CN110607291B - Heat-resistant xylanase mutant - Google Patents
Heat-resistant xylanase mutant Download PDFInfo
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Abstract
The invention provides a heat-resistant xylanase mutant, which has an amino acid sequence of SEQ ID NO:1 by substituting the xylanase at amino acid position 28 and/or 37. The xylanase mutants Xyn1-28, Xyn1-37 and Xyn1-28/37 provided by the invention have obviously improved heat resistance. Compared with xylanase Xyn1, after treatment for 5min at 80 ℃, the enzyme activity residual rates of the three xylanase mutants are generally improved by 18.83-33.91%; after the treatment for 3min at 85 ℃, the residual rate of the enzyme activity is generally improved by 25.50-74.87 percent, and unexpected technical effects are achieved. The xylanase mutant has high heat resistance, is beneficial to the application of the xylanase mutant in feed, and has wide market prospect.
Description
Technical Field
The invention belongs to the technical field of protein engineering modification, and particularly relates to a xylanase mutant obtained by heat resistance screening.
Background
Xylan is the main component of plant hemicellulose and widely exists in crop wastes such as corncobs, bagasse, wheat bran, straws and the like, and xylanase can decompose xylan into xylo-oligosaccharides and xylose with different lengths, so that the product has important economic value, the usable resources are fully utilized through xylanase, the potential application value of the xylanase is exerted, and the research of xylanase is also fully paid attention.
Xylanases are glycosyl hydrolases that hydrolyze beta-1, 4-linked pyranoside chains, have been demonstrated to exist in at least hundreds of different organisms, and can be economically produced on a large scale. Together with other glycosyl hydrolases, they form a superfamily comprising over 40 different enzyme families. Trichoderma reesei is known to produce three different xylanases, of which xylanase I and II (XynI and XynII) have the best characteristics. The XynI has a molecular weight of 19kDa, and has a lower isoelectric point and an optimal pH (pI 5.5, pH 3-4) compared with XynII. The XynII has a molecular weight of 20kDa, an isoelectric point of 9.0 and an optimal pH of 5.0-5.5.
Xylanases are now widely used in pulp bleaching, textile fibre modification and in animal feed and human food production. A common problem in all these applications is the extreme conditions that xylanases face. The high temperature in industrial applications and the different pH optimum from many xylanases from the pH conditions in industrial applications reduce the effective industrial utilization of the existing xylanases.
In applications in pulp bleaching, the material from the caustic wash has a high temperature (>80 ℃) and pH (> 10). Most xylanases will be inactivated under such conditions, and the pulp must therefore be cooled and alkaline neutralized, which means an increase in costs.
In feed applications, the xylanase is exposed to a brief high temperature (e.g., 2-5 minutes at 90 ℃) during feed preparation. However, the temperature required for catalytic activity of xylanases is relatively low (e.g.about 37 ℃). Thus, the xylanase will be irreversibly inactivated at high temperatures.
Although much research has been carried out to improve the stability of xylanase, the improved industrial production requirements cannot be met, and therefore, the important practical significance of providing a high-temperature resistant xylanase suitable for industrial production is achieved.
Disclosure of Invention
In view of the above, the invention provides a xylanase mutant, which can obtain mutant protein and improve the heat resistance thereof, thereby being beneficial to the wide application of xylanase in the field of feed.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a xylanase mutant, which has any one of amino acid sequences shown in (I), (II) or (III):
(I) a sequence having at least 95% homology with the amino acid sequence of xylanase SEQ ID NO: 1;
(II) has at least one immune epitope of the xylanase, and the amino acid sequence of the xylanase is an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids in SEQ ID NO 1;
(III) an amino acid sequence encoded by a nucleotide sequence shown by SEQ ID NO:2 or a complementary sequence thereof or a sequence that differs from the nucleotide sequence shown by SEQ ID NO:2 or a complementary sequence thereof due to degeneracy of the genetic code;
in some embodiments of the invention, the xylanase mutant is a xylanase having an amino acid sequence of SEQ ID NO:1 by substituting the amino acid at position 28 of the xylanase.
In a preferred embodiment, the 28 th amino acid of the xylanase mutant is mutated from Asn to Leu.
In some embodiments of the invention, the xylanase mutant is a xylanase with the amino acid sequence of SEQ ID NO:1 by substituting the 37 th amino acid of the xylanase.
