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CN-122012549-A - Recombinant poria cocos chitin endonuclease and gene, preparation method and application thereof

CN122012549ACN 122012549 ACN122012549 ACN 122012549ACN-122012549-A

Abstract

The invention discloses a recombinant tuckahoe chitinase and a gene, a preparation method and application thereof, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. The nucleotide sequence shown in SEQ ID NO.1 can realize high-level recombinant secretion expression of target protein in pichia pastoris host bacteria by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a vector. Under the condition of shaking flask fermentation, the average secretion expression quantity can reach 300.3 mg/L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloid chitin to generate chitosan oligosaccharide (Chitooligosaccharides, COS), but also can directly degrade chitin-containing raw materials such as shrimps and crabs to generate COS. In addition, the enzyme also shows the activity of hydrolyzing cellulose, pectin, agar and other polysaccharide, and has important potential value in the fields of functional oligochitosan preparation, biological energy production, medicine development, feed, 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 tuckahoe chitinase 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 expression vector, an expression cassette, or a recombinant bacterium.
  3. 3. The biomaterial of claim 2, wherein the recombinant expression vector consists of an empty vector and the gene of claim 1 inserted into the empty vector, the empty vector being a secretory expression vector.
  4. 4. The biomaterial of claim 3, wherein the secretory expression vector is any one of the group consisting of ppiczα A, pPIC K and pHIL-S1.
  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 secretion type 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 process according to claim 5, wherein the fermentation in step 3) is further supplemented with methanol.
  7. 7. The method according to claim 5, further comprising a step of purifying the supernatant obtained in the step 4) by using a nickel affinity column, sequentially equilibration the column with equilibration buffer, loading the column, rinsing the column with a pH 8.0 buffer containing 10 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole, and finally eluting the target protein with a pH 6.0 buffer containing 20 mM monosodium phosphate, 100 mM NaCl and 150 mM imidazole.
  8. 8. The method according to any one of claims 5 to 7, wherein the secretory expression vector comprises any one of pPICZ alpha A, pPIC K and pHIL-S1, and the Pichia host strain comprises any one of strains X33, GS115, SMD1168 and KM 71.
  9. 9. The method of claim 8, wherein the secretory expression vector is pPICZ alpha A and the Pichia host is strain X33.
  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

Recombinant poria cocos chitin endonuclease and gene, preparation method and application thereof Technical Field The invention relates to the technical field of bioengineering, in particular to a construction of artificially synthesized tuckahoe source chitinase gene, optimization of a pichia pastoris expression system, high-level secretion expression under shake flask culture condition, efficient purification process and enzymatic characteristic analysis and application thereof. Background Chitin is an important substance in nature, and is an N-acetylglucosamine polymer formed by interconnecting beta-1, 4 glycosidic bonds. It is worth emphasizing that chitin is a secondary renewable resource in nature and is widely found in various biological structures such as crustacean shells, fungal cell walls and insect exoskeletons. The biodegradation process of chitin relies on specific hydrolase-endochitinase (Endochitinase, EC 3.2.1.14). The enzyme generates chitosan oligosaccharide with low polymerization degree by breaking beta-1, 4 glycosidic bond inside chitin chain. Many researches show that the chitosan oligosaccharide has multiple important functions, such as antibacterial property, can inhibit the growth of harmful bacteria, has antioxidation property, is favorable for resisting oxidative stress, has immunoregulation function, and can effectively regulate the immune system of an organism. In view of the functions of the chitosan oligosaccharide, the chitosan oligosaccharide has important application value in a plurality of fields, can assist crops to resist fungal attack in the aspect of agricultural antifungal preparations, can promote wound healing in the field of medical healing promoting materials, and can play a certain role in the field of food processing. Currently, natural strains such as trichoderma and the like are mostly adopted for producing chitinase in industrial production. However, this production method has problems such as low enzyme yield, complicated extraction process, high time and effort consumption, and poor enzyme activity stability, and the enzyme activity is easily affected under different environmental conditions. Currently, in the field of chitin-related research, most chitinases obtained by recombinant expression techniques exhibit a more typical property. In particular, these chitinases generally have only a certain degree of hydrolytic activity towards colloidal chitin, which, during its interaction with colloidal chitin, is able to promote a certain degree of decomposition reactions. However, when the prawn shell element or other type of polysaccharide, the chitinase hardly shows any degradation capability, and the chemical structure of the prawn shell element and other polysaccharide is difficult to be changed substantially. In view of the wide application prospect of chitin in various fields, the effective utilization of chitin in industries such as agriculture, medicine, food and the like is urgently required. Therefore, it is important to actively search and develop a novel high-activity endo-chitinase gene and a preparation method of recombinant protein thereof. The novel high-activity endo-chitinase needs to have the characteristic of high activity, and can efficiently catalyze the hydrolysis reaction of chitin under relatively mild conditions. Meanwhile, the chitosan has the advantage of wide selectivity on polysaccharide substrates, can have good hydrolysis effect on colloidal chitin, and can also degrade prawn chitin and other various polysaccharides. Therefore, the utilization rate of the chitin can be greatly improved, a more solid technical support is provided for the application of the chitin in various fields, and the further development of related industries is promoted. In the process of mass production of chitinase, the existing recombinant expression technology faces a series of complex technical challenges, and the technical challenges are embodied in the following key aspects. First, a prokaryotic expression system typified by E.coli is extremely susceptible to inclusion body formation during expression. After inclusion bodies are formed, the enzyme activity loss rate can be up to 60% -80% through renaturation treatment. Furthermore, prokaryotic expression systems lack eukaryotic post-translational modification functions, rendering their expression products significantly less active than the native enzyme. Thirdly, the cost of the downstream purification link is high, the proportion of the impurity protein in the crude enzyme solution exceeds 80 percent, and a multi-step purification process is needed to meet certain purity standards. Even so, the yield is still lower than 40%, and the active half-life of the enzyme preparation is short, which has adverse effects on practical application and storage. In order to solve the technical problems, there is a need to develop a recombinant expression system which is efficient and suitable for industrial producti