JPS62112049A - Method of measuring alcohol content in water liquation organic system - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for determining alcohol concentration in water-immiscible organic systems.

The use of alcohol oxidase test probes to measure lower chain length alcohol concentrations is known to those skilled in the art. Such enzyme probes are useful for measuring alcohol levels in biological fluids such as blood and the like.

However, the ability of such enzyme probes to measure alcohol content in organic systems is limited by the fact that the structure of the enzymes is substantially altered in non-aqueous systems and thus their characteristic catalytic activity v1. This is severely limited by the inability to expose live bamboo.

There is currently a need for a simple, rapid, and accurate method for measuring alcohol content ω in organic systems. Such analytical methods are particularly desired for the determination of alcohol type S in gasohol (ie, a blend of gasoline and ethyl alcohol).

It is therefore an object of the present invention to provide an analytically accurate method for determining the amount of alcohol present in water-immiscible systems.

In accordance with the present invention, it has been found that the alcohol content of substantially water-immiscible organic systems can be accurately determined in a simple and rapid manner. This method according to the present invention involves preparing an organic sample containing alcohol at a concentration of less than about 0.01% (weight/volume) using a dissolved oxygen electrode having alcohol oxidase immobilized therein.
This method involves contacting an enzyme probe consisting of an alcohol oxidase (oxygen electron) and then measuring changes in oxygen concentration in an immobilized alcohol oxidase enzyme layer.

In the present invention, the alcohol to be measured is a linear C1-C4 alcohol, most typically methanol or J
: and ethanol.

For purposes of this invention, the term substantially water-immiscible organic system is defined as any liquid organic material that has a solubility in water of less than about 5% (weight/volume). Examples of such substantially water-immiscible systems include chloroform, ethers, alcohol esters of aliphatic acids, hydrocarbons, and the like. A typical substantially water-immiscible organic material that can contain alcohol is the gasoline listed above. Gasoline is typically refined naphtha obtained by processes such as distillation of crude oil and stripping or aggregation of natural gas.

In the present invention, a ChelliCal Comp
any, st.

Any alcohol oxidase available from commercial sources such as Louis V. Louis, Hissouri) can be used.

Alternatively, alcohol oxidase can be obtained from Pichia pastoris with TI
You can also build J. To manufacture this solution, @
The aqueous fluid containing the cell suspension is homogenized to produce a homogenate. Removal of suspended solids from this homogenate produces a 111 solution containing soluble alcohol oxidase. Crystalline forms of the enzyme can be obtained by methods of ultrafiltration and preferably by dialysis. Dialysis is from 0.05M
Approximately 0. Recovery range with a molar ionic strength in the range of OIM Alcohol in the solution The crude solution produced by homogenization of an aqueous fluid with a cell concentration effective to crystallize the oxidase is on the enzyme side of the membrane. The alcohol oxidase is then dialyzed against the dialysis solvent through a membrane that is impermeable to the alcohol oxidase but permeable to the water in the dialysis solvent and to the slowing agent molecules if present, thus freeing the alcohol oxidase from the crystals. and then separating and collecting the resulting crystalline alcohol oxidase from the dialysis solvent. Such an enzyme isolation method is shown in Example 1.

The enzyme thus produced has the following characteristics: It has 8 subunits with an estimated molecular weight of 78,000 per subunit as determined by SDS gel electrophoresis. This enzyme is a flavoprotein with FAD (flavin adenine dinucleotide) coenzyme. The apparent Michaelis coefficient (Km) for methanol is approximately 0.7. This enzyme is also characterized by its reaction with various alcohols.

The alcohol oxidase of the present invention catalyzes the following reaction: PCI-10f-1+o −+RCHO+H202(
where R is hydrogen or lower alkyl, generally H-1CH
-1OH-CH,2- and CI-1(CH) --
(selected from the group consisting of).

The alcohols catalyzed by yeast alcohol oxidase include propargyl alcohol, allyl alcohol, and formaldehyde hydrates.

During this reaction, oxygen is consumed and aldehydes and hydrogen peroxide are produced, so alcohol oxidase can be used for one-time determination of short-chain alcohols (RCH20H) in fluid samples under conditions compatible with enzyme activity. Can be used. More particularly, alcohol oxidase can be used to measure the concentration of lower alcohols such as methanol or ethanol in gasohol.

