Pla depolymerase mutants with high catalytic activity and thermal stability and uses thereof

Abstract

The application discloses a PLA depolymerase mutant with high catalytic activity and thermal stability and application thereof. By means of rational design and site-directed mutagenesis technology, one or more amino acid residues in positions 128, 151, 153, 202, 245, 267 and 276 in the amino acid sequence of the PLA depolymerase shown in SEQ ID NO: 2 are mutated to obtain a mutant with enhanced catalytic activity and thermal stability. The PLA depolymerase mutant solves the problem of poor stability of the PLA depolymerase in a high-temperature environment in the prior art and can be applied to enzymatic depolymerization of PLA plastics in the field of enzyme engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a PLA depolymerase mutant with high activity and thermal stability and application thereof. BACKGROUND

[0002] With the escalation of the plastic ban and plastic limit order at home and abroad, bioplastics have become the main substitute for the current traditional petroleum-based plastic market. Bioplastics represented by polylactic acid (PLA) meet the world's demand for environmentally friendly and safe materials and are favored by the industry. According to the European Bioplastics Conference report, PLA accounted for 19.2% of the global bio-based plastic production capacity in 2021, and will achieve stable growth in the next five years. PLA is derived from renewable biological resources such as corn and sugarcane, and has excellent biodegradability and good mechanical properties, and has become an industry with great growth potential.

[0003] However, there are still many misunderstandings about bioplastics among the public. Although bioplastics have certain advantages in biodegradability compared to petroleum-based plastics, it does not mean that they can be discarded at will or ignore the importance of recycling. In fact, bioplastics can only be completely degraded under high-temperature industrial composting conditions, and under household composting and especially natural environmental conditions, they still degrade slowly, and discarding them at will will still pollute and harm the environment like petroleum-based plastics. The most commonly used landfill and incineration methods for waste PLA bioplastics do not differ from the environmental pollution caused by traditional plastics, and cannot reflect the excellent biodegradability of PLA.

[0004] At the same time, great progress has been made in the enzymatic depolymerization of polyethylene terephthalate (PET) plastics. A research team in Japan reported that a bacterium from Ideonella sakaiensis can secrete a new type of PET depolymerase PETase on PET film, which has great market application prospects. Enzymatic depolymerization of PET provides new ideas and technical guidance for the biodegradation of other polyester plastics.

[0005] In order to further promote the emerging bioplastic PLA, the development of the PLA market in the future needs to pay more attention to the research on the recycling mode of bioplastic PLA. However, the depolymerization effect of the existing PLA depolymerase is still at a low level and needs to be further optimized and improved. For example, for the esterase RPA1511 from Rhodopseudomonas palustris, 50 μg of the esterase was reacted with 20 mg of PLA powder in 1 mL of the system for 36 h, and the degradation rate of PLA was about 40%, that is, although the esterase has certain depolymerization activity on solid PLA, the depolymerization effect needs to be improved. SUMMARY

[0006] A first object of the present application is to provide a mutant protein having high depolymerization activity and thermal stability to PLA plastic.

[0007] To achieve the above technical objects, the present application adopts the following solutions:

[0008] A PLA depolymerase mutant obtained by mutating one or more of the amino acid residues at positions 128, 151, 153, 202, 245, 267, and 276 in the PLA depolymerase amino acid sequence shown in SEQ ID NO: 2.

[0009] wherein the serine at position 128 is mutated to tyrosine;

[0010] the phenylalanine at position 151 is mutated to leucine;

[0011] the serine at position 153 is mutated to leucine;

[0012] the valine at position 202 is mutated to tryptophan;

[0013] the isoleucine at position 245 is mutated to valine;

[0014] the alanine at position 267 is mutated to glycine;

[0015] the arginine at position 276 is mutated to aspartic acid.

[0016] As a preferred embodiment, the mutant is obtained by mutating two of the amino acid residues at positions 128, 151, 153, 245, 267, and 276 in the PLA depolymerase amino acid sequence shown in SEQ ID NO: 2.

[0017] As a preferred embodiment, the mutant is:

[0018] S153L / R276D, S153L / S128Y, S153L / I245V, I245V / R276D, I245V / S128Y, or F151L / A267G.

[0019] As a preferred embodiment, the mutant is obtained by mutating four or five of the amino acid residues at positions 128, 153, 202, 245, and 276 in the PLA depolymerase amino acid sequence shown in SEQ ID NO: 2.

[0020] As a preferred embodiment, the mutant is:

[0021] S153L+I245V+R276D+S128Y;

[0022] S153L+I245V+R276D+S128Y+V202W;

[0023] or S153L+I245V+S128Y+V202W.

