CN110607292B - High specific activity xylanase mutant - Google Patents
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Abstract
The invention relates to the technical field of protein engineering modification, and particularly provides a xylanase mutant with high specific activity. Compared with xylanase Xyn1, the specific activity of the xylanase mutant is generally improved by 16.30-41.29%, wherein the xylanase mutant containing F13Y and S79T two-point mutation has the highest specific activity of 1-13/79 which reaches 1083.3U/mg, and unexpected technical effects are achieved. After fermentation in a 10L fermentation tank, the specific activity of the xylanase mutant Xyn1-13/79 is further improved to 1414.3U/mg, which is 41.4% higher than that of xylanase Xyn 1. The xylanase mutant provided by the invention has improved specific activity, is beneficial to reducing the production cost of xylanase, accelerates the wide application of the xylanase in the field of 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 with improved specific activity.
Background
Xylan is the main component of plant hemicellulose and widely exists in corn cob, bagasse, wheat bran, straw and other crop wastes. Xylanase can degrade xylan into xylo-oligosaccharides and xylose with different lengths, the product has important economic value, the xylanase can fully utilize the available resources to exert the potential application value, and the research of xylanase is also fully paid attention.
Xylanases are glycosyl hydrolases that hydrolyze β -1, 4-linked xylopyranoside 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 (Xyn I and Xyn II) have the best characteristics. The Xyn I has a molecular weight of 19kDa, and has a lower isoelectric point and an optimal pH (pI 5.5, pH 3-4) compared with Xyn II. The Xyn II has a molecular weight of 20kDa, an isoelectric point of 9.0 and an optimal pH of 5.0-5.5.
The xylanase produced by different microorganisms has great difference in structure and property, and some xylanases only contain a single region, namely a catalytic region, and some xylanases simultaneously have a catalytic region and a plurality of non-catalytic regions. The functional regions in the xylanase molecule are connected by a connecting sequence, and the sequence contains more serine residues or proline and threonine residues. The xylanase is widely applied to a plurality of fields such as paper pulp industry, feed industry, food industry, energy industry and the like.
With the wide application of xylanase in the fields of feed industry, food industry, paper industry and the like, further improving the fermentation titer of xylanase to reduce the production and use cost becomes one of the hot spots of xylanase research at present. The expression of target protein by using a foreign protein high-efficiency expression system or the improvement of the structure of xylanase protein to improve the specific activity are effective ways for further improving the application potential of xylanase. In the aspect of high-efficiency expression of xylanase, great progress has been made in recent years, the expression level in recombinant bacteria reaches more than mg/mL level, for example, xylanase gene xynB derived from Streptomyces olivaceus (Streptomyces peridoticus) A1 is efficiently expressed in pichia pastoris, the absolute expression level of xylanase XYNB in a 3L fermentation tank reaches 1.4mg/mL fermentation liquor, the enzyme activity reaches 1200U/mL fermentation liquor, and good materials are provided for further improving the expression level of xylanase and reducing the cost. The specific activity of the enzyme molecule is an important index for measuring the property of the enzyme, and the enzyme with high specific activity has wider prospect in practical application.
Disclosure of Invention
In view of the above, the invention provides a xylanase mutant, which can obtain mutant protein, improve the specific activity of the mutant protein, greatly reduce the production cost of xylanase and is beneficial to the wide application of the 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 the complementary sequence thereof due to the degeneracy of the genetic code.
In some embodiments of the invention, the xylanase mutant is an enzyme protein having the amino acid sequence SEQ ID NO. 1 with a substitution at amino acid position 13.
In a preferred embodiment, the 13 th amino acid of the xylanase mutant is mutated from Phe to Tyr.
In some embodiments of the invention, the xylanase mutant is an enzyme protein having the amino acid sequence SEQ ID NO. 1 with a substitution at amino acid position 79.
In a preferred embodiment, the 79 th amino acid of the xylanase mutant is mutated from Ser to Thr.
In some embodiments of the invention, the xylanase mutant is an enzyme protein having the amino acid sequence SEQ ID NO. 1 in which amino acids 13 and 79 are simultaneously substituted.
In a preferred embodiment, the 13 th amino acid of the xylanase mutant is mutated from Phe to Tyr, and the 79 th amino acid of the xylanase mutant is mutated from Ser to Thr.
