CN113584010A - Bacterial biofilm core extracellular polysaccharide lyase PelAN as well as preparation method and application thereof - Google Patents
Bacterial biofilm core extracellular polysaccharide lyase PelAN as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bacterial biofilm core extracellular polysaccharide lyase PelAN as well as a preparation method and application thereof, belonging to the technical field of microbial engineering. The preparation method of the PelAN comprises the steps of designing a gene sequence of a coded bacterial biofilm core extracellular polysaccharide lyase PelAN, cloning the gene sequence into a pET28b expression vector, introducing the cloned gene into a DE3 competent cell to obtain a DE3 engineering strain, carrying out amplification culture, carrying out induction expression, centrifugally collecting thalli, suspending the thalli in a cell disruption solution, carrying out ice bath ultrasonic disruption, carrying out high-speed centrifugation, collecting a supernatant, purifying and collecting the PelAN protein. The invention constructs the recombinant expression PelAN protein through a prokaryotic expression system, has simple steps and convenient operation, is suitable for extracting a large amount of high-purity and high-activity PelAN protein, makes up for the technical blank of industrial preparation, and lays a foundation for targeted removal of gram-negative bacteria biofilm by PelAN.
Description
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a bacterial biofilm core extracellular polysaccharide lyase PelAN as well as a preparation method and application thereof.
Background
Most gram-negative bacteria can form structurally strong aggregates, i.e. biofilm, with their secretions such as exopolysaccharides, extracellular proteins and extracellular DNA, etc. The biomembrane is considered as one of important factors causing antibiotic resistance, can be widely present in water sources, soil and various foods, and causes serious public health problems.
Pel is a cationic exopolysaccharide composed of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, is an essential component for forming a pseudomonad biofilm, is closely related to the biofilm thickness, adhesiveness and cell aggregation of the pseudomonas and is transported and synthesized by the Pel system. The chaperonin PelA in the Pel system contains 1 glycoside hydrolase structural domain PelAN, and the recombinant protein (PelAN) can radically break Pel polysaccharide and inhibit and eliminate gram-negative bacteria biofilm. At present, researches on the Pel exopolysaccharide inhibitor are relatively few, and particularly, related reports in the field of food science are provided.
Disclosure of Invention
Based on the biological characteristics of PelAN and the technical blank of industrial preparation of the PelAN, the invention mainly aims to provide the preparation method of the bacterial biofilm core extracellular polysaccharide lyase PelAN, which has simple steps and convenient operation and is suitable for extracting a large amount of high-purity and high-activity PelAN protein.
The invention also aims to provide the application of the bacterial biofilm core extracellular polysaccharide lyase PelAN obtained by the method in inhibiting the formation of the bacterial biofilm or in targeted removal of the bacterial biofilm.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a preparation method of bacterial biofilm core extracellular polysaccharide lyase PelAN, which comprises the following steps:
(1) obtaining a whole genome sequence of bacteria, designing a gene sequence for coding bacterial biofilm core extracellular polysaccharide lyase PelAN, and cloning the gene sequence into a pET28b expression vector to obtain a Pet28b-PelAN recombinant expression vector;
(2) after sequencing and confirming the Pet28b-PelAN recombinant expression vector in the step (1), introducing the recombinant expression vector into E.coli BL21(DE3) competent cells to obtain a DE3 engineering strain;
(3) carrying out amplification culture on the DE3 engineering strain in the step (2), adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression, and centrifuging to collect thalli;
(4) resuspending the thalli collected in the step (3) in cell disruption solution, and ultrasonically disrupting the thalli under ice bath;
(5) centrifuging the crushing liquid in the step (4) at a high speed, and collecting supernatant;
(6) and (4) sequentially separating and purifying the supernatant obtained in the step (5) by an NI-NTA affinity chromatography column and an AKTA protein purification system, collecting PelAN protein, and verifying and purifying through SDS-PAGE to obtain the PelAN protein.
Preferably, the bacteria are gram-negative bacteria selected from pseudomonas aeruginosa PA14, pseudomonas fluorescens NBRC15842 or pseudomonas putida JBC 17.
Preferably, in the step (1), the gene sequence of the PelAN encoding bacterial biofilm core extracellular polysaccharide lyase is shown as SEQ ID NO:2, 4 or 6, and the PelAN amino acid site and the rare codon preference optimization are determined by Phyre2 homology modeling analysis after the whole genome sequence of the bacteria is obtained from an NCBI database.
Preferably, the step (2) further comprises the step of positive screening the DE3 engineering strain by kanamycin with the final concentration of 50 mug/ml.
Preferably, in the step (3), the DE3 engineering strain is expanded and cultured to the bacterial liquid concentration OD600Is 0.55-0.6.
Preferably, in step (3), IPTG induction expression is carried out until the final concentration of the thalli is 1mmol/L, and the induction condition is 20 ℃ for overnight induction culture for 18 h.
Preferably, in step (4), the cell disruption solution has a composition of 20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol, 30mg/mL lysozyme, a pad of Roche protease inhibitor and a scoop of DNase.
Preferably, in step (6), the NI-NTA affinity chromatography column contains buffer 1, buffer 2 and buffer 3; wherein the composition of the buffer 1 is 20mM Tris, 300mM NaCl, 10mM Imidazole and 5% Glycerol; the composition of the buffer 2 is 20mM Tris, 500mM NaCl, 35mM Imidazole and 5% Glycerol; the composition of buffer 3 was 20mM Tris, 500mM NaCl, 300mM Imidazole and 5% Glycerol.
Preferably, in step (6), the AKTA protein purification system contains buffer 4 with a composition of 25mM Tris, 200mM NaCl, and 5% glycol.
The invention also provides a bacterial biofilm core extracellular polysaccharide lyase PelAN which is obtained by any one of the preparation methods of the bacterial biofilm core extracellular polysaccharide lyase PelAN.
