PET polylipase LCC-D, coding gene, recombinant plasmid, recombinant engineering bacterium and application

Abstract

The invention belongs to the technical field of enzyme engineering and bioengineering, and discloses PET polylipase LCC-D, a coding gene, a recombinant plasmid, a recombinant engineering bacterium and application. The amino acid sequence of the PET polylipase LCC-D provided by the invention is as shown in SEQ ID NO.1, the PET polylipase LCC-D is obtained by mutating Asn at the 213th site of the PET polylipase LCC into Asp, and the nucleotide sequence for coding the PET polylipase LCC-D is as shown in SEQ ID NO.2. The invention also provides a preparation method of the PET polylipase LCC-D. The invention also provides an application of the PET polylipase LCC-D in hydrolysis of PET. Compared with the PET polylipase LCC, the activity of the PET polylipase LCC-D and the efficiency of degrading PET with various lengths are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and bioengineering, and specifically relates to PET polyester enzyme LCC-D, encoding gene, recombinant plasmid and recombinant engineering bacteria and application. Background Art

[0002] PET (polyethylene terephthalate) is the most widely used polyester plastic, composed of terephthalic acid (TPA) and ethylene glycol (EG) derived from crude oil connected by ester bonds. PET has the characteristics of simple synthesis, low price, strong and durable. PET products include fiber-grade PET and non-fiber-grade PET (such as bottles, films and engineering plastics, etc.), among which packaging is the largest non-fiber-grade application market for PET and the fastest growing field. Currently, most bottled water and soft drinks are filled with PET, and PET is also more widely used in the packaging of food, medicine, cosmetics and daily chemicals. However, a large amount of PET is thrown into the environment through production or waste treatment, accumulating in the global ecosystem and causing serious environmental damage.

[0003] PET has a high proportion of aromatic terephthalate units, which reduces chain mobility. Therefore, it is a polyester that is extremely difficult to hydrolyze and can exist in nature for 16-48 years. At present, the main treatment methods for PET plastics are landfill, incineration, pyrolysis and chemical degradation, which all cause secondary pollution to the environment. More than 40 million tons of PET plastics are abandoned or landfilled in nature each year, causing extremely serious global pollution.

[0004] In recent years, biological treatment technology has been gradually applied to the treatment of waste plastics due to its high efficiency, low economic cost, and green environmental protection. At present, good research progress has been made in the field of enzymatic degradation of PET plastics, which provides the possibility for further industrial recycling. In addition, researchers such as Avantika Singh believe that TPA (rTPA) recovered by enzymatic method may be cost competitive. In addition to favorable long-term socioeconomic benefits, rTPA can also reduce total supply chain energy use by 69%-83% and greenhouse gas emissions by 17%-43% per kilogram of TPA. An overall economic assessment for the United States estimates that the TPA recovery process can reduce environmental impact by up to 95% compared with non-enzymatic rTPA production, while generating up to 45% socioeconomic benefits.

[0005] To date, many PET polyesterases have been identified, such as esterases, lipases or cutinases LCC, among which cutinases LCC are the most widely studied plastic degrading enzymes. In order to improve the efficiency of PET polyesterases for recycling PET and reduce costs, we need to further increase the reaction rate of PET polyesterases. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the present invention provides PET polyester enzyme LCC-D, encoding gene, recombinant plasmid and recombinant engineering bacteria and application.

[0007] Based on the above purpose, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a PET lipase LCC-D, the amino acid sequence of the PET lipase LCC-D is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1:

[0010]

[0011] Preferably, the PET lipase LCC-D is prepared by mutating the Asn at position 213 of the PET lipase LCC to Asp.

[0012] The second aspect of the present invention provides a nucleic acid molecule encoding the PET lipase LCC-D described in the first aspect.

[0013] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2.

[0014] SEQ ID NO.2:

[0015]

[0016] The third aspect of the present invention provides an expression cassette, a recombinant vector or a recombinant engineered bacterium containing the nucleic acid molecule described in the second aspect.

[0017] Preferably, the recombinant vector is the recombinant plasmid pET-22b-LCC-D.

[0018] Preferably, the recombinant engineered bacteria is Escherichia coli or Vibrio natriuresis; more preferably, the recombinant engineered bacteria is BL21(DE3) / pET-22b-LCC-D.

