KR20200107176A - A novel bacillus amyloliquefaciens MKSE strain, an incubation method of the strain and a producing method of asparaginase using the strain - Google Patents

Abstract

The present invention relates to a novel Bacillus amyloliquefaciens MKSE strain (KCTC18753P) that produces asparaginase with improved heat and pH stability, a method of culturing the strain, and a method of producing asparaginase using the strain.

Description

A novel bacillus amyloliquefaciens MKSE strain, an incubation method of the strain and a producing method of asparaginase using the strain, a novel Bacillus amyloliquefaciens MKSE strain, a method for culturing the strain and a method for producing asparaginase using the strain }

The present invention relates to a novel Bacillus amyloliquefaction MKSE strain (KCTC18753P) that produces asparaginase with improved heat and pH stability, a method for culturing the strain, and a method for producing asparaginase using the strain.

Asparaginase is widely used as an anticancer agent and is also used as a treatment for acute lymphocytic leukemia, lymphosarcoma, and Hodgkin's disease, and it is highly valued in the pharmaceutical market, contributing to about 40% of global enzyme sales. Has become.

In addition, asparaginase is also used to reduce the content of acrylamide, which is a potential carcinogen.When a large amount of acrylamide contained in food is consumed, the incidence of kidney cancer increases, and acrylamide reduces neurotoxicity. As the results of the study, the International Cancer Research Institute, classified acrylamide as a carcinogenicity class 2A group, that is, a suspected carcinogen.

Acrylamide is produced by the mylard reaction between reducing sugar and asparagine during high temperature processing of starchy foods in the food industry, and the Swedish National Food Service provides acrylamide when cooking vegetable foods with high carbohydrate content and low protein content at a high temperature of 120℃ or higher. It has been the first reported that is produced naturally. In the case of the general population, acrylamide exposure through food accounts for the largest proportion, and in recognition of the potential risk through food intake, countries around the world are making efforts, such as setting standards and regulating acrylamide among foods.

In particular, the European Union (EU) has become a hot topic for the first time in the world to legally regulate the residual amount of acrylamide in food, which has been identified as a potential carcinogen, and the limits are potato chips 750 μg/kg, French fries 500 μg/kg. , Infant biscuits are known as 150 μg/kg. It is known that the EU intends to impose a duty to reduce the residual amount of acrylamide in foods on imported food companies as well as food operators through this regulation.

Korea is also making continuous efforts to reduce the amount of acrylamide in foods below the recommended standard (1,000 μg/kg) set by the Ministry of Food and Drug Safety, and especially since 2013, the Ministry of Food and Drug Safety has been working to reduce unintentional hazardous chemical substances in food. Under the five-year plan, efforts were also made to reduce acrylamide.

The degree of acrylamide contamination of domestic processed foods analyzed using LC-MS/MS was 40.2-1428.7 μg/kg for potato snacks, 26.4-692.9 μg/kg for snacks other than potatoes, 15.1-713.3 μg/kg for biscuits, and 39.1 for infant biscuits. -289.2 μg/kg, processed chocolate 11.5-231.6 μg/kg, roasted coffee 76.8-244 μg/kg, instant coffee 318.3-989.4 μg/kg, prepared coffee 96.2-476.3 μg/kg, french fries 92.8-1079.9 μg/kg Among them, the foods that exceeded the provisional standard content of the Ministry of Food and Drug Safety (KFDA) were potato snacks and french fries. In addition, when compared with the EU's acrylamide index standard, the foods that exceeded the standard were potato snacks, biscuits, baby biscuits, french fries, and instant coffee.

The addition of substances such as fungi, antioxidants, and allicin can lower the content of acrylamide in food, which is produced by the mylar reaction of reducing sugar and asparagine, but other than asparagine in food The easiest and most effective way to maintain organoleptic properties without affecting the substance is to use an enzyme, i.e. asparaginase.

It has been reported that when asparaginase is applied, acrylamide reduction effects of 90-95% for cereal products and 70-80% for coffee were obtained. When asparaginase derived from bacteria is applied, asparaginase derived from Bacillus licheniformis and enzymes derived from Bacillus subtilis and Thermococcus zilligii can reduce acrylamide by 80% in French fries, potato chips, and French fries. It has been reported that there was.