Preferably, the bacterial biofilm exocellular polysaccharide lyase PelAN is selected from Pseudomonas aeruginosa PA14 biofilm exocellular polysaccharide lyase PelAN-PA, Pseudomonas fluorescens NBRC15842 biofilm exocellular polysaccharide lyase PelAN-PF or Pseudomonas putida JBC17 biofilm exocellular polysaccharide lyase PelAN-PP.
The invention also provides application of any one of the bacterial biofilm core extracellular polysaccharide lyase PelAN in inhibiting the formation of a bacterial biofilm or in targeted removal of the bacterial biofilm.
Preferably, the bacteria are gram-negative bacteria selected from pseudomonas aeruginosa PA14, pseudomonas fluorescens NBRC15842 or pseudomonas putida JBC 17.
Preferably, the bacterial biofilm core extracellular polysaccharide lyase PelAN is a bacterial biofilm scavenger or a bacterial biofilm disinfectant.
Compared with the prior art, the invention has the beneficial effects that:
(1) the preparation method of the bacterial biofilm core extracellular polysaccharide lyase PelAN is constructed based on the biological characteristics of PelAN, has simple steps and convenient operation, is suitable for extracting a large amount of high-purity and high-activity PelAN protein, and fills up the technical blank of industrial preparation of the PelAN protein.
(2) The bacterial biofilm core extracellular polysaccharide lyase PelAN prepared by the invention can be used for inhibiting the formation of a bacterial biofilm or removing the bacterial biofilm in a targeted manner, lays a foundation for the targeted removal of the gram-negative bacterial biofilm by PelAN, and is used for controlling the risk and harm of the biofilm in industries such as food and the like.
The conception and the resulting technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features and effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of the preparation process of bacterial biofilm core extracellular polysaccharide lyase PelAN of the present invention.
FIG. 2 is a schematic diagram of the construction of the Pet28b-PelAN expression vector of the present invention.
FIG. 3 shows gel filtration chromatography (A) and SDS-PAGE analysis (B) of the Pseudomonas aeruginosa PA14 lyase PelAN-PA of example 1.
FIG. 4 shows gel filtration chromatography (A) and SDS-PAGE analysis (B) of Pseudomonas fluorescens NBRC15842 lyase PelAN-PF in example 2.
FIG. 5 shows gel filtration chromatography (A) and SDS-PAGE analysis (B) of the Pseudomonas putida JBC17 lyase PelAN-PP in example 3.
FIG. 6 is a graph showing the inhibition of Pseudomonas aeruginosa PA14 biofilm by exopolysaccharide-cleaving enzymes PelAN-PA, PelAN-PF and PelAN-PP in example 4.
Detailed Description
The technical scheme of the invention is further explained by the concrete examples and the attached drawings. It should be understood that the following specific examples are illustrative only and are not limiting upon the present invention. The described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained without any inventive work by those skilled in the art are within the scope of the present invention.
Unless otherwise indicated, reagents, methods and equipment used in the present invention are conventional in the art. All reagents used were analytically pure, and water was deionized water.
As shown in FIGS. 1 and 2, the preparation method of the bacterial biofilm extracellular polysaccharide lyase PelAN in the invention is described, which comprises the following steps:
(1) obtaining a whole genome sequence of bacteria, designing a gene sequence for coding bacterial biofilm core extracellular polysaccharide lyase PelAN, and cloning the gene sequence into a pET28b expression vector to obtain a Pet28b-PelAN recombinant expression vector;
(2) after sequencing and confirming the Pet28b-PelAN recombinant expression vector in the step (1), introducing the recombinant expression vector into E.coli BL21(DE3) competent cells to obtain a DE3 engineering strain;
(3) carrying out amplification culture on the DE3 engineering strain in the step (2), adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression, and centrifuging to collect thalli;
(4) resuspending the thalli collected in the step (3) in cell disruption solution, and ultrasonically disrupting the thalli under ice bath;
(5) centrifuging the crushing liquid in the step (4) at a high speed, and collecting supernatant;
(6) and (4) sequentially separating and purifying the supernatant obtained in the step (5) by an NI-NTA affinity chromatography column and an AKTA protein purification system, collecting PelAN protein, and verifying and purifying through SDS-PAGE to obtain the PelAN protein.
In some embodiments, the bacteria are gram-negative bacteria selected from pseudomonas aeruginosa PA14, pseudomonas fluorescens NBRC15842, or pseudomonas putida JBC 17.
In some embodiments, in step (1), the gene sequence encoding bacterial biofilm core extracellular polysaccharide lyase PelAN is obtained by obtaining the whole genome sequence of bacteria from NCBI database and then determining PelAN amino acid sites and rare codon preference optimization through Phyre2 homology modeling analysis.
In some embodiments, step (2) further comprises the step of positive selection of the engineering strain DE3 by kanamycin to a final concentration of 50. mu.g/ml.
In some embodiments, in step (3), the engineering strain DE3 is expanded to OD6000.55-0.6; IPTG induction expression is carried out until the final concentration of the thalli is 1mmol/L, and the induction condition is 20 ℃ for overnight induction culture for 18 h.
In some embodiments, in step (4), the cell disruption solution is composed of 20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol, 30mg/mL lysozyme, a pad of Roche protease inhibitor and a scoop of DNase.
In some embodiments, in step (6), the NI-NTA affinity chromatography column comprises buffer 1, buffer 2, and buffer 3; wherein the buffer 1 comprises the components of 20mM Tris, 300mM NaCl, 10mM Imidazole and 5% Glycerol; the composition of buffer 2 was 20mM Tris, 500mM NaCl, 35mM Imidazole and 5% Glycerol; buffer 3 had a composition of 20mM Tris, 500mM NaCl, 300mM Imidazole and 5% Glycerol; the AKTA protein purification system contained buffer 4, which consisted of 25mM Tris, 200mM NaCl, and 5% glycol.
