CN116622683A - Specific bacillus lyase and preparation method and application thereof - Google Patents

Specific bacillus lyase and preparation method and application thereof Download PDF

Info

Publication number
CN116622683A
CN116622683A CN202310308686.XA CN202310308686A CN116622683A CN 116622683 A CN116622683 A CN 116622683A CN 202310308686 A CN202310308686 A CN 202310308686A CN 116622683 A CN116622683 A CN 116622683A
Authority
CN
China
Prior art keywords
bacillus
lyase
primer
acetylmuramyl
alanine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202310308686.XA
Other languages
Chinese (zh)
Other versions
CN116622683B (en
Inventor
梁小波
王振宇
刘贻坦
冯应山
唐若诗
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kunming University of Science and Technology
Original Assignee
Kunming University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kunming University of Science and Technology filed Critical Kunming University of Science and Technology
Priority to CN202310308686.XA priority Critical patent/CN116622683B/en
Publication of CN116622683A publication Critical patent/CN116622683A/en
Application granted granted Critical
Publication of CN116622683B publication Critical patent/CN116622683B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01028—N-Acetylmuramoyl-L-alanine amidase (3.5.1.28)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The application provides a specific bacillus lyase and a preparation method and application thereof, wherein a genome DNA of bacillus belicus FS001 is used as a template, primers are designed and synthesized, an xlyB (BSFS 001GM 003346) gene is amplified by PCR, the gene is recombined with a pET-23b vector, induced expression is carried out in E.coli BL21, and Ni-Sepharose affinity chromatography is adopted for purification to obtain recombinant N-acetylmuramyl-L-alanine-amidase protein with higher purity. The lyase can hydrolyze cell walls of a variety of bacillus species. And has great application prospect in inhibiting the growth of bacillus and in the production of biological medicines and foods.

