A method for producing and extracting N-acetylmuramoyl-L-alanine amidase by microbial fermentation

Abstract

This invention relates to the field of microbial technology, specifically to a method for the production and extraction of N-acetylmuroic acid-L-alanine amidase via microbial fermentation. The method for preparing N-acetylmuroic acid-L-alanine amidase provided by this invention utilizes *Clostridium beijerinckii* XH0906 for fermentation to produce N-acetylmuroic acid-L-alanine amidase. As the main extracellular protein of this bacterium, N-acetylmuroic acid-L-alanine amidase has a high yield and is beneficial for subsequent separation and purification. This method has advantages such as simplicity, efficiency, and low cost, providing an effective method for the preparation of N-acetylmuroic acid-L-alanine amidase, and has good application prospects in the fields of microbial food, biomedicine, and antibacterial applications.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for the production and extraction of N-acetylmucol-L-alanine amidase by microbial fermentation. Background Technology

[0002] Peptidoglycan is a major component of bacterial cell walls, primarily composed of a peptidoglycan backbone made up of N-acetylglucosamine and N-acetylmuramic acid units, along with short peptides (also known as tetrapeptide side chains) connecting the backbone, forming a stable network structure. Lysozyme is a hydrolytic enzyme that cleaves the sugar chains in peptidoglycan. The breaking of these sugar chains leads to cell wall lysis, resulting in bacterial death. Therefore, hydrolytic enzymes such as lysozyme, which can cleave peptidoglycan, have wide applications in food preservation and biomedicine. Theoretically, enzymes that degrade peptide chains and the bonds between peptide and sugar chains can also cause the peptidoglycan structure to collapse, leading to bacterial death. Among these, amidases are enzymes that degrade peptide and sugar chain bonds. Currently, there are few reports on the use of amidases for antibacterial activity or cell wall treatment, and there are no reports on the preparation of amidases for cleaving peptidoglycan structures using microbial fermentation. Summary of the Invention

[0003] This invention provides a method for the production and extraction of N-acetylmucol-L-alanine amidase by microbial fermentation.

[0004] Specifically, the present invention provides the following technical solutions.

[0005] This invention provides a method for preparing N-acetylmuroic acid-L-alanine amidase, the method comprising: fermenting Clostridium beijerincki XH0906 and extracting N-acetylmuroic acid-L-alanine amidase from the fermentation broth; The Clostridium beijerincki XH0906 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9124.

[0006] N-acetylmuroic acid-L-alanine amidase is an amidase with peptidoglycan-cleaving activity, belonging to the class of lysozymes. Currently, there are no reports of preparing N-acetylmuroic acid-L-alanine amidase using microbial fermentation. *Clostridium beijerincki* is the model strain for fermenting pentose and hexoses to produce butanol. Its main products are solvents such as acetone and butanol; hydrogen is also one of its products and can be used as a bioenergy source. *Clostridium beijerinckii* XH0906 is a high-butanol-producing *Clostridium beijerinckii* strain previously screened by the applicant. This strain was deposited on May 4, 2014, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Clostridium beijerinckii*, with accession number CGMCC No. 9124. This strain has been disclosed in patent application CN106554931A. When fermented with glucose as the carbon source, this strain can produce high concentrations of butanol, as well as small amounts of acetic acid and butyric acid. Current research on this strain focuses on the fermentation production of small molecule compounds such as butanol. During the identification of extracellular products of *Clostridium beijerincki* XH0906, this invention unexpectedly discovered that the strain can express high levels of amidase in its extracellular secretion during fermentation. Mass spectrometry and sequencing analysis identified it as N-acetylmullate-L-alanine amidase. Activity analysis confirmed that this amidase possesses the activity to cleave peptide and glycan linkages in peptidoglycan, thereby cleaving the peptidoglycan structure. Based on these findings, this invention develops a method for producing N-acetylmullate-L-alanine amidase using *Clostridium beijerincki* XH0906 through fermentation.

[0007] The N-acetylmuroic acid-L-alanine amidase described above is any one or more of the following (1)-(4): (1) The amino acid sequence is shown in SEQ ID NO.1; (2) The amino acid sequence is a sequence obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1; (3) The amino acid sequence is shown in SEQ ID NO.2; (4) The amino acid sequence is obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.2.

