CN108956500A - A kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method - Google Patents
A kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method Download PDFInfo
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- CN108956500A CN108956500A CN201810895330.XA CN201810895330A CN108956500A CN 108956500 A CN108956500 A CN 108956500A CN 201810895330 A CN201810895330 A CN 201810895330A CN 108956500 A CN108956500 A CN 108956500A
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Abstract
The present invention is a kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method.The kit is by 5~50U/L of 5~50U/L of acetyl-CoA-synthetase, 0.5~2.5g/L of adenosine triphyosphate disodium salt and coacetylase, 0.5~2.5g/L of L MALIC ACID, NADH 7.5mmol/L, low temperature 5~500U/L of malic dehydrogenase, 5~500U/L of citrate synthase composition；Detection method are as follows: acetyl-CoA-synthetase, ATP and coacetylase is added in acetic acid sample and is reacted, NAD+, L MALIC ACID and low temperature malic dehydrogenase are sequentially added in above-mentioned reaction solution, citrate synthase is added immediately after mixing, whole process measures the absorbance value of NADH at 340nm, obtains acetate concentration according to formula.Kit of the present invention can be used in 20 DEG C of room temperature environments and under hypersaline environment, easy to operate, and detection acetate concentration accuracy is high, and stability is good.
Description
Technical field
The present invention relates to acetate concentration detection method, specifically a kind of low temperature malic dehydrogenase acetate concentration detection reagent
Box and its detection method.
Background technique
Acetic acid is colourless hygroscopic liquid, is clear crystal after solidification.Although according to the dissociation of acetic acid in aqueous solution
It is a weak acid to ability, but acetic acid has corrosivity, and steam is to eye and the irritant effect of nose.Acetic acid is a kind of simple
Carboxylic acid, be an important chemical reagent.Acetic acid is also used to cellulose acetate and timber required for manufacture cinefilm
With the polyvinyl acetate in adhesive, and many synthetic fibers and fabric.Acetic acid is uniquely may be used in all chemical products
With the coalification chemical product competed with petrochemical industry.Meanwhile acetic acid is widely present in food, fruit juice, papermaking, pharmacy and other industry
In product, it is important one of parameter in production.The method of existing measurement acetic acid content is broadly divided into chemical method.Chemical method
Predominantly acid-base titration, this method is easy to operate, and cost is relatively low, but there are biggish error, detection inaccuracy.
Summary of the invention
To solve defect of the existing technology, the present invention provides one kind
For achieving the above object, the invention adopts the following technical scheme:
A kind of low temperature malic dehydrogenase acetate concentration detection kit, which is characterized in that the kit is auxiliary by acetyl
Enzyme A synzyme, adenosine triphyosphate disodium salt, coacetylase, L MALIC ACID, NADH, low temperature malic dehydrogenase, citric acid
Synthase, pH8Tris-Hcl composition.
Above-mentioned low temperature malic dehydrogenase acetate concentration detection kit, 5~50U/L of acetyl-CoA-synthetase, adenine
5~50U/L of 0.5~2.5g/L of ribonucleoside triphosphote disodium salt and coacetylase, L MALIC ACID 0.5~2.5g/L, NADH 7.5mmol/
L, low temperature 5~500U/L of malic dehydrogenase, 5~500U/L of citrate synthase.
Above-mentioned low temperature malic dehydrogenase acetate concentration detection kit, the low temperature malic dehydrogenase are the micro- life in ocean
The thermophilic salt enzyme of the low temperature extracted in object, specific screening technique are as follows: the collecting sample from deep-sea ooze, seawater, by containing 0.1-
The beef extract-peptone solid medium of 0.2g/L bromocresol green carries out primary dcreening operation, and acid stronger bacterial strain is produced in screening, using containing
The beef extract-peptone solid medium of MTT0.015g/L detects the dynamic property of bacterial strain, carries out programmed screening, selects power
Property stronger bacterial strain survey enzyme activity, colloid bacillus cereus (Bacillus is accurately filtered out by Double Selection
Mucilaginosus) and sticky Serratieae (Serratia marcescens), the as low temperature bacterial strain of high yield MDHs.
