A kind of crude enzyme preparation, its preparation method and application

A preparation and crude enzyme technology, applied in the field of bioengineering, can solve the problems of energy consumption, ATP consumption, high price, etc., and achieve the effect of reducing cost and enzyme amount

Active Publication Date: 2020-11-03
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrate glutamine used by γ-glutamyl transpeptidase and glutaminase is relatively expensive, and it will hydrolyze the substrate, and it is necessary to add an excess of another substrate ethylamine to inhibit
Glutamine synthetase and γ-glutamine synthetase can convert cheap sodium glutamate and equivalent ethylamine to synthesize L-theanine, but it is an energy-consuming reaction that consumes equivalent ATP
However, there is no report on the application of ATP-specific regeneration enzymes to the theanine synthesis system.

Method used

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  • A kind of crude enzyme preparation, its preparation method and application
  • A kind of crude enzyme preparation, its preparation method and application
  • A kind of crude enzyme preparation, its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The construction of embodiment 1 genetic engineering bacterial classification

[0037] (1) Construction of pET21a(+)-ppk

[0038] Extract the plasmid pET-21a(+) with a plasmid extraction kit, and digest the plasmid with NdeI / SalI double enzymes at 37°C for 2 hours. The enzyme digestion system is: 50 μL of plasmid, 10 μL of 10×buffer, 3 μL of NdeI, 3 μL of SalI, 34 μL of water, and the 5.4 kb pET-21a (+) fragment was recovered by electrophoresis.

[0039] The synthesized plasmid containing phosphokinase (US20050287627 A1, SEQ ID NO 125) encoding gene ppk was digested with NdeI / SalI double enzymes at 37°C for 2h, the digestion system was the same as above, and a 1 kb ppk fragment was recovered by electrophoresis.

[0040] The 5.4 kb pET-21a(+) fragment recovered above was ligated with the 1 kb ppk fragment under the action of T4 ligase at 22° C. for 5 h. The ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformant...

Embodiment 2

[0049] Expression of embodiment 2 recombinant protein

[0050] The recombinant bacteria BL21(DE3) / pET21a(+)-ppk+gams were picked and inoculated in LB medium containing ampicillin, and cultured overnight at 37°C with shaking. The culture was transferred to fresh LB medium containing ampicillin at 1% inoculum size, and cultured with shaking at 37°C until OD 600 =0.6-0.8, IPTG induction (final concentration 0.1mM) at 20°C overnight, centrifuged to collect cells, resuspended cells in 20mM sodium phosphate buffer (pH 8.0), ultrasonically disrupted, centrifuged and freeze-dried the supernatant to obtain a crude lyophilized powder Enzyme preparations, protein expression such as image 3 shown.

[0051] From image 3 It can be seen that the prepared crude enzyme preparation contains two proteins with sizes of 38kDa and 49kDa, respectively, which is consistent with the prediction.

Embodiment 3

[0052] Example 3 Enzyme Activity Determination

[0053] Use the following method to measure the enzyme activity of gained freeze-dried powder in embodiment 2:

[0054] The enzyme activity assay detection system of γ-glutamine synthetase is: the 3mL reaction system contains final concentrations of 50mM sodium glutamate, 15mM hydroxylamine hydrochloride, 7.5mM adenosine triphosphate disodium (ATP), 30mM magnesium chloride hexahydrate and 100mM Imidazole buffer (pH 8.0), add an appropriate amount of crude enzyme preparation enzyme solution at 30°C, react for 30 minutes, add 1mL GS color reagent (3.32g trichloroacetic acid, 6.06g ferric chloride and 5mL concentrated hydrochloric acid dissolved in water , and set the volume to 100 mL) to terminate the reaction, and measure the absorbance at 540 nm in a spectrophotometer. The amount of enzyme required to generate 1 μmol γ-glutamylhydroxamic acid per minute under these conditions was defined as 1U.

[0055] The enzyme activity dete...

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Abstract

The invention relates to the technical field of biological engineering and particularly relates to a crude enzyme preparation as well as a preparation method and application thereof. The crude enzyme preparation comprises gamma-glutamyl-methylamine synthetase and phosphokinase, wherein genes of the gamma-glutamyl-methylamine synthetase and the phosphokinase are cloned on one expression vector. According to the crude enzyme preparation provided by the invention, a recombinant vector containing a gamma-glutamyl-methylamine synthetase encoding gene and a phosphokinase encoding gene is constructed for the first time; the recombinant vector is used for converting host bacteria to obtain an engineering strain; and when the engineering strain is fermented, the crude enzyme preparation containing the gamma-glutamyl-methylamine synthetase and the phosphokinase can be obtained.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a crude enzyme preparation, its preparation method and application, in particular to a crude enzyme preparation prepared by gene cloning and expression and its application for synthesizing theanine. Background technique [0002] L-theanine (C 7 h 14 o 3 N 2 , also known as γ-glutamyl ethylamine) is a special amino acid contained in tea, which has many uses: it is used in ordinary food to improve food flavor and strengthen nutrition, etc.; in health food, it is used for calming and relaxing, Improve sleep, anti-fatigue, improve learning and memory ability, etc.; in the field of medicine, it has been used to lower blood pressure, prevent cancer, prevent Alzheimer's disease, control depression, etc. Therefore, L-theanine has a wide demand in beverages, functional foods, and functional medicines. [0003] Enzymatic method is a new trend in the synthesis of L-theanine in r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N9/00C12N15/52C12N15/54C12N15/70C12N1/21C12P13/04
CPCC12N9/1229C12N9/93C12P13/04C12Y603/04012
Inventor 祝俊刘珊李元
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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