Application of chitinase with cross-species activity and related biomaterials

Abstract

The present invention discloses a chitinase KPGH3 for inhibiting biofilm synthesis, destroying biofilm structure, or in antibacterial application. KPGH3 can be any of the following proteins: A1) a protein whose amino acid sequence is sequence 3 in a sequence list; A2) a protein whose amino acid sequence is sequence 5 in a sequence list; A3) a protein having more than 90% identity with the protein shown in A1) and having chitinase activity obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein of A1) or A2); A4) a fusion protein obtained by connecting a protein tag at the N-terminus or / and C-terminus of A1) or A2). KPGH3 has the potential to inhibit and disperse biofilms of various pathogenic microorganisms and the potential chitin biotransformation ability, and has the feasibility of using it to develop new anti-biofilm therapeutic agents.

Description

Technical Field

[0001] The invention relates to application of chitinase with cross-species activity in the field of biotechnology and related biological materials. Background Art

[0002] In recent years, biofilm formation has been identified as a key factor in most microbial infections and drug resistance. Approximately 80% of chronic and recurrent microbial infections in humans are caused by bacterial biofilms [1]. Biofilms provide many survival advantages for pathogens encapsulated therein, including but not limited to: protecting pathogens from the host immune system and antimicrobial drugs / antibiotics, conserving water, increasing microbial tolerance to desiccation, absorbing and storing nutrients, maintaining high extracellular enzyme activity, adhering to the site of infection, cell aggregation, and coordinating the expression of virulence factors through quorum sensing [2]-[4]. Traditional methods for treating bacterial infections directly target pathogens, but the presence of biofilms raises the effective concentration of antimicrobial drugs to dangerous levels. In addition, pathogens encapsulated in biofilms may also produce metabolic dormant cells or molecular persistence mechanisms to resist drug treatment, which is also the reason why biofilm infections recur after a period of clinical treatment has been suspended.

[0003] The tenacity of biofilms is the result of the complex physical and biological properties created by multiple microorganisms. It often involves the interaction of multiple microorganisms, and the effect of single antibiotic treatment is unsatisfactory. Therefore, in recent years, some researchers have turned their attention to the study of biofilm dispersion, hoping to improve the efficacy of traditional antimicrobial drugs by inhibiting biofilm formation and dispersing mature biofilms. Biofilms are formed when microorganisms irreversibly attach to the surface of a physical object, begin cell division, and provide more adhesion sites to recruit other microorganisms [5]. Biofilms are layers synthesized by microorganisms to protect microbial communities. They are composed of complex polysaccharides, proteins, lipids, and extracellular DNA (eDNA). These components can be collectively referred to as extracellular polymeric substances (EPS) [2]. The composition of the EPS matrix is ​​variable in time and space, which mainly depends on the microbial species, local mechanical shear force, matrix availability, and host environment [6]. An important mechanism for bacteria to actively disperse biofilms is to produce extracellular enzymes. These enzymes can act on EPS components such as proteins, eDNA, and extracellular polysaccharides to promote biofilm dispersion and transform bacteria into a more fragile planktonic state. Targeting EPS components can also destroy the viscoelastic properties of biofilms, further weaken the cohesion of biofilms, and enhance host-mediated antimicrobial responses and antimicrobial effects. In most biofilms, the main components of EPS are highly dependent on secreted exopolysaccharides [2][7][8]. Exopolysaccharides provide many important functions for the establishment and persistence of biofilms, including but not limited to: structural stability, physical and chemical defense against antimicrobial drugs and the host immune system, cell-to-cell adhesion and aggregation, tolerance to drying, absorption of organic and inorganic compounds, and provision of carbon sources when nutrients are insufficient [2][9]

[10] . Previous researchers have found that glycoside hydrolase genes exist in the biosynthetic pathway of bacterial exopolysaccharides, which have the ability to inhibit biofilm synthesis and destroy mature biofilm structures. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to improve the efficiency of inhibiting biofilm synthesis and destroying mature biofilm structure.

[0005] 1. The first object of the present invention is to provide an application of KPGH3, wherein the application is any one or more of the following:

[0006] Y1. Application of KPGH3 protein in the preparation of antibacterial agents;

[0007] Y2. Application of KPGH3 protein in the preparation of products for enhancing the bactericidal and / or antibacterial activity of antibacterial drugs;

[0008] Y3. Application of KPGH3 protein in antibacterial activities;

[0009] Y4. Application of KPGH3 protein in inhibiting Pseudomonas aeruginosa;

[0010] Y5. Application of KPGH3 protein in inhibiting Klebsiella pneumoniae;

[0011] Y6. Application of KPGH3 protein in the preparation of anti-biofilm therapeutic agents;

[0012] Y7. Application of KPGH3 protein in inhibiting biofilm synthesis;

[0013] Y8. Application of KPGH3 protein in destroying biofilm structure;

[0014] Y9. Application of KPGH3 protein as chitinase;

[0015] Y10. Application of KPGH3 protein in the transformation of chitin;

[0016] The KPGH3 protein is any of the following proteins:

[0017] A1) The amino acid sequence is the protein of sequence 3 in the sequence listing;

[0018] A2) The amino acid sequence is the protein of sequence 5 in the sequence listing;

[0019] A3) a protein obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein of A1) or A2) and having more than 90% identity with the protein of A1) and having chitinase activity;

[0020] A4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0021] The protein of A1) is named KPGH3, which is derived from Klebsiella bacteria. The protein of A2) is named his-KPGH3, which is a fusion protein obtained by fusing the 21 amino acid residues MGSSHHHHHHSSGLVPRGSHM encoded at the amino terminus of the protein of A1).

[0022] In the above application, the KPGH3 protein can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

[0023] In the above application, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0024] In the above applications, the above 90% identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0025] In the above application, the antibacterial agent is a preparation that inhibits bacteria.

[0026] In the above application, the bacteria are Gram-negative bacteria.

[0027] In the above application, the bacteria is any of the following:

[0028] 1) Gram-negative bacteria;

[0029] 2) Proteobacteria;

[0030] 3) Gammaproteobacteria;

[0031] 4) Pseudomonas bacteria:

[0032] 5) Pseudomonas bacteria;

[0033] 6) Pseudomonas bacteria;

[0034] 7) Pseudomonas aeruginosa (also known as Pseudomonas aeruginosa);

[0035] 8) Enterobacteriales;

[0036] 9) Enterobacteriaceae;

[0037] 10) Klebsiella bacteria;

[0038] 11) Klebsiella pneumoniae.

[0039] The present invention also provides any of the following applications of the biological material related to the KPGH3 protein:

[0040] X1. Application in the preparation of antibacterial agents;

[0041] X2. Application in the preparation of anti-biofilm therapeutic agents;

[0042] X3. Application in the preparation of chitinase;

[0043] The biological material is any one of the following B1) to B5):

[0044] B1) a nucleic acid molecule encoding the protein;

[0045] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0046] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0047] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0048] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0049] In the above application, in the biological material, the nucleic acid molecule in B1) is the gene shown in b1) or b2) below:

[0050] b1) The coding sequence of the coding strand is a cDNA molecule or a DNA molecule of sequence 1 or sequence 2 in the sequence list;

[0051] b2) The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of sequence 1 or sequence 2 in the sequence list.

