CN113337001B - Method for degrading polyethylene glycol terephthalate by combining bacteria and enzyme - Google Patents

Abstract

The invention discloses a method for degrading polyethylene glycol terephthalate by combining bacterial enzymes, which takes cell suspension obtained by amplification culture of Klebsiella variicola SY1 (Klebsiella variicola SY 1) and Candida antarctica cutinase HiC as catalysts, takes polyethylene glycol terephthalate as a substrate, takes a buffer solution with pH of 7.0-9.0 as a reaction medium to form a reaction system, carries out degradation reaction at 30-60 ℃, and degrades high polymer PET into monomer compounds; by combined application of bacterial enzymes, product inhibition of PET degrading enzyme is relieved, and degradation efficiency is improved.

Description

Method for degrading polyethylene glycol terephthalate by combining bacteria and enzyme

(I) technical field

The invention relates to a method for degrading polyethylene glycol terephthalate by combining bacteria and enzymes.

(II) background of the invention

Polyethylene terephthalate (PET) is a polyester of terephthalic acid (TPA) and ethylene glycol, and is one of the most widely used thermoplastic polyester materials, accounting for about 1/5 of the global plastic production, and its waste accumulation in large quantities has caused serious environmental problems. At present, the treatment of PET waste is mainly divided into a physical method, a chemical method and a biological method. The physical recycling method is to sort the recycled PET plastics, mechanically process the sorted PET plastics into chips, further wash and dry the chips, and re-granulate the chips by a process such as heating. Although physical recycling is widely used, the method often causes the conditions of PET molecular chain breakage, molecular weight reduction, incapability of removing impurity components and the like, and the service performance of PET is greatly reduced. The chemical method mainly comprises glycolysis, alcoholysis, hydrolysis, ammonolysis and the like, and high-temperature conditions and extreme chemical reagents are needed for carrying out depolymerization reaction on the PET high polymer, so that partial oligomers and monomers are generated, the degradation cost is high, and additional environmental pollutants are easily generated. The biological rule has mild reaction conditions, and the PET polymer is degraded into water-soluble micromolecules with small molecular weight mainly by secreting some extracellular degrading enzymes by microorganisms and then is further eliminatedDissolving, and hydrolyzing to CO ₂ And H ₂ O and the like, which is an environment-friendly and green technology.

The enzymatic hydrolysis of PET is a surface erosion process, the degree of crystallinity is also a crucial factor influencing the speed of enzymatic degradation efficiency of PET, and PET materials with high degree of crystallinity have negative influence on the activity of cutinase in the hydrolysis process of PET. In addition, the enzymatic degradation products of PET also have a certain inhibitory effect on the degradation process. It was found that the enzymatic degradation intermediates of PET, bis (2-hydroxyethyl) terephthalate (BHET) and 2-hydroxyethyl monoester terephthalate (MHET), are competitive inhibitors of PET degrading enzymes, whereas MHET is hydrolyzed much more slowly to TPA than BHET, and accumulation of the degradation product TPA leads to a decrease in the pH of the system, thereby affecting the activity of the enzyme. Therefore, the products such as BHET, MHET, TPA and the like can be degraded by using enzyme or cells, so that the inhibition of PET degrading enzyme is reduced or relieved, and the PET degrading efficiency is improved.

The invention inspects the degradation condition of candida antarctica cutinase (Humicola insolens cutinase, hiC) to two kinds of PET with different crystallinity, and compares the effect of HiC in degrading PET respectively by combining with lipase CALB and Klebsiella variicola SY1.

Disclosure of the invention

The invention aims to provide a method for degrading polyethylene glycol terephthalate by combining bacterial enzymes, which can effectively reduce the contents of PET degradation products BHET, MHET and TPA by degrading PET by combining Candida antarctica cutinase and Klebsiella variicola SY1, thereby relieving the inhibition of the products on the cutinase and improving the PET degradation efficiency.

The technical scheme adopted by the invention is as follows:

the invention provides a method for degrading polyethylene glycol terephthalate by combining bacteria and enzymes, which comprises the following steps: using cell suspension obtained by amplification culture of Klebsiella variicola SY1 (Klebsiella variicola SY 1) and Candida antarctica cutinase as catalysts, using polyethylene terephthalate (PET) as a substrate, using a buffer solution with pH of 7.0-9.0 (preferably pH of 8.0) as a reaction medium to form a reaction system, and performing degradation reaction at 30-60 ℃ to degrade polyethylene terephthalate high polymer into monomer compounds; the Klebsiella variicola SY1 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020198, preservation date 2020, 6.10.8, address Wuhan, wuhan university, zip code 430072.

