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Aminopeptidase for catalytic synthesis of carnosine, and preparation method and application thereof

A technology of aminopeptidase and carnosine, which is applied in the field of aminopeptidase for catalyzing the synthesis of carnosine and its preparation, achieving the effects of high enzymatic reaction generation rate, reduced production cost and less pollution

Inactive Publication Date: 2017-09-29
JIANGSU CHENGXIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Reaction without direct participation of β-alanine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the genetic engineering strain construction of aminopeptidase

[0047] The biological enzyme fragment synthesized from the whole gene (sequence shown in SEQ ID NO: 1, synthesized by Changzhou Jiyu Biotechnology Co., Ltd.) was treated with restriction endonuclease Eco RI and not Ⅰ (purchased from New England Biolabs, operated according to the instructions) was digested and recombined into the yeast expression vector pPIC9k (invitrogen), transformed into E.coli Top10 competent (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the E.coli Top10 was placed in LB liquid medium, 37°C, 160rpm, shaking culture overnight, and the recombinant plasmid was extracted. The recombinant plasmid was linearized with restriction endonuclease SalI (purchased from New England Biolabs, operated according to the instructions).

[0048] Competent cells of Pichia pastoris GS115 (invitrogen company): Pick a single colony of Pichia Pastoris GS115 into YPD medium for...

Embodiment 2

[0051] Embodiment 2: the fermentation preparation of aminopeptidase

[0052] Streak the original strains on YPD and culture them upside down at 30°C overnight. Pick a single colony (diameter 1mm) on the plate and put it in 50ml YPD liquid medium (yeast powder 10g, peptone 10g, glucose 10g, add water to 1L), 30°C, 200rpm shaking culture overnight (24h), OD600 increased to 4 -5. Inoculate 10% of the inoculum into a shaker flask containing 300ml of YPD liquid medium (1L Erlenmeyer flask), shake the culture at 30°C and 200rpm on a shaker, and the OD600 will grow to about 12 after about 24 hours. After the fermentation medium was prepared, it was transferred into a fermenter (30L), sterilized at 121°C for 30 minutes; the temperature was lowered to 30°C, and the pH value was adjusted to 5.0 with ammonia water. Inoculate the cultured strain into the fermenter with an inoculation amount of 5%. Adjust the rotation speed and ventilation according to the dissolved oxygen, and control th...

Embodiment 3

[0053] Embodiment 3 Aminopeptidase catalyzes the synthesis of carnosine

[0054] Including the following steps:

[0055] 1) Synthesis of β-alanine methyl ester hydrochloride:

[0056] 1.1) Add methanol to the reaction kettle, control the temperature at 0-20°C, and drop thionyl chloride into it. After the dropwise addition, add β-alanine, and keep stirring at 30-60°C;

[0057] 1.2) Reduce pressure at 30-50°C, vacuum degree ≤ -0.09MPa, concentrate until no fraction flows out to obtain β-alanine methyl ester hydrochloride;

[0058] 2) Enzyme-catalyzed reaction:

[0059] 2.1 Add L-histidine, deionized water, and β-alanine methyl ester hydrochloride prepared by the above reaction into the reactor;

[0060] 2.2 At a temperature of 15-45°C, adjust the pH value to 7.0-9.0 with an alkaline solution;

[0061] 2.3 Add the aminopeptidase prepared as claimed in claim 4, the amount of the aminopeptidase is 1% to 3% of the volume of the reaction solution, and the reaction is stirred at 2...

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PUM

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Abstract

The invention provides aminopeptidase for catalytic synthesis of carnosine, and a preparation method and application thereof. The aminopeptidase has the amino acid sequence shown by SEQ ID NO:2. For the preparation of biological enzymes, firstly, a gene engineering strain of the biological enzymes is built; the biological enzyme gene fragment obtained through full gene synthesis is subjected to linearized plasmid recombination by using restriction enzymes SalI. The built gene-engineered strain is suitable for high-density culture; the exoenzyme is biologically synthesized; solid-liquid separation is performed; ultrafiltration concentration is performed to obtain enzyme liquid; histidine and beta-alanine can be catalyzed in normal-temperature and normal-pressure water solution to efficiently synthesize carnosine; the reaction environment is friendly; the production cost is low; the conversion time is short; the process operation is simple; the reaction system impurity content is low; the post treatment is easy; the wide prospects of large-scale industrial application are realized.

Description

technical field [0001] The invention relates to a method for synthesizing carnosine, in particular to a method for synthesizing carnosine using highly efficient aminopeptidase catalysis and modern separation technology, in particular to an aminopeptidase that catalyzes the synthesis of carnosine and a preparation method thereof. Background technique [0002] L-carnosine was first discovered in beef in 1900 and widely exists in the brain, muscle and other tissues of mammals. Its structure is mainly composed of β-alanine and L-histidine. It is a natural active Dipeptide, easily soluble in water, 0.1mol / L hydrochloric acid and 0.1mol / L sodium hydroxide, almost insoluble in methanol, acetonitrile, absolute ethanol, chloroform and acetone. Studies have proved that L-carnosine has anti-oxidation and anti-aging functions. It has therapeutic effects on hypertension, heart disease, senile cataracts, ulcers, etc. It also has biological activities such as anti-tumor, which can delay sk...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12P17/10
CPCC12N9/485C12P17/10
Inventor 黄科学祝俊吴锋王峰峰张超华俊国余玉奎徐飞
Owner JIANGSU CHENGXIN PHARMA
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