CN113584057A - ICCG expression element, expression vector, bacillus subtilis recombinant strain and method for degrading PET or monomer thereof - Google Patents
ICCG expression element, expression vector, bacillus subtilis recombinant strain and method for degrading PET or monomer thereof Download PDFInfo
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Abstract
The invention relates to the technical field of recombinant strains, in particular to an ICCG expression element, an expression vector, a bacillus subtilis recombinant strain and a method for degrading PET or monomers thereof. The expression element comprises a signal peptide, an ICCG gene and a hydrophobic module. The bacillus subtilis recombinant strain is assembled together through the signal peptide, the ICCG and the hydrophobic module, so that the secretion amount of degrading enzyme and the contact capacity with a substrate can be effectively improved, and the PET degrading efficiency is improved; according to the invention, through screening different signal peptides and hydrophobic modules, the recombinant bacillus subtilis capable of effectively degrading PET or monomers thereof is obtained; the invention applies high temperature resistant degrading enzyme to a normal temperature degrading system to obtain the recombinant strain capable of efficiently degrading PET under the normal temperature condition.
Description
Technical Field
The invention relates to the technical field of recombinant strains, in particular to an ICCG expression element, an expression vector, a bacillus subtilis recombinant strain and a method for degrading PET or monomers thereof.
Background
Polyethylene terephthalate (PET) is a condensation polymer of ethylene glycol and terephthalic acid, and is currently the most widely used thermoplastic polymer material. Due to the characteristics of good mechanical property, safety, transparency and the like, the composite material is widely applied to fibers, films and engineering plastics. But the mass production of plastics also poses a serious environmental risk to us. Although large quantities of plastic are produced, recycling is extremely inefficient. At present, methods for recycling PET mainly comprise physical degradation, chemical degradation and biodegradation methods. Among them, most of the physical degradation and chemical degradation methods require high temperature and high pressure conditions, and are liable to cause secondary pollution, and it is necessary to explore the biodegradation method in order to avoid pollution in a real sense.
PET is a high molecular weight polymer that cannot enter cells, and its degradation requires secretion of PET degrading enzyme (PETase). LCC (leaf-bridge composition cutinase) is found to have the fastest initial depolymerization rate of PET at 65 ℃, the highest efficiency of the LCC is 33 times that of other tested enzymes, and simultaneously, the LCC has the highest thermal stability and is known to be an enzyme capable of maximally improving the fluidity and depolymerization effect of PET chains. However, even so, the LCC reaction stops after 3 days at 65 ℃ and the conversion only reaches 31%. Therefore, to increase depolymerization yield, researchers have attempted to further increase LCC activity and thermostability by enzyme engineering.
Four variants performed well after analysis by the investigator comparing activity and melting temperature: F243I/D238C/S283C/Y127G (ICCG), F243I/D238C/S283C/N246M (ICCM), F243W/D238C/S283C/Y127G (WCCG) and F243W/D238C/S283C/N246M (WCCM), which retain specific activities similar to or higher than wild-type LCC. After pretreatment of PET waste materials, researchers evaluate the performances of the four variants (ICCG, ICCM, WCCG and WCCM), and finally find that the conversion rate can be maximally improved under the condition of 72 ℃, and the ICCG variant obtains 90% depolymerization effect in 9.3 hours and becomes the best choice.
The modified cutinase LCC variant ICCG (F243I/D238C/S283C/Y127G) can depolymerize at least 90% of PET polymers into monomers within 10 hours, the efficient depolymerization performance of the improved PET hydrolase is more excellent than that of all previously reported PET hydrolases, and in addition, the modified cutinase ICCG has higher heat stability and has the highest capacity (at least 33 times of the enzyme activity of other enzymes) of degrading amorphous PET plastics at 65 ℃. At present, the research mainly focuses on the modification of protein, and the application of the enzyme to the microbial degradation process is not researched. In addition, the high temperature degradation conditions are limited by environmental conditions and production costs, and the degradation ability of applying the high temperature degrading enzyme to a normal temperature degradation system is not verified.
Most of microorganisms which are separated from the environment and can secrete PET degrading enzymes are not model organisms, the degradation rate is possibly low when various environmental conditions are met, the genetic background is not clear, and genetic modification is difficult. Therefore, it is necessary to obtain engineering strains capable of secreting and expressing plastic degrading enzymes by using model organisms as underplate cells.