Commercialized asparaginases include PreventASe™ made by DSM (Heerlen, Netherlands) and Acrylaway® made by Novozyme (Bagsvaerd, Denmark). PreventASe™ is an enzyme derived from Aspergillus niger with an optimum temperature of 50℃ and an optimum pH of 4 While being an acidic enzyme at -5, Acrylaway® is a neutral enzyme with an optimum temperature of 37°C and pH 7 derived from Aspergillus oryzae. However, these enzymes can be inactivated during high temperature heating, making it difficult to use them throughout the food industry.

Recently, Korean Patent No. 10-1207287 and Korean Patent No. 10-1777904 disclose asparaginase derived from a new strain for decomposing asparagine, and asparaginase derived from a strain that is stable under high temperature heat and a wide pH range is disclosed. There is a need to develop a product.

Korean Patent Registration No. 10-1207287 Korean Patent Registration No. 10-1777904

An object of the present invention is to obtain asparaginase with improved heat and pH stability from a novel Bacillus amyloliquefaciens MKSE strain (KCTC18753P) isolated from Korean traditional soybean fermented food.

In addition, it is an object of the present invention to reduce acrylamide in food by using the asparaginase having improved heat and pH stability.

To achieve the above object,

The present invention provides a Bacillus amyloliquefaciens MKSE strain (KCTC18753P).

In addition, the present invention is sucrose (Sucrose), asparagine (Asparagine), glutamine (Glutamine), yeast extract, peptone (Peptone), Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, CaCl _{2 ·} 2H ₂ O and MgSO _{4 It} provides a method of culturing the Bacillus amyloliquefaciens MKSE strain (KCTC18753P) using a medium containing at least one selected from the group consisting of 7H ₂ O.

In addition, the present invention provides a method for producing asparaginase from the culture by culturing the Bacillus amyloliquefaciens MKSE strain (KCTC18753P).

The novel strain of the present invention, Bacillus amyloliquefaciens , MKSE strain (KCTC18753P) can produce asparaginase with improved thermal and pH stability capable of decomposing asparagine, a precursor of acrylamide, and thermal processing. It can reduce acrylamide, a carcinogen produced in food, etc.

1 is a diagram showing the nucleotide sequence of Bacillus amyloliquefaciens MKSE strain (KCTC18753P) of the present invention.
Figure 2 is a view showing the result of identification by comparing the 16s rRNA sequence of Bacillus amyloliquefaciens MKSE strain (KCTC18753P) and Bacillus amyloliquefaciens FZB42 strain of the present invention.
3 is a graph showing the results of measuring the enzyme activity of asparaginase derived from Bacillus amyloliquefactions MKSE strain (KCTC18753P) according to the composition of the medium.
4 is a view showing the results of SDS-PAGE analysis of asparaginase derived from Bacillus amyloliquefactions MKSE strain (KCTC18753P) of the present invention.
5 is a graph showing the results of analyzing the optimum temperature for enzyme activity of asparaginase derived from Bacillus amyloliquefactions MKSE strain (KCTC18753P) of the present invention.
6 is a graph comparing the enzymatic activity of asparaginase derived from Bacillus amyloliquefactions MKSE strain (KCTC18753P) of the present invention and asparaginase derived from various Bacillus genus strains.
7 is a graph showing the results of analyzing the optimum pH of the enzyme activity of asparaginase derived from Bacillus amyloliquefactions MKSE strain (KCTC18753P) of the present invention.
8 is a graph showing the activity or inhibitory effect of metal ions and compounds on the enzyme activity of asparaginase derived from Bacillus amyloliquefactions MKSE strain (KCTC18753P) of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention relates to Bacillus amyloliquefaciens MKSE strain (KCTC18753P). The MKSE strain is a strain directly isolated from Korean traditional fermented soybean food, and does not degrade starch, so it does not deteriorate the food, and the MKSE strain-derived asparaginase can have excellent safety in application to food. have.

Bacillus amyloliquefactions MKSE strain (KCTC18753P), a novel strain of the present invention, has the nucleotide sequence of SEQ ID NO: 1 shown in FIG. 1, and produces an enzyme asparaginase, and the asparaginase is The thermal and pH stability is improved, so it is stable at high temperatures and a wide pH range.

Asparaginase is an enzyme that degrades asparagine, a precursor of acrylamide, a carcinogen in food.Asparagine is decomposed into aspartic acid and ammonia as shown in Scheme 1 below. Given, acrylamide can be reduced.