Description

Specific bacillus lyase and preparation method and application thereof
Technical Field
The application relates to the technical field of bioengineering, in particular to a specific bacillus lyase and a preparation method and application thereof.
Background
Bacillus is a gram positive bacterium, and its cell wall is composed of thick and compact peptidoglycan and teichoic acid, so it has strong stress-resistance action and can survive in high-temp. and acid-base polar environments. Therefore, in the prior art, when bacillus is killed by using antibiotics, problems such as resistance of bacteria to antibiotics are likely to occur. The study of the use of bacterial lytic enzymes to lyse and kill bacillus under the specific and highly active characteristics of bacterial lytic enzymes has become of increasing interest.
Disclosure of Invention
The embodiment of the application aims to provide a specific bacillus lyase, a preparation method and application thereof, which can provide a molecular basis for exploring bacterial apoptosis and a theoretical basis for developing and utilizing efficient bacillus hydrolase.
According to a first aspect of the present application there is provided a specific bacillus lyase, said lyase being an N-acetylmuramyl-L-alanine-amidase, said lyase being obtained based on analysis of sequencing data of bacillus bailii FS001 genome.
According to a second aspect of the present application, there is also provided a method for preparing a specific bacillus lyase, comprising the steps of:
s1, designing and synthesizing a primer for amplifying N-acetylmuramyl-L-alanine-amidase, wherein the primer comprises an upstream primer and a downstream primer.
The sequence of the upstream primer is as follows: CGGGATCCGGTGGGGATTGAAGTGAAAAA.
The sequence of the downstream primer is as follows: CCGCTCGAGCAGTTTTTCTTCAATTTTCG.
S2, amplifying by using the bacillus bailii FS001 genome as a template based on the upstream primer and the downstream primer to obtain a target gene fragment.
S3, connecting the target gene fragment with a plasmid to complete construction of a recombinant plasmid; the recombinant plasmid is transformed into escherichia coli for expression.
S4, purifying to obtain the N-acetylmuramyl-L-alanine-amidase.
In one embodiment, in step S2, the conditions of amplification include at least: pre-denaturation at 98℃for 2min, denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 10s, for a total of 30 cycles.
In one embodiment, after step S3, a recombinant plasmid containing the N-acetylmuramyl-L-alanine-amidase gene is selected and subjected to double cleavage and sequencing verification.
In one embodiment, in step S4, the purifying comprises at least: protein purification was performed using a protein purification system.
According to a third aspect of the present application there is also provided the use of a lyase for specific lysis of bacillus using the lyase provided in the first aspect.
In one embodiment, the bacillus includes at least bacillus subtilis, bacillus belicus, bacillus aryabhattai.
In one embodiment, the reaction conditions suitable for the specific cleavage include at least: the pH is 6.5-7.5, and the temperature is 30-37 ℃.
In one embodiment, the reaction conditions suitable for the specific cleavage include: the pH was 6.5 and the temperature was 37 ℃.
In one embodiment, the effect of the lyase on bacillus is determined by absorbance measurement.
Compared with the prior art, the application has the beneficial effects that:
in the technical scheme of the application, the lyase provided by the application has specificity and high activity, has strong specificity to pathogenic bacteria and does not interfere with normal flora; can kill pathogenic bacteria colonized on mucosal surfaces; the probability of bacterial resistance to the lyase is low. Can hydrolyze cell walls of various bacillus and can crack bacillus efficiently. Has great application prospect in inhibiting the growth of bacillus and in the production of biological medicines and foods.
Drawings
FIG. 1 is an electrophoresis chart of the results of the gene polymerase chain reaction in the method for preparing a lyase according to the embodiment of the application.
FIG. 2 is a diagram showing construction of a recombinant vector in the method for producing a lyase according to an embodiment of the present application.
FIG. 3 is a polyacrylamide gel electrophoresis of the purified enzyme protein in the method for preparing a lyase according to an embodiment of the application.
FIG. 4 is a schematic diagram showing the comparison of the effects of the lyase in the embodiment of the application on hydrolyzing Bacillus subtilis under different pH values;
FIG. 5 is a schematic view showing the effect of the lyase of the present application on hydrolyzing Bacillus at 37℃and pH 6.5;
FIG. 6 is a photograph of a sample of the lyase of the present application taken before and after hydrolysis of Bacillus subtilis.
Detailed Description