Example 1
A preparation method of Pseudomonas aeruginosa PA14 biofilm core extracellular polysaccharide lyase PelAN-PA comprises the following steps:
1) obtaining and designing bacterial PelAN-PA genome DNA: a whole genome sequence (protein _ ID: EOT14801.1) of pseudomonas aeruginosa PA14 is downloaded from an NCBI database, and is subjected to homology modeling analysis through Phyre2, a glycoside hydrolase domain of the pseudomonas aeruginosa PA14 is found to be positioned at amino acids 47-303 (SEQ ID NO: 1: GGPSSVAFWYAERPPLAELSQFDWVVLEAAHLKPADVGYLKEQGSTPFAYLSVGEFDGDAAAIADSGLARGKSAVRNQAWNSQVMDLAAPSWRAHLLKRAAELRKQGYAGLFLDTLDSFQLQAEERREGQRRALASFLAQLHRQEPGLKLFFNRGFEVLPELPGVASAVAVESIHAGWDAAAGQYREVPQDDRDWLKGHLDALRAQGMPIVAIDYLPPERRDEARALAARLRSEGYVPFVSTPALDYLGVSDVEVQP), and rare Codon preference optimization is carried out on the sequence through online Codon optimization software E.coli Codon Usage Analyzer 2.1 according to an Escherichia coli genetic Codon frequency table, so that a nucleotide sequence (SEQ ID NO: 2: ggcgggccgtccagcgtggcgttctggtacgccgagcggccgccgctggccgagctttcccagttcgactgggtggtgctcgaagcggcgcacctcaagccggccgatgtcgggtatctgaaagagcagggcagcacgcccttcgcctatctgtcggtcggcgagttcgacggcgacgccgccgccatcgccgatagcggcctggcccggggcaagagcgcggtccgcaaccaagcctggaacagccaggtaatggacctcgccgcgccgagttggcgggcgcacctgctcaagcgcgccgcggagctgcgcaaacagggctacgccggcctgttcctcgataccctggacagcttccagctacaggccgaggagcgccgcgagggccagcgccgggcgctggccagtttcctcgcccagctgcatcgccaggagccgggcctcaagctgtttttcaatcgcggtttcgaagtgctgccggagttgcccggcgtcgcgtcggcggtggccgtggagtcgatccatgccggttgggacgccgctgccgggcaataccgcgaggtgccccaggacgatcgcgattggctgaagggtcacctggatgccctgcgcgcccagggcatgcccatcgtcgccatcgactacctgccgccggagcggcgcgacgaggcgcgcgcgctcgctgcgcgcctgcgtagcgaaggctacgtgccgttcgtcagcaccccggcgctggactacctgggggtgagcgacgtcgaggtgcaaccg) for coding pseudomonas aeruginosa PA14 biofilm core extracellular polysaccharide lyase PelAN-PA is obtained.
2) PelAN-PA PCR amplification and pET28b expression vector construction: PCR amplification and primer pair design synthesis are completed by Suzhou Jinzhi company, NdeI and Hind III enzyme cutting sites are selected to connect the PelAN-PA fragment to pET28b vector, Pet28b-PelAN-PA recombinant expression vector is prepared, and sequencing verification is carried out by Suzhou Jinzhi company.
3) Transformation of the expression vector: the Pet28b-PelAN-PA recombinant expression vector of step 2) was introduced into E.coli BL21(DE3) competent cells, and positive selection was performed on LB solid medium containing kanamycin to a final concentration of 50. mu.g/ml.
4) And (3) carrying out amplification culture on the engineering strain: selecting the positive single colony in the step 3) to be cultured in 100ml LB liquid culture medium containing 50 mu g/ml kanamycin at the final concentration overnight, taking 5ml to be inoculated in 500ml LB liquid culture medium containing 50 mu g/ml kanamycin at the final concentration, and carrying out amplification culture at 37 ℃ and 180r/min until OD is achieved600Is 0.55-0.6.
5) Inducible expression of PelAN-PA: adding IPTG with the final concentration of 1mmol/L into the bacterial liquid amplified in the step 4), carrying out induction culture at 20 ℃ overnight for 18h, and centrifuging at 4 ℃ and 5000rpm for 30min to collect thalli.
6) Cell disruption: the cells collected in step 5) were resuspended in 80mL of cell disruption solution (20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol, 30mg/mL lysozyme, Roche protease inhibitor and DNase), sonicated under ice bath conditions, and the supernatant was collected by centrifugation at 12000 rmp.
7) NI-NTA affinity chromatography column separation: the Ni-NTA affinity chromatography column was equilibrated with 50mL of buffer 1(20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol); passing the supernatant collected in step 6) through a Ni-NTA affinity chromatography column; the Ni-NTA affinity chromatography column was washed thoroughly with 50mL of buffer 2(20mM Tris, 500mM NaCl, 35mM Imidazole, 5% Glycerol); the target protein was eluted with buffer 3(20mM Tris, 500mM NaCl, 300mM Imidazole, 5% Glycerol).
8) And (3) AKTA system purification: balancing 120mL gel chromatography column Superdex 7510/300 GL with buffer solution 4(25mM Tris, 200mM NaCl, 5% Glycerol), performing gel chromatography experiment (molecular sieve) on the protein purified by the Ni-NTA column in the step 7) by an AKTA protein purification system, collecting a sample corresponding to a molecular sieve peak diagram, and verifying the purified target protein by SDS-PAGE.
As shown in figure 3, the preparation method of the pseudomonas aeruginosa PA14 biofilm extracellular core polysaccharide lyase PelAN-PA is successful in obtaining the recombinant PelAN-PA protein through gel filtration chromatography (figure 3A) and SDS-PAGE analysis (figure 3B).