In some embodiments of the invention, the xylanase mutant has an amino acid sequence as shown in SEQ ID NO 3 or SEQ ID NO 5 or SEQ ID NO 7.
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 shown as SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8.
The invention also provides a vector having 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, and the specific activity of the xylanase mutant subjected to recombinant expression is obviously improved.
The specific activity of the xylanase mutants Xyn1-13, Xyn1-79 and Xyn1-13/79 is remarkably improved. Compared with xylanase Xyn1, the specific activity of the xylanase mutant is generally improved by 16.30-41.29%, wherein the xylanase mutant containing F13Y and S79T two-point mutation has the highest specific activity of 1-13/79 which reaches 1083.3U/mg, and unexpected technical effects are achieved. After fermentation in a 10L fermentation tank, the specific activity of the xylanase mutant Xyn1-13/79 is further improved to 1414.3U/mg, which is 41.4% higher than that of xylanase Xyn 1. The xylanase mutant has improved specific activity, is favorable for reducing the production cost of xylanase, accelerates the wide application of the xylanase in the field of feed, and has wide market prospect.
Detailed Description
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. DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, second edition, by Singleton et al, John Wiley AND Sons, 1994, AND 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; amino acid sequences are written from left to right in the amino to carboxyl direction. 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 DNA segment 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.
As used herein, "Specific Activity" refers to the number of units of enzyme Activity per weight of protein, generally expressed as U/mg protein.
The experimental materials and reagents used in the specific examples of the present invention are as follows:
bacterial strain and carrier: coli DH5 α, Pichia pastoris GS115, vector pPIC9K, Amp, G418 were purchased from Invitrogen.
Enzyme and kit: PCR enzymes and ligases were purchased from Takara, restriction enzymes from Fermentas, plasmid extraction kits and gel purification recovery kits from Omega, GeneMorph II random mutagenesis kit from Beijing Bomais Biotechnology Ltd.
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 present invention will be described in detail with reference to examples.
Example 1 screening of high specific Activity xylanase mutants
In order to further improve the specific activity of the heat-resistant xylanase Xyn1 (the nucleotide sequence is SEQ ID NO:2, and the coded amino acid sequence is SEQ ID NO: 1), the applicant screens a large number of mutations of the xylanase by a directed evolution technology.
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; adding 30ul of substrate into a 96-well plate, reacting at 37 ℃ for 30min, determining the generated reducing sugar by using a DNS method, adding 150ul of Coomassie brilliant blue solution into the other plate, standing for 10min, determining the protein content by using a Coomassie brilliant blue (Bradford) combination method, and respectively calculating the enzyme activity levels and the protein content of different mutagens. Finally, applicants screened mutation sites that significantly improved the specific activity of xylanase mutant Xyn1 from more than twenty thousand transformants: F13Y, S79T.
The xylanase mutant containing F13Y point mutation is named as Xyn1-13, and the amino acid sequence of the xylanase mutant is SEQ ID NO:3, obtaining a coding nucleotide sequence of SEQ ID NO: 4;
the xylanase mutant containing S79T point mutation is named as Xyn1-79, and the amino acid sequence of the xylanase mutant is SEQ ID NO: and 5, obtaining a coding nucleotide sequence of SEQ ID NO: 6.
the xylanase mutant containing two mutations of F13Y and S79T is named as Xyn1-13/79, and the amino acid sequence of the xylanase mutant is SEQ ID NO: and 7, obtaining a coding nucleotide sequence of SEQ ID NO: 8.
the nucleotide sequence of the xylanase mutant is synthesized by Shanghai Czeri organism.
Example 2 construction of Pichia engineering Strain
The xylanase mutant gene Xyn1-13/79 fragment obtained above is connected with an expression vector pPIC9K through EcoRI and Not I sites to construct an expression vector pPIC9K-Xyn 1-13/79.
Linearizing the mutant expression plasmid by Sal I, transforming Pichia pastoris GS115 by the linearized fragment of the expression plasmid through an electroporation method, respectively screening Pichia pastoris recombinant strains GS115/pPIC9K-Xyn1-13/79 on an MD plate, and then screening multicopy transformants on YPD plates containing different concentrations of geneticin.
