by using T4 DNA ligase, wherein the reaction system is 10 mu L: 6 mu L of target fragment; 2. Mu.L of pET-28a plasmid; 10x T4 DNA Ligase Buffer1 μl; t4 DNA LIGASE. Mu.L was placed in a metal bath at 16℃overnight for 10-12h. After ligation, the ligation product of the target gene and the vector was purified using a PCR product purification kit, E.coli JM109 was transformed, the transformed solution was plated on LB plates containing kanamycin sulfate (50. Mu.g/mL), and single colonies were obtained by culturing. Picking single colony transformants, inoculating the single colony transformants into LB culture medium containing kanamycin, shake culturing for 6-8h, taking bacterial liquid, performing bacterial liquid PCR verification and sequencing verification, and verifying that the correct recombinants are used for subsequent experiments.
Example 2 selection of mutation sites
The invention takes the crystal structure (PDB id:2UWF; as shown in figure 1) of wild xylanase as an initial MODEL, adopts SWISS-MODEL to carry out homologous modeling, uses Gromacs 5.0.3 to carry out high-pressure-based molecular dynamics simulation, determines 5 key areas based on isothermal compression coefficient perturbation engineering strategy screening analysis, and carries out combined prediction of four heat stability algorithms of FoldX, I-Mutant, dDFIRE and Rosetta Cartesian _ddG on the 5 key areas;
Then, on the premise of keeping the stability of the enzyme, the surface charge of the enzyme is reasonably designed and mutated, the Rosetta software is utilized to calculate and design the surface charge of the enzyme, and positively charged lysine, arginine and histidine are used for replacing negatively charged glutamic acid and aspartic acid, so that the optimal pH value of the mutated enzyme is improved, and the enzyme activity in an alkaline environment is enhanced; running Rosetta supercharge allows the protein to be more positively charged ：supercharge.mpi.macosclangrelease-s 2B3P_A_min.pdb-use_input_sc-ignore_unrecognized_res-jd2:no_output-dont_mutate_glyprocys true-dont_mutate_correct_charge true-dont_mutate_hbonded_sidechains true-include_arg-include_lys-refweight_arg-1.98-refweight_lys-1.65-surface_residue_cutoff 16-jd2:no_output-nstruct 1>logCOPY;SuperCharge for a faster calculation time without concern for computational resource issues. Furthermore SuperCharge need not calculate multiple repetitions, rather should calculate as many as possible of a series of net charge data; seven potentially effective mutants of R79L, H136K, I197K, G213L, R247L, Q329K and S349F were finally obtained.
EXAMPLE 3 construction of mutant recombinant plasmid
Primers shown in Table 1 were designed. And carrying out full plasmid PCR amplification by taking the recombinant plasmid pET-28a-XY as an original template, and constructing to obtain a mutant recombinant plasmid. The PCR reaction system was 50. Mu.L: ddH 2 O18 μL;2x Max Buffer 25 μL; dNTP Mix (10 mM) 1. Mu.L; 1 μl of pET-28a-XY template; 2. Mu.L of each of the upstream and downstream primers (10 mM); phanta Max Super-FIDELITY DNA Ploymerase. Mu.L. PCR reaction conditions: 95 ℃ for 30s; 15s at 95 ℃, 15s at 68 ℃, 5min at 72 ℃,30 cycles; stored at 72℃for 5min and at 4 ℃. After the reaction is finished, the PCR product is digested by using Dpn I enzyme, the digested product is transferred into E.coli JM109, and finally, the recombinants with correct sequencing are amplified and cultured, plasmids are extracted, and transferred into E.coli BL21 (DE 3) and stored at the temperature of minus 20 ℃.
TABLE 1 mutant primer design
EXAMPLE 4 preparation and purification of mutants
Plasmid transformation: the xylanase plasmid is added into the competence of escherichia coli BL21, water bath is carried out at 42 ℃ for 30-45 seconds, cooling is carried out on ice for 2-3 minutes (note that the process needs to be kept stable and can not shake vigorously), 500 mu LLB (pre-high-pressure high-temperature sterilization) is added, shaking is carried out at 37 ℃ and 200rpm/min for 1 hour, the xylanase plasmid is coated on an LB plate containing 50 mu g/mL kanamycin, the plate is firstly cultivated at 37 ℃ for 1 hour in a normal position, and then the plate is cultivated for 12 hours in an inverted mode under the same conditions.
E.coli BL21 (DE 3) transformants carrying mutant recombinant plasmids are selected for small-scale expansion culture, and bacterial liquid PCR and sequencing verification are carried out. After verification of correctness, E.coli BL21 (DE 3) with mutant recombinant plasmid is subjected to expansion culture, and the expansion culture step is as follows: the invention selects 1L basic culture medium as unit to carry out cell culture, the escherichia coli containing xylanase genes is enriched and cultured before a large amount of basic culture medium is cultured, single bacterial colony is selected from the LB agarose plate cultured before, and inoculated into 50mL LB culture solution (containing 50ug/ML KANAMYCIN), and cultured for 3 hours at 37 ℃ until the OD600 value is 0.6-0.8; after the amplification culture, the combined mutant xylanase was induced to be expressed, and after the completion of the induction culture, the cells were collected, and the cells were washed with 50mM Tris (pH 8.0) to remove the residual medium as much as possible, and then the cells were resuspended in the buffer solution and subjected to cell disruption using an ultrasonic breaker (450 w,5s/5s,25 min). After the cell disruption, the color of the cell fluid is changed from milky to transparent, after the disruption, the disrupted fluid is centrifuged at 4 ℃ (10000 r.min < -1 >) for 1h, the supernatant is collected, and the supernatant is filtered by a 0.22 mu m microporous filter, thus obtaining crude enzyme fluid.