Preferably, the buffer is a Tris-HCl buffer, pH8.0, 50-100 mM.

The crystallinity of the polyethylene terephthalate is 10 to 30%, and preferably lcPET (low-crystallinity PET) or boPET (biaxially oriented PET). Where lcPET was 3.0mm by 2.5mm by 2.0mm plastic pellets with a crystallinity of 11.62%. The boPET is a plastic film with the thickness of 75 mu m, and the tensile strength is 200Mpa in the longitudinal direction and 240Mpa in the transverse direction; elongation at break 173 in the transverse direction and 122% in the longitudinal direction; the thermal expansion and contraction rate (150 ℃,30 min) is 1.25 percent in the longitudinal direction and-0.11 percent in the transverse direction; the crystallinity is 26.49%.

The substrate is added in an amount of 1-5mg/mL, preferably 3.17mg/mL, based on the volume of the buffer.

The candida antarctica cutinase is candida antarctica cutinase HiC (Novozym 51032), the enzyme activity is 15kU/g, the protein content is 1.424mg/mL, and the adding amount of the candida antarctica cutinase HiC is 10-50 mu g/mL buffer solution, preferably 0.019mg/mL calculated by the protein content.

The cell suspension is obtained by inoculating Klebsiella variicola SY1 into an LB culture medium, culturing at 30 ℃ for 1 day to logarithmic growth phase, washing with PBS buffer solution (pH 7.4) for three times, and finally suspending the thallus in the PBS buffer solution; the adding amount of the cell suspension in the buffer solution is 2-8 multiplied by 10 ⁹ cfu/mL, preferably 6X 10 ⁹ cfu/mL。

In the degradation reaction process, a substrate and candida antarctica cutinase are firstly added into a buffer solution, klebsiella variicola SY1 cell suspension is added after 5 days of degradation reaction at 60 ℃, the temperature is increased to 60 ℃ after 1 day of degradation at 30 ℃ for continuous degradation, and concretely, the method for degrading the polyethylene glycol terephthalate by the combination of the bacterial enzymes comprises the following steps: adding substrate and Candida antarctica cutinase in 50mM Tris-HCl (pH 8.0) buffer, and treating at 60 deg.C for 5 days; the temperature was then lowered to 30 ℃ and Klebsiella variicola SY1 cell suspension was added, after 1 day of treatment at 30 ℃ the temperature was raised again to 60 ℃ and treatment continued for 5 days to degrade the PET to monomeric compounds.

Compared with the prior art, the invention has the following beneficial effects: according to the invention, firstly Candida antarctica cutinase HiC is adopted to degrade lcPET with the crystallinity of 11.62 percent for 5 days (60 ℃), and Klebsiella sp.SY1 cell suspension (30 ℃) is added for treatment for 1 day, so that the contents of MHET and TPA can be obviously reduced, when the reaction temperature is recovered to 60 ℃, and enzymolysis is continued for 5 days, the quality of lcPET is reduced by 19.80 percent, which is superior to that of enzyme group addition, and therefore, the accumulation of TPA and MHET is reduced through Klebsiella sp.SY1, the product inhibition of cutinase HiC is favorably removed, and the degradation reaction of PET catalyzed by HiC after re-heating is favorably realized.

At present, the biological treatment method researches on PET are all researches on the single use of enzymatic degradation or microbial degradation and the combined degradation of sterile enzymes. The product inhibition of PET degrading enzyme can be relieved and the degrading efficiency can be improved by combined application of the bacterial enzymes. Description of the drawings

FIG. 1, liquid phase diagram of strain SY1 degrading TPA.

FIG. 2 phylogenetic tree of the strain SY1.

Fig. 3, TPA standard curve.

FIG. 4, BHET standard curve.

Figure 5 MHET standard curve.

Figure 6, HPLC profile of products of cutinase HiC degradation of lcPET samples.

FIG. 7 is a graph showing the amount of products generated by degradation of different PET samples by cutinase HiC; a is BHET; b is MHET; c is TPA.

FIG. 8, SEM scanning electron micrograph of lcPET sample after cutinase HiC degradation; (A) lcPET 60 ℃ control group, 50x; (B) lcPET treatment at 60 ℃ HiC for 120h,50x; (C) lcPET 60 ℃ control group, 200x; (D) lcPET 60 ℃ HiC treatment 120h,200x.