Method using above-mentioned low temperature malic dehydrogenase acetate concentration detection kit detection acetate concentration includes following
Step:
(1) acetyl-CoA-synthetase, ATP and coacetylase is added in the acetic acid sample of measurement, is urged in acetyl-CoA-synthetase
Under change effect, CoA converting acetic acid is acetyl coenzyme A, and generates AMP and pyrophosphoric acid simultaneously；
(2) NAD+, L MALIC ACID and malic dehydrogenase (MDH) are sequentially added in the reaction solution of step (1)；
(3) after mixing step 2 reaction solution, it is added immediately citrate synthase, acetyl coenzyme A converts oxaloacetic acid and generates
Citric acid, the oxaloacetic acid in reaction derive from step 2；
(4) it is dense that acetic acid can be obtained according to formula (1) and formula (2) in the whole absorbance value that NADH is measured at 340nm
It spends (mol/ml)；
In formula: △ A340nm/min: the decreasing value of absorbance per minute
VT: reaction total volume, mL
VS: sample volume, mL
Df: extension rate
ε: the NADH molar absorptivity coefficient at 340nm, 6.22/ μm of olcm
B: cuvette optical path, cm.
Acetate concentration measuring method principle of the present invention is as follows:
Under acetyl-CoA-synthetase (ACS) catalytic action, CoA converting acetic acid is acetyl coenzyme A, and is generated simultaneously
AMP and pyrophosphoric acid, under the action of citrate synthase, acetyl coenzyme A converts oxaloacetic acid and generates citric acid, the oxalyl in reaction
Acetic acid derives from the product of L MALIC ACID and NAD+ under the action of EC 1.1.1.39 (L-MDH), the NAD+ in this reaction
It is reduced into NADH, the amount of the NADH of generation depends on the amount of acetic acid, and the absorbance value of NADH can be measured at 340nm.
Compared with prior art, the invention has the benefit that the present invention is for measuring acetate concentration method in fermentation liquid,
Used kit configuration is simple, and raw material sources are extensive, at low cost, Environmental Safety, do not need using toxic organic solvent；It is different
In relevant high temperature kit in the market, kit of the present invention is by low temperature malic dehydrogenase as coenzyme for measuring vinegar
The content of acid can be used in 20 DEG C of room temperature environments, easy to operate.Through actually detected verifying, this method stability is good, measurement
Accuracy and precision it is higher.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment 1
Low temperature malic dehydrogenase acetate concentration detection kit, specific as follows:
Wherein, low temperature malic dehydrogenase is the thermophilic salt enzyme of low temperature extracted in marine microorganism, specific screening technique are as follows:
The collecting sample from deep-sea ooze, seawater, by the beef extract-peptone solid medium of the bromocresol green containing 0.1-0.2g/L into
Row primary dcreening operation, screening are produced acid stronger bacterial strain, are detected using the beef extract-peptone solid medium containing MTT0.015g/L
The dynamic property of bacterial strain carries out programmed screening, selects the stronger bacterial strain of dynamic property to survey enzyme activity, is accurately screened by Double Selection
Colloid bacillus cereus (Bacillus mucilaginosus) and sticky Serratieae (Serratia marcescens) out, i.e.,
For the low temperature bacterial strain of high yield MDHs.
Acetate concentration is measured using mentioned reagent box, specific as follows:
(1) acetyl-CoA-synthetase, ATP (adenosine triphyosphate disodium salt) is added in acetic acid sample to be determined
And coacetylase, under acetyl-CoA-synthetase catalytic action, CoA converting acetic acid be acetyl coenzyme A, and generate simultaneously AMP and
Pyrophosphoric acid；
(2) NAD+, L MALIC ACID and malic dehydrogenase (MDH) are sequentially added in the reaction solution of step (1)；
(3) after mixing step 2 reaction solution, it is added immediately citrate synthase, acetyl coenzyme A converts oxaloacetic acid and generates
Citric acid, the oxaloacetic acid in reaction derive from step (2)；
(4) it is dense that acetic acid can be obtained according to formula (1) and formula (2) in the whole absorbance value that NADH is measured at 340nm
It spends (mol/ml)；
In formula: △ A340nm/min: the decreasing value of absorbance per minute
VT: reaction total volume, m L
VS: sample volume, m L
Df: extension rate
ε: the NADH molar absorptivity coefficient at 340nm, 6.22/ μm of olcm
B: cuvette optical path, cm
N: acetate concentration, mol/ml
U:MDH enzyme activity, U/ml
T: reaction time, min
Wherein, NADH measures mixed liquor are as follows: 0.1mL7.5mmol/L NADH, 0.1m L 0.5g/L L MALIC ACID,
2.8mL0.1mol/L pH8Tris-Hcl buffer and 0.1mL malate dehydrogenase enzyme solution exist in 25 DEG C of monitoring reaction systems
340nm absorbance change can calculate enzyme activity according to the decreasing value of absorbance per minute, then to aoxidize 1 μm of ol's per minute
Thus enzyme amount needed for NADH calculates the content of acetic acid.