Example 2
A preparation method of Pseudomonas fluorescens NBRC15842 biofilm core extracellular polysaccharide lyase PelAN-PF comprises the following steps:
1) obtaining and designing bacterial PelAN-PF genome DNA: the whole genome sequence (protein _ ID ═ GED 73779.1) of pseudomonas fluorescens NBRC15842 is downloaded from NCBI database and is subjected to homology modeling analysis by Phyre2, the glycoside hydrolase domain of the pseudomonas fluorescens is found to be positioned at amino acids 37 to 288 (SEQ ID NO: 3: PASVGFWYAEQPPLQELAQFEWAVVEPGHMASADVATLRKLGSQPFAYLSVGEFDGNRAALAKQALAQGASPVRNKAWDSQVMDIATPAWREHLFKRAKALQDQGYAGLFLDTLDSFQLLPEADREPQRKALASFLRELHSRLPNLKLFFNRGFEVLGELDGVASAVAVESIHAGWDASAKRYRPVSEADRTWLEGELKPLRARNIPLVAIDYLPANRREEARKLVRQLSQEGFIPVVTTPDLNALSMSTVE), and the sequence is subjected to rare Codon preference optimization by online Codon optimization software E.coli Codon Usage Analyzer 2.1 according to the frequency table of Escherichia coli genetic codons, so as to obtain the nucleotide sequence (SEQ ID NO: 4: cccgccagcgtcgggttctggtacgccgagcagccacccttgcaggagctggcgcagttcgaatgggcagtggtcgagcccggccatatggccagcgccgatgtcgccactttgcgcaagctcggcagccagccgttcgcctacctgtcggtaggggagttcgacggcaaccgcgctgccctggccaagcaggccctggcccagggcgcgagccccgtgcgcaacaaggcctgggacagccaggtgatggacatcgccaccccggcctggcgcgaacacctgttcaagcgtgccaaggcgctgcaggaccagggctacgccggcctgttcctggacaccctggacagtttccagttgctccccgaagccgatcgcgaaccgcaacgcaaggccctggcctcgttcctgcgggaactgcacagccggctgcccaacctcaagctgttcttcaaccggggcttcgaggtactgggcgagctcgatggcgtcgcctcggccgtggcggtggaatccatccacgccggctgggatgcctcggccaagcgttaccgcccggtttccgaagccgaccgcacctggcttgaaggcgaactcaagccgctgcgcgcacgcaacatcccgctggtggccatcgattacctgccggccaaccgtcgggaagaggcacgcaagctggtccggcaattgagccaggaaggctttataccggtggtcaccaccccggatctgaacgccctgagcatgagcaccgtggaa) for coding pseudomonas fluorescens NBRC15842 biofilm core extracellular polysaccharide lyase PelAN-PF.
2) PelAN-PF PCR amplification and pET28b expression vector construction: PCR amplification, primer pair design synthesis by Suzhou Jinzhi company, selection of NdeI and Hind III enzyme cutting sites and PelAN-PF fragment connected to pET28b vector, preparation of recombinant expression vector, and by Suzhou Jinzhi company sequencing verification.
3) Transformation of the expression vector: the expression vector in step 2) was introduced into E.coli BL21(DE3) competent cells and positive selection was performed on LB solid medium containing kanamycin to a final concentration of 50. mu.g/ml.
4) And (3) carrying out amplification culture on the engineering strain: the positive single colonies described in step 3) were picked and cultured overnight in 100ml of LB liquid medium containing kanamycin to a final concentration of 50. mu.g/ml. Inoculating 5ml of LB liquid medium containing 50. mu.g/ml kanamycin to 500ml, and performing amplification culture at 37 ℃ and 180r/min to OD600Is 0.55-0.6.
5) Inducible expression of PelAN-PF: adding IPTG with the final concentration of 1mmol/L into the bacterial liquid amplified in the step 4), carrying out induction culture at 20 ℃ overnight for 18h, and centrifuging at 4 ℃ and 5000rpm for 30min to collect thalli.
6) Cell disruption: the cells collected in step 5) were resuspended in 80mL of cell disruption solution (20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol, 30mg/mL lysozyme, Roche protease inhibitor and DNase), sonicated under ice bath conditions, and the supernatant was collected by centrifugation at 12000 rmp.
7) NI-NTA affinity chromatography column separation: the Ni-NTA affinity chromatography column was equilibrated with 50mL of buffer 1(20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol); passing the supernatant collected in step 6) through a Ni-NTA affinity chromatography column; the Ni-NTA affinity chromatography column was washed thoroughly with 50mL of buffer 2(20mM Tris, 500mM NaCl, 35mM Imidazole, 5% Glycerol); the target protein was eluted with buffer 3(20mM Tris, 500mM NaCl, 300mM Imidazole, 5% Glycerol).
8) And (3) AKTA system purification: balancing 120mL gel chromatography column Superdex 7510/300 GL with buffer solution 4(25mM Tris, 200mM NaCl, 5% Glycerol), performing gel chromatography experiment (molecular sieve) on the protein purified by the Ni-NTA column in the step 7) by an AKTA protein purification system, collecting a sample corresponding to a molecular sieve peak diagram, and verifying the purified target protein by SDS-PAGE.
As shown in FIG. 4, the recombinant PelAN-PF protein was successfully obtained by the above-mentioned Pseudomonas fluorescens NBRC15842 biofilm core extracellular polysaccharide lyase PelAN-PF preparation method through gel filtration chromatography (FIG. 4A) and SDS-PAGE analysis (FIG. 4B).