In a preferred embodiment, the xylanase mutant has a mutation from Asn to Ile at amino acid position 37.
In some embodiments of the invention, the xylanase mutant is a xylanase with the amino acid sequence of SEQ ID NO:1, the xylanase of position 28 and position 37 being substituted simultaneously.
In a preferred embodiment, the xylanase mutant has the mutation from Asn to Leu at amino acid 28 and from Asn to Ile at amino acid 37.
The xylanase mutant containing the N28L single-point mutation is named as Xyn1-28, the amino acid sequence of the xylanase mutant is SEQ ID NO. 3, and a coding nucleotide sequence obtained by referring to the sequence is SEQ ID NO. 4.
The xylanase mutant containing the N37I single-point mutation is named as Xyn1-37, the amino acid sequence of the xylanase mutant is SEQ ID NO. 5, and a coding nucleotide sequence obtained by referring to the sequence is SEQ ID NO. 6.
The xylanase mutant containing N28L and N37I point mutations is named as Xyn1-28/37, the amino acid sequence of the xylanase mutant is SEQ ID NO. 7, and a coding nucleotide sequence obtained by referring to the sequence is SEQ ID NO. 8.
The invention also provides a DNA molecule for coding the xylanase mutant.
In some embodiments of the invention, the DNA molecule encoding the xylanase mutant described above has a nucleotide sequence as shown in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8.
The invention also provides a recombinant expression vector carrying the DNA molecule.
The invention also provides a host cell comprising the recombinant expression vector.
The host cell is preferably Pichia pastoris (Pichia pastoris).
The host cell is preferably Trichoderma reesei (Trichoderma reesei).
The host cell is preferably Aspergillus niger.
The recombinant expression vector is transferred into the host cell, so that the heat resistance of the xylanase mutant subjected to recombinant expression is obviously improved.
The xylanase mutants Xyn1-28, Xyn1-37 and Xyn1-28/37 provided by the invention have obviously improved heat resistance. Compared with xylanase Xyn1, after treatment for 5min at 80 ℃, the enzyme activity residual rates of the three xylanase mutants are generally improved by 18.83-33.91%; after being treated for 3min at 85 ℃, the residual rate of enzyme activity is generally improved by 25.50-74.87 percent. The xylanase mutant Xyn1-28/37 has the strongest heat resistance, and after treatment for 5min at 80 ℃, the residual enzyme activity is respectively improved by 8.2% and 12.7% compared with xylanase mutants Xyn1-28 and Xyn 1-37; after treatment for 3min at 85 ℃, the residual enzyme activity is respectively improved by 27.2 percent and 39.3 percent compared with xylanase mutants Xyn1-28 and Xyn1-37, and unexpected technical effects are achieved. The xylanase mutant has high heat resistance, is beneficial to the application of the xylanase mutant in feed, and has wide market prospect.
The specific implementation mode is as follows:
the invention discloses a xylanase mutant, which can be realized by appropriately improving process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention will now be described in detail by way of reference only using the definitions and examples given below. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, second edition, by Singleton et al, John Wiley AND Sons, 1994, AND the THE HARPER COLLINS DICTIONARY OF BIOLOGY, by Hale AND Marham, by Harper Perennial, New York, 1991, provide the artisan with a comprehensive DICTIONARY OF many OF the terms used in this invention. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction, respectively; the amino acid sequence is written from left to right in the direction from amino to carboxyl. In particular, the practitioner can refer to Sambrook et al, 1989 and Ausubel FM et al, 1993 to understand the definitions and terminology in the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.
The headings provided herein are not limitations of the various aspects and embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.
As used herein, the term "expression" refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene. The process includes transcription and translation.
As used herein, the term "gene" means a segment of DNA involved in the production of a polypeptide chain, which may or may not include regions preceding or following the coding region.
As used herein, "enzyme" refers to a protein or polypeptide that catalyzes a chemical reaction.
As used herein, the term "activity" refers to a biological activity associated with a particular protein, such as an enzymatic activity associated with a protease. Biological activity refers to any activity that one of skill in the art would ordinarily attribute to the protein.
As used herein, the term "xylanase" refers to a glycosyl hydrolase that hydrolyzes the β -1, 4-linked xylopyranoside chain.
As used herein, "point mutations" refers to changes in a single nucleotide in DNA, particularly where such changes would result in a change in a protein.