It is detection sample with the acetic acid detection kit bought in the market and kit of the present invention according to the above measuring method
This, carries out stability trace detection respectively, obtain the sample stability with high salt of Self-made reagent and contrast agents, 37 DEG C and 20 DEG C plus
Fast stability, long-term stable experiment result (being shown in Table 1).
Table 1 and contrast agents performance for stability index results of comparison
The present invention is using concentration of component as acetyl-CoA-synthetase 5U/L as can be seen from Table 1, adenosine triphyosphate two
Sodium salt 0.5g/L, coacetylase 5U/L, L MALIC ACID 0.5g/L, NADH 7.5mmol/L, low temperature malic dehydrogenase 100U/L, lemon
When lemon acid synthase 100U/L, pH8Tris-Hcl 0.1mol/L, compared to control group, high stability, sensitivity and accuracy
It is all relatively high；And can use at a lower temperature, detect the acetate concentration in sample with high salt.
Embodiment 2
Low temperature malic dehydrogenase acetate concentration detection kit, specific as follows:
Wherein, low temperature malic dehydrogenase is the thermophilic salt enzyme of low temperature extracted in marine microorganism, specific screening technique are as follows:
The collecting sample from deep-sea ooze, seawater, by the beef extract-peptone solid medium of the bromocresol green containing 0.1-0.2g/L into
Row primary dcreening operation, screening are produced acid stronger bacterial strain, are detected using the beef extract-peptone solid medium containing MTT0.015g/L
The dynamic property of bacterial strain carries out programmed screening, selects the stronger bacterial strain of dynamic property to survey enzyme activity, is accurately screened by Double Selection
Colloid bacillus cereus (Bacillus mucilaginosus) and sticky Serratieae (Serratia marcescens) out, i.e.,
For the low temperature bacterial strain of high yield MDHs.
It is detected according to the method for embodiment 1.
Table 2 and contrast agents performance for stability index results of comparison
The present invention is using concentration of component as acetyl-CoA-synthetase 20U/L, adenosine triphyosphate as can be seen from Table 2
Disodium salt 1.5g/L, coacetylase 20U/L, L MALIC ACID 1.5g/L, NADH 7.5mmol/L, low temperature malic dehydrogenase 200U/
When L, citrate synthase 200U/L, pH8Tris-Hcl 0.1mol/L, compared to control group, compared to control group, stability is
Highest in three groups of examples, sensitivity and accuracy are all with respect to highest；And can use at a lower temperature, it detects in sample with high salt
Acetate concentration.
Embodiment 3
Low temperature malic dehydrogenase acetate concentration detection kit, specific as follows:
Wherein, low temperature malic dehydrogenase is the thermophilic salt enzyme of low temperature extracted in marine microorganism, specific screening technique are as follows:
The collecting sample from deep-sea ooze, seawater, by the beef extract-peptone solid medium of the bromocresol green containing 0.1-0.2g/L into
Row primary dcreening operation, screening are produced acid stronger bacterial strain, are detected using the beef extract-peptone solid medium containing MTT0.015g/L
The dynamic property of bacterial strain carries out programmed screening, selects the stronger bacterial strain of dynamic property to survey enzyme activity, is accurately screened by Double Selection
Colloid bacillus cereus (Bacillus mucilaginosus) and sticky Serratieae (Serratia marcescens) out, i.e.,
For the low temperature bacterial strain of high yield MDHs.
