Hydrophobicity-enhanced LCCICCG mutant protein and application thereof

Abstract

The invention discloses a hydrophobicity-enhanced LCCICCG mutant protein and application thereof, the 53th, 81th, 179th, 197th, 228th, 229th, 266 or 278th amino acid residues of the amino acid sequence of the LCCICCG protein are mutated into phenylalanine, and mutants D53F, G81F, P179F, N197F, S228F, T229F, N266F or N278F, namely the mutant protein, are obtained. The hydrophobicity of the mutant protein is remarkably enhanced compared with that of wild type protein, PET depolymerization experiments prove that the PET depolymerization efficiency of the mutant protein is remarkably improved compared with that of the wild type protein under the condition of low enzyme load, and the mutant protein has economic feasibility and product selectivity.

Description

Technical Field

[0001] The invention belongs to the field of bioenzymatic depolymerization of PET waste plastics, and particularly relates to LCCICCG mutant proteins with enhanced hydrophobicity and applications thereof. Background Art

[0002] Polyethylene terephthalate (PET) is a semi-crystalline polyester material. Due to its good heat resistance, processability, biosafety and other advantages, it is widely used in many fields such as textile manufacturing, packaging materials, and medical consumables. With the expansion of its application fields, the degradation of PET waste has become one of the global problems. Compared with physical and chemical methods, bioenzymatic hydrolysis is the most environmentally friendly method for treating PET waste due to its mild reaction conditions, strong specificity, and few by-products. At present, how to achieve cost-effectiveness and improve depolymerization efficiency in the process of polyethylene terephthalate (PET) depolymerization is a key goal. Appropriately reducing the enzyme concentration is an effective strategy. By reducing the amount of enzyme used, it can reduce the substrate or product inhibition caused by excessive enzyme concentration while reducing production costs, thereby optimizing the overall reaction rate. In addition, this strategy also helps to maintain the efficiency of the catalytic process and ensure the best results in terms of economic and environmental sustainability. Summary of the invention

[0003] The first object of the present invention is to solve the problems existing in the above-mentioned prior art and to provide a LCC ICCG The variants were optimized for hydrophobicity by site-directed mutagenesis, providing a hydrophobicity-enhanced LCC ICCG Mutant protein.

[0004] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: Hydrophobicity-enhanced LCC ICCG Mutant protein, LCC ICCG The amino acid residue at position 53, 81, 179, 197, 228, 229, 266 or 278 of the protein amino acid sequence is mutated to phenylalanine to obtain mutants D53F, G81F, P179F, N197F, S228F, T229F, N266F or N278F, i.e. the mutant protein.

[0005] The second object of the present invention is to provide the use of the mutant protein in PET depolymerization.

[0006] As a preferred embodiment, the enzyme solution of the mutant protein is mixed with the PET to be degraded to perform PET depolymerization.

[0007] As a preferred embodiment, the temperature for depolymerization of PET is 70-75°C.

[0008] As a preferred embodiment, the enzyme solution of the mutant protein during PET depolymerization is added once at the beginning of the reaction.

[0009] As a preferred embodiment, the concentration of mutant protein in the PET depolymerization system is ≤0.8 mg / L, and the protein / substrate mass ratio is ≤0.0004.

[0010] As a preferred embodiment, the PET is in powder form.

[0011] As a preferred embodiment, the mutant protein is mutant S228F or N266F.

[0012] The present invention has the following beneficial effects: (1) LCC of the present invention ICCG Mutant protein compared to LCC ICCG The hydrophobicity of the variant was significantly enhanced, and the PET depolymerization experiment verified that LCC ICCG The efficiency of mutant protein in depolymerizing PET at low enzyme loading is lower than that of original protein LCC. ICCG The variants have been significantly improved to achieve a balance between cost-effectiveness and catalytic efficiency, making them economically viable.

[0013] (2) The mutant hydrophobic protein has product selectivity for PET depolymerization, especially at low enzyme loading, the TPA / MHET ratio in the yield is significantly increased. The product selectivity of the protein is of great significance, which can ensure the effective conversion of depolymerization monomers and optimize the utilization efficiency of resources. It provides a new idea for promoting the biodegradation and bioconversion of PET waste, thereby promoting the development of environmentally friendly PET waste bioenzymatic treatment technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effect of enzyme addition method on depolymerization efficiency.

[0015] Figure 2 This is a diagram of the depolymerization effect at different enzyme loadings.

[0016] Figure 3 This is a comparison chart of the depolymerization effect of hydrophobic proteins.

[0017] Figure 4 A comparison chart of the selectivity effects of hydrophobic proteins. DETAILED DESCRIPTION

[0018] The following examples will further illustrate the method provided by the present invention, but the present invention is not limited to the listed examples, and should also include any other known changes within the scope of the rights claimed by the present invention.

[0019] Example 1 An optimization method for enhancing the hydrophobicity of LCC protein to affect the depolymerization performance of PET comprises the following steps: (1) Construction of hydrophobic mutant proteins: First, LCC was calculated using NetSurfP-2.0 LCCG Surface amino acids (LCC LCCG The amino acid sequence is shown in SEQ ID NO: 1), and S36, D53, G81, S83, T144, S148, A152, N157, P179, N197, S199, Q224, S228, T229, N239, N266, T268, N278 and R290 with RSA (Relative Surface Accessibility) scores higher than 65% were selected for subsequent experiments. In order to ensure that the hydrophobicity is increased while the protein activity is not affected as much as possible, it is necessary to remove the specific sites that destroy the secondary structure and are located at the N-terminus and C-terminus from the above-obtained sites. After removal, D53, G81, S83, P179, N197, S199, S228, T229, N239, N266 and N278 were selected for subsequent experiments. Then, site-directed mutations were constructed and screened, and the eleven sites selected were mutated into highly hydrophobic phenylalanine, and protein expression and purification were performed. The surface hydrophobicity of the eight successfully expressed mutant proteins D53F, G81F, P179F, N197F, S228F, T229F, N266F and N278F was detected by the CPA probe method.