As used herein, "mutant" refers to a type of organism or protein that is different from the wild type. Such changes can be effected by methods known to those skilled in the art, for example, by point mutations, where the resulting protein can then be referred to as mutants.
As used herein, "modified" refers to a sequence, such as an amino acid sequence comprising a polypeptide, that includes deletions, insertions, substitutions or truncations to the naturally occurring sequence.
As used herein, "substituted" shall mean a substitution to a naturally occurring residue.
The experimental materials and reagents selected in the preferred embodiment of the invention:
bacterial strain and carrier: coli DH5 α, Pichia pastoris GS115, vector pPIC9K, Amp, G418 were purchased from Invitrogen.
Enzyme and kit: PCR enzyme and ligase were purchased from Takara, restriction enzyme was purchased from Fermentas, plasmid extraction kit and gel purification recovery kit were purchased from Omega, and GeneMorph II random mutagenesis kit was purchased from Beijing Bomais Biotech.
The formula of the culture medium is as follows:
coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, ph 7.0);
LB-AMP medium: adding 100 mu g/mL ampicillin into LB culture medium;
yeast medium (YPD medium): 1% yeast extract, 2% peptone, 2% glucose;
yeast screening medium (MD medium): 2% glucose, 2% agarose, 1.34% YNB, 4X 10-5Biotin;
BMGY medium: 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4X 10-5Biotin, 1% glycerol;
BMMY medium: 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4X 10-5Biotin, 0.5% methanol.
The raw materials and reagents used in the xylanase mutant provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 screening of thermostable xylanase mutants
Based on a wild-type xylanase from Trichoderma reesei (Trichoderma reesei), two unnatural disulfide bridges Q33C-T187C and S109C-N153C and three mutation sites Q51N, H143K and Q161F are introduced to obtain a xylanase mutant with remarkably improved heat resistance, wherein the enzyme activity residual rate of the xylanase mutant is up to 61.28% after the xylanase mutant is treated at 80 ℃ for 5min, and 26.89% of enzyme activity residual is remained after the xylanase mutant is treated at 85 ℃ for 3 min. Applicants named this mutant as Xyn1, the nucleotide sequence of which is SEQ ID NO:2, the encoded amino acid sequence is SEQ ID NO: 1.
in order to further improve the heat resistance of the xylanase Xyn1, the applicant screens a large number of mutations of the xylanase Xyn1 by a directed evolution technology.
Xyn-F1：5’—CGCGAATTCACTATTCAACCTGGAACTGGATAC-3' (recognition site for restriction enzyme ECORI is underlined)
Xyn-R1：5’—CTCGCGGCCGCTTATGAGACTGTGATAGAGGCAG-3' (restriction endonuclease Not I recognition site underlined)
Taking Xyn1 gene as a template, carrying out PCR amplification by using primers Xyn-F1 and Xyn-R1 in example 1 and using a GeneMorph II random mutation PCR kit (Stratagene), carrying out gel recovery on PCR products, carrying out enzyme digestion treatment on EcoRI and Not I, connecting the PCR products with pET21a carriers subjected to the same enzyme digestion, transforming the PCR products into escherichia coli BL21(DE3), coating the escherichia coli BL21 in an LB + Amp plate, carrying out inversion culture at 37 ℃, after transformants appear, picking the escherichia coli in a 96-well plate one by using toothpicks, adding 150ul LB + Amp culture medium containing 0.1mM IPTG into each well, carrying out culture at 37 ℃ and 220rpm for about 6 hours, centrifuging, discarding supernatant, carrying out resuspension on thalli by using buffer solution, and repeatedly freezing and thawing to obtain an escherichia coli cell lysate containing xylanase.
Respectively taking out 30ul of lysate to two new 96-well plates; treating one of the above materials at 75 deg.C for 8 min; then adding 30ul of substrate into both 96-well plates, reacting for 30min at 37 ℃, determining the generated reducing sugar by using a DNS method, and calculating the enzyme activity level of different mutagens after high-temperature treatment. Finally, the applicant obtains a mutation site which can obviously improve the heat resistance of the xylanase mutant Xyn1 and can not obviously influence the enzyme activity and the original enzymology property: N28L, N37I.
The xylanase mutant containing the N28L single-point mutation is named as Xyn1-28, the amino acid sequence of the xylanase mutant is SEQ ID NO. 3, and a coding nucleotide sequence obtained by referring to the sequence is SEQ ID NO. 4.