A particularly preferred method for measuring such Artur enrichment by enzymatic methods is to immobilize alcohol oxidase on top of a dissolved oxygen electrode.

In such a configuration, this analytical method can be considered a reagent-free method, since the fluorophore can be reused many times without the use of any added chemical reagents. A few such polarographic dissolved oxygen electrodes are commercially available and are suitable for use with alcohol oxidase enzymes. for example,
A Clark dissolved oxygen electrode can be used.

Based on the principles of chemical batteries (combustible batteries), another DO
Probes can also be used. Additionally, one product for the catalytic oxidation of alcohol is an electrochemical probe which has an immobilized alcohol oxidase on its sensing region.

The alcohol oxidase can be immobilized on the top of the electrode by any suitable method. For example, enzymes can be mixed with suitable supporting materials to form a paste;
This is carried out as a thin film by a membrane (typically in hydrated form) that is permeable to the compound whose concentration is to be measured but impermeable to the enzyme itself. It can be placed on top of the electrode. - As an example, the support material may be manufactured by Pharmacia Fine Chemical Company (Phari
acia Fine Chemicals, Sweden
Hydrated DEAE, a polysaccharide ion exchange resin from en)
Sephadex (Seladex). A suitable membrane for ethanol measurement is a cellulose acetate-1 film, through which the mobility of ethanol is sufficient. Of course, the enzyme can also be covalently bound to a suitable galvanic membrane, or physically incorporated into a suitable small composite film. However, the enzyme must be present in the aqueous phase so that it can be immobilized and partition the alcohol in the water-immiscible organic solvent into the aqueous enzyme phase and participate in the catalytic oxidation of 5% of the alcohol. This hydration environment is typically provided either by a hydrated shell, such as cellulose acetate, or by a hydrated immobilizer, such as hydrated DEAE Sephadex.

Conditions for the fluid that are compatible with enzymatic activity include that the fluid contains alcohol at a concentration of less than 0.01% (ffl!fi/volume ffi), and that the temperature of the fluid sample is between about 25°C and 45°C. preferably about 25°C.

In order to obtain an alcohol concentration of less than o, oi% (wt/vol) in the system, 1
A dilution of several powers of 0 is required.

For example, in the case of gasohol, where the ethanol content is typically about 10% (by volume), the concentration of ethanol is 0.01% by diluting it at least 103 times with gasoline. (weight/capacity).

The concentration is 0.01% (weight/volume).
This is a practical limit rather than an arbitrary limit, as alcohol oxidase probes can be too sensitive to be accurate analyzers for the analyte (alcohol) above the i/volume level. be.

Preferably, a series of known temperatures of the alcohol to be analyzed are used to create the evaluation curve. To measure the alcohol concentration, the sample electrode is immersed in the thus prepared fluid to be tested (ie, alcohol in a water-immiscible solvent). The substrate alcohol diffuses across the membrane and reacts in the presence of oxygen to form aldehyde products and hydrogen peroxide. This reaction can be tracked by looking at the absolute change or rate of change in the oxygen concentration of the enzyme layer. Since this catalytic reaction is stoichiometric, alcohol (RCI-
1201-1> can be determined from the absolute change or rate of change in the M elementary concentration by techniques known to those skilled in the art.

Eight further illustrate the invention.

Example 1 In a continuous aerated fermentation method, methanol and an aqueous mineral salts medium were added to yeast strain Pichia in a ratio of about 40:60.
Pastoris (Pichia pastoris) NR
Fermenters inoculated with RL Y-114,30 are each charged at a rate such that methanol is the growth limiting factor. This fermenter is a 15001 foam-filled fermenter with approximately 6101 liquid volume, automatic DH, temperature and level control. Stirring is provided by two conventional paddle-type turbines driven at 1000 rpm. The aeration rate is approximately 4 volumes of air per volume of fermented product in the fermenter per minute (
at about 3 s psig, 13 and about 25°C). Anhydrous ammonia is added at a rate to maintain the pH of the fermentation mixture at a pH of about 3.5.

Water mineral? ! l Salt medium is 75% Hl) O per tap water 11
15,86 stable, K5o49.53gMgno-7
1-1207,8y, CaCf -2N20 0.6!
l? j5 and 85% KOI- (2.6 g). The trace mineral solution → - biotin additive is charged separately by means of a methanol stream at the rate of 10 d/11 methanol. This trace mineral solution → - biotin Additives are 780 d of mineral solution, 200 d of methanol, and 20 ml of water.
and 0.032 g of biotin.