[0019] The fourth aspect of the present invention provides the use of the PET polyesterase LCC-D described in the first aspect in catalytic degradation of polyester plastics.

[0020] Preferably, the polyester plastic is 3PET or PET.

[0021] Preferably, the reaction conditions for the PET polyesterase LCC-D to catalyze the degradation of 3PET are: pH 6.0-10.0, temperature 50°C-65°C.

[0022] Preferably, the 3PET is 3PET gel.

[0023] Preferably, the operation of catalyzing the degradation of 3PET by the PET polyesterase LCC-D is:

[0024] 3PET was added to a buffer solution with a pH value of 6.0-10.0, followed by PET lipase LCC-D, and the mixture was incubated at 50°C-65°C.

[0025] Preferably, the buffer is a potassium phosphate buffer with a pH value of 6.0-8.0 or a glycine buffer with a pH value of 9.0-10.0; more preferably, the buffer is a potassium phosphate buffer with a pH value of 8.0.

[0026] Preferably, the incubation temperature is 65°C.

[0027] Preferably, the incubation time is 4h-168h; more preferably, the incubation time is 40h.

[0028] Preferably, the buffer concentration is 5mM-500mM; more preferably, the buffer concentration is 100mM.

[0029] Preferably, after the addition of PET polyester lipase LCC-D, the final concentration of PET polyester lipase LCC-D in the reaction system is 10nM-1000nM; more preferably, after the addition of PET polyester lipase LCC-D, the final concentration of PET polyester lipase LCC-D in the reaction system is 400nM.

[0030] Preferably, the reaction conditions for the PET polyesterase LCC-D to catalyze the degradation of PET are: pH 6.0-10.0, temperature 55°C-85°C.

[0031] Preferably, the PET is commercial packaging PET.

[0032] Preferably, the operation of catalyzing the degradation of PET by the PET polyesterase LCC-D is:

[0033] Add PET fragments to a buffer solution with a pH value of 6.0-10.0, then add PET lipase LCC-D, and incubate at 55°C-85°C.

[0034] Preferably, the buffer is a potassium phosphate buffer with a pH value of 6.0-8.0 or a glycine buffer with a pH value of 9.0-10.0.

[0035] Preferably, the incubation time is 4h-48h; more preferably, the incubation time is 20h.

[0036] Preferably, the incubation speed is 60rpm-600rpm; more preferably, the incubation speed is 300rpm.

[0037] Preferably, the buffer concentration is 5mM-500mM; more preferably, the buffer concentration is 100mM.

[0038] Preferably, after the addition of PET polyester lipase LCC-D, the final concentration of PET polyester lipase LCC-D in the reaction system is 10nM-1000nM; more preferably, after the addition of PET polyester lipase LCC-D, the final concentration of PET polyester lipase LCC-D in the reaction system is 400nM.

[0039] The fifth aspect of the present invention provides the use of the expression cassette, recombinant vector or recombinant engineering bacteria described in the third aspect in catalytic degradation of polyester plastics.

[0040] Preferably, the recombinant vector is the recombinant plasmid pET-22b-LCC-D.

[0041] Preferably, the recombinant engineered bacteria is Escherichia coli or Vibrio natriuresis; more preferably, the recombinant engineered bacteria is BL21(DE3) / pET-22b-LCC-D.

[0042] Preferably, the polyester plastic is 3PET or PET.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The invention uses PET polyester lipase LCC for molecular modification, uses recombinant plasmid pET-22b-LCC as a template to obtain recombinant plasmid pET-22b-LCC-D, transforms the recombinant plasmid pET-22b-LCC-D into BL21 (DE3) competent cells, successfully constructs expression engineering bacteria BL21 (DE3) / pET-22b-LCC-D containing recombinant plasmid pET-22b-LCC-D, and then induces expression of the obtained BL21 (DE3) / pET-22b-LCC-D, breaks the cells, and adsorbs and purifies the target protein to finally obtain a PET polyester lipase LCC mutant enzyme named PET polyester lipase LCC-D, wherein the PET polyester lipase LCC-D mutates the Asn at position 213 of the PET polyester lipase LCC to Asp. Compared with the PET polyester lipase LCC, the activity of the PET polyester lipase LCC-D and the efficiency of degrading PET of various lengths are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pET-22b-LCC;