The xylanase mutant containing the N37I single-point mutation is named as Xyn1-37, the amino acid sequence of the xylanase mutant is SEQ ID NO. 5, and a coding nucleotide sequence obtained by referring to the sequence is SEQ ID NO. 6.
The xylanase mutant containing N28L and N37I point mutations is named as Xyn1-28/37, the amino acid sequence of the xylanase mutant is SEQ ID NO. 7, and a coding nucleotide sequence obtained by referring to the sequence is SEQ ID NO. 8.
The nucleotide sequence of the xylanase mutant is synthesized by Shanghai Czeri Bio.
Example 3 construction of Pichia engineering Strain
The xylanase mutant genes Xyn1-28, Xyn1-37 and Xyn1-28/37 obtained in the way are connected with an expression vector pPIC9K through EcoR I and Not I sites to construct expression vectors pPIC9K-Xyn1-28, pPIC9K-Xyn1-37 and pPIC9K-Xyn 1-28/37.
The mutant expression plasmid is linearized by Sal I, the linearized fragment of the expression plasmid is transformed into pichia pastoris GS115 by an electroporation method, pichia pastoris recombinant strains GS115/pPIC9K-Xyn1, GS115/pPIC9K-Xyn1-28 and GS115/pPIC9K-Xyn1-28/37 are obtained by screening on an MD plate respectively, and then multiple copies of transformants are screened on YPD plates containing geneticin with different concentrations respectively.
The screened positive transformants of the recombinant expression xylanase mutants Xyn1-28, Xyn1-37 and Xyn1-28/37 are named as Pichia pastoris Xyn1-28(Pichia pastoris Xyn1-28) and Pichia pastoris Xyn1-37 respectively
(Pichia pastoris Xyn1-37) and Pichia pastoris Xyn1-28/37(Pichia pastoris Xyn1-28/37), and then respectively transferred into a BMGY culture medium, and subjected to shaking culture at 30 ℃ and 250rpm for 1 d; then transferring the strain into a BMMY culture medium, and carrying out shaking culture at 30 ℃ and 250 rpm; adding 0.5% methanol every day to induce expression for 4 d; centrifuging to remove thalli, and respectively obtaining fermentation supernatant containing xylanase mutants; the xylanase mutant in the fermentation supernatant is analyzed by SDS-PAGE electrophoresis detection, and the molecular weight of the xylanase mutant in the fermentation supernatant is about 20.7 kDa.
The xylanase gene Xyn1 is cloned into a Pichia pastoris GS115 host by the same enzyme digestion connection method, and a Pichia pastoris engineering bacterium for recombining and expressing the xylanase Xyn1 is constructed and named as Pichia pastoris Xyn1(Pichia pastoris Xyn 1). Horizontally fermenting pichia pastoris Xyn1 in a shaking flask, and carrying out shaking culture at 30 ℃ and 250 rpm; adding 0.5% methanol every day to induce expression for 4 d; and (4) centrifuging to remove the thallus to obtain fermentation supernatant containing xylanase Xyn 1.
(1) Definition of xylanase Activity units
The enzyme amount required for releasing 1 mu mol of reducing sugar from 5mg/ml xylan solution per minute at 37 ℃ and pH5.5 is an enzyme activity unit U.
(2) Enzyme activity measuring method
Taking 2ml of xylan substrate with the concentration of 1% (prepared by a pH5.5 acetic acid-sodium acetate buffer solution), adding the xylan substrate into a colorimetric tube, balancing for 10min at 37 ℃, adding 2ml of acidic xylanase enzyme solution which is properly diluted by the pH5.5 acetic acid-sodium acetate buffer solution and well balanced at 37 ℃, uniformly mixing, and accurately preserving the temperature at 37 ℃ for reaction for 30 min. After the reaction was completed, 5ml of DNS reagent was added and mixed well to terminate the reaction. Boiling in boiling water bath for 5min, cooling to room temperature with tap water, adding distilled water to constant volume of 25ml, mixing, and measuring light absorption value AE at 540nm with standard blank as blank control.