[0024] The present application also aims to protect the genes encoding the above mutants, and the recombinant plasmids connected to the encoding genes.

[0025] The present application also aims to provide the use of the above mutants in PLA degradation.

[0026] As a preferred embodiment, the crude enzyme solution or purified protein of the mutant is added to the PLA degradation system to achieve PLA degradation.

[0027] As a preferred embodiment, the amount of the crude enzyme solution or purified protein added is: the protein concentration in the PLA degradation system is 4‰ of the substrate concentration in terms of protein mass.

[0028] The present application uses structure analysis and site-directed mutagenesis technology to mutate wild-type PLA depolymerase (RPA1511) to obtain a plurality of mutants, which improves the low activity and stability of the wild-type PLA depolymerase, effectively improves the activity of RPA1511 in degrading PLA, and improves the degradation effect of RPA1511. PLA is a difficult-to-degrade insoluble high polymer bioplastic, and the plurality of mutants greatly improves the depolymerization efficiency and thermal stability of PLA plastic, and has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Relative enzyme activity of all mutants relative to wild-type protein RPA1511 tested at 55℃.

[0030] Figure 2 Lactic acid monomer concentration produced by mutant proteins R4-1, R4-2, R5 and RPA1511 in degrading PLA at 65℃.

[0031] Figure 3 Liquid chromatography detection results of product lactic acid after mutant proteins R4-1, R4-2, R5 and wild type degrade PLA for 24h and 72h at 65℃. A, liquid chromatography detection graph of 25mM lactic acid monomer standard. B, liquid chromatography detection graph after RPA1511 degradation. C, liquid chromatography detection graph of mutant R4-2. D, liquid chromatography detection graph of R4-1 mutant. E, liquid chromatography detection graph of R5 mutant. B, C, D, and E are diluted 3 times in concentration when detected.

[0032] Figure 4 T of mutant R5 and wild type RPA1511 mValue detection result. DETAILED DESCRIPTION

[0033] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0034] The raw materials and equipment used in the specific embodiments of the present application are known products, which are obtained by purchasing commercially available products.

[0035] In order to increase the industrial application value of PLA depolymerase, the gene of PLA depolymerase derived from Rhodopseudomonas palustris is synthesized, and the gene is expressed and purified. After studying the structure of the PLA depolymerase, the amino acids in the active region participating in the interaction with the substrate are mutated to increase the activity of the enzyme on the substrate PLA.

[0036] Example 1 Construction of wild-type PLA depolymerase recombinant plasmid and its mutant recombinant plasmid

[0037] 1. In this embodiment, RPA1511 (nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2) is used as a template to further improve the activity of RPA1511 by directional evolution, temperature, so that it can be better applied to depolymerization reaction after modification.

[0038] The RPA1511 gene is derived from Rhodopseudomonas palustris with accession number WP_011157072.1. The rpa1511 gene is inserted into the plasmid pET-29a(+) with Nde I and Xho I as the restriction sites and transformed into E. coli DH5α. 1 μL of pET-29a(+)-rpa1511 pure plasmid is added to E. coli BL21(DE3) competent cells for transformation. Single colonies are picked into LB liquid test tubes containing kanamycin, and the plasmid is extracted as a genomic template for PCR with the above primers to obtain plasmids expressing different PLA depolymerase mutants. The purified reaction product is transformed into E. coli competent cells, and kanamycin is used for preliminary screening. DNA sequencing is performed to determine the successfully mutated genes, and plasmids expressing different PLA depolymerase mutants are obtained.

[0039] RPA1511 is rationally designed for site-directed mutagenesis primers to perform site-directed mutagenesis at 9 sites. The mutants are S128Y, S153L, V202W, P232A, I245V, R276D, G139P, F151L and A267G.

[0040] Table 1 Site-directed mutagenesis primers

[0041]

[0042]

[0043] 2. In order to further evolve the enzyme with better stability and tolerance, the present study combines multiple mutations in the sites that have positive effects on the activity and stability of the depolymerase mutants, and obtains multiple mutants with further improved activity and stability through the method of directed screening. The mutation sites for combination mutation are shown as follows:

[0044] G139P+F151L, G139P+A267G, F151L+A267G, G139P+F151L+A267G, S153L+I245V, S153L+R276D, S153L+S128Y, I245V+R276D, I245V+S128Y, S153L+I245V+R276D, S153L+I245V+R276D+S128Y (abbreviated as R4-1), S153L+I245V+R276D+S128Y+V202W (abbreviated as R5), S153L+I245V+S128Y+V202W (abbreviated as R4-2).

