WO2014163479A1 - Procédé de détection de l'activité de dépolymérase de lipases - Google Patents

Procédé de détection de l'activité de dépolymérase de lipases Download PDF

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Publication number
WO2014163479A1
WO2014163479A1 PCT/MY2013/000205 MY2013000205W WO2014163479A1 WO 2014163479 A1 WO2014163479 A1 WO 2014163479A1 MY 2013000205 W MY2013000205 W MY 2013000205W WO 2014163479 A1 WO2014163479 A1 WO 2014163479A1
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WIPO (PCT)
Prior art keywords
polymer film
transparent polymer
detection
hydrolysis
lipases
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PCT/MY2013/000205
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English (en)
Inventor
K. Sudesh Kumar C. KANAPATHI PILLAI
Diana Hooi Ean CH'NG
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Universiti Sains Malaysia
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Publication of WO2014163479A1 publication Critical patent/WO2014163479A1/fr

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    • 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

Definitions

  • This invention relates to a polymer film formed on a solid support for the use of detection and quantification of depolymerase activity of lipases.
  • PHAs Polyhydroxyalkanoates
  • PHA Polyhydroxyalkanoates
  • PHA polyhydroxybutyrate
  • Incorporation of ⁇ - ⁇ into a polymer chain containing ⁇ will result in the formation of poly(p-hydroxybutyrate-co-y-hydroxybutyrate) copolymer.
  • the conventional method used for the quantification of depolymerase activity by lipases uses a weight loss measurement of polymer films incubated in buffered solution of lipases for hours to days, depending on the amount and activity of the enzymes involved.
  • these prior arts have several disadvantages.
  • US patent 3817838 discloses a method for detecting the presence of lipase activity in fat-containing food.
  • the method starts with dispersing and agitating the fat-containing food in water .to facilitate extraction of water-soluble components.
  • the dispersion is then filtered and the filtrate reserved.
  • a solution of polyoxyethylene and sorbitanmonolaurate is added with the filtrate.
  • Hydroxylamine assay is performed wherein an aqueous alkaline solution of hydroxylamine is first added and followed by an acidic solution of ferric chloride to form a colored solution in the presence of ester material, wherein the density of the color is related to the amount of ester material in the solution.
  • the drawback of this invention is that it is limited to only fat containing food and cannot be used for test against lipase activity extracted from bacteria, fungi, plants and animals.
  • US patent 20080193958 A1 disclosed a method for the detection of lipase or phospholipase activity in a sample. The method includes the steps of adding the sample in aqueous into the wells of microtitration plates coated with a layer of lipid substrate approximately 0.5 to 5pm in thickness, which is able to be hydrolyzed by said lipase or phospholipase by releasing a-eleostearic acid which solubilises in the micellar phase in said aqueous solution. UV absorption spectrum of the a-eleostearic acid released is detected, which indicates lipase or phospholipase activity in the sample.
  • the drawback of the invention is the use of aqueous reactant, which is hard to handle.
  • a research article disclosed a method to study the degradation of poly(L-lactide)s (PLLA) films. Hydrolytic degradation of the films is performed at 37°C in 0.01 M NaOH solution. After a predetermined period of time, the films are removed from the incubation medium, washed with distilled water, and dried under vacuum to constant weight. Reduction in transparency of the PLLA films is measured by a spectrophotometer at 570nm. The transparency of the film is found to reduce with the increase of alkaline solution incubation time. The drawback of this system is that it uses multiple solutions, making it harder and complicated to use.
  • the present invention seeks to address the above mentioned disadvantages of the prior arts. Accordingly, it can be seen in the prior arts that there exist a need to provid a simpler method for the detection of lipase that uses a solid support frame instead of liquid base and has a larger detection range of samples.
  • the present invention relates to a method for detection of depolymerase activity of a lipase, characterized by contacting the lipase to a transparent polymer film, wherein the transparent polymer film comprises a polyhydroxyalkanoate polymer.
  • the surface of the transparent polymer film having a hydrolysis mark is scanned against a dark background to obtain an image of the transparent polymer film with a dark background.
  • the scanned image of the transparent polymer film is colour inverted to obtain an image with light background; and analysing the hydrolysis mark on the image of the transparent polymer film using densitometry analysis, wherein the lipase is determined to have depolymerase activity against the transparent polymer film if the density of an area with the hydrolysis mark is higher than the density of the area without the hydrolysis mark.
  • Figure 1 is a diagram showing a scanned picture of polymer dish with hydrolysis spots produced by lipases from (A) P. cepacia, (B) R. arrhizus, (C) R. oryzae and (D) A. oryzaewtih concentrations of 0.125 mg/mL (a), 0.25 mg/mL (b) and 0.5 mg/mL (c). Each row with 3 adjacent spots indicated triplicates.