The enzyme activity calculation formula is as follows:
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is an enzymeThe solution reaction time is min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
(3) Results of enzyme Activity measurement
And (3) detecting the xylanase enzyme activity in the fermentation supernatant of the pichia pastoris Xyn1, the pichia pastoris Xyn1-28, the pichia pastoris Xyn1-37 and the pichia pastoris Xyn1-28/37 respectively according to the method. The results show that: the enzyme activity of the fermentation supernatant of the pichia pastoris Xyn1 is 105U/mL, the enzyme activity of the fermentation supernatant of the pichia pastoris Xyn1-28 is 91U/mL, and the enzyme activities of the fermentation supernatants of the pichia pastoris Xyn1-37 and Xyn1-28/37 are 99U/mL and 83U/mL respectively.
Example 4 fermentation validation
Fermentation of pichia pastoris Xyn1, pichia pastoris Xyn1-28, pichia pastoris Xyn1-37 and pichia pastoris Xyn1-28/37 is respectively carried out on a 10-liter fermentation tank, and the formula of a culture medium used for fermentation is as follows: 1.1g/L of calcium sulfate, 5.5g/L of potassium dihydrogen phosphate, 55g/L of ammonium dihydrogen phosphate, 20.3g/L of potassium sulfate, 16.4g/L of magnesium sulfate, 1.65g/L of potassium hydroxide and 0.05% of defoaming agent.
The fermentation production process comprises the following steps: the pH value is 5.0, the temperature is 30 ℃, the stirring speed is 300rpm, the ventilation volume is 1.0-1.5 (v/v), and the dissolved oxygen is controlled to be more than 20%.
The whole fermentation process is divided into three stages: the first stage is a thallus culture stage, seeds are inoculated according to the proportion of 7 percent, and the mixture is cultured for 24-26 h at the temperature of 30 ℃ with the mark of complete glucose supplementation; the second stage is a starvation stage, when the glucose is supplemented, no carbon source is added, when the dissolved oxygen rises to more than 80%, the stage is ended, and the period is about 30-60 min; the third stage is an induction expression stage, methanol is fed for induction, dissolved oxygen is kept to be more than 20%, and the culture time is 150-180 h. After the fermentation is finished, the fermentation liquor is processed by a plate and frame filter to obtain a crude enzyme liquid.
The xylanase enzyme activity determination method of embodiment 3 is adopted to detect the crude enzyme solution, and the result shows that the final fermentation enzyme activity of pichia pastoris Xyn1 for recombinant expression of xylanase Xyn1 is 5730U/ml, and the final fermentation enzyme activities of pichia pastoris Xyn1-28, pichia pastoris Xyn1-37 and pichia pastoris Xyn1-28/37 for recombinant expression of xylanase mutants are 5573U/ml, 5645U/ml and 5474U/ml respectively.
Example 5 thermotolerance analysis of xylanases
The crude enzyme solutions obtained by fermentation in example 4 were diluted to about 20U/ml with acetic acid-sodium acetate buffer solution of pH5.5, and after treatment at 80 ℃ and 85 ℃ for 5min, respectively, the residual enzyme activities were measured, and the enzyme activity residual rates were calculated as 100% of the enzyme activity of the untreated samples. Specific results are shown in table 1.
TABLE 1 comparison of xylanase mutants for Heat resistance
According to the invention, 2 screened mutation sites N28L and N37I are introduced into xylanase Xyn1, so that the heat resistance of the xylanase can be effectively improved, after the xylanase is treated at 80 ℃ for 5min, the enzyme activity residual rate of the xylanase mutant introduced with the mutation sites is generally improved by 18.8-33.9%, and after the xylanase is treated at 85 ℃ for 3min, the enzyme activity residual rate is generally improved by 25.5-74.9%. The xylanase mutant Xyn1-28/37 has the strongest heat resistance, and after treatment for 5min at 80 ℃, the residual enzyme activity is respectively improved by 8.2% and 12.7% compared with xylanase mutants Xyn1-28 and Xyn 1-37; after treatment for 3min at 85 ℃, the residual enzyme activity is respectively improved by 27.2 percent and 39.3 percent compared with xylanase mutants Xyn1-28 and Xyn1-37, and unexpected technical effects are achieved.