The following describes the embodiments of the present application in further detail with reference to the accompanying drawings. These embodiments are merely illustrative of the present application and are not intended to be limiting.
According to a first aspect of the present application there is first provided a specific bacillus lyase (wp_ 015417280.1, protein molecular weight 35.11 KDa) designated xlyB as N-acetylmuramyl-L-alanine-amidase. The lyase is obtained based on sequencing data analysis of the FS001 genome of Bacillus subtilis.
Biological preservation unit: china general microbiological culture Collection center, preservation address: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute. Deposit No.: 17946, date of preservation 2019.06.17.
According to a second aspect of the present application, there is also provided a method for preparing a specific bacillus lyase.
It should be noted that, the biological materials involved in the preparation method provided in this embodiment include: bacillus belicus (Bacillus velezensis) FS001 is selected in the laboratory, and E.coli JM109, E.coli E.coilBL21 and expression vector pET-23b are selected; bacterial genomic DNA extraction kits were purchased from BioTeke; DNA polymerase is purchased from Shanghai chemical company; restriction enzymes BamHI, xhoI and T4 ligase were purchased from Takara; each liter of LB culture medium contains 10g of peptone, 5g of yeast extract and 10g of NaCl; the binding buffer included 300mM NaCl, 50mM disodium hydrogen phosphate, 10mM imidazole, and the pH of the binding buffer was 7.4; the rinse buffer included 300mM NaCl, 50mM disodium hydrogen phosphate, 20mM imidazole, and the pH of the rinse buffer was 7.4; the elution buffer included 300mM NaCl, 50mM disodium hydrogen phosphate, 500mM imidazole, and the pH of the elution buffer was 7.4; phosphate buffer (1 XPBS buffer) including 10mM phosphate, 1XPBS buffer pH 7.2-7.4; ampicillin and lysozyme were purchased from Shanghai. Ampicillin-containing LB medium used in this example had ampicillin concentrations of 100. Mu.g/mL.
The preparation method comprises the following steps:
s1, designing and synthesizing a primer for amplifying and removing N-acetylmuramyl-L-alanine-amidase gene xlyB of a stop codon coding sequence, wherein the primer comprises an upstream primer and a downstream primer.
The sequence of the upstream primer (xlyB-F) is as follows:
the sequence of the downstream primer (xlyB-R) is:
s2, amplifying by using the bacillus bailii FS001 genome as a template based on the upstream primer and the downstream primer to obtain a target gene fragment.
Specifically, the genomic DNA of bacillus belay FS001 was extracted using a bacterial genomic DNA extraction kit. Amplification was performed using the Bacillus belicus FS001 genomic DNA as a template, and the xlyB-F and x1yB-R primers.
The amplification system is as follows: the concentration of xlyB-F was 10. Mu.M, 1. Mu.L was added, the concentration of xlyB-R was 10. Mu.M, 1. Mu.L was added, 10 XPCR buffer was 5. Mu.L, dNTPMmix was 2.5mM, 4. Mu.L was added, 2. Mu.L of genomic DNA was added, and high fidelity enzyme (rTaq enzyme) was added to 0.25. Mu.L, dd H 2 O was added at 36.75. Mu.L.
The amplification conditions were as follows: the results of 2min pre-denaturation at 98 ℃,10 s denaturation at 98 ℃, 15s annealing at 60 ℃ and 10s extension at 72 ℃ for 30 cycles are shown in FIG. 1. FIG. 1 is an electrophoresis chart of the results of a gene polymerase chain reaction (PCR for short). In FIG. 1, lane 1 shows the result of electrophoresis of a nucleic acid standard, lane 2 shows the result of PCR of the lyase gene provided in this example, and lane 3 shows the result of PCR of a negative control group to which Bacillus belicus genome was not added.
S3, connecting the target gene fragment with a plasmid to complete construction of a recombinant plasmid.
Specifically, the PCR product is subjected to glue recovery, the pET23b vector and the corresponding target fragment are subjected to enzyme digestion by restriction enzymes BamHI and XhoI, and then are connected by T4 ligase to obtain a recombinant vector, wherein the structure of the recombinant vector is shown in figure 2; the ligation product was mixed with recipient E.coli JM109 competent, placed on ice for 30min, heat-shocked at 42℃for 90s, then added with 900. Mu.L of 37℃temperature-bath SOC liquid medium, resuscitated at 37℃for 45min at 100rpm, centrifuged and spread on ampicillin-containing LB solid medium, cultured overnight at 37℃to screen recombinant plasmids, and subjected to double digestion and sequencing verification. And transforming the recombinant plasmid which is verified to be correct into E.coli BL21 to obtain the recombinant strain containing the N-acetylmuramyl-L-alanine-amidase gene.
S4, the recombinant plasmid is transformed into escherichia coli for expression.