FIG. 9 SEM scanning electron micrograph of boPET sample after degradation of cutinase HiC; (A) boPET 60 ℃ control, 10,000x; (B) boPET treatment at 60 ℃ with HiC 120h,10,000x.

FIG. 10, graph of degradation BHET of Klebsiella variicola SY1.

Fig. 11 is a graph of the production of TPA with different treatment regimes.

FIG. 12 graph of MHET production under different treatment regimes.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1 screening and identification of strains

1. Experimental materials and methods

1.1 culture Medium:

LB liquid Medium (g/L): yeast extract (5.0), peptone (10.0), naCl (10.0) and solvent water, adjusting pH to 7.0, sterilizing at 115 deg.C for 30min;

the LB solid medium was prepared by adding 20g/L agar to LB liquid medium.

BSM medium (i.e., basic mineral salts medium, g/L): k ₂ HPO ₄ ·3H ₂ O(1.0)，NaCl(1.0)，(NH ₄ ) ₂ SO ₄ (0.5)，MgSO ₄ ·7H ₂ O(0.4)，CaCl ₂ (0.0755)，FeCl ₃ ·6H ₂ O (0.0143), with water as solvent, adjusting pH to 7.5, and sterilizing at 115 deg.C for 30min;

BSM solid Medium 20g/L agar was added to the BSM medium.

TPA liquid medium: terephthalic acid (TPA) (dissolved in DMSO, with a mother liquor concentration of 250 mg/mL) was added to BSM medium to give a final TPA concentration of 250mg/L in the solution.

TPA solid culture medium: 20g/L agar was added to the TPA broth.

1.2 screening of TPA-degrading bacteria

Enrichment culture of the strain: 100 mu L of Hangzhou city pond river bottom sludge water sample is taken and dripped into 50ml of LB liquid culture medium, and the mixture is cultured overnight at 30 ℃ and 180rpm to obtain an enrichment culture solution.

Subjecting the enriched culture solution to sterile water treatment 10 ^-3 ～10 ^-6 And (3) diluting in a gradient mode, then coating the gradient dilution liquid on TPA solid culture medium, and performing inverted culture at the temperature of 30 ℃. Performing primary screening according to colony morphology and size, inoculating strain with good growth vigor into TPA liquid culture mediumActivating at the temperature of 30 ℃; streaking the activated strain on TPA solid culture medium plate for further purification; inoculating the purified strain into a TPA liquid culture medium for re-screening comparison, sampling at 0h and 48h, measuring TPA degradation capacity by using an HPLC method, and screening to obtain a strain SY1.

1.3 determination of TPA by HPLC

Preparing TPA standard solution and preparing a standard curve: accurately weighing a certain amount of TPA standard sample, dissolving the TPA standard sample by using DMSO (dimethyl sulfoxide), preparing a standard solution with the concentration of 125mg/mL, diluting the standard solution by using a 70% (v/v) methanol-water mixed solvent, and respectively preparing the standard solution with the final concentrations of 250mg/L,200mg/L,150mg/L,100mg/L and 50mg/L. The sample amount is 10 mu L, peak areas of TPA at various concentrations are respectively measured by HPLC, and a standard curve is drawn by taking the TPA concentration as a horizontal ordinate and the corresponding TPA peak area as a vertical coordinate.

Chromatographic analysis conditions: the sample was taken 10 μ L by Agilent 1260HPLC assay, mobile phase methanol: water =70%:30% (volume concentration), isocratic elution, flow rate of 0.5mL/min, column of Diamonsil 5 μm C18, 250X 4.6mm, column temperature of 30 deg.C, using Diode Array (DAD) detector, detection wavelength of 254nm.

1.4 sequencing of the 16S rRNA Gene of Strain SY1

Extraction of genomic DNA: extracting genome DNA of the strain SY1 by using an Ezup columnar bacteria genome DNA extraction kit. 16S rDNA amplification: the 16S rDNA sequence of the strain was amplified using high fidelity polymerase 2 × Phanta Master Mix using bacterial universal primers with the extracted total DNA as template. The PCR fragment is recovered and purified and then sent to Hangzhou Zhixixi biotechnology limited company for sequencing, and the nucleotide sequence is shown as SEQ ID NO. 1.