[0008] Clostridium beijerinckii XH0906 can secrete and express two N-acetylmuroic acid-L-alanine amidases, whose amino acid sequences are shown in SEQ ID NO. 1 and 2, respectively. Those skilled in the art will understand that, to promote the secretory expression of N-acetylmuroic acid-L-alanine amidases, a signal peptide sequence can be added to the N-terminus and / or C-terminus of their amino acid sequences. The addition of the signal peptide generally does not affect the activity of N-acetylmuroic acid-L-alanine amidases. Furthermore, for ease of purification, protein tags (e.g., histidine tags, GST tags, etc.) can be added to the N-terminus and / or C-terminus of their amino acid sequences. The addition of protein tags generally does not affect the activity of N-acetylmuroic acid-L-alanine amidases. Therefore, the aforementioned N-acetylmuroic acid-L-alanine amidases with added protein tags or signal peptides are also within the scope of protection of this invention.

[0009] Based on the amino acid sequence and codon rules of N-acetylmuramic acid-L-alanine amidase provided above, those skilled in the art can obtain its encoding gene sequence. Due to codon degeneracy, its encoding gene sequence is not unique and can be optimized according to the host's codon preference. All gene sequences capable of encoding the above-mentioned N-acetylmuramic acid-L-alanine amidase are within the protection scope of this invention.

[0010] Preferably, a feed solution is added during the fermentation process, the feed solution containing a nitrogen source and an alkali; the feed solution flow rate is adjusted to control the pH of the fermentation system to 6.8-7.2.

[0011] This invention reveals that the supply of nitrogen source and pH control during fermentation significantly affect the growth of *Clostridium beijerincki* XH0906, the yield of N-acetylmuroic acid-L-alanine amidase, and the types of extracellular proteins. By employing the aforementioned method for nitrogen supplementation and pH control, the key element nitrogen is added to the alkaline solution for feeding, and nitrogen supplementation is linked to pH control. By controlling the pH, nitrogen is automatically added to the fermentation system, making N-acetylmuroic acid-L-alanine amidase the main extracellular protein product, significantly increasing the yield of N-acetylmuroic acid-L-alanine amidase and reducing the difficulty of subsequent purification. Experimental verification shows that during fermentation, *Clostridium beijerincki* XH0906 consumes 82 g / L of glucose and produces 40 g / L of amidase (wet weight). After freeze-drying, the amidase yield is 7.4 g / L.

[0012] The alkali described above is preferably potassium hydroxide and / or sodium hydroxide. As an example, the alkali is potassium hydroxide.

[0013] The preferred nitrogen source is ammonia.

[0014] Preferably, the concentration of alkali in the feed solution is 1.6-2.2M, and the concentration of nitrogen source is 0.5-1M.

[0015] Preferably, during the fermentation process, glucose, ferrous salt, manganese salt, magnesium salt, sodium salt, vitamin B1, biotin, and para-aminobenzoic acid are added to the fermentation system.

[0016] Adding the above-mentioned raw materials during fermentation is beneficial to further increase the yield of N-acetylmuroic acid-L-alanine amidase.

[0017] The supplementation of glucose, as well as ferrous salts, manganese salts, magnesium salts, sodium salts, vitamin B1, biotin, and para-aminobenzoic acid, is preferably carried out in a single dose.

[0018] Preferably, during the 10-14 hours of fermentation, glucose is added to the fermentation system until the total sugar concentration in the fermentation system reaches 80-100 g / L, and ferrous sulfate, manganese sulfate, magnesium sulfate, sodium sulfate, vitamin B1, biotin, and para-aminobenzoic acid are added in amounts equivalent to: 0.15-1.25 g / L ferrous sulfate heptahydrate, 0.05-0.3 g / L manganese sulfate monohydrate, 1-6 g / L magnesium sulfate heptahydrate, 0.05-0.6 g / L sodium chloride, 0.8-1.2 mg / L vitamin B1, 0.008-0.012 mg / L biotin, and 0.8-1.2 g / L para-aminobenzoic acid.

