CN113528487B - A method to improve the thermostability of xylanase by iterative saturation mutation - Google Patents

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

A method to improve the thermostability of xylanase by iterative saturation mutation

technical field

The invention relates to a method for improving the thermal stability of xylanase through iterative saturation mutation, which belongs to the field of genetic engineering.

Background technique

The xylanase enzyme system includes β-1,4-D-xylanase (EC 3.2.1.8), β-D-1,4 xylosidase (EC 3.2.1.37), α-L-arabinoside enzymes (EC 3.2.1.55) and α-L-glucuronidase (EC 3.2.1.139), among them, β-1,4-D-xylanase is the most critical enzyme, which will β-1,4-glycosidic bond breaks. The enzyme is widely used in food, medicine, feed and new energy, but its use is limited in the production process due to its poor thermal stability. Among them, family 11 xylanases have great potential due to their pH tolerance and substrate specificity, but poor thermal stability is an urgent problem to be solved.

With the development of protein engineering technology and molecular biology, many scholars have used this technology to successfully modify the thermal stability of proteins. For example, in the patent CN104911163B, the inventor constructed 5 mutants, the optimum temperature was increased by 2-17°C, and the half-life in the range of 65-80°C was increased by 2-16 times. Li Zhihong mutated T152 in the loop region of family 11 xylanases to Phe, and the activity at 70°C was 27% higher than that of the wild type. However, there is currently no xylanase that maintains pH stability while improving thermostability.

Contents of the invention

[technical problem]

The technical problem to be solved by the present invention is to provide a xylanase with improved thermostability and pH stability.

[Technical solutions]

In order to solve the above technical problems, the present invention provides a mutant xylanase, the amino acid sequence of which is shown in SEQ ID NO.3.

The present invention also provides the gene encoding the above mutant xylanase.

The present invention also provides a vector carrying the above gene.

In one embodiment, the vector uses pET-22b(+) as the expression vector.

The present invention also provides host cells expressing the above-mentioned genes or the above-mentioned vectors.

The present invention also provides a recombinant bacterium expressing the above-mentioned xylanase mutant.

In one embodiment, the recombinant bacteria use pET-22b(+) as the expression vector.

In one embodiment, the recombinant bacteria use Escherichia coli as the host cell.

In one embodiment, the Escherichia coli includes E. coli JM109 and E. coli BL21.

The present invention also provides a method for improving the thermostability of xylanase, the method is to mutate the 121st threonine of the xylanase whose amino acid sequence is as shown in SEQ ID NO.2 to valine acid, alanine at position 124 to proline, isoleucine at position 126 to valine, threonine at position 129 to leucine and/or alanine at position 130 Acid mutation to asparagine.

The present invention also provides a composition for degrading xylan, the composition contains the above-mentioned xylanase mutant as an active ingredient, based on the total weight of the composition, the xylanase The content is 10-90% by weight.

The present invention also provides the above-mentioned xylanase mutant, or the above-mentioned gene, or the above-mentioned vector, or the above-mentioned recombinant bacteria, or the application of the above-mentioned composition in degrading xylan.

[beneficial effect]

1. The present invention adopts the 121st threonine, the 124th alanine, the 126th isoleucine, and the 129th threonine of the xylanase whose amino acid sequence is as shown in SEQ ID NO.2 and the 130th alanine were mutated, and the obtained mutant enzymes were incubated at 55°C, 60°C and 70°C for 5 minutes respectively, and still maintained 76.8%, 68.4% and 35.5% of the enzyme activities; the mutant enzymes were incubated at 50°C After incubation for 1200min, the enzyme activity did not decrease.

2. The xylanase mutant is very stable at pH 2.0-9.0, and can still maintain more than 70% of the enzyme activity after incubation for 1 hour.

Description of drawings

Figure 1: Schematic diagram of the construction of the recombinant plasmid pET-22b(+)-xynA.

Figure 2: Iterative saturation mutation circuit diagram.

Figure 3: Thermostability map of xynA and mutants.

Detailed ways

Determination of xylanase activity: a method for determining xylanase activity by using 3,5-dinitrosalicylic acid. Mix 500 μL of enzyme solution or fermentation supernatant diluted to an appropriate concentration with 500 μL of birch wood xylan substrate (pH 4.0) at a concentration of 10 mg/mL, react at 50°C for 10 min, and then use 3,5-dinitrosalicyl Acid reagents terminate the reaction. After the reaction was terminated, the samples were reacted in boiling water for 5 minutes and immediately cooled to room temperature with water, and the absorbance was measured at 545 nm, and the inactivated enzyme solution was used as the blank group.