[0046] Figure 2 This is a schematic diagram of the structure of the recombinant plasmid pET-22b-LCC-D;

[0047] Figure 3 Schematic diagram of PET polyesterase LCC-D hydrolyzing 3PET;

[0048] Figure 4 This is the result diagram of the content change of 3PET during the degradation of 3PET by PET lipase LCC and PET lipase LCC-D;

[0049] Figure 5 It is the standard curve diagram of standard substances MHET and TPA;

[0050] Figure 6 This is a graph showing the results of MHET and TPA concentration detection in the reaction system when PET polyester enzyme LCC and PET polyester enzyme LCC-D catalyze the degradation of commercial packaging PET under different temperature conditions;

[0051] Figure 7 This is the result of detecting MHET and TPA concentrations in the reaction system when PET lipase LCC and PET lipase LCC-D catalyze the degradation of commercial packaging PET under different pH conditions. DETAILED DESCRIPTION

[0052] 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 through embodiments in combination with the accompanying drawings. 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.

[0053] The test methods described in the following examples are all conventional techniques in the art, or are based on the conditions recommended by the manufacturers unless otherwise specified; the reagents, materials and instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0054] Among them, 3PET is ethylene terephthalate trimer; PET is polyethylene terephthalate; BHET is dihydroxyethyl terephthalate; MHET is mono(2-hydroxyethyl) terephthalate; TPA is terephthalic acid; HEB is hydroxyethyl benzoate; BA is benzoic acid; EG is ethylene glycol.

[0055] Example 1: Preparation of recombinant plasmid pET-22b-LCC-D containing the gene encoding PET lipase LCC-D

[0056] The preparation steps of the recombinant plasmid pET-22b-LCC-D containing the gene encoding PET polylipase LCC-D are as follows:

[0057] According to the requirements of Agilent Technologies QuikChange II Site-Directed Mutagenesis Kit, the PET lipase LCC gene (wherein the amino acid sequence of PET lipase LCC is shown in SEQ ID NO.3, and the gene sequence is shown in SEQ ID NO.4) was used as a template to design primers as follows:

[0058] N213D-F: 5′-TCATTTTGCGCCGAACAGTGATAACGCGGCGATTAGTGTG-3′ (introducing a mutation at position 213) (SEQ ID NO. 5),

[0059] N213D-R: 5′-CACACTAATCGCCGCGTTATCACTGTTCGGCGCAAAATGA-3′ (mutation introduced into position 213) (SEQ ID NO. 6).

[0060] SEQ ID NO.3:

[0061]

[0062] SEQ ID NO.4:

[0063]

[0064] According to the requirements of the recombination kit, PCR technology is used to construct a recombinant plasmid containing a mutant gene, which is mainly divided into three steps:

[0065] (1) The recombinant plasmid pET-22b-LCC (purchased from Gene Universal, catalog number 7A03986) (SEQ ID NO. 4) was used as a template. Its structure is as follows Figure 1 As shown, N213D-F (SEQ ID NO.5) and N213D-R (SEQ ID NO.6) were used as primers for PCR amplification; (2) DpnI enzyme was added to digest the PCR product; (3) the product was transformed into DH5a competent cells, and the recombinant plasmid pET-22b-LCC-D encoding PET polyesterase LCC-D was obtained by ampicillin plate culture medium screening, plasmid extraction and sequencing verification. Its structure is shown in Figure 2 shown.

[0066] The formula of the ampicillin plate culture medium is: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 15 g / L agar powder, and 50 mg / L ampicillin; the PCR reaction system and PCR program are carried out according to the requirements of the Agilent Technologies QuikChange II Site-Directed Mutagenesis Kit, as shown in Table 1:

[0067] Table 1

[0068]

[0069] Example 2

[0070] This embodiment provides a method for constructing an engineered bacterium BL21(DE3) / pET-22b-LCC-D.

