PET hydrolase variant and application thereof

Abstract

The invention relates to the field of biotechnology and enzyme engineering, and discloses a PET hydrolase variant and application thereof. Compared with a sequence 2, the amino acid sequence of the PET hydrolase mutant has more than 96% of identity, mutations of W104L, D238K, A251C and A281C exist at the 104th site, the 238th site, the 251th site and the 281th site of the sequence 2 at the same time, and compared with PET hydrolase shown in the sequence 2, the PET hydrolase mutant has improved PET degradation catalysis activity. Compared with a wild type PET hydrolase, the mutant type PET hydrolase provided by the invention has the advantages that the PET degradation activity is obviously improved, and the mutant type PET hydrolase has application value.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and enzyme engineering, and relates to a PET hydrolase variant and application thereof. Background Art

[0002] Polyethylene terephthalate (PET) is one of the most widely used plastics. From the characteristics of PET materials, there are a certain amount of ester bonds in its molecular chain, a crystalline region inside, a high melting temperature, and its aromatic structure greatly reduces the biodegradability of PET. At the glass transition temperature (Tg = 75°C), the flexibility of the polymer chain in the amorphous region of the polymer increases, making it easier to be nucleophilically attacked by the catalytic triad. To ensure continuous degradation, most PET degrading enzymes need to exhibit extremely strong thermal stability near the glass transition temperature.

[0003] The Marty team at the University of Toulouse in France (V. Tournier, An engineered PET depolymerase to break down and recycle plastic bottles, Nature volume 580, pages 216–219 (2020)) modified the thermophilic cutinase LCC identified by the metagenomic method, improved the stability of LCC, and achieved 90% degradation of PET products. However, the remaining 10% PET residues cannot be biodegraded due to the sharp increase in crystallinity, which is an important bottleneck in the current industrial application of PET degrading enzymes.

[0004] Therefore, it is now necessary to modify PET hydrolase to significantly improve its degradation activity while ensuring its thermal stability, so as to enhance the industrial application potential of PET degrading enzyme. Summary of the invention

[0005] The purpose of the present invention is to transform PET hydrolase and improve its catalytic activity of promoting PET hydrolysis to generate TPA, MHET and BHET. Based on the previous transformation of PET hydrolase, the present invention is completed by further predicting mutants that can improve enzyme activity according to the prediction results of latent space calculation, and verifying them.

[0006] The PET hydrolase variant provided by the present invention has an amino acid sequence that is more than 96% identical to sequence 2 (a wild-type PET hydrolase derived from bacteria HR29) in the sequence list, and has mutations of W104L, D238K, A251C and A281C at positions 104, 238, 251 and 281 in sequence 2, and has improved catalytic PET degradation activity compared to the PET hydrolase shown in sequence 2;

[0007] Further, there is at least one of the following mutations: T85E, A209R, H218S, F222I, F243T, P248D and / or S287A;

[0008] More preferably, the amino acid sequence of the PET hydrolase variant, compared with Sequence 2, has or only has any one of the following mutation combinations 1)-61):

[0009] 1)T85E / W104L / A209R / H218S / D238K / F243T / A251C / A281C

[0010] 2)T85E / W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C

[0011] 3)T85E / W104L / A209R / H218S / D238K / F243T / A251C / A281C / S287A

[0012] 4)T85E / W104L / A209R / H218S / D238K / P248D / A251C / A281C

[0013] 5)T85E / W104L / A209R / H218S / D238K / P248D / A251C / A281C / S287A

[0014] 6)T85E / W104L / A209R / H218S / D238K / A251C / A281C / S287A

[0015] 7)T85E / W104L / A209R / H218S / F222I / D238K / A251C / A281C

[0016] 8)T85E / W104L / A209R / F222I / D238K / F243T / A251C / A281C

[0017] <h2 style=";text-align:left;direction:ltr">9)T85E / W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0018] <h2 style=";text-align:left;direction:ltr"> 10)T85E / W104L / A209R / F222I / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0019] <h2 style=";text-align:left;direction:ltr"> 11)T85E / W104L / A209R / F222I / D238K / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0020] <h2 style=";text-align:left;direction:ltr"> 12)T85E / W104L / A209R / F222I / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0021] <h2 style=";text-align:left;direction:ltr"> 13)T85E / W104L / A209R / F222I / D238K / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0022] <h2 style=";text-align:left;direction:ltr"> 14)T85E / W104L / A209R / D238K / F243T / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0023] <h2 style=";text-align:left;direction:ltr"> 15)T85E / W104L / A209R / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0024] <h2 style=";text-align:left;direction:ltr"> 16)T85E / W104L / A209R / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0025] <h2 style=";text-align:left;direction:ltr"> 17)T85E / W104L / A209R / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0026] <h2 style=";text-align:left;direction:ltr"> 18)T85E / W104L / H218S / F222I / D238K / F243T / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0027] <h2 style=";text-align:left;direction:ltr"> 19)T85E / W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0028] <h2 style=";text-align:left;direction:ltr"> 20)T85E / W104L / H218S / F222I / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0029] <h2 style=";text-align:left;direction:ltr"> 21)T85E / W104L / H218S / F222I / D238K / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr">