[0052] Wherein, Sequence 1 or Sequence 2 in the sequence listing encodes the protein shown in Sequence 3 in the sequence listing.

[0053] In the above application, the expression cassette containing the nucleic acid molecule described in biological material B2) refers to a nucleic acid molecule capable of expressing kpgh3 in a host cell, which may include not only a promoter for initiating transcription of the kpgh3 gene, but also a terminator for terminating transcription of the kpgh3 gene. Further, the expression cassette may also include an enhancer sequence.

[0054] Existing expression vectors can be used to construct a recombinant expression vector containing the kpgh3 gene expression cassette.

[0055] In the above applications, the recombinant microorganism in the biological material can specifically be yeast, bacteria, algae and fungi.

[0056] The present invention also protects KPGH3 protein, specifically any one of the following proteins:

[0057] A1) The amino acid sequence is the protein of sequence 3 in the sequence listing;

[0058] A2) The amino acid sequence is the protein of sequence 5 in the sequence listing;

[0059] A3) a protein obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein of A1) or A2) and having more than 90% identity with the protein of A1) and having chitinase activity;

[0060] A4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0061] The present invention also protects biological materials related to KPGH3 protein; the biological material is any one of the following B1) to B5):

[0062] B1) a nucleic acid molecule encoding the protein;

[0063] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0064] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0065] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0066] Wherein, the nucleic acid molecule described in B1) is specifically the following b1) or b2):

[0067] b1) The coding sequence of the coding strand is a cDNA molecule or a DNA molecule of sequence 1 or sequence 2 in the sequence list;

[0068] b2) The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of sequence 1 or sequence 2 in the sequence list.

[0069] In order to solve the above technical problems, the present invention also provides an antibacterial agent. The antibacterial agent provided by the present invention contains the protein.

[0070] The active ingredient of the above-mentioned antibacterial agent may be the protein, and the active ingredient of the above-mentioned antibacterial agent may also contain other biological components and / or non-biological components. The other active ingredients of the above-mentioned antibacterial agent can be determined by those skilled in the art according to the antibacterial effect.

[0071] In order to solve the above technical problems, the present invention also provides an anti-biofilm therapeutic agent. The antibacterial agent provided by the present invention contains the protein.

[0072] The examples of the present invention prove that KPGH3 can effectively inhibit the formation of biofilms of Pseudomonas aeruginosa PAO1, PA14 strains, and Klebsiella pneumoniae (ATCC700603) strains under a 12-hour action time, and effectively destroy the formed biofilms. 50 The damage effect EC 50 The concentrations of KPGH3 and 50nM were 36.85nM, 43.34nM and 82.58nM, respectively. At a concentration of 50nM, KPGH3 was able to enhance the sensitivity of Pseudomonas aeruginosa PAO1 strain to tobramycin. In addition, sequence analysis results showed that KPGH3 had chitinase activity. In summary, KPGH3 has the potential to inhibit and disperse biofilms of various pathogenic microorganisms, as well as potential chitin biotransformation ability, and is feasible for developing new anti-biofilm therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The figure is the SDS-PAGE electrophoresis result of the expression and purification of 13 proteins in Example 1. Among them, lane M is 170KDa Protein Ladder, and lanes 1-13 are the induced expression products of the hypothetical glycoside hydrolase proteins, which are ECGH1, ECGH2, ECGH4, KPGH9, KPGH10, KPGH1, KPGH2, KPGH3, KPGH4, KPGH5, KPGH6, KPGH7, and KPGH8 from left to right.

[0074] Figure 2 This is an example of the treatment distribution of the 96-well culture plate in the biofilm inhibition experiment in Example 1.

[0075] Figure 3 The results of the preliminary screening experiment of biofilm activity in Example 1. The residual biofilm was stained and quantified using crystal violet, and the data were standardized using the blank control as the standard. Each data point was averaged from three groups of experimental data. In the figure, ECGH2 represents ECGH2-His, KPGH1 represents KPGH1-His, KPGH2 represents KPGH2-His, KPGH3 represents KPGH3-His, KPGH4 represents KPGH4-His, KPGH5 represents KPGH5-His, KPGH6 represents KPGH6-His, and KPGH7 represents KPGH7-His. Figure 3 a is the treatment with PAO1 strain, Figure 3 b is the treatment with strain PA14, Figure 3c is the treatment with K. pneumoniae ATCC700603 strain; the number of replicates was 3, the data were expressed as mean ± standard deviation, and significance analysis was performed using SPSS, ** indicates that the result of the significant difference analysis was p < 0.01, and * indicates that the result of the significant difference analysis was p < 0.05.

[0076] Figure 4 The figure is the experimental result of the inhibitory effect of KPGH3 protein on biofilm in Example 1. The residual biofilm was stained and quantified using crystal violet, and the data was standardized using the blank control as the standard. Each data point was averaged from three groups of experimental data. Figure 4 a is the treatment with PAO1 strain, Figure 4 b is the treatment with strain PA14, Figure 4 c refers to the treatment with K. pneumoniae ATCC700603 strain; the number of replicates was 3, and the data are expressed as mean ± standard deviation.

[0077] Figure 5 The figure is the experimental result of the KPGH3 protein on the biofilm destruction in Example 1. The residual biofilm was stained and quantified using crystal violet, and the data was standardized using the blank control as the standard. Each data point was averaged from three groups of experimental data. Figure 5 a is the treatment with PAO1 strain, Figure 5 b is the treatment with strain PA14, Figure 5 c refers to the treatment with K. pneumoniae ATCC700603 strain; the number of replicates was 3, and the data are expressed as mean ± standard deviation.

[0078] Figure 6 The diagram shows the steps of the antibiotic sensitization reaction of KPGH3 in Example 1.

[0079] Figure 7 This is a photograph of the results of the KPGH3 sensitization experiment on the PAO1 strain antibiotic tobramycin in Example 1.

[0080] Figure 8 The figure is the result of sequence bioinformatics analysis of KPGH3 in Example 1. Figure 8 Figure a is a diagram of the KPGH3 protein glycoside hydrolase family analyzed by Blast. Figure 8 Figure b is the result of calculating the theoretical Mw and pI using Compute pI / Mw on the ExPAXy website. Figure 8 Figure c is the result of analyzing protein domain and enzyme activity information through InterPro. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are conventional biochemical reagents and can be obtained from commercial sources unless otherwise specified.

[0083] 1. Strains and plasmids

[0084] In the following examples, the Pseudomonas aeruginosa PAO1 strain and PA14 strain were kindly donated by Researcher Wang Xiaoxue of the South China Sea Institute of Oceanology, Chinese Academy of Sciences, and are recorded in the non-patent literature "Li, Y.; Liu, X.; Tang, K.; Wang, P.; Zeng, Z.; Guo, Y.; Wang, X. Excisionase in Pf filamentous prophage controls lysis-lysogeny decision-making in Pseudomonas aeruginosa. Molecular microbiology 2019, 111, 495-513, doi: 10.1111 / mmi.14170.". The public can obtain them from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences to repeat the experiments of this application, and cannot be used for other purposes.

[0085] The Klebsiella pneumoniae (ATCC700603) strain in the following examples is a product of ATCC.

