阅读说明：本技术 一株海洋假单胞菌产出高含量环二肽类化合物方法 (Method for producing high-content cyclic dipeptide compound by using marine pseudomonas ) 是由 杜风强 邵海峰 于 2021-08-25 设计创作，主要内容包括：一株海洋假单胞菌产出高含量环二肽类化合物方法,其包括如下步骤,1)将一株海洋假单胞菌即F-Q127菌种从固体斜面培养基中取适量接种到内装100mL液体培养基的三角瓶中,在28℃、180r·min－1的条件下摇床培养48h,作为种子培养液；2)将该种子培养液按体积分数7%的接种量接种于含150mL液体培养基的三角瓶中,共接种15L,在28℃180r·min－1的条件下摇床培养5d,再对培养液进行萃取,得到菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物；经薄层检查,菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物主要斑点相似,将两部分的氯仿萃取物合并得氯仿萃取物；3)对氯仿萃取物采用流式细胞术测定化合物的细胞周期抑制活性,得到环二肽类化合物。(A method for producing high-content cyclic dipeptide compounds by using marine pseudomonas comprises the following steps of 1) taking a proper amount of marine pseudomonas, namely F-Q127 strain from a solid slant culture medium, inoculating the strain into a triangular flask filled with 100mL of liquid culture medium, and performing shake cultivation for 48 hours at 28 ℃ and 180 r.min < -1 > to obtain a seed culture solution; 2) inoculating the seed culture solution into a triangular flask containing 150mL of liquid culture medium according to the inoculation amount of 7% of volume fraction, inoculating 15L of the seed culture solution, performing shake culture at 28 ℃ for 5d under the condition of 180 r.min < -1 >, and extracting the culture solution to obtain a chloroform extract of strain fermentation liquor and a chloroform extract of mycelia; performing thin layer inspection to obtain chloroform extract of strain fermentation liquid and main spot of chloroform extract of mycelium similar, and mixing the two chloroform extracts to obtain chloroform extract; 3) and measuring the cell cycle inhibitory activity of the compound by adopting flow cytometry on the chloroform extract to obtain the cyclic dipeptide compound.)

1. The method for producing the high-content cyclic dipeptide compound by using the marine pseudomonas is characterized by comprising the following steps of: comprises the following steps of 1) taking a proper amount of marine pseudomonas, namely F-Q127 strain from a solid slant culture medium, inoculating the strain into a triangular flask filled with 100mL of liquid culture medium, and performing shake culture for 48 hours at 28 ℃ under the condition of 180 r.min < -1 > to obtain a seed culture solution;

2) inoculating the seed culture solution into a triangular flask containing 150mL of liquid culture medium according to the inoculation amount of 7% of volume fraction, inoculating 15L of the seed culture solution, performing shake culture at 28 ℃ for 5d under the condition of 180 r.min < -1 >, and extracting the culture solution to obtain a chloroform extract of strain fermentation liquor and a chloroform extract of mycelia; performing thin layer inspection to obtain chloroform extract of strain fermentation liquid and main spot of chloroform extract of mycelium similar, and mixing the two chloroform extracts to obtain chloroform extract;

3) the cell cycle inhibitory activity of the compound was determined by flow cytometry on the chloroform extract to obtain cyclic dipeptide compounds, i.e., 6 cyclic dipeptide compounds were isolated from the chloroform extract and identified as cyclo (prop-Leu) [ cyclo (Ala-Leu), 1 ], cyclo (prop-Leu) [ cyclo (Ala-Ile), 2 ], cyclo (prop-Val) [ cyclo (Ala-Val), 3 ], cyclo (phenylprop-Leu) [ cyclo (Phe-Leu), 4 ], cyclo (prop-Pro) [ cyclo (Ala-Pro), 5 ] and cyclo (phenylprop-valine) [ cyclo (Phe-Val), respectively.

Technical Field

The invention relates to preparation of cyclic dipeptide compounds, in particular to a method for producing a high-content cyclic dipeptide compound by using marine pseudomonas.

Background

Proteins can be hydrolysed by acid, alkali or enzymes, during which the protein is gradually degraded into smaller and smaller protein fragments such as peptones, polypeptides, tripeptides and dipeptides, and so on, until finally into an amino acid mixture.