[0030] 22)T85E / W104L / H218S / F222I / D238K / P248D / A251C / A281C / S287A

[0031] 23)T85E / W104L / H218S / F222I / D238K / A251C / A281C / S287A

[0032] 24)T85E / W104L / H218S / D238K / F243T / P248D / A251C / A281C

[0033] 25)T85E / W104L / H218S / D238K / F243T / P248D / A251C / A281C / S287A

[0034] 26)T85E / W104L / H218S / D238K / F243T / A251C / A281C / S287A

[0035] 27)T85E / W104L / H218S / D238K / P248D / A251C / A281C / S287A

[0036] 28)T85E / W104L / F222I / D238K / F243T / P248D / A251C / A281C

[0037] 29)T85E / W104L / F222I / D238K / F243T / P248D / A251C / A281C / S287A

[0038] 30)T85E / W104L / F222I / D238K / F243T / A251C / A281C / S287A

[0039] 31)T85E / W104L / F222I / D238K / P248D / A251C / A281C / S287A

[0040] 32)T85E / W104L / D238K / F243T / P248D / A251C / A281C / S287A

[0041] 33)W104L / A209R / H218S / F222I / D238K / F243T / A251C / A281C

[0042] 34)W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C <h2 style=";text-align:left;direction:ltr">

[0043] <h2 style=";text-align:left;direction:ltr"> 35) W104L / A209R / H218S / F222I / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0044] <h2 style=";text-align:left;direction:ltr"> 36) W104L / A209R / H218S / F222I / D238K / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] <h2 style=";text-align:left;direction:ltr"> 37) W104L / A209R / H218S / F222I / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] <h2 style=";text-align:left;direction:ltr"> 38) W104L / A209R / H218S / F222I / D238K / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> 39)W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> 40)W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> 41) W104L / A209R / H218S / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> 42)W104L / A209R / H218S / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] <h2 style=";text-align:left;direction:ltr"> 43) W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0052] <h2 style=";text-align:left;direction:ltr"> 44) W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0053] <h2 style=";text-align:left;direction:ltr"> 45) W104L / A209R / F222I / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0054] <h2 style=";text-align:left;direction:ltr"> 46) W104L / A209R / F222I / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0055] <h2 style=";text-align:left;direction:ltr"> 47) W104L / A209R / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0056] <h2 style=";text-align:left;direction:ltr"> 48)W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0057] <h2 style=";text-align:left;direction:ltr"> 49)W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0058] <h2 style=";text-align:left;direction:ltr"> 50) W104L / H218S / F222I / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0059] <h2 style=";text-align:left;direction:ltr"> 51)W104L / H218S / F222I / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0060] <h2 style=";text-align:left;direction:ltr"> 52)W104L / H218S / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0061] <h2 style=";text-align:left;direction:ltr"> 53) W104L / F222I / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0062] <h2 style=";text-align:left;direction:ltr"> 54) W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0063] <h2 style=";text-align:left;direction:ltr"> 55)T85E / W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0064] <h2 style=";text-align:left;direction:ltr"> 56) T85E / W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0065] <h2 style=";text-align:left;direction:ltr"> 57)T85E / W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0066] <h2 style=";text-align:left;direction:ltr"> 58) T85E / W104L / A209R / H218S / F222I / D238K / P248D / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0067] <h2 style=";text-align:left;direction:ltr"> 59) T85E / W104L / A209R / H218S / F222I / D238K / F243T / A251C / A281C / S287A<h2 style=";text-align:left;direction:ltr">

[0068] 60)T85E / W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C

[0069] 61)T85E / W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A.

[0070] Among them, the wild-type PET hydrolase shown in sequence 2 is a mature protein: the term "mature protein" means a protein in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In other words, the mature protein refers to the portion of the precursor protein that is inherited after the signal peptide portion and the propeptide portion are cut off. The signal peptide portion can be predicted by programs known in the art (such as SignalP). The amino acid residues 34 to 293 of sequence 2 are the expected mature portion. In general, the first amino acid of the mature portion of the enzyme can be completed by N-terminal sequencing of the purified enzyme.