Example 3
A preparation method of Pseudomonas putida JBC17 biofilm core extracellular polysaccharide lyase PelAN-PP comprises the following steps:
1) obtaining and designing bacterial PelAN-PP genome DNA: the whole genome sequence (ID: AWY42675.1) of Pseudomonas putida JBC17 is downloaded from NCBI database, and is subjected to homology modeling analysis by Phyre2, the glycoside hydrolase domain of the sequence is found to be positioned at amino acids 38-289 (SEQ ID NO: 5: PSSVSFWYADEPPLAELAQFAWTVVEPGHMTAADVKTLRKLGSEPFAYLSVGEFDGSKADITKAGLTKAVSPVRNDSWNSQVMDLTSPAWRDHLLGRAKQLQAQGYGGLFLDTLDSFTLLPQAAQEAQRAGLASFLRELHKQQPQLKLFFNRGFEVLPELDGVAAAVAFESLYAGWDAAAKRYRPVPEADRQWLLGQLQPLRAKGIPLVAIDYLPPERRDEARKLAKRLRDEGFIPFISTPDLNSMGISTVE), and the sequence is subjected to rare Codon preference optimization by online Codon optimization software E.coli Codon Usage Analyzer 2.1 according to the frequency table of Escherichia coli genetic codons, so that the nucleotide sequence (SEQ ID NO: 6: ccttccagtgtgtccttctggtatgccgacgagccgccgctggccgagctggcgcagttcgcctggacggtggtcgagccggggcatatgacggcggcggatgtcaaaaccctgcgcaagctgggcagcgagccgttcgcctatctgtcggtcggcgagttcgacggatccaaggctgacatcaccaaagcgggcctcaccaaggccgtttccccggtgcgcaacgactcgtggaacagtcaggtcatggacctcacttctccagcctggcgcgaccacttgctgggtcgggccaagcaattgcaggcccagggctatggcggcctgttcctcgataccctcgacagtttcacgctgttgccgcaagccgcgcaggaagcgcagcgcgcggggctggccagtttcctgcgcgaactgcacaagcagcagcctcagctgaagctgttctttaaccgtggctttgaagtgttgcccgagctcgacggcgtggcggcagcggtggcattcgagtcgctgtatgccggctgggatgcggcggccaagcgttatcgcccggtgccggaagccgatcgccagtggctgctgggccagttgcaaccgttgcgcgccaagggcattccactggtggccatcgattatttgccgccggagcgtcgcgacgaggcccgcaagctggccaagcgtctgcgtgacgaaggcttcattcctttcatcagcacccctgacctcaactcgatgggcatcagcaccgtcgaa) for coding Pseudomonas putida JBC17 biofilm core extracellular polysaccharide lyase PelAN-PP is obtained.
2) PelAN-PP PCR amplification and pET28b expression vector construction: PCR amplification, primer pair design and synthesis were performed by Suzhou Jinzhi corporation, NdeI and Hind III enzyme cleavage sites were selected to ligate the PelAN-PP fragment to pET28b vector, recombinant expression vector was prepared, and sequencing was performed by Suzhou Jinzhi corporation.
3) Transformation of the expression vector: the expression vector in step 2) was introduced into E.coli BL21(DE3) competent cells and positive selection was performed on LB solid medium containing kanamycin to a final concentration of 50. mu.g/ml.
4) And (3) carrying out amplification culture on the engineering strain: picking the positive single colony described in step 3) in 100ml LB containing a final concentration of 50. mu.g/ml kanamycinCultured in liquid medium overnight. Inoculating 5ml of LB liquid medium containing 50. mu.g/ml kanamycin to 500ml, and performing amplification culture at 37 ℃ and 180r/min to OD600Is 0.55-0.6.
5) Inducible expression of PelAN-PP: adding IPTG with the final concentration of 1mmol/L into the bacterial liquid amplified in the step 4), carrying out induction culture at 20 ℃ overnight for 18h, and centrifuging at 4 ℃ and 5000rpm for 30min to collect thalli.
6) Cell disruption: the cells collected in step 5) were resuspended in 80mL of cell disruption solution (20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol, 30mg/mL lysozyme, Roche protease inhibitor and DNase), sonicated under ice bath conditions, and the supernatant was collected by centrifugation at 12000 rmp.
7) NI-NTA affinity chromatography column separation: the Ni-NTA affinity chromatography column was equilibrated with 50mL of buffer 1(20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol); passing the supernatant collected in step 6) through a Ni-NTA affinity chromatography column; the Ni-NTA affinity chromatography column was washed thoroughly with 50mL of buffer 2(20mM Tris, 500mM NaCl, 35mM Imidazole, 5% Glycerol); the target protein was eluted with buffer 3(20mM Tris, 500mM NaCl, 300mM Imidazole, 5% Glycerol).
8) And (3) AKTA system purification: balancing 120mL gel chromatography column Superdex 7510/300 GL with buffer solution 4(25mM Tris, 200mM NaCl, 5% Glycerol), performing gel chromatography experiment (molecular sieve) on the protein purified by the Ni-NTA column in the step 7) by an AKTA protein purification system, collecting a sample corresponding to a molecular sieve peak diagram, and verifying the purified target protein by SDS-PAGE.
As shown in FIG. 5, the preparation method of Pseudomonas putida JBC17 biofilm core extracellular polysaccharide lyase PelAN-PP successfully obtains the recombinant PelAN-PP protein through gel filtration chromatography (FIG. 5A) and SDS-PAGE analysis (FIG. 5B).
Example 4
The bacterial biofilm core extracellular polysaccharide lyases PelAN-PA, PelAN-PF and PelAN-PP prepared in examples 1 to 3 were used for inhibiting Pseudomonas aeruginosa PA14 biofilm by the following steps:
1) culture of pseudomonas aeruginosa PA 14: pseudomonas aeruginosa PA14 was cultured overnight at 37 ℃ at 200 r/min.
2) Biofilm formation: standardizing the bacterial liquid cultured in the step 1) to OD6000.5, then diluted 1:100 with LB liquid medium; mu.l of the dilution and 5. mu.l of 5. mu.M PelAN-PA or PelAN-PF or PelAN-PP were taken, and 100. mu.l of the dilution was added to a sterile 96-well plate as a positive control, and incubated at 25 ℃ for 24 hours to form a biofilm.
3) Biofilm amount measurement: the formation of the biofilm is determined by a crystal violet method, namely, a pore plate is thoroughly washed by sterile water, planktonic cells are washed off, then the pore plate is stained by 0.1% (W/V)150 mu l of crystal violet for 10min, the crystal violet is washed off by water, the rest part is dissolved by 95% (V/V)150 mu l of ethanol for 10min, and finally the absorbance is determined under OD 595.