The primer sequences are listed below:

F:5’-AGA GTT TGA TCC TGG CTC AG-3’(E.coli 27F)

R:5’-TPAC CTT GTT ACG ACT T-3’(E.coli 1492R)

SEQ ID NO.1

cagtcgagcggtagcacagagagcttgctctcgggtgacgagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaagtgggggaccttcgggcctcatgccatcagatgtgcccagatgggattagctggtaggtggggtaacggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcgatgaggttaataacctcatcgattgacgttacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttaggccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcatatacaaagagaagcgacctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggaggg。

1.5 physiological and biochemical characterization of Strain SY1

The purified strain SY1 obtained by separation is streaked on a TPA plate, and a single colony is picked for gram staining. Biochemical identification of the strain SY1 using a biochemical identification strip of bacteria of the enterobacteriaceae family: picking single colony on nutrient agar plate to 2ml sterile physiological saline, and suspending to uniform cell suspension. The following media were inoculated (100. Mu.L of bacterial suspension per well): semi-solid agar, ornithine decarboxylase broth, lysine decarboxylase broth, amino acid decarboxylase control, simmons citrate, hydrogen sulfide, urease, peptone water (tryptophan broth), MR, VP, phenylalanine, mannitol, inositol, sorbitol, melibiose, ribitol, raffinose. Wherein the semisolid agar is inoculated by puncture. Lysine, ornithine and amino acid controls were sealed with 200 μ L sterile paraffin. After inoculation, the cover is covered, and the mixture is placed in a constant temperature incubator at 37 ℃ for culture. After 24h incubation, the indicator was added dropwise and the results recorded, wherein the Methyl Red (MR) test was judged after 48h incubation.

2 results and analysis

2.1TPA Standard Curve

TPA solutions with the concentrations of 50, 100, 150, 200 and 250mg/L are respectively prepared, and the peak areas of TPA with each concentration are respectively measured by HPLC; the TPA standard curve was plotted with TPA concentration as the abscissa and the corresponding TPA peak area as the ordinate (FIG. 3). The conversion equation of the peak area (y) to the TPA concentration (x) is as follows: y =57.87x-52.28, coefficient of correlation R of the curve ² =0.9945, the linear relationship is good.

2.2 screening and preliminary identification of TPA-degrading Strain

The bacterial colony of the strain SY1 is white and round, the surface is smooth, and the edge is neat; the TPA degradation capability is detected by HPLC, and the TPA is degraded almost completely after 48 hours of degradation (figure 1), which shows that the TPA degradation capability is good.

Extracting genome DNA of the strain SY1, amplifying a 16S rRNA gene by PCR and sequencing, wherein the accession number of the sequence (SEQ ID NO. 1) on NCBI is as follows: MN822704. Performing homologous comparison by using an RDP database to obtain a 16S rDNA sequence of 20 strains with the highest similarity to the strain SY 1; a phylogenetic tree is constructed by MEGA7.0 software by using a Neighbor-joining method (figure 2); the results show that the strain SY1 is associated with Klebsiella variicola F2R9 ^T The homology was the highest at 99.93%. Gram stain microscopy shows: SY1 thallus is in a short rod shape and is gram negative; the results of physiological and biochemical identification are shown in Table 1, and with reference to the results of the above identification, the strain SY1 was named Klebsiella variicola SY1 (Klebsiella variicola SY 1) deposited in the China center for type culture Collection with the preservation number of CCTCC NO: M2020198, the preservation date of 2020,6 months and 10 days, address: wuhan, wuhan university, post 430072, china.

TABLE 1 physiological and biochemical identification results of the Strain SY1

Example 2

1. Experimental Material

Klebsiella variicola SY1 (Klebsiella variicola SY 1) is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020198, preservation date 2020, 6.10.8, address Wuhan, wuhan university, zip code 430072.

Candida antarctica cutinase HiC (Novozym 51032) was purchased from Dowana chemical Co., ltd, and had a nominal esterase activity of 15kU/g and a protein content of 1.424mg/mL; lipase CALB (Candida antarctica lipase B, CALB) was purchased from Hopkinson technologies, inc., and its nominal esterase activity was 5kU/g and protein content was 1.8mg/mL.

lcPET (low-crystalline PET, available from Shaoxing Xiangyu Green packaging Co., ltd.) was 3.0mm by 2.5mm by 2.0mm plastic pellets with a crystallinity of 11.62%.

boPET (biaxially oriented PET, available from Shaoxing Xiangyu Green packaging Co., ltd.) is a plastic film having a thickness of 75 μm; the tensile strength is 200MPa in the longitudinal direction and 240MPa in the transverse direction; elongation at break 173 in the transverse direction and 122% in the longitudinal direction; the thermal expansion and contraction rate (150 ℃,30 min) is 1.25 percent in the longitudinal direction and-0.11 percent in the transverse direction; the crystallinity is 26.49%.