[0071] Example 2-1

[0072] The construction of the BL21 (DE3) / pET-22b-LCC-D engineering bacteria comprises the following steps: transforming the recombinant plasmid pET-22b-LCC-D constructed in Example 1 into BL21 (DE3) competent cells, spreading them on ampicillin plate culture medium, and obtaining positive recombinants BL21 (DE3) / pET-22b-LCC-D, that is, successfully constructing an expression engineering bacteria containing the recombinant plasmid pET-22b-LCC-D.

[0073] Comparative Example 2-1

[0074] The method of Comparative Example 2-1 is similar to that of Example 2-1, except that the recombinant plasmid pET-22b-LCC-D is replaced with pET-22b-LCC, and finally the expression engineering bacteria BL21 (DE3) / pET-22b-LCC containing the recombinant plasmid pET-22b-LCC are obtained.

[0075] Example 3

[0076] This embodiment provides a method for obtaining PET lipase LCC-D.

[0077] Example 3-1

[0078] Engineering bacteria BL21 (DE3) / pET-22b-LCC-D induced expression and target protein purification:

[0079] The engineered bacteria BL21(DE3) / pET-22b-LCC-D obtained in Example 2-1 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 r / min. The overnight culture solution was inoculated into fresh LB liquid medium at 1.5% inoculum, cultured at 37°C and 220 r / min until OD600 was about 0.6, 0.5% (v / v) IPTG was added, and the temperature was lowered to 20°C to induce expression for 16 hours. The wet cells of the engineered bacteria were collected by centrifugation at 8000g for 5 minutes.

[0080] The wet cells of the engineered bacteria were resuspended in a lysis buffer and the resuspended wet cells were lysed using an ultrasonic cell disruptor. After lysis, the bacterial solution was centrifuged at 18000g for 45 minutes to remove cell debris, the supernatant was adsorbed with a Ni-NTA column, and the non-specifically adsorbed impurities were washed with a washing buffer. The target protein was eluted with an elution buffer, and the eluate was concentrated with a protein concentrator to obtain PET polylipase LCC-D.

[0081] Among them, the formula of the LB liquid culture medium is: 5g / L yeast extract, 10g / L tryptone, and 10g / L sodium chloride; the formula of the lysis buffer is: 100mM potassium phosphate, 30mM imidazole, pH 8.0; the formula of the wash buffer is: 100mM potassium phosphate, 30mM imidazole, pH 8.0; the formula of the elution buffer is: 100mM potassium phosphate, 30mM to 300mM imidazole, pH 8.0.

[0082] Comparative Example 3-1

[0083] Comparative Example 3-1 is similar to Example 3-1, except that the engineered bacteria BL21 (DE3) / pET-22b-LCC-D is replaced with the engineered bacteria BL21 (DE3) / pET-22b-LCC obtained in Comparative Example 2-1, and PET polyesterase LCC is finally obtained.

[0084] Example 4

[0085] This example discusses the application of PET polyester lipase LCC-D in catalytic degradation of 3PET, and the reaction rate is tested using PET polyester lipase LCC as a control.

[0086] Example 4-1

[0087] Add 200 μL of 100 mM potassium phosphate buffer with a pH value of 8.0 to the 3PET gel; add the PET polylipase LCC-D obtained in Example 3-1 to a final concentration of 400 nM; incubate at 65°C for 40 hours, and detect the absorbance of the reaction solution at 405 m after incubation for 4, 8, 16, 20, 24, and 40 hours.

[0088] The schematic diagram of the process of PET polyesterase LCC-D degrading 3PET is as follows: Figure 3 shown.

[0089] The preparation method of 3PET gel is as follows:

[0090] 50 mL of acetone was added to 1.2 g of 3PET, and then Triton X-10 was added to make the final concentration of 2%, and then heated until dissolved. After dissolution, 50 mL of 100 mM potassium phosphate buffer with a pH value of 8.0 was added, and ultrasonic treatment was performed. After ultrasonication, it was placed at room temperature and stirred until the acetone was completely evaporated, and then agar was added to make the final concentration of 2%, and a mixed solution was obtained by heating; then 100 μL of the prepared mixed solution was added to each well of a 96-well plate, and solidified into 3PET gel at room temperature. Among them, 3PET was produced by Shanghai Runnuo Biotechnology Co., Ltd., and the nature and purity of the product were verified by mass spectrometry and HPLC.