Dipeptides are generally hydrolyzed by dipeptidases in vivo to two free amino acids. There is also an energy-consuming active transport system for the absorption of dipeptides or tripeptides on intestinal mucosal cells in vivo, so it is said that dipeptides are absorbed into cells before free amino acids.

Dipeptides are compounds obtained by dehydration condensation of the amino group of a natural amino acid, i.e., an alpha amino acid (amino group is linked to a carbon to which a carboxyl group is linked) with a carboxyl group. Which contains only one peptide bond, i.e., -CO-NH-. Can be hydrolyzed.

The cyclic dipeptide essence-compound active peptide formed by amino acid peptide bonds has direct effects of animal nutrition, hormone and enzyme inhibition, immunity regulation, antibiosis, antivirus and antioxidation.

The prior cyclic dipeptide compound is inconvenient to prepare, the yield is difficult to promote, and the preparation method is complicated.

Disclosure of Invention

Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the method for producing the high-content cyclic dipeptide compound by using the marine pseudomonas, and the problems that the existing cyclic dipeptide compound is inconvenient to prepare, the yield is difficult to improve, and the preparation method is complicated are effectively solved.

In order to achieve the purpose, the invention provides the following technical scheme: the method comprises the following steps of 1) taking a proper amount of marine pseudomonas, namely F-Q127 strain from a solid slant culture medium, inoculating the strain into a triangular flask filled with 100mL of liquid culture medium, and performing shake cultivation for 48 hours at 28 ℃ under the condition of 180 r.min < -1 > to obtain a seed culture solution;

2) inoculating the seed culture solution into a triangular flask containing 150mL of liquid culture medium according to the inoculation amount of 7% of volume fraction, inoculating 15L of the seed culture solution, performing shake culture at 28 ℃ for 5d under the condition of 180 r.min < -1 >, and extracting the culture solution to obtain a chloroform extract of strain fermentation liquor and a chloroform extract of mycelia; performing thin layer inspection to obtain chloroform extract of strain fermentation liquid and main spot of chloroform extract of mycelium similar, and mixing the two chloroform extracts to obtain chloroform extract;

3) and measuring the cell cycle inhibitory activity of the compound by adopting flow cytometry on the chloroform extract to obtain the cyclic dipeptide compound.

Has the advantages that: according to the preparation method, a strain of marine pseudomonas can produce high-content cyclic dipeptide compounds which are respectively identified as cyclo (propyl-bright) [ cyclo (Ala-Leu), 1 ], cyclo (propyl-isoleucine) [ cyclo (Ala-Ile), 2 ], cyclo (propyl-valine) [ cyclo (Ala-Val), 3 ], cyclo (phenylpropyl-bright) [ cyclo (Phe-Leu), 4 ], cyclo (propyll-Pro) [ cyclo (Ala-Pro), 5 ] and cyclo (phenylpropyl-Val) [ cyclo (Phe-Val), and the cyclic dipeptide compounds are obtained by expression experimental methods of target proteins at different induction times: when the marine pseudomonas F-Q127 grows for 5 hours, adding an inducer IPTG with the final concentration of 0.3mmol/L for inducing for 4 hours, which is the optimal condition for expressing the target protein.

Detailed Description

The following provides a more detailed description of the embodiments of the present invention.

The embodiment provides a method for producing high-content cyclic dipeptide compounds by using marine pseudomonas, which comprises the following steps of 1) taking a proper amount of marine pseudomonas, namely F-Q127 strain from a solid slant culture medium, inoculating the marine pseudomonas into a triangular flask filled with 100mL of liquid culture medium, and performing shake culture for 48 hours at 28 ℃ under the condition of 180 r.min < -1 > to obtain seed culture solution;

2) inoculating the seed culture solution into a triangular flask containing 150mL of liquid culture medium according to the inoculation amount of 7% of volume fraction, inoculating 15L of the seed culture solution, performing shake culture at 28 ℃ for 5d under the condition of 180 r.min < -1 >, and extracting the culture solution to obtain a chloroform extract of strain fermentation liquor and a chloroform extract of mycelia; and performing thin layer inspection to obtain chloroform extract of strain fermentation liquid and mycelium, wherein the main spots of the chloroform extract are similar, and mixing the chloroform extracts to obtain chloroform extract.