The crude enzyme solution of mutant xylanase is purified by using a nickel ion affinity chromatography column (1 mL His Trap FF) with Ni-NTA as filler and an AKTA protein purifier, and the purification steps are as follows:
(1) Balance column: the column (20 column volumes) was equilibrated with 50mM Tris (pH 8.0) and then equilibrated with a final concentration of imidazole of 20mM binding solution (20 column volumes).
(2) Loading: and (3) adopting a sample injection pump to automatically sample, and loading the crude enzyme liquid at a flow rate of 1 mL/min.
(3) Eluting: washing 10 column volumes with buffer solution with imidazole final concentration of 20mM to remove part of the impurity protein, washing 30 column volumes with eluent with imidazole final concentration of 500mM, collecting the eluted product under the target peak and labeling.
(4) Column regeneration: due to the loss of nickel ions in the purification process, after the purification is finished, the nickel column is regenerated by the pre-prepared regeneration solution, so that the next use is convenient.
(5) And performing SDS-PAGE verification and enzyme activity detection on the purified and collected enzyme solution.
EXAMPLE 5 enzymatic Properties of mutant enzymes
And measuring xylanase activity parameters by adopting a DNS reagent chromogenic reaction method. The color development reaction method of the DNS reagent is characterized in that xylanase can be utilized to hydrolyze xylan into reducing sugar, the DNS reagent and the reducing sugar are subjected to color development reaction under the high-temperature condition, the color development intensity can be metered by ultraviolet spectrophotometry, and the color development intensity is in direct proportion to the amount of the reducing sugar generated by hydrolysis.
Xylanase activity refers to the ability of catalyzing the degradation of a xylan solution to release reducing sugars within a certain period of time. The study defines 1mg enzyme, and under the optimal condition, 1 mu mol of reducing sugar is released from xylan solution with the concentration of 2mg/mL in a degradation way for 1min, namely, the enzyme activity unit is expressed as mu/mg.
The half-unfolding temperatures (T m) of wild-type xylanases and mutants were analyzed using a MOS-450 circular dichroism spectrometer available from biological company, france. The purified enzyme solution is dialyzed in phosphate buffer solution with pH of 7.4 overnight to remove chloride ions in the enzyme solution, so as to prevent interference to the unfolding temperature value measurement result. Diluting the dialyzed enzyme solution to a concentration of 0.01-0.1 mg.ml -1, determining the unfolding temperature (T m) of the sample by using a circular dichroism chromatograph, setting the temperature gradient to 20-100 ℃, and taking phosphate buffer as a blank control.
Xylanase enzyme reaction kinetic parameter determination: the initial enzyme activity of the betulinic xylan with the mass concentration of 0-25 mg/mL is measured under the optimal condition of the enzyme. And obtaining single-point mutant kinetic parameters K m、Kcat and K cat/Km by measuring xylanase enzyme activities corresponding to different substrate concentrations.
The specific enzyme activities, half unfolding temperatures (T m), reaction kinetic parameters K m、Kcat and K cat/Km of the wild-type xylanase and the seven mutants are shown in Table 2.
TABLE 2 enzymatic Properties of mutant enzymes
By mutation, the T m value of mutant H136K, G213L, R247L, S349F is respectively increased by 5.94 ℃, 1.50 ℃, 3.60 ℃ and 0.43 ℃; the specific enzyme activities of the mutant H136K, I197K, G213L, Q329K are respectively improved by 120.94%, 53.12%, 47.78% and 43.25%; the catalytic efficiency of the mutant H136K, I197K, G213L, R247L, Q329K, S349F is respectively improved by 36.58%, 15.80%, 56.24%, 3.91%, 9.97% and 26.19%; the half-lives of mutant H136K, I197K, G213L, R247L, S349F at 70 ℃ and pH 9.0 are respectively improved by 18.95 times, 2.83 times, 4.51 times, 11.14 times and 0.17 times; wherein the T m, specific enzyme activity, catalytic efficiency and half-life of the mutant H136K, G L are all improved, and the H136K has the best performance effect in combination, and is the mutant with the greatest improvement of specific enzyme activity, heat resistance and alkali resistance.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A heat-resistant alkali-resistant xylanase mutant is characterized in that the xylanase mutant has the following mutation relative to xylanase with an amino acid sequence shown as SEQ ID NO. 1:
Histidine 136 was mutated to lysine.
2. A gene encoding the xylanase mutant of claim 1.
3. A recombinant plasmid carrying the gene of claim 2.
4. A host cell expressing the xylanase mutant of claim 1.
5. The host cell of claim 4, wherein: the host cell is a bacterial, fungal, plant cell or animal cell.
6. Use of the xylanase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3 or the host cell of claim 4 or 5 for hydrolyzing xylan.
7. The use according to claim 6, characterized in that: the application is to add xylanase mutants or an expression system containing the xylanase mutants into a system containing xylan for reaction.
8. A method of hydrolyzing xylan, characterized by: hydrolysis using the xylanase mutant of claim 1.
9. The method according to claim 8, wherein: the reaction temperature is not higher than 80 ℃.
10. The method according to claim 8, wherein: the reaction pH is 7.0-10.0.
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