PET-degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and application

Abstract

The present invention belongs to the field of enzyme engineering technology, and specifically relates to a PET degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and application. The PET degrading enzyme PET-mh mutant provided by the present invention is obtained by mutating the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the protein with an amino acid sequence such as that shown in SEQ ID No.1. Compared with the protein with an amino acid sequence such as that shown in SEQ ID No.1, the activity or thermal stability of the mutant is significantly improved, and it has a high application value for realizing efficient degradation of PET. The present invention also provides a coding gene encoding the mutant, a recombinant plasmid containing the coding gene, and an engineering bacteria containing the recombinant plasmid and an application.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a PET degradation enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and application. Background Art

[0002] Polyethylene terephthalate (PET) is one of the most widely used plastics at present. It has the advantages of strong chemical resistance, high thermal stability, light weight, and convenient storage. It can be used to produce packaging materials, mineral water bottles, mobile phone cases and many other daily necessities. It is a common engineering polymer with an annual output of about 50 million tons. Because the sub-substrate of polyethylene terephthalate contains covalent bonds that are difficult to hydrolyze, PET plastic is difficult to degrade. With the increase in the use of PET plastics, PET waste has accumulated in the environment in large quantities, which is one of the culprits causing the "white pollution" problem. It is reported that micron-sized PET has been found in food and has become part of the human food chain, which may threaten human health.

[0003] At present, the recycling strategies for PET waste mainly include physical recycling and chemical recycling, but both are prone to cause secondary pollution to the environment. The biological method of using enzymes or microorganisms to degrade PET into monomer molecules of terephthalic acid (TPA) and ethylene glycol has the advantages of normal temperature and pressure, single degradation products and easy recycling, and green environmental protection. It conforms to the concept of sustainable development and is considered to be the most promising method to reduce PET plastic pollution. However, there are problems such as poor enzyme stability and low catalytic efficiency in the biological degradation of PET plastics. Through biosynthesis technology, researchers have obtained some PET degradation enzymes with improved degradation activity, but when using these degradation enzymes to degrade PET, it is necessary to first reduce the high crystallinity and hydrophobic surface of PET on PET enzymatic hydrolysis through high-temperature melt recrystallization and crusher micronization, making the process complicated and costly. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a PET degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineered bacteria and application. The PET degrading enzyme PET-mh mutant has high activity and thermal stability, can improve the degradation efficiency of PET, and can also efficiently degrade high-crystallinity PET, thereby simplifying the PET degradation process and reducing costs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a PET degrading enzyme PET-mh mutant, which is obtained by mutating at least one of the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the protein with an amino acid sequence as shown in SEQ ID No. 1.

[0007] The protein with an amino acid sequence as shown in SEQ ID No. 1 has good degradation activity against PET degrading enzymes. The present invention can obtain a mutant with better activity or thermal stability than the protein by mutating specific amino acids in the amino acid sequence. The obtained mutant can improve the efficiency of PET degradation, providing a new option for PET bio-scale conversion and monomer recovery.

[0008] In conjunction with the first aspect, it is preferred that the amino acid at position 29 is mutated to L-alanine.

[0009] In combination with the first aspect, it is preferred that the amino acid at position 62 is mutated to L-threonine.

[0010] In combination with the first aspect, it is preferred that the amino acid at position 63 is mutated to L-threonine.

[0011] In conjunction with the first aspect, it is preferred that the amino acid at position 68 is mutated to L-asparagine.

[0012] In combination with the first aspect, it is preferred that the amino acid at position 124 is mutated to S-glycine.

[0013] In conjunction with the first aspect, it is preferred that the amino acid at position 152 is mutated to L-methionine.

[0014] In conjunction with the first aspect, it is preferred that the amino acid at position 176 is mutated to L-arginine.

[0015] In conjunction with the first aspect, it is preferred that the amino acid at position 177 is mutated to L-isoleucine.