[0045] In addition, four groups of disulfide bond mutations M34C+S63C, A97C+S128C, L257C+A260C, A51C+G77C are additionally designed to improve the thermal stability of the depolymerase.

[0046] Table 2 Disulfide bond primers

[0047]

[0048]

[0049] The mutants of the present application can also be mutants with the above mutation sites and having 85% or more (preferably 90% or more, or 95% or more, or 99% or more, or 99.95% or more, or even 99.99% or more) homology with the amino acid sequence in which the mutation occurs, while having depolymerase activity. More preferably, these mutants are all derived from Rhodopseudomonas palustris and have depolymerase activity.

[0050] In order to verify the differences between the wild-type PLA depolymerase and the PLA depolymerase mutants, the present embodiment preliminarily uses the soluble substrate 1-naphthyl propionate to determine the enzyme activity of the mutants.

[0051] The reaction system was 1 mL: 10 μL of 10 mM 1-naphthalene propionate, 10 μL of enzyme solution, 960 μL of 50 mM phosphate buffer solution (PBS buffer, pH 8.0). After heating at 55°C for 10 min, 4-amino-phenylhydrazine and potassium ferricyanide solution were added to make the final concentration 1 mM and 2 mM, respectively. After 2 min of reaction, the absorbance was detected at 510 nm by using an enzyme marker. The enzyme activity was defined as follows: the amount of enzyme required to generate 1 μmol of 1-naphthalenol per minute at 55°C was taken as one enzyme activity unit (U).

[0052] Example 2 Preparation of PLA depolymerase mutant and wild-type PLA depolymerase

[0053] The mutant plasmid with the target gene was obtained by using pET-29a(+) as the expression vector. The mutant plasmid was transformed into E. coli cells, and the optimal conditions for inducing expression of the PLA depolymerase were as follows: 18°C, 0.1 mM IPTG induction for 20-24 h. The crude enzyme was obtained by ultrasonic cell disruption.

[0054] In order to obtain high-purity enzyme protein, the bacterial solution was ultrasonically broken, and then centrifuged at 9000 rpm for 10 min. The broken supernatant was filtered by using a 0.22 μm filter head, and purified by using an AKTA pure protein purification instrument and a Ni-NTA column. The target protein was eluted by using a buffer containing 50 mM Na2HPO4, 50 mM KH2PO4, 300 mM NaCl and different concentrations of imidazole. The purified mutant protein eluted was concentrated by using an ultrafiltration centrifuge tube and dialyzed to remove imidazole, and stored at 4°C.

[0055] Example 3 Relative activity and thermal stability analysis of PLA depolymerase mutant and wild-type RPA1511 depolymerase

[0056] In order to verify the difference between the wild-type PLA depolymerase and the PLA depolymerase mutant, the degradation activity of the two on PLA was further determined in this embodiment. The activity test method of the PLA depolymerase mainly included:

[0057] The reaction system was 5 mL: the reaction temperature for enzyme depolymerization was 55°C, and the buffer was a phosphate buffer with a concentration of 50 mM and pH 8.0. The addition amount of the PLA substrate was 4 mg / mL, and the addition amount of the enzyme was 4‰ of the substrate. Therefore, the final enzyme depolymerization system was 5 mL of phosphate buffer, 0.08 mg of pure enzyme and 20 mg of PLA powder (purchased from Sigma Aldrich, Resomer R202H, M w10,000-18,000) as substrate. Different temperature was set in the metal bath for the enzymatic degradation reaction, and the rotation speed was set at 400 r / min for 3 d. After the reaction, the mixture was centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter; high performance liquid chromatography (HPLC, Agilent 1260Series) was used for product determination and analysis, and the analysis column was Agilent Polaris C18-A (4.6 mm x 250 mm, 5 um). The detection conditions were as follows: column temperature 30 ℃; the mobile phase was methanol and 0.02 M potassium dihydrogen phosphate solution (phosphoric acid was used to adjust the pH to 2.1), and the volume ratio was 5:95; the flow rate was 0.5 mL / min; the detection wavelength was 210 nm; and the injection volume was 10 μL.

[0058] The activity of the wild type and mutant enzymes was determined by comparing the peak area of the hydrolysis product lactic acid monomer of the wild type PLA depolymerase or its mutants. In the HPLC experiment, the amount of the compound in the solution was linearly related to the peak area, so the amount of the compound in the solution could be calculated by the peak area; in this experiment, the peak area of the product was used to define the catalytic effect of the mutant protein on the substrate; the more the product, the better the activity of the mutant protein.