[0086] Escherichia coli Trans5ɑ and BL21 (DE3) in the following examples were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0087] The pET 28a(+) plasmid in the following examples was kindly donated by our researcher He Weiqing and is recorded in the non-patent literature "Lu Zhili. Study on the regulation of biosynthesis of bitespiramycin. Master, Peking Union Medical College, 2017." The public can obtain it from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences to repeat the experiments of this application, but it cannot be used for other purposes.

[0088] 2 Culture medium

[0089] Unless otherwise specified, the solvent used in the preparation of the culture medium in the following examples is deionized water (ddH2O).

[0090] The LB medium (Cat No. 22700-025) in the following examples was purchased from Invitrogen, BHI medium was purchased from Solarbio, and CAMHB medium (MB2439) was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0091] In the following examples, the preparation method of M63 medium supplemented with magnesium sulfate, glucose and casamino acids is as follows:

[0092] 1) Prepare 5×M63 solution: add 15g KH2PO4, 35g K2HPO4, and 10g (NH4)2SO4 to 1L water. No need to sterilize. Store at room temperature.

[0093] 2) Prepare 1M MgSO4 solution, sterilize by high pressure, and store at room temperature;

[0094] 3) Prepare 20% (g / mL weight-to-volume ratio) glucose storage solution, sterilize by high pressure, and store at room temperature;

[0095] 4) Prepare 20% (g / mL weight-to-volume ratio) casamino acid solution, sterilize by high pressure, and store at room temperature;

[0096] 5) 5×M63 was diluted to 1×M63, sterilized by autoclave, and cooled to room temperature. Then, 1M MgSO4 solution, 20% glucose stock solution, and 20% casamino acid solution were added thereto, so that the final concentrations of MgSO4, glucose, and casamino acid in the solution were 1 mM MgSO4, 0.2% (g / mL mass volume ratio) glucose, and 0.5% (g / mL mass volume ratio) casamino acid, respectively.

[0097] 3 Solutions and reagents

[0098] In the following examples, the working concentration and storage concentration of antibiotics are shown in Table 1.

[0099] Table 1

[0100] antibiotic Solvents Use concentration (μg / mL) Storage concentration (mg / mL) Kanamycin water 25 25 Tobramycin water 100 2

[0101] In the following examples, the concentrations used and the storage and usage methods of the protease inhibitors are shown in Table 2.

[0102] Table 2

[0103] Inhibitors Solvents Use concentration Storage (-20℃) PMSF Ethanol 1mM Reusable Aprotinin water 2 μg / mL 10 mg / mL, aliquots, do not reuse thawed samples Leupeptin water 5-10 μg / mL Once thawed, it cannot be reused

[0104] In the following examples, the working concentration and storage concentration of IPTG are shown in Table 3.

[0105] Table 3

[0106] Reagents Solvents Use concentration (mM) Storage concentration (M) IPTG water 0.5 1

[0107] In the following examples, PageRuler Prestained Protein Ladder was purchased from ThermoScientific.

[0108] In the following examples, the plasmid miniprep kit (Trans EasyPure Plasmid MiniPrep Kit) (Cat. No.: L51214) was purchased from Quanshijin Biotechnology Co., Ltd. The protein quantification kit (BCA method) was purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0109] In the following examples, the formula of the Binding buffer is: 20 mM imidazole, 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 2% (V / V) glycerol.

[0110] In the following examples, the formula of Elution Buffer is: 250 mM imidazole, 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 2% (V / V) glycerol.

[0111] In the following examples, the formula of Final wash Buffer is: 500 mM imidazole, 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 2% (V / V) glycerol.

[0112] In the following examples, QuickBlue rapid gel staining solution (BF06152) was purchased from Beijing Biolong Immunotechnology Co., Ltd. Ultrafiltration centrifuge tubes were purchased from Merk Millipore, USA. 0.22 μm and 0.45 μm filter membranes were purchased from Millipore, USA. 96-well culture plates were purchased from Corning Incorporated, USA. Affinity chromatography columns (cat#DS0106) and 10×PBS (cat.No.P1022-500) were purchased from Solarbio.

[0113] 4 Instruments

[0114] In the following examples, the models of the instruments used are shown in Table 4.

[0115] Table 4

[0116]

[0117] In the following examples, the gene sequences were synthesized by BGI.

[0118] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0119] Example 1

[0120] The Blast (Basic Local Alignment Search Tool) analysis tool was used to perform similarity comparisons on DNA sequences or protein amino acid sequences. The protein-related tools of the online website ExPAXy were used to predict protein molecular weights, and InterPro was used to predict structural domains and protein functions. Through gene sequence analysis and sequence similarity comparisons, a total of 13 putative glycoside hydrolases were selected, specifically ECGH1, ECGH2, ECGH4, KPGH9, KPGH10, KPGH1, KPGH2, KPGH3, KPGH4, KPGH5, KPGH6, KPGH7, and KPGH8. Among them, KPGH3 comes from Klebsiella bacteria, and the sequence of the gene kpgh3 encoding KPGH3 is sequence 1 in the sequence list, and the gene sequence after optimization for the Escherichia coli expression system is sequence 2 in the sequence list. The amino acid sequence of the KPGH3 protein is shown in sequence 3 in the sequence list.

[0121] The selected 13 putative glycoside hydrolases were purified and expressed, and the subsequent biofilm-related experiments were carried out with the successfully purified and expressed proteins. The specific methods are as follows:

[0122] 1. Expression and purification of putative glycoside hydrolases

[0123] 1.1 Construction of KPGH3 recombinant expression vector pET-KPGH3

[0124] The kpgh3 gene shown in SEQ ID No. 2 was used to replace the fragment between the NdeI and XhoI recognition sites of pET-28a(+) (EMD Biosciences, size 5369 bp), and the other sequences of pET-28a(+) were kept unchanged to obtain a recombinant expression vector, which was named pET-KPGH3. pET-KPGH3 contains the His tag fusion protein encoding gene shown in Sequence 4 of the sequence list and the kpgh3 gene shown in Sequence 2 of the sequence list. The amino acid sequence of the protein KPGH3-His encoded by the KPGH3-His encoding gene is shown in Sequence 5 of the sequence list. KPGH3-His is a fusion protein obtained by connecting MGSSHHHHHHHSSGLVPRGSHM to the N-terminus of KPGH3 shown in Sequence 3 of the sequence list.

[0125] 1.2 Construction of recombinant expression vectors for ECGH1, ECGH2, ECGH4, KPGH9, KPGH10, KPGH1, KPGH2, KPGH4, KPGH5, KPGH6, KPGH7, and KPGH8 genes

[0126] The kpgh3 gene shown in sequence 2 of the sequence table in pET-KPGH3 of 1.1 was replaced with the coding gene of ECGH1, the coding gene of ECGH2, the coding gene of ECGH4, the coding gene of KPGH9, the coding gene of KPGH10, the coding gene of KPGH1, the coding gene of KPGH2, the coding gene of KPGH4, the coding gene of KPGH5, the coding gene of KPGH6, the coding gene of KPGH7 and the coding gene of KPGH8, respectively, to obtain the recombinant expression vector pET-ECGH1 of ECGH1, the recombinant expression vector pET-ECGH2 of ECGH4, and the recombinant expression vector pET-ECGH7 of ECGH8. The recombinant expression vector of GH4 is pET–ECGH4, the recombinant expression vector of KPGH9 is pET–KPGH9, the recombinant expression vector of KPGH10 is pET–KPGH10, the recombinant expression vector of KPGH1 is pET–KPGH1, the recombinant expression vector of KPGH2 is pET–KPGH2, the recombinant expression vector of KPGH4 is pET–KPGH4, the recombinant expression vector of KPGH5 is pET–KPGH5, the recombinant expression vector of KPGH6 is pET–KPGH6, the recombinant expression vector of KPGH7 is pET–KPGH7, and the recombinant expression vector of KPGH8 is pET–KPGH8.