Specifically, the recombinant strain is inoculated in LB liquid medium containing 100 mu g/mL ampicillin for overnight culture, and then inoculated in 500mL of LB liquid medium containing ampicillin according to the ratio of 1:100 for culturing at 37 ℃ and 200rpm until OD 600 About 0.6 to 0.8, addIPTG is added to lead the final concentration to be 0.8-1 mmol/L for induction, the induction expression is carried out for 12-16 h under the condition of 20 ℃ and 150rpm, and the bacterial cells are collected by centrifugation for 10min at 5000 rpm. Suspending the bacterial body in 30ml of binding buffer solution, adding a proper amount of lysozyme, standing and lysing the cell wall, performing ultrasonic disruption for 3s-5s, centrifuging at 4 ℃ and 10000rpm for 20min after cell disruption is completed, separating supernatant and precipitate, and collecting supernatant to obtain crude enzyme solution.
S4, purifying to obtain the N-acetylmuramyl-L-alanine-amidase.
Protein purification was performed using a protein purification system. The system program was started and the purification system and A/B pump were thoroughly rinsed with suction, and the columns were equilibrated with 5 column volumes of rinse buffer at a flow rate of 1 mL/min. Cell disruption supernatant was fed automatically from the A-feed inlet at a flow rate of 1 mL/min. After all samples were fed into the system, the a sample inlet was moved into the elution buffer and washed at about 2CV at the same flow rate. The impurity proteins were removed by washing with rinse buffer and elution buffer in linear low to high imidazole content 2CV. The cloned protein containing the histidine tag adsorbed to the Ni-affinity chromatography column was eluted with elution buffer at a flow rate of 1 mL/min. And (5) paying attention to the peak time of the system at moment, judging and collecting filtrate. The purity of the eluate was checked by polyacrylamide gel electrophoresis (SDS-PAGE). The eluate was dialyzed overnight against 1XPBS buffer to remove imidazole and high concentration sodium chloride NaCl using a dialysis bag, and the purified enzyme solution was obtained as shown in FIG. 3, in which FIG. 3, lane 1 is a standard protein electrophoresis pattern, lane 2 is a purified electrophoresis pattern of the disrupted supernatant after induction of the expression strain, and lane 3 is an electrophoresis pattern of the lyase provided in the purification example.
According to a third aspect of the present application there is also provided the use of a lyase for specific lysis of bacillus using the lyase provided in the first aspect.
In one embodiment, the bacillus includes at least bacillus subtilis, bacillus belicus, and bacillus aryabhattai.
Specifically, bacillus subtilis, bacillus belgium, bacillus alnicosum,Culturing the bacillus megaterium strain in 100mL LB culture medium at 37 ℃ under the condition of 200 revolutions per minute until logarithmic growth phase, wherein the absorbance value of bacterial liquid meets OD 600 =about 1.0. The cells were collected by centrifugation at 8000 Xs for 5min and washed twice with 1.0M phosphate buffer, pH 6.5. Finally, the bacterial strain is resuspended in PBS to make OD 600 To about 0.6. Purified 22.5. Mu.g of XlyB protein was added to 3mL of the bacterial suspension and incubated at 37 ℃.
In one embodiment, the reaction conditions suitable for the specific cleavage include at least: the pH is 6.5-7.5, and the temperature is 30-37 ℃.
Preferably, the reaction conditions for the specific cleavage include: the pH was 6.5 and the temperature was 37 ℃.
Specifically, the optimal reaction pH for N-acetylmuramyl-L-alanine-amidase is 6.5. To determine the optimal conditions for the bactericidal activity of X1yB, the conditions were optimized in terms of pH of the buffer. The activity of XlyB-hydrolyzed Bacillus subtilis (B.subtilisRIK1285) cells was examined in buffers with pH values of 6.5, 6.8, 7.0 and 7.5, respectively, and the results showed that the pH range of 6.5 to 6.8 was the best, and the results are shown in FIG. 4.
The control group (CK group) in FIG. 4 shows absorbance of the strain of Bacillus without XlyB protein.
In one embodiment, the effect of the lyase on bacillus is determined by absorbance measurement.
Specifically, 200. Mu.L was taken after 60min of cleavage for absorbance measurement. The results show that XlyB has good hydrolysis effect on Bacillus subtilis, bacillus belicus and Bacillus aryabhattai in logarithmic growth phase, and slightly poor hydrolysis effect on Bacillus megaterium, and the result is shown in FIG. 5, and the control group (CK group) in FIG. 5 is the absorbance of the Bacillus strain without XlyB protein. The effect of XlyB on hydrolyzing bacillus subtilis is shown in fig. 6.
The foregoing is merely a preferred embodiment of the present application, and it should be noted that modifications and substitutions can be made by those skilled in the art without departing from the technical principles of the present application, and these modifications and substitutions should also be considered as being within the scope of the present application.