Bacillus subtilis is a gram-positive bacterium and is widely used as a host bacterium for producing medicaments, foods, beverages and feed additives due to strong secretion capacity and safety of foreign proteins. The bacillus subtilis has the advantages of large secretion amount, no outer membrane and the like, and can effectively secrete heterologous proteins to the outside of cells and play a role. Meanwhile, in order to realize high-level production of target protein, the selection of a proper signal peptide to improve the secretion of foreign protein is also a common metabolic production strategy, and a hydrophobic module is added to improve the contact area between enzyme and a substrate, so that the degradation efficiency can be effectively improved.
Disclosure of Invention
In view of the above, the invention provides an ICCG expression element, an expression vector, a bacillus subtilis recombinant strain and a method for degrading PET or monomers thereof. The bacillus subtilis recombinant strain can effectively degrade PET or a monomer thereof, and realizes efficient degradation of PET at normal temperature.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an ICCG expression element, which comprises a signal peptide, an ICCG gene and a hydrophobic module.
The invention constructs the bacillus subtilis capable of secreting the PET degrading enzyme ICCG. The invention selects bacillus subtilis BS168 as a chassis cell, and obtains the recombinant bacillus subtilis secreting plastic degrading enzyme by introducing ICCG coding gene into the chassis cell. Through screening different signal peptides and hydrophobic modules, the bacillus subtilis capable of effectively degrading PET is obtained.
Preferably, the signal peptide is one or more of wild-type signal peptides pp2, AmyE, XynA, BglS, BglC and Lip.
Preferably, the signal peptide is the wild-type signal peptide pp 2.
Preferably, the hydrophobic module is one or more of PBM, HFB4, HFB7 and CBM.
Preferably, the hydrophobic moiety is PBM.
The invention also provides an ICCG expression vector, which comprises the ICCG expression element and a plasmid vector.
Preferably, the plasmid vector is pHP 13-P43.
The invention also provides a bacillus subtilis recombinant strain which comprises the ICCG expression vector.
The invention also provides a culture method of the bacillus subtilis recombinant strain, wherein the culture temperature of the bacillus subtilis recombinant strain is 37 ℃.
The invention also provides a method for degrading PET or monomer BHET thereof, ICCG secreted by the bacillus subtilis recombinant strain is adopted to degrade the PET or monomer BHET thereof, and the temperature of a degradation system is 37 ℃.
The invention provides an ICCG expression element, an expression vector, a bacillus subtilis recombinant strain and a method for degrading PET or monomers thereof. The expression element comprises a signal peptide, an ICCG gene and a hydrophobic module. The invention has the technical effects that:
the bacillus subtilis recombinant strain is assembled together through the signal peptide, the ICCG and the hydrophobic module, so that the secretion amount of degrading enzyme and the contact capacity with a substrate can be effectively improved, and the PET degrading efficiency is improved;
according to the invention, through screening different signal peptides and hydrophobic modules, the recombinant bacillus subtilis capable of effectively degrading PET or monomers thereof is obtained;
the invention applies high temperature resistant degrading enzyme to a normal temperature degrading system to obtain the recombinant strain capable of efficiently degrading PET under the normal temperature condition.
Drawings
FIG. 1 shows the optimal degradation temperature of the recombinant strain 168-Lip-ICCG on BHET;
FIG. 2 shows the effect of different signal peptides on the rate of degradation of recombinant strain BHET;
FIG. 3 shows the effect of different signal peptides on the concentration of the degradation product TPA;
FIG. 4 shows the effect of different signal peptides on the concentration of degradation products MHET;
FIG. 5 shows the effect of different hydrophobic modules on the rate of degradation of recombinant strain BHET;
FIG. 6 shows the effect of different hydrophobic modules on the concentration of the degradation product TPA;
FIG. 7 shows the effect of different hydrophobic moieties on the concentration of degradation products MHET;
FIG. 8 shows the BHET degrading ability of the recombinant strain 168-pp 2-ICCG-PBM.