As shown in figure 6, the biofilm formation amount of the pseudomonas aeruginosa PA14 pretreated by the bacterial biofilm exocellular polysaccharide lyase PelAN-PA, PelAN-PF and PelAN-PP is obviously lower than that of a control group, which proves that the bacterial biofilm exocellular polysaccharide lyase PelAN can be used as a green and efficient biofilm scavenger.
Sequence listing
<110> Shanghai ocean university
<120> bacterial biofilm core extracellular polysaccharide lyase PelAN as well as preparation method and application thereof
<141> 2021-08-30
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 257
<212> PRT
<213> Pseudomonas aeruginosa PA14()
<400> 1
Gly Gly Pro Ser Ser Val Ala Phe Trp Tyr Ala Glu Arg Pro Pro Leu
1 5 10 15
Ala Glu Leu Ser Gln Phe Asp Trp Val Val Leu Glu Ala Ala His Leu
20 25 30
Lys Pro Ala Asp Val Gly Tyr Leu Lys Glu Gln Gly Ser Thr Pro Phe
35 40 45
Ala Tyr Leu Ser Val Gly Glu Phe Asp Gly Asp Ala Ala Ala Ile Ala
50 55 60
Asp Ser Gly Leu Ala Arg Gly Lys Ser Ala Val Arg Asn Gln Ala Trp
65 70 75 80
Asn Ser Gln Val Met Asp Leu Ala Ala Pro Ser Trp Arg Ala His Leu
85 90 95
Leu Lys Arg Ala Ala Glu Leu Arg Lys Gln Gly Tyr Ala Gly Leu Phe
100 105 110
Leu Asp Thr Leu Asp Ser Phe Gln Leu Gln Ala Glu Glu Arg Arg Glu
115 120 125
Gly Gln Arg Arg Ala Leu Ala Ser Phe Leu Ala Gln Leu His Arg Gln
130 135 140
Glu Pro Gly Leu Lys Leu Phe Phe Asn Arg Gly Phe Glu Val Leu Pro
145 150 155 160
Glu Leu Pro Gly Val Ala Ser Ala Val Ala Val Glu Ser Ile His Ala
165 170 175
Gly Trp Asp Ala Ala Ala Gly Gln Tyr Arg Glu Val Pro Gln Asp Asp
180 185 190
Arg Asp Trp Leu Lys Gly His Leu Asp Ala Leu Arg Ala Gln Gly Met
195 200 205
Pro Ile Val Ala Ile Asp Tyr Leu Pro Pro Glu Arg Arg Asp Glu Ala
210 215 220
Arg Ala Leu Ala Ala Arg Leu Arg Ser Glu Gly Tyr Val Pro Phe Val
225 230 235 240
Ser Thr Pro Ala Leu Asp Tyr Leu Gly Val Ser Asp Val Glu Val Gln
245 250 255
Pro
<210> 2
<211> 771
<212> DNA/RNA
<213> Artificial sequence ()
<400> 2
ggcgggccgt ccagcgtggc gttctggtac gccgagcggc cgccgctggc cgagctttcc 60
cagttcgact gggtggtgct cgaagcggcg cacctcaagc cggccgatgt cgggtatctg 120
aaagagcagg gcagcacgcc cttcgcctat ctgtcggtcg gcgagttcga cggcgacgcc 180
gccgccatcg ccgatagcgg cctggcccgg ggcaagagcg cggtccgcaa ccaagcctgg 240
aacagccagg taatggacct cgccgcgccg agttggcggg cgcacctgct caagcgcgcc 300
gcggagctgc gcaaacaggg ctacgccggc ctgttcctcg ataccctgga cagcttccag 360
ctacaggccg aggagcgccg cgagggccag cgccgggcgc tggccagttt cctcgcccag 420
ctgcatcgcc aggagccggg cctcaagctg tttttcaatc gcggtttcga agtgctgccg 480
gagttgcccg gcgtcgcgtc ggcggtggcc gtggagtcga tccatgccgg ttgggacgcc 540
gctgccgggc aataccgcga ggtgccccag gacgatcgcg attggctgaa gggtcacctg 600
gatgccctgc gcgcccaggg catgcccatc gtcgccatcg actacctgcc gccggagcgg 660
cgcgacgagg cgcgcgcgct cgctgcgcgc ctgcgtagcg aaggctacgt gccgttcgtc 720
agcaccccgg cgctggacta cctgggggtg agcgacgtcg aggtgcaacc g 771
<210> 3
<211> 252
<212> PRT
<213> Pseudomonas fluorescens NBRC15842()
<400> 3
Pro Ala Ser Val Gly Phe Trp Tyr Ala Glu Gln Pro Pro Leu Gln Glu
1 5 10 15
Leu Ala Gln Phe Glu Trp Ala Val Val Glu Pro Gly His Met Ala Ser
20 25 30
Ala Asp Val Ala Thr Leu Arg Lys Leu Gly Ser Gln Pro Phe Ala Tyr
35 40 45
Leu Ser Val Gly Glu Phe Asp Gly Asn Arg Ala Ala Leu Ala Lys Gln
50 55 60
Ala Leu Ala Gln Gly Ala Ser Pro Val Arg Asn Lys Ala Trp Asp Ser
65 70 75 80
Gln Val Met Asp Ile Ala Thr Pro Ala Trp Arg Glu His Leu Phe Lys
85 90 95
Arg Ala Lys Ala Leu Gln Asp Gln Gly Tyr Ala Gly Leu Phe Leu Asp
100 105 110
Thr Leu Asp Ser Phe Gln Leu Leu Pro Glu Ala Asp Arg Glu Pro Gln
115 120 125
Arg Lys Ala Leu Ala Ser Phe Leu Arg Glu Leu His Ser Arg Leu Pro
130 135 140
Asn Leu Lys Leu Phe Phe Asn Arg Gly Phe Glu Val Leu Gly Glu Leu
145 150 155 160
Asp Gly Val Ala Ser Ala Val Ala Val Glu Ser Ile His Ala Gly Trp
165 170 175
Asp Ala Ser Ala Lys Arg Tyr Arg Pro Val Ser Glu Ala Asp Arg Thr
180 185 190
Trp Leu Glu Gly Glu Leu Lys Pro Leu Arg Ala Arg Asn Ile Pro Leu
195 200 205
Val Ala Ile Asp Tyr Leu Pro Ala Asn Arg Arg Glu Glu Ala Arg Lys
210 215 220
Leu Val Arg Gln Leu Ser Gln Glu Gly Phe Ile Pro Val Val Thr Thr
225 230 235 240
Pro Asp Leu Asn Ala Leu Ser Met Ser Thr Val Glu
245 250
<210> 4
<211> 756
<212> DNA/RNA
<213> Artificial sequence ()
<400> 4
cccgccagcg tcgggttctg gtacgccgag cagccaccct tgcaggagct ggcgcagttc 60
gaatgggcag tggtcgagcc cggccatatg gccagcgccg atgtcgccac tttgcgcaag 120