[0127] The detailed process of extraction and transformation of each of the above recombinant expression vectors is as follows:

[0128] Use the plasmid extraction kit of Quanshijin Company to extract E. coli plasmid DNA. The steps are as follows:

[0129] 1) Pick a single colony of Escherichia coli Trans5ɑ and inoculate it into 3-5 mL of liquid LB medium, add kanamycin at a final concentration of 25 μg / mL, and culture overnight at 37°C and 220 rpm in a constant temperature shaker.

[0130] 2) Take the overnight culture solution, centrifuge it at 10,000×g for 1 min, and discard the supernatant. If the amount of bacterial solution is too large, collect it by multiple centrifugation.

[0131] 3) Add colorless solution RB (containing RNaseA) according to Table 5, and vortex to suspend the bacterial precipitate. No small bacterial clumps should remain.

[0132] Table 5

[0133] LB Media (mL) RB(μL) LB (μL) NB(μL) ≤5 250 250 350 5～10 500 500 700 10～15 750 750 1050 15～20 1000 1000 1400

[0134] 4) Add the blue solution LB and gently mix it upside down 4-6 times to fully lyse the bacteria and form a blue transparent solution. When the color changes from semi-transparent to transparent blue, it indicates complete lysis (the time should not exceed 5 minutes).

[0135] 5) Add yellow solution NB and mix gently for 5-6 times until the color changes completely from blue to yellow and a compact yellow agglomerate is formed, indicating uniform mixing. Let stand at room temperature for 2 minutes.

[0136] 6) Centrifuge at 12000×g for 5 minutes, carefully pipette the supernatant and add it to the centrifuge column. Centrifuge the centrifuge column at 12000×g for 1 minute, and discard the flow-through. If the supernatant volume is greater than 800μL, add it to the column several times and centrifuge it as above, and discard the flow-through.

[0137] 7) Add 650 μL of WB solution, centrifuge at 12,000 × g for 1 min, and discard the flow-through.

[0138] 8) Centrifuge at 12000×g for 1-2 min to completely remove residual WB.

[0139] 9) Place the centrifugal column in a clean 1.5 ml EP tube, add 30-50 μL of preheated deionized water to the center of the column, and let it stand at room temperature for 1 min.

[0140] 10) Centrifuge at 10000×g for 1 min to elute the DNA, and store it at -20°C to obtain the corresponding plasmid DNA in Escherichia coli.

[0141] 1.3 Construction of gene expression strains

[0142] pET–KPGH3 in 1.1, pET–ECGH1, pET–ECGH2, pET–ECGH4, pET–KPGH9, pET–KPGH10, pET–KPGH1, pET–KPGH2, pET–KPGH4, pET–KPGH5, pET–KPGH6, pET–KPGH7 and pET–KPGH8 in 1.2 were transformed into competent E. coli BL21 (DE3) cells, and the recombinant E. coli containing pET–KPGH3 was named KPGH3-BL21 strain, the recombinant E. coli containing pET–ECGH1 was named ECGH1-BL21 strain, the recombinant E. coli containing pET–ECGH2 was named ECGH2-BL21 strain, the recombinant E. coli containing pET–KPGH4 was named KPGH4-BL21 strain, and the recombinant E. coli containing pET–KPGH9 was named KPGH3-BL21 strain. PGH9-BL21 strain, the recombinant Escherichia coli containing pET–KPGH10 was named KPGH10-BL21 strain, the recombinant Escherichia coli containing pET–KPGH1 was named KPGH1-BL21 strain, the recombinant Escherichia coli containing pET–KPGH2 was named KPGH2-BL21 strain, the recombinant Escherichia coli containing pET–KPGH4 was named KPGH4-BL21 strain, the recombinant Escherichia coli containing pET–KPGH5 was named KPGH5-BL21 strain, the recombinant Escherichia coli containing pET–KPGH6 was named KPGH6-BL21 strain, the recombinant Escherichia coli containing pET–KPGH6 was named KPGH6-BL21 strain, the recombinant Escherichia coli containing pET–KPGH7 was named KPGH7-BL21 strain, and the recombinant Escherichia coli containing pET–KPGH8 was named KPGH8-BL21 strain.

[0143] The process of obtaining each recombinant E. coli is as follows:

[0144] 1) Take 50 μL of freshly thawed Escherichia coli BL21 competent cells in a clean bench and add 1 μL of the extracted pET recombinant expression vector of the corresponding protein (see 1.1 and 1.2). Flick to mix and place on ice for 30 minutes.

[0145] 2) Heat shock in 42℃ water bath for 90s and then immediately place in ice bath for 2min.

[0146] 3) Add 250 μL of LB medium equilibrated to room temperature and culture in a constant temperature shaker at 200 rpm and 37°C for 1 h.

[0147] 4) Take 50-100 μL of the bacterial solution from step 3) and spread it evenly on a solid LB medium plate, then incubate it upside down in a 37°C constant temperature incubator overnight.

[0148] 2. Induced expression of proteins

[0149] 2.1 Seed culture

[0150] In the clean bench, take ECGH1-BL21 strain, ECGH2-BL21 strain, ECGH4-BL21 strain, KPGH9-BL21 strain, KPGH10-BL21 strain, KPGH1-BL21 strain, KPGH2-BL21 strain, KPGH3-BL21 strain, KPGH4-BL21 strain, KPGH5-BL21 strain, KPGH6-BL21 strain, KPGH7-BL21 strain, and KPGH8-BL21 strain, and separately inoculate them into LB medium containing 25 μg / mL kanamycin (a medium obtained by adding kanamycin to LB medium to a final concentration of kanamycin of 25 μg / mL), and culture at 37°C with shaking at 220 rpm for 12 h.

[0151] 2.2 Shake flask fermentation

[0152] In a clean bench, inoculate the overnight cultured bacteria in step 2.1 into 100 mL of LB medium containing 25 μg / mL kanamycin at a 1% inoculum volume, and culture at 37°C and 220 rpm with shaking. When the OD600 of the bacterial solution is between 0.5 and 0.6, add IPTG to a final concentration of 0.5 mM, and culture at 18°C ​​and 220 rpm with shaking for 12 h.

[0153] 2.3 Bottom Fermentation

[0154] 1) Collect the cells by centrifugation at 5000 g for 30 min at 4°C, remove the liquid, resuspend the cells in Binding buffer and centrifuge again to wash the LB medium.

[0155] 2) Add PMSF, Aprotinin, and Leupeptin to the Binding buffer (see Table 2 for added concentrations). Use this buffer to resuspend the bacteria. Add 3-4 ml per gram of wet bacteria and mix well.

[0156] 3) The bacterial solution was disrupted by an ultrasonic disruptor, and the program was set as follows: disruption for 10 seconds, rest for 10 seconds, as one cycle, and this was repeated 30 times in total at a power of 28%.