[0019] The amounts of ferrous salt, manganese salt, magnesium salt, sodium salt, vitamin B1, biotin, and para-aminobenzoic acid added are relative to the total volume of the fermentation system.

[0020] The function of the aforementioned salts is to provide the corresponding metal ions; therefore, the present invention does not impose any special restrictions on the type of salt. The term "equivalent to" refers to the mass concentration, calculated based on the molecular weight of the selected salt, that is equivalent to the molar concentration of the specific salt type (ferrous sulfate heptahydrate, manganese sulfate monohydrate, etc.) provided above.

[0021] In the above method, the culture medium used for fermentation preferably comprises the following components: glucose 55-65 g / L, yeast powder 0.8-1.2 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.4-0.6 g / L, ammonium acetate 2-3 g / L, magnesium sulfate heptahydrate 1-6 g / L, manganese sulfate monohydrate 0.05-0.3 g / L, ferrous sulfate heptahydrate 0.15-1.25 g / L, sodium chloride 0.05-0.6 g / L, para-aminobenzoic acid 0.8-1.2 g / L, vitamin B1 0.8-1.2 mg / L, and biotin 0.008-0.012 mg / L.

[0022] More preferably, the culture medium used for fermentation comprises the following components: glucose 55-65 g / L, yeast powder 0.8-1.2 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.4-0.6 g / L, ammonium acetate 2-3 g / L, magnesium sulfate heptahydrate 1-1.5 g / L, manganese sulfate monohydrate 0.04-0.06 g / L, ferrous sulfate heptahydrate 0.15-0.25 g / L, sodium chloride 0.04-0.06 g / L, para-aminobenzoic acid 0.8-1.2 g / L, vitamin B1 0.8-1.2 mg / L, and biotin 0.008-0.012 mg / L.

[0023] The fermentation culture temperature is 35-37℃.

[0024] The fermentation culture was carried out under anaerobic conditions.

[0025] The optimal fermentation speed is 300-400 rpm.

[0026] The inoculum size for the fermentation culture is preferably 1-3%. The seed culture used for inoculation can be obtained using conventional methods for culturing *Clostridium beyerii*. Preferably, a single colony is selected on a plate and cultured in an anaerobic tube, then transferred to an anaerobic flask for further culture to obtain the seed culture.

[0027] The fermentation time is preferably 30-40 hours.

[0028] In the above method, after the fermentation culture is completed, the fermentation broth is separated and the bacterial cells are removed to obtain a fermentation supernatant. The pH of the supernatant is adjusted to 4.3-4.6 with acid, and after standing at low temperature, a protein precipitate containing N-acetylmuramic acid-L-alanine amidase is obtained.

[0029] By optimizing the extraction method of N-acetylmuroic acid-L-alanine amidase, this invention provides a simple and easy extraction method. The pH of the fermentation supernatant is adjusted to 4.3-4.6 (preferably pH 4.5) with acid, and N-acetylmuroic acid-L-alanine amidase will automatically settle. N-acetylmuroic acid-L-alanine amidase can be obtained by simple separation methods such as centrifugation.

[0030] Preferably, the acid is sulfuric acid. The concentration of the sulfuric acid is 45%-55%.

[0031] Preferably, the low-temperature settling is performed at 2-6°C. The preferred low-temperature settling time is 8-16 hours.

[0032] The methods for separating and removing bacterial cells and separating protein precipitates from fermentation broth include, but are not limited to, separation methods such as centrifugation.

[0033] This invention also provides the application of Clostridium beijerincki XH0906 in the preparation or selection of production strains for the fermentation production of N-acetylmuroic acid-L-alanine amidase. The Clostridium beijerinckii XH0906 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9124.

[0034] This invention also provides the application of Clostridium beijerinckii XH0906 in the preparation of lysozyme or the selection of production bacteria for fermentation production of lysozyme; The Clostridium beijerincki XH0906 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9124.

[0035] In the above applications, the N-acetylmuroic acid-L-alanine amidase is any one or more of the following (1)-(4): (1) The amino acid sequence is shown in SEQ ID NO.1; (2) The amino acid sequence is a sequence obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1; (3) The amino acid sequence is shown in SEQ ID NO.2; (4) The amino acid sequence is obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.2.

