Method for preparing L-tryptophan-L-alanine cyclic dipeptide by using aspergillus oryzae

A tryptophan and alanine technology, applied in the field of biomedicine, can solve the problems of difficult chemical synthesis, unstable fermentation, high price, etc., and achieve the effects of simple and easy separation and purification, breaking through resource limitations, and low cost.

Active Publication Date: 2022-06-28
NORTHEAST FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although the L-tryptophan-L-alanine cyclic dipeptide has been commercialized at present, its CAS registration number: 17079-37-7, but its price is extremely expensive
The main reason for the high price is that its sources are limited. On the one hand, due to its special structure, the chemical synthesis of L-tryptophan-L-alanine cyclic dipeptide is difficult; on the other hand, although it can be obtained from Aspergillus It is obtained from the metabolites of fungi related to the genus Eurotium, but a series of problems such as unstable fermentation, low yield, and extraction and purification severely limit the source of its microbial production

Method used

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  • Method for preparing L-tryptophan-L-alanine cyclic dipeptide by using aspergillus oryzae
  • Method for preparing L-tryptophan-L-alanine cyclic dipeptide by using aspergillus oryzae
  • Method for preparing L-tryptophan-L-alanine cyclic dipeptide by using aspergillus oryzae

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specific Embodiment approach 1

[0027] Embodiment 1: In this embodiment, a method for preparing L-tryptophan-L-alanine cyclic dipeptide using Aspergillus oryzae is carried out according to the following steps:

[0028] Step S1: construction of expression plasmid pMA-criC;

[0029] Using the genomic DNA of Eurotium cristatum as the template, using P1F as the forward primer and P1R as the reverse primer, the fragment 1 of the cyclic dipeptide synthase encoding gene criC was obtained by amplification, and then P2F was used as the forward primer. The primer and P2R are reverse primers, and the second fragment of the cyclic dipeptide synthase encoding gene criC is obtained by amplification; then the genomic DNA of Eurotium cristatum is used as the template, and the pMA plasmid is cut and linearized with KpnI enzyme. The fragment is a vector, and the fragment 1 and fragment 2 of the cyclic dipeptide synthase encoding gene criC are inserted into the restriction site KpnI of the pMA plasmid to obtain the expression ...

specific Embodiment approach 2

[0036] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is: in step S1, fragment 1 and fragment 2 of the cyclic dipeptide synthase encoding gene criC and the pMA plasmid fragment that has been linearized by KpnI digestion are according to 1: After the ligation was completed, the ligation system was transformed into Escherichia coli DH5α, and positive clones were obtained after overnight culture. Finally, after PCR verification and enzyme digestion verification, the expression plasmid was obtained. pMA-criC.

[0037] Other steps are the same as in the first embodiment.

specific Embodiment approach 3

[0038] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is: in step S2, the step of culturing a high-yielding strain based on the L-tryptophan-L-alanine cyclic dipeptide expressing the plasmid pMA-criC As follows: take the spore preservation solution of Aspergillus oryzae and insert it into the DPY medium containing 100 mL, and at 28-30° C., shake and culture at 180-200 rpm for 2-3 days to obtain Aspergillus oryzae. ) of the thalli; the thalli of Aspergillus oryzae were collected by filtration and washed with sterile water for 3 to 5 times, and then 20 mL of cell wall lysing solution was added, and at a temperature of 25 to 28 ° C, gently shake for 2 to 3 hours to obtain protoplasts; wash the protoplasts twice with sterilized 0.8M NaCl solution, add buffer II and buffer III, and then add 10 μg of the expression plasmid pMA-criC, after mixing, ice bath for 18-20 min, and then add Add 1 mL of buffer III to the mixed system, incubate at room temperature ...

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Abstract

The invention discloses a method for preparing L-tryptophan-L-alanine cyclic dipeptide by using aspergillus oryzae, and relates to the technical field of biological medicines. The invention aims to solve the problems of unstable fermentation, low yield and difficulty in extraction and purification in the traditional synthesis method of L-tryptophan-L-alanine cyclic dipeptide. Aspergillus oryzae is taken as a host, and host cells have strong amino acid synthesis capability and can provide abundant L-tryptophan and L-alanine; the expression plasmid pMA-criC used in the invention carries a pAmyB amylase promoter, and when rice is used as a culture medium, the efficient expression of the cyclic dipeptide synthase coding gene criC can be efficiently driven; the high-yield strain takes rice as a culture medium, and is low in cost, simple to operate, short in culture period and simple and feasible to separate and purify. The invention can obtain the method for preparing the L-tryptophan-L-alanine cyclic dipeptide by using the aspergillus oryzae.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for large-scale preparation of L-tryptophan-L-alanine cyclic dipeptide by using Aspergillus oryzae. Background technique [0002] L-tryptophan-L-alanine cyclic dipeptide (Cyclo(L-Trp-L-Ala)) is a small molecule compound containing an indole ring and a 2,5-diketopiperazine ring. Important building blocks of alkaloid natural products. Compounds with L-tryptophan-L-alanine cyclic dipeptide as the structural backbone often have important biological activities and have potential application value in clinical drug development. [0003] Although, L-tryptophan-L-alanine cyclic dipeptide has been commercialized at present, its CAS registration number: 17079-37-7, but its price is extremely expensive. The main reason for the high price is its limited source. On the one hand, due to its special structure, the chemical synthesis of L-tryptophan-L-alanine cyclic dipeptide is di...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/66C12N15/52C12N1/15C12P17/12C12R1/69
CPCC12N15/80C12N9/93C12P17/12C12Y603/02
Inventor 刘成伟祁建钊刘长莉
Owner NORTHEAST FORESTRY UNIVERSITY
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