Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase

Definitions

• the present invention relates to a method of determining amylase concentrations, especially the amylase concentrations of amylase-containing wash liquor applied in a machine warewashing process. More in particular, the invention relates to a method of determining amylase concentrations, wherein a starch solution and an iodine solution are applied.
• enzymes particularly amylase enzymes, have been known as effective ingredients of mechanical warewashing compositions for domestic use.
• amylase enzymes have also been disclosed as components in mechanical warewashing compositions, for use in industrial multi-tank or single-tank machines.
• WO-96/16152 discloses a cleaning system for a multi-tank warewashing machine, wherein amylase enzyme and bleach are dosed into different wash tanks or zones of the machine.
• WO-99/34723 discloses a cleaning system for an industrial warewashing machine, having a bleach component and an enzyme component, wherein the enzyme component is introduced into a wash zone or step and the bleach component is introduced into a post-wash rinse zone or step.
• the optical density of the starch-iodine solution containing the specimen is compared against the optical density of the starch-iodine solution without the specimen.
• the difference in optical densities is used as a relative measure of how much amylose (present in the starch) has reacted with the amylase, which is a function of the amylase concentration of the specimen.
• the entire method of US-A-3, 888 , 739, as described above, starting from the time the freeze-dried starch is reconstituted until the amylase concentration is determined, is performed in about 15 minutes. It is an object of the present invention to further shorten the time period for amylase activity determination.
• the present invention provides a method for determining amylase concentrations, comprising the steps of:
• the method of the invention can be carried out in a short time period.
• Reason is that said method has been developed for use in industrial cleaning processes, particularly those applied in multi-tank or single-tank warewashing machines, wherein amylase enzymes are used. In those processes, it is desirable to be able to immediately adjust the enzyme dosage on the basis of the outcome of the amylase activity determination.
• the present invention provides a method for accurately and simply determining the amylase activity within a sufficiently short time period for enabling a proper and timely adjustment of the enzyme dosage if needed.
• the method of the invention is carried out within a time period of 10 minutes.
• the duration of said method is less than 8 minutes, more preferably less than 6 minutes, a time period of less than 4 minutes being most preferred.
• a first type of sample being the 'test' sample, in which a part of specimen is allowed to react with a first portion of buffered starch solution for a specified time period before an aqueous iodine solution is added
• a second type of sample which is used as a reference sample.
• Said second type of sample is prepared by adding the rest of the specimen to a mixture of iodine solution and buffered starch solution, whereby the presence of the iodine solution prevents any reaction to take place between the specimen and the starch solution.
• both the 'test' sample and the reference sample contain equal concentrations of buffered starch solution and specimen.
• the presence of equal amounts of specimen in both types of sample is needed in order to be able to correct for any interference of the optical density measurement caused by the presence of soil and enzyme stabilizing species such as sulfite and other reducing materials.
• the buffered starch solution used in the method of the invention is a starch solution containing a suitable buffer and having a pH in the range of 5-11, preferably 6-8, a pH of around 7 being most preferred.
• An effective buffer for said buffered starch solution is a phosphate buffer, preferably containing a mixture of disodiu hydrogen orthophosphate and monosodium dihydrogen orthophosphate .
• Said buffered starch solution is preferably an aqueous solution.
• the starch solution is desirably an isotropic aqueous solution.
• the buffered starch solution is an aqueous solution containing starch at a concentration of 40-100 mg/liter. This starch is preferably readily soluble in water at room temperature, and will normally produce a definite blue color upon the addition of an aqueous iodine solution thereto.
• steps (a) and (b) thereof can be suitably carried out at a temperature between about 10°C and 60°C, preferably at a temperature of at most 30°C, more preferably at room temperature.
• step (b) of the method of the invention between the specimen and the buffered starch solution is preferably carried out for a time period of 1-5 minutes, more preferably 2-4 minutes, a time period of about 3 minutes being most preferred.
• This reaction time period can be adjusted by selecting the moment of starting step (c) by adding the aqueous iodine solution, since addition of said iodine solution will stop said reaction immediately.