[0157] 4) After the bacterial suspension was disrupted, it was centrifuged at 20,000 rpm and 4°C for 50 min to collect the supernatant (soluble), which was filtered through a 0.45 μm filter membrane to obtain a protein supernatant for later use.

[0158] 3. Protein purification

[0159] 3.1 Pretreatment of affinity chromatography column

[0160] 1) Prepare an affinity chromatography column from Solarbio, an appropriate volume of Millipore Novagen Ni-NTA The column is packed with resin filler.

[0161] 2) Use 5 times the volume of filler to wash away the alcohol with deionized water, and use 5 times the volume of filler to activate the matrix with Binding buffer, and place it at 4°C for use.

[0162] 3.2 Protein purification

[0163] The following steps were all carried out at 4°C

[0164] 1) Add the protein supernatant extracted in step 2.3 to the chromatography column prepared in step 3.1 and mix at 4°C for 30 minutes using a tumbling shaker.

[0165] 2) The column was vertically set up and eluted by gravity. When the interface dropped to the surface of the medium, the column was washed with Binding buffer until the filtrate was clear, and then the protein was eluted with Elution Buffer. The eluate containing the target protein was collected by 1.5 mL EP tubes for SDS-PAGE analysis. QuickBlue fast gel staining solution (BF06152, purchased from Beijing Biolong Immunotechnology Co., Ltd.) was used for staining.

[0166] The results of SDS-PAGE analysis are shown in Figure 1 The results showed that eight soluble glycoside hydrolases were successfully expressed and purified, namely, soluble glycoside hydrolase KPGH3-His expressed by KPGH3-BL21 strain, soluble glycoside hydrolase ECGH2-His expressed by ECGH2-BL21 strain, soluble glycoside hydrolase KPGH1-His expressed by KPGH1-BL21 strain, soluble glycoside hydrolase KPGH2-His expressed by KPGH2-BL21 strain, and soluble glycoside hydrolase KPGH3-His expressed by KPGH3-BL21 strain. is, the soluble glycoside hydrolase KPGH4-His expressed by KPGH4-BL21 strain, the soluble glycoside hydrolase KPGH5-His expressed by KPGH5-BL21 strain, the soluble glycoside hydrolase KPGH6-His expressed by KPGH6-BL21 strain, and the soluble glycoside hydrolase KPGH7-His expressed by KPGH7-BL21 strain. The other five strains did not express soluble glycoside hydrolase.

[0167] The eluates of the above 8 target proteins were collected respectively, ultrafiltered in Millipore ultrafiltration tubes using 1×PBS solution, and frozen at -20°C after being divided. Finally, the chromatography column was washed with Final wash Buffer with a concentration of 500mM imidazole, and then the chromatography column was treated with Milli-Q ultrapure water, sealed with 20% alcohol and stored in a refrigerator at 4°C.

[0168] 4. BCA protein quantification

[0169] 1) Working solution preparation: BCA solution and Cu solution were prepared in a ratio of 50:1, and the working solution was added to a 96-well culture plate at a volume of 200 μL per well.

[0170] 2) Protein standard curve determination: 30 μL 1×PBS was added with 30 μL BSA (4000 μg / mL) for serial dilution. 25 μL of the diluted sample was added to each well of a 96-well plate, and then 200 μL of the working solution was added to each well. Incubate in a 37°C incubator for 30 min, and use an ELISA reader to measure the absorbance value at OD562 to draw a standard curve.

[0171] 3) Dilute the 8 protein samples in appropriate proportions according to the situation, add 25 μL per well to a 96-well plate in triplicate, add 200 μL of working solution to each well, and incubate at 37°C for 30 min. Determine the OD562 absorbance value and calculate the original protein concentration based on the standard curve.

[0172] 5. Determination of biofilm content by 96-well ELISA plate crystal violet method

[0173] The following experiments were carried out in a biological safety cabinet in a P2 laboratory.

[0174] 5.1 Biofilm inhibition experiment

[0175] 1) The extracted protein was diluted on ice to 8 concentrations Pr1-Pr8. The final concentration of protein is as follows Figure 4 , and save for later use to obtain a protein sample.

[0176] 2) One day before the experiment, add 3 mL of LB medium to a shaking tube and inoculate an appropriate amount of glycerol culture of Klebsiella pneumoniae (ATCC700603) and Pseudomonas aeruginosa PAO1 and PA14, and culture at 37°C with shaking at 220 rpm for 12 h.

[0177] 3) Dilute the Klebsiella pneumoniae culture and Pseudomonas aeruginosa culture culture overnight at a ratio of 1:100 with M63 medium supplemented with magnesium sulfate, glucose and casamino acids and BHI medium, respectively, and dilute the culture solutions evenly.

[0178] 4) Take a 96-well culture plate, add 200 μL sterile ddH2O or PBS to each well around the edges to prevent edge effects. Add 5 μL of gradient diluted protein sample to each well in the remaining rows from left to right, in triplicate. Add 5 μL 1×PBS to the 10th column as a negative control, and add 5 μL tobramycin to the 11th column as a positive control. Then add the diluted bacterial solution to the 96-well plate, 95 μL per well. Seal the periphery of the 96-well plate with sealing film, and place the plate at a constant temperature of 25.5°C for 12 hours. The specific distribution is as follows: Figure 2 shown.

[0179] 5) After 12 hours of incubation, wash the 96-well culture plate with ddH2O to remove residual bacterial solution and debris, clean the plate, and place it in a 37°C incubator to dry or air dry.

[0180] 6) Add 200 μL of 0.1% (w / v) crystal violet to each well for staining for 15-20 minutes. Note that the crystal violet solution should be added slowly to avoid unnecessary errors.

[0181] 7) Use ddH2O to wash the crystal violet, reduce the background color, and observe the adhesion circle formed by Pseudomonas aeruginosa at the air-liquid interface. Since the Klebsiella pneumoniae strain is a non-motile strain, a biofilm is formed at the bottom of the well. Place the well plate in a 37°C incubator to dry or dry naturally, then add 150 μL of 33% glacial acetic acid to each well to dissolve for 10-15 minutes, measure the absorbance at 595nm or 600nm, and standardize the data with the blank control (1×PBS instead of protein treatment) as the standard.

[0182] The results of screening the activities of 8 proteins by Klebsiella pneumoniae ATCC700603 and Pseudomonas aeruginosa PAO1 and PA14 are shown in Figure 3 , indicating that the activity of KPGH3-His protein was the best among the eight proteins and was active against all three strains, that is, it had cross-species activity.

[0183] The inhibitory effect of KPGH3-His protein on biofilm formation of three strains was determined. KPGH3-His protein was co-cultured with the strains in a 96-well plate in the form of concentration gradient for 12 h, and the biofilm residue was determined and quantified after crystal violet staining. Figure 4 Through data calculation, we can conclude that KPGH3-His has a consistent effect on the biofilm formation of Pseudomonas aeruginosa PAO1 strain, PA14 strain, and Klebsiella pneumoniae ATCC700603 strain. 50 They are 36.85nM, 43.34nM and 82.58nM respectively.

