Search

CN-122012455-A - Tannase mutant delta Tan, coding gene, vector, engineering bacteria, preparation method and application

CN122012455ACN 122012455 ACN122012455 ACN 122012455ACN-122012455-A

Abstract

The invention discloses a tannase mutant delta Tan, a coding gene, a vector, engineering bacteria, a preparation method and application thereof, wherein the tannase mutant delta Tan is obtained by mutating amino acids at 179 th, 217 th and 339 th positions of wild tannase Tan from lysine, glutamine and aspartic acid into leucine, serine and tyrosine respectively, and the amino acid sequence and the nucleotide sequence of the tannase mutant delta Tan are SEQ ID NO.1 and SEQ ID NO.2 respectively. The specific enzyme activity of the tannin enzyme mutant delta Tan is 5268.1U/mL under the conditions of pH 7.0 and 50 ℃ and is 1.89 times of that of the wild enzyme Tan, the residual enzyme activity of the tannin enzyme mutant delta Tan is 4382.9U/mL under the conditions of 60 ℃ and is 2.98 times of that of the wild enzyme Tan, and the tannin enzyme mutant delta Tan has important application prospect under the neutral condition.

Inventors

  • LI HUAN
  • LI GUOGAO
  • LI QI

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 tannin enzyme mutant delta Tan is characterized in that, The amino acids at positions 179, 217 and 339 of wild-type tannase Tan are mutated from lysine, glutamine and aspartic acid to leucine, serine and tyrosine, respectively.
  2. 2. The tannase mutant Δtan according to claim 1, characterized in that, The amino acid sequence is shown as SEQ ID NO. 1.
  3. 3. The tannase mutant Δtan encoding gene Δtan of any of claims 1-2.
  4. 4. The coding gene Deltatan 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 Deltatan according to any of claims 3 to 4.
  6. 6. A host cell comprising a gene Δtan encoding any of claims 3-4 or a vector according to claim 5.
  7. 7. An engineering bacterium comprising the coding gene Δtan of any of claims 3 to 4 or the vector of claim 5.
  8. 8. A process for producing tannase mutant ΔTan according to any one of claim 1 to 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 tannase mutant Δtan according to claim 8, characterized in that, The expression vector is preferably ppiczαa; and/or the expression host is preferably pichia pastoris.
  10. 10. Use of the coding gene Δtan according to any of claims 3-4 and the enzymes encoded thereby in food processing and feed processing.

Description

Tannase mutant delta Tan, coding gene, vector, engineering bacteria, preparation method and application Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a tannin enzyme mutant delta Tan, a coding gene, a vector, engineering bacteria, a preparation method and application. Background Tannase (Tannase, EC 3.1.1.20) is a single Ning Xianji hydrolase, can specifically hydrolyze ester bonds and dephenolization carboxyl bonds in tannin to generate products such as gallic acid and glucose, and has wide application in the fields of food industry, pharmaceutical manufacturing, biodegradation and the like. Tannase is reported to be useful for removing tannin astringency in tea processing, clarifying juice in wine brewing, reducing tannin content in persimmon juice processing, and the like. At present, the tannase is mainly derived from microorganisms such as aspergillus niger (Aspergillus niger) and aspergillus oryzae (Aspergillus oryzae), and the wild type tannase has the defects of low catalytic activity and sensitivity to environmental conditions (such as pH and temperature). In detail, the catalytic activity of tannase is obviously affected by pH environment, and part of wild type tannase has low activity under acidic or neutral conditions (pH 5.0-7.0) and is difficult to meet the requirement of efficient and stable enzyme preparation in industrial production, and in addition, the insufficient thermal stability is another bottleneck affecting the application of tannase, and researches show that most of wild type tannase is easy to inactivate at the temperature higher than 60 ℃ and limit the application of the wild type tannase in a high-temperature processing technology. Up to now, no reports have been made about mutant engineering tannase. Disclosure of Invention In view of the above, the invention provides a tannin enzyme mutant delta Tan, a coding gene, a vector, engineering bacteria, a preparation method and application. The invention designs potential mutation tannase based on wild type tannase sequences in a database through structural biology and energy calculation, obtains tannase mutation gene delta tan through site-directed mutation according to the wild type gene sequences, adopts a eukaryotic expression system to carry out efficient expression, and obtains excellent tannase mutants with improved enzyme activity and heat resistance, thereby reducing production cost and expanding application range, and being suitable for being applied to food and feed processing. In order to achieve the above purpose, the technical scheme of the invention is as follows: The invention provides a tannase mutant delta Tan, which mutates amino acids at 179 th, 217 th and 339 th of wild tannase Tan from lysine, glutamine and aspartic acid into leucine, serine and tyrosine respectively. The invention utilizes a combined site-directed mutagenesis technology to carry out molecular transformation on wild tannase, specifically designs mutants through a molecular dynamics simulation combination calculation program FoldX, screens out site mutations such as K179L, Q217S, D339Y and the like, and remarkably improves the catalytic activity, thermal stability and other enzymatic properties of the enzyme. In detail, in the technical scheme, after the 179 th amino acid is mutated from positively charged hydrophilic residue lysine to nonpolar leucine, unstable charge interaction on the surface is eliminated, hydrophobic core stacking efficiency of the region is enhanced, integral thermal stability of the protein is improved, conformational instability of enzyme caused by charge repulsion at a higher pH is reduced, and the pH tolerance range is widened. In detail, in the technical scheme, glutamine at 217 is mutated into serine with similar structure and stable chemical property, a thermal induced degradation path is eliminated, and the hydroxyl of serine can maintain necessary polar interaction, so that the site is endowed with higher chemical and conformational rigidity, and the thermal stability and the tolerance under a neutral pH environment of the enzyme are obviously enhanced. In detail, in the technical scheme, aspartic acid at 339 is a negatively charged residue, and the existence of the aspartic acid is a key structural factor for limiting the optimal pH value of enzyme to be acidic, firstly, the introduction of tyrosine benzene rings can form new pi-pi accumulation or hydrophobic interaction in the protein, so that the stability of the tertiary structure of the protein is greatly enhanced, the improvement of heat resistance is directly contributed, secondly, the elimination of negative charges directly changes the electrostatic network of an active microenvironment, the dependence of an active center on protons is reduced, and the optimal pH value required by enzyme catalysis is stably expanded from an acidic region to a neutral region. In the technical scheme, the comb