Search

CN-122012550-A - Mutant gene of tuckahoe endo-chitinase and preparation method and application of recombinant protein thereof

CN122012550ACN 122012550 ACN122012550 ACN 122012550ACN-122012550-A

Abstract

The invention provides recombinant bacteria for highly expressing tuckahoe chitinase mutant and a preparation method of protein thereof. The method comprises the steps of integrating an optimally designed tuckahoe chitinase target gene mutant SEQ ID NO.1 into a pichia pastoris expression vector, and then converting the tuckahoe chitinase target gene mutant SEQ ID NO.1 into a pichia pastoris expression strain. Recombinant bacteria expressing active tuckahoe chitinase are obtained through YPD plate screening with high Zeocin resistance, and high expression pichia pastoris transformant is further selected from high Zeocin resistance transformant. Performing amplification culture under shake flask condition, wherein the expression level of recombinant tuckahoe chitinase reaches 214 mg/L, and protein electrophoresis shows single band. The high-efficiency purification of the enzyme is realized by adopting ammonia sulfate fractional precipitation and ion exchange technology. The recombinant enzyme mutant can efficiently hydrolyze colloid chitin to generate chitin oligosaccharide, and has important potential value in the fields of functional oligosaccharide preparation, bioenergy development, medical research, feed industry, environmental protection, agricultural application and the like.

Inventors

  • LI HONGBO

Assignees

  • 湖南医药学院

Dates

Publication Date
20260512
Application Date
20260121

Claims (10)

  1. 1. A high-expression tuckahoe endochitinase mutant gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO.1 or the amino acid sequence encoded by the gene is shown as SEQ ID NO. 2.
  2. 2. A biological material comprising the gene of claim 1, wherein the biological material comprises a recombinant vector, an expression cassette, or a recombinant bacterium.
  3. 3. The biomaterial according to claim 2, wherein the recombinant expression vector consists of an empty vector and the gene of interest according to claim 1 inserted into the empty vector, and the empty vector is a constitutive expression vector.
  4. 4. The biomaterial of claim 3, wherein the constitutive expression vector is pGAPZB.
  5. 5. The preparation method of the recombinant tuckahoe chitinase is characterized by comprising the following steps of: 1) Constructing the gene of claim 1 into a constitutive expression vector to obtain a recombinant expression vector; 2) Transforming the recombinant expression vector obtained in the step 1) into a pichia pastoris host cell to obtain recombinant bacteria; 3) Fermenting the recombinant bacterium obtained in the step 2), wherein the obtained supernatant contains recombinant tuckahoe chitinase.
  6. 6. The method of claim 5, wherein the fermentation in step 3) is further supplemented with glycerol and potassium phosphate buffer.
  7. 7. The method according to claim 5, further comprising a protein purification step of precipitating the target protein with 90% saturated ammonium sulfate, desalting by dialysis with 10 mM Tris-HCl buffer at pH 8.3, equilibration of the DEAE column with 10 mM Tris-HCl buffer at pH 8.3, loading the dialyzed and centrifuged filtrate, washing the column with 20 mM sodium dihydrogen phosphate buffer at pH 6.0, and eluting the target protein with 20 mM sodium dihydrogen phosphate buffer at pH 6.0 at 200 mM NaCl to obtain a high purity product.
  8. 8. The method of any one of claims 5-7, wherein the constitutive expression vector is pGAPZB and the pichia host is strain X33.
  9. 9. Use of the tuckahoe chitinase mutant gene of claim 1 in the preparation of recombinant tuckahoe chitinase.
  10. 10. The recombinant enzyme prepared by the preparation method of claim 5 is applied to the hydrolysis of polysaccharide or the preparation of functional oligochitosan, biological energy, feed, medicine, environmental protection and agricultural fields.