In addition to Pichia pastoris (Pichia pastoris), applicants further recombinantly expressed xylanase Xyn1 and the mutants Xyn1-28, Xyn1-37, Xyn1-28/37 using Trichoderma reesei (Trichoderma reesei) or Aspergillus niger (Aspergillus niger) host cells and compared the thermotolerance of recombinant proteins. The results show that the heat resistance of the obtained mutant proteins Xyn1-28, Xyn1-37 and Xyn1-28/37 is obviously higher than that of xylanase Xyn1, wherein the mutant Xyn1-28/37 has the highest heat resistance, and unexpected technical effects are achieved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Islands blue biological group Co Ltd
<120> thermostable xylanase mutants
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Gly Trp Asn Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly Ser
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Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg
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Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Cys Asp Gly Ser
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Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile Ile
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Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn Lys Arg
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Ser Ser Gly Ser Val Asn Thr Ala Cys His Phe Asn Ala Trp Ala Gln
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Phe Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val Glu
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Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Cys Val Ser
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<400> 2
actattcaac ctggaactgg atacaataac ggttatttct actcttactg gaacgatgga 60
catggaggtg tcacatacac taacggtcca ggtggatgtt tctcagttaa ttggtctaac 120
tcaggaaatt tcgtcggagg taaaggatgg aacccaggaa ctaagaataa ggtcattaac 180
ttctcaggtt catataatcc aaacggaaac tcctacttgt ccgtttacgg ttggtcccgt 240
aaccctttga tcgaatatta cattgttgaa aacttcggta cttataatcc ttccaccgga 300
gccactaagc tgggtgaagt cacctgtgat ggttcagttt atgatatata tagaacacaa 360
cgtgttaatc aaccatccat catcggtaca gctacatttt accaatattg gtctgttagg 420
cgtaacaagc gtagctccgg ttccgtcaac accgcatgtc atttcaatgc ttgggcccaa 480
ttcggactga ccttaggtac tatggattat caaatcgtcg ctgtcgaagg atacttctcc 540
tctggatctg cctctatctg tgtctca 567
<210> 3
<211> 189
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser Tyr
1 5 10 15
Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Leu Gly Pro Gly Gly
20 25 30
Cys Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly Lys
35 40 45
Gly Trp Asn Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly Ser
50 55 60
Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg
65 70 75 80
Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn
85 90 95
Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Cys Asp Gly Ser
100 105 110
Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile Ile
115 120 125
Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn Lys Arg
130 135 140
Ser Ser Gly Ser Val Asn Thr Ala Cys His Phe Asn Ala Trp Ala Gln
145 150 155 160
Phe Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val Glu
165 170 175
Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Cys Val Ser
180 185
<210> 4
<211> 567
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
actattcaac ctggaactgg atacaataac ggttatttct actcttactg gaacgatgga 60
catggaggtg tcacatacac tttgggtcca ggtggatgtt tctcagttaa ttggtctaac 120
tcaggaaatt tcgtcggagg taaaggatgg aacccaggaa ctaagaataa ggtcattaac 180
ttctcaggtt catataatcc aaacggaaac tcctacttgt ccgtttacgg ttggtcccgt 240
aaccctttga tcgaatatta cattgttgaa aacttcggta cttataatcc ttccaccgga 300
gccactaagc tgggtgaagt cacctgtgat ggttcagttt atgatatata tagaacacaa 360
cgtgttaatc aaccatccat catcggtaca gctacatttt accaatattg gtctgttagg 420
cgtaacaagc gtagctccgg ttccgtcaac accgcatgtc atttcaatgc ttgggcccaa 480
ttcggactga ccttaggtac tatggattat caaatcgtcg ctgtcgaagg atacttctcc 540
tctggatctg cctctatctg tgtctca 567
<210> 5
<211> 189
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser Tyr
1 5 10 15
Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro Gly Gly
20 25 30
Cys Phe Ser Val Ile Trp Ser Asn Ser Gly Asn Phe Val Gly Gly Lys
35 40 45
Gly Trp Asn Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly Ser
50 55 60
Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg
65 70 75 80
Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn
85 90 95
Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Cys Asp Gly Ser
100 105 110
Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile Ile
115 120 125
Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn Lys Arg
130 135 140
Ser Ser Gly Ser Val Asn Thr Ala Cys His Phe Asn Ala Trp Ala Gln
145 150 155 160
Phe Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val Glu
165 170 175
Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Cys Val Ser
180 185
<210> 6
<211> 567
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
actattcaac ctggaactgg atacaataac ggttatttct actcttactg gaacgatgga 60
catggaggtg tcacatacac taacggtcca ggtggatgtt tctcagttat ctggtctaac 120
tcaggaaatt tcgtcggagg taaaggatgg aacccaggaa ctaagaataa ggtcattaac 180
ttctcaggtt catataatcc aaacggaaac tcctacttgt ccgtttacgg ttggtcccgt 240
aaccctttga tcgaatatta cattgttgaa aacttcggta cttataatcc ttccaccgga 300
gccactaagc tgggtgaagt cacctgtgat ggttcagttt atgatatata tagaacacaa 360
cgtgttaatc aaccatccat catcggtaca gctacatttt accaatattg gtctgttagg 420
cgtaacaagc gtagctccgg ttccgtcaac accgcatgtc atttcaatgc ttgggcccaa 480
ttcggactga ccttaggtac tatggattat caaatcgtcg ctgtcgaagg atacttctcc 540
tctggatctg cctctatctg tgtctca 567
<210> 7
<211> 189
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser Tyr
1 5 10 15
Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Leu Gly Pro Gly Gly
20 25 30
Cys Phe Ser Val Ile Trp Ser Asn Ser Gly Asn Phe Val Gly Gly Lys
35 40 45
Gly Trp Asn Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly Ser
50 55 60
Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg
65 70 75 80
Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn
85 90 95
Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Cys Asp Gly Ser
100 105 110
Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile Ile
115 120 125
Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn Lys Arg
130 135 140
Ser Ser Gly Ser Val Asn Thr Ala Cys His Phe Asn Ala Trp Ala Gln
145 150 155 160
Phe Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val Glu
165 170 175
Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Cys Val Ser
180 185
<210> 8
<211> 567
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
actattcaac ctggaactgg atacaataac ggttatttct actcttactg gaacgatgga 60
catggaggtg tcacatacac tttgggtcca ggtggatgtt tctcagttat ctggtctaac 120
tcaggaaatt tcgtcggagg taaaggatgg aacccaggaa ctaagaataa ggtcattaac 180
ttctcaggtt catataatcc aaacggaaac tcctacttgt ccgtttacgg ttggtcccgt 240
aaccctttga tcgaatatta cattgttgaa aacttcggta cttataatcc ttccaccgga 300
gccactaagc tgggtgaagt cacctgtgat ggttcagttt atgatatata tagaacacaa 360
cgtgttaatc aaccatccat catcggtaca gctacatttt accaatattg gtctgttagg 420
cgtaacaagc gtagctccgg ttccgtcaac accgcatgtc atttcaatgc ttgggcccaa 480
ttcggactga ccttaggtac tatggattat caaatcgtcg ctgtcgaagg atacttctcc 540
tctggatctg cctctatctg tgtctca 567
Claims (5)
1. A xylanase mutant characterized in that the xylanase mutant has an amino acid sequence of SEQ ID NO:1, mutation of amino acid 28 from Asn to Leu.
2. A xylanase mutant characterized in that the xylanase mutant has an amino acid sequence of SEQ ID NO:1, the 28 th amino acid of the xylanase is mutated from Asn to Leu, and the 37 th amino acid is mutated from Asn to Ile.
3. A DNA molecule encoding the xylanase mutant of claim 1.
4. A recombinant expression vector carrying the DNA molecule of claim 3.
5. A host cell carrying the recombinant expression vector of claim 4; the host cell is Pichia pastoris (Pichia pastoris), Trichoderma reesei (Trichoderma reesei) or Aspergillus niger (Aspergillus niger).
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| CN109402091B (en) * | 2017-08-18 | 2022-02-11 | 潍坊康地恩生物科技有限公司 | Xylanase mutants |
| CN110607292B (en) * | 2018-06-14 | 2022-05-31 | 青岛蔚蓝生物集团有限公司 | High specific activity xylanase mutant |
| WO2023014081A1 (en) * | 2021-08-06 | 2023-02-09 | 씨제이제일제당 (주) | Variant polypeptide having xylanase activity |
| CN113528486B (en) * | 2021-08-19 | 2023-10-20 | 广东众汇食品科技有限公司 | Method for improving xylanase thermal stability by introducing disulfide bond |
| CN114457059B (en) * | 2022-01-21 | 2024-03-19 | 青岛尚德生物技术有限公司 | Xylanase-containing enzyme preparation and application thereof in production of xylooligosaccharide |
| CN117384888A (en) * | 2022-07-12 | 2024-01-12 | 青岛蔚蓝生物集团有限公司 | High-temperature-resistant xylanase mutant and application thereof |
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