@Hanged mineral solution is per solution 11, FeSO4・7HO65
! 7, Zn5O-7H20 209, Mn5O-HO3,Og, CuS0 ・5H○ 6. (1,aH,2So45.
0d and enough deionized water to bring the total volume to 11.

Aqueous mineral salt medium is charged at a rate of 31.51 per hour and methanol at a rate of 211 per hour.

Fermentation is conducted at about 30° C. and about 38 psig pressure;
Reaction time is 11.6 hours.

Pichia pastoris NRRL Y-11430 no 11
! aFlj is performed according to the representative method described above. A portion of the fermentation effluent was removed and adjusted to pH 7 with ammonium hydroxide.
, 5 and then using a 0.61 container.
el KDL) in continuous operation at 30 DEG C. with belt combination No. 3 and a flow rate of 20-30 d/h. Beads in the mill are 0.3-0
．． The lead-containing glass beads have a diameter of 5 Irun. The resulting homogenate was heated to 5°C and 20,000 ml.
Centrifuge for 30 minutes at
II/d. In this way, it's gross ¥'J! l?
Produce Ffi homogenate.

Six test stones, each containing 130 parts of the supernatant, were placed in a cellulose acetate dialysis bag and dialyzed against 81 liters of distilled water at 5°C. After 4 days, the aqueous phase of the pouch is separated by decantation. The solids remaining in the bag consist of two types of solids. Carefully remove and discard the thin white layer on top. The solid at the bottom is brown-yellow and is crystalline alcohol oxidase.

Add a portion of this crystalline alcohol oxidase to distilled water (
(approximately 10 times the volume of the solid material) and dye as described below
Analyze by peroxidase method.

94EIl/7 activity. The specific activity of this alcohol oxidase is 10.41 EU/protein η.

A sample of solid alcohol oxidase was detected by SDS gel electrophoresis and showed a homogeneous and pure enzyme. Comparing this mobility with the electrophoretic mobility of a protein with a known molecular weight, it is approximately 72.000±30
It was found to have a partial unit molecular weight of 0.00 (estimated). Thus, Vitia Bath 1 ~ Pure from the squirrel/:
i: M m alcohol oxidase is produced.

Example 2 Alcohol oxidase/dissolved oxygen pro[]-buoxidase was prepared as follows to obtain dissolved oxygen sovereignty [Beckman Model 777]
A concentrated solution of alcohol oxidase in two bottles (~1001+19/d> and hydrated DE
AEt? Mix Fadex (approximately 1:1 weight pressure),
Teflon (1
'el'1on) applied to the membrane.

A cellulose acetate-1-dialysis membrane with a molecular weight cut-off of 12,000-14,000 is used to retain the alcohol oxidase-DEAE-Sephadex mixture in the electrode compartment. The electrode circuit consists of an analog differential circuit (d
if feren-tiator). The initial rate of separation of dissolved oxygen is directly proportional to the amount of alcohol in the sample.

The electrodes are calibrated using a series of standard ethanol solutions. In each case 0,05 M' aerated at 25 °C
) > Proof strength IJ Ram slack tj'a (DII7, 5 >
3. The internal solution in the electrode chamber containing 1M (bulkS
Place the sample into the OltltiOn'). The peak height versus alcohol concentration evaluation curve is linear up to a final concentration in the internal solution of approximately 0.001% alcohol by volume (10 ppm). The electrode exhibits a rapid response with an analysis time of approximately 15 seconds), 0.211t) II (by volume) and is sensitive down to low ethanol concentrations.

Example 3 Dye-Peroxidase Method Alcohol Agysidase activity is determined using a leuco dye and peroxidase and by determining the amount of 1"202 produced.

Prepare the dye-buffer mixture by mixing 12 d of O-dianisidine solution (1% O-dianisidine in water > 0.1 d with aerated 0,1 M sodium phosphate buffer (DH 7,5). The assay mixture consisted of 2.5 liters of dye-buffer mixture, 50 µl of methanol and 10 µl of peroxidase solution (1 mg of horseradish peroxidase-Sigma, type II).
and alcohol oxidase solution at 25 μC. The assay compound is kept at 25°C in a 4 x 1 x 1 cm cubera 1- and the increase in absorbance by the dye at 460 nm is recorded for 2-4 minutes. Enzyme activity is calculated using the following formula: Activity (μmol/min/d or Koki/me) = coefficient based on the standard curve prepared using H2O2, and ΔA is the experimental period. ).

