CN113528487B - Method for improving xylanase thermal stability through iterative saturation mutation - Google Patents

Method for improving xylanase thermal stability through iterative saturation mutation Download PDF

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CN113528487B
CN113528487B CN202110952917.1A CN202110952917A CN113528487B CN 113528487 B CN113528487 B CN 113528487B CN 202110952917 A CN202110952917 A CN 202110952917A CN 113528487 B CN113528487 B CN 113528487B
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CN113528487A (en
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刘松
李阳阳
陈坚
堵国成
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Jiangnan University
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Abstract

The invention discloses a method for improving xylanase thermal stability through iterative saturation mutation, and belongs to the field of genetic engineering. The invention obtains mutant A124P/A130N/T121V/T129L/I126V through 5 rounds of screening, the optimal reaction temperature is 60 ℃, and the temperature is 10 ℃ higher than xynA. Half-life t of wild-type enzyme xynA 1/2 50℃ For 18min, the mutants were incubated at 50℃for 1200min, with no decrease in enzyme activity. The enzyme activity of 76.8%, 68.4% and 35.5% can be maintained after incubation for 5min at 55 ℃, 60 ℃ and 70 ℃, and the enzyme activity can be maintained after incubation for 1h, wherein the enzyme activity is very stable at pH 2.0-9.0, and the enzyme activity can be maintained above 70%, thus the method has wide application prospect.