[0059] The main method for testing the thermal stability of the PLA depolymerase is as follows:

[0060] During the heating process, the protein will undergo thermal denaturation and unfolding. The melting temperature (T m ) of the protein refers to the temperature corresponding to 50% protein unfolding. DSF method is used to determine the melting temperature (T m ) of the protein. The reaction system includes protein sample (PLA depolymerase RPA1511 and its mutants), reaction buffer and fluorescent dye (SYPRO TM Protein Gel Stains). First, the protein to be tested is concentrated to the desired concentration (about 0.4 mg / mL), mixed with 1x dye probe, and then transferred to a 96-well PCR plate for detection. The sample volume of each detection well is 20-25 μL, and the buffer is used as a blank control group. Three sets of repeated experiments are set for each detection group, and three sets of repeated experiments are also set for each detection group. Then, the 96-well PCR plate containing the control group and the detection group is placed in a fluorescence quantitative PCR instrument (7300Plus Real-Time PCR system), and the temperature is changed from 25 ℃ to 99 ℃ at a rate of 1% of the instrument setting value. Finally, the data is exported by software, and the fluorescence intensity-temperature curve is drawn, and the temperature corresponding to 50% protein unfolding is calculated, so as to determine the T m value of the sample protein.

[0061] The results are shown in Figure 1 It can be seen that the relative enzyme activity of all mutants relative to wild-type RPA1511.

[0062] The results are shown in Figure 2 It can be seen that the three listed mutants have higher PLA depolymerization activity than the wild-type protein, and the R5 catalyzed product lactic acid monomer concentration is nearly 4 times higher than the wild-type WT.

[0063] The results are shown in Figure 3 It can be seen that the high performance liquid chromatography (HPLC) detection results of wild-type RPA1511 PLA depolymerase and various PLA depolymerases are shown in Figure 2 The results are shown in Figure 2 It can be seen that the peak at a retention time of 9.4 min; the peak at a retention time of 9.4 min is consistent with the peak time of the standard lactic acid (sigma, cas: 50-21-5) Figure 3 It can be seen that the peak at a retention time of 9.4 min; the peak at a retention time of 9.4 min is consistent with the peak time of the standard lactic acid (sigma, cas: 50-21-5)

[0064] The results are shown in Figure 4 It can be seen that the wild-type protein T m value is 70.18℃, the T m value of R4-1 is 72.82℃, the T m value of R4-2 is 73.83℃, the T m value of mutant R5 is 78.75℃, and the T m value of mutant R5 relative to the wild-type protein WT is increased by 8.57℃.

Claims

1. A PLA depolymerase mutant, characterized in that, The PLA depolymerase amino acid sequence shown in SEQ ID NO: 2 was obtained by mutating one or more amino acid residues as follows: Mutating serine at position 153 to leucine yields the mutant S153L; or, The mutant S153L+R276D was obtained by mutating serine at position 153 to leucine and arginine at position 276 to aspartic acid; or, The mutant S153L+S128Y was obtained by mutating serine at position 153 to leucine and serine at position 128 to tyrosine; or, The mutant S153L+I245V was obtained by mutating serine at position 153 to leucine and isoleucine at position 245 to valine; or, The mutant S153L+I245V+R276D+S128Y was obtained by mutating serine at position 153 to leucine, isoleucine at position 245 to valine, serine at position 128 to tyrosine, and arginine at position 276 to aspartic acid; or, The mutant S153L+I245V+S128Y+V202W was obtained by mutating serine at position 153 to leucine, isoleucine at position 245 to valine, serine at position 128 to tyrosine, and valine at position 202 to tryptophan; or, The mutant S153L+I245V+R276D+S128Y+V202W was obtained by mutating serine at position 153 to leucine, isoleucine at position 245 to valine, arginine at position 276 to aspartic acid, serine at position 128 to tyrosine, and valine at position 202 to tryptophan.

2. The gene encoding the mutant of claim 1.

3. A recombinant plasmid containing the gene of claim 2.

4. The application of a PLA depolymerase mutant in PLA degradation, characterized in that, The mutant is the mutant S153L+I245V+R276D+S128Y, mutant S153L+I245V+S128Y+V202W or mutant S153L+I245V+R276D+S128Y+V202W as described in claim 1.

5. The application according to claim 4, characterized in that, The crude enzyme solution or purified protein of the mutant is added to the PLA degradation system to achieve PLA degradation.

6. The application according to claim 5, characterized in that, The amount of crude enzyme solution or purified protein added is: based on protein mass, the protein concentration in the PLA degradation system is 4‰ of the substrate concentration.

Images