Example 4 Standard aqueous solution of alcohol oxidase (~100 μ/w) in the presence of gasoline
Dilute with 0x water.

The resulting ˜10 μ/− alcohol oxidase-containing solution is mixed with a volume of gasoline sufficient to saturate the aqueous phase with hydrocarbons and then incubated at room temperature in an enzyme activity assay lock.

In this way, alcohol oxidase-containing solution 0
．． Enzyme activity was assayed for 5Id and then gasoline 2
The alcohol oxidase activity of this sample was evaluated over several hours.

The enzyme units ([0)/d measured by the dye-peroxidase method are shown in the following table.

Elapsed time [0/d 5 minutes 125 10 minutes 140 30 minutes 133 1 hour 151 2 hours 140 3 hours 129 4 hours 129 The results of this example show that the enzyme alcohol oxidase was converted to a relatively low concentration of hydrophobic hydrocarbons such as gasoline. The results show that enzyme activity is not significantly affected by exposure over an extended period of time.

Example 5 A chemical cell-type dissolved oxygen probe is made using silver wire as the anode and aluminum as the cathode. The top of the anode was coated with a fluorocarbon film and then alcohol in a DEAE-Sephadex gel.
A layer of oxidase is applied and finally a layer of cellulose acetate membrane as described in Example 2 is applied.

The probe is then placed in a stirred solvent 40d of either water or cyclohexane contained in a small open beaker.

A fixed amount of 0.1% (vol/vol) or 1.0% (vol/vol) ethanol is added to this solvent and the relative decrease in dissolved oxygen signal (ΔH) is recorded.

The results obtained are shown in the table below.

Experiment Addition number of 1.0% ethanol in water solvent
(μj ΔH1101,6 2508,7 310017,4 Addition of 0.1% ethanol in cyclohexane (μm) Not only is it practical for measuring the concentration of ethanol in water, but the sensitivity of the probe to ethanol was also measured in the case of an aqueous solvent (
This has been proven to be approximately 30 times larger than in Experiments 1 to 3).

Claims (10)

[Claims] (1) A method for determining the concentration of C_1-C_4 alcohols in a substantially water-immiscible organic system, the method comprising: (a) containing C_1-C_4 alcohols at a concentration of less than about 0.01% (weight/volume); (b) contacting a substantially water-immiscible organic sample with an enzyme probe comprising an aqueous layer of immobilized alcohol oxidase on a dissolved oxygen electrode; A method comprising the steps of: determining alcohol concentration in a substantially water-immiscible organic system by measuring a change in concentration. (2) the electrode has a sensing region for measuring dissolved oxygen content, and the alcohol oxidase is immobilized on the electrode by a layer of hydrated support material bound to the sensing region of the electrode; provides an alcohol oxidase-containing aqueous phase so that the alcohol in the water-immiscible organic system can be partitioned into this aqueous phase and can be oxidized in the aqueous phase by oxygen under catalysis by the alcohol oxidase. Method in section 1. 3. The method of claim 1 or claim 2, wherein the organic system has a solubility in water of less than about 5% (weight/volume). (4) The method according to any one of claims 1 to 3, wherein the alcohol is methanol. (5) The method according to any one of claims 1 to 3, wherein the alcohol is ethanol. (6) The method according to any one of claims 1 to 5, wherein the organic system is cyclohexane. (7) The method according to any one of claims 1 to 5, wherein the organic system is gasoline. (8) The method according to any one of claims 1 to 7, wherein the alcohol oxidase is derived from Pichia pastoris. (9) A probe for measuring alcohol concentration in substantially water-immiscible organic systems, characterized in that it comprises an aqueous layer of immobilized alcohol oxidase on a dissolved oxygen electrode. (10) the electrode has a sensing region for measuring dissolved oxygen content, and alcohol oxidase is immobilized on the electrode by a layer of hydrated support material bound to the sensing region of the electrode; The support material layer provides an aqueous phase containing alcohol oxidase so that the alcohol in the water-miscible organic system can be partitioned into the aqueous phase and the alcohol can be oxidized in the aqueous phase by oxygen under catalysis by the alcohol oxidase. Claim No. 9 has become
term probe.