[0184] 5.2 Biofilm destruction experiment

[0185] After preliminary activity screening, we selected the KPGH3-His protein with the best activity and cross-species activity for subsequent activity determination experiments. The biofilm disruption experiment is as follows:

[0186] 1) Add 3 mL of LB medium into an inoculation tube and inoculate an appropriate amount of Klebsiella pneumoniae (ATCC700603) and Pseudomonas aeruginosa PAO1 and PA14 glycerol stocks, and culture at 37°C in a shaker at 220 rpm for 12 h.

[0187] 2) Dilute the Klebsiella pneumoniae culture and Pseudomonas aeruginosa culture overnight at a ratio of 1:100 with M63 medium supplemented with magnesium sulfate, glucose and casamino acids and BHI medium, respectively, and dilute the culture evenly.

[0188] 3) Take a 96-well culture plate and add 200 μL sterilized ddH2O or PBS to each well around the edges to prevent edge effects. Add 100 μL of diluted bacterial solution to each well in the remaining rows from left to right, in triplicate. Add 5 μL of antibiotic tobramycin to the 11th column as a positive control. Seal the periphery of the 96-well plate with sealing film and place the plate at a constant temperature of 25.5℃ for 12 hours.

[0189] 4) After 12 hours of incubation, wash the 96-well culture plate with ddH2O to remove residual bacterial solution and debris, clean the plate, and place it in a 37°C incubator to dry or air dry.

[0190] 5) The extracted KPGH3-His protein was diluted with 1×PBS on ice to 8 concentrations of Pr1-Pr8. The final concentration of the protein is as follows Figure 5 , save for future use.

[0191] 6) Add 150 μL of gradient diluted protein to each well, add 150 μL of 1× PBS to the 10th and 11th columns respectively, and place the plate at 25.5°C for 1 hour.

[0192] 7) Wash the 96-well culture plate with ddH2O, clean the plate, and place it in a 37°C incubator to dry or air dry.

[0193] 8) Add 200 μL of 0.1% (w / v) crystal violet to each well for staining for 15-20 minutes. Be careful to add the crystal violet solution slowly to avoid unnecessary errors.

[0194] 9) Wash the crystal violet with ddH2O to reduce the background color. Place the well plate in a 37°C incubator to dry or air dry, then add 150 μL of 33% glacial acetic acid to each well to dissolve for 10-15 minutes, measure the absorbance at 595nm or 600nm, and standardize the data using the blank control (1×PBS instead of KPGH3-His protein treatment) as the standard.

[0195] After crystal violet staining, the Figure 5 The data showed that KPGH3-His protein had a destructive effect on the biofilm formed by the three strains after 12 hours of culture for 1 hour. By calculating the data to determine the biofilm residue, we can obtain the EC value of KPGH3-His on the biofilm destruction of Pseudomonas aeruginosa PAO1 strain, PA14 strain, and Klebsiella pneumoniae ATCC700603 strain. 50 They are 45.35nM, 29.3nM and 18.23nM respectively.

[0196] 6. Antibiotic sensitization reaction of KPGH3-His

[0197] The following experiments were performed in a P2 laboratory biosafety cabinet, and all steps involving proteins were handled on low-temperature ice. Antibiotic sensitization experiments were performed on Pseudomonas aeruginosa PAO1 strains. On a 96-well culture plate, the concentration of antibiotic tobramycin was gradient diluted from left to right (distributed in columns), and the concentration of KPGH3-His protein was gradient diluted from top to bottom (distributed in rows) with 1× PBS. The distribution of the culture plate for the antibiotic sensitization experiment is shown in Table 6, and the steps are detailed in the diagram. Figure 6 .

[0198] Table 6

[0199]

[0200]

[0201] 1) Take the strain frozen at -80℃ and streak it onto a nutrient agar plate and culture it at 37℃ overnight.

[0202] 2) Pick well-separated and well-shaped colonies from the agar culture dish and inoculate them into a shake tube containing 3 mL of CAMHB medium. Culture at 37°C, 220 rpm for about 6-7 hours; or culture at 37°C overnight. Shake the culture solution well before diluting it.

[0203] During the bacterial culture process, the following preparations must be completed:

[0204] 3) Calculate the final concentration of the protein and the initial concentration, and dilute the protein on ice using CAMHB medium. Calculate the initial tobramycin concentration and prepare the solution.

[0205] 4) Take a round-bottom 96-well plate and add 90 μL of CAMHB medium to each well in rows A and H.

[0206] 5) Add 1 μL of antibiotic to each well in the first column of row BG.

[0207] 6) Add 180 μL of CAMHB medium to each well in the first column of rows F and G, and add 90 μL of CAMHB medium to each well in the remaining columns.

[0208] 7) In row BE, add 180 μL of the previously gradient diluted protein to each well in the first column, and 90 μL to each well thereafter. That is, in row BE, each row contains different concentrations of protein.

[0209] 8) Rows BG: Start with the first column to perform serial dilution of antibiotics. Column 12 is not involved in the dilution and the antibiotic concentration is 0.

[0210] Keep the prepared plates on ice.

[0211] 9) Take the bacterial solution in the exponential growth phase and measure the McFarland concentration of each bacterial strain using a McFarland turbidimeter. Dilute the bacterial solution with sterile saline to a McFarland concentration of 0.5, i.e., 1.5×10 8 CFU / mL, the bacterial solution should be used within 15 minutes after preparation.

[0212] 10) Add 10 μL of the test strain solution to each well in rows BF, and 10 μL of the CLSI quality control strain solution to each well in row G. Seal the periphery of the plate with sealing film and transfer to a 37°C incubator for 18-20 hours.

[0213] 11) Read the results visually and record them. The MIC value is the minimum concentration of protein required to inhibit visible growth of microorganisms.

[0214] 12) The above experiment needs to be carried out three times in parallel, and the results are read as the final results.

[0215] Results Figure 7 The original tobramycin MIC value of PAO1 strain was 2μg / mL, and the minimum sensitization concentration of KPGH3 protein was 50nM; at a concentration of 200nM, the MIC of PAO1 strain could be reduced to 0.5μg / mL.

[0216] 7. KPGH3 protein domain and function analysis

[0217] Through BLAST analysis, we found that KPGH3 protein belongs to the glycoside hydrolase 19 family (see Figure 8The theoretical Mw of KPGH3 protein was calculated by using the Compute pI / Mw in the ExPAXy website to be about 22.9 KDa and the theoretical pI was 9.84 (see Figure 8 (b).

[0218] We then used InterPro to analyze the entire sequence, analyzed the KPGH3 protein domain, and obtained KPGH3 enzyme activity information, which showed that it had chitinase activity. Figure 8 Figure c. Chitin, composed of β-(1,4) linked N-acetyl-D-glucosamine (GlcNAc) units, is one of the most abundant polymers in nature and is the main component of crab, shrimp and lobster shells. Crabs, shrimp and lobsters are the main types of seafood, and the global seafood industry produces approximately 6–8 million tons of shell waste each year

[11] . This waste stream represents a cheap, renewable source of chitin that can be used to produce value-added chemicals. In addition to crustacean waste, chitin can also be isolated from insects and fungi. Products made from chitin and its deacetylated derivative chitosan can be used in medical applications, packaging, food and nutrition, biotechnology, agriculture and environmental protection

[12]

[13] . Notably, water-soluble chitosan oligosaccharides can also act as antimicrobial agents

[14] , antitumor agents and anti-inflammatory drugs

[15] . Chitooligosaccharides can be produced by chemical or enzymatic depolymerization of chitin and chitosan. The current conventional methods for producing chitooligosaccharides mainly rely on acid catalysis, which is characterized by low yields and high environmental impact. Enzyme catalysis for the depolymerization of chitin and chitosan is a promising alternative because it allows us to produce specific chitin and chitooligosaccharides in a controllable and environmentally friendly manner.

