Novel polyurethane degrading enzyme MBX1 and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a novel polyurethane plastic degrading enzyme and application thereof. The amino acid sequence of the novel polyurethane plastic degrading enzyme MBX1 provided by the invention is as shown in SEQ ID No. 1; the sequence of the coding gene is as shown in SEQ ID No.2. Escherichia coli engineering bacteria constructed by using the coding gene sequence can efficiently express the novel polyurethane plastic degrading enzyme. The method specifically comprises the following steps: cloning an MBX1 gene; the preparation method comprises the following steps: constructing a pET28a-MBX1 expression vector; expression, purification and functional verification of MBX1 protein; the degradation rate of the novel polyurethane plastic degrading enzyme to polyether polyol type polyurethane within 8 hours at the temperature of 50 DEG C and the pH value of 7.5 is 98%. The novel polyurethane plastic degrading enzyme can be applied to biological treatment and related research and development of polyurethane plastics, and has a huge application prospect in biodegradation of the polyurethane plastics.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and enzyme engineering, and relates to a novel polyurethane degrading enzyme MBX1 and application thereof. Background Art

[0002] Waste plastic pollution has become a global environmental problem, and the development of technologies that can achieve high-value utilization of plastic waste has become particularly critical. In recent years, microbial enzyme-mediated biodegradation methods have attracted much attention due to their mild reaction conditions, directional controllability, and low carbon emissions, and have become the first choice for solving the environmental pollution crisis in the plastic circular economy. At present, a series of breakthroughs have been made in the biodegradation research of polyester plastics represented by polyethylene terephthalate (PET), realizing food-grade recycling from "bottle" to "bottle". Polyurethane plastic (Polyurethane, PUR), which is the same polyester plastic as PET, is known as the "sixth plastic". It is a polymer compound formed by the condensation of polyisocyanates and polyols and contains multiple repeated carbamate segments (-NHCOO-) on the main chain. It is widely used in thermal insulation building materials, furniture cushions, footwear materials and other scenarios. Compared with PET bioenzymatic recycling technology, the development of PUR waste biodegradation technology is seriously lagging behind. Its research has long been limited by the lack of PUR degradation enzyme resources and the unclear molecular mechanism of enzyme-catalyzed polyurethane depolymerization.

[0003] However, there are currently very limited reports on polyurethane-degrading microorganisms and enzymes, and new polyurethane-degrading microorganisms and enzymes are urgently needed to enhance the industrial application potential of polyurethane biodegradation. Summary of the invention

[0004] In order to solve the defects of the prior art that polyurethane plastic degrading enzymes are less and have poor degradation activity, the present invention provides a novel polyurethane plastic degrading enzyme and application thereof.

[0005] The present invention provides a novel polyurethane plastic degrading enzyme MBX1, which has: 1) the amino acid sequence shown in SEQ ID No.1; or 2) An amino acid sequence as shown in SEQ ID No. 1, with one or more amino acids substituted, deleted and / or added without changing the function, and having the same function as 1).

[0006] The present invention also provides a gene encoding the novel polyurethane plastic degrading enzyme MBX1, and preferably the nucleotide sequence thereof is shown as SEQ ID No.2.

[0007] The present invention also provides a recombinant vector, an expression cassette or a recombinant cell containing the nucleic acid molecule.

[0008] Preferably, the coding gene is inserted between the Nde I and Xho I sites of pET-28a(+).

[0009] In addition, preferably, the expression strain of the genetically engineered bacteria is E. coli BL21(DE3).

[0010] The present invention provides an enzyme preparation for degrading polyurethane plastics, which is prepared from the novel polyurethane plastic degrading enzyme MBX1, specifically prepared from the product expressed by the genetically engineered bacteria.

[0011] The present invention also provides the use of the novel polyurethane plastic degrading enzyme MBX1 or the encoding gene or the recombinant expression vector, the genetically engineered bacteria, and the enzyme preparation in preparing products having catalytic polyurethane degrading enzyme activity, or in degrading polyurethane plastics, or in producing the target product 2,4-TDA or 2,6-TDA.

[0012] Specifically, the polyurethane plastic is selected from polyester polyurethane or polyether polyurethane.

[0013] The present invention also provides a method for degrading polyurethane plastics, which comprises the steps of contacting the novel polyurethane plastic degrading enzyme MBX1 with the polyurethane plastic to be treated, and optionally comprises the step of obtaining the target product 2,4-TDA or 2,6-TDA.

[0014] Specifically, the degradation reaction temperature is 30-60° C.; specifically, the enzyme preparation according to claim 6 is used for degradation.