Bis (2-hydroxyethyl) terephthalate (BHET) (analytical grade) was purchased from Tianjin Xiansi Biotech, inc.

Terephthalic acid 2-hydroxyethyl Monoester (MHET) is prepared by BHET hydrolysis, and the preparation method of MHET comprises the following steps: adding purified recombinant esterase EstB 0.4mg/mL and BHET with the final concentration of 250mg/mL into 50mmol/L Tris-HCl (pH8.0) buffer solution, reacting for 6h at 37 ℃ in a total reaction system of 2mL, stopping the reaction in a boiling water bath for 10min, centrifuging the reaction solution at 10000rpm for 10min, filtering the supernatant, and detecting the BHET and the MHET by HPLC. Since hydrolysis of 1mol of BHET corresponds to the production of 1mol of MHET, the MHET content can be calculated from the amount of reduction in BHET. Measuring the peak areas corresponding to the concentrations of BHET and MHET by HPLC (see method 2.2 for HPLC conditions), converting the reduced BHET into MHET with corresponding content, and then drawing an MHET standard curve by taking the concentration of MHET as the abscissa and the corresponding peak area of MHET as the ordinate.

Esterase EstB (Genbank Access Number: MK 681857) is derived from Enterobacter sp.HY1, and can hydrolyze BHET to generate MHET, which is exogenously expressed and functionally verified in E.coli BL21 (DE 3); (see Lequan Qiu, xinge Yin, tengfei Liu, handyu Zhang, gumei Chen, shijin Wu. Biogradation of bis (2-hydroxyhexyl) terephthalic by a new isolated organism sp.HY1 and chromatography of interest properties [ J ]. Journal of Basic Microbiology 2020,60 (3): 699-711.)

2. Experimental method

2.1 preparation of standard curves for BHET, MHET and TPA

Preparation of BHET standard solution and determination of standard curve: a certain amount of BHET standard sample (HPLC pure) is accurately weighed, dissolved in DMSO to prepare a standard solution with the concentration of 125mg/mL, and then the standard solution is diluted by a 70% methanol-water mixed solvent (V/V) to prepare standard solutions with the final concentrations of 250mg/L,200mg/L,150mg/L,100mg/L and 50mg/L respectively. The amount of the sample was 10. Mu.L, and the peak area of BHET at each concentration was measured by HPLC (conditions 2.2), and a standard curve was drawn with the concentration as the abscissa and the peak area as the ordinate.

Standard curves for MHET were made the same as BHET.

TPA standard curve is the same as in example 1.

2.2 analysis of PET degradation products (MHET, BHET, TPA)

Metabolites were analyzed by HPLC, chromatographic conditions: the sample was taken 10 μ L by Agilent 1260HPLC assay, mobile phase methanol: water =70%:30%, isocratic elution, flow rate of 0.5mL/min, column of Diamonsil 5 μm C18 (250X 4.6 mm), column temperature of 30 ℃, using Diode Array (DAD) detector, detection wavelength of 254nm.

According to MHET, BHET and TPA standard curves, obtaining the contents of MHET, BHET and TPA in the metabolite.

2.3 SEM Electron microscopy examination of PET samples

Sample treatment: au is sprayed on the surface of the cleaned and dried PET sample, and the PET sample is placed under a FEI Nova Nano SEM field emission scanning electron microscope (Thermo Fisher science and technology Co., ltd., USA) for observation and imaging.

SEM parameters: residence time (dwell): 20 mu s; acceleration voltage (HV): 5.0kV; interfacial resolution (HFW): 1.04nm; magnification (mag): 200-10000X; working Distance (WD): 6.8mm; illumination mode (mode): secondary electron SE mode.

2.4 degradation of PET of different crystallinity by Cutinase HiC

PET sample (boPET film, lcPET particles) pretreatment: (1) pretreating a boPET film: the boPET film was cut into 3cm × 1.5cm rectangular pieces (two pieces of lcPET were used, the mass of the pieces was equal), immersed in absolute ethanol at room temperature overnight, washed with 2% Tween 80 for 30min, then washed with sterile water, dried and weighed for use, and the PET sample was UV-sterilized in a clean bench for several hours before use. (2) pretreatment of lcPET particles: the boPET film was replaced with two lcPET pellets, and other operations were performed in the same manner as the pretreatment of the boPET film.

