Bio-enzyme for synthesis of N(2)-L-alanyl-L-glutamine by catalysis as well as preparation method and application thereof

A technology of glucuronide dipeptide and bio-enzyme, which is applied in the field of catalyzing and synthesizing glucoglutide dipeptide and its preparation, can solve the problems of high process cost, long reaction synthesis route, and large environmental pollution, and achieve simple equipment requirements, high solvent recycling rate, Post-processing simple effects

Active Publication Date: 2015-04-01
JIANGSU CHENGXIN PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CN101659691A and CN1786019A disclose a kind of chemical synthesis and industrialized production method (following route

Method used

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  • Bio-enzyme for synthesis of N(2)-L-alanyl-L-glutamine by catalysis as well as preparation method and application thereof
  • Bio-enzyme for synthesis of N(2)-L-alanyl-L-glutamine by catalysis as well as preparation method and application thereof
  • Bio-enzyme for synthesis of N(2)-L-alanyl-L-glutamine by catalysis as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: the genetic engineering strain construction of biological enzyme

[0071] The biological enzyme gene fragment synthesized by the whole gene (sequence shown in SEQ ID NO: 1, synthesized by Nanjing GenScript Biotechnology Co., Ltd.), was subjected to restriction endonucleases EcoRI and Not I (purchased from New England Biolabs Company, Operate according to the instructions) after enzyme digestion, recombine into yeast expression vector pPIC9K (invitrogen company), transform into E.coli TOP10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), put E.coli TOP10 in LB liquid medium Incubate overnight at 37°C with shaking at 160 rpm, and extract the recombinant plasmid. Using the restriction endonuclease SalI (purchased from New England Biolabs company, operate according to the instructions) to linearize the recombinant plasmid.

[0072] The preparation of Pichia pastoris GS115 competent cell (invitrogen company): Pichia Pastoris GS115 single bacteriu...

Embodiment 2

[0075] Embodiment 2: the fermentation preparation of biological enzyme

[0076] Streak the original strain on the YPD plate, and culture it upside down overnight at 30°C. Pick a single colony (1 mm in diameter) from the plate and put it in 50 ml of YPD liquid medium (10 g of yeast powder, 10 g of peptone, 10 g of glucose, add water to make up to 1 L), shake overnight at 30°C and 200 rpm for 24 hours. OD600 grows to 4-5. Inoculate 10% of the inoculum into 300ml YPD liquid medium (1L Erlenmeyer flask) shaker flask, shake culture at 30°C and 200rpm on a shaker, and the OD600 grows to about 12 after about 24h. Will Fermentation medium After the material is configured per liter, it is poured into a fermenter (30L), and sterilized at 121°C for 30 minutes; after cooling down, the temperature is controlled at 30°C, and the pH value is adjusted to 5.0 with ammonia water. Inoculate the well-grown seed solution into the tank, and the inoculum amount is 5%. Adjust the rotation speed ...

Embodiment 3

[0078] Add 58g of L-glutamine (397mmol) into 400mL of water, cool down to 10°C, adjust the pH to 9.0 with NaOH aqueous solution (2M), add 110mg of dry enzyme powder into the reaction system, and then add L-alanine methyl ester aqueous solution (Dissolve 82g of L-alanine methyl ester hydrochloride (590mmol) in 100mL of water) dropwise into the reaction system, while adding dropwise NaOH aqueous solution (2M) to keep the pH at 8.9-9.0; when the pH remains unchanged, the reaction At the end, heat up to 60°C to inactivate for 30 minutes, add 20g of activated carbon, stir for 10 minutes and then filter; the filtrate is desalted by nanofiltration, and distilled under reduced pressure until a large amount of solids are precipitated; the temperature is lowered and filtered, and the solids are dried under reduced pressure at 50°C to obtain crude dipeptide 60g.

[0079] Refining: Dissolve 60g of crude dipeptide in 60mL of water, add 300mL of methanol at 50°C, slowly cool down to 5-10°C ...

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Abstract

The invention provides a bio-enzyme for synthesis of proglumetacin dipeptide N-(2)-L-alanyl-L-glutamine by catalysis as well as a preparation method and an application thereof. The amino acid sequence of the bio-enzyme is shown as SEQID NO: 2. The preparation method of the bio-enzyme comprises the following steps: firstly, constructing a genetically engineered strain of the bio-enzyme, and recombining plasmids by cutting the gene segment of the bio-enzyme linearly by using a restriction endonuclease SalI, wherein the gene segment of the bio-enzyme is totally synthesized by genes; and then selecting a positive clone, adding the positive clone into a YPD liquid culture medium, activating, inoculating the positive clone in a BMGY liquid culture medium, culturing the positive clone until the OD reaches 1, inoculating the positive clone in 1% methanol, inducing for 72 hours to express the bio-enzyme, and further fermenting to obtain the bio-enzyme for synthesis of proglumetacin dipeptide N-(2)-L-alanyl-L-glutamine by catalysis. The synthesis of N-(2)-L-alanyl-L-glutamine from bio-enzyme by catalysis is as follows: heating to deactivate, filtering, removing the generated salt by adopting a nanofiltration technology, concentrating, and crystallizing with methanol and water to obtain the high-purity N-(2)-L-alanyl-L-glutamine. The synthesis method of N-(2)-L-alanyl-L-glutamine is simple, efficient and environment-friendly and has extremely high economic value and market competitiveness.

Description

technical field [0001] The invention relates to a method for synthesizing dipeptide, in particular to a biosynthetic method for dipeptide using efficient bio-enzyme catalysis and modern separation technology, in particular to a bio-enzyme that catalyzes the dipeptide synthesis and its preparation method and application. Background technique [0002] N(2)-L-alaninephthalein-L-glutamic acid amine is a derivative of amino acid, referred to as proglycine dipeptide, which is mainly used in the human health care industry and is an ideal substitute for the most abundant amino acid glutamine in the human body. [0003] Due to the extremely important physiological functions and pharmacological effects of glutamine, its application in parenteral nutrition has received widespread attention. However, due to its low solubility and instability in solution, toxic pyroglutamic acid and ammonia are generated under heat sterilization conditions, so commercial amino acid preparations do not co...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12P21/02C12R1/19C12R1/125C12R1/84
CPCC07K5/06026C12N9/00
Inventor 邢将军
Owner JIANGSU CHENGXIN PHARMA
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