[0016] In conjunction with the first aspect, it is preferred that the amino acid at position 183 is mutated to L-tyrosine.

[0017] In conjunction with the first aspect, it is preferred that the amino acid at position 208 is mutated to L-threonine.

[0018] In combination with the first aspect, it is preferred that the amino acid at position 219 is mutated to L-methionine.

[0019] In combination with the first aspect, as a more preferred technical solution, at least one of the amino acids at positions 63, 68, 152, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated.

[0020] Preferably, at least the 183rd amino acid in the amino acid sequence shown in SEQ ID No. 1 is mutated. Experiments have shown that even if only the 183rd amino acid is mutated, the performance of the resulting mutant is greatly improved relative to the protein. Moreover, mutating the 183rd amino acid together with the amino acids at other positions may also produce a synergistic effect.

[0021] Specifically, as a more preferred technical solution, any one of the amino acids at positions 63, 68, 124, 152, 176 and 177 and the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. The above combined mutations can further improve the efficiency of PET degradation.

[0022] Preferably, the amino acid at position 124 or 152 and the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together.

[0023] As a more preferred technical solution, any one of the amino acids at positions 63, 68, 152, 176 and 208 and the amino acids at positions 124 and 183 in the amino acid sequence as shown in SEQ ID No. 1 are mutated together. More preferably, the amino acid at position 208 and the amino acids at positions 124 and 183 in the amino acid sequence as shown in SEQ ID No. 1 are mutated together.

[0024] High crystallinity PET is more difficult to degrade than low crystallinity PET, and has a worse degradation effect under the same conditions. The amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 is mutated, or the amino acid at position 124 or 152 and the amino acid at position 183 are mutated together, or the amino acids at positions 124, 183, and 208 are mutated together. The resulting mutant can significantly improve the degradation efficiency of low crystallinity PET and the degradation efficiency of high crystallinity PET relative to the protein, and has better practicality.

[0025] In a second aspect, the present invention also provides the use of the above-mentioned PET degrading enzyme PET-mh mutant in hydrolyzing PET: adding the PET degrading enzyme PET-mh mutant to a potassium phosphate buffer of 80-120 mM and a pH value of 7.5-8.5 to a final concentration of 5-15 μg / mL, and performing PET degradation at 65-75° C. and 100-220 rpm.

[0026] In a third aspect, the present invention also provides a gene encoding the above-mentioned PET degrading enzyme PET-mh mutant.

[0027] In combination with the third aspect, the amino acid sequence of the PET degrading enzyme PET-mh mutant is shown as SEQ ID No.2 to SEQ ID No.23, and the nucleotide sequence thereof is shown as SEQ ID No.24 to SEQ ID No.45, respectively.

[0028] In a fourth aspect, the present invention also provides a recombinant plasmid containing the nucleotide sequence of the above gene.

[0029] In a fifth aspect, the present invention also provides a method for constructing the above-mentioned recombinant plasmid: using the above-mentioned nucleotide sequence as a template, a single point mutation is constructed using corresponding primers, the target gene is first exponentially amplified by a PCR reaction, and then the template chain is removed using the restriction endonuclease Dpn I, and the PCR product is quickly circularized using a DNA ligase to obtain a recombinant plasmid.

[0030] In a sixth aspect, the present invention also provides an engineered bacterium constructed using the above-mentioned recombinant plasmid.

[0031] In combination with the sixth aspect, the engineered bacteria is Escherichia coli.

[0032] In a seventh aspect, the present invention also provides a method for constructing the above-mentioned engineered bacteria: transforming the above-mentioned recombinant plasmid into BL21 (DE3) competent cells, spreading them on ampicillin plate culture medium, and obtaining positive recombinants, namely the engineered bacteria.

[0033] In an eighth aspect, the present invention also provides the use of the above-mentioned engineered bacteria in the degradation of PET.