[0020] (2) Method for determining the hydrophobicity of mutant proteins: The CPA fluorescent probe method was used to determine the hydrophobicity of proteins. Different amounts of enzyme solution were added to a light-proof 96-well plate, and different amounts of PBS buffer were added to dilute the protein concentration to 0.05-0.5 μmol / L. The total volume of the solution was 150 μL. 1.5 μL of CPA solution with a concentration of 10 mmol / L was added, and the mixture was allowed to stand for 3 minutes after shaking. Using a fluorescence spectrophotometer, the excitation wavelength λ ex =325nm (slit correction 2nm), emission wavelength λ em =340-550nm (slit correction 5nm), measure the fluorescence intensity. Plot a curve of fluorescence intensity versus protein concentration, and the slope of the initial stage of the curve is the surface hydrophobicity index (H) of the protein molecule. 0 ). LCC ICCG The hydrophobicity measurement results of the mutant protein are shown in Table 1.

[0021] Table 1 Results of protein hydrophobicity determination Protein name D53F G81F P179F N197F S228F T229F N266F N278F <![CDATA[LCC ICCG ]]> Hydrophobicity 210718 167069 164612 102680 224489 252642 108098 101973 36015 Increase multiple 5.85 4.64 4.57 2.85 6.23 7.01 3.00 2.83 —— (3) Effect of enzyme addition method on PET depolymerization: reaction time was 12 h, reaction temperature was 72 °C, substrate concentration was 2 g / L (PET powder), enzyme amount was 4 mg / L, and reaction system was 25 mL. The experimental group added the enzyme in batches at 0 / 2 / 4 / 6 / 8 h of reaction, and the control group added it once at the beginning of the reaction. It can be observed that during the reaction, the enzyme addition method had some effect on the depolymerization effect, but did not change the final depolymerization efficiency.

[0022] The effect of enzyme addition method on depolymerization efficiency in Example 1 is as follows Figure 1 shown.

[0023] Example 2 The difference between this example and Example 1 is that the amount of enzyme added to the experimental group is shown in Table 2. The enzyme loadings of the three experimental groups were 1 / 2, 1 / 5, and 1 / 10 of those of the control group, respectively. The depolymerization effect was different under different enzyme loadings, but the depolymerization efficiency was not proportional to the multiple of the enzyme loading reduction, and there was a significant difference in the degradation effect of the wild type (ICCG) and hydrophobic protein (S228F) under different enzyme loadings.

[0024] Table 2 Different enzyme loadings Group <![CDATA[Enzyme amount (g 酶 / g 底物 )]]> Enzyme amount (mg / L) Enzyme dosage ratio (compared to the control group dosage) Control group (CK) 0.002 4 1 Experimental Group 1 0.001 2 1 / 2 Experimental Group 2 0.0004 0.8 1 / 5 Experimental Group 3 0.0002 0.4 1 / 10 The depolymerization effects of different enzyme loadings in Example 2 are as follows Figure 2 As shown, it can be seen that under the enzyme loading of the control group, there was no significant difference in the depolymerization efficiency of ICCG and S228F, but under low enzyme loading (≤0.8 mg / L), the depolymerization efficiency of S228F was significantly better than that of ICCG.

[0025] Example 3 Based on Example 2, this example further compares the depolymerization effects of S228F, N266F and ICCG on PET when the enzyme loading is 1 / 10 of that of the control group.

[0026] like Figure 3 As shown, under low enzyme loading conditions, the hydrophobic mutant proteins S228F and N266F have a much higher depolymerization effect on PET than the wild type.

[0027] Example 4 Based on Example 2, this example compares the product composition of the mutant protein and the wild-type protein at different enzyme loadings (1 / 2, 1 / 5, and 1 / 10 of the control group, respectively). Figure 4 As shown, it can be seen that under low enzyme loading conditions, the hydrophobic mutant proteins S228F and N266F show completely different product selectivity for the depolymerization of PET than the wild type, and this phenomenon is more significant when the enzyme loading is lower.

[0028] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Hydrophobicity-enhanced LCC ICCG A mutant protein, characterized in that LCC ICCG The amino acid residue at position 53, 81, 179, 197, 228, 229, 266 or 278 of the protein amino acid sequence is mutated to phenylalanine to obtain mutants D53F, G81F, P179F, N197F, S228F, T229F, N266F or N278F, i.e. the mutant protein.

2. Use of the mutant protein according to claim 1 in PET depolymerization.

3. The application according to claim 2, characterized in that: The enzyme solution of the mutant protein is mixed with the PET to be degraded to perform PET depolymerization.

4. The use according to claim 3, characterized in that: The temperature for PET depolymerization is 70-75°C.

5. The application according to claim 3, characterized in that: When PET is depolymerized, the enzyme solution of the mutant protein is added once at the beginning of the reaction.

6. The use according to claim 3, characterized in that: The mutant protein concentration in the PET depolymerization system is ≤0.8 mg / L, and the protein / substrate mass ratio is ≤0.0004.

7. The use according to claim 3, characterized in that: The PET is in powder form.

8. The use according to claim 3, characterized in that: The mutant protein is mutant S228F or N266F.

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