CN102732421B - Detection apparatus for rapid calculation of generation amount of gamma-aminobutyric acid in biotransformation - Google Patents

Detection apparatus for rapid calculation of generation amount of gamma-aminobutyric acid in biotransformation Download PDF

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CN102732421B
CN102732421B CN 201210172827 CN201210172827A CN102732421B CN 102732421 B CN102732421 B CN 102732421B CN 201210172827 CN201210172827 CN 201210172827 CN 201210172827 A CN201210172827 A CN 201210172827A CN 102732421 B CN102732421 B CN 102732421B
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gaba
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CN102732421A (en
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江波
缪铭
张天萌
张涛
沐万孟
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Jiangnan University
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Abstract

The invention provides a detection apparatus for rapid calculation of generation amount of gamma-aminobutyric acid (GABA) in biotransformation, which belongs to the technical field of chemical apparatuses. The main body of the apparatus comprises 1) a pH induction device, 2) a digital transformation device, 3) a liquid dropping flask, 4) a controller and 5) a reactor. The digital transformation device can record the volume of an added acid, the controller can set two parameters, i.e., the pH value of a reaction and the concentration of the added acid, and the generation amount of GABA can be calculated by a program based on the volume of the added acid and is directly displayed on the controller. Protons are irreversibly consumed in a glutamic acid decarboxylase catalytic reaction; and it is proportional relationship between the number of consumed protons and GABA that the apparatus is used. The apparatus provided in the invention overcomes the defects of a long detection period, high cost and complicated operation in conventional detection of GABA in biotransformation. When the apparatus provided in the invention is used for detection of GABA in biotransformation, the advantages of no need for treatment of samples, simple and practicable operation, a short detection period and low cost are obtained.

