JPS6043399A - Measurement of amylase activity - Google Patents

Abstract

PURPOSE:To measure directly amylase activity from a test specimen containing amylase, by converting glucose and/or maltose to be an obstacle for measuring directly amylase activity into glucono-delta-lactone, etc. in the test specimen. CONSTITUTION:Maltose and/or glucose already existing in a test specimen is converted with an alkalophilic glucose dehydrogenase and/or maltose dehydrogenase, showing activity in a basic range, slightly showing activity approximately in a neutral range, into glucono-delta-lactone and/or glucose-glucono-delta-lactone in an alkalie range. The treated substance is then restored to a neutral pH, and amylase activity is directly measured by using an excess amount of one or more selected from starch, dextrin, etc. or its derivative as a substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring the activity of α-amylase, β-amylase or glucoamylase.

More specifically, the present invention relates to a method for directly measuring amylase activity such as α-amylase present in samples such as saliva, blood, and plants.

Conventionally, in order to measure the activity of α-amylase, etc. present in samples such as saliva and blood derived from living organisms, the maltose and glucose already present in the sample are first subjected to dialysis treatment or adsorption treatment with a resin. The alpha-amylase activity of the sample was measured by either removing the sample or blindly testing the sample in advance. However, the dialysis treatment of the specimen is complicated and has the disadvantage that the specimen is diluted, and in the adsorption treatment with a resin, although glucose is adsorbed and separated, α-amylase itself may also be adsorbed. Both are good.

In addition, the method of blind testing the specimen was not preferred because it required double effort and also resulted in the inability to perform measurements when the amount of specimen was small.

As a result of intensive research into a method for directly measuring amylase activity from amylase-containing samples, the present inventors found that glucose and/or maltose, which is an obstacle to directly measuring amylase activity, was detected in the sample by gluconose δ. -lactone and /-f*-glucose-gluconose δ-lactone.

The present invention involves adding glucose dehydrogenase and/or maltose dehydrogenase and NAr) + and/or NA, DP'' to a sample containing α-amylase, β-amylase or glucoamylase in an alkaline range, converting glucose and/or Matahamaltose present in the sample into glucono-δ-lactone and/or glucose-glucono-δ-lactone, then returning the pi of the sample to the neutral range and adding an excess amount of amylase reaction substrate; This is an amylase activity measuring method characterized by measuring amylase activity.

The present invention is characterized by alkalophilic glucose dehydrogenase and/or glucose dehydrogenase that is active in an alkaline range and exhibits almost no activity near neutrality in maltose and/or glucose already present in a sample. Maltose is converted to glucono-δ-lactone and/or tahaganglucose-glucono-δ-lactone by maltose dehydrogenase, the pH is returned to neutral, and excess starch, dextrin, amylose, amylopectin, oligosaccharide or their derivatives are removed. This is a method for measuring amylase activity using one or more selected substrates.

According to the amylase activity measurement method of the present invention, since the maltose and glucose already present in the sample are consumed in advance, glucose is directly generated from the amylase substrate added as is in the same sample. Amylase activity can often be measured accurately because glucose groups can be measured as glucose degraded by amylase.

Method for measuring amylase activity of the present invention α-amylase activity in saliva, blood, Swiss fluid, urine, etc., glucoamylase activity in blood, β-amylase activity in plant tissues, and other α-amylase activities
- Used for measuring samples containing amylase, β-amylase or glucoamylase.

Usually, these samples already contain signs of maltose,
Since glucose is constantly present, if the activity of each amylase is measured by direct glucose production reaction, the activity of each amylase will be measured by adding trace amounts of maltose and/or glucose, making it impossible to obtain an accurate titer.

In the present invention, glucose and/or maltose are converted into glucono-δ-lactone and/or glucose-glucono-δ-lactone in advance, so that glucose and/or maltose are converted into glucono-δ-lactone and/or glucose-glucono-δ-lactone. Instead, the amount of glucose produced is accurately measured as amylase activity.

Glucose dehydrogenase and maltose dehydrogenase used in the present invention are in an alkaline range, for example, pH=9 to 1.
It exhibits activity when it is 1 to 2, and shows no activity when it is neutral. Therefore, if the sample is returned to neutrality after consuming glucose and/or maltose in an alkaline region using glucose dehydrogenase and/or maltose dehydrogenase, both enzymes will produce no amount of glucose. It has no effect.

As the glucose dehydrogenase and/or maltose dehydrogenase used in the present invention, a wide variety of conventionally known enzymes can be used. Preferably, glucose dehydrogenase and/or maltose dehydrogenase produced by an alkaline bacterium of the genus Corynebacterium are used.

Although this enzyme is a single enzyme, it can act on glucose and maltose simultaneously and convert them into glucono-δ-lactone and glucose-glucono-δ-lactone.

In the present invention, first, pi-T = 1 F is applied to the specimen.
About 100% of a buffer solution is added, and it is preferable to add glucose dehydrogenase and/or maltose dehydrogenase and NAD+ and/or NAI)P'' to this buffer solution.