[0034] Exemplarily, the method for degrading PET using the above-mentioned engineered bacteria can refer to the following operation: the above-mentioned engineered bacteria are cultured in LB liquid culture medium until the OD600 value reaches the range of 0.8-1.0, and the inducer IPTG (isopropyl β-D-1-thiogalactopyranoside) is added to a concentration of 0.1-0.15 mM, and the expression is induced at 15-17° C. for 20-24 hours;

[0035] The fermentation culture is centrifuged, resuspended by pipetting with a bacteria-breaking buffer, and centrifuged again to obtain a crude enzyme solution containing a PET-mh mutant of a PET-degrading enzyme; the crude enzyme solution is purified from proteins and then concentrated to serve as a biocatalyst for degrading PET;

[0036] The PET film substrate to be degraded is added to the PET reaction solution, and a biocatalytic reaction is carried out at 65-75° C. The PET reaction solution contains a potassium phosphate buffer solution with a pH of 8.0 and a biocatalyst for degrading PET.

[0037] The beneficial effects of the present invention are as follows: the PET-mh mutant of the PET degrading enzyme provided by the present invention has higher activity and thermal stability than the protein with an amino acid sequence as shown in SEQ ID No. 1, and the catalytic efficiency is greatly improved, and it can more effectively degrade PET plastic waste, which is beneficial to the resource regeneration and secondary development of PET waste, and can not only alleviate the pressure of petroleum resources required for the production of PET, but also solve the serious environmental pollution problem caused by PET waste, which is in line with the concept of sustainable development, and can also improve the recovery value of PET and promote the realization of plastic economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The degradation effect of the 63rd, 68th, 124th and 183rd amino acids on low crystallinity PET after mutation in the test example 1 of the present invention;

[0039] Figure 2 The degradation effect of the amino acids 63, 68, 124, 152, 176, 177 and 183 on low crystallinity PET after the common mutation in the test example 1 of the present invention;

[0040] Figure 3 The degradation effect of the amino acids at positions 63, 68, 152, 176, 208 and 124 and 183 on low crystallinity PET after the mutation in Example 1 of the present invention;

[0041] Figure 4 In Example 2 of the present invention, the degradation effect on high-crystallinity PET was tested by mutating the amino acid at position 183 alone, mutating the amino acids at positions 124, 152 and 183 together, and mutating the amino acids at positions 124, 183 and 208 together. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] With the rapid development of my country's economy, PET products are being produced and used in large quantities due to their low price, light weight, convenience, and strong plasticity. The resulting PET waste will cause unimaginable pollution. The biological method of using enzymes or microorganisms to degrade PET into monomer molecules terephthalic acid (TPA) and ethylene glycol is considered to be the most promising method to reduce PET plastic pollution. However, there are problems such as poor enzyme stability and low catalytic efficiency in the biological degradation of PET plastics.

[0044] Modifying the amino acid sequence of an enzyme is a common way to obtain new functions, but not all modifications can produce mutants with ideal functions.

[0045] In order to solve the above problems, the embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, which is obtained by mutating at least one of the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1. Mutating the amino acid at position 177 can increase the Tm to 99.6°C; mutating other amino acids can improve the thermal stability to varying degrees while also improving the efficiency of PET degradation.

[0046] The embodiments of the present invention also provide a method for constructing the above-mentioned PET degrading enzyme PET-mh mutant and its application in hydrolyzing PET.

[0047] The present invention also provides a gene encoding the mutant, and the nucleotide sequence thereof is shown in SEQ ID No. 24 to SEQ ID No. 45.

[0048] The embodiments of the present invention also provide a recombinant plasmid containing the above nucleotide sequence and a construction method thereof, as well as an engineered bacterium constructed using the recombinant plasmid.

[0049] The embodiment of the present invention also provides a method for constructing the engineered bacteria and its application in degrading PET.

[0050] The scheme of the present invention is described below through specific embodiments.

[0051] Unless otherwise specified, the reagents in the following examples are commercially available or obtained according to methods known in the art.

[0052] Example 1

[0053] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-serine at position 29 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-alanine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.2, and the nucleotide sequence is shown in SEQ ID No.24.

