Background of the Invention Field of the Invention

This invention relates to a polymer film formed on a solid support for the use of detection and quantification of depolymerase activity of lipases.

Description of Related Arts

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters produced as carbon storage by some bacteria. The most common type of PHA is polyhydroxybutyrate (PHB). Incorporation of γ-ΗΒ into a polymer chain containing βΗΒ will result in the formation of poly(p-hydroxybutyrate-co-y-hydroxybutyrate) copolymer. The higher the y-HB monomer composition in the copolymer, the more stretchable is the resulting polymer. 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. However, these prior arts have several disadvantages.

For example, 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. Furthermore, in 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.

In Cam et. al., 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.

Therefore 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.

References

• Cam, et. al., 1995, "Degradation of high molecular weight poly(L-lactide) in alkaline medium, Biomaterials, vol 16, pg 833-843. 000205

Summary of Invention

It is an objective of the present invention to provide a method for the efficient assay of depolymerase activity of enzymes through densitometry analysis. It is also another objective of the present invention to provide a method which is capable of sensitive and rapid evaluation of hydrolytic activity of lipase droplets towards polymer-coated surface.

Accordingly, these objectives may be achieved by following the teachings of the present invention. 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.

Brief Description of the Drawings

The features of the invention will be more readily understood and appreciated from the following detailed description when read in conjunction with the accompanying drawings of the preferred embodiment of the present invention, in which:

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 Ρ(β-ΗΒ-οο-92 mol% γ-ΗΒ) film.

Detailed Description of the Invention

As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a .basis, for claims It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modification, equivalents and alternatives falling within the scope of the present invention as defined by the appended claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean including, but not limited to. Further, the words "a" or "an" mean "at least one" and the word "plurality" means one or more, unless otherwise mentioned. Where the abbreviations or technical terms are used, these indicate the commonly accepted meanings as known in the technical field. For ease of reference, common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures. The present invention will now be described with reference to Figs. 1 -6.

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.

In a preferred embodiment of the method for detection of depoiymerase activity of lipases, wherein the polyhydroxyalkanoate polymer is preferably obtained from Delftiaacidovorans.

In a preferred embodiment of the method for detection of depoiymerase activity of lipases, wherein the transparent polymer film is preferably cast by melt casting or solvent casting.

In a preferred embodiment of the method for detection of depolymerase activity of lipases, wherein the transparent polymer film is preferably scanned by against a dark background.

In a preferred embodiment of the method for detection of depolymerase activity of lipases, wherein the scanned image of the transparent polymer film is preferably analyzed by densitometry software program.

In a preferred embodiment of the method for detection of depolymerase activity of lipases, wherein the transparent polymer film is preferably cased with a group of polyesters consisting of β-hydroxypropionate, γ-hydroxybutyrate, δ-hydroxyvalerate, ε-hydroxyhexanoate, ε-caprolactone, lactic acid, butylene succinate, butylene adipate, or ethylene succinate.

In a preferred embodiment of the method for detection of depolymerase activity of lipases, wherein the lipase is preferably incubated with the transparent polymer film until the appearance of hydrolysis mark on the film surface.

Examples

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the scope of the present invention is not limited thereto. - ~-

Biosynthesis of P(B-HB-co-92mol%v-HB) copolymer

1 , 4-butanediol is autoclaved separately and added aseptically to the mineral medium 1 % (v/v) as the sole carbon source to promote synthesis of the polymer. 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. To promote synthesis of PHA, the harvested cells are transferred into a second medium, which contain 100 mL of nitrogen free mineral (NM) medium (pH 7.0). The 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. 1 .98g MnCI ₂ -4H ₂ 0, 2.81 g CoS0 ₄ -7H ₂ 0, 1 .67g CaCI ₂ -2H ₂ 0, 0.17g CuCI ₂ -2H ₂ 0 and 0.29g ZnS0 ₄ -7H ₂ 0 (per litre of 0.1 M HCI) were included in the TE solution. After 48 hours, 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.

To determine the composition and content of P(p-HB-co-v-HB) copolymer produced by the cells, 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. 1 filter paper and the collected filtrate was concentrated by using an Eyela rotary evaporator N-1000 until a volume of about 10 mL. A rapidly stirring cold methanol was used to precipitate the PHA from the concentrated filtrate by drop-wise method. Finally the pure polymer was scooped out from the methanol solution and dried overnight at room temperature.

P(3-HB-co-92mo /oV-HB) copolymer thin film

20 mg of P(P-HB-co-92mol%v-HB) polymer 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. 20 μΙ_ of lipase solution or control solution was pipetted onto the polymer film surface to form semispherical droplets. Markings were made at the bottom surface of the glass petri dish by a marker pen prior to dropping to assist in the location of droplets and separation of samples and controls. The polymer dish with droplets was then covered with aluminium foil and incubated at 37°C for 30 minutes. After incubation, the droplets may be recovered by pipetting from the polymer film or immediately flooded with distilled water to remove the enzymes from the film surface. The film was then allowed to dry at room temperature before observations were recorded.

Quantitative analysis of hydrolysis spot density

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. For further analysis of white hydrolysis spots, 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.

Result of using P(S-HB-co-92mol%v-HB) copolymer thin film on different types of lipases.

The results of the study are shown in Table 1 , where the types of lipases and their ability to hydrolyze thin films of Ρ(β-ΗΒ-οο-92ηιοΙ%γ-ΗΒ) are listed. The hydrolytic ability of the lipases from Aspergillusoryzae, A, niger, Candida antarctica, C. rugosa, Mucorjavanicus, M. miehei, Rhizopusarrhizus, R. niveus, R. oryzae, Chromobacteriumviscosum, Pseudomonas cepacia, P. fluorescens, Porcine pancreas and wheat germ on Ρ(β-ΗΒ-οο-92ιτιοΙ%γ-ΗΒ) was investigated. 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.

Table 1 : Types of lipases and their ability to hydrolvze thin films of

P(B-HB-co-92mol%v-HB)

Table 2: Effect of lipase concentration on patterns of hydrolysis spots

Although the present invention has been described with reference to specific embodiments, also shown in the appended figures, it will be apparent for those skilled in the art that many variations and modifications can be done within the scope of the invention as described in the specification and defined in the following claims.