Chassis system for ATP (Adenosine Triphosphate) regeneration and application

A chassis, glucose phosphate technology, applied in biochemical equipment and methods, enzymes, isomerases, etc., can solve problems such as increasing the production cost of target products, and achieve sustainable ATP regeneration, low production costs, and low separation costs.

Inactive Publication Date: 2018-03-20
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 reaction systems in the above studies all require the participation of coenzyme CoA and NAD, thus increasing the production cost of the target product

Method used

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  • Chassis system for ATP (Adenosine Triphosphate) regeneration and application
  • Chassis system for ATP (Adenosine Triphosphate) regeneration and application
  • Chassis system for ATP (Adenosine Triphosphate) regeneration and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The enzyme involved in the ATP regeneration system of embodiment 1 and the enzyme used to produce L-theanine - Preparation of glutamine synthetase.

[0043] The construction of the expression vector of the enzyme of the ATP regeneration chassis system: using the corresponding genomic DNA (purchased from ATCC) as a template, using PCR to obtain the gene of the enzyme, and by simple cloning (Simple Cloning) method (You et al.ApplEnviron Microbiol 2012 78(5):1593-5) respectively cloned the genes into pET vectors (Novagen, Madison, WI) to obtain the corresponding expression vectors for each enzyme.

[0044] - Glutamine synthetase from Methylovorus mays. After preparing the expression vector gmas / pET21a according to the method of Liu et al. (ProcessBiochemistry 2016 51(10):1458-63), the terminator of the gmas gene was removed by site-directed mutagenesis, and the C-terminus of GMAS with a histidine tag was obtained. Expression vector. The primers used for site-directed...

Embodiment 2

[0054] Example 2 uses fructose 6-phosphate and phosphate as energy sources to regenerate ATP and produce L-theanine. Fructose 6-phosphate is one of the intermediate products of the ATP regeneration chassis system in the present invention, and can replace maltodextrin as an energy substrate for ATP regeneration.

[0055] Configure a 1ml reaction system in a screw bottle, the system contains 200mM HEPES buffer at pH 7.2, 5mM sodium phosphate at pH 7.2, 10mM magnesium chloride, 0.5mM manganese chloride, 1mM thiamine pyrophosphate, 1mM ADP, 30 mM sodium glutamate, 60 mM ethylamine hydrochloride, 5 mM fructose 6-phosphate. Experimental group 1 was an ATP regeneration system containing only PKL, AK, and GMAS enzymes ( figure 2 ), the concentration of each enzyme in the reaction system (concentration of enzyme activity unit) is 2U / ml. Experimental group 2 is an ATP regeneration system with carbon rearrangement modules added. In addition to the above three enzymes at the same conce...

Embodiment 3

[0057] Example 3 uses maltodextrin and phosphate as energy sources to regenerate ATP and produce L-theanine.

[0058] Configure a 1ml reaction system in a screw bottle, the system contains 200mM HEPES buffer at pH 7.2, 5mM sodium phosphate at pH 7.2, 10mM magnesium chloride, 0.5mM manganese chloride, 1mM thiamine pyrophosphate, 1mM ADP, 30 mM sodium glutamate, 60 mM ethylamine hydrochloride, contains 6.3 mM glucose equivalent maltodextrin (DE 4-7). Experimental group 1 was an ATP regeneration system containing only αGP, PGM, PGI, PKL, AK and GMAS ( Figure 5 ), the concentration of each enzyme in the reaction system was 2U / ml. Experimental group 2 is an ATP regeneration system with carbon rearrangement modules added. In addition to the above 6 enzymes at the same concentration, TAL, TK, RPI, RPE, TIM, ALD, FBP ( Figure 6), the concentration of each enzyme in the carbon rearrangement module in the reaction system is 1U / ml. Samples were taken at 37°C for 0, 1, 3, 6, and 8 ho...

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Abstract

The invention provides a chassis system for ATP (Adenosine Triphosphate) regeneration and application. The chassis system for ATP regeneration comprises the following five enzymes: alpha-glucan phosphorylase, glucophosphomutase, phosphoglucose Isomerase, phosphoketolase and acetokinase. In addition, the chassis system can further comprise the following seven enzymes: transaldolase, transketolase,ribose-5-phosphate isomerase, ribulose-3 epimerase, triosephosphate isomerase, fructose-bisphosphate aldolase and fructose-1,6-bisphosphatase. The chassis system can be coupled to an enzymic catalyticreaction needing ATP, utilizes starch or maltodextrin as energy, is not added with any coenzyme, can efficiently regenerate the ATP at low cost through a one-pot reaction and is an economical, highlyefficient, stable and sustainable ATP regeneration system.

Description

technical field [0001] The invention relates to the technical field of biocatalysis, in particular to a chassis system and application for ATP regeneration. Background technique [0002] Adenosine triphosphate (ATP) is a high-energy phosphate compound, and its interconversion with adenosine diphosphate (ADP) realizes energy storage and release, thereby ensuring the energy supply for various biocatalytic processes. ATP is unstable and expensive, and is not suitable for directly adding a large amount to the industrial enzyme reaction production process. Therefore, building a cost-effective ATP regeneration system is one of the focuses in the field of enzyme catalysis. [0003] According to the substrate used, the ATP regeneration system can be divided into a reaction system that transfers high-energy phosphate bonds and a system that utilizes carbohydrate degradation to generate energy. Compounds containing high-energy phosphate bonds mainly include phosphoenolpyruvate (PEP),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N9/90C12N9/92C12N9/88C12N9/12C12N9/16C12P19/32
CPCC12N9/1022C12N9/1051C12N9/1217C12N9/16C12N9/88C12N9/90C12N9/92C12P19/32C12Y202/01001C12Y202/01002C12Y204/01001C12Y207/02001C12Y301/03011C12Y401/01022C12Y501/03001C12Y503/01001C12Y503/01006C12Y503/01009C12Y504/02002
Inventor 游淳魏欣蕾
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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