Detailed Description
The invention discloses an ICCG expression element, an expression vector, a bacillus subtilis recombinant strain and a method for degrading PET or a monomer thereof, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The gene sequence or protein sequence used by the invention is as follows:
one, ICCG sequence
TCAAATCCGTATCAAAGAGGCCCGAATCCGACAAGATCAGCACTGACAGCAGATGGCCCGTTTTCAGTTGCAACATATACAGTTTCAAGACTGTCAGTTAGCGGCTTTGGCGGAGGCGTTATTTATTATCCGACGGGCACATCACTGACATTTGGCGGCATTGCAATGTCACCGGGCTATACAGCAGATGCATCATCACTGGCATGGCTGGGCAGAAGACTGGCATCACATGGCTTTGTTGTTCTGGTTATTAATACAAATTCAAGATTTGATGGCCCGGATTCAAGAGCATCACAACTGTCAGCAGCACTGAATTATCTGAGAACATCATCACCGTCAGCAGTTAGAGCAAGACTGGATGCAAATAGACTGGCAGTTGCGGGCCATTCAATGGGCGGAGGCGGCACACTGAGAATTGCAGAACAAAATCCGTCACTGAAAGCAGCAGTTCCGCTGACACCGTGGCATACAGATAAAACATTTAATACATCAGTTCCGGTTCTGATTGTTGGCGCAGAAGCAGATACAGTTGCACCGGTTTCACAACATGCAATTCCGTTTTATCAAAATCTGCCGTCAACAACACCGAAAGTTTATGTTGAACTGTGCAATGCATCACATATTGCACCTAATTCAAATAATGCAGCAATTTCAGTTTATACAATTTCATGGATGAAACTGTGGGTTGATAATGATACAAGATATAGACAATTTCTGTGCAATGTTAATGATCCGGCACTGTGCGATTTTAGAACAAATAATAGACATTGCCAA
II, signal peptide sequence
XynA：MFKFKKNFLVGLSAALMSISLFSATASA
BglS：MPYLKRVLLLLVTGLFMSLFAVTATASA
BglC：MKRSISIFITCLLITLLTMGGMIASPASA
LipB：LAKKDEHLRKPEWLKIKLNTNENYTGLKKLMREN
AmyE：MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA
pp2：MDGVLWRVRTAALMAALLALAAWALVWASPSVEAQ
Three, hydrophobic module sequence
PBM：
AFTCTATTASNYAHVQAGRAHDSGGIAYANGSNQSMGLDNLFYTSTLAQTAAGYYIVGNCP*
CBM：
TQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL*
HFB4：
MQYSAIVALFATLAVAAPAQEAAADIAILDGPCTAGVTNNIPMCCGSGILDLLYLDCETPTQATSVLNPLSAVCGRVGLQAKCCTLGIADLGVLCQDALPE*
HFB7：
MKFFAVAALFVASAMASPMGSEGCPGGLTNTVPLCCATNVLGVATLDCSTPTVPVPNVGIFQAHCASKGKQPVCCTVPVAGLGLLCQKPTGAQ*
In the study, bacillus subtilis was selected as the underpant cell to express the LCC cutinase variant ICCG. The bacillus subtilis is a gram-positive bacterium, is used as a model organism, is widely applied to expressing various proteins, and utilizes the characteristics of quick growth and strong secretion capacity of foreign proteins of the bacillus subtilis to efficiently secrete ICCG. In view of the characteristics of stable property and difficult degradation of PET plastics, monomer BHET of the PET plastics is selected as a research object, BHET is esterified from terephthalic acid and ethylene glycol and is an important chemical, BHET is selected as an early-stage degradation substrate in view of high crystallinity of PET, and the optimal degradation strain is screened by comparing the degradation capability difference of different strains on BHET.
The reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1 determination of optimal degradation temperature of recombinant Strain
ICCG is a high temperature stable enzyme with improved heat resistance, while the optimum growth temperature of Bacillus subtilis BS168 is 37 deg.C, and in order to determine the culture temperature of the recombinant strain, the degradation ability of the recombinant strain at 25 deg.C, 30 deg.C, 37 deg.C, 39 deg.C and 40 deg.C was tested. The results in FIG. 1 show that the optimal degradation temperature of the recombinant strain is 37 ℃ which is the optimal growth temperature of the recombinant strain, and under the condition, 2g/L of BHET can be completely degraded within 22 h.