[Scheme 1]

In more detail, the asparaginase produced by the MKSE strain is characterized by being stable at a temperature of 40°C to 95°C, and specifically, 57% to 94% even after 6 hours at a temperature of 40°C to 95°C It can maintain the enzyme activity of. In addition, the asparaginase produced by the MKSE strain is characterized by maintaining the enzymatic activity of 90% to 99% even after 4 hours at pH 3.0 to 11.0, and even after 10 hours at pH 3.0 to 11.0. It can maintain the enzyme activity of 45% to 70%.

In addition, the present invention is sucrose (Sucrose), asparagine (Asparagine), glutamine (Glutamine), yeast extract, peptone (Peptone), Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, CaCl _{2 ·} 2H ₂ O and MgSO _{4 It} provides a method of culturing the MKSE strain using a medium containing at least one selected from the group consisting of 7H ₂ O. Preferably, the medium is sucrose (Sucrose) 1 to 35g / L, asparagine (Asparagine) 1 to 14g / L, glutamine (Glutamine) 2 to 14g / L, yeast extract 1 to 5g / L, peptone (Peptone) 1 To 5g/L, Na ₂ HPO ₄ 6g/L, KH ₂ PO ₄ 3g/L, NaCl 0.5g/L, CaCl ₂ 2H ₂ O 0.012 g/L and MgSO ₄ 7H ₂ O 0.24 g/L, and more preferably, the medium is sucrose 5 g/L, asparagine 7 g/L, glutamine 10 g/L, yeast extract 1 g/L, peptone 1g/L, Na ₂ HPO ₄ 6g/L, KH ₂ PO ₄ 3g/L, NaCl 0.5g/L, CaCl ₂ 2H ₂ O 0.012 g/L and MgSO ₄ 7H ₂ O It may contain 0.24g/L.

The MKSE strain is characterized in that it is cultured for 24 to 48 hours at a temperature of 25 ℃ to 55 ℃ using the medium.

In addition, the present invention provides a method of culturing the MKSE strain to produce asparaginase from the culture, and specifically, culturing the MKSE strain; And purifying asparaginase from the culture medium, and at this time, the method of culturing the above-described MKSE strain may be used.

Bacillus amyloliquefactions MKSE strain (KCTC18753P), a novel strain of the present invention, can produce asparaginase with improved heat and pH stability, thereby stably reducing acrylamide at high temperatures and a wide pH range. By using the asparaginase, it is possible to stably and efficiently reduce acrylamide in foods prepared at high temperatures and in a wide pH range.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for explaining the present invention more specifically, and the scope of the present invention is not limited by the following examples.

Culture of Bacillus amyloliquefactions MKSE strain (KCTC18753P)

To screen for bacteria producing asparaginase, 28 fermented Korean traditional soybean foods were diluted with 0.85% (w/v) physiological saline, and ammonia detection medium (NaCl 2.25g, L-asparagine 5g, MgSO ₄ 7H ₂₎ O 0.5g, CaCl _{2 ·} 2H ₂ O 0.11g, glucose 2g, phenol red 0.025g, agar 15g _· L ^-1 ) after inoculation, incubated for 48 hours at 30 ℃, by hydrolysis of L-asparagine Colonies having a formed pink region were selected as strains.

After incubating the strain at 30° C. for 24 hours using Nutrient agar (NA) and Nutrient broth (NB) medium, LB medium (tryptone 7.7g, yeast extract 9g, peptone 5.25g, CaCl _2· 2H ₂ O 0.76 g, L-apsaragine _· L ^-1 7g) was inoculated (1%), and incubated at 37°C at 180 rpm for about 12 hours.

The nucleotide sequence of the 16s rRNA of the cultured strain was analyzed, and as a result, Bacillus amyloliquefaciens showed 99% homology with FZB42 (FIG. 2).

In addition, using API 50CH KIT to analyze the ability of the strain to degrade carbohydrates (sugars), and as a result, it shows the decomposition ability for Glycerol, L-arabinose and 17 other carbohydrates (sugars) (Table 1), Bacillus subtilis ) and Bacillus amyloliquefaciens showed 94.8% homology.