ctcggcagcc agccgttcgc ctacctgtcg gtaggggagt tcgacggcaa ccgcgctgcc 180
ctggccaagc aggccctggc ccagggcgcg agccccgtgc gcaacaaggc ctgggacagc 240
caggtgatgg acatcgccac cccggcctgg cgcgaacacc tgttcaagcg tgccaaggcg 300
ctgcaggacc agggctacgc cggcctgttc ctggacaccc tggacagttt ccagttgctc 360
cccgaagccg atcgcgaacc gcaacgcaag gccctggcct cgttcctgcg ggaactgcac 420
agccggctgc ccaacctcaa gctgttcttc aaccggggct tcgaggtact gggcgagctc 480
gatggcgtcg cctcggccgt ggcggtggaa tccatccacg ccggctggga tgcctcggcc 540
aagcgttacc gcccggtttc cgaagccgac cgcacctggc ttgaaggcga actcaagccg 600
ctgcgcgcac gcaacatccc gctggtggcc atcgattacc tgccggccaa ccgtcgggaa 660
gaggcacgca agctggtccg gcaattgagc caggaaggct ttataccggt ggtcaccacc 720
ccggatctga acgccctgag catgagcacc gtggaa 756
<210> 5
<211> 252
<212> PRT
<213> Pseudomonas putida JBC17()
<400> 5
Pro Ser Ser Val Ser Phe Trp Tyr Ala Asp Glu Pro Pro Leu Ala Glu
1 5 10 15
Leu Ala Gln Phe Ala Trp Thr Val Val Glu Pro Gly His Met Thr Ala
20 25 30
Ala Asp Val Lys Thr Leu Arg Lys Leu Gly Ser Glu Pro Phe Ala Tyr
35 40 45
Leu Ser Val Gly Glu Phe Asp Gly Ser Lys Ala Asp Ile Thr Lys Ala
50 55 60
Gly Leu Thr Lys Ala Val Ser Pro Val Arg Asn Asp Ser Trp Asn Ser
65 70 75 80
Gln Val Met Asp Leu Thr Ser Pro Ala Trp Arg Asp His Leu Leu Gly
85 90 95
Arg Ala Lys Gln Leu Gln Ala Gln Gly Tyr Gly Gly Leu Phe Leu Asp
100 105 110
Thr Leu Asp Ser Phe Thr Leu Leu Pro Gln Ala Ala Gln Glu Ala Gln
115 120 125
Arg Ala Gly Leu Ala Ser Phe Leu Arg Glu Leu His Lys Gln Gln Pro
130 135 140
Gln Leu Lys Leu Phe Phe Asn Arg Gly Phe Glu Val Leu Pro Glu Leu
145 150 155 160
Asp Gly Val Ala Ala Ala Val Ala Phe Glu Ser Leu Tyr Ala Gly Trp
165 170 175
Asp Ala Ala Ala Lys Arg Tyr Arg Pro Val Pro Glu Ala Asp Arg Gln
180 185 190
Trp Leu Leu Gly Gln Leu Gln Pro Leu Arg Ala Lys Gly Ile Pro Leu
195 200 205
Val Ala Ile Asp Tyr Leu Pro Pro Glu Arg Arg Asp Glu Ala Arg Lys
210 215 220
Leu Ala Lys Arg Leu Arg Asp Glu Gly Phe Ile Pro Phe Ile Ser Thr
225 230 235 240
Pro Asp Leu Asn Ser Met Gly Ile Ser Thr Val Glu
245 250
<210> 6
<211> 756
<212> DNA/RNA
<213> Artificial sequence ()
<400> 6
ccttccagtg tgtccttctg gtatgccgac gagccgccgc tggccgagct ggcgcagttc 60
gcctggacgg tggtcgagcc ggggcatatg acggcggcgg atgtcaaaac cctgcgcaag 120
ctgggcagcg agccgttcgc ctatctgtcg gtcggcgagt tcgacggatc caaggctgac 180
atcaccaaag cgggcctcac caaggccgtt tccccggtgc gcaacgactc gtggaacagt 240
caggtcatgg acctcacttc tccagcctgg cgcgaccact tgctgggtcg ggccaagcaa 300
ttgcaggccc agggctatgg cggcctgttc ctcgataccc tcgacagttt cacgctgttg 360
ccgcaagccg cgcaggaagc gcagcgcgcg gggctggcca gtttcctgcg cgaactgcac 420
aagcagcagc ctcagctgaa gctgttcttt aaccgtggct ttgaagtgtt gcccgagctc 480
gacggcgtgg cggcagcggt ggcattcgag tcgctgtatg ccggctggga tgcggcggcc 540
aagcgttatc gcccggtgcc ggaagccgat cgccagtggc tgctgggcca gttgcaaccg 600
ttgcgcgcca agggcattcc actggtggcc atcgattatt tgccgccgga gcgtcgcgac 660
gaggcccgca agctggccaa gcgtctgcgt gacgaaggct tcattccttt catcagcacc 720
cctgacctca actcgatggg catcagcacc gtcgaa 756
Claims (10)
1. A preparation method of bacterial biofilm core extracellular polysaccharide lyase PelAN is characterized by comprising the following steps:
(1) obtaining a whole genome sequence of bacteria, designing a gene sequence for coding bacterial biofilm core extracellular polysaccharide lyase PelAN, and cloning the gene sequence into a pET28b expression vector to obtain a Pet28b-PelAN recombinant expression vector;
(2) after sequencing and confirming the Pet28b-PelAN recombinant expression vector in the step (1), introducing the recombinant expression vector into E.coli BL21(DE3) competent cells to obtain a DE3 engineering strain;
(3) carrying out amplification culture on the DE3 engineering strain in the step (2), adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression, and centrifuging to collect thalli;
(4) resuspending the thalli collected in the step (3) in cell disruption solution, and ultrasonically disrupting the thalli under ice bath;
(5) centrifuging the crushing liquid in the step (4) at a high speed, and collecting supernatant;
(6) and (4) sequentially separating and purifying the supernatant obtained in the step (5) by an NI-NTA affinity chromatography column and an AKTA protein purification system, collecting PelAN protein, and verifying and purifying through SDS-PAGE to obtain the PelAN protein.