Example 2 construction and screening of Signal peptides
Because different signal peptides have strong influence on protein secretion, in order to select the optimal secretion signal peptide aiming at ICCG, the secretion signal peptides of five common proteins of AmyE, XynA, BglS, BglC and Lip and the LCC cutinase wild-type signal peptide pp2 are selected in the genome of the bacillus subtilis to optimize the secretion of the ICCG. First, a signal peptide was obtained from the Bacillus subtilis genome using PCR, while introducing the homology arm of ICCG during PCR. Then, gene segments of different signal peptides and ICCG are constructed by using an overlap extension PCR method. The resulting gene fragment and pHP13-P43 vector were digested with BamHI and EcoRI, and purified by agarose gel electrophoresis. The obtained linearized pHP13-P43 vector and the gene fragment digested by the endonuclease are connected by T4 DNA ligase and transformed into an escherichia coli body, and after colony PCR, enzyme digestion and sequencing verification are successful, the vector is used as a bacillus subtilis expression vector, and a recombinant strain is obtained through bacillus subtilis transformation.
The degradation experiment is carried out by taking 2g/L BHET as a substrate, the result of figure 2 shows that the secretion effect of the wild-type signal peptide pp2 is the best, 2g/L BHET can be completely degraded within 22h, and the degradation rate is improved compared with that of the room-temperature degradation enzyme Lip-PETase constructed in the laboratory. In addition, the best degradation effect of 168-pp2-ICCG can be further proved by detecting the concentrations of MHET and TPA which are degradation products, and the highest concentrations of MHET and TPA which are degradation products are detected. MHET reached 1.37g/L at 22h and TPA reached 0.32g/L at 22h (FIGS. 3 and 4).
Example 3 construction and screening of hydrophobic modules
The hydrophobin double-layer structure can form an amphiphilic membrane on a hydrophobic/hydrophilic interface, and the amphiphilic membrane is used as a biosurfactant to improve the contact area with a substrate, so that the degradation efficiency is improved. In the research, three hydrophobic modules, namely a substrate binding module (PBM) of polyhydroxy butyrate depolymerizing enzyme from alcaligenes faecalis, hydrophobin from trichoderma, class II hydrophobin HFB4 and HFB7 and cellobiohydrolase I (CBM) from hypocrea jecorina are screened, and are connected with ICCG through a Linker to realize fusion expression. The recombinant strain is obtained by transforming bacillus subtilis, and a fermentation degradation experiment is carried out by taking 2g/L BHET as a substrate and compared with the recombinant strain without the hydrophobic module.
The degradation of BHET results indicated the fastest degradation rate for the ICCG fused PBM module followed by the CBM module (figure 5). Strains fused with PBM module produced higher concentrations of TPA and MHET (fig. 6, fig. 7). Compared with the strain without the hydrophobic module, the degradation rate is further improved.
Example 4 combination of optimal Signal peptide and hydrophobic Module
Based on the experiment, an ICCG optimal signal peptide pp2 and an optimal hydrophobic module PBM are selected and assembled to obtain 168-pp2-ICCG-PBM, and BHET degradation experiments prove that 2g/LBHET is completely degraded within 20 h.
Through optimization, the bacillus subtilis capable of efficiently expressing the PET degrading enzyme ICCG is obtained, the optimal signal peptide is the wild-type signal peptide pp2, the optimal hydrophobic module is PBM, and the bacillus subtilis can completely degrade 2g/L BHET within 20h (figure 8).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An ICCG expression element, wherein the expression element comprises a signal peptide, an ICCG gene and a hydrophobic module.
2. The ICCG expression element of claim 1 wherein the signal peptide is one or more of wild type signal peptides pp2, AmyE, XynA, BglS, BglC, Lip.
3. An ICCG expression element according to claim 2, characterised in that the signal peptide is the wild type signal peptide pp 2.
4. An ICCG expression element according to claim 1 wherein the hydrophobic moiety is one or more of PBM, HFB4, HFB7, CBM.
5. An ICCG expression element according to claim 4, wherein the hydrophobic moiety is PBM.
6. An ICCG expression vector comprising the ICCG expression element of any one of claims 1 to 5 and a plasmid vector.
7. The ICCG expression vector of claim 6, wherein the plasmid vector is pHP 13-P43.
8. A recombinant strain of Bacillus subtilis comprising the ICCG expression vector of claim 6.
9. The method for culturing the recombinant strain of Bacillus subtilis according to claim 8, wherein the recombinant strain of Bacillus subtilis is cultured at a temperature of 37 ℃.
10. A method for degrading PET or monomer BHET thereof, which is characterized in that the ICCG secreted by the bacillus subtilis recombinant strain of claim 7 is used for degrading the PET or monomer BHET thereof, and the temperature of the degradation system is 37 ℃.
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