[0071] In the present invention, the "96% or more identity" can be understood as 96%, 97%, 98% or 99% sequence identity; it can also be further understood as 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.1%, 99.2%, 99.3% or 99.4%, but less than 100% sequence identity.

[0072] Here, "improved catalytic PET hydrolysis activity" means a variant that exhibits higher catalytic PET hydrolysis activity than the parent (wild-type PET hydrolase shown in Sequence 2) after storage for a period of time under conditions that exist during industrial use or transportation of the variant.

[0073] The present invention further provides a nucleic acid molecule encoding the PET hydrolase variant.

[0074] The present invention also provides a recombinant vector, an expression cassette or a recombinant cell containing the nucleic acid molecule.

[0075] The present invention further provides a method for producing the PET hydrolase variant, comprising the steps of culturing recombinant cells containing the nucleic acid molecule and then recovering the PET hydrolase variant from the cell culture.

[0076] The present invention also provides the use of the PET hydrolase variant or the nucleic acid molecule or the recombinant vector, expression cassette or recombinant cell in preparing a product having catalytic PET degradation activity.

[0077] The present invention also provides the use of the PET hydrolase variant or the nucleic acid molecule or the recombinant vector, expression cassette or recombinant cell in producing target products TPA, MHET and / or BHET.

[0078] The present invention further provides a method for producing a target product, comprising the following steps: using a PET material to react under the catalysis of the PET hydrolase variant to obtain the target product;

[0079] The target product is any one or more of the following: TPA, MHET and BHET.

[0080] Specifically, the pH value of the reaction is 7-8-8.2.0, and the temperature is 60-70° C. Preferably, the pH value of the reaction is 8.0, and the temperature is 65° C. Optionally, the step of isolating the target product is also included.

[0081] The PET hydrolase variants of the present invention may have improved catalytic PET hydrolysis activity compared to the parent PET hydrolase, wherein the improved catalytic PET hydrolysis activity is determined as an increased relative activity. In one aspect, when compared using the assay for determining the hydrolysis activity in the examples, the enzyme activity of the variant with improved catalytic hydrolysis activity is at least 1.2 times, up to about 4.4 times, that of the parent enzyme, and has great application value and prospects. DETAILED DESCRIPTION

[0082] 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.

[0083] 1. Construction of PET hydrolase engineered bacteria

[0084] The wild-type PET hydrolase gene was codon optimized using E. coli as a host cell to obtain an optimized PET hydrolase gene (i.e., the target gene), and a recombinant plasmid was constructed by chemical synthesis, the nucleotide sequence of which was SEQ ID No. 1, and the expressed amino acid sequence was SEQ ID No. 2. The chemically synthesized plasmid was transferred into E. coli BW25113 ((DE3)) competent cells and spread on an ampicillin plate culture medium to obtain a positive recombinant expression engineered bacterium.

[0085] 2. Construction of PET hydrolase mutants:

[0086] The wild-type PET hydrolase was used as a template, and its nucleotide sequence was SEQ ID No. 1, and the expressed amino acid sequence was SEQ ID No. 2. According to the prediction results of latent space calculation, a pair of complementary oligonucleotides with mutation sites were used as amplification primers (primers are shown in Table 1), and site-directed mutagenesis technology was used to perform whole plasmid PCR amplification with purchased primerstar high-fidelity enzyme (Takara) to obtain a recombinant plasmid with a specific mutation site; the constructed recombinant plasmid was transformed into E. coli Top10 to obtain a transformation product, which was spread on an ampicillin plate culture medium and inverted in a 37°C constant temperature incubator for 8 to 12 hours to obtain a transformant. The transformant was picked and inoculated into LB liquid culture medium, and the plasmid was extracted for sequencing verification after shaking the flask at 37°C and 200rpm for 8 to 12 hours to obtain a recombinant plasmid expressing different PET hydrolase mutants.

[0087]

[0088]