[0036] In the above-described applications for preparing N-acetylmuroic acid-L-alanine amidase or lysozyme, *Clostridium beijerinckii* XH0906 is used as the strain for fermentation production of N-acetylmuroic acid-L-alanine amidase. The applications include a fermentation culture of *Clostridium beijerinckii* XH0906.

[0037] In the above-mentioned application of the selected production strains, Clostridium beijerinckii XH0906 is used as the starting strain for selecting production strains of N-acetylmucolonic acid-L-alanine amidase or lysozyme. The selection methods include, but are not limited to, genetic engineering modification, mutagenesis, and adaptive evolution.

[0038] The beneficial effects of this invention include at least the following: The method for preparing N-acetylmuroic acid-L-alanine amidase provided by this invention utilizes *Clostridium beijerinckii* XH0906 for fermentation to produce N-acetylmuroic acid-L-alanine amidase. As the main extracellular protein of this bacterium, N-acetylmuroic acid-L-alanine amidase has a high yield and is beneficial for subsequent separation and purification. This method has advantages such as simplicity, high efficiency, and low cost, providing an effective method for the preparation of N-acetylmuroic acid-L-alanine amidase. As a specific enzyme that can disrupt the glycan and peptide chains of bacterial cell wall peptidoglycan, N-acetylmuroic acid-L-alanine amidase has good application prospects in the fields of microbial food, biomedicine, and antibacterial applications. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 This is an electrophoresis image of extracellular proteins from Clostridium beyerrix in Example 2 of the present invention; wherein, M: marker (KDa): from top to bottom: 180, 130, 95, 70 (orange), 53, 40, 33, 25, 17, 10; lanes 1 and 2 are extracellular protein samples.

[0041] Figure 2 The images show the enzyme reaction kinetic curves of the purified proteins OD350_21025 and OD350_20815 in Example 3 of this invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] Example 1: Fermentation culture of Clostridium beyerii XH0906

[0044] 1. Seed culture of Carboxycarbazium baijellii XH0906

[0045] Frozen *Bacillus baijieri* XH0906 was spread on agar plates and incubated statically at 36°C in an anaerobic flask until colonies grew. The colonies were then transferred to anaerobic tubes and incubated overnight at 36°C. The inoculum from the anaerobic tubes was further transferred to a medium anaerobic flask and incubated overnight at 36°C to obtain the seed culture.

[0046] The culture medium used in the anaerobic tubes was a carboxyl-enhanced medium, with 1L of medium containing 10.0g peptone, 10.0g beef meal, 3.0g yeast powder, 5.0g glucose, 1.0g soluble starch, 5.0g sodium chloride, 3.0g sodium acetate, 0.5g L-cysteine ​​hydrochloride, and 0.5g agar.

[0047] The plate culture medium is based on the above-mentioned carboxyl bacteria-enhanced culture medium with the addition of 2% agar.

[0048] The culture medium used for anaerobic culture was a modified P2 medium with the following components: 1L of medium contains 60g glucose, 1g yeast extract, 10mL acetate stock solution, 10mL macronutrient salt stock solution, 1mL ferrous sulfate stock solution, and 1mL vitamin stock solution.

[0049] The preparation method of the macro-element salt storage solution is as follows: 0.5g manganese sulfate, 10g magnesium sulfate heptahydrate, and 0.5g sodium chloride are dissolved in boiling pure water, and the volume is adjusted to 100mL. The solution is then filtered through a 0.22μm filter membrane for sterilization and stored at room temperature.

[0050] The preparation method of acetate storage solution is as follows: 22g ammonium acetate, 5g potassium dihydrogen phosphate, and 5g dipotassium hydrogen phosphate; dissolve in boiling pure water, bring the volume to 100mL, filter through a 0.22μm filter membrane for sterilization, and store at room temperature.

[0051] The preparation method of vitamin storage solution is as follows: Biotin 1mg, Vitamin B1 100mg, Para-aminobenzoic acid 100g; Dissolve in boiling pure water (70-80℃), bring the volume to 100mL, filter through a 0.22μm filter membrane for sterilization, and store at room temperature.