[0091] Comparative Example 4-1

[0092] Comparative Example 4-1 is substantially the same as Example 4-1, except that PET lipase LCC-D is replaced by PET lipase LCC obtained in Comparative Example 3-1.

[0093] The absorbance test results are as follows Figure 4 As shown, the absorbance at 405 nm of the reaction solution treated with PET polyester enzyme LCC-D was 1.214, 1.118, 0.916, 0.816, 0.786 and 0.504 respectively when incubated for 4, 8, 16, 20, 24 and 40 hours, and the standard curve equation was y = -0.0197x + 1.2592, with a slope of 0.0197, so the initial rate was 0.0197 / hr. When the reaction solution treated with PET polyesterase LCC was incubated for 4, 8, 16, 20, 24, and 40 hours, the absorbance at 405 nm was 1.181, 1.097, 1.071, 0.936, 0.949, and 0.708, respectively, and the standard curve equation y=-0.0127x+1.2282 was obtained, and its slope was 0.0127, so its initial rate was 0.0127 / hr.

[0094] The results showed that the efficiency of PET lipase LCC-D in degrading 3PET was increased by about 55% compared with that of PET lipase LCC.

[0095] Example 5

[0096] This example discusses the application of PET polyester lipase LCC-D in catalytic degradation of commercial packaging PET under different temperature conditions, and the production of MHET and TPA is tested with PET polyester lipase LCC as a control.

[0097] Example 5-1

[0098] Two pieces of PET with a diameter of 6 mm were added to a 100 mM potassium phosphate buffer solution with a pH value of 8.0, and the PET polylipase LCC-D obtained in Example 3-1 was added to make the final concentration 400 nM; the mixture was incubated at 55°C and 300 rpm for 20 hours. After the incubation, the yields of MHET and TPA in the reaction solution were detected by HPLC, and the detection conditions of MHET and TPA were the same.

[0099] Example 5-2

[0100] The contents of Example 5-2 are basically the same as those of Example 5-1, except that the incubation temperature of 55°C is replaced by 65°C.

[0101] Example 5-3

[0102] The contents of Example 5-3 are basically the same as those of Example 5-1, except that the incubation temperature of 55°C is replaced by 75°C.

[0103] Example 5-4

[0104] The content of Example 5-4 is basically the same as that of Example 5-1, except that the incubation temperature of 55°C is replaced by 85°C.

[0105] Comparative Example 5-1

[0106] Comparative Example 5-1 is substantially the same as Example 5-1, except that PET polyester lipase LCC-D is replaced by PET polyester lipase LCC obtained in Comparative Example 3-1.

[0107] Comparative Example 5-2

[0108] Comparative Example 5-2 is substantially the same as Example 5-2, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0109] Comparative Example 5-3

[0110] Comparative Example 5-3 is substantially the same as Example 5-3, except that PET lipase LCC-D is replaced by PET lipase LCC obtained in Comparative Example 3-1.

[0111] Comparative Example 5-4

[0112] Comparative Example 5-4 is substantially the same as Example 5-4, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0113] The HPLC detection conditions are: UV detector, characteristic absorption peak of 240nm, ZORBAX Eclipse PlusC18 reverse phase chromatographic column (5μL, 250mm×4.6mm), mobile phase of 60% methanol, running for 60min, column temperature of 25°C, injection volume of 5μL, flow rate of 0.1mL / min.

[0114] Among them, the concentration gradient of standard MHET and TPA is set as follows:

[0115] MHET was purchased from BLD pharm, with 100% methanol as the solvent, and the MHET concentration gradient was set to 25uM, 50uM, 100uM, 200uM, 400uM, 800uM, 1.6mM, and 3.2mM; TPA was purchased from BLD pharm, with 100% methanol as the solvent, and the TPA concentration gradient was set to 25uM, 50uM, 100uM, 200uM, 400uM, 800uM, 1.6mM, and 3.2mM.

