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CN-122012467-A - Xylanase mutant delta Xyn, coding gene, vector, engineering bacteria, preparation method and application

CN122012467ACN 122012467 ACN122012467 ACN 122012467ACN-122012467-A

Abstract

The invention discloses a xylanase mutant delta Xyn, a coding gene, a vector, engineering bacteria, a preparation method and application thereof, wherein the xylanase mutant delta Xyn is obtained by mutating amino acids at 56 th, 152 th and 204 th positions of wild xylanase Xyn from valine, asparagine and lysine into isoleucine, aspartic acid and glutamic acid respectively, and the amino acid sequence and the nucleotide sequence of the xylanase mutant delta Xyn are SEQ ID NO.1 and SEQ ID NO.2 respectively. The specific enzyme activity of the xylanase mutant delta Xyn is 9951.5U/mL at the pH of 10.0 and 60 ℃ and is 2.2 times of that of the wild enzyme Xyn, the heat preservation is carried out at 70 ℃ for 40. 40 min, the residual enzyme activity of the xylanase mutant delta Xyn is 8312U/mL and is 4.1 times of that of the wild enzyme Xyn, and the xylanase mutant delta Xyn has important application prospect under alkaline conditions.

Inventors

  • LI GUOGAO
  • LI HUAN
  • SONG JUAN

Assignees

  • 湖南利尔康生物股份有限公司

Dates

Publication Date
20260512
Application Date
20251229

Claims (10)

  1. 1. A xylanase mutant delta Xyn is characterized in that, The amino acids at positions 56, 152 and 204 of the wild-type xylanase Xyn were mutated from valine, asparagine and lysine to isoleucine, aspartic acid and glutamic acid, respectively.
  2. 2. The xylanase mutant ΔXyn according to claim 1, characterised in that, The amino acid sequence is shown as SEQ ID NO. 1.
  3. 3. The xylanase mutant Δxyn encoding gene Δxyn according to any one of claims 1-2.
  4. 4. The coding gene Deltaxyn according to claim 3, characterized in that, The nucleotide sequence is shown as SEQ ID NO. 2.
  5. 5. A vector comprising the coding gene Deltaxyn according to any one of claims 3 to 4.
  6. 6. A host cell comprising the coding gene Δxyn according to any one of claims 3-4 or the vector according to claim 5.
  7. 7. An engineering bacterium comprising the coding gene Δxyn according to any one of claims 3 to 4 or the vector according to claim 5.
  8. 8. A process for producing a xylanase mutant DeltaXyn as defined in any one of claim 1-2, characterized in that, The nucleotide sequence shown in SEQ ID NO.2 takes a plasmid capable of expressing the enzyme as an expression vector, and takes a strain capable of expressing the enzyme as an expression host, so that the efficient expression of the mutant shown in SEQ ID NO.1 is realized.
  9. 9. The method for producing a xylanase mutant ΔXyn according to claim 8, wherein, The expression vector is preferably ppiczαa; and/or the expression host is preferably pichia pastoris.
  10. 10. Use of the coding gene Δxyn according to any one of claims 3-4 and the enzyme encoded thereby in cotton and hemp processing, food processing, feed processing and pulping and papermaking.

Description

Xylanase mutant delta Xyn, coding gene, vector, engineering bacteria, preparation method and application Technical Field The invention belongs to the technical field of molecular biology, and in particular relates to a xylanase mutant delta Xyn, a coding gene, a vector, engineering bacteria, a preparation method and application. Background Xylanase (xylanases) is a hydrolase capable of catalyzing hydrolysis of xylan 1, 4-beta-D-xyloside bonds, and is widely applied to the fields of papermaking, food processing, feed, biological energy sources, environmental protection and the like. In the prior art, the use of xylanases in industry has faced multiple challenges: (1) The heat stability is insufficient, most wild xylanases are easy to inactivate under high temperature conditions (such as above 60 ℃), and the enzyme activity residual rate of partial xylanases is less than 50% after the xylanase is treated at 70 ℃ for 30 min, so that the requirements of high-temperature industrial processes (such as biological bleaching and high-temperature fermentation) are difficult to meet; (2) The alkali stability is poor, the catalytic efficiency of wild xylanase is obviously reduced in an alkaline environment (pH 9-11), and the specific activity of some xylanases under the alkaline condition is only 30-50% under the neutral condition, so that the application of the xylanase in the high alkaline scene such as biological bleaching in the paper industry is limited; (3) The heat resistance and alkali resistance are difficult to cooperatively optimize, in the existing research, the transformation of the heat stability or the alkali stability is mostly in a single direction, and a systematic strategy for improving the performances of the two is lacked, part of mutants obviously improve the heat resistance through site-directed mutagenesis (such as N165H, H L), but the performance of the mutants under an alkaline condition is not clear, and the activity of other mutants is improved in an alkaline environment, but the heat stability is not synchronously optimized. Up to now, no report has been seen on optimizing both the thermostability and the alkaline stability of xylanases. Disclosure of Invention In view of the above, the invention provides a xylanase mutant delta Xyn, a coding gene, a vector, engineering bacteria, a preparation method and application. The invention designs potential mutant xylanase based on wild xylanase sequences in a database through structural biology and energy calculation, obtains xylanase mutant genes delta xyn through site-directed mutagenesis according to wild gene sequences, adopts a eukaryotic expression system to carry out efficient expression, obtains xylanase mutants with improved heat stability, alkali stability and specific activity, can reduce production cost, expands application range and enhances application potential in industrial scenes such as papermaking, food, feed and the like. In order to achieve the above purpose, the technical scheme of the invention is as follows: The invention provides a xylanase mutant delta Xyn, which mutates amino acids at 56 th, 152 th and 204 th positions of wild xylanase Xyn into isoleucine, aspartic acid and glutamic acid from valine, asparagine and lysine respectively. The invention utilizes a combined site-directed mutagenesis technology to carry out molecular transformation on wild xylanase, specifically designs mutants through a molecular dynamics simulation combination calculation program FoldX, screens out site mutations such as V56I, N152D, K E and the like, and remarkably improves enzyme thermal stability, alkali stability and other enzymatic properties of the enzyme. In detail, in the technical scheme, valine at position 56 is mutated into isoleucine, so that the side chain of the isoleucine is longer and has stronger hydrophobicity, the beta-sheet barrel interaction of the hydrophobic region of the core of the enzyme molecule can be enhanced, the probability of conformational unfolding at high temperature is reduced, and the thermal stability of mutant xylanase is obviously improved. In detail, in the technical scheme, the 152 th asparagine is mutated into aspartic acid, negative charge is introduced into the surface, so that hydrophilicity under alkaline conditions can be enhanced, enzyme protein aggregation is reduced, and meanwhile, the Asp side chain carboxyl can form an additional hydrogen bond with an adjacent residue, so that the spatial conformational stability is enhanced, and the heat resistance of mutant xylanase is improved. In detail, in the technical scheme, the 204 th lysine is mutated into glutamic acid, so that the surface negative charge density is increased, the stability of mutant xylanase in an alkaline environment is enhanced, and a Glu204 side chain can form a salt bridge with an adjacent Arg180, so that the conformation at a high temperature is further stabilized. In the technical scheme, the combined mutation of three key