[0052] The preparation method of ferrous sulfate stock solution is as follows: 20g of ferrous sulfate heptahydrate; dissolve in sterile pure water while hot and bring the volume to 100mL, filter through a 0.22μm filter membrane for sterilization, and store frozen.

[0053] 2. Fermentation culture of Clostridium beyerii XH0906

[0054] The seed culture of Clostridium beyerrix XH0906 was inoculated into a fermenter containing fermentation medium for fermentation culture. The fermentation conditions were as follows: 1L of liquid was added to a 2L fermenter for fermentation, the medium used was the modified P2 medium mentioned above, the temperature was maintained at 36℃, the rotation speed was 300rpm, and the inoculum size was 2% (20mL). The pH was controlled at 7.0 throughout the fermentation process with an alkaline solution (400mL of 2M potassium hydroxide solution mixed with 20mL of ammonia water (from Sinopharm)). After 12 hours of fermentation, ferrous sulfate storage solution, vitamin storage solution, and macronutrient salt storage solution were added, with the same amount as the corresponding components in the initial medium. At the same time, glucose was added to make the total sugar concentration in the fermentation system reach 90g / L.

[0055] The growth of Clostridium beyerrix XH0906 in modified P2 medium was measured by the light absorbance density (OD) of the fermentation broth at 600 nm. 600 This indicates that, through calculation, 1 OD of Clostridium difficile cells is equivalent to 0.2 g / L of biomass dry weight. Adding twice the volume of ethanol to the supernatant after centrifugation of the fermentation broth yields extracellular protein precipitate. The weight after centrifugation to remove the supernatant is the wet weight, and the weight after 12 hours of freeze-drying is the dry weight.

[0056] 3. Fermentation results

[0057] Clostridium beyerrix XH0906 was fermented using glucose as the carbon source. The entire fermentation process was automatically controlled at pH 7.0, and the fermentation ended in 36 hours. The total consumption of glucose was 82 g / L, producing 16 L of carbon dioxide and 7 L of hydrogen, and generating 5.2 g / L of acetic acid, 1.1 g / L of isopropanol, 2 g / L of butyric acid, and 2.6 g / L of butanol. The supernatant of the fermentation broth was mixed with twice its volume of ethanol, allowed to stand overnight, and centrifuged to obtain 40 g / L of extracellular protein (wet weight). After freeze-drying, 7.4 g / L of protein was obtained after dehydration. The fermentation results show that Clostridium beyerrix XH0906 is not only an isopropanol and butanol-ethanol (BI) fermentation strain, but extracellular protein is also an important product of this bacterium.

[0058] Example 2: Identification of extracellular proteins of Carboxycarbazium baijellii XH0906

[0059] Fermentation culture of *Carboxycarbae* XH0906 was performed according to the method in Example 1. The supernatant of the fermentation broth was directly subjected to SDS-PAGE protein electrophoresis. The results showed that ( Figure 1 The extracellular proteins of *Clostridium beyere* XH0906 mainly consist of two bands, approximately 150 kDa and 110 kDa in size, respectively. These two protein bands were subjected to mass spectrometry sequencing. The characteristic peptide sequences obtained from the mass spectrometry sequencing were compared with the functional genome of *Clostridium beyere*, identifying the functional gene of the first band protein as OD350_21025, named N-acetylmuramoyl-L-alanineamidase family protein. The protein sequence is shown in SEQ ID NO.1, as follows: MFKRANKITSLLVAAASVMALVPAYAADVKKVDSEDGTVYNAVAYKDGKFYVDGEINDD EEAYYVADGKMNKLEDVDSGDDAVLFGEKYLDISDGDYTVDLDKGSVTDDDVKGDTAD DAASALRKKAKDDTDDRYLDKGTNSTDTVKDENKTDDFKADELNIIPGAKYSEPWYATT FAKGDGSTANGAADSFNVFTDTKGNYIDADYNLGKIKVGTTGDSVNVENTNDKYDLKGI DDVLTAKVRNSVVLTQDKDYIYRKAEVTLTIDTTKTGASDLKITTINGVKISGTTSALGTA DAASVTFNVVQKISKAQASGDVDGAKYAKSVTTYVISDDKGVANDDYTIVTDATNFAHT KYTVAGGKLIAYNTVDDSNANKKVTARAYSLKTSGGYYYLDAEDKSSEDCETKDDVAAV QTDVDGNLWRLDGGYIYKFDNTDDWDKVYKVDGSFDELQVYDKDNMVAWSEDDDVY SLIGGKSADDNKGNDTPAVTAGWTQTSAGWTYTKADGTKATGWLQDGGAWYYLKAD GVMATGWVQDGATWYYLNGSGAMQTGWFNDNGTWYYLNGSGAMLANTTTPDGYYVGANGAWVK.