Enzyme activity unit (U/mL) definition: the amount of enzyme required to hydrolyze 1 μmol of reducing sugar produced by xylan per minute.

The specific enzyme activity represents the catalytic ability per unit mass of protein, which can reflect the size of the enzyme activity. The larger the value, the higher the enzyme activity. The calculation formula of the specific activity is: specific enzyme activity (U/mg) = total number of enzyme activity units /mg total protein.

Aspergillus niger (A. niger): disclosed in CN110438018B, and the preservation number is CCTCC M 2018881.

The preparation of embodiment 1 xylanase mutant

(1) Construction of recombinant plasmid pET-22b(+)-xynA

Using the genome of Aspergillus niger (A. niger) as a template, the gene fragment xynA was obtained by PCR amplification with primers F1 and R1. Using the pET-22b(+) vector as a template, the pET-22b(+) vector fragment was amplified with intron primeF and intro primerR, and the gene fragment xynA and the pET-22b(+) vector fragment were subjected to agarose gel electrophoresis respectively, The product was recovered by gelling, and the recovered gene fragment xynA was inserted between the restriction sites NcoI and XhoI of the pET-22b(+) vector, and the intron was removed with phosphorylase and Solution I to obtain the expression vector pET-22b(+ )-xynA (Figure 1). The expression vector pET-22b(+)-xynA was transformed into E.coli JM109, cultured overnight in LB solid medium containing 50 μg/mL ampicillin, and cultured in LB liquid medium containing 50 μg/mL ampicillin for 12 After ~16 hours, the plasmid was extracted and verified by sequencing, and the recombinant plasmid pET-22b(+)-xynA expressing the wild-type xylanase gene xynA (nucleotide sequence shown in SEQ ID NO.1) was successfully constructed.

The PCR reaction system is: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, 25 μL of 2×Phanta Max Master Mix, and add double distilled water to 50 μL.

The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 50 s, and 34 cycles; finally, extension at 72°C for 5 min.

(2) Construction and screening of recombinant plasmids containing xylanase mutants

Using the pET-22b(+)-xynA constructed in step (1) as a template, design degenerate primers (Table 1) for a single amino acid site at positions 121-130 of xylanase xynA, and perform saturation mutation by PCR , to construct a recombinant plasmid containing xylanase mutants.

The PCR reaction system is: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, 25 μL of 2×Phanta Max Master Mix, and add double distilled water to 50 μL.

The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 50 s, and 34 cycles; finally, extension at 72°C for 5 min.

Transfer the recombinant plasmid containing the xylanase mutant into Escherichia coli E.coli JM109 competent cells and culture at 37°C for 12-16h, pick different transformants and put them in 96-well plates containing LB medium for fermentation and culture at 37°C , 750r/min at a constant temperature shaker culture to OD ₆₀₀ to 0.8, add IPTG (Isopropylβ-D-Thiogalactoside) with a final concentration of 0.5mM, induce 24h at 25°C and 750rpm The enzyme activity was firstly measured. Then the supernatant was incubated at 55°C for 10 min, and then the enzyme activity was measured again, and the relative value of the enzyme activity of the mutant enzyme measured at 50°C and 55°C was calculated to characterize the thermal stability of the mutant enzyme.

When the 121st, 124th, 126th, 127th, 129th, and 130th amino acid positions of xylanase xynA have single-point mutations, the thermal stability of xylanase can be improved, and the rest of the positions cannot improve the stability of xylanase xynA. thermal stability. On the basis of the best mutants A124P and I126V, the remaining forward mutation sites 121, 127, 129, and 130 were subjected to saturation mutations in sequence to gradually form a more stable structure (Figure 2), and each mutation was thermally stable Sexual screening to get the best mutants. After five rounds of saturation mutation and thermostability screening, the recombinant plasmid pET-22b(+)-A124P/A130N/T121V/T129L/I126V was finally obtained. The mutant PCR reaction system and amplification program are the same as step (1).

Table 1 The list of primers constructed by each plasmid

Note: The underline is the restriction site.

Example 2: Expression and purification of XynA and mutants and determination of enzymatic properties

(1) Expression and purification

The recombinant plasmids pET-22b(+)-xynA and pET-22b(+)-A124P/A130N/T121V/T129L/I126V constructed in Example 1 were respectively transferred into E.coli BL21 competent cells and spread on LB Cultivate the solid medium overnight at 37°C, pick a single colony and inoculate it in the LB liquid medium, and cultivate it overnight at 37°C and 220rpm to obtain the seed liquid, inoculate the seed liquid at a volume ratio of 2% in 30mL of fresh PDA liquid In the medium, cultured at 37°C, 220rpm until the OD ₆₀₀ was 0.8, induced with IPTG at a final concentration of 0.5mM and cultured at 25°C, 220rpm for 24h. The fermentation broth was collected by centrifugation at 12000 rpm to collect the supernatant, and the supernatant was filtered through a 0.22 μM filter membrane, purified by HisTrap ^TM FF, desalted with a desalting column Sephadex G25, and purified wild-type enzyme xynA and mutant enzymes were obtained. SDS-PAGE electrophoresis showed that the molecular weight of the purified wild-type enzyme and the mutant enzyme were consistent, and the size was the same as the theoretical value.