NAD+ and/or NAT)P+ acts as a coenzyme for glucose dehydrogenase and/or maltose dehydrogenase, oxidizing glucose and/or maltose and converting it to NAT)H and/or NAT')PIT.

To consume glucose and/or maltose in the sample, the sample is heated to about 25° C., and the reaction continues while there is a change in absorbance at 64° C., and is stopped when the change ends.

After all the glucose and maltose in the sample has been changed to glucono-δ-lactone and glucose-glucono-δ-lactone, the pH of the sample is
Returns lA α-amylase, β-amylase to the neutral range,
Alternatively, glucoamylase activity can be measured.

As a measurement thread for measuring the activity of α-amylase, maltose produced from the substrate of glucan or its conductor added after consumption of glucose and/or maltose is converted to glucose, and then glucose is fixed at a constant level. Any measuring thread will suffice. For example, such systems include glucan or its t4 conductor and maltase, hexokinase, glucose 6-phosphate dehydrogenase and A, TT' and NAI')+ or NAT)P4 total addition i~ to produce NAT)T( Alternatively, there is a method of measuring amylase activity by the amount of NADP)T.

Also, after consuming glucose and maltose, maltase, mutarotase, glucose oxotase,
A method of measuring amylase activity by adding peroxidase, 4-aminoantipyrine, and phenol and measuring the amount of guinone color, and others (converting maltose produced by the action of amylase to glucose (~, converting sonoglucose to glucose) A chair may be used as long as it is a stable system.

Furthermore, the measurement of β-amylase activity can be carried out in the same manner as the measurement of α-amylase activity. Glucoamylase can be measured in exactly the same way as α-amylase, except that maltase is not required.

In this way, the present invention enables direct measurement of amylase activity in the same sample by consuming maltose and glucose in the sample in advance in the measurement of amylase activity. It provides an extremely suitable method for automatic analysis.

Next, test examples and examples of the present invention will be shown.

Test Example 6 Stability of saliva and pancreatic juice amylase at pH 10 at 67°C. Dilute saliva and pancreatic juice amylase to approximately 6 u/me with 1 part 1 mM glycine buffer (p110) and incubate with inkinipation 1 in a water bath at 37°C.
, 2. Activity was measured every hour.

The results are shown in Table 1 below, but as is clear from this table, even if α-amylase is temporarily under the condition of pH=10, ? It can be seen that there are significant changes in the activity of 4 and X7.

Table 1 Stability of amylase at pH 1o at 37°C 0
5.86 5.67 10 5.57 5.85 20 6.01 5.86 30 6.35 5.72 60 6, nl 5.91 Example NAD" 3.OnM Glucose dehydrogenase 411/g or more 001M glycine-KO buffer containing (pH
10) Put /m/ into 4 tubes, and put 5 into each 4 tubes.
Prepare 4 parts of 7 μt of human saliva containing mM glucose adjusted to different concentrations, add these, incubate at 25°C for 10 minutes, and add 3400 μt each.
Measurement 1 with absorbance of 300 mM NaC! , 30mMfC1
200 mM PTPES slow solution containing pT+ 7
) 0.15-1 and 7A/tase 1000o/mf! 20111 hexokinase 1 [100 u/mf, 5111 glucose-6-phospho-dehydrogenase 1000 u/m
e 5μt ATP 72mM 15μt Maltopentaose 1 [1oJrrf//me 1
01tL was added, and the increase in absorbance at 1.340 nm was followed for about 2 minutes to measure 1-1α-amylase activity.

The increase in absorbance for 1 minute for the 4 types of samples is A = 0.26
4 B = 0.2280 = [+,! 112D
= 0.240.

Calculating α-amylase activity using the following formula:
A near, 36 B = 6.36 C = 8.70 respectively
T) = 6.69.

(International units) △E = NAT) Increase in absorbance due to increase in H 6.2
= Extinction coefficient of 1mM of NADH 1.21 = Total reaction volume 0007 - Saliva - where α-amylase Iu (International Units) 1 at 25°C
It refers to the activity of producing 1 μmole of maltose per minute.

Agent Patent Attorney 1) Parent Male (11) Continued from page 1 0 Inventor Isamu Takakawahara Yamato Higashi, Kawanishi City Inventor Shinji Iwasaki 612 Hino Minami Toyoda: 5-7-13 12-21 -17

Claims (1)

[Claims] For samples containing α-amylase, β-amylase or glucoamylase, in an alkaline range, glucose dehydrogenase and/or maltose dehydrogenase and NAT) +
and/or NAr) P+; 7. Glucose and/or maltose present in the sample are converted into glucono-δ-lactone and/or glucose-glucono-
A method for measuring amylase activity, which comprises converting the sample into δ-lactone, returning the pH of the sample to a neutral range, adding a 1-1 excess amount of amylase reaction substrate, and measuring amylase activity.