No. Carbohydrate (sugar) Fermentation No. Carbohydrate (sugar) Fermentation 0 Control - 26 Salicin + One Glycerol + 27 Celiobiose + 2 Erythrytol - 28 Maltose + 3 D-arabinose - 29 Lactose - 4 L-arabinose + 30 Melibiose - 5 Ribose + 31 Sucrose + 6 D-xylose + 32 Trehalose + 7 L-xylose - 33 Inulin - 8 Adonitol - 34 Melezitose - 9 β-Mcthyl-D-Xylose - 35 Raffinose - 10 Galactose - 36 Starch - 11 Glucose + 37 Glycogen - 12 Fructose + 38 Xylitol - 13 Mannose + 39 Gentobiose - 14 Sorbose - 40 D-Turanose - 15 Rhamnose - 41 D-Lyxose - 16 Dulcitol - 42 D-Tagatose - 17 Inositol + 43 D-fucose - 18 Manitol + 44 L-fucose - 19 Sorbitol + 45 D-Arabitol - 20 α-Methyl-D-Mannoside - 46 L-Arabitol - 21 α-Methyl-D-Glucoside + 47 Gluconate - 22 N-Acctyl-Glucosamine - 48 2-Keto-Gluconate - 23 Amygdalin + 49 5-Keto-Gluconate - 24 Arbutin + Identification (Similarity %) Bacillus subtilis / amyloliquefaciens (94.8%) 25 Esculin +

Therefore, the strain was named Bacillus amyloliquefaciens MKSE (hereinafter, MKSE strain), and on December 11, 2018, it was registered at the Korea Research Institute of Bioscience and Biotechnology Life Resource Center and was given a KCTC18753P number.

The MKSE strain differs from other Bacillus strains in fermentable carbohydrate sources (No. 22, 33, 35, 36, 37, 39), and thus, it can be seen that a mycological difference exists.

In particular, the MKSE strain cannot ferment starch (No. 36), whereas other Bacillus strains ferment starch.If the strain ferment starch, that is, hydrolyze, the quality of food Since it means deterioration, it is not preferable apart from removing acrylamide.

Measurement of enzyme activity of L-asparaginase according to medium composition

The MKSE strain produces asparaginase, and the asparaginase degrades asparagine into aspartic acid and ammonia. After culturing the MKSE strain in a medium having each composition shown in Table 2, the amount of ammonia was measured to measure the enzyme activity of asparaginase, and the results are shown in FIG. 3, and the specific measurement method is as follows. .

45 μL of the supernatant of the MKSE strain was added to 650 μL of 50 mM Tris-HCl Buffer (pH 8.6) containing 10 mM L-apsaragine, and then reacted at 37° C. for 10 minutes. Thereafter, 200 μL of 1.5M TCA Solution was added to stop the reaction, centrifuged at 10,000 rpm for 8 minutes, and then 100 μL of Nessler's reagent was added, followed by reaction at room temperature for 10 minutes.

The color change indicated by the reaction between the produced ammonia and Nessler's reagent was measured at 410 nm using a spectrophotometer, and the amount of asparaginase producing 1 μM of ammonia per minute was defined as 1 IU. I did.

Medium composition Non Asn-Gln Medium (=Control) Asn-sucrose Medium Gln-sucrose Medium Asn-Gln (half) Medium Asn-Gln (full) Medium Sucrose 5 5 5 5 5 Asparagine - 7 - 3.5 7 Glutamine - - 10 5 10 Yeast extract One One One One One Peptone One One One One One Na2HPO4 6 6 6 6 6 KH2PO4 3 3 3 3 3 NaCl 0.5 0.5 0.5 0.5 0.5 CaCl22H2O 0.012 0.012 0.012 0.012 0.012 MgSO47H2O 0.24 0.24 0.24 0.24 0.24

Referring to Tables 2 and 3, compared to a control that does not contain both asparagine and glutamine, when containing both asparagine and glutamine in a high content as a medium composition for culturing the MKSE strain (Asn-Gln ( full) Medium) It can be seen that the enzyme activity of asparaginase is the best.

Purification of asparaginase

After centrifuging the MKSE strain cultured in Asn-Gln (full) Medium of Table 2 at 8,000 rpm for 20 minutes, cell lysis (cell lysis buffer (1mM Na-EDTA, 50mM NaCl, 1mM PMSF, pH 8.0) 50mM Tris-HCl, glass beads 0.4-0.6mm, lysozyme 2mg/mL, on ice), centrifuged at 8,000rpm for 20 minutes to remove cell debris, and the crude extract of the MKSE strain ( crude extract) was prepared.