Grouping reaction systems: (1) lcPET particle group: to 15mL of 0.1M Tris-HCl (pH 8.0) buffer was added 47.5mg of the pretreated lcPET particles, and 200. Mu.L of cutinase HiC was added (the protein concentration was 0.019mg/mL based on the volume of the buffer); (2) boPET film group: to 15mL of 0.1M Tris-HCl (pH 8.0) buffer, 47.5mg of the pretreated lcPET particles and the pretreated boPET film of 3 cm. Times.1.5 cm were added, followed by 200. Mu.L of cutinase HiC (protein concentration added was 0.019mg/mL based on the volume of the buffer); (3) control group: to 15mL of 0.1M Tris-HCl (pH 8.0) buffer, 47.5mg of the pretreated lcPET particles and the pretreated boPET film of 3 cm. Times.1.5 cm were added, and 200. Mu.L of cutinase HiC inactivated after addition in a boiling water bath for 15min (the protein concentration was 0.019mg/mL based on the volume of the buffer) was added.

Reaction conditions are as follows: at 60 ℃,120h;

3 groups are paralleled, reaction liquid is sampled every 24h in the period, and the generation condition of the product is detected by HPLC (same as 2.2); after 120h, the sample was recovered, weighed and examined by SEM (same as 2.3).

Degradation of BHET by 2.5Klebsiella variicola SY1

(1) The strain SY1 was inoculated to an LB medium (composition same as example 1), and cultured at 30 ℃ for 1 day to logarithmic phase to obtain a seed solution.

(2) The seed liquid was inoculated at an inoculum size of 2% by volume into BSM medium (composition as in example 1) containing 250mg/L BHET (added as a DMSO solution) and 0.5g/L yeast extract, and cultured in a 30 ℃ constant temperature shaker at 180 rpm. Each set of three parallel. Sampling every 24h, and performing HPLC liquid phase detection on the contents of BHET, MHET and TPA in the culture process, wherein the detection method is the same as 2.2.

2.6 degradation of lcPET by the combination of cutinase HiC and Klebsiella variicola SY1

Preparation of a suspension of Klebsiella variicola SY1 cells: the strain SY1 is inoculated to LB culture medium, cultured at 30 ℃ for 1 day to logarithmic growth phase, then washed three times with PBS buffer (pH 7.4), and finally the thalli is resuspended in PBS buffer to make the thalli concentration be 3X 10 ¹⁰ cfu/mL, overnight at 4 ℃, was starved to obtain a cell suspension.

The enzymolysis reaction system is as follows: to a solution of 60mL of 50mM Tris-HCl (pH 8.0) buffer, 8 pieces (190 mg by mass) of lcPET particles pretreated in step 2.4 were added; adding 800 μ L cutinase HiC (protein concentration is 0.019mg/mL based on buffer solution volume), and treating at 60 deg.C for 5 days; the temperature was then reduced to 30 ℃ and three treatments (3 replicates per group) were used: (1) control group: hiC (i.e. no added substances); (2) adding an enzyme group: then 100 mu L of lipase CLAB is added, and the concentration of the added protein is 0.003mg/mL based on the volume of the buffer solution; (3) bacterium and enzyme combination group: klebsiella variicola SY1 cell suspension was added to a concentration of 6X 10 by volume of buffer ⁹ cfu/mL. After 1 day of treatment at 30 ℃ each group was again warmed to 60 ℃ and treatment continued for 5 days. Samples were taken on days 1, 3, 5, 6, 7, 9, 11, respectively, and the content change and pH change of the degradation products (BHET, MHET, TPA) were examined. And samples were recovered on day 11 and tested for mass change.

3. Results and analysis

3.1BHET, MHET Standard curves

1. Preparing BHET solutions with the concentrations of 50, 100, 150, 200 and 250mg/L respectively, and measuring peak areas of the BHET with the concentrations by HPLC respectively; by BHThe ET concentration is the abscissa, the corresponding BHET peak area is the ordinate, and a BHET standard curve is drawn. The conversion formula of the peak area to the BHET concentration is as follows: y =56.06x+489.72, the curve correlation coefficient R ² =0.9994, the linear relationship is good (fig. 4).

2. And (3) drawing an MHET standard curve by taking the concentration of MHET as an abscissa and the corresponding peak area of MHET as an ordinate, wherein the conversion formula of the peak area and the concentration of MHET is as follows: y =23.28x-9.36, coefficient of correlation R of the curve ² =0.99518, the linear relationship is good (fig. 5).