The screened positive transformant of the recombinant expression xylanase mutant Xyn1-13/79 is named as Pichia pastoris Xyn1-13/79(Pichia pastoris Xyn1-13/79), and then is transferred into a BMGY culture medium, and is 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 to obtain fermentation supernatant containing xylanase mutants; the xylanase mutant Xyn1-13/79 in the fermentation supernatant is analyzed by SDS-PAGE electrophoresis detection, and the molecular weight of the xylanase mutant is about 20.7 kDa.
Xylanase gene Xyn1, xylanase single-point mutant genes Xyn1-13 and Xyn1-79 are respectively cloned into a Pichia pastoris GS115 host by the same enzyme digestion connection method, and Pichia pastoris engineering bacteria for recombining and expressing the xylanase are constructed and named as Pichia pastoris Xyn1(Pichia pastoris Xyn1), Pichia pastoris Xyn1-13(Pichia pastoris Xyn1-13) and Pichia pastoris Xyn1-79(Pichia pastoris Xyn 1-79). Horizontally fermenting the pichia pastoris engineering bacteria in a shaking bottle, and carrying out shaking culture at 30 ℃ and 250 rpm; adding 0.5% methanol every day to induce expression for 4 days; and (3) centrifuging to remove bacteria to respectively obtain fermentation supernatants containing xylanase Xyn1, Xyn1-13 and Xyn 1-79.
(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 determination 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 to 25ml, mixing, and measuring absorbance 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 enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
(3) Results of enzyme Activity measurement
And respectively detecting the xylanase enzyme activity in the fermentation supernatant of the pichia pastoris engineering bacteria according to the method. The results show that: the enzyme activities of fermentation supernatants of the pichia pastoris Xyn1, the pichia pastoris Xyn1-13, the pichia pastoris Xyn1-79 and the pichia pastoris Xyn1-13/79 are respectively 115U/mL, 107U/mL, 121U/mL and 130U/mL.
(4) Protein content determination method
The Coomassie brilliant blue (Bradford) binding method for determining protein content is a combined method of a colorimetric method and a pigment method. Coomassie Brilliant blue G-250 is reddish brown in acidic solution, turns blue when combined with protein, conforms to beer's law in a certain concentration range of protein, and can be measured colorimetrically at 595 nm. Absorbing a large amount of the active ingredients within 3-5 minutes, and stabilizing for at least 1 hour. Within the range of 10-1000 mug/mL, the light absorption value is in direct proportion to the protein concentration.
According to the volume ratio of the enzyme solution to the Coomassie brilliant blue solution of 1: 5, standing for 10 mm, and measuring the protein content by Coomassie brilliant blue (Bradford) binding method
(5) Protein content measurement results
And respectively detecting the xylanase protein content in the fermentation supernatant of the pichia pastoris engineering bacteria according to the method. The results show that: the protein contents of the fermentation supernatants of the pichia pastoris Xyn1, the pichia pastoris Xyn1-13, the pichia pastoris Xyn1-79 and the pichia pastoris Xyn1-13/79 are respectively 0.15mg/mL, 0.12mg/mL, 0.13mg/mL and 0.12 mg/mL.
(6) Calculation of specific Activity
"Specific Activity" means: the number of units of enzyme activity per weight of protein is generally expressed as U/mg protein. In general, the higher the specific activity of the enzyme, the purer the enzyme.
Specific activity calculation formula: specific activity (U/mg) ═ enzyme activity (U/mL)/protein content (mg/mL)
The specific calculation results are shown in table 1.
TABLE 1 comparison of specific Activity of xylanase mutants
| Mutants | Enzyme activity (U/mL) | Protein content (mg/mL) | Specific activity (U/mg) |
| Xyn1 | 115 | 0.15 | 766.7 |
| Xyn1-13 | 107 | 0.12 | 891.7 |
| Xyn1-79 | 121 | 0.13 | 930.8 |
| Xyn1-13/79 | 130 | 0.12 | 1083.3 |
As can be seen from the results in Table 1, compared with xylanase Xyn1, the specific activities of the xylanase mutants Xyn1-13, Xyn1-79 and Xyn1-13/79 provided by the invention are generally improved by 16.30% -41.29%. The xylanase mutant Xyn1-13/79 containing two point mutations of F13Y and S79T has the highest specific activity which reaches 1083.3U/mg, and is respectively improved by 21.5% and 16.4% compared with xylanase mutants Xyn1-13 and Xyn1-79, thereby achieving unexpected technical effects.