[0015] The present invention identifies sequences with a sequence homology of more than 50% with the Aes72 enzyme through sequence comparison analysis, chemically synthesizes the obtained 10 sequences to construct recombinant plasmids, screens the obtained 10 genes for expression purification and activity determination, and finally finds the polyurethane degradation enzyme gene mbx1, whose activity is increased by 16 times, and the degradation rate of polyether polyol type polyurethane is 98%. Therefore, it can be used as an efficient degradation enzyme for polyurethane plastics and has great uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The 10 genes obtained were expressed, purified and tested for activity.

[0017] Figure 2 Show plasmid map.

[0018] Figure 3 The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to detect the expression level and purity of the protein.

[0019] Figure 4 Displays the results of the determination of the optimal reaction temperature.

[0020] Figure 5 Shows the results of organic solvent resistance tests.

[0021] Figure 6 The results of the assay showing the activity of catalyzing polyurethane degradation. DETAILED DESCRIPTION

[0022] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0023] 1. Discovery of the polyurethane-degrading enzyme MBX1 Using the enzyme sequence of the reported polyurethane degrading enzyme Aes72 as a template, a multiple sequence alignment (MSA) was performed in a non-redundant protein sequence database. Through sequence alignment analysis, sequences with a sequence homology greater than 50% with the Aes72 enzyme were identified, and the 10 sequences obtained were chemically synthesized to construct recombinant plasmids. The 10 genes obtained were expressed, purified, and their activities were measured. HPLC calculations revealed that the activity of the polyurethane degrading enzyme gene mbx1 was increased by 16 times ( Figure 1 ).

[0024] 2. Construction of engineered bacteria producing polyurethane-degrading enzyme MBX1 The polyurethane degrading enzyme MBX1 gene was codon optimized using Escherichia coli as the host cell to obtain the optimized polyurethane degrading enzyme gene mbx1 (i.e., the target gene). The recombinant plasmid was constructed by chemical synthesis. Its nucleotide sequence is SEQ ID No. 2, and the expressed amino acid sequence is SEQ ID No. 1. The plasmid map is shown in the figure below ( Figure 2 ). The chemically synthesized plasmid was transferred into E. coli BL21 (DE3) competent cells were spread on kanamycin sulfate plate culture medium to obtain positive recombinant expression engineering bacteria.

[0025] 3. Expression and purification of polyurethane-degrading enzyme MBX1 protein Pick some engineered bacterial colonies and inoculate them in 5 mL Luria-Bertani liquid medium (containing Kana), shake and culture at 37°C, 200 rpm for 4-6 h, then inoculate them in 2YT medium (containing Amp) at 1% inoculation, place them on a shaker at 37°C, 200 rpm, culture for about 5 h (OD600 is 0.8-1.0), add 1mM IPTG, and induce at 20°C for 18-20 h. Centrifuge the overnight induced culture at 4°C, 10,000 rpm for 10 min, discard the supernatant, collect the engineered bacterial wet cells, resuspend the cells with protein purification Buffer A, break them by low-temperature ultrasonication for 15-30 min, centrifuge at 4°C, 10,000 rpm for 1 h, remove the cell debris, collect the supernatant, filter with a 0.22 μm filter, and place on ice. Use His Trap HP column for purification. After washing with deionized water for 5 column volumes, balance the column with protein purification buffer A for 5-10 column volumes. Load the filtered supernatant at a flow rate of 5 mL / min, wash the unbound protein with protein purification buffer A, and then wash away the impurities with low-concentration imidazole protein purification buffer B. Elute the target protein with high-concentration imidazole protein purification buffer B and collect the target protein. Use HiPrep desalting column to exchange the buffer of the target protein with protein purification buffer C to remove imidazole. After collecting the eluate, use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to detect the expression level and purity of the protein ( Figure 3 ), the results showed that the purified protein showed a single band on the SDS-PAGE electrophoresis spectrum, with a size of about 35 kDa, which was consistent with the predicted theoretical value of 32.1 kDa. The collected protein was concentrated using an ultrafiltration concentrator with a molecular cutoff of 10 KDa, and the protein concentration was determined by the BCA method. The solution was placed at 4 °C for later use. The new polyurethane plastic degrading enzyme was abbreviated as MBX1.

[0026] Among them, the formula of the 2YT liquid culture medium is: 10g / L yeast extract, 16g / L tryptone, and 5g / L sodium chloride; the formula of the lysis buffer is Buffer A: 50 mM Na2HPO4·12H2O, 100 mM NaCl, 30 mMImidazole, pH=7.5; the formula of the washing buffer is Buffer B: 50 mM Na2HPO4·12H2O, 100 mMNaCl, 300 mM Imidazole, pH=7.5; the formula of the elution buffer is Buffer C: 50 mM Na2HPO4·12H2O, 100 mM NaCl, pH=7.5.