Using Ni-sepharose 6 FF affinity resin (GE healthcare, USA), asparaginase was purified from the crude extract, and the crude extract was compared with specific activity and purification yield. It is shown in Table 3 below.

At this time, it was confirmed whether asparaginase obtained in the form of extracellular enzyme in addition to asparaginase obtained in the form of intracellular enzyme through cell lysis of the MKSE strain was present. In the case of the extracellular asparaginase, ammonium sulfate The crude protein component (crude enzyme) in the culture medium was precipitated using (ammonium sulfate), and then purified through Ni-affinity column chromatography.

Referring to Table 3, in the case of the asparaginase, the purification yield was measured to be 30.8%, and it was confirmed that the specific enzyme activity increased by about 8.26 times compared to the crude extract.

On the other hand, in the case of the extracellular asparaginase, the specific enzyme activity compared to the crude protein component (crude enzyme) in the culture medium increased by about 6.08 times, but was lower than the specific enzyme activity of the asparaginase, and the purification yield This is very low, and in purifying asparaginase from the MKSE strain, it was confirmed that it is more preferable to purify the asparaginase in the form of an intracellular enzyme.

Thus, the asparaginase was analyzed by SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the results are shown in FIG. 4. Referring to Figure 4, compared to the standard marker (M) and the crude extract (CE), the intracellular asparaginase (N) has a single band, and the protein molecular weight is the actual protein molecular weight of the asparaginase It can be seen that the measurement was measured at 38.2 kDa similar to (39.5 kDa). Therefore, it can be seen that the purification of asparaginase was preferably performed.

Measurement of enzyme activity of asparaginase according to temperature

The asparaginase derived from the crude extract of the MKSE strain was incubated for 6 hours at 40°C to 95°C in 50mM Tris-HCl Buffer (pH 8.6) containing 10mM L-apsaragine for 6 hours, depending on the temperature. The residual enzyme activity of the asparaginase was measured and the results are shown in FIG. 5.

Referring to FIG. 5, using the enzyme activity of the asparaginase not applied with heat as a standard (100%), the residual enzyme activity of the asparaginase applied with heat was measured. Although there was a decrease in enzyme activity due to heat treatment under all temperature conditions, it was confirmed that 57% to 94% of enzyme activity remained in all even when heat was applied at 40°C to 95°C for 6 hours. In particular, the asparaginase had a residual activity of about 57% even when heat was applied at 95°C for 6 hours. To] Referring to Table 4, wherein the MKSE strain derived, better than the aspartate agent as compared with the better than aspartate other Bacillus (Bacillus) derived from genus to check that the highest optimal temperature, so that the excellent thermal stability can see.

Asparaginase producing strain Optimal temperature(℃) Optimal pH References Bacillus amyloliquefaciens MKSE 65 8.5 This study Bacillus subtilis B11-06 40 7.5 Jia et al, 2013 Bacillus subtilis strain R5 35 9.0 Chohan et al, 2018 Bacillus megaterium H-1 40 7.0 Zhang et al, 2015

More specifically, referring to FIG. 6, a result of comparing the enzyme activity of asparaginase according to the strain can be confirmed. Bacillus sp . strains were extracted from Korean traditional soybean fermented foods such as soybean paste and soy sauce, and 27 strains were selected through Asparaginase assay, and the asparaginase derived therefrom was not heat treated (control), Enzyme activities were compared when heat treatment was performed at 60°C or 95°C for 10 minutes.

It can be seen that the asparaginase derived from the crude extract of the MKSE strain has remarkably superior enzyme activity compared to the asparaginase derived from other strains even when heat treatment is performed up to 95°C.

Measurement of enzyme activity of asparaginase according to pH

The asparaginase derived from the crude extract of the MKSE strain, 50mM Citrate-Sodium citrate buffer (pH 3.0-6.0), 50mM Sodium phosphate buffer (pH 6.0-7.5), 50mM Tris-HCl buffer (pH 7.5- 9.0) and 50mM Sodium bicarbonate-NaOH buffer (pH 9.5-11.0) at pH 3.0 to 11.0 for 10 hours at 4°C, followed by 50mM Tris-HCl Buffer (pH 8.6) containing 10mM L-apsaragine. ) Was reacted at 65° C. for 10 minutes, and the residual enzyme activity of the asparaginase according to the pH was measured, and the results are shown in FIG. 7.