Description

Method for improving xylanase thermal stability through iterative saturation mutation
Technical Field
The invention relates to a method for improving xylanase thermal stability through iterative saturation mutation, belonging to the field of genetic engineering.
Background
Xylanase enzymes include beta-1, 4-D-xylanase (EC 3.2.1.8), beta-D-1, 4 xylosidase (EC 3.2.1.37), alpha-L-arabinosidase (EC 3.2.1.55) and alpha-L-glucuronidase (EC 3.2.1.139), of which beta-1, 4-D-xylanase is the most critical enzyme, which cleaves the beta-1, 4-glycosidic bond in an endo-type manner. The enzyme has wide application in food, medicine, feed, new energy and other fields, but the use of the enzyme in the production process is limited due to the poor thermal stability of the enzyme. Among them, family 11 xylanases have great potential due to their pH tolerance and substrate specificity, but poor thermostability is a problem that needs to be addressed.
With the development of protein engineering technology and molecular biology, many students have successfully modified the thermal stability of proteins by applying the technology. As in patent CN104911163B, the inventor constructs 5 mutants, the optimum temperature is increased by 2-17 ℃, and the half life in the range of 65-80 ℃ is increased by 2-16 times. Li Zhihong mutation of T152 of the loop region of the 11 family xylanase to Phe increased activity by 27% compared to the wild-type at 70 ℃. However, there are currently no xylanases with improved thermostability while maintaining pH stability.
Disclosure of Invention
[ technical problem ]
The invention aims to provide xylanase with improved heat stability and maintained pH stability.
Technical scheme
In order to solve the technical problems, the invention provides a xylanase mutant, and the amino acid sequence of the xylanase mutant is shown as SEQ ID NO. 3.
The invention also provides a gene for encoding the xylanase mutant.
The invention also provides a vector carrying the gene.
In one embodiment, the vector is pET-22b (+) as an expression vector.
The invention also provides a host cell for expressing the gene or the vector.
The invention also provides a recombinant bacterium which expresses the xylanase mutant.
In one embodiment, the recombinant bacterium uses pET-22b (+) as an expression vector.
In one embodiment, the recombinant bacterium is a host cell of E.coli.
In one embodiment, the E.coli includes E.coli JM109 and E.coli BL21.
The invention also provides a method for improving the heat stability of xylanase, which comprises the steps of mutating threonine at 121 th position, alanine at 124 th position, isoleucine at 126 th position, threonine at 129 th position and/or alanine at 130 th position of the xylanase with an amino acid sequence shown as SEQ ID NO.2 into valine, mutating alanine at 124 th position into proline, mutating isoleucine at 126 th position into valine, mutating threonine at 129 th position into leucine and/or mutating alanine at 130 th position into asparagine.
The invention also provides a composition for degrading xylan, which contains the xylanase mutant as an active ingredient, wherein the xylanase is 10-90 wt% based on the total weight of the composition.
The invention also provides application of the xylanase mutant, the gene, the vector, the recombinant strain or the composition in degrading xylan.
[ advantageous effects ]
1. According to the invention, the mutant enzyme obtained by mutating the 121 th threonine, the 124 th alanine, the 126 th isoleucine, the 129 th threonine and the 130 th alanine of xylanase with the amino acid sequence shown as SEQ ID NO.2 is incubated for 5min at 55 ℃, 60 ℃ and 70 ℃ respectively, and the enzyme activities of 76.8%, 68.4% and 35.5% can be still maintained; the mutant enzyme was incubated at 50℃for 1200min, and the enzyme activity did not tend to decrease.
2. The xylanase mutant is very stable at pH 2.0-9.0, and can still maintain the enzyme activity of more than 70% after 1h of incubation.
Drawings
Fig. 1: construction schematic of recombinant plasmid pET-22b (+) -xynA.
Fig. 2: iterative saturation mutation circuit diagram.
Fig. 3: thermal stability profile of xynA and mutants.
Detailed Description
Xylanase enzyme activity assay: a method for determining xylanase enzyme activity by using 3, 5-dinitrosalicylic acid. 500. Mu.L of the enzyme solution or fermentation supernatant diluted to an appropriate concentration was mixed with 500. Mu.L of a betulinic xylan substrate (pH 4.0) at a concentration of 10mg/mL, reacted at 50℃for 10min, and then the reaction was terminated with a 3, 5-dinitrosalicylic acid reagent. Immediately after the reaction was terminated, the sample was allowed to react in boiling water for 5 minutes and then cooled to room temperature with water, and absorbance was measured at 545nm to obtain a blank of inactive enzyme solution.
Enzyme activity unit (U/mL) definition: the amount of enzyme required to hydrolyze xylan per minute to produce 1. Mu. Mol of reducing sugar.
Specific enzyme activity represents the catalytic capability of protein per unit mass, and the larger the value of the specific enzyme activity is, the higher the specific enzyme activity is, and the calculation formula of the specific activity is as follows: specific enzyme activity (U/mg) =total enzyme activity units/mg total protein.
Aspergillus niger (A.niger): is disclosed in CN110438018B, and the preservation number is CCTCC M2018881.
EXAMPLE 1 preparation of xylanase mutants
(1) Construction of recombinant plasmid pET-22b (+) -xynA
PCR amplification is performed by using the genome of Aspergillus niger (A. Niger) as a template and using primers F1 and R1 to obtain a gene fragment xynA. The pET-22b (+) vector is used as a template, the intron primeF and the intro primerR are used for amplification to obtain pET-22b (+) vector fragments, the gene fragments xynA and pET-22b (+) vector fragments are respectively subjected to agarose gel electrophoresis, products are recovered by gel, the recovered gene fragments xynA are inserted between enzyme cleavage sites NcoI and XhoI of the pET-22b (+) vector, and the introns are removed by phosphorylase and Solution I to obtain the expression vector pET-22b (+) -xynA (figure 1). The expression vector pET-22b (+) -xynA is transformed into E.coli JM109, cultured overnight in LB solid medium containing 50 mug/mL ampicillin, then monoclonal cultured in LB liquid medium containing 50 mug/mL ampicillin for 12-16 h, plasmids are extracted, and sequencing verification is carried out, thus successfully constructing recombinant plasmid pET-22b (+) -xynA for expressing wild xylanase gene xynA (the nucleotide sequence of which is shown as SEQ ID NO. 1).
The PCR reaction system is as follows: 1. Mu.L of forward primer (10. Mu.M), 1. Mu.L of reverse primer (10. Mu.M), 1. Mu.L of template DNA, 2X Phanta Max Master Mix. Mu.L, and double distilled water was added to 50. Mu.L.
The PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 55℃for 15s, extension at 72℃for 50s, and cycling 34 times; finally, the extension is carried out for 5min at 72 ℃.
(2) Construction and screening of recombinant plasmids containing xylanase mutants
And (3) taking the pET-22b (+) -xynA constructed in the step (1) as a template, designing degenerate primers (table 1) aiming at single amino acid sites of the 121 th to 130 th positions of xylanase xynA, respectively carrying out saturation mutation by PCR, and constructing and obtaining the recombinant plasmid containing the xylanase mutant.
The PCR reaction system is as follows: 1. Mu.L of forward primer (10. Mu.M), 1. Mu.L of reverse primer (10. Mu.M), 1. Mu.L of template DNA, 2X Phanta Max Master Mix. Mu.L, and double distilled water was added to 50. Mu.L.
The PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 55℃for 15s, extension at 72℃for 50s, and cycling 34 times; finally, the extension is carried out for 5min at 72 ℃.
