CN-121406605-B - KL-CCF-MHETase mutant and related biological materials and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly discloses a KL-CCF-MHETase mutant and related biological materials and application thereof. The amino acid sequence of the KL-CCF-MHETase mutant provided by the invention is shown as SEQ ID No. 3. Compared with the KL-MHETase mutant, the degradation activity of the BHET of the KL-CCF-MHETase mutant is obviously improved, and the BHET can be rapidly converted into a target product TPA. Meanwhile, the invention provides a PET degradation method, which is characterized in that PET is subjected to chemical depolymerization to prepare an intermediate and then is matched with KL-MHETase mutant to be efficiently converted, and under the strategy of a crude substrate-crude enzyme, the efficient degradation of high-crystallinity PET and the green recovery of TPA are realized. The invention can be applied to the fields of degrading PET or BHET, preparing degradation agent of PET or BHET, recovering TPA and the like.

Inventors

• YOU SHENGPING
• ZHANG LIN
• GUO YIWEI
• WANG MENGFAN
• QI WAI

Assignees

• 源天生物科技(天津)有限公司

Dates

Publication Date

20260512

Application Date

20251230

Claims (9)

1. 1. A KL-CCF-MHETase mutant is characterized in that the amino acid sequence is shown as SEQ ID No. 3.
2. 2. A gene encoding the KL-CCF-MHETase mutant according to claim 1, characterized in that its nucleotide sequence is shown in SEQ ID No. 4.
3. 3. A recombinant plasmid comprising the coding gene according to claim 2.
4. 4. A recombinant strain, characterized in that: the recombinant strain comprises the recombinant plasmid of claim 3.
5. 5. The recombinant strain according to claim 4, wherein the host cell of the recombinant strain is Vibrio natrii Vibrio natriegens.
6. 6. Use of the KL-CCF-MHETase mutant according to claim 1, the encoding gene according to claim 2, the recombinant plasmid according to claim 3 or the recombinant strain according to claim 4 or 5 for degrading PET degradation products, the degradation agent for producing PET degradation products, or recovering terephthalic acid, wherein the PET degradation products are MHET.
7. 7. A PET degradation method is characterized by comprising the following steps: S1, under the catalysis of bicarbonate, carrying out glycolysis reaction on PET to obtain a crude BHET product; s2, mixing the lysate of the recombinant strain according to claim 4 or 5 and FAST-PETase with the BHET crude product, and carrying out hydrolysis reaction at 40-50 ℃ to obtain PET degradation products.
8. 8. The method for degrading PET according to claim 7, wherein the preparation method of the lysate of the recombinant strain comprises the following steps: step one, inoculating the recombinant strain in claim 4 or 5 into an LB3 culture medium for expansion culture to obtain recombinant strain seed solution; Inoculating the recombinant strain seed solution into LBv-sodium formate culture medium, culturing until OD 600 reaches 0.8-1.0, and adding isopropyl-beta-D-thiogalactoside for induction culture; And thirdly, after the induction culture is finished, collecting thalli, re-suspending the thalli, and crushing cells to obtain lysate of the recombinant strain.
9. 9. A PET degradation method according to claim 8, wherein in the first step, the conditions of the expansion culture are a culture temperature of 35℃to 39℃and a rotation speed of 180rpm to 240rpm for 10 hours to 14 hours, and/or In the second step, the inoculation amount of the recombinant strain seed solution is 1% -5%, and/or In the second step, the LBv-sodium formate culture medium is LBv culture medium added with 20g/L to 35g/L sodium formate, and/or In step two, the LBv sodium formate medium comprises at least one of a LBv sodium formate medium which is not sterilized and contains no antibiotics or a LBv sodium formate medium which is not sterilized and contains antibiotics, and/or In the second step, the conditions of the induction culture are that the culture temperature is 14-18 ℃, the rotating speed is 150pm-170rpm, and the culture time is 26-32 h.