[0060] Similarly, the second band was also identified as N-acetylmuramoyl-L-alanine amidase family protein, with the corresponding functional gene number OD350_20815. The protein sequence is shown in SEQ ID NO.2, as follows: MIRGMGKVTSLLVAAATVASLVPFSGANAAEVKRISSDDGTIYNAIAYKDGKAYIDGEIND DEEAYYLSNGKFNKLDDVDSGDSAALFGEKYLDISDGDYTVDLDKGSVTDDDIKGDTEDD AAAALRKKIKDDTDDRYNETEANTIKDSNHGDLFDLIPGAKYNKVWYYTQYKAAQKSID KNVNGLNGLDSAHQIFNVFTDDKGNYIDADYNLGKVKVTTTASSASGTTLTKTDTIENT NDAYDAADGIINGTSVSGSDKLSASVVQDRVLTQDKDYIYRLATVKITITTGAAATISEING VKVDPNNSNDIFKVENNGQVVSFKAIQKISKTQASGDIDDAKYAKTVTTYALSDKDGKKL DAEDLFINTSGNVVTTTNYTVGSGKLIAYNSEINNNDKVTVRAYTLKSSSGFYYADEEDQS KEDCENSKNQGAAVQTDVDGNLWRLDGGYIYKFDNTDDWDKVYRVDGSFFDEFSVYDK DNIVAWSEDDDVYSLIGGKQSNTDPDDTPVVKTGWVQAADGTWSYIKADGNKATGWV QDGSTWYYFKADGSMATGWVQDGSTWYYFQSWGGMQTGWLNNNGTWYYLQSWGG MKTGWLNDNGAWYFLNGSGAMQTGWLNDNGTWYYLYSNGVMAANTVVNGYNLSASGAWV.

[0061] Based on the extracellular protein electrophoresis and content determination results of Example 1, the extracellular proteins of Clostridium beyere XH0906 are mainly the two N-acetylmuroic acid-L-alanine amidases shown in SEQ ID NO.1 and 2 above. Therefore, the yield of N-acetylmuroic acid-L-alanine amidase (dry weight after freeze-drying) of Clostridium beyere XH0906 can reach about 7.4 g / L.

[0062] Example 3: Expression and enzyme activity determination of extracellular proteins in *Bacillus baijieri* XH0906

[0063] Based on the extracellular protein proteomic identification results of Example 2, the encoding gene for N-acetylmuroic acid-L-alanine amidase in the genome of Clostridium beyerrix XH0906 was amplified, an expression vector for this gene was constructed, and the gene was introduced into Escherichia coli for protein expression. The purpose was to determine whether the N-acetylmuroic acid-L-alanine amidase produced by Clostridium beyerrix XH0906 has peptidoglycan cleaving activity. The specific method is as follows: Gene sequences encoding two N-acetylmuramic acid-L-alanine amidases, OD350_21025 and OD350_20815 (signal peptide removed), were amplified from the genomic DNA of Clostridium beijerinckii XH0906 by polymerase chain reaction (PCR). The primer sequences for amplifying the protein-coding genes are shown below.