Example 3 fermentation validation
Fermentation of pichia pastoris Xyn1 and pichia pastoris Xyn1-13/79 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 and protein content determination method of embodiment 2 is adopted to detect the enzyme solution, and the result shows that the final fermentation enzyme activity of pichia pastoris Xyn1 for recombinant expression of xylanase Xyn1 is 6020U/ml, and the fermentation enzyme activity of pichia pastoris Xyn1-13/79 for recombinant expression of mutant Xyn1-13/79 is 9954U/ml.
The protein content identification is carried out after the enzyme solution is respectively diluted to 500U/ml, and the results show that the protein content in the pichia pastoris Xyn1 and Xyn1-13/79 fermentation enzyme solution is respectively 0.5mg/ml and 0.35 mg/ml. The results of the specific activity calculations are shown in Table 2.
TABLE 2 comparison of specific Activity of xylanase mutants
| Mutants | Enzyme activity (U/mL) | Protein content (mg/mL) | Specific activity (U/mg) |
| Xyn1 | 500 | 0.5 | 1000 |
| Xyn1-13/79 | 495 | 0.35 | 1414.3 |
The results in Table 2 show that the specific activity of the xylanase Xyn1 introduced with the screened 2 mutation sites F13Y and S79T can be remarkably improved and reaches 1414.3U/mg. The xylanase mutant has improved specific activity, is beneficial to reducing the production cost of xylanase, accelerates the wide application of the xylanase in the field of feed, and has wide market prospect.
Besides Pichia pastoris, the applicant further transforms genes of xylanase Xyn1 and the mutants Xyn1-13, Xyn1-79 and Xyn1-13/79 into Trichoderma reesei (Trichoderma reesei) or Aspergillus niger (Aspergillus niger) host cells respectively to construct recombinant strains for recombinant expression of xylanase Xyn1 and the mutants, respectively detects the enzyme activity and protein content of the xylanase in fermentation liquor after fermentation under the same conditions, and calculates and compares the specific activities. The results show that the specific activities of the obtained mutant proteins Xyn1-13, Xyn1-79 and Xyn1-13/79 are all obviously higher than those of xylanase Xyn1, wherein the specific activity of the mutant Xyn1-13/79 is the highest, 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> high specific activity xylanase mutant
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 189
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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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 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> 2
<211> 567
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<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 Tyr 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 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 ggttattact 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> 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 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 Thr 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 tctcagttaa ttggtctaac 120
tcaggaaatt tcgtcggagg taaaggatgg aacccaggaa ctaagaataa ggtcattaac 180
ttctcaggtt catataatcc aaacggaaac tcctacttgt ccgtttacgg ttggacccgt 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 Tyr 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 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 Thr 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 ggttattact actcttactg gaacgatgga 60
catggaggtg tcacatacac taacggtcca ggtggatgtt tctcagttaa ttggtctaac 120
tcaggaaatt tcgtcggagg taaaggatgg aacccaggaa ctaagaataa ggtcattaac 180
ttctcaggtt catataatcc aaacggaaac tcctacttgt ccgtttacgg ttggacccgt 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 is characterized in that the 13 th amino acid of the xylanase with an amino acid sequence of SEQ ID NO. 1 is mutated from Phe to Tyr.
2. A xylanase mutant is characterized in that the 13 th amino acid of the xylanase with an amino acid sequence of SEQ ID NO. 1 is mutated from Phe to Tyr, and the 79 th amino acid is mutated from Ser to Thr.
3. A DNA molecule encoding a xylanase mutant according to any one of claims 1-2.
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 (A)Pichia pastoris) Trichoderma reesei (T. reesei) ((T. 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 |
| CN112322604B (en) * | 2020-11-03 | 2022-05-17 | 南京工业大学 | Xylanase mutant with high specific enzyme activity and application thereof |
| CN115612682A (en) * | 2021-07-14 | 2023-01-17 | 青岛蔚蓝生物集团有限公司 | Alkaline xylanase mutants |
| CN115704018A (en) * | 2021-08-06 | 2023-02-17 | 青岛蔚蓝生物集团有限公司 | Alkaline xylanase mutant and application thereof |
| 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 |
| CN115029334B (en) * | 2022-07-12 | 2024-04-19 | 潍坊康地恩生物科技有限公司 | High specific activity alkaline xylanase mutant |
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