LCC with enhanced hydrophobicity ICCG Mutated proteins and uses thereof

Abstract

The application discloses a LCC with enhanced hydrophobicity ICCG The application discloses a mutant protein and application thereof ICCG The mutant protein is obtained by mutating an amino acid residue at the 266th amino acid residue in a protein amino acid sequence into phenylalanine, and is a mutant N266F. Compared with a wild-type protein, the mutant protein has significantly enhanced hydrophobicity, and is verified by a PET depolymerization experiment. The mutant protein has significantly improved PET depolymerization efficiency compared with the wild-type protein under the condition of low enzyme load, is economically feasible, and has product selectivity.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biocatalytic depolymerization of PET waste plastics, and particularly relates to a LCCICCG mutant protein with enhanced hydrophobicity and application thereof. BACKGROUND

[0002] Polyethylene terephthalate (PET) is a semi-crystalline plastic material that is widely used in textile manufacturing, packaging materials, medical consumables and many other fields due to its good heat resistance, processability, biological safety and other advantages. 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, biocatalytic depolymerization is the most environmentally friendly method for treating PET waste, as it has the advantages of mild reaction conditions, strong specificity, and less by-products. At present, how to achieve cost-effectiveness and improve the depolymerization efficiency in the process of polyethylene terephthalate (PET) depolymerization is the key goal. Properly reducing the enzyme concentration is an effective strategy, which can reduce the production cost while reducing the substrate or product inhibition caused by high enzyme concentration, 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

[0003] The first object of the present application is to solve the above-mentioned problems of the prior art by optimizing the LCC ICCG variant through site-directed mutagenesis, and providing a LCC ICCG mutant protein with enhanced hydrophobicity.

[0004] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0005] The LCC ICCG mutant protein with enhanced hydrophobicity, wherein the 53rd, 81st, 179th, 197th, 228th, 229th, 266th or 278th amino acid residue in the amino acid sequence of the LCC ICCG protein is mutated to phenylalanine to obtain a mutant D53F, G81F, P179F, N197F, S228F, T229F, N266F or N278F, i.e. the mutant protein.

[0006] The second object of the present application is to provide the application of the mutant protein in PET depolymerization.

[0007] As a preferred embodiment, the enzyme solution of the mutant protein is mixed with the PET to be degraded for PET depolymerization.

[0008] As a preferred embodiment, the temperature for PET depolymerization is 70-75℃.

[0009] As a preferred embodiment, the enzyme solution of the mutant protein in PET depolymerization is added at once at the beginning of the reaction.

[0010] As a preferred embodiment, the concentration of the mutant protein in the PET depolymerization system is ≤0.8 mg / L, and the mass ratio of protein to substrate is ≤0.0004.

[0011] As a preferred embodiment, the PET is in the form of powder.

[0012] As a preferred embodiment, the mutant protein is mutant S228F or N266F.

[0013] The present application has the following beneficial effects:

[0014] (1) The LCC of the present application ICCG The mutant protein has a higher hydrophobicity than the LCC ICCG The variant has a significantly enhanced hydrophobicity, and the PET depolymerization experiment has verified that the LCC ICCG The mutant protein has a higher efficiency in PET depolymerization than the original protein LCC ICCG The variant significantly improves the balance between cost efficiency and catalytic efficiency, and is economically feasible.

[0015] (2) The mutant hydrophobic protein has product selectivity in PET depolymerization, especially at low enzyme load, 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, and provides a new idea for promoting the biodegradation and bioconversion of PET waste, thereby promoting the development of the biodegradation method of environmentally friendly PET waste. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The effect of enzyme addition mode on depolymerization efficiency.

[0017] Figure 2 The depolymerization effect diagram of different enzyme load.

[0018] Figure 3 The comparison diagram of the depolymerization effect of hydrophobic proteins.

[0019] Figure 4 The comparison diagram of the product selectivity effect of hydrophobic proteins. DETAILED DESCRIPTION

[0020] The following examples will further illustrate the method provided by the present application, but the present application is not limited to the listed examples, and any other known changes within the scope of the rights claimed by the present application should also be included.