Description

KL-CCF-MHETase mutant and related biological materials and application thereof Technical Field The invention belongs to the technical field of enzyme engineering, relates to PET degrading enzyme, and in particular relates to a KL-CCF-MHETase mutant, a related biological material and application thereof. Background The increasing waste polyethylene terephthalate (polyethylene terephthalate, PET) poses serious threat to the ecological environment, and efficient recovery and high-value utilization of waste PET have become the current research focus. At present, the recovery method of PET mainly comprises a mechanical method, a chemical method and a biological enzyme method. Among them, the bio-enzyme method is receiving attention by virtue of its advantages of mild reaction conditions, high purity of the product, etc. However, the method still faces the problems of low enzymolysis efficiency, limited degradation capability to high-crystallinity PET and the like, and formate-containing wastewater generated in the product purification process also increases the treatment cost. The method comprises the steps of (1) waste PET generally has higher crystallinity, so that biological enzyme is difficult to effectively contact and act, degradation efficiency is low, (2) the conventional PET degrading enzyme is mostly intracellular enzyme, the multi-step operation such as aseptic fermentation, thallus breakage, protein purification and the like is needed, the whole process is high in energy consumption, long in period and low in equipment utilization rate, and (3) terephthalic acid (TPA) in an enzymolysis product is recovered after acid precipitation, and the concentration of a byproduct Ethylene Glycol (EG) in a reaction system is low, so that separation and purification are difficult. In the existing method, the waste water is often converted into formic acid or formate through electrocatalytic oxidation, but salt-containing waste water is still generated, so that the burden and the environmental risk of subsequent treatment are increased. The above problems limit the industrial application of the biological recovery process. Disclosure of Invention The invention provides a KL-CCF-MHETase mutant capable of obviously improving the degradation activity of BHET and related biological materials and application thereof by modifying the KL-MHETase mutant. When the KL-CCF-MHETase mutant provided by the invention degrades BHET, the yield of a target product TPA is obviously higher than that of the KL-MHETase mutant. Furthermore, the invention provides biological materials such as the coding genes, the recombinant plasmids, the recombinant strains and the like, which support industrial application. When the recombinant strain is constructed, V.natriegins are used as host cells, so that formate wastewater treatment and resource utilization are realized. The invention also provides a PET degradation method, which realizes high-efficiency degradation of high-crystallinity PET and TPA green recovery through a chemical depolymerization and enzymolysis cooperative process, adopts a strategy of 'crude substrate-crude enzyme' in the enzymolysis stage, adopts a non-sterile culture mode in the fermentation stage, greatly reduces cost, energy consumption and operation difficulty, and improves process economy and scale potential. In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme: in a first aspect, the invention provides a KL-CCF-MHETase mutant, which is obtained by site-directed mutagenesis of valine at position 62, phenylalanine at position 78 and methionine at position 161 in the amino acid sequence of the KL-MHETase mutant into cysteine, and the amino acid sequence of the KL-CCF-MHETase mutant is shown as SEQ ID No. 3. Compared with the KL-MHETase mutant, the KL-CCF-MHETase mutant provided by the invention has obviously improved BHET degradation activity and can rapidly convert BHET into a target product TPA. In a second aspect, the invention provides a coding gene of the KL-CCF-MHETase mutant, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 4. In a third aspect, the present invention provides a recombinant plasmid comprising the coding gene provided in the second aspect. In a fourth aspect, the present invention provides a recombinant strain comprising the recombinant plasmid provided in the third aspect. Preferably, the host cell of the recombinant strain is vibrio natrii Vibrio natriegens. The formate-containing wastewater generated in the PET degradation process is used as a carbon source to be reused in the fermentation process of vibrio natrii so as to produce PET degrading enzyme. The method not only avoids the terminal treatment cost of formate-containing wastewater, but also realizes the resource utilization of wastes in a process system. The coding gene of the KL-CCF-MHETase mutant, the recombinant plasmid, the recombinant strain and other relat