  • Figure 2 is a diagram showing the concentration profiles of lipases from (A) A. oryzaeand (B) P. cepacia. The concentrations of lipases increase from number 2 (0 ⁇ 03125 mg/mL) to number 8 (2 mg/mL). Number 1 shows control without enzyme. Each row with 3 adjacent spots indicates triplicates.
  • Figure 3 shows the cropped, aligned and color-inverted picture of hydrolysis spots by P. cepacia lipase from low (2) to high concentration (8). Each column shows triplicates. Column (1 ) shows control without enzyme.
  • Figure 4 is a graph showing the plot of relative density of hydrolysis spots versus concentration of P. cepacia lipase. Points 1 -8 correspond to the spots on Figure 3.
  • Figure 5 is a graph showing the model for the effect of lipase concentration on relative density of hydrolysis spots by P. cepacia lipase.
  • Figure 6 is a graph showing the range of concentration of different lipases with measurable opacity by using thin ⁇ ( ⁇ - ⁇ - ⁇ -92 mol% ⁇ - ⁇ ) film.
  • the present invention relates to a method for detection of depoiymerase activity of a lipase, characterized by: contacting the lipase to a transparent polymer film, wherein the transparent polymer film comprises a polyhydroxyalkanoate polymer; scanning a surface of the transparent polymer film having a hydrolysis marks against a dark background to obtain an image of the transparent polymer film with a dark background; colour inverting the scanned image of the transparent polymer film to obtain an image with light background; and analysing the hydrolysis mark on the image of the transparent polymer film using densitometry analysis, wherein the lipase is determined to have depoiymerase activity against the transparent polymer film if the density of an area with the hydrolysis mark is higher than the density of the area without the hydrolysis mark.
  • the polyhydroxyalkanoate polymer is preferably obtained from Delftiaacidovorans.
  • the transparent polymer film is preferably cast by melt casting or solvent casting.
  • the transparent polymer film is preferably scanned by against a dark background.
  • the transparent polymer film is preferably cased with a group of polyesters consisting of ⁇ -hydroxypropionate, ⁇ -hydroxybutyrate, ⁇ -hydroxyvalerate, ⁇ -hydroxyhexanoate, ⁇ -caprolactone, lactic acid, butylene succinate, butylene adipate, or ethylene succinate.
  • the lipase is preferably incubated with the transparent polymer film until the appearance of hydrolysis mark on the film surface.
  • the method used to culture the bacteria D. acidovorans for the biosynthesis of copolymer consists of two steps.
  • the first step of cultivation comprises the inoculation of 3% (w/v) of fresh culture into 100 ml_ 2x nutrient broth (NB) medium (pH 7.0) with 1 % (w/v) glucose in a 500 mL flask. After 24 hours, the cells were harvested by centrifugation at 4000 g, 16°C for 10 minutes.
  • the harvested cells are transferred into a second medium, which contain 100 mL of nitrogen free mineral (NM) medium (pH 7.0).
  • NM medium consisted of 0.37g K 2 HP0 4 , 0.58g KH 2 P0 4 , 0.1 mMMgSO4.7H 2 O, supplemented with 0.1 ml trace elements (TE) solution.
  • TE trace elements
  • the cells are harvested by centrifugation at 4000 g, 16°C for 10 minutes. The cells were then frozen at -20°C overnight and the frozen cells were then subjected to lyophilisation for 48 hours by using Labconco 4.5 freezone apparatus.
  • methanolysis was carried out by the addition of methanolysis solution [15% (v/v) of sulphuric acid and 85% (v/v) of methanol] to about 20 mg of freeze-dried cells and the mixture was heated to 100°C for 140 minutes.
  • the hydroxyacyl methyl esters then formed was subjected to gas chromatography analysis by using a Shimadzu GC-2010 gas chromatograph.
  • 150 ml of chloroform was added to 1.0g of the cells and stirred for about 4-5 days at room temperature. Cell debris was then filtered out using a Whatman No.
  • P(P-HB-co-92mol%v-HB) polymer 20 mg was dissolved in 20 mL of chloroform by stirring for about 15 minutes at room temperature.
  • the polymer solution was poured into a 9 cm glass Petri dish and covered with perforated aluminium foil. The solvent was allowed to evaporate by leaving the Petri dish on a well balanced surface to prevent unequal thickness of film.
  • the transparent polymer film formed on the dish was aged for 3 days and used directly for enzyme assay without peeling off the dish.
  • the lyophilized powder of lipases was dissolved in phosphate buffer solution (100 mM, pH 7.4) to the desired concentration in 1 .5 ml_ Eppendorf tubes and vortexed until they are completely dissolved.
  • a scanned picture of the hydrolysis spots on polymer film was obtained by placing the petri dish facing upwards on a CanoScanLiDE 20 paper scanner.
  • the dish was scanned against a black background with the lid of the scanner left open to obtain an image with a completely dark background.
  • the picture was then colour-inverted and subjected to densitometry analysis by using the ImageJ densitometry software.
  • Table 1 The results of the study are shown in Table 1 , where the types of lipases and their ability to hydrolyze thin films of ⁇ ( ⁇ - ⁇ - ⁇ -92 ⁇ % ⁇ - ⁇ ) are listed.
  • Table 2 shows the observation of 4 types of lipases to study the effect of lipase concentration on hydrolysis patterns obtained on a single film. The lipase solutions were serial diluted 2 times to obtain 3 different concentrations and were dropped on a film and incubated.