• step (e) of the method of the invention the optical density or remission of both types of samples (expressed as % transmission or % remission) is measured at a wavelength of 300-800 ran, preferably 500-650 nm.
• the light remission is preferably measured using a portable reflectometer .
• step (f) the delta optical density or remission being the difference between the optical density or remission values obtained with the test sample respectively the reference sample, is calculated.
• step (f) the amylase activity of the specimen concerned is calculated by interpolation using the thus-obtained optical density or remission and a calibration curve showing the relationship between delta optical density and amylase activity (for instance, expressed in Maltose Units (MU) /liter) .
• the buffered starch solution is prepared by dissolving 50 mg/liter of starch (ex Merck KGaA, Darmstadt, art. nr.1252) in 200 mM phosphate buffer at room temperature and a pH of 6.8.
• This phosphate buffer consists of 100 mM disodium hydrogen phosphate (Na 2 HP0 4 .7H 2 0) and 100 mM sodium dihydrogen phosphate (Na H 2 P0 4 ) .
• the aqueous iodine solution for use in this preferred embodiment is prepared by dissolving 1.2 mg/ml of a complex of iodine and polyvinyl pyrrolidone (PVP) (ex Aldrich) in 400 mM glycine and 20% vol. ethanol at a pH of 1.5. Said PVP, glycine and ethanol assist in stabilising and solubilising the iodine.
• PVP polyvinyl pyrrolidone
• the calibration curve used in this preferred embodiment is prepared as follows. Standard enzyme activity solutions having alpha-amylase concentrations ranging from 0-60 ppm Termamyl 300 LDX (ex NOVO) , are prepared and the activity thereof is checked using a pocket reflectometer (RQflex® ex Merck KGaA, art. nr. 16970) or an enzyme analyzer, e.g. Cobas Mira, ex Roche Diagnostics. 100 ⁇ l samples of each of these standard enzyme solutions are added to test tubes filled with a buffered starch solution as prepared above, having a temperature of 20 °C. Immediately after the addition of the standard solutions the timer is started. After exactly 3 minutes, 1 ml of iodine solution, as prepared above, is added. Thus standard test samples having varying alpha-amylase activities are obtained. In addition, 'blank' samples are prepared by adding to new test tubes 1 ml of iodine solution and lOO ⁇ l of each of the standard enzyme solutions.
• the remission or optical density of all thus-prepared samples is measured at a wavelength of 576nm and is plotted (see Figure 1).
• the delta optical densities for each of the standard enzyme solutions being the difference between the optical densities obtained with the standard test sample concerned and the 'blank' sample concerned- is calculated.
• the delta optical densities so obtained are plotted as a function of the alpha-amylase activity of the standard enzyme solutions, so as to obtain a calibration curve for alpha amylase. Via linear regression, a best fit can be calculated for said calibration curve.
• This calibration curve can be used in the method of the invention, when applying a reaction time of 3 minutes and a temperature of 20° C in step (b) of said method.
• the actual activity determination of alpha-amylase present in the wash liquor of a warewashing machine is performed as follows. A sample is taken from this wash liquor. A test tube containing a buffered starch solution as prepared above and having a temperature of 20 °C is taken and 100 ⁇ l of this wash liquor sample is added to the starch solution. Immediately, the timer is started. The test tube is swirled gently. After exactly 3 minutes, 1 ml of iodine solution as prepared above is added to the contents of the test tube, so as to obtain the "test" sample.
• Another test tube containing the same starch solution and 1 ml of the same iodine solution is taken, and lOO ⁇ l of the wash liquor sample is added so as to obtain the 'reference' sample.
• the optical densities of both the 'test' sample and the 'reference' sample are measured and the delta optical density being the difference between the thus-obtained values is calculated.
• the activity of the alpha-amylase present in the wash liquor sample is determined by interpolation using this delta optical density and the above-prepared calibration curve .

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Physics & Mathematics (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Analytical Chemistry (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)

Priority Applications (1)

Applications Claiming Priority (4)

Publications (1)

Family

ID=8171410

Family Applications (1)

Country Status (7)

Families Citing this family (4)

Family Cites Families (2)

• 2001
  • 2001-04-17 TR TR2002/02424T patent/TR200202424T2/xx unknown
  • 2001-04-17 JP JP2001578688A patent/JP2003530891A/ja active Pending
  • 2001-04-17 CA CA002407563A patent/CA2407563A1/en not_active Abandoned
  • 2001-04-17 EP EP01933841A patent/EP1278888A1/de not_active Withdrawn
  • 2001-04-17 AU AU2001260219A patent/AU2001260219A1/en not_active Abandoned
  • 2001-04-17 WO PCT/EP2001/004360 patent/WO2001081617A1/en not_active Application Discontinuation
  • 2001-04-17 BR BR0110289-3A patent/BR0110289A/pt not_active IP Right Cessation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events