Transferring recombinant plasmid containing xylanase mutant into E.coli JM109 competent cells of Escherichia coli, culturing at 37deg.C for 12-16 hr, selecting different transformants, fermenting and culturing in 96-well plate containing LB culture medium, shaking culture at 37deg.C under 750r/min to OD 600 To 0.8, IPTG (Isopropyl Thiogalactoside) was added at a final concentration of 0.5mM, and the supernatant was taken after induction at 25℃and 750rpm for 24 hours to initially determine the enzyme activity. The enzyme activity was measured again after incubating the supernatant at 55℃for 10min, and the relative values of the enzyme activities of the mutant enzymes measured at 50℃and 55℃were calculated to characterize the thermostability of the mutant enzymes.
When single point mutations occur at amino acid positions 121, 124, 126, 127, 129, 130 of xylanase xynA, the thermostability of xylanase can be improved, and the remaining positions cannot improve the thermostability of xylanase xynA. The remaining forward mutation sites 121, 127, 129, 130 were again subjected to saturation mutation in sequence based on the optimal mutant a124P, I V, gradually forming a more stable structure (fig. 2), and each mutation was subjected to selection of thermal stability to obtain the optimal mutant. After 5 rounds of saturation mutation and thermal stability screening, the recombinant plasmid pET-22b (+) -A124P/A130N/T121V/T129L/I126V is finally obtained. The mutant PCR reaction system and amplification procedure were the same as in step (1).
TABLE 1 construction of primers for each plasmid
Note that: underlined are cleavage sites.
Example 2: expression purification and enzymatic Property determination of XynA and mutants
(1) Expression and purification
Recombinant plasmids pET-22b (+) -xynA and pE constructed in example 1 were usedT-22b (+) -A124P/A130N/T121V/T129L/I126V are respectively transferred into E.coli BL21 competent cells, and are coated on LB solid medium for overnight culture at 37 ℃, single colony is selected and inoculated on LB liquid medium, seed liquid is obtained by overnight culture under the condition of 37 ℃ and 220rpm, the seed liquid is inoculated in 30mL fresh PDA liquid medium with the volume ratio of 2 percent, and the seed liquid is cultured to OD under the condition of 37 ℃ and 220rpm 600 At 0.8, induction was performed with IPTG at a final concentration of 0.5mM and incubation at 25℃and 220rpm for 24h. Collecting the fermentation broth, centrifuging at 12000rpm to collect supernatant, filtering the supernatant with 0.22 μm filter membrane, and treating with HisTrap TM And (3) FF purification, desalting by a desalting column Sephadex G25, and obtaining purified wild-type enzyme xynA and mutant enzyme. SDS-PAGE electrophoresis shows that the purified wild-type enzyme and mutant enzyme have the same molecular weight and the same size as the theoretical value.
(2) Enzymatic Property determination
Determination of the optimum reaction temperature: with NaH at pH 7.5 at a concentration of 50mM 2 PO 4 -Na 2 HPO 4 The buffer system is used for carrying out enzymatic reactions at different temperatures (40-60 ℃) to respectively determine the enzyme activities so as to determine the optimal temperatures of the purified wild-type enzyme xynA and the mutant enzyme. The relative enzyme activities at each temperature were calculated with the highest enzyme activity being 100%. The results showed that the optimal reaction temperature for mutant A124P/A130N/T121V/T129L/I126V was 60℃which was 10℃higher than that of the wild-type enzyme xynA.
Determination of temperature stability: the purified wild-type enzyme xynA and mutant enzyme are respectively incubated at 50-70 ℃ for 0-80min and then are preserved on ice, and the enzyme activity under each condition is measured. The relative enzyme activity at each incubation time was calculated with the enzyme activity at 0min incubation as 100%. The mutant enzyme remained 76.8% enzyme activity at 55℃for 5min, 68.4% enzyme activity at 60℃for 5min, 35.5% enzyme activity at 70℃and almost all of the wild-type enzyme xynA was inactivated at 55℃for 10min (FIG. 3).
Half-life is calculated after fitting regression equation of relative enzyme activities of wild-type enzyme xynA and mutant enzyme at different temperatures, and the result shows that half-life t of wild-type enzyme xynA 1/2 50℃ For 18min, the mutant enzyme was incubated at 50℃for 1200min, with no decrease in enzyme activity.
Determination of optimal reaction pH: the purified wild-type enzyme xynA and mutant enzyme were enzymatically reacted in buffers of different pH values (pH 2.0-9.0) at 40 ℃ to determine their optimum pH values, using Na as the buffer 2 HPO 4 Citric acid (pH 2.0-5.0), na 2 HPO 4 -NaH 2 PO 4 (pH 6.0-7.0), tris-HCl (pH 8.0) and glycine-NaOH (pH 9.0) buffers. The results showed that the optimal pH values of mutant A124P/A130N/T121V/T129L/I126V and wild-type enzyme xynA were kept identical and were both 4.0.
Determination of pH stability: the enzyme solution was treated in buffers of different pH (pH 2.0-9.0) at 40℃for 1 hour, and the enzyme activity was measured to investigate the pH stability of the enzyme, using the buffers as described above. The result shows that the mutant enzyme is very stable at pH 2.0-9.0, and the enzyme activity is maintained above 70%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> a method for improving xylanase thermostability by iterative saturation mutagenesis
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<170> PatentIn version 3.3
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gtgggcaact ttgtcggtgg aaagggctgg aaccccggaa gtgcgcagga catcacctac 180
agcggcacct tcacccctag cggcaacggc tatctctccg tctatggctg gaccactgac 240
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Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe Tyr Tyr Ser Phe
1 5 10 15
Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn Gly Asp Ala Gly
20 25 30
Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe Val Gly Gly Lys
35 40 45
Gly Trp Asn Pro Gly Ser Ala Gln Asp Ile Thr Tyr Ser Gly Thr Phe
50 55 60
Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly Trp Thr Thr Asp
65 70 75 80
Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Asp Tyr Asn Pro
85 90 95
Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser Asp Gly Ser Val
100 105 110
Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala Ser Ile Gln Gly
115 120 125
Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Asn Lys Arg Val
130 135 140
Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala Trp Ala Lys Leu
145 150 155 160
Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu Gly
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Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln
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Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe Tyr Tyr Ser Phe
1 5 10 15
Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn Gly Asp Ala Gly
20 25 30
Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe Val Gly Gly Lys
35 40 45
Gly Trp Asn Pro Gly Ser Asn Gln Ser Ile Asn Tyr Ser Gly Thr Phe
50 55 60
Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly Trp Thr Thr Asp
65 70 75 80
Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Asp Tyr Asn Pro
85 90 95
Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser Asp Gly Ser Val
100 105 110
Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala Ser Ile Gln Gly
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Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Asn Lys Arg Val
130 135 140
Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala Trp Ala Lys Leu
145 150 155 160
Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu Gly
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Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln
180 185