3.2 analysis of degradation products of lcPET and boPET by Cutinase HiC

The crystallinity is an important index of PET performance and also an important factor influencing PET degradation. The larger the crystallinity, the larger the crystalline region of PET, and the more difficult the macromolecular chain moves, possibly affecting the binding of the enzyme to the substrate. In order to examine the influence of PET crystallinity on the degradation effect of cutinase HiC, lcPET and boPET samples were reacted with cutinase HiC at 60 ℃ for 120h, and liquid phase detection was performed every 24 h. The results show that cutinase HiC enzyme has better effect on the degradation of low crystallinity lcPET, and the main degradation products are terephthalic acid 2-hydroxyethyl Monoester (MHET), terephthalic acid (TPA) (fig. 6, fig. 7). After 120h of degradation, the yields of MHET and TPA reach 266.88mg/L and 97.63mg/L respectively (Table 2), and the mass is reduced by 8.28 +/-0.64%; in contrast, for boPET, the degradation of the cutinase HiC was poor, and the yields of MHET and TPA after 120h were only 5.16mg/L and 2.48mg/L, and no significant change in mass was detected.

The above results show that the crystallinity is a key factor influencing the effect of HiC in degrading PET, and that HiC has a significantly higher degradation effect on low-crystallinity PET (11.62%) than on high-crystallinity PET (26.49%) and has substantially no degradation effect on high-crystallinity PET. The subsequent experiments used lcPET as the main experimental material.

TABLE 2PET enzymatic products and their quality changes

3.3 SEM scanning Electron microscopy of cutinase HiC degraded PET

Enzymatic hydrolysis of PET is a process of surface erosion that also leaves traces of erosion on the PET surface. In order to more intuitively understand the degradation condition of the cutinase HiC on the PET, the PET sample after HiC treatment and a control group sample are detected by using an SEM scanning electron microscope.

The lcPET sample results are shown in fig. 8: the lcPET samples (B, D in FIG. 8) after 120h treatment with cutinase HiC showed a clear trace of erosion at 50-fold compared to the control (A, C in FIG. 8); at 200x, lcPET was shown to degrade as if rock weathering was generally laminar, with degradation occurring primarily at the junction of the smooth and rough sides of lcPET.

The results for the boPET samples are shown in fig. 9: the cutinase HiC has no good degradation effect on the boPET, and SEM (scanning electron microscope) results of samples treated by the cutinase for 120h show no obvious erosion trace under the magnification of 10,000.

Degradation of BHET by 3.4Klebsiella variicola SY1

As a result of examining the BHET degrading ability of Klebsiella variicola SY1 (FIG. 10), it was found that the strain SY1 can degrade BHET rapidly when 0.5g/L yeast extract is added to BSM medium, and 250mg/L of BHET is degraded substantially completely after 48 hours, at which time the MHET yield is significantly reduced and begins to decrease, indicating that the strain SY1 can also degrade MHET.

3.5 Complex degradation of PET by Cutinase HiC and Klebsiella variicola SY1

The literature reports that BHET and MHET which are enzymatic degradation products of PET have certain inhibiting effect on the degradation process of PET. And the decrease of the pH value of the system caused by the accumulation of a large amount of degradation end product TPA is not beneficial to the further action of the enzyme. Driano et al found that when the cutinase HiC was used in conjunction with another esterase CALB, the intermediate MHET was removed by CALB, resulting in a 7.7-fold increase in the yield of TPA.

In this study, the lcPET was degraded with cutinase HiC for 5 days at 60 ℃; the samples were then divided into three groups, (1) control: hiC; (2) adding an enzyme group: hiC + CALB; (3) bacterium and enzyme complex group: hiC + Klebsiella variicola SY1 cell suspension. After three groups of samples were treated at 30 ℃ for 1 day, they were again warmed to 60 ℃. Samples were taken on days 1, 3, 5, 6, 7, 9, and 11 to detect changes in the content of degradation products and changes in pH, respectively. And samples were recovered on day 11 and tested for mass change.

The results are shown in FIGS. 11 and 12: the TPA and MHET content decreased rapidly one day after the addition of Klebsiella variicola SY1 cell suspension; after 5 days of treatment at 60 ℃ after re-warming, the percentage increase of TPA in three groups was 102.14%, 103.44% and 148.88%, respectively, and the percentage increase of MHET was 66.94%, 62.71% and 102.56%, respectively (Table 3), the percentage increase of TPA and MHET in the suspension of Klebsiella variicola SY1 cells was significantly higher than in the other groups, and was reflected in the weight loss of the final lcPET, with a mass loss of 19.80% and also higher than in the other groups, indicating that degradation of TPA by Klebsiella variicola SY1 reduced the accumulation of TPA and MHET, and facilitated the degradation reaction of the PET by HiC after re-warming.