[0116] The standard curve is as follows Figure 5 As shown, when the MHET concentration is 25uM, 50uM, 100uM, 200uM, 400uM, 800uM, 1.6mM, 3.2mM, the peak area of ​​absorbance at 240nm is 401.1, 777.8, 1540.2, 2944.7, 5523.9, 10338.6, 20273.3, 38061.8. The standard curve equation y=11882x+513.94 is obtained. When the TPA concentration is 25uM, 50uM, 100uM, 200uM, 400uM, 800uM, 1.6mM, 3.2mM, the peak area of ​​absorbance at 240nm is 298.5, 615.2, 1202.4, 2292.4, 4654.6, 9252.1, 16799.8, 33174.8. The standard curve equation y=10323x+309.94 is obtained.

[0117] The yield results of MHET and TPA are as follows Figure 6As shown, it can be seen that under the conditions of 55°C, 65°C, 75°C and 85°C, the MHET yields in the reaction solution obtained by degrading PET using PET polyester lipase LCC-D were 275uM, 591uM, 796uM and 0.0uM, respectively, and the TPA yields were 344uM, 536uM, 1.209mM and 27uM, respectively, and the total yields of MHET and TPA were 619uM, 1.127mM, 2.006mM and 27uM; in the reaction solution obtained by degrading PET using PET polyester lipase LCC, the MHET yields were 246uM, 612uM, 657uM and 0.0uM, respectively, and the TPA yields were 266uM, 441uM, 1.255mM and 19uM, respectively, and the total yields of MHET and TPA were 512uM, 1.053mM, 1.912mM and 19uM.

[0118] It can be seen that the optimum temperature of PET lipase LCC-D and PET lipase LCC is 75°C; and when the temperature is 65°C, the output of MHET is higher than that of TPA, and when the temperature is 55°C, 75°C, and 85°C, the output of TPA is higher than that of MHET; at the same time, at the temperature of 55°C, 65°C, 75°C, and 85°C, compared with PET lipase LCC, the degradation efficiency of PET lipase LCC-D increased by about 21%, 7%, 5%, and 42%, respectively.

[0119] The results showed that at temperatures of 55℃, 65℃, 75℃ and 85℃, the activity of PET lipase LCC-D was higher than that of PET lipase LCC.

[0120] Example 6

[0121] This example discusses the application of PET lipase LCC-D in catalytic degradation of commercial packaging PET under different pH conditions, and the production of MHET and TPA is tested using PET lipase LCC as a control.

[0122] Example 6-1

[0123] Two pieces of PET with a diameter of 6 mm were added to a 100 mM potassium phosphate buffer solution with a pH value of 6.0, and the PET polylipase LCC-D obtained in Example 3-1 was added to make the final concentration 400 nM; the mixture was incubated at 75°C and 300 rpm for 20 hours. After the incubation, the yields of MHET and TPA in the reaction solution were detected by HPLC.

[0124] Example 6-2

[0125] The content of Example 6-2 is basically the same as that of Example 6-1, except that the potassium phosphate buffer with a pH of 6.0 is replaced with a potassium phosphate buffer with a pH of 7.0.

[0126] Example 6-3

[0127] The content of Example 6-3 is basically the same as that of Example 6-1, except that the potassium phosphate buffer with a pH of 6.0 is replaced with a potassium phosphate buffer with a pH of 8.0.

[0128] Example 6-4

[0129] The content of Example 6-4 is basically the same as that of Example 6-1, except that the potassium phosphate buffer with a pH of 6.0 is replaced by a glycine buffer with a pH of 9.0.

[0130] Example 6-5

[0131] The content of Example 6-5 is basically the same as that of Example 6-1, except that the potassium phosphate buffer with a pH of 6.0 is replaced by a glycine buffer with a pH of 10.0.

[0132] Comparative Example 6-1

[0133] Comparative Example 6-1 is substantially the same as Example 6-1, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0134] Comparative Example 6-2

[0135] Comparative Example 6-2 is substantially the same as Example 6-2, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0136] Comparative Example 6-3

[0137] Comparative Example 6-3 is substantially the same as Example 6-3, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0138] Comparative Example 6-4

[0139] Comparative Example 6-4 is substantially the same as Example 6-4, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0140] Comparative Example 6-5

[0141] Comparative Example 6-5 is substantially the same as Example 6-5, except that PET lipase LCC-D is replaced with PET lipase LCC obtained in Comparative Example 3-1.

[0142] The detection conditions and standard curve of HPLC are the same as those in Example 5.