Description

Calculate the proofing unit of γ-An Jidingsuan growing amount in a kind of bioconversion reaction fast
Technical field
Calculate the proofing unit of γ-An Jidingsuan growing amount in a kind of bioconversion reaction fast, belong to the chemical apparatus technical field.
Background technology
γ-An Jidingsuan (GABA, Gamma-aminobutyric acid) is that a kind of naturally occurring non-protein groups becomes amino acid, and GABA is a kind of inhibitory nerve mediator in mammalian body, the inhibitory nerve signal of mediation more than 40%.GABA has and improves the brain vigor, brings high blood pressure down, stable spirit, improves function such as liver kidney function, thereby it is with a wide range of applications in functional food.
L-Glutamic decarboxylase (GAD, Glutamate decarboxylase) is a kind of pyridoxal phosphate (PLP) fermentoid, and it is the important biomolecule enzyme in the GABA biosynthetic process.GAD extensively is present in microorganism, plant tissue in senior mammalian body, but its amount in cell is very little.
In the GABA biosynthetic process, mensuration to GABA has several different methods and instrument at present, and different measuring methods relative merits difference, thereby be necessary according to practical situation, by experiment research set up a kind of targetedly fast, reliably, measuring method easily, existing detection method report has following several:
Double-enzyme method: use the two enzyme reagent kits of two enzyme reagent kit Gabase(, i.e. [α-Tong Wuersuan: GABA, transaminase (E.C.2.6.19)] and [NAD (P): succsinic acid hemiacetal, oxydo-reductase (E.C.1.2.1.16)]), reaction principle is: α-Tong Wuersuan+GABA → succsinic acid hemiacetal+L-L-glutamic acid; NADP ++ succsinic acid hemiacetal → NADPH+H ++ succsinic acid.This method is carried out quantitative analysis to GABA, and by the variable quantity of fluoroscopic examination NADH or NAD, required time is short, and is highly sensitive, reaches 1 * 10 -11Mol/L is usually used in the trace analysis to contained GABA in the nervous tissue in medical research.But the cost of determination of this detection method is relatively more expensive, is unsuitable for industrial application.
Amino acidanalyser assay method or high performance liquid phase assay method: adopt analytical instrument quantitative assay GABA.These class methods be widely used at present and accuracy very high, but analysis cost is higher, needs that sometimes sample is compared loaded down with trivial details pre-treatment or derivatize and prepares since the accuracy height usually as a kind of with reference to the comparative standard method.
Ply of paper is analysed or thin layer chromatography scanning: these class methods mainly utilize amino acid and ninhydrin reaction to generate coloring matter, carry out separation detection.These class methods are both economical fast, but sensitivity is lower, usually are used as the qualitative detection of GABA.
Colorimetry: ultimate principle is to utilize the colour developing difference of biological respinse front and back Berthelot colorimetry to measure enzyme work.The Berthelot reaction utilizes phenol, clorox and free ammonia reaction, the developing sensitivity height, be commonly used to ammonia in the mensuration system and the content of its esters, amino acid has free amine group, in the Berthelot reaction, a provisioning response is arranged, a rule is, the reaction sensitivity of omega-amino acid is very high, and the a-amino acid response is low.For L-L-glutamic acid and the GABA in the system, they are response difference great disparity in the Berthelot reaction, be that the response of enzyme reaction product GABA is very high and the response of substrate L-L-glutamic acid is low, so enzyme reaction can change and carry out enzyme activity determination when taking place according to the system colored intensity.Johnson and Tsushida etc. have reported that the enzyme that utilizes colorimetry to measure L-Glutamic decarboxylase in cowpea and the tealeaves respectively is alive.The required sample size of these class methods is few, and fast, but accuracy is not high, and reaction system is little, is unsuitable for industrial applications.
In addition, the characteristics of reacting according to L-Glutamic decarboxylase: GAD belongs to lyase, reacts for acting on L-L-glutamic acid to produce CO 2And GABA, CO in the reaction process 2Constantly effusion, so GAD decarboxylic reaction is a non-reversible process, constantly consumes proton in the reaction.By calculating the CO that discharges in the reaction 2Amount is calculated enzyme and lived report is also arranged: adopt electrochemical appliance, principle is for detecting the CO that quantitatively discharges in the decarboxylic reaction 2Ling Da Renhe Wu state fine jade utilizes microcomputer ion analyser and CO 2Gas sensing electrodes is measured the L-Glutamic decarboxylase vigor, can also calculate the growing amount of GABA accordingly, and this element method data appliance computer record is very convenient, easy handling.But facility investment is big, and is strict to reaction system.
Anthracometry.The enzyme that adopts the radiation quantitative method to measure GAD is lived, and principle is to have radioelement 14The L-L-glutamic acid of C is substrate, can quantitatively discharge in the GAD decarboxylic reaction 14CO 2, detect 14CO 2, then according to discharging in the unit time in the reaction 14CO 2Determine that the GAD enzyme is alive, can also calculate the growing amount of GABA accordingly.This method accuracy is high, but cost of determination is higher, and complex operation is to the equipment requirements strictness.
Summary of the invention
The technical problem to be solved in the present invention is: at the time-consuming length of present GABA measuring method, and the cost height, complicated operation, defectives such as small scale have been invented a kind of being specifically designed to and have been measured the proofing unit that generates GABA content in the bio-transformation.
Technical scheme of the present invention: industrial biological transforms to be produced in the GABA process, live in order to take full advantage of enzyme, generally answer the portion-wise addition substrate, the irreversible consumption proton of GAD decarboxylation, need online adjusting pH, draw the proportionlity that consumes proton number and γ-An Jidingsuan growing amount by experiment, but invented the new device of rapid detection GABA in a kind of biotransformation accordingly.This proofing unit main body comprises: 1) pH inductance gauge; 2) digital conversion instrument; 3) Liquid dropping bottle; 4) controller; 5) reactor.The interior placing response liquid of reactor (5), in pH inductance gauge (1) insertion reaction device (5) the internal reaction liquid, pH inductance gauge (1) is the online pH value that is detecting reaction solution all the time, pH inductance gauge (1) and controller (4) join, and controller (4) adds acid according to setting pH value control Liquid dropping bottle (3) to reaction solution; PH inductance gauge (1) joins with digital conversion instrument (2) again, numeral conversion instrument (2) record adds the volume of acid, the volume that numeral conversion instrument (2) will add acid amount is converted into electrical signal and passes to controller (4), and controller (4) can directly demonstrate the γ-An Jidingsuan growing amount according to computation program then.
The detection operation steps is:
A, set reaction pH(4.6-4.8) value, two parameters of concentration n (0.1-0.5M) of adding hydrochloric acid, add glutamic acid solution, with the wet cell that adds L-Glutamic decarboxylase solution or produce L-Glutamic decarboxylase in reactor, water is supplied reaction volume between 100-500mL; The L-glutamic acid total amount of adding between 5-100mmol, the L-Glutamic decarboxylase solution of interpolation or the wet cell amount (total enzyme is lived be 100-2000U) of producing L-Glutamic decarboxylase;
Should transfer to set(ting)value before B, the reaction system reaction;
C, pH inductance gauge be the pH of on-line monitoring reaction solution all the time, and is that the HCl of n transfers to the suitableeest set(ting)value with concentration, and record accumulative total is added the volume V of hydrochloric acid;
D, using formula W=(n * V * k * 103.1) calculate γ-An Jidingsuan growing amount W;
The unit of n is mol/L, and the unit of V is mL, and the unit of W is mg;
kBe defined as: in the L-Glutamic decarboxylase decarboxylic reaction, the ratio of γ-An Jidingsuan growing amount and proton consumption number is an eigenwert of L-Glutamic decarboxylase.Mainly be that this device of application and high-efficient liquid phase technique mensuration GABA contrast obtains, numerical value is 0.5.
Beneficial effect of the present invention: the γ-An Jidingsuan growing amount need not sample is handled operation in this device mensuration biotransformation, and operation is simple, weak point consuming time, and cost is low.
Description of drawings
Fig. 1 structural representation of the present invention.
Embodiment
Embodiment 1: operation: a. sets the parameter of controller: pH4.6 as follows, adds the 0.1M hydrochloric acid soln; B. add the L-glutamic acid of 20mmol to reactor, add deionized water to reaction volume 100mL; C. transfer to pH4.6; D. add 10mL L-Glutamic decarboxylase solution (enzyme 100U alive) and in reactor, begin reaction.Behind the 10min, the volume that record accumulative total is added hydrochloric acid is 79.5mL, using formula W=(n * V * k* 103.1) calculate generation GABA amount and be 409.8mg, it is 417mg that the method for high performance liquid phase detects GABA result, and specific inaccuracy is 1.8%.
Embodiment 2: operation: a. sets the parameter of controller: pH4.8 as follows, adds the 0.5M hydrochloric acid soln; B. add the L-glutamic acid of 5mmol to reactor, add deionized water to 200mL; C. transfer to pH4.8; D. add 20g wet cell (enzyme 500U alive) and in reactor, begin reaction.Behind the 10min, the volume that record accumulative total is added hydrochloric acid is 57.3mL, using formula W=(n * V * k* 103.1) calculate generation GABA amount and be 1477mg, it is 1489.4mg that the method for high performance liquid phase detects GABA result, and specific inaccuracy is 0.9%.
Embodiment 3: operation: a. sets the parameter of controller: pH4.7 as follows, adds the 0.25M hydrochloric acid soln; B. add the L-glutamic acid of 100mmol to reactor, add deionized water to 500mL; C. transfer to pH4.7; D. add 80g wet cell (enzyme 2000U alive) and in reactor, begin reaction.Behind the 10min, the volume that record accumulative total is added hydrochloric acid is 331.3mL, using formula W=(n * V * k* 103.1) calculate generation GABA amount and be 4.2696g, it is 4.32g that the method for high performance liquid phase detects GABA result, and specific inaccuracy is 1.1%.