In addition, the pH change during the experiment was between 8.0 and 7.3 for each group (table 3), with no significant difference. This is because the Tris-HCl (pH 8.0) buffer in the system has a certain regulating ability, and the TPA generated by 5 days of HiC degradation of lcPET is not enough to cause enough pH reduction, so that the elimination of TPA in the system does not cause obvious pH value change. Nevertheless, the reduction of TPA helped shift the reaction equilibrium towards degradation, so the mass of lcPET in the bacterial enzyme complex decreased more than in the enzyme addition group after re-warming.

TABLE 3 quality variation of lcPET under different treatment regimes

4. Conclusion

(1) The present inventors carried out degradation studies on lcPET with a crystallinity of 11.62% and boPET with a crystallinity of 26.49% using the cutinase HiC from Humicola insolens. The results found that the products of cutinase HiC degradation of lcPET were mainly MHET and TPA, and the samples lost 8.28 ± 0.64% after 120h; but has no degradation effect on the boPET basically; SEM scanning electron microscope results show that the lcPET has obvious erosion traces, while the boPET has no erosion traces; the above results indicate that crystallinity of PET is an important factor affecting enzymatic degradation of PET.

(2) The Klebsiella variicola SY1 and HiC are adopted to carry out enzyme composite degradation of PET, the results show that after the strain SY1 is added, the contents of MHET and TPA are both obviously lower than those of an enzyme adding group, when the reaction temperature is recovered to 60 ℃, and enzymolysis is continued for 5 days, the quality of lcPET of the enzyme composite group is reduced by 19.80 percent and is higher than those of a control group and a CALB adding group (18.61 percent and 18.38 percent), which shows that the strain SY1 effectively degrades the MHET and TPA in the system, is favorable for enzymatic hydrolysis of HiC after re-heating, and therefore, better degradation effect is shown.

Sequence listing

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<120> method for degrading polyethylene glycol terephthalate by combining bacterial enzymes

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Claims (7)

1. A method for degrading polyethylene glycol terephthalate by combining bacteria and enzymes is characterized by comprising the following steps: using a cell suspension obtained by amplification culture of Klebsiella variicola SY1 (Klebsiella variicola SY 1) and Candida antarctica cutinase as catalysts, using polyethylene glycol terephthalate as a substrate, and using a buffer solution with pH of 7.0-9.0 as a reaction medium to form a reaction system for degradation reaction, wherein in the degradation reaction process, the substrate and the Candida antarctica cutinase are firstly added into the buffer solution, after the degradation reaction is carried out for 5 days at 60 ℃, the Klebsiella variicola SY1 cell suspension is added, after the degradation is carried out for 1 day at 30 ℃, the temperature is increased to 60 ℃, the degradation is continued for 5 days, so that the polyethylene glycol terephthalate high polymer is degraded into a monomer compound; the Klebsiella variicola SY1 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020198, preservation date 2020, 6.10.8, address Wuhan, wuhan university, zip code 430072.

2. The method according to claim 1, wherein the buffer is a 50-100mM Tris-HCl buffer, pH 8.0.

3. The process of claim 1, wherein the polyethylene terephthalate has a crystallinity of 10 to 30%.

4. The method according to claim 3, wherein the polyethylene terephthalate is lcPET or boPET, wherein lcPET is plastic granules with a crystallinity of 11.62%; boPET is a plastic film with a crystallinity of 26.49%.

5. The method of claim 1, wherein the substrate is added in an amount of 1 to 5mg/mL based on the volume of the buffer.

6. The method according to claim 1, wherein the candida antarctica cutinase is candida antarctica cutinase HiC, and the candida antarctica cutinase HiC is added in an amount of 10 to 50 μ g/mL buffer in terms of protein content.

7. The method as claimed in claim 1, wherein the cell suspension is obtained by inoculating Klebsiella variicola SY1 into LB culture medium, culturing at 30 ℃ for 1 day to logarithmic growth phase, washing with PBS buffer solution for three times, and finally suspending the thallus in PBS buffer solution; the adding amount of the cell suspension in the buffer solution is 2-8 multiplied by 10 ⁹ cfu/mL。

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