Claims (10)

1. A xylanase mutant with improved heat stability is characterized in that the amino acid sequence is obtained by mutating threonine at 121 th position, alanine at 124 th position, isoleucine at 126 th position, leucine at 129 th position and alanine at 130 th position of xylanase with amino acid sequence shown as SEQ ID NO.2 into valine, mutating alanine at 124 th position into proline, mutating isoleucine at 126 th position into valine, mutating threonine at 129 th position into leucine at 130 th position into asparagine.
2. A gene encoding the xylanase mutant of claim 1.
3. A vector carrying the gene of claim 2.
4. A host cell expressing the gene of claim 2 or the vector of claim 3.
5. A recombinant bacterium, wherein the xylanase mutant of claim 1 is expressed.
6. The recombinant bacterium according to claim 5, wherein pET-22b (+) is used as an expression vector.
7. A recombinant bacterium according to claim 5, wherein E.coli is used as host cell.
8. A method for improving the heat stability of xylanase is characterized in that threonine at position 121, alanine at position 124, isoleucine at position 126, threonine at position 129, leucine and alanine at position 130 of the xylanase with an amino acid sequence shown in SEQ ID NO.2 are mutated into valine, proline and valine.
9. A composition for degrading xylan, characterized in that it comprises the xylanase mutant according to claim 1 as an active ingredient in an amount of 10-90% by weight, based on the total weight of the composition.
10. Use of the xylanase mutant of claim 1, the gene of claim 2, the vector of claim 3, the recombinant bacterium of any one of claims 5-7 and the composition of claim 9 for degrading xylan.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109355272A (en) * 2018-12-28 2019-02-19 江南大学 A kind of xylanase mutant that catalytic efficiency improves

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109355272A (en) * 2018-12-28 2019-02-19 江南大学 A kind of xylanase mutant that catalytic efficiency improves

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