[0054] Example 2

[0055] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 62 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.25.

[0056] Example 3

[0057] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.4, and the nucleotide sequence is shown in SEQ ID No.26.

[0058] Example 4

[0059] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.27.

[0060] Example 5

[0061] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.6, and the nucleotide sequence is shown in SEQ ID No.28.

[0062] Example 6

[0063] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 152 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.29.

[0064] Example 7

[0065] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-threonine at position 176 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-arginine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.8, and the nucleotide sequence is shown in SEQ ID No.30.

[0066] Example 8

[0067] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 177 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-isoleucine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.31.

[0068] Example 9

[0069] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-histidine at position 183 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-tyrosine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.10, and the nucleotide sequence is shown in SEQ ID No.32.

[0070] Example 10

[0071] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-isoleucine at position 208 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.11, and the nucleotide sequence is shown in SEQ ID No.33.

[0072] Embodiment 11

[0073] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 219 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.12, and the nucleotide sequence is shown in SEQ ID No.34.

[0074] Example 12

[0075] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.13, and the nucleotide sequence is shown in SEQ ID No.35.

[0076] Example 13

[0077] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.14, and the nucleotide sequence is shown in SEQ ID No.36.

[0078] Embodiment 14

[0079] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.15, and the nucleotide sequence is shown in SEQ ID No.37.

[0080] Embodiment 15

[0081] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 152 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.16, and the nucleotide sequence is shown in SEQ ID No.38.

[0082] Example 16

[0083] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-threonine at position 176 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-arginine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.17, and the nucleotide sequence is shown in SEQ ID No.39.

[0084] Embodiment 17

[0085] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 177 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-isoleucine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.18, and the nucleotide sequence is shown in SEQ ID No.40.

[0086] Embodiment 18

[0087] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the L-leucine at position 124 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.19, and the nucleotide sequence is shown in SEQ ID No.41.

[0088] Embodiment 19

[0089] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, the L-leucine at position 124 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.20, and the nucleotide sequence is shown in SEQ ID No.42.

[0090] Embodiment 20

[0091] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-alanine at position 152 is mutated to L-asparagine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.21, and the nucleotide sequence is shown in SEQ ID No.43.

[0092] Embodiment 21

[0093] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-threonine at position 176 is mutated to L-arginine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.22, and the nucleotide sequence is shown in SEQ ID No.44.

[0094] Embodiment 22

[0095] An embodiment of the present invention provides a PET degrading enzyme PET-mh mutant, wherein the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No. 1 is mutated to S-glycine, the L-histidine at position 183 is mutated to L-tyrosine, and the L-isoleucine at position 208 is mutated to L-threonine. The amino acid sequence of the obtained mutant is shown in SEQ ID No. 23, and the nucleotide sequence is shown in SEQ ID No. 45.

[0096] Embodiment 23

[0097] The present invention provides the use of the PET-mh mutant of the PET degrading enzyme in Examples 1 to 22 in hydrolyzing PET:

[0098] The PET to be degraded was used as a substrate, and the PET-mh mutant of the PET degrading enzyme of each example was added to 300 μL of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as the PET reaction solution, and PET degradation was performed in a water bath at 60-75° C.

[0099] Embodiment 24

[0100] The present invention provides a recombinant plasmid containing a gene encoding the PET-mh mutant of the PET degrading enzyme in Examples 1 to 22 and a construction method thereof:

[0101] Take the recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Example 1 as an example: the nucleotide sequence encoding the PET-mh mutant of the PET degrading enzyme in Example 1 is used as a template, and the corresponding primers are used to construct a single point mutation. The target gene is first exponentially amplified by PCR reaction, and then the template chain is removed by restriction endonuclease Dpn I, and the PCR product is quickly cyclized by DNA ligase to obtain a recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Example 1.

[0102] The recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Examples 2 to 22 can be constructed in the same manner.

