• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    This invention relates to a method for producing peptides, especially Z-ALA-ALA-LEN-P-nitroanilide and Z-L-ASP-L-PHEOME (aspartame) in the presence of metalloproteinases. The purpose of the invention is to increase the yield of tripeptides (Z-aspartame). A way to connect Z-ALA-ALA .
    公开号:SU1664845A1
    申请号:SU867774327
    申请日:1986-11-03
    公开日:1991-07-23
    发明作者:Корн Ульрих；Андрей Львович Остерман；Валентин Михайлович Степанов；Татьяна Львовна Воюшина；Людмила Ароновна Люблинская；Грунов Райнер
    申请人:Научный Центр По Биотехнологии (Инопредприятие)；Всесоюзный научно-исследовательский институт генетики и селекции промышленных микроорганизмов；
    IPC主号:
    专利说明:

    1 (89) DD 257174 (48) 08.06.88
    (21) 7774327/13
    (22) 11/03/86
    (31JWPC 12 P / 284889
    (32) 12.20.85
    (33) DD
    (46) 07.23.91. Bul №27
    (71) Biotechnology Research Center (DD), All-Union Scientific Research Institute of Genetics and Selection of Industrial Microorganisms (51) 5
    (72) Ulrich Korn (DD), A.L. Osterman, V.M. Stepanov, T.L. Voyushin L.A. Lublin (SU), Rainer Grunov (DD)
    (53) 668.392 (088.8)
    (57) METHOD FOR OBTAINING PEPTIDES (57) The invention relates to a method for producing peptides, especially Z-Ala-A a-Leu-p-nitroanilide and Z-L-Asp-L-Phe OMe (aspartame) in the presence of metalloprotein. The purpose of the invention is to increase the yield of tripeptides (Z-aspartame). The method involves binding Z-Ala-Aia with Leu-p-nitro-anilide and ZL-Asp with L-PheOMe-HCI in the presence of a proteinase from Bacillus cereus ZIMET 10700 Metalloproteinase used in the form of a filtrate of culture liquid or purified by bacinitacin-silochrom affinity chromatography . 1 z p f-ly
    The invention relates to a process for the preparation of peptides, especially Z-Ala-Ala-Leu-p-nitroanilide and Z-L-Asp-L-PheoMe (aspartame), in the presence of metalloproteinis.
    From the review of ISOWA, V, Vu ki, Case Kagaku Kycka aishi, 36, 1978, 195-20, there is known a method for producing peptides using proteinases as catalysts. In this case, serine proteinases and metalloproteinases of different biological origin from Bacillus polymyxa, Bacillus subtilis, Bacillus thermoproteolyticus, etc. are used.
    The disadvantage of this method is that these enzymes require deep cleaning.
    From the description of the patent of Germany No. 3203292, cl. C 12 P 21/00, published 1985, a method for producing peptides by binding Z-Ala-Ala to Leu-p-nitroanilide and Z-L (L
    WITH
    Asp with Zthe-ome-NS in the presence of metalloproteinase.
    The disadvantage of this method is that it produces a small amount of tripeptides.
    The aim of the invention is to increase the yield of target products.
    The metalloproteinase from B. cereus ZIMET 10700 is used to prepare the peptide Z-Ala-Ala-Leu-p-nitroanilide or the peptide ZL-Asp-L-PheOMe, and it has been established that bacitracin-silochrome
    The efficiency of the synthesis is higher if the purified enzyme is used. An effective purification method for metalloproteinases from you. cereus ZIMET 10700 is a bacitracin-silochrome affinity chromatography. As a result of purification,
    about
    four
    oh
    4 SL

    an enzyme almost free of carbohydrates and foreign proteins. This enzyme improves the reaction rate and the yield of the final product.