[0219] The above results have proven that KPGH3 can effectively inhibit the formation of biofilms of Pseudomonas aeruginosa PAO1, PA14 strains, and Klebsiella pneumoniae (ATCC700603) strains within 12 hours of action, and effectively destroy the formed biofilms. 50 The damage effect EC 50 The concentrations of KPGH3 and 50nM were 36.85nM, 43.34nM and 82.58nM respectively. At a concentration of 50nM, KPGH3 could enhance the sensitivity of Pseudomonas aeruginosa PAO1 strain to tobramycin. In addition, sequence analysis results showed that KPGH3 had chitinase activity, which also represented the potential application ability of KPGH3 in chitin biotransformation.

[0220] In summary, KPGH3 has the potential to inhibit and disperse biofilms of various pathogenic microorganisms, as well as potential chitin biotransformation ability, and it is feasible to use it to develop new anti-biofilm therapeutic agents.

[0221] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

[0222] References

[0223] 1. Sharma, D.; Misba, L.; Khan, AUAntibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrobial resistance and infectioncontrol 2019,8,76,doi:10.1186 / s13756-019-0533-3.

[0224] 2.Flemming,HC; Wingender,J.The biofilm matrix.Naturereviews.Microbiology 2010,8,623-633,doi:10.1038 / nrmicro2415.

[0225] 3. Rumbaugh, KP; Diggle, SP; Watters, CM; Ross-Gillespie, A.; Griffin, AS; West, SAQuorum Sensing and the Social Evolution of BacterialViruulence. Current Biology 2009, 19, 341-345, doi: 10.1016 / j.cub.2009.01.050.

[0226] 4.Karatan,E.;Watnick,P.Signals,Regulatory Networks,and Materials ThatBuild and Break Bacterial Biofilms.Microbiology and Molecular Biology Reviews2009,73,310,doi:10.1128 / MMBR.00041-08.

[0227] 5.Hall-Stoodley,L.;Costerton,J.W.;Stoodley,P.Bacterial biofilms:fromthe natural environment to infectious diseases.Nature reviews.Microbiology2004,2,95-108,doi:10.1038 / nrmicro821.

[0228] 6.Koo,H.;Allan,R.N.;Howlin,R.P.;Stoodley,P.;Hall-Stoodley,L.Targetingmicrobial biofilms:current and prospective therapeutic strategies.Naturereviews.Microbiology 2017,15,740-755,doi:10.1038 / nrmicro.2017.99.

[0229] 7.Bales,P.M.;Renke,E.M.;May,S.L.;Shen,Y.;Nelson,D.C.Purification andCharacterization ofBiofilm-Associated EPS Exopolysaccharides from ESKAPEOrganisms and Other Pathogens.PloS one 2013,8,e67950,doi:10.1371 / journal.pone.0067950.

[0230] 8.Wingender,J.;Strathmann,M.;Rode,A.;Leis,A.;Flemming,H.C.Isolationand biochemical characterization of extracellular polymeric substances fromPseudomonas aeruginosa.Methods in enzymology 2001,336,302-314,doi:10.1016 / s0076-6879(01)36597-7.

[0231] 9.Limoli,D.H.;Jones,C.J.;Wozniak,D.J.Bacterial ExtracellularPolysaccharides in Biofilm Formation and Function.Microbiology Spectrum 2015,3,doi:doi:10.1128 / microbiolspec.MB-0011-2014.

[0232] 10.Watters,C.;Fleming,D.;Bishop,D.;Rumbaugh,K.P.Host Responses toBiofilm.Progress in molecular biology and translational science 2016,142,193-239,doi:10.1016 / bs.pmbts.2016.05.007.

[0233] 11.Gao,X.;Chen,X.;Zhang,J.;Guo,W.;Jin,F.;Yan,N.Transformation ofChitin and Waste Shrimp Shells into Acetic Acid and Pyrrole.ACS SustainableChemistry&Engineering 2016,4,3912-3920,doi:10.1021 / acssuschemeng.6b00767.

[0234] 12.Kardas,I.;Struszczyk,M.H.;Kucharska,M.;van den Broek,L.A.M.;vanDam,J.E.G.;Ciechańska,D.Chitin and Chitosan as Functional Biopolymers forIndustrial Applications.In The European Polysaccharide Network ofExcellence(EPNOE):Research Initiatives and Results,Navard,P.,Ed.Springer Vienna:Vienna,2012;10.1007 / 978-3-7091-0421-7_11pp.329-373.

[0235] 13.vandenBroek,L.A.M.;Knoop,R.J.I.;Kappen,F.H.J.;Boeriu,C.G.Chitosanfilms and blends for packaging material.Carbohydrate Polymers 2015,116,237-242,doi:https: / / doi.org / 10.1016 / j.carbpol.2014.07.039.

[0236] 14.No,H.K.;Young Park,N.;Ho Lee,S.;Meyers,S.P.Antibacterial activityof chitosans and chitosan oligomers with different molecularweights.International journal offood microbiology 2002,74,65-72,