2. The method for preparing the bacterial biofilm core exopolysaccharide lyase PelAN of claim 1, wherein the bacteria is gram-negative bacteria selected from Pseudomonas aeruginosa PA14, Pseudomonas fluorescens NBRC15842 or Pseudomonas putida JBC 17.
3. The method for preparing the bacterial biofilm core extracellular polysaccharide lyase PelAN according to claim 2, wherein in the step (1), the gene sequence coding the bacterial biofilm core extracellular polysaccharide lyase PelAN is shown in SEQ ID NO 2, 4 or 6, the whole genome sequence of the bacteria is obtained from NCBI database, and then the preference optimization of amino acid sites and rare codons of the PelAN is determined through Phyre2 homology modeling analysis; and/or step (2), further comprising the step of carrying out positive screening on the DE3 engineering strain by kanamycin with the final concentration of 50 mu g/ml.
4. The method for preparing bacterial biofilm core extracellular polysaccharide lyase PelAN according to claim 1, wherein in the step (3), the DE3 engineering strain is subjected to amplification culture until the bacterial liquid concentration OD6000.55-0.6; and/or the IPTG is induced and expressed until the final concentration of the thalli is 1mmol/L, and the induction condition is 20 ℃ for overnight induction culture for 18 h.
5. The method for preparing bacterial biofilm core extracellular polysaccharide lyase PelAN according to claim 1, wherein in the step (4), the components of the cell disruption solution are 20mM Tris, 300mM NaCl, 10mM Imidazole, 5% Glycerol, 30mg/mL lysozyme, one-piece Roche protease inhibitor and one scoop of DNase.
6. The method for preparing bacterial biofilm core extracellular polysaccharide lyase PelAN according to claim 1, wherein in step (6), the NI-NTA affinity chromatography column contains buffer 1, buffer 2 and buffer 3; wherein the composition of the buffer 1 is 20mM Tris, 300mM NaCl, 10mM Imidazole and 5% Glycerol; the composition of the buffer 2 is 20mM Tris, 500mM NaCl, 35mM Imidazole and 5% Glycerol; the composition of the buffer 3 is 20mM Tris, 500mM NaCl, 300mM Imidazole and 5% Glycerol; and/or the AKTA protein purification system contains buffer 4 with a composition of 25mM Tris, 200mM NaCl and 5% glycol.
7. A bacterial biofilm extracellular polysaccharide lyase PelAN obtained by the method for preparing the bacterial biofilm extracellular polysaccharide lyase PelAN according to any one of claims 1 to 6.
8. The bacterial biofilm exocellular polysaccharide lyase PelAN of claim 7, wherein said bacterial biofilm exocellular polysaccharide lyase PelAN is selected from Pseudomonas aeruginosa PA14 biofilm exocellular polysaccharide lyase PelAN-PA, Pseudomonas fluorescens NBRC15842 biofilm exocellular polysaccharide lyase PelAN-PF or Pseudomonas putida JBC17 biofilm exocellular polysaccharide lyase PelAN-PP.
9. Use of the bacterial biofilm core extracellular polysaccharide lyase PelAN of claim 7 or 8 for inhibiting bacterial biofilm formation or targeted removal of a bacterial biofilm.
10. The use according to claim 9, wherein the bacteria are gram-negative bacteria selected from the group consisting of pseudomonas aeruginosa PA14, pseudomonas fluorescens NBRC15842, or pseudomonas putida JBC 17; and/or the bacterial biofilm core extracellular polysaccharide lyase PelAN is a bacterial biofilm scavenger or a bacterial biofilm disinfectant.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170216410A1 (en) * | 2014-06-06 | 2017-08-03 | The Hospital For Sick Children | Soluble bacterial and fungal proteins and methods and uses thereof in inhibiting and dispersing biofilm |
| CN110651029A (en) * | 2017-04-04 | 2020-01-03 | 诺维信公司 | Glycosyl hydrolase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170216410A1 (en) * | 2014-06-06 | 2017-08-03 | The Hospital For Sick Children | Soluble bacterial and fungal proteins and methods and uses thereof in inhibiting and dispersing biofilm |
| CN110651029A (en) * | 2017-04-04 | 2020-01-03 | 诺维信公司 | Glycosyl hydrolase |
Non-Patent Citations (1)
| Title |
|---|
| PERRIN BAKER等: "Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms", 《SCI. ADV》 * |
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