    Example 1. 74 mg of Z-A a-Ala and 62 mg of Leu-p-nitroanilide are dissolved in 250 µl of dimethylformamide and mixed with 500 µl
    50 mM Tris-HCl, pH 7.5, with 1 mm Ca-acetate, at 35 ° C and 180 µl of the culture fluid filtrate from you is added. cereus ZIMET 10700, containing 140 μg of enzyme. The concentration of the substrates is 200 µmol each. With stirring for 25 h at 35 ° C obtained
    51% of the product. Qualitative analysis of the final product is carried out by thin layer chromatography on silica gel plates.
    in the system n-butanol: pyridine: acetic acid: water in the ratio 10: 15: 3: 12, and detection in ultraviolet light after treatment with ninhydrin and using KJ after chlorination. In addition, HPLC is used for the qualitative and quantitative determination of the final product. Detection is carried out at the absorption peak of p-nitroaniline (315 nm) after separation of the substrate from the product in the C18 column in the acetonitrile gradient. Additionally, the yield of Z-Ala-Ala-Leu-p-nitroanilide is determined by a gravimetric method after repeated washing with NaHCO3 and water.
    Example 2. 74 mg of Z-Ala-Ala and 62 mg of Leu-p-nitroanilide are dissolved in 250 µl of dimethylformamide and mixed with 300 µl of 50 mM Tris-HCl, pH 7.5, with 1 mM Ca-acetate. The total volume is 900 µl and the substrate concentration is 250 µmol. Then bring the temperature to 35 ° C, add 10 µg of the enzyme from you, cereus ZIMET 10700, purified by affinity chromatography, and stir for 18 hours at 35 ° C. The yield of Z-Ala-Ala-Leu-p-nitroanilide is 72%.
    Example 3. 24.4 mg of Z-Asp are dissolved in 30 µl of 6N. NaOH and 42.2 mg of PheOMe-HCl is dissolved in 200 µl of dimethylformamide and mixed with 300 µl of 50 mM Tris-HCl, pH
    7.5, with 1 mM Ca-acetate. After heating to 35 ° C, 100 µl of the culture fluid filtrate containing 110 µg of the enzyme is added. After stirring for 24 hours, the yield is 38%. The qualitative and quantitative determination of Z-aspartame corresponds to the method of Example 1, and the HPLC-separated product is detected at 220 nm.
    Example 4, 122 mg of Z-Asp is dissolved in 160 µl of 6N. NaOH and 211 mg of PheOMe-HCI in 800 μl of 50 mM Tris-HCl, pH 7.5, with 1 mM Ca-acetate. After heating to 35 ° C, 1 mg of an enzyme purified by affinity chromatography is added in 100 µl of 50 mM Tris-HCl, pH 7.5, with 1 mM Ca-acetate. As a result of stirring for 24 hours, a yield of Z-aspartame of 54% is obtained.
    Example 5. The method is carried out as in Example 4, with a quarter amount of reagents being dissolved in 2.5 ml of 50 mM Tris-HCl buffer, pH 7.0, with 1.0 mM Ca-acetate. After raising the temperature to 35 ° C, 3 mg of the enzyme purified by affinity chromatography is added. After 5 hours of stirring, the solid precipitate is removed by centrifugation, and the supernatant is again incubated. The precipitate formed over the next 5 hours is centrifuged again and the amount of Z-aspartame determined. The overall yield is 85%.
    权利要求:
    Claims (2)
    [1]
    1. A method for producing Z-Ala-Ala-Leu-p-nitroanilide peptides or ZL-Asp-PheOMe by linking Z-Ala-Ala to Leu-p-nitroanilide or ZL-Asp, respectively, with L-PheOMe-HCI in the presence of metalloproteinases from Bacillus cereus, characterized in that, in order to increase the yield of the target products, the metalloproteinase from Bacillus cereus ZIMET 10700 is used.
    [2]
    2. The method according to claim 1, wherein metalloproteinase is used as a filtrate of the culture fluid or purified by affinity chromatography with bacitracin-silochrom.
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    同族专利:
    公开号 | 公开日
    DD257174A3|1988-06-08|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DD28488985A|DD257174A3|1985-12-20|1985-12-20|PROCESS FOR PREPARING THE PEPTIDES Z ALA ALA LON P NITRANILIDE OR Z ASP ASP PHE OME WITH METALOPROTEASE|
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