[0064] 21025F (SEQ ID NO.3): TTCCAGGGGCCCGGATCCGCAGACGTAAAGAAAGTTGATTCAGAAG; 21025R (SEQ ID NO.4): TTCTTTACCAGACTCGAGCTATTTAACCCAAGCTCCATTAGTCCCTA; 20815F (SEQ ID NO.5): TTCCAGGGGCCCGGATCCGCTGAGGTGAAAAGAATTAGTTCTGATG; 20815R (SEQ ID NO.6): TTCTTTACCAGACTCGAGTTATACCCCAAGCACCGCTTGC.

[0065] The amplification products of the two genes were ligated into the pGLO1 expression vector (plasmid reference: Liu Nan, Application of glycoside hydrolase DexA70 in hydrolysis of Streptococcus mutans biofilm, doctoral dissertation, November 2021). The plasmid was transformed into E. coli BL21(DE3) and cultured. The bacterial cells were collected and resuspended in resuspension buffer (25 mM Tris pH 8.0, 500 mM NaCl) and then autoclaved. The resulting bacterial culture was centrifuged (14000 rpm, 4℃, 45 min), and the supernatant was loaded onto a Ni-NTA affinity chromatography column. The column was washed with resuspension buffer for about 10 column volumes to remove non-specifically adsorbed proteins. Finally, the target protein was eluted from the nickel column with 2 column volumes of elution buffer. The protein sample purified by nickel column was concentrated to 2 mL by ultrafiltration, centrifuged (14000 rpm, 4℃, 10 min), and then loaded onto a equilibrated Superdex-200 chromatography column. The elution program was set, eluted for 1.5 column volumes, and the purified protein was collected.

[0066] 5 μM of the purified protein was added to 0.1 M sodium phosphate buffer (pH 8.0) along with 500 μM of the synthetic substrate MDP (carbamoyl dipeptide, N-acetyl-carbamoyl-L-alanyl-D-isoglutamine) and incubated at 37 °C for 15, 30, 60, 90, and 120 minutes, respectively. The reaction was then stopped by placing the tubes on ice, and the product was separated from the enzyme using an ultrafiltration tube. 20 μL of the product was mixed with 100 μL of OPA solution (1.5 mM o-phthalaldehyde, 85 mM sodium borate buffer (pH 10), and 1% v / v 2-mercaptoethanol). The mixture was incubated at room temperature for 5 minutes, and the absorbance was measured at 340 nm using a microplate reader. Enzyme activity assay results showed (…). Figure 2 Clostridium beyerrix extracellular proteins possess enzymatic activity that cleaves peptidoglycan side chains; they are amidases that can act on the glycan and peptide chain linkages of peptidoglycan.

[0067] Example 4: Rapid Extraction of N-acetylmuramic Acid-L-alanine Aminoase

[0068] This embodiment provides a method for rapidly extracting extracellular N-acetylmucol-L-alanine amidase produced by Clostridium beyerrix XH0906, as detailed below: After centrifuging the fermentation broth of *Clostridium beyere* XH0906 to remove bacterial cells, the supernatant was adjusted to pH 4.5 with 50% sulfuric acid (v / v), mixed, and incubated overnight at 4°C to obtain protein precipitate. After centrifugation, the protein precipitate was resuspended in phosphate buffer (pH 10). The sample was then subjected to SDS-PAGE electrophoresis to obtain the protein... Figure 1The same electrophoresis pattern shows that the protein precipitate contains N-acetylmuramic acid-L-alanine amidase produced by Clostridium beyerridis XH0906, indicating that the extracellular amidase of Clostridium beyerridis XH0906 can be rapidly extracted by adjusting the pH.

[0069] Clostridium beyerrix XH0906 was fermented using the method of Example 1, and the extracellular amidase obtained from the fermentation culture was extracted using the rapid extraction method of extracellular amidase in this example. The yield of N-acetylmuramic acid-L-alanine amidase (dry weight) from Clostridium beyerrix XH0906 was measured to be 7.2 g / L.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing N-acetylmuroic acid-L-alanine amidase, characterized in that, The method includes: fermenting Clostridium beijerinckii XH0906 and extracting N-acetylmucol-L-alanine amidase from the fermentation broth; The Clostridium beijerinckii XH0906 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9124.