[0143] The yield results of MHET and TPA are as follows Figure 7 As shown, it can be seen that under the conditions of pH 6.0, pH 7.0, pH 8.0, pH 9.0, and pH 10.0, the MHET production in the reaction solution obtained by degrading PET using PET lipase LCC-D was 169uM, 722uM, 796uM, 394uM, and 230uM, and the TPA production was 49uM, 459uM, 1.209mM, 1.204mM, and 96uM, respectively. The total production of MHET and TPA was 218uM, 1.181mM, 2.006mM, 1.634mM, and 325uM; in the reaction solution obtained by degrading PET using PET polyesterase LCC, the production of MHET was 99uM, 352uM, 657uM, 118uM, and 167uM, and the production of TPA was 21uM, 191uM, 1.255mM, 1.002mM, and 127uM, respectively; the total production of MHET and TPA was 120uM, 543uM, 1.912mM, 1.120mM, and 294uM.

[0144] It can be seen that the optimum pH value of PET lipase LCC-D and PET lipase LCC is 8.0; and when the pH value is 6.0 and 7.0, the output of MHET is higher than that of TPA, and when the pH value is 8.0 and 9.0, the output of TPA is higher than that of MHET; at the same time, under the conditions of pH 6.0, pH 7.0, pH 8.0, pH 9.0, and pH 10.0, compared with PET lipase LCC, the degradation efficiency of PET lipase LCC-D increased by 82%, 118%, 5%, 46%, and 11%, respectively.

[0145] The results showed that at 70℃ and pH 6.0, pH 7.0, pH 8.0, pH 9.0 and pH 10.0, the activity of PET lipase LCC-D was higher than that of PET lipase LCC.

[0146] In summary, the PET lipase LCC-D obtained by the present invention mutates the Asn at position 213 of the PET lipase LCC to Asp. Compared with the PET lipase LCC, the activity of the PET lipase LCC-D and the efficiency of degrading PET of various lengths are improved.

[0147] The above is an explanation of the embodiments of the present invention. By describing the disclosed embodiments, professionals in the field can implement or use the present invention, but it is not used to limit the present invention. It is impossible to fully reflect the various contents involved in the present invention in the above embodiments. Any equivalent changes or modifications made by any professional familiar with this technology without departing from the spirit or scope of the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. A PET polyesterase LCC-D, characterized in that The amino acid sequence of the PET lipase LCC-D is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the PET polyesterase LCC-D according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. An expression cassette, recombinant vector or recombinant engineered bacterium containing the nucleic acid molecule according to claim 2 or 3.

5. Use of the PET polyesterase LCC-D according to claim 1 in catalytic degradation of polyester plastics.

6. The use according to claim 5, characterized in that: The polyester plastic is 3PET or PET.

7. The use according to claim 5, characterized in that: The reaction conditions for the PET polyester lipase LCC-D to catalyze the degradation of 3PET are: pH 6.0-10.0, temperature 50°C-65°C; the reaction conditions for the PET polyester lipase LCC-D to catalyze the degradation of PET are: pH 6.0-10.0, temperature 55°C-85°C.

8. The use according to claim 7, characterized in that: The operation of catalytic degradation of 3PET by the PET polyesterase LCC-D is as follows: 3PET was added to a buffer solution with a pH value of 6.0-10.0, and then PET lipase LCC-D was added, and the mixture was incubated at 50°C-65°C. The operation of PET polyesterase LCC-D catalyzing the degradation of PET is as follows: Add PET fragments to a buffer solution with a pH value of 6.0-10.0, then add PET lipase LCC-D, and incubate at 55°C-85°C.

9. The use according to claim 8, characterized in that: In the operation of catalytic degradation of 3PET by the PET polyester lipase LCC-D, after the addition of the PET polyester lipase LCC-D, the final concentration of the PET polyester lipase LCC-D in the reaction system is 10nM-1000nM; in the operation of catalytic degradation of PET by the PET polyester lipase LCC-D, after the addition of the PET polyester lipase LCC-D, the final concentration of the PET polyester lipase LCC-D in the reaction system is 10nM-1000nM.

10. Use of the expression cassette, recombinant vector or recombinant engineered bacteria described in claim 4 in catalytic degradation of polyester plastics.

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