Claims (10)

1. A specific bacillus lyase, characterized in that the lyase is an N-acetylmuramyl-L-alanine-amidase, the lyase being obtained based on analysis of sequencing data of bacillus bailii FS001 genome; the biological preservation number is CGMCC No.17946.
2. The preparation method of the specific bacillus lyase is characterized by comprising the following steps of:
s1, designing and synthesizing a primer for amplifying N-acetylmuramyl-L-alanine-amidase, wherein the primer 1 comprises an upstream primer and a downstream primer;
the sequence of the upstream primer is as follows: CGGGATCCGGTGGGGATTGAAGTGAAAAA;
the sequence of the downstream primer is as follows: CCGCTCGAGCAGTTTTTCTTCAATTTTCG;
s2, amplifying by taking the bacillus bailii FS001 genome as a template based on the upstream primer and the downstream primer to obtain a target gene fragment;
s3, connecting the target gene fragment with a plasmid to complete construction of a recombinant plasmid; the recombinant plasmid is transformed into escherichia coli for expression;
s4, purifying to obtain the N-acetylmuramyl-L-alanine-amidase.
3. The method of preparing a lyase according to claim 1, wherein in step S2, the conditions for amplification comprise at least: pre-denaturation at 98℃for 2min, denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 10s,30 cycles.
4. The method for producing a lyase according to claim 2, wherein after step S3, a recombinant plasmid containing an N-acetylmuramyl-L-alanine-amidase gene is selected and subjected to double cleavage and sequencing.
5. A method of preparing a lyase according to claim 3, wherein in step S4 the purification comprises at least: protein purification was performed using a protein purification system.
6. Use of a lyase according to claim 1 for the specific lysis of bacillus.
7. The use of the lyase according to claim 6, wherein the bacillus comprises at least bacillus subtilis, bacillus belicus, bacillus aryabhattai.
8. Use of a lyase according to claim 7, wherein the reaction conditions suitable for specific cleavage comprise at least: the pH is 6.5-7.5, and the temperature is 30-37 ℃.
9. Use of a lyase according to claim 8, wherein the reaction conditions suitable for specific cleavage comprise: the pH was 6.5 and the temperature was 37 ℃.
10. Use of a lyase according to claim 9, wherein the effect of the lyase on the lysis of bacillus is determined by absorbance measurement.
CN202310308686.XA 2023-03-27 2023-03-27 Specific bacillus lyase and preparation method and application thereof Active CN116622683B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310308686.XA CN116622683B (en) 2023-03-27 2023-03-27 Specific bacillus lyase and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310308686.XA CN116622683B (en) 2023-03-27 2023-03-27 Specific bacillus lyase and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN116622683A true CN116622683A (en) 2023-08-22
CN116622683B CN116622683B (en) 2024-08-23