Claims (2)

1. calculate the proofing unit of γ-An Jidingsuan growing amount in the bioconversion reaction fast, it is characterized in that this proofing unit main body by pH inductance gauge (1), digital conversion instrument (2), Liquid dropping bottle (3), controller (4) and reactor (5) formation; Two parameters of concentration that controller (4) is used for setting pH value in reaction, adds acid, the interior placing response liquid of reactor (5), in pH inductance gauge (1) insertion reaction device (5) the internal reaction liquid, pH inductance gauge (1) is the online pH value that is detecting reaction solution all the time, pH inductance gauge (1) and controller (4) join, and controller (4) adds acid according to setting pH value control Liquid dropping bottle (3) to reaction solution; PH inductance gauge (1) joins with digital conversion instrument (2) again, numeral conversion instrument (2) record adds the volume of acid, the volume that numeral conversion instrument (2) will add acid amount is converted into electrical signal and passes to controller (4), controller (4) can directly draw the γ-An Jidingsuan growing amount according to computation program then, and directly is presented on the controller (4).
2. in bioconversion reaction, calculate the method for γ-An Jidingsuan growing amount with the described proofing unit of claim 1 fast, it is characterized in that carrying out according to the following steps:
A, two parameters of concentration n:0.1-0.5M of setting reaction pH4.6-4.8 value, adding hydrochloric acid, add glutamic acid solution and add L-Glutamic decarboxylase solution or the wet cell of product L-Glutamic decarboxylase in reactor, water is supplied reaction volume between 100-500mL; The L-glutamic acid total amount of adding is between 5-100mmol, and the wet cell amount of the L-Glutamic decarboxylase solution of interpolation or product L-Glutamic decarboxylase is total enzyme 100-2000U alive;
Should transfer to set(ting)value before B, the reaction system reaction;
C, pH inductance gauge be the pH of on-line monitoring reaction solution all the time, and is that the hydrochloric acid of n transfers to the suitableeest set(ting)value with concentration, and record accumulative total is added the volume V of hydrochloric acid;
D, using formula W=(n * V * k * 103.1) calculate γ-An Jidingsuan growing amount W;
The unit of n is mol/L, and the unit of V is mL, and the unit of W is mg;
K is defined as: in the L-Glutamic decarboxylase decarboxylic reaction, the ratio of γ-An Jidingsuan growing amount and proton consumption number is an eigenwert of L-Glutamic decarboxylase, and it is to use this device and high-efficient liquid phase technique mensuration GABA to contrast and obtain, and numerical value is 0.5.
CN 201210172827 2012-05-30 2012-05-30 Detection apparatus for rapid calculation of generation amount of gamma-aminobutyric acid in biotransformation Expired - Fee Related CN102732421B (en)

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