3) The cell cycle inhibitory activity of the compound was determined by flow cytometry on the chloroform extract to obtain cyclic dipeptide compounds, i.e., 6 cyclic dipeptide compounds were isolated from the chloroform extract and identified as cyclo (prop-Leu) [ cyclo (Ala-Leu), 1 ], cyclo (prop-Leu) [ cyclo (Ala-Ile), 2 ], cyclo (prop-Val) [ cyclo (Ala-Val), 3 ], cyclo (phenylprop-Leu) [ cyclo (Phe-Leu), 4 ], cyclo (prop-Pro) [ cyclo (Ala-Pro), 5 ] and cyclo (phenylprop-valine) [ cyclo (Phe-Val), respectively.

Identifying metabolites cultured by a strain of marine pseudomonas: to give compound 1: white amorphous powder, positive ion TOF-MS gives a pseudo molecular ion peak [ M + H ] + at M/z185, and formula C₉H₁₆O₂N₂In agreement, the infrared spectrum has absorption peaks at 3193 cm-1 (m) and 1689 cm-1(s) showing imide groups, the 1H-NMR (600MHz, CD3OD) spectrum gives δ:3.99(1H, qd, J =6.2, 0.8Hz, H-6), 3.93(1H, ddd, J =8.5, 4.8, 0.8Hz, H-3), 1.84(1H, m, H-8), 1.72(1H, m, H-7), 1.63(1H, m, H-7), 1.44(3H, d, J =7.0Hz, H-11), 0.97(3H, d, J =6.6Hz, H-9) and 0.95(3H, d, J =6.6Hz, H-10) proton signals, identified as ring (propane-bright) compounds;

the preparation method can also be adopted as follows: inoculating the activated seed bacteria into 10 test tubes containing ampicillin LB liquid medium with the inoculum size of 1:100 respectively, carrying out shake culture at 37 ℃ and 200r/min, taking out one test tube every 1h, measuring OD590 value by an ultraviolet spectrophotometer, and simultaneously culturing E.coliTB1 and pMAL-c2X-TB1 strains by the same method as a control to obtain the cyclodipeptide compound.

Examples of the experiments

An experimental method for optimizing the growth of target protein expression conditions of marine pseudomonas F-Q127 includes such steps as culturing marine pseudomonas F-Q127 in different culture media at different growth rates, inoculating correctly identified transformed bacteria in selective TB culture media and GS culture media (both containing 100ug/ml ampicillin and 0.2% (w/v) glucose), measuring the growth conditions (OD) of said bacteria in different culture media in the same time by UV spectrophotometer, plotting growth curve, inducing the target protein expression at different time, inoculating activated seeds in 5ml tubes containing ampicillin LB liquid culture medium at 1:100 ratio, culturing at 37 deg.C and 200r/min by shaker at 0.5 hr, and culturing at 2 hr, 3 hr and 4 hr, 5h) Adding inducer IPTG (final concentration is 0.3mmol/L), continuing shaking for 4h, taking 1ml of bacterial liquid, and performing SDS-PAGE electrophoresis;

the results show that: when the marine pseudomonas F-Q127 grows for 5 hours, adding an inducer IPTG with the final concentration of 0.3mmol/L for inducing for 4 hours, which is the optimal condition for expressing the target protein.

Has the advantages that: according to the preparation method, a strain of marine pseudomonas can produce high-content cyclic dipeptide compounds which are respectively identified as cyclo (propyl-bright) [ cyclo (Ala-Leu), 1 ], cyclo (propyl-isoleucine) [ cyclo (Ala-Ile), 2 ], cyclo (propyl-valine) [ cyclo (Ala-Val), 3 ], cyclo (phenylpropyl-bright) [ cyclo (Phe-Leu), 4 ], cyclo (propyll-Pro) [ cyclo (Ala-Pro), 5 ] and cyclo (phenylpropyl-Val) [ cyclo (Phe-Val), and the cyclic dipeptide compounds are obtained by expression experimental methods of target proteins at different induction times: when the marine pseudomonas F-Q127 grows for 5 hours, adding an inducer IPTG with the final concentration of 0.3mmol/L for inducing for 4 hours, which is the optimal condition for expressing the target protein.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.