[0103] Embodiment 25

[0104] The present invention provides an engineering bacterium containing the recombinant plasmid of Example 24 and a method for constructing the same:

[0105] The recombinant plasmid of Example 24 was transformed into BL21 (DE3) (full gold, CD601) competent cells and spread on ampicillin plate culture medium to obtain positive recombinants, which are engineered bacteria containing the recombinant plasmids in Example 24.

[0106] Embodiment 26

[0107] The present invention provides an application of the engineering bacteria in Example 25 in the degradation of PET:

[0108] The E. coli engineered bacteria constructed in Example 25 were first cultured in 5 mL LB liquid medium (containing 100 mg / L ampicillin resistance) for 12-16 h, and then transferred to a shake flask at a 1% inoculum. When the OD600 value reached the range of 0.8-1.0, the inducer IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a concentration of 0.1 mM, and the expression was induced at 16°C for 22 h.

[0109] The fermentation culture was centrifuged at 4000rpm for 20min to obtain a bacterial slurry containing the PET-mh mutant, which was resuspended by suction with a bacterial lysis buffer (each liter of bacterial lysis buffer contained 50mM Tris-HCl, 150mM NaCl and 10mM imidazole, pH = 7.5), and the cells were broken by an ultra-high pressure continuous flow cell disruptor to obtain a broken bacterial solution. The broken bacterial solution was centrifuged at 4°C and 10000rpm for 1h, and the supernatant was taken to obtain a crude enzyme solution containing the PET-mh mutant. The crude enzyme solution was filtered with a 0.45μm filter membrane, mixed with the Ni medium, and placed in a 4°C, 180rpm full-temperature shaking incubator for at least 1h. Then, the protein was purified by Ni-NTA column, and the impurities were washed with washing buffer (each liter of washing buffer contained 50mM Tris-HCl, 150mM NaCl and 20mM imidazole, pH = 7.5) to remove impurities, and then eluted with elution buffer (each liter of elution buffer contained 50mM Tris-HCl, 300mM NaCl and 300mM imidazole, pH = 7.5), and the eluted liquid was collected, i.e., the protein purified liquid. The protein purified liquid was concentrated to 5mg / mL through a protein concentrator tube, collected and stored in a 4°C refrigerator. It was then added to the catalytic system (final concentration: 10μg / mL) as a biocatalyst for degrading PET.

[0110] The PET substrate to be degraded was added to 300 μL of PET reaction solution (each liter of PET reaction solution contained 100 mM potassium phosphate buffer, pH=8.0, and PET-mh mutant protein was added to a final concentration of 10 μg / mL), and placed at 65-75°C for biocatalytic reaction.

[0111] Comparative Example 1

[0112] This comparative example provides a PET degrading enzyme mutant, in which the L-alanine at position 211 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.

[0113] Comparative Example 2

[0114] This comparative example provides a PET degrading enzyme mutant, in which the L-serine at position 212 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.

[0115] Comparative Example 3

[0116] This comparative example provides a PET degrading enzyme mutant, in which the L-proline at position 179 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-glutamic acid.

[0117] Comparative Example 4

[0118] This comparative example provides a PET degradation enzyme mutant, in which the L-serine at position 66 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-alanine.

[0119] Comparative Example 5

[0120] This comparative example provides a PET degradation enzyme mutant, in which the L-alanine at position 66 in the amino acid sequence shown in SEQ ID No. 1 is mutated to S-glycine.

[0121] Test Example 1

[0122] According to the method of Example 23, the effects of the protein with the amino acid sequence shown in SEQ ID No. 1 and the PET degrading enzyme mutants of Examples 1 to 7, 9 to 25 and Comparative Examples 1 to 5 on the degradation of low-crystallinity PET for 5 h were investigated.