[0027] 4. Determination of the optimal reaction temperature Using 2,4-TDA-DEG ​​(2,4-TDA-DEG ​​is a small molecule analog of polyurethane plastic substrate) as the substrate, high performance liquid chromatography was used to determine at 30°C, 37°C, 50°C, and 60°C. The reaction system was 100 μL, including 10 μL enzyme solution, 10 μL 2,4-TDA-DEG ​​(50 mM), and 80 μL 0.1 M phosphate buffer. The generation rate of 2,4-TDA at different temperatures was recorded at 254 nm. The optimal temperature for MBX1 is 60°C ( Figure 4 ).

[0028] 5. Organic solvent tolerance test Using 2,4-TDA-DEG ​​as substrate, high performance liquid chromatography was used to measure the diethylene glycol under different concentrations (1~10 M). The reaction system was 100 μL, including 10 μL enzyme solution, 10 μL 2,4-TDA-DEG ​​(50 mM), 80 μL 0.1 M phosphate buffer, and the generation rate of 2,4-TDA at different temperatures was recorded at 254 nm. The results showed that MBX1 can tolerate 6MDEG ( Figure 5 ).

[0029] 6. Application of new polyurethane plastic degrading enzyme The polyether polyurethane foam plastics involved in the following examples were purchased from BASF, and 2,4-diaminotoluene (2,4-TDA) and 2,6-diaminotoluene (2,6-TDA) were purchased from Sigma. Phosphate buffer (0.1 M pH 7.5): Accurately weigh 2.28 g of K2HPO4·3H2O and 1.36 g of KH2PO4, add about 100 mL of deionized water and stir to dissolve, respectively weigh 96 mL of 0.1 M K2HPO4 and 4 mL of 0.1 M KH2PO4 and dilute to 100 mL. Add 200 μL of polyether polyurethane foam alcoholysis solution to 0.1 M phosphate buffer with a pH value of 7.5, mix 1.2 μg of amine degrading enzyme MBX1 with the substrate, stir and react at 50 °C for 8.0 h, and detect the yield of 2,4-TDA and 2,6-TDA in the obtained reaction solution by HPLC to determine its catalytic polyurethane degradation activity. The degradation rate of polyether polyol type polyurethane calculated by HPLC was 98% ( Figure 6 ). The results showed that MBX1 enzyme can effectively degrade polyether PUR plastics, so it has great application potential in biodegradation of PUR plastics.

Claims

1. A novel polyurethane plastic degrading enzyme MBX1, characterized in that: It has: 1) the amino acid sequence shown in SEQ ID No.1; or 2) An amino acid sequence as shown in SEQ ID No. 1, with one or more amino acids substituted, deleted and / or added without changing the function, and having the same function as 1).

2. The gene encoding the novel polyurethane plastic degrading enzyme MBX1 according to claim 1, preferably the nucleotide sequence of which is shown as SEQ ID No.

2.

3. A recombinant expression vector containing the gene encoding mbx1 according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that it is obtained by inserting the coding gene according to claim 2 between the Nde I and Xho I sites of pET-28a(+).

5. A genetically engineered bacterium containing the coding gene according to claim 2, preferably, the expression strain of the genetically engineered bacterium is E. coli BL21(DE3).

6. An enzyme preparation for degrading polyurethane plastics, characterized in that: It is prepared from the novel polyurethane plastic degrading enzyme MBX1 as claimed in claim 1, and specifically prepared from the product expressed by the genetically engineered bacteria as claimed in claim 5.

7. Use of the novel polyurethane plastic degrading enzyme MBX1 according to claim 1, the encoding gene according to claim 2, or the recombinant expression vector according to claim 3 or 4, the genetically engineered bacteria according to claim 5, or the enzyme preparation according to claim 6 in the preparation of a product having catalytic polyurethane degrading enzyme activity, or in the degradation of polyurethane plastics, or in the production of the target product 2,4-TDA or 2,6-TDA.

8. The use according to claim 7, characterized in that: The polyurethane plastic is selected from polyester polyurethane or polyether polyurethane.

9. A method for degrading polyurethane plastics, characterized in that: The method comprises the steps of contacting the novel polyurethane plastic degrading enzyme MBX1 described in claim 1 with the polyurethane plastic to be treated, and optionally comprises the step of obtaining the target product 2,4-TDA or 2,6-TDA.

10. The method according to claim 9, characterized in that The reaction temperature of the degradation is 30-60° C.; specifically, the enzyme preparation as claimed in claim 6 is used for degradation.

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