Family

ID=87590847

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310308686.XA Active CN116622683B (en) 2023-03-27 2023-03-27 Specific bacillus lyase and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN116622683B (en)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006262730A (en) * 2005-03-22 2006-10-05 Kao Corp Method for collective substitution of host dna subjected to preliminary depletion of substitution object region
CN101003569A (en) * 2007-01-19 2007-07-25 中国人民解放军军事医学科学院生物工程研究所 Antigen epitope and mutant of lyase in gamma bacteriophage of anthrax bacillus, and application
US20100297703A1 (en) * 2006-10-16 2010-11-25 Katsutoshi Ara Lytic Enzyme Inhibitor, Lysis Inhibitor, Inhibitor of Poly-y-glutamic Acid Degradation, and Method for Producing Poly- y-glutamic Acid
US20120244595A1 (en) * 2008-08-19 2012-09-27 Martin Loessner Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins
CN106755048A (en) * 2017-01-20 2017-05-31 颜萌 A kind of method that recombined bacillus subtilis produce hyaluronate lyase
CN106811416A (en) * 2017-02-24 2017-06-09 中国农业科学院植物保护研究所 Applications of the hydrolase CwlC in the cracking of bacillus mother cell
CN108441462A (en) * 2018-04-16 2018-08-24 武汉珈创生物技术股份有限公司 One bacillus subtilis and preparation method thereof
CN110452848A (en) * 2019-08-20 2019-11-15 昆明理工大学 One plant of Bei Laisi bacillus and its application
CN110846296A (en) * 2019-11-13 2020-02-28 三峡大学 Cloning expression and application of bacillus subtilis β -mannase
AU2019263941A1 (en) * 2018-05-03 2020-11-12 Crigasseni Ag Probiotic bacterial strains producing antimicrobial proteins and compositions comprising these for use in the treatment of diarrheal and other microbial diseases
WO2020244031A1 (en) * 2019-06-03 2020-12-10 中国海洋大学 Ulva lactuca polysaccharide lyase, encoding gene thereof, and application thereof
WO2021001397A1 (en) * 2019-07-04 2021-01-07 Basf Se A recombinant cell for producing proteins
CN113301804A (en) * 2018-09-17 2021-08-24 美国宝微技术股份有限公司 Antifungal compositions and methods of use
CN114958661A (en) * 2022-05-14 2022-08-30 昆明理工大学 High-temperature-resistant antibacterial active fermentation liquor and application thereof in storage of cash crops
CN116333957A (en) * 2023-03-13 2023-06-27 华中农业大学 Recombinant bacillus for displaying streptococcus suis prophage lyase, construction method and application thereof
CN117645967A (en) * 2023-11-28 2024-03-05 江南大学 Bacillus subtilis chassis cell suitable for high-density fermentation enzyme production

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006262730A (en) * 2005-03-22 2006-10-05 Kao Corp Method for collective substitution of host dna subjected to preliminary depletion of substitution object region
US20100297703A1 (en) * 2006-10-16 2010-11-25 Katsutoshi Ara Lytic Enzyme Inhibitor, Lysis Inhibitor, Inhibitor of Poly-y-glutamic Acid Degradation, and Method for Producing Poly- y-glutamic Acid
CN101003569A (en) * 2007-01-19 2007-07-25 中国人民解放军军事医学科学院生物工程研究所 Antigen epitope and mutant of lyase in gamma bacteriophage of anthrax bacillus, and application
US20120244595A1 (en) * 2008-08-19 2012-09-27 Martin Loessner Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins
CN106755048A (en) * 2017-01-20 2017-05-31 颜萌 A kind of method that recombined bacillus subtilis produce hyaluronate lyase
CN106811416A (en) * 2017-02-24 2017-06-09 中国农业科学院植物保护研究所 Applications of the hydrolase CwlC in the cracking of bacillus mother cell
CN108441462A (en) * 2018-04-16 2018-08-24 武汉珈创生物技术股份有限公司 One bacillus subtilis and preparation method thereof
AU2019263941A1 (en) * 2018-05-03 2020-11-12 Crigasseni Ag Probiotic bacterial strains producing antimicrobial proteins and compositions comprising these for use in the treatment of diarrheal and other microbial diseases
CN113301804A (en) * 2018-09-17 2021-08-24 美国宝微技术股份有限公司 Antifungal compositions and methods of use
WO2020244031A1 (en) * 2019-06-03 2020-12-10 中国海洋大学 Ulva lactuca polysaccharide lyase, encoding gene thereof, and application thereof
WO2021001397A1 (en) * 2019-07-04 2021-01-07 Basf Se A recombinant cell for producing proteins
CN110452848A (en) * 2019-08-20 2019-11-15 昆明理工大学 One plant of Bei Laisi bacillus and its application
CN110846296A (en) * 2019-11-13 2020-02-28 三峡大学 Cloning expression and application of bacillus subtilis β -mannase
CN114958661A (en) * 2022-05-14 2022-08-30 昆明理工大学 High-temperature-resistant antibacterial active fermentation liquor and application thereof in storage of cash crops
CN116333957A (en) * 2023-03-13 2023-06-27 华中农业大学 Recombinant bacillus for displaying streptococcus suis prophage lyase, construction method and application thereof
CN117645967A (en) * 2023-11-28 2024-03-05 江南大学 Bacillus subtilis chassis cell suitable for high-density fermentation enzyme production

