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CN-122012426-A - Laccase and application thereof

CN122012426ACN 122012426 ACN122012426 ACN 122012426ACN-122012426-A

Abstract

The invention discloses laccase and application thereof, and belongs to the field of bioengineering. Compared with the existing high-activity laccase BstCotA, the high-activity laccase Lac-11 obtained by database mining has the advantages that 1. The enzyme activity is obviously improved by about 50% under the condition of 37℃, pH and 5.0, and the cost of industrial enzyme is reduced. The pH stability is stronger, the enzyme activity retention rate is higher within the pH range of 5.0-8.0, and the method is suitable for wider acid-base environments. 3. The thermal stability is better, the stability of the high temperature range of 50-80 ℃ is obviously higher than that of the control enzyme, and the method is suitable for industrial high-temperature processes. 4. The dye decolorization efficiency is higher, the decolorization rate of the dye to the amino black 10B and the crystal violet reaches 31 percent and 37 percent respectively, and the dyeing wastewater can be treated efficiently. 5. The lignin degradation capability is outstanding, and the degradation rates of 1 hour, 2 hours and 24 hours are respectively 22%, 25% and 58%, which is beneficial to pulping bleaching and biomass recycling.

Inventors

  • TANG JIN
  • MA HONGRU
  • LIANG QI
  • LI KE
  • YANG CHUNHONG
  • SUN CHANGHUI

Assignees

  • 杭州努咖智能科技有限公司

Dates

Publication Date
20260512
Application Date
20260415

Claims (8)

  1. 1. A laccase is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
  2. 2. A nucleic acid molecule encoding the laccase of claim 1.
  3. 3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No. 2.
  4. 4. A vector comprising the nucleic acid molecule of claim 2 or 3.
  5. 5. A host cell comprising the vector of claim 4.
  6. 6. Use of the laccase of claim 1, the nucleic acid molecule of claim 2 or 3, the vector of claim 4 or the host cell of claim 5 in lignin degradation.
  7. 7. Use of the laccase of claim 1, the nucleic acid molecule of claim 2 or 3, the vector of claim 4 or the host cell of claim 5 for decolorizing a synthetic dye selected from at least one of amino black 10B, crystal violet, congo red, neutral red, alizarin red, methyl orange.
  8. 8. The use according to claim 7, characterized in that the use is carried out in a dye waste water treatment system.

Description

Laccase and application thereof Technical Field The invention belongs to the field of bioengineering, and particularly relates to laccase and application thereof. Background 1. Overview of laccase Laccase (lacase, EC 1.10.3.2) is a copper-containing polyphenol oxidase, belonging to the family of blue-colored multi-copper oxidases, capable of catalyzing the oxidation of a variety of phenolic and non-phenolic compounds, while reducing molecular oxygen to water. Laccase has the characteristics of broad substrate spectrum, mild catalytic condition, environmental protection, no pollution and the like, and has important application value in the fields of bioremediation, textile industry, pulp bleaching, bioenergy and the like. 2. Problems of the prior art (1) The activity is insufficient, the activity of laccase reported in the prior art is generally low, and the requirement of industrial application is difficult to meet; (2) The stability is poor, the tolerance of most laccase to temperature and pH is limited, and the application range is limited; (3) The production cost is high, the discovery of the laccase with high activity mainly depends on the traditional screening method, and the efficiency is low and the cost is high. In the prior art, huang et al (2021) at Shandong university report a laccase BstCotA derived from termite intestinal Bacillus stratosphericus, which has good heat stability and alkali resistance, and specific activity of 554.1U/mg. This enzyme is representative of the higher activity of the natural laccase, but there is still room for further improvement. And the patent application with the publication number of CN116064605A discloses laccase, and a gene and application thereof, wherein the laccase gene is obtained by carrying out codon optimization on the Cerrena unicolor laccase gene. The laccase gene provided by the invention can realize large-scale production of laccase with high enzyme activity in industry by recombinant expression of Aspergillus niger, and the highest enzyme activity reaches 39U/ml in shake flask culture. Disclosure of Invention The invention provides a novel laccase with activity higher than that reported in the prior literature, and the enzyme activity, pH stability and thermal stability of the laccase are obviously improved. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: Firstly, the invention adopts database mining to quickly obtain the laccase candidate protein with high activity. In order to verify the functions and enzymatic properties, the laccase candidate protein Lac-11 is obtained by fusing the laccase to the downstream of the His tag through gene synthesis, carrying out recombinant expression by using escherichia coli, and carrying out affinity chromatography by using Ni-NTA. The amino acid sequence of the laccase candidate protein is shown as SEQ ID NO. 1. The Protein Language Model (PLM) is used for deep searching of the remote homologous protein, the obtained Lac-11 has 53.19 percent of sequence similarity with BstCotA, and has 71.51 percent and 72.59 percent of sequence similarity with the closest protein sequence of NCBI protein library, thus having good novelty. In another aspect, the invention also provides nucleic acid molecules encoding the laccase enzymes. Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2. In yet another aspect, the invention also provides a vector comprising said nucleic acid molecule. Further, in some embodiments of the invention, the above-described vector is a cloning vector or an expression vector, more preferably a recombinant expression vector for expression in bacteria, such as E.coli (E.coli). Preferably, in some embodiments of the invention, the expression vector is pRSFDuet a 1. In yet another aspect, the invention also provides a host cell comprising the vector. The invention also provides the use of said laccase, said nucleic acid molecule, said vector or said host cell in oxidizing a substrate. The substrate comprises a non-phenolic substrate ABTS (2, 2' -biazo-bis (3-ethylbenzothiazoline-6-sulfonic acid)). The invention explores the enzyme activity of Lac-11 to the ABTS substrate, and the result shows that Lac-11 has better enzyme activity to the ABTS substrate, and the enzyme activity is improved by about 50% under the conditions of 37 ℃ and pH5.0 compared with laccase BstCotA. The invention respectively explores the optimal pH and the temperature of Lac-11, discovers that the optimal pH of Lac-11 is 4.0, the enzyme activity retention rate of Lac-11 is higher than BstCotA after the Lac-11 is maintained for 1 hour in the environment of pH5.0-8.0, the optimal temperature is 70 ℃, and the enzyme activity retention rate of Lac-11 is higher than BstCotA after the Lac-11 is maintained for 1 hour in the environment of 50-80 ℃. The invention also provides application of the laccase, the nucleic acid molecule, the vector or the host