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[0238] 15. Azuma, K.; Osaki, T.; Minami, S.; Okamoto, Y. Anticancer and anti-inflammatory properties of chitin and chitosan oligosaccharides. Journal of functional biomaterials 2015, 6, 33-49, doi:10.3390 / jfb6010033. Sequence Listing <110> Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences <120> Application of chitinase with cross-species activity and related biomaterials <130> GNCSY202233 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 627 <212> DNA <213> Klebsiella pneumoniae subsp. Pneumoniae <400> 1 atgaatcaaa cacaattcca gaaggcggct ggcatcagcg ccgggttagc tgcgcgctgg 60 tttccgcata ttacagccgc gatgaaagag tttggcatca cttccgctat cgaccaggca 120 atgttcattg cccaggtagg gcatgaaagc acgggattta cccagcttgt tgagagcttc 180 aattatagcg tggctggctt gaatagtttt gtccgcgccg ggcggctgac gcagggtcag 240 gctaattcgc tcggccgccg gcagggtgaa ccatcgttgc cactggaaag gcaacgagcg 300 atcgccaatc tggtgtacag caaacgcatg gggaataacg gggcaacaga cggctggttt 360 taccgcgggc gcgggctcat ccagaccacc ggcctgaaca attaccgcga atgcgggaat 420 gctttgaaga ttgatctggt taaacagcct gaattgctgg cacaggatga gtatgcggcg 480 cgcagtgctg cctggttcta tacctcacgc ggctgtttgc gttatcccgg tgaccttaca 540 cgcgtcactc agattatcaa cggcggacag aacggcattg atgaccgtaa agcccgctat 600 ctcctggcaa aaagtattct tgtttga 627 <210> 2 <211> 627 <212> DNA <213> Artificial Sequence <400> 2 atgaaccaga cccagtttca gaaagcagca ggtattagcg ccggtctggc agcccgctgg 60 tttccgcata ttaccgcagc aatgaaagaa tttggtatta ccagcgccat tgatcaggca 120 atgtttattg cccaggttgg tcatgaaagc accggcttta cccagctggt ggaaagcttt 180 aattatagtg ttgcaggtct gaatagtttt gtgcgcgcag gccgcctgac ccagggtcaa 240 gcaaatagcc tgggccgtcg tcagggtgaa ccgagcctgc cgctggaacg tcagcgcgca 300 attgcaaatc tggtttatag taaacgtatg ggcaataatg gtgccaccga tggttggttt 360 tatcgtggcc gtggcctgat tcagaccacc ggcctgaata attatcgcga atgtggcaat 420 gccctgaaaa ttgatctggt gaaacagccg gaactgctgg cacaggatga atatgccgcc 480 cgtagcgcag cctggtttta taccagccgt ggctgcctgc gctatccggg cgatctgacc 540 cgcgttaccc agattattaa tggtggccag aatggcattg atgatcgcaa agcccgttat 600 ctgctggcca aaagcattct ggtttaa 627 <210> 3 <211> 208 <212> PRT <213> Artificial Sequence <400> 3 Met Asn Gln Thr Gln Phe Gln Lys Ala Ala Gly Ile Ser Ala Gly Leu 1 5 10 15 Ala Ala Arg Trp Phe Pro His Ile Thr Ala Ala Met Lys Glu Phe Gly 20 25 30 Ile Thr Ser Ala Ile Asp Gln Ala Met Phe Ile Ala Gln Val Gly His 35 40 45 Glu Ser Thr Gly Phe Thr Gln Leu Val Glu Ser Phe Asn Tyr Ser Val 50 55 60 Ala Gly Leu Asn Ser Phe Val Arg Ala Gly Arg Leu Thr Gln Gly Gln 65 70 75 80 Ala Asn Ser Leu Gly Arg Arg Gln Gly Glu Pro Ser Leu Pro Leu Glu 85 90 95 Arg Gln Arg Ala Ile Ala Asn Leu Val Tyr Ser Lys Arg Met Gly Asn 100 105 110 Asn Gly Ala Thr Asp Gly Trp Phe Tyr Arg Gly Arg Gly Leu Ile Gln 115 120 125 Thr Thr Gly Leu Asn Asn Tyr Arg Glu Cys Gly Asn Ala Leu Lys Ile 130 135 140 Asp Leu Val Lys Gln Pro Glu Leu Leu Ala Gln Asp Glu Tyr Ala Ala 145 150 155 160 Arg Ser Ala Ala Trp Phe Tyr Thr Ser Arg Gly Cys Leu Arg Tyr Pro 165 170 175 Gly Asp Leu Thr Arg Val Thr Gln Ile Ile Asn Gly Gly Gln Asn Gly 180 185 190 Ile Asp Asp Arg Lys Ala Arg Tyr Leu Leu Ala Lys Ser Ile Leu Val 195 200 205 <210> 4 <211> 690 <212> DNA <213> Artificial Sequence <400> 4 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgatgaacc agacccagtt tcagaaagca gcaggtatta gcgccggtct ggcagcccgc 120 tggtttccgc atattaccgc agcaatgaaa gaatttggta ttaccagcgc cattgatcag 180 gcaatgttta ttgcccaggt tggtcatgaa agcaccggct ttacccagct ggtggaaagc 240 tttaattata gtgttgcagg tctgaatagt tttgtgcgcg caggccgcct gacccagggt 300 caagcaaata gcctgggccg tcgtcagggt gaaccgagcc tgccgctgga acgtcagcgc 360 gcaattgcaa atctggttta tagtaaacgt atgggcaata atggtgccac cgatggttgg 420 ttttatcgtg gccgtggcct gattcagacc accggcctga ataattatcg cgaatgtggc 480 aatgccctga aaattgatct ggtgaaacag ccggaactgc tggcacagga tgaatatgcc 540 gcccgtagcg cagcctggtt ttataccagc cgtggctgcc tgcgctatcc gggcgatctg 600 acccgcgtta cccagattat taatggtggc cagaatggca ttgatgatcg caaagcccgt 660 tatctgctgg ccaaaagcat tctggtttaa 690 <210> 5 <211> 229 <212> PRT <213> Artificial Sequence <400> 5 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Met Asn Gln Thr Gln Phe Gln Lys Ala Ala Gly 20 25 30 Ile Ser Ala Gly Leu Ala Ala Arg Trp Phe Pro His Ile Thr Ala Ala 35 40 45 Met Lys Glu Phe Gly Ile Thr Ser Ala Ile Asp Gln Ala Met Phe Ile 50 55 60 Ala Gln Val Gly His Glu Ser Thr Gly Phe Thr Gln Leu Val Glu Ser 65 70 75 80 Phe Asn Tyr Ser Val Ala Gly Leu Asn Ser Phe Val Arg Ala Gly Arg 85 90 95 Leu Thr Gln Gly Gln Ala Asn Ser Leu Gly Arg Arg Gln Gly Glu Pro 100 105 110 Ser Leu Pro Leu Glu Arg Gln Arg Ala Ile Ala Asn Leu Val Tyr Ser 115 120 125 Lys Arg Met Gly Asn Asn Gly Ala Thr Asp Gly Trp Phe Tyr Arg Gly 130 135 140 Arg Gly Leu Ile Gln Thr Thr Gly Leu Asn Asn Tyr Arg Glu Cys Gly 145 150 155 160 Asn Ala Leu Lys Ile Asp Leu Val Lys Gln Pro Glu Leu Leu Ala Gln 165 170 175 Asp Glu Tyr Ala Ala Arg Ser Ala Ala Trp Phe Tyr Thr Ser Arg Gly 180 185 190 Cys Leu Arg Tyr Pro Gly Asp Leu Thr Arg Val Thr Gln Ile Ile Asn 195 200 205 Gly Gly Gln Asn Gly Ile Asp Asp Arg Lys Ala Arg Tyr Leu Leu Ala 210 215 220 Lys Ser Ile Leu Val 225

Claims

1. Application of protein, characterized in that: The application is any one or more of the following: Y1. Use of KPGH3 protein in the preparation of a product for enhancing the bactericidal and / or antibacterial activity of an antibacterial drug; the bactericidal and / or antibacterial activity is directed against Pseudomonas aeruginosa and / or Klebsiella pneumoniae; Y2. Application of KPGH3 protein in the preparation of an anti-biofilm therapeutic agent; the anti-biofilm therapeutic agent is directed against biofilms of Pseudomonas aeruginosa and / or biofilms of Klebsiella pneumoniae; The KPGH3 protein is any of the following proteins: A1) The amino acid sequence is the protein of sequence 3 in the sequence listing; A2) The amino acid sequence is the protein of sequence 5 in the sequence listing; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. Use of the biological material related to the KPGH3 protein described in claim 1 in the preparation of an anti-biofilm therapeutic agent; the anti-biofilm therapeutic agent is directed against the biofilm of Pseudomonas aeruginosa and / or the biofilm of Klebsiella pneumoniae; The biological material is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

3. The use according to claim 2, characterized in that: In the biological material, the nucleic acid molecule in B1) is the gene shown in b1) or b2) below: b1) The coding sequence of the coding chain is the cDNA molecule or DNA molecule of sequence 1 or sequence 2 in the sequence list; b2) The nucleotides of the coding chain are the cDNA molecule or DNA molecule of sequence 1 or sequence 2 in the sequence list.

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