[0123] A PET film (6 mm in diameter) with a crystallinity of 7.3% was used as a substrate, and a protein with an amino acid sequence as shown in SEQ ID No. 1, a PET degrading enzyme PET-mh mutant of each embodiment, or a PET degrading enzyme mutant of each comparative example was added to 300 μL of a potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution, and PET degradation was performed under a water bath condition of 60-75° C. After reacting for 5 hours, methanol was added to terminate the reaction, and the generation of TPA, BHET, and MHET was tested by reverse phase HPLC analysis.

[0124] The results are shown in Table 1.

[0125] Table 1 TPA, BHET and MHET production (low crystallinity, 5h)

[0126]

[0127]

[0128] From the above results, it can be seen that by mutating the amino acids at positions 63, 68, 124, and 183 individually, or mutating the amino acids at positions 63, 68, 124, 152, 176, 177, and 183 together, or mutating the amino acids at positions 63, 68, 152, 176, 208, and 124 and 183 together, the mutants obtained can achieve a higher degradation effect than the protein with the amino acid sequence shown in SEQ ID No. 1 when degraded for 5 hours. The yields of BHET and TPA are shown in Figure 1 to Figure 3 .

[0129] Test Example 2

[0130] This test example investigated the effect of the protein with an amino acid sequence as shown in SEQ ID No. 1 or the PET-mh mutant of the PET degrading enzyme of Examples 9, 14, 15, and 22 on the degradation of high-crystallinity PET for 5 hours according to the method of Example 23.

[0131] PET particles (100 mg) obtained by grinding a PET plastic bottle with a crystallinity of 26% were used as a substrate, and a protein with an amino acid sequence as shown in SEQ ID No. 1 or a PET degrading enzyme PET-mh mutant of each embodiment was added to 300 μL of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution. PET degradation was carried out in a water bath at 70° C. After the reaction for 5 hours, methanol was added to terminate the reaction, and the production of TPA, BHET and MHET was tested by reverse phase HPLC analysis.

[0132] The results are shown in Table 2.

[0133] Table 2 TPA, BHET and MHET production (high crystallinity, 5h)

[0134]

[0135] From the above results, it can be seen that the mutants obtained by mutating the amino acid at position 183 alone, or mutating the amino acids at positions 124, 152 and 183 together, or mutating the amino acids at positions 124, 183 and 208 together can achieve a higher degradation effect on high-crystallinity PET than the protein with the amino acid sequence shown in SEQ ID No. 1 when degrading for 5 hours. The yields of BHET and TPA are shown in Figure 4 .

[0136] Test Example 3

[0137] This test example investigates the effect of the protein with an amino acid sequence as shown in SEQ ID No. 1 or the PET-mh mutant of the PET degrading enzyme of Examples 1, 2, 6, 7, 10, and 11 on the degradation of PET with different crystallinity for 24 hours according to the method of Example 23.

[0138] Using PET with different crystallinity as a substrate, a protein with an amino acid sequence as shown in SEQ ID No. 1 or a PET-mh mutant of the PET degrading enzyme of each embodiment was added to 300 μL of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution, and PET degradation was performed in a water bath at 60-75° C. After reacting for 24 hours, methanol was added to terminate the reaction, and the amount of TPA and MHET generated was tested by reverse phase HPLC analysis.

[0139] The results are shown in Table 3.

[0140] Table 3 TPA and MHET generation (24h)

[0141]