(2) Determination of enzymatic properties

Determination of the optimum reaction temperature: use pH 7.5, NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer system with a concentration of 50mM, carry out enzymatic reactions at different temperatures (40-60°C), and measure the enzyme activity to determine the purification Temperature optima of the wild-type enzyme xynA and mutant enzymes. Taking the highest enzyme activity as 100%, calculate the relative enzyme activity at each temperature. The results showed that the optimal reaction temperature of the mutant A124P/A130N/T121V/T129L/I126V was 60°C, which was 10°C higher than that of the wild-type enzyme xynA.

Measurement of temperature stability: the purified wild-type enzyme xynA and the mutant enzyme were incubated at 50-70° C. for 0-80 min, respectively, and stored on ice, and the enzyme activity under each condition was measured. Taking the enzyme activity at the time of incubation for 0 min as 100%, the relative enzyme activity at each incubation time was calculated. The mutant enzyme can still maintain 76.8% of the enzyme activity when incubated at 55°C for 5 minutes, 68.4% of the enzyme activity when incubated at 60°C for 5 minutes, and 35.5% of the enzyme activity when incubated at 70°C for 5 minutes The wild-type enzyme xynA was almost completely inactivated when incubated at 55° C. for 10 min ( FIG. 3 ).

The half-life is calculated by fitting the regression equation of the relative enzyme activities of the wild-type enzyme xynA and the mutant enzyme at different temperatures. The results show that the half-life t _1/2 of the wild-type enzyme xynA is 18min at ^50°C , and the mutant enzyme is at After incubation at 50°C for 1200 min, the enzyme activity did not decrease.

Determination of the optimum reaction pH: at 40°C, the purified wild-type enzyme xynA and mutant enzymes were enzymatically reacted in buffers with different pH values (pH2.0-9.0) to determine their optimum pH values. The buffer is Na 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) liquid. The results showed that the optimal pH values of the mutant A124P/A130N/T121V/T129L/I126V and the wild-type enzyme xynA were consistent at 4.0.

Determination of pH stability: Treat the enzyme liquid in buffers of different pH (pH 2.0-9.0) at 40°C for 1 hour, and then measure the enzyme activity to study the pH stability of the enzyme. The buffers used are as described above. The results show that the mutant enzyme is very stable between pH 2.0-9.0, and more than 70% of the enzyme activity is maintained.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A method to improve the thermostability of xylanase by iterative saturation mutation

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<170> PatentIn version 3.3

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tcgaccccga gctcgaccgg cgagaacaac ggcttctact actccttctg gaccgacggc 60

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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

165 170 175

Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln

180 185

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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

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

165 170 175

Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln

180 185

Claims (10)

1. A xylanase mutant whose thermostability is improved is characterized in that its amino acid sequence is that the threonine at the 121st position of the xylanase whose amino acid sequence is shown in SEQ ID NO.2 is mutated into Valine, alanine at position 124 to proline, isoleucine at position 126 to valine, threonine at position 129 to leucine and alanine at position 130 Acid mutation to asparagine was obtained. 2. The gene encoding the xylanase mutant described in claim 1. 3. A carrier 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, characterized in that it expresses the xylanase mutant according to claim 1. 6. A recombinant bacterium as claimed in claim 5, wherein pET-22b(+) is used as the expression vector. 7. A kind of recombinant bacterium as claimed in claim 5, is characterized in that, take Escherichia coli as host cell. 8. A method for improving the thermostability of xylanase, characterized in that the threonine at position 121 of the xylanase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to valine, and the Mutation of alanine at position 124 to proline, isoleucine at position 126 to valine, threonine at position 129 to leucine and alanine at position 130 to aspartame amides. 9. A composition for degrading xylan, characterized in that, the composition contains the xylanase mutant according to claim 1 as an active ingredient, based on the total weight of the composition, the The content of the mutant xylanase is 10-90% by weight. 10. the xylanase mutant described in claim 1, the gene described in claim 2, the carrier described in claim 3, the recombinant bacterium described in any one of claim 5-7 and claim 9 described The application of said composition in degrading xylan.