[0089] 3. PET hydrolase protein expression and purification

[0090] Pick some engineering bacterial colonies and inoculate them in 5mL Luria-Bertani liquid medium (containing Amp), shake and culture at 37°C, 200rpm for 4-6h, then inoculate them in 2YT medium (containing Amp) at 1% inoculum, place them on a shaker at 37°C, 200rpm, culture for about 5h (OD600 is 0.8-1.0), add 0.2% (w / v) L-arabinose, and induce at 20°C for 18-20h. Centrifuge the overnight induced cultured bacterial solution, centrifuge at 4°C, 10000rpm for 10min, discard the supernatant to collect the wet cells of the engineering bacteria, resuspend the bacteria with protein purification Buffer A, and then centrifuge at 4°C, 10000rpm for 1h to remove cell debris and collect the supernatant. Filter with a 0.22μm filter and place on ice. Use His Trap HP chromatographic column for purification, wash with deionized water for 5 column volumes, and then balance the column with protein purification Buffer A for 5-10 column volumes. The filtered supernatant was loaded at a flow rate of 5 mL / min, and the unbound proteins were washed with protein purification Buffer A, and then the impurities were washed with low-concentration imidazole protein purification Buffer B. The target protein was eluted with high-concentration imidazole protein purification Buffer B and collected. The buffer of the target protein was exchanged with protein purification Buffer C using a HiPrep desalting column to remove imidazole. 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 and placed at 4°C for later use.

[0091] 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: 50mM Na2HPO4·12H2O, 100mM NaCl, 30mMImidazole, pH=7.5; the formula of the washing buffer is Buffer B: 50mM Na2HPO4·12H2O, 100mMNaCl, 300mM Imidazole, pH=7.5; the formula of the elution buffer is Buffer C: 50mM Na2HPO4·12H2O, 100mM NaCl, pH=7.5.

[0092] 4. Relative enzyme activity determination

[0093] The polyethylene terephthalate (PET) plastic involved in the following examples was purchased from Goodfellow, and polyethylene terephthalate (BHET), hydroxyethyl terephthalate (MHET) and terephthalic acid (TPA) were purchased from Sigma. Phosphate buffer (1M pH 8.0): Accurately weigh 2.8 g of K2HPO4·3H2O2 and 13.6 g of KH2PO4, add about 100 mL of deionized water and stir to dissolve, and weigh 96 mL of 1M K2HPO4 and 4 mL of 1M KH2PO4 to 100 mL. PET was added to 1M phosphate buffer with a pH value of 8.0 to a final concentration of 30 g / L, 30 μg of wild-type PET hydrolase or its variant protein was mixed with substrate PET, and the reaction was stirred at 65°C for 3.0 h. The yields of TPA, MHET and BHET in the obtained reaction solution were detected by HPLC to determine its catalytic PET degradation activity.

[0094] Relative enzyme activity is the ratio of the enzyme activity of the PET hydrolase variant to the enzyme activity of the wild-type PET hydrolase. The relative enzyme activity results of the PET hydrolase variants are shown in Table 2.

[0095] Table 2. Mutation sites of PET hydrolase variant proteins and relative activity determination results of PET degradation

[0096]

[0097]

[0098] The results showed that compared with the wild-type PET hydrolase, the enzymatic activity of the obtained mutants increased by at least 1.4 times, many mutants increased by 2-4 times, and some mutants increased by more than 4 times, with the highest reaching nearly -4.4 times.

Claims

1. A PET hydrolase variant, characterized in that Its amino acid sequence has more than 96% identity with that of sequence 2, and mutations W104L, D238K, A251C and A281C exist simultaneously at positions 104, 238, 251 and 281 of sequence 2, and it has improved catalytic PET degradation activity compared with the PET hydrolase shown in sequence 2.