Referring to FIG. 7, the residual enzyme activity of the asparaginase according to pH was measured. Although there was a decrease in enzyme activity over time in all pH conditions, 90% to 99% of enzyme activity remained even after 4 hours, and 45% to 70% of enzyme activity remained even after 10 hours. , It can be seen that the asparaginase has very high pH stability.

Measurement of enzyme activity of asparaginase according to metal ions and compounds

To the asparaginase derived from the crude extract of the MKSE strain, a metal ion and a compound as shown with reference to FIG. 8 were added at 1 mM or 10 mM to measure enzyme activity, and the results are shown in FIG. Done.

Referring to FIG. 8, at all concentrations, manganese ions, magnesium ions, and divalent iron ions acted as major activators, and zinc ions and copper ions acted as major inhibitors.

Therefore, in the case of using the asparaginase derived from the MKSE strain as an additive, when manganese ions, magnesium ions, and divalent iron ions acting as activators are used together, the activity of the asparaginase may be improved It can be confirmed that it is preferable because it can reduce the activity of the asparaginase when used together with zinc ions and copper ions acting as inhibitors.

Therefore, it was found through experiments that Bacillus amyloliquefactions MKSE strain, a novel strain of the present invention, can produce asparaginase excellent in heat and pH stability, maintaining enzymatic activity at high temperatures and a wide pH range. .

Korea Research Institute of Bioscience and Biotechnology KCTC18753P 20181211

<110> Industry-Academic Cooperation Foundation, Dankook University <120> A novel bacillus amyloliquefaciens MKSE strain, an incubation method of the strain and a producing method of asparaginase using the strain <130> HY180631 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1421 <212> DNA <213> Bacillus amyloliquefaciens MKSE <400> 1 tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60 cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120 tggttgtctg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180 gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240 cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300 gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360 atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420 ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480 gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540 ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600 acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660 ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720 cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780 gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840 agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900 atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960 cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080 cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140 gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200 aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260 cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320 ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380 ccgaagtcgg tgaggtaacc tttaggagcc agccgccgaa g 1421

Claims (12)

Bacillus amyloliquefaciens MKSE strain (KCTC18753P).
The method according to claim 1,
The strain is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that producing asparaginase (asparaginase).
The method according to claim 2,
The asparaginase is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that it is stable at a temperature of 40 ℃ to 95 ℃.
The method of claim 3,
The asparaginase Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that maintaining the enzyme activity of 57% to 94% even after 6 hours at a temperature of 40 ℃ to 95 ℃.
The method according to claim 2,
The asparaginase Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that maintaining the enzymatic activity of 90% to 99% even after 4 hours at pH 3.0 to 11.0.
The method according to claim 2,
The asparaginase Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that maintaining the enzyme activity of 45% to 70% even after 10 hours at pH 3.0 to 11.0.
The method according to claim 1,
The strain is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that it does not degrade starch.
The method according to claim 1,
Bacillus amyloliquefaciens having a nucleotide sequence of SEQ ID NO: 1 MKSE strain (KCTC18753P).
Sucrose, Asparagine, Glutamine, Yeast Extract, Peptone, Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, CaCl ₂ 2H ₂ O and MgSO ₄ 7H ₂ O Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P), characterized in that the culture using a medium containing one or more selected from the group consisting of culture method.
The method of claim 8,
The medium is sucrose (Sucrose) 1 to 35g / L, Asparagine (Asparagine) 1 to 14g / L, Glutamine (Glutamine) 2 to 14g / L, yeast extract 1 to 5g / L, Peptone (Peptone) 1 to 5g / L, Na ₂ HPO ₄ 6g/L, KH ₂ PO ₄ 3g/L, NaCl 0.5g/L, CaCl ₂ 2H ₂ O 0.012 g/L and MgSO ₄ 7H ₂ O Characterized in that containing 0.24g / L, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MKSE strain (KCTC18753P) culture method.
The method of claim 8,
The Bacillus amyloliquefaciens MKSE strain (KCTC18753P) is characterized in that cultured for 24 to 48 hours at a temperature of 25 ℃ to 55 ℃ using the medium, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Culture method of MKSE strain (KCTC18753P).
Culturing the strain of claim 1;
Recovering the culture medium in which the strain of claim 1 is cultured; And
Purifying asparaginase from the culture medium; Method for producing asparaginase comprising a.

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