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  • 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)
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  • Physics & Mathematics (AREA)
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  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
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  • 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)

Abstract

La présente invention concerne un procédé de détection de l'activité de dépolymérase d'une lipase, caractérisé par la mise en contact de la lipase avec un film polymère transparent, où le film polymère transparent comprend un polymère de polyhydroxyalcanoate. La surface du film polymère transparent ayant une marque d'hydrolyse est scannée contre un fond sombre pour obtenir une image du film polymère transparent avec un fond sombre. On inverse la couleur de l'image scannée du film polymère transparent pour obtenir une image avec un fond clair; et l'analyse de la marque d'hydrolyse sur l'image du film polymère transparent en utilisant une analyse de densitométrie, où la lipase est déterminée comme ayant une activité de dépolymérase contre le film polymère transparent si la densité d'une zone avec la marque d'hydrolyse est supérieure à la densité de la zone sans la marque d'hydrolyse.
PCT/MY2013/000205 2013-04-01 2013-11-26 Procédé de détection de l'activité de dépolymérase de lipases WO2014163479A1 (fr)

Applications Claiming Priority (2)

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MYPI2013700510 2013-04-01
MYPI2013700510 2013-04-01

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160061830A1 (en) * 2014-08-27 2016-03-03 The Governors Of The University Of Alberta Bacteriophage-based biosensor for microbial detection

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CH'NG, D.H-E. ET AL.: "Densitometry based microassay for the determination of lipase depolymerizing activity on polyhydroxyalkanoate", AMB EXPRESS, vol. 3, 2013, pages 22 *
JAEGER, K-E. ET AL.: "Substrate Specificities of Bacterial Polyhydroxyalkanoate Depolymerases and Lipases: Bacterial Lipases Hydrolyze Poly(omega-Hydroxyalkanoates", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, no. 8, 1995, pages 3113 - 3118 *
SCHIRMER, A. ET AL.: "Degradation of Poly(3-Hydroxyoctanoic Acid) [P(3HO)] by Bacteria: Purification and Properties of a P(3HO) Depolymerase from Pseudomonas fluorescens GK13", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 59, no. 4, 1993, pages 1220 - 1227 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160061830A1 (en) * 2014-08-27 2016-03-03 The Governors Of The University Of Alberta Bacteriophage-based biosensor for microbial detection
US9921219B2 (en) * 2014-08-27 2018-03-20 Anastasia Elias Bacteriophage-based biosensor for microbial detection

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