2. The PET hydrolase variant according to claim 1, characterized in that There is also at least one of the following mutations: T85E, A209R, H218S, F222I, F243T, P248D and / or S287A; More preferably, the amino acid sequence of the PET hydrolase variant, compared with Sequence 2, has or only has any one of the following mutation combinations 1)-61): 1) T85E / W104L / A209R / H218S / D238K / F243T / A251C / A281C 2) T85E / W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C 3) T85E / W104L / A209R / H218S / D238K / F243T / A251C / A281C / S287A 4) T85E / W104L / A209R / H218S / D238K / P248D / A251C / A281C 5) T85E / W104L / A209R / H218S / D238K / P248D / A251C / A281C / S287A 6) T85E / W104L / A209R / H218S / D238K / A251C / A281C / S287A 7) T85E / W104L / A209R / H218S / F222I / D238K / A251C / A281C 8) T85E / W104L / A209R / F222I / D238K / F243T / A251C / A281C 9) T85E / W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C 10) T85E / W104L / A209R / F222I / D238K / F243T / A251C / A281C / S287A 11) T85E / W104L / A209R / F222I / D238K / P248D / A251C / A281C 12) T85E / W104L / A209R / F222I / D238K / P248D / A251C / A281C / S287A 13) T85E / W104L / A209R / F222I / D238K / A251C / A281C / S287A 14) T85E / W104L / A209R / D238K / F243T / P248D / A251C / A281C 15) T85E / W104L / A209R / D238K / F243T / P248D / A251C / A281C / S287A 16)T85E / W104L / A209R / D238K / F243T / A251C / A281C / S287A 17)T85E / W104L / A209R / D238K / P248D / A251C / A281C / S287A 18)T85E / W104L / H218S / F222I / D238K / F243T / A251C / A281C 19)T85E / W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C 20)T85E / W104L / H218S / F222I / D238K / F243T / A251C / A281C / S287A 21)T85E / W104L / H218S / F222I / D238K / P248D / A251C / A281C 22)T85E / W104L / H218S / F222I / D238K / P248D / A251C / A281C / S287A 23)T85E / W104L / H218S / F222I / D238K / A251C / A281C / S287A 24)T85E / W104L / H218S / D238K / F243T / P248D / A251C / A281C 25)T85E / W104L / H218S / D238K / F243T / P248D / A251C / A281C / S287A 26)T85E / W104L / H218S / D238K / F243T / A251C / A281C / S287A 27)T85E / W104L / H218S / D238K / P248D / A251C / A281C / S287A 28)T85E / W104L / F222I / D238K / F243T / P248D / A251C / A281C 29)T85E / W104L / F222I / D238K / F243T / P248D / A251C / A281C / S287A 30)T85E / W104L / F222I / D238K / F243T / A251C / A281C / S287A 31)T85E / W104L / F222I / D238K / P248D / A251C / A281C / S287A 32)T85E / W104L / D238K / F243T / P248D / A251C / A281C / S287A 33)W104L / A209R / H218S / F222I / D238K / F243T / A251C / A281C 34)W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C <h2 style=";text-align:left;direction:ltr">35)W104L / A209R / H218S / F222I / D238K / F243T / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 36)W104L / A209R / H218S / F222I / D238K / P248D / A251C / A281C <h2 style=";text-align:left;direction:ltr"> 37)W104L / A209R / H218S / F222I / D238K / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 38)W104L / A209R / H218S / F222I / D238K / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 39)W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C <h2 style=";text-align:left;direction:ltr"> 40)W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 41)W104L / A209R / H218S / D238K / F243T / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 42)W104L / A209R / H218S / D238K / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 43)W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C <h2 style=";text-align:left;direction:ltr"> 44)W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 45)W104L / A209R / F222I / D238K / F243T / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 46)W104L / A209R / F222I / D238K / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 47)W104L / A209R / D238K / F243T / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 48)W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C <h2 style=";text-align:left;direction:ltr"> 49)W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 50)W104L / H218S / F222I / D238K / F243T / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 51)W104L / H218S / F222I / D238K / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 52)W104L / H218S / D238K / F243T / P248D / A251C / A281C / S287A <h2 style=";text-align:left;direction:ltr"> 53)W104L / F222I / D238K / F243T / P248D / A251C / A281C / S287A 54) W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A 55) T85E / W104L / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A 56) T85E / W104L / A209R / F222I / D238K / F243T / P248D / A251C / A281C / S287A 57) T85E / W104L / A209R / H218S / D238K / F243T / P248D / A251C / A281C / S287A 58) T85E / W104L / A209R / H218S / F222I / D238K / P248D / A251C / A281C / S287A 59) T85E / W104L / A209R / H218S / F222I / D238K / F243T / A251C / A281C / S287A 60) T85E / W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C 61) T85E / W104L / A209R / H218S / F222I / D238K / F243T / P248D / A251C / A281C / S287A.

3. A nucleic acid molecule encoding the PET hydrolase variant according to claim 1 or 2.

4. A recombinant vector, expression cassette or recombinant cell containing the nucleic acid molecule of claim 3.

5. A method for producing the PET hydrolase variant of claim 1 or 2, comprising the steps of culturing a recombinant cell containing the nucleic acid molecule of claim 3, and then recovering the PET hydrolase variant from the cell culture.

6. Use of the PET hydrolase variant according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the recombinant vector, expression cassette or recombinant cell according to claim 4 in the preparation of a product having catalytic PET degradation activity.

7. Use of the PET hydrolase variant according to claim 1 or 2, or the nucleic acid molecule according to claim 3, or the recombinant vector, expression cassette or recombinant cell according to claim 4 in producing target products TPA, MHET and / or BHET.

8. A method for producing a target product, characterized in that: The method comprises the following steps: using PET material to react under the catalysis of the PET hydrolase variant according to claim 1 or 2 to obtain the target product; The target product is any one or more of the following: TPA, MHET and BHET.

9. The method according to claim 8, characterized in that The pH value of the reaction is 7-8-8.2.0 and the temperature is 60-70°C.

10. The method according to claim 9, characterized in that The pH value of the reaction was 8.0 and the temperature was 65°C; The method also includes the step of isolating the target product.

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