It is detected according to the method for embodiment 1.
Table 3 and contrast agents performance for stability index results of comparison
The present invention is using concentration of component as acetyl-CoA-synthetase 50U/L, adenosine triphyosphate as can be seen from Table 3
Disodium salt 2.5g/L, coacetylase 50U/L, L MALIC ACID 2.5g/L, NADH 7.5mmol/L, low temperature malic dehydrogenase 500U/L,
When citrate synthase 500U/L, pH8Tris-Hcl 0.1mol/L, compared to control group, compared to control group, high stability,
Sensitivity and accuracy are all relatively high；And can use at a lower temperature, detect the acetate concentration in sample with high salt.
Acetate concentration can accurately be measured using detection kit of the present invention and measuring method by verification experimental verification.
Claims (4)
1. a kind of low temperature malic dehydrogenase acetate concentration detection kit, which is characterized in that the kit is by acetyl coenzyme A
Synzyme, adenosine triphyosphate disodium salt, coacetylase, L MALIC ACID, NADH, low temperature malic dehydrogenase, citric acid close
Enzyme, pH8Tris-Hcl composition.
2. low temperature malic dehydrogenase acetate concentration detection kit as described in claim 1, which is characterized in that acetylcoenzyme
5~50U/L of A synzyme, 5~50U/L of 0.5~2.5g/L of adenosine triphyosphate disodium salt and coacetylase, L MALIC ACID 0.5
~2.5g/L, NADH 7.5mmol/L, low temperature 5~500U/L of malic dehydrogenase, 5~500U/L of citrate synthase.
3. low temperature malic dehydrogenase acetate concentration detection kit as claimed in claim 1 or 2, which is characterized in that described
Low temperature malic dehydrogenase is the thermophilic salt enzyme of low temperature extracted in marine microorganism, specific screening technique are as follows: from deep-sea ooze, sea
Collecting sample in water carries out primary dcreening operation by the beef extract-peptone solid medium of the bromocresol green containing 0.1-0.2g/L, filters out
Acid stronger bacterial strain is produced, the dynamic property of bacterial strain is detected using the beef extract-peptone solid medium containing MTT0.015g/L,
Programmed screening is carried out, selects the stronger bacterial strain of dynamic property to survey enzyme activity, colloid gemma bar is accurately filtered out by Double Selection
Bacterium (Bacillus mucilaginosus) and sticky Serratieae (Serratia marcescens), as high yield MDHs's
Low temperature bacterial strain.
4. the side of application low temperature malic dehydrogenase acetate concentration detection kit described in claim 1 detection acetate concentration
Method, which is characterized in that the described method comprises the following steps:
(1) acetyl-CoA-synthetase, ATP and coacetylase is added in the acetic acid sample of measurement, is catalyzed and makees in acetyl-CoA-synthetase
Under, CoA converting acetic acid is acetyl coenzyme A, and generates AMP and pyrophosphoric acid simultaneously；
(2) NAD+, L MALIC ACID and malic dehydrogenase (MDH) are sequentially added in the reaction solution of step (1)；
(3) after mixing step (2) reaction solution, it is added immediately citrate synthase, acetyl coenzyme A converts oxaloacetic acid and generates lemon
Lemon is sour, and the oxaloacetic acid in reaction derives from step (2)；
(4) acetate concentration can be obtained according to formula (1) and formula (2) in the whole absorbance value that NADH is measured at 340nm
(mol/ml)；
In formula: △ A340nm/min: the decreasing value of absorbance per minute
VT: reaction total volume, mL
VS: sample volume, mL
Df: extension rate
ε: the NADH molar absorptivity coefficient at 340nm, 6.22/ μm of olcm
B: cuvette optical path, cm.
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| CN201810895330.XA CN108956500B (en) | 2018-08-08 | 2018-08-08 | Low-temperature malate dehydrogenase acetic acid concentration detection kit and detection method thereof |
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| CN201810895330.XA CN108956500B (en) | 2018-08-08 | 2018-08-08 | Low-temperature malate dehydrogenase acetic acid concentration detection kit and detection method thereof |
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