Description

Mutant gene of tuckahoe endo-chitinase and preparation method and application of recombinant protein thereof Technical Field The invention belongs to the technical field of bioengineering, and particularly discloses a synthesis construction method of a chitinase mutant gene based on a poria cocos source, recombinant expression and secretion of the gene in pichia pastoris, an efficient purification process of the obtained recombinant protein, and systematic analysis of enzymatic properties of the recombinant protein. Background Chitin is a linear polymer of N-acetylglucosamine linked by beta-1, 4 glycosidic bonds, and is widely distributed in crustacean shells, fungal cell walls and insect exoskeletons as the second largest renewable resource with reserves next to cellulose in nature. The chitin endonuclease (Endochitinase, EC 3.2.1.14) can specifically hydrolyze beta-1, 4 glycosidic bonds in chitin molecules, and has important application value in the fields of biodegradation, industrial production and biological control. The enzyme is widely existing in organisms such as microorganisms, plants, insects, arthropods and the like. The current research focus is on the use of metagenomic technology to dig novel extreme condition resistant endonuclease genes from extreme environments such as deep sea, polar regions, etc. The main application value of the chitinase is shown in (1) the antifungal agent can effectively inhibit the growth of pathogenic fungi (such as botrytis cinerea and Pyricularia oryzae) by degrading chitin components in the cell walls of the pathogenic fungi. For example, endoenzymes produced by Trichoderma harzianum (Trichoderma harzianum) have been used in the development of biopesticides. (2) Promoting plant immunity, inducing plant to produce systemic acquired resistance, and enhancing disease resistance of crops. (3) The chitosan oligosaccharide is prepared by degrading chitin with enzyme method, and the chitosan oligosaccharide has various biological activities such as antibacterial, antioxidant and immunoregulation. (4) The chitosan oligosaccharide can promote tissue repair and has application potential in the development of medical dressing. (5) Food processing, namely, the method is applied to shelling crustaceans and extracting flavor substances. Despite the remarkable potential of the chitinase application, the industrialization process of the chitinase still has a plurality of challenges (1) the expression system has low efficiency, inclusion bodies are easy to form in a prokaryotic expression system (such as escherichia coli), the enzyme activity loss is remarkable after renaturation, and the stability of the enzyme can be influenced due to the difference of protein modification modes in a eukaryotic expression system (such as yeast and insect cells). (2) The fermentation process is difficult to optimize, enzyme-producing strains are easy to be subjected to feedback inhibition by carbon and nitrogen sources, and parameters such as dissolved oxygen, pH and the like are complicated to control in the fermentation process, so that the yield is unstable after large-scale amplification. (3) The downstream purification cost is high, the content of the impurity protein in the crude enzyme solution is high, the crude enzyme solution needs to be purified by multi-step chromatography (such as ion exchange chromatography and affinity chromatography), and the recovery rate is always lower than 50 percent. In addition, enzyme preparations have a short shelf life and lyophilization or immobilization techniques further increase production costs. The natural chitinase has the common problems of low yield, difficult extraction, unstable enzyme activity and the like. The existing recombinant expression systems (such as escherichia coli and saccharomyces cerevisiae) have the defects of low secretion efficiency, complex products, high purification cost and the like. The traditional fermentation process is difficult to realize high-density culture and large-scale production. The Pichia pastoris (Pichia pastoris) methanol induction type expression system has the remarkable limitations that the expression of the Pichia pastoris is strictly dependent on methanol with toxicity as an inducer, so that the production safety risk is increased, the application of the Pichia pastoris in the field of medicines/foods is limited, a staged culture strategy (such as glycerol growth followed by methanol induction) is adopted, the operation flow is complex, the dissolved oxygen, pH and methanol concentration gradient are required to be precisely controlled, the industrial amplification difficulty is high, the methanol metabolism depends on a peroxisome approach, the bacterial growth is stagnated in the induction stage, more energy is used for the expression of target proteins rather than biomass accumulation, and the integral fermentation efficiency is limited. In contrast, pichia pastoris constitutive expression systems