2. The method for preparing N-acetylmuroic acid-L-alanine amidase according to claim 1, characterized in that, The N-acetylmuroic acid-L-alanine amidase is any one or more of the following (1)-(4): (1) The amino acid sequence is shown in SEQ ID NO.1; (2) The amino acid sequence is a sequence obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1; (3) The amino acid sequence is shown in SEQ ID NO.2; (4) The amino acid sequence is obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.

2.

3. The method for preparing N-acetylmuroic acid-L-alanine amidase according to claim 1 or 2, characterized in that, During the fermentation process, a feed solution is added, the feed solution containing a nitrogen source and an alkali; The flow rate of the feed solution is adjusted to control the pH of the fermentation system to 6.8-7.

2.

4. The method for preparing N-acetylmuroic acid-L-alanine amidase according to claim 3, characterized in that, The alkali is potassium hydroxide and / or sodium hydroxide; the nitrogen source is ammonia water; Preferably, the concentration of alkali in the feed solution is 1.6-2.2M, and the concentration of nitrogen source is 0.5-1M.

5. The method for preparing N-acetylmuroic acid-L-alanine amidase according to claim 3 or 4, characterized in that, During the fermentation process, glucose, ferrous salt, manganese salt, magnesium salt, sodium salt, vitamin B1, biotin, and para-aminobenzoic acid are added to the fermentation system. Preferably, during the 10-14 hours of fermentation, glucose is added to the fermentation system until the total sugar concentration in the fermentation system reaches 80-100 g / L, and ferrous sulfate, manganese sulfate, magnesium sulfate, sodium sulfate, vitamin B1, biotin, and para-aminobenzoic acid are added in amounts equivalent to: 0.15-1.25 g / L ferrous sulfate heptahydrate, 0.05-0.3 g / L manganese sulfate monohydrate, 1-6 g / L magnesium sulfate heptahydrate, 0.05-0.6 g / L sodium chloride, 0.8-1.2 mg / L vitamin B1, 0.008-0.012 mg / L biotin, and 0.8-1.2 g / L para-aminobenzoic acid.

6. The method for preparing N-acetylmuroic acid-L-alanine amidase according to any one of claims 1 to 5, characterized in that, The culture medium used for fermentation includes the following components: glucose 55-65 g / L, yeast extract 0.8-1.2 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, potassium dihydrogen phosphate 0.4-0.6 g / L, ammonium acetate 2-3 g / L, magnesium sulfate heptahydrate 1-6 g / L, manganese sulfate monohydrate 0.05-0.3 g / L, ferrous sulfate heptahydrate 0.15-1.25 g / L, sodium chloride 0.05-0.6 g / L, para-aminobenzoic acid 0.8-1.2 g / L, vitamin B1 0.8-1.2 mg / L, and biotin 0.008-0.012 mg / L. And / or, the fermentation culture temperature is 35-37℃; And / or, the fermentation culture is carried out under anaerobic conditions.

7. The method for preparing N-acetylmuroic acid-L-alanine amidase according to any one of claims 1 to 6, characterized in that, After the fermentation culture is completed, the fermentation broth is separated to remove the bacterial cells, and the fermentation supernatant is obtained. The pH of the supernatant is adjusted to 4.3-4.6 with acid, and after standing at low temperature, a protein precipitate containing N-acetylmuramic acid-L-alanine amidase is obtained. Preferably, the acid is sulfuric acid; More preferably, the concentration of the sulfuric acid is 45%-55%.

8. Application of Clostridium beijerinckii XH0906 in the preparation or selection of production strains for the fermentation production of N-acetylmuroic acid-L-alanine amidase; The Clostridium beijerinckii XH0906 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9124.

9. Application of Clostridium beijerinckii XH0906 in the preparation or selection of lysozyme-producing bacteria for fermentation production; The Clostridium beijerinckii XH0906 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9124.

10. The application according to claim 8 or 9, characterized in that, The N-acetylmuroic acid-L-alanine amidase is any one or more of the following (1)-(4): (1) The amino acid sequence is shown in SEQ ID NO.1; (2) The amino acid sequence is a sequence obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1; (3) The amino acid sequence is shown in SEQ ID NO.2; (4) The amino acid sequence is obtained by adding a protein tag and / or signal peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.2.

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