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHENG, X. 等: "MULTISPECIES: N-acetylmuramoyl-L-alanine amidase [Bacillus]", GENBANK DATABASE, 18 May 2022 (2022-05-18), pages 015417280 *
REGAMEY A等: "The N-acetylmuramoyl-L-alanine amidase encoded by the Bacillus subtilis 168 prophage SP beta", MICROBIOLOGY, 30 April 1998 (1998-04-30), pages 885 - 893 *
ZHANG J等: "Identification and investigation of the effects of N-acetylmuramoyl-L-alanine amidase in Bacillus amyloliquefaciens for the cell lysis and heterologous protein production", INT J BIOL MACROMOL., 29 November 2023 (2023-11-29), pages 10 - 16 *
方圆子;王琰;孙建和;: "噬菌体裂解酶――现状与未来", 微生物学通报, no. 12, 20 December 2009 (2009-12-20), pages 90 - 95 *
李辉梅等: "一种新型褐藻胶裂解酶及其截短体对酶学性质的影响", 生物加工过程, 19 February 2024 (2024-02-19), pages 1 - 12 *
雷阳;张倩;陈钰;范玲玉;郑毅;: "对虾养殖高效降解氨氮微生态菌的筛选与鉴定", 福建农业科技, no. 10, 28 October 2019 (2019-10-28), pages 1 - 8 *

Also Published As

Publication number Publication date
CN116622683B (en) 2024-08-23

Similar Documents

Publication Publication Date Title
CN102199581B (en) Zearalenone toxin degradation enzyme and coding gene and application thereof
CN110564707B (en) Zearalenone hydrolase mutant ZHDM1 and coding gene and application thereof
CN110527677B (en) Zearalenone hydrolase mutant ZHDM2 and coding gene and application thereof
CN109402032B (en) Genetic engineering bacterium for producing resuscitation promoting factor RpfE and application thereof
CN109022390B (en) Method for separating and purifying escherichia coli recombinant phospholipase D
CN110862978B (en) Preparation method of recombinant halophilic archaea protease
CN116622683B (en) Specific bacillus lyase and preparation method and application thereof
CN102367448A (en) Construction method of genetic engineering strain for high expression and easy purification of beta-mannanase
CN117264975A (en) Cloning, expression and application of vomitoxin detoxification enzyme gene
CN108410843B (en) New pullulanase, and coding gene and application thereof
CN115960879A (en) High-throughput screening method of D-psicose 3-epimerase mutant library and obtained mutant
CN115058408B (en) Metagenome-derived high-specific-activity acid-resistant D-psicose 3-epimerase and encoding gene and application thereof
CN113999826B (en) Bacterial laccase allosteric and preparation method thereof
CN114045241B (en) Bacillus subtilis HKS018 and application thereof in production of beta-mannase
CN108103050A (en) A kind of Pseudomonas aeruginosa phage lyases, its encoding gene, recombinant expression carrier and its preparation method and application
CN107201354A (en) A kind of neutral proteinase and its gene and application
CN113817758A (en) Chitosan enzyme gene for encoding Bacillus belgii, chitosanase, preparation method and application thereof
CN102174553A (en) Preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri
US10351857B2 (en) Marine bacterial gene LfliZ and use
CN115747187B (en) Recombinase UvsX and expression gene and application thereof
CN110791513B (en) Codon-optimized holothuria leucospilota alpha-amylase gene, recombinant expression vector and application thereof
CN110066814B (en) beta-D-glucosidase gene and encoding protein thereof
CN114686500B (en) 1, 4-alpha-glucan branching enzyme, coding gene, engineering strain and application
CN107574174B (en) Construction method of plasmid expression vector for improving yield of rhodobacter sphaeroides coenzyme Q10
CN105950597B (en) Method for improving thermal stability of acid urease

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant