Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase

A glutamine and synthetase technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as no breakthrough progress, achieve good development prospects, increase production, and simple cultivation conditions Effect

Inactive Publication Date: 2012-10-10
HUNAN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the limitations of research methods and technical means, key technical difficulties such as the enzyme source screening, cultivation and immobilization of γ-glutamine synthetase and its enzymatic synthesis of L-theanine have not yet been achieved at home and abroad. breakthrough

Method used

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  • Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 Preparation of bacterial strain of the present invention

[0023] (1) Strain domestication

[0024] Samples were taken from the soil at 15-25 cm of the rhizosphere of tea trees in good growth conditions in the Gaoqiao Experimental Tea Field of the Hunan Tea Research Institute, and 10 g of fresh soil samples were put into a triangular flask containing 90 mL of sterile water, dispersed with glass beads, and placed in a In a constant temperature shaker, shake at 25°C and 150 r / min for 1 hour to make a soil suspension and let it stand. Add the soil suspension supernatant to the screening medium at a volume ratio of 2%, and after 5-14 days of shaking culture at 30°C and 150 r / min, transfer the turbid medium (OD610 value greater than 0.1) to a fresh screening medium , and then cultivated until turbid, and this process was repeated 3-5 times to obtain the acclimatized bacteria liquid.

[0025] (2) Isolation and purification of bacterial strains

[0026] The acc...

Embodiment 2

[0027] Example 2 Preparation of γ-glutamine synthetase

[0028] Take the above-obtained slant strain, pick a ring and put it in 40mL enrichment medium, culture it with shaking at 30°C and 150r / min for 3 days, and check that the OD value is greater than 0.1. Then centrifuge the enriched culture solution at 4°C and 12000r / min for 18min, discard the supernatant, soak it twice with 10 mmol / L phosphate buffer solution of pH 6.0, then centrifuge at 4°C and 12000r / min for 18min, discard Supernatant, then resuspend the obtained cells in a small amount of buffer (it is enough to cover the cells), and then perform ultrasonic cell disruption on ice (20 kHZ, 2-3 min) to make a crude enzyme solution.

[0029] Adjust the pH value of the crude enzyme solution to 8, add ammonium sulfate with 30% saturation at 30°C for salting out, redissolve with the heavy protein precipitation solution to the original volume, and perform SephadexG-75 column chromatography after ultrafiltration and desalinati...

Embodiment 3

[0031] Example 3 Synthesis of L-theanine catalyzed by γ-glutamine synthetase

[0032] Add the purified γ-glutamine synthetase to the test tube equipped with the L-theanine synthesis system at the ratio of 0.02 mL of γ-glutamine synthetase per liter of L-theanine synthesis system, After reacting for 12 hours at 30°C and a pH value of 8, the test tube was immersed in boiling water to terminate the reaction. After cooling to room temperature, centrifuge at 12000r / min for 5 minutes, and take the supernatant to obtain L-theanine, which was stored at -20°C.

[0033] Among them, the synthesis system of L-theanine is: 30 mmol / L glutamic acid, 150 mmol / L ethylamine hydrochloride, 15 mmol / L ATP, 30 mmol / L MgCl 2 , 100 mmol / L imidazole buffer (pH7.75), and 0.1 mg / mL CTAB.

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Abstract

The invention discloses a catalytic synthesis method of L-theanine by using a microorganism-produced gamma-glutamyl amino carboxamide synthase. The catalytic synthesis method comprises the following steps of: screening from tea tree rhizosphere soil to obtain gamma-glutamyl amino carboxamide synthase-sourced strain, nearly Sporidiobolus pararoseus T-C2, with the preservation number of CCTCC NO: M2012232, so as to obtain a purified gamma-glutamyl amino carboxamide synthase; and performing enzymatic synthesis by using the gamma-glutamyl amino carboxamide synthase to obtain the L-theanine, thereby obtaining a method for synthesizing the L-theanine by using the gamma-glutamyl amino carboxamide synthase with high enzyme activity. The synthesis efficiency is much higher than that of the existing enzymatic synthesis technology of the L-theanine by using a microbial enzyme; the safe, simple, convenient, efficient and economical production is realized; and a theoretical basis and a practical basis are provided for subsequent large-scale microbial enzymatic fermentation production of the L-theanine.

Description

technical field [0001] The invention relates to a Sporidiobolus pararoseus T-C2, in particular to a method for catalyzing the synthesis of L-theanine by using the gamma-glutamine synthetase produced by fermentation of the microorganism. Background technique [0002] L-Theanine (L-Theanine) is a unique non-protein free amino acid in tea tree. It is one of the main quality and functional components in tea. In 2002, it was rated as one of the most potential natural products by the American health food industry. [0003] There are very few natural sources of L-theanine. The L-theanine in the tea tree is based on glutamic acid and ethylamine, and is formed under the action of theanine synthase. It is related to the nitrogen metabolism of the tea tree. closely related. At present, its preparation mainly includes several methods such as direct separation and purification from tea leaves, chemical synthesis and biosynthesis: [0004] (1) Separation and purification: Since there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N9/00C12P13/04C12R1/645
Inventor 肖文军张玥龚志华
Owner HUNAN AGRICULTURAL UNIV
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