[0142] From the above results, it can be seen that by mutating the amino acid at position 29, 62, 152, 176, 208 or 219 alone, the mutants obtained can achieve a higher degradation effect on PET of different morphologies and crystallinities than the protein with the amino acid sequence shown in SEQ ID No. 1 when degrading for 24 hours.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A PET degrading enzyme PET-mh mutant, characterized in that: The PET degrading enzyme PET-mh mutant is obtained by mutation based on the amino acid sequence shown in SEQ ID No. 1, and the mutation method is as follows: the amino acid at position 29 is mutated to L-alanine; or the amino acid at position 62 is mutated to L-threonine; or the amino acid at position 63 is mutated to L-threonine; or the amino acid at position 68 is mutated to L-asparagine; or the amino acid at position 124 is mutated to S-glycine; or the amino acid at position 152 is mutated to L-methionine; or the amino acid at position 176 is mutated to L-arginine; or the amino acid at position 20 ...177 is mutated to L-arginine; or the amino acid at position 178 is mutated to L-arginine; or the amino acid at position 179 is mutated to L-arginine; or the amino acid at position 18 The amino acid at position 8 is mutated to L-threonine; or the amino acid at position 219 is mutated to L-methionine; or the amino acid at position 63 is mutated to L-threonine and the amino acid at position 183 is mutated to L-tyrosine; or the amino acid at position 68 is mutated to L-asparagine and the amino acid at position 183 is mutated to L-tyrosine; or the amino acid at position 124 is mutated to S-glycine and the amino acid at position 183 is mutated to L-tyrosine; or the amino acid at position 152 is mutated to L-methionine and the amino acid at position 153 is mutated to L-methionine The amino acid at position 183 is mutated to L-tyrosine; or the amino acid at position 177 is mutated to L-isoleucine and the amino acid at position 183 is mutated to L-tyrosine; or the amino acid at position 124 is mutated to S-glycine, the amino acid at position 183 is mutated to L-tyrosine and the amino acid at position 63 is mutated to L-threonine; or the amino acid at position 124 is mutated to S-glycine, the amino acid at position 183 is mutated to L-tyrosine and the amino acid at position 68 is mutated to L-asparagine; or The amino acid at position 124 is mutated to S-glycine, the amino acid at position 183 is mutated to L-tyrosine, and the amino acid at position 152 is mutated to L-methionine; or the amino acid at position 124 is mutated to S-glycine, the amino acid at position 183 is mutated to L-tyrosine, and the amino acid at position 176 is mutated to L-arginine; or the amino acid at position 124 is mutated to S-glycine, the amino acid at position 183 is mutated to L-tyrosine, and the amino acid at position 208 is mutated to L-threonine.

2. Use of the PET-mh mutant of the PET degrading enzyme according to claim 1 in hydrolyzing PET, characterized in that: The PET-mh mutant of the PET degrading enzyme is added to a potassium phosphate buffer having a pH value of 7.5 to 8.5 and a final concentration of 5 to 15 μg / mL, and PET degradation is performed at 65 to 75° C. and 100 to 220 rpm.

3. A gene encoding the PET-mh mutant of the PET degrading enzyme according to claim 1.

4. The gene according to claim 3, characterized in that The amino acid sequences of the PET degrading enzyme PET-mh mutant are shown in SEQ ID No.2 to SEQ ID No.8, SEQ ID No.11 to SEQ ID No.16 and SEQ ID No.18 to SEQ ID No.23, and the nucleotide sequences thereof are shown in SEQ ID No.24 to SEQ ID No.30, SEQ ID No.33 to SEQ ID No.38 and SEQ ID No.40 to SEQ ID No.45, respectively.

5. A recombinant plasmid containing the nucleotide sequence of the gene according to claim 3 or 4.

6. The method for constructing the recombinant plasmid according to claim 5, characterized in that: Using the nucleotide sequence described in claim 4 as a template, a single point mutation is constructed using corresponding primers, the target gene is first exponentially amplified by PCR reaction, and then the template chain is removed by restriction endonuclease Dpn I, and then the PCR product is quickly cyclized using DNA ligase to obtain a recombinant plasmid.

7. An engineered bacterium constructed using the recombinant plasmid according to claim 5.

8. The engineered bacteria according to claim 7, characterized in that: The engineered bacteria is Escherichia coli.

9. The method for constructing the engineering bacteria according to claim 8, characterized in that: The recombinant plasmid described in claim 5 is transformed into BL21 (DE3) competent cells, and spread on ampicillin plate culture medium to obtain positive recombinants, which are the engineered bacteria.

10. Use of the engineered bacteria according to claim 7 or 8 in degrading PET.

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