[0021] Example 1

[0022] An optimization method for LCC protein hydrophobicity enhancement affecting PET depolymerization performance includes the following steps:

[0023] (1) Construction of hydrophobic mutant protein: first, calculate the LCC LCCG surface amino acid (LCC LCCG amino acid sequence shown as SEQ ID NO: 1), and select 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) score higher than 65% for subsequent experiments. To ensure the increase of hydrophobicity while trying not to affect the protein activity, remove the specific sites that destroy the secondary structure and are located at the N- and C-termini from the above obtained sites. After removal, select D53, G81, S83, P179, N197, S199, S228, T229, N239, N266 and N278 for subsequent experiments. Then construct and screen site-directed mutation, mutate the eleven selected sites to strong hydrophobicity phenylalanine, and perform protein expression and purification. Eight successfully expressed mutant proteins D53F, G81F, P179F, N197F, S228F, T229F, N266F and N278F are detected for surface hydrophobicity by CPA probe method.

[0024] (2) Mutant protein hydrophobicity determination method:

[0025] The CPA fluorescence probe method is used to determine the hydrophobicity of the protein. Different amounts of enzyme solution are added to a non-transparent 96-well plate, different amounts of PBS buffer are added, the protein concentration is diluted to 0.05-0.5 μmol / L, and the total volume of the solution is 150 μL. Add 1.5 μL of CPA solution with a concentration of 10 mmol / L, shake and stand for 3 min. Use a fluorescence spectrophotometer, set the excitation wavelength λ ex = 325 nm (slit correction 2 nm), the emission wavelength λ em = 340-550 nm (slit correction 5 nm), and measure the fluorescence intensity. Plot the fluorescence intensity against the protein concentration, and the slope of the initial stage of the curve is the surface hydrophobicity index (H0) of the protein molecule. The hydrophobicity of LCC ICCG and the mutant protein is determined as shown in Table 1.

[0026] Table 1 Protein hydrophobicity determination results

[0027] Protein name D53F G81F P179F N197F S228F T229F N266F N278F LCC ICCG ]]> Hydrophobicity 210718 167069 164612 102680 224489 252642 108098 101973 36015 Fold increase 5.85 4.64 4.57 2.85 6.23 7.01 3.00 2.83 ——

[0028] (3) Effect of enzyme addition mode 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 enzyme amount of the experimental group was added in five batches at 0 / 2 / 4 / 6 / 8 h, and the control group was added at the beginning. It can be observed that the enzyme addition mode has an effect on the depolymerization effect during the reaction, but does not change the final depolymerization efficiency.

[0029] The effect of enzyme addition mode on depolymerization efficiency in Example 1 is shown in Figure 1 .

[0030] Example 2

[0031] The difference between this example and Example 1 is only that the enzyme amount added in the experimental group is shown in Table 2. The enzyme load of the three experimental groups is 1 / 2, 1 / 5, and 1 / 10 of the control group, respectively. The depolymerization effect is different under different enzyme loads, but the depolymerization efficiency is not proportional to the enzyme load reduction factor, and the degradation effect of wild type (ICCG) and hydrophobic protein (S228F) under different enzyme loads is significantly different.

[0032] Table 2 Different enzyme loads

[0033] Group Enzyme amount (g 酶 / g 底物 )]]> Enzyme amount (mg / L) Enzyme amount ratio (compared with the amount used in the control group) 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

[0034] The depolymerization effect of different enzyme loads in Example 2 is shown in Figure 2 . It can be seen that under the enzyme load of the control group, there is no significant difference in the depolymerization efficiency of ICCG and S228F, but under low enzyme load (≤0.8 mg / L), the depolymerization efficiency of S228F is significantly better than that of ICCG.

[0035] Example 3

[0036] This example further compares the depolymerization effect of S228F, N266F, and ICCG on PET under the condition that the enzyme load is 1 / 10 of the control group based on Example 2.

[0037] As shown in Figure 3 , under low enzyme load, the depolymerization effect of hydrophobic mutant proteins S228F and N266F on PET is much higher than that of the wild type.

[0038] Example 4

[0039] This example compares the product composition of mutant proteins and wild type proteins under different enzyme loads (1 / 2, 1 / 5, and 1 / 10 of the control group) based on Example 2. The results are shown in Figure 4As shown, it can be seen that, in the case of low enzyme load, the hydrophobic mutant S228F, N266F shows a completely different product selectivity for PET depolymerization than the wild type, and this phenomenon is more significant when the enzyme load is lower.

[0040] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. LCC with enhanced hydrophobicity ICCG A mutant protein, characterized in that LCC ICCG The amino acid residue at position 266 of the protein amino acid sequence is mutated to phenylalanine to obtain the mutant N266F, i.e., the mutant protein; The LCC ICCG The amino acid sequence of the protein is shown in SEQ ID NO:

1.

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 application 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 concentration of mutant protein in the PET depolymerization system was ≤0.8 mg / L, and the protein / substrate mass ratio was ≤0.0004.

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

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