Rapid and visual test panel for transesterification activity of lipase as well as preparation method and application of rapid and visual test panel

Abstract

The invention discloses a lipase transesterification activity rapid visual test plate and a preparation method and application thereof, the lipase transesterification activity test plate comprises animal fat or animal and vegetable fat, tert-butyl alcohol, methanol and a solid medium, the volume ratio of tert-butyl alcohol to animal fat or animal and vegetable fat is 1: 1-1: 5, and the volume ratio of tert-butyl alcohol to methanol is 1: 1-1: 5. The volume ratio of the animal fat to the vegetable fat is (1: 0)-(5: 1), and the volume ratio of the methanol to the animal fat or the animal fat and the vegetable fat is (1: 2)-(1: 5). The lipase transesterification activity detection flat plate is suitable for efficiently and visually detecting the lipase transesterification activity, meanwhile, compared with a traditional liquid transesterification method for detecting the catalytic effect, the method for detecting the lipase transesterification activity is visual, simple and convenient, qualitative detection and quantitative detection of the lipase transesterification activity can be achieved at the same time, and the method is suitable for large-scale popularization and application. And moreover, the required amount of lipase is small, efficient screening can be realized on the same inspection flat plate or a few inspection flat plates, and manpower and time are greatly saved.

Description

Technical Field

[0001] The invention belongs to the technical field of lipase activity testing, and in particular relates to a lipase transesterification activity rapid visual testing plate and a preparation method and application thereof. Background Art

[0002] Lipase is a type of serine hydrolase that can catalyze the breaking and forming of ester bonds and participate in a variety of reactions such as esterification, transesterification and transesterification. They are widely found in animals, plants and microorganisms. In terms of industrial applications, lipase is widely used in the synthesis of ester compounds, oil modification, and flavor improvement of wine and other beverages due to its high catalytic efficiency and mild reaction conditions.

[0003] The process of lipase-catalyzed biodiesel production mainly involves lipase-catalyzed reaction of triglycerides with methanol to produce glycerol and fatty acid methyl esters. Lipase-catalyzed transesterification is particularly important in biofuel production. Testing the transesterification activity of lipase can evaluate its potential and efficiency in the production of biofuels such as biodiesel. At present, the liquid reaction method is usually selected in conjunction with gas chromatography to detect the formation of fatty acid methyl esters, and then judge the strength of the lipase transesterification activity. This method requires the use of precision instruments and needs to be calculated by the peak area. Therefore, a method for quickly and intuitively testing the transesterification activity of lipase is particularly important for quickly evaluating the transesterification activity of lipase.

[0004] Chinese patent CN117987407A discloses a method for high-throughput intuitive screening of enzyme mutants, which is aimed at testing the hydrolytic activity of lipase by a lipase activity plate test method. It uses agar as a coagulant and adds a grease substrate, then adds lipase droplets to an emulsified agar plate, and then puts the plate into a constant temperature incubator for reaction for a period of time, and evaluates the lipase hydrolytic activity by observing the size of the hydrolysis transparent circle that appears. Usually, the larger the hydrolysis transparent circle, the higher the lipase hydrolytic activity. The advantages of the plate test method are simple operation, low cost, and suitable for preliminary screening and qualitative analysis. However, this existing method can only test the hydrolytic activity of lipase, and cannot test its transesterification activity. Therefore, establishing a method for rapid and intuitive testing of lipase transesterification activity is the unanimous pursuit and goal of the majority of relevant scientific research workers. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide a rapid and intuitive test plate for lipase transesterification activity, which can achieve efficient screening on the same or a few test plates, greatly saving manpower and time.

[0006] The present invention also provides a preparation method of a lipase transesterification activity rapid visual test plate and its application, which can rapidly and visually test the strength of the lipase transesterification activity, shorten the test time and be intuitive and clear.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a lipase transesterification activity test plate, which comprises animal fat or animal and vegetable fat, tert-butanol, methanol and a solid medium, wherein the volume ratio of tert-butanol to animal fat or animal and vegetable fat is 1:1-1:5, the volume ratio of animal fat to vegetable fat is 1:0-5:1, and the volume ratio of methanol to animal fat or animal and vegetable fat is 1:2-1:5.

[0008] The test plate of the present invention comprises animal fat or animal and vegetable fat, tert-butyl alcohol, methanol, dye and solid medium, wherein the volume ratio of tert-butyl alcohol to animal fat or animal and vegetable fat is 1:1-1:5, the volume ratio of animal fat to vegetable fat is 1:0-5:1, and the volume ratio of methanol to animal fat or animal and vegetable fat is 1:2-1:5.

[0009] Furthermore, the animal fat includes any one of lard, beef tallow or chicken fat, and the vegetable fat includes any one or more of soybean oil, peanut oil or rapeseed oil.

[0010] Furthermore, the solid medium includes any one of gauze, sponge, non-woven fabric, printing paper, facial tissue or filter paper.

[0011] Furthermore, the gauze includes any one of single-layer gauze, double-layer gauze or triple-layer gauze.

[0012] The method for preparing the lipase transesterification activity test plate of the present invention comprises the following steps:

[0013] (1) Place solid medium in a culture dish;

[0014] (2) After heating animal fat or animal and vegetable fat, tert-butyl alcohol is added for emulsification, and before the mixture is cooled, methanol is added for emulsification;

[0015] (3) Pour the mixed solution from step (2) onto the solid medium in the culture dish, seal it, and place it at low temperature to cool.

[0016] Preferably, in step (2) of the method for preparing the test plate, when the mixed solution has not yet cooled, methanol and dye are added for emulsification, and the dye includes any one of rhodamine B, methyl red or Nile red.

[0017] Preferably, the method for preparing the colorable lipase transesterification activity test plate of the present invention comprises the following steps:

[0018] (1) Place solid medium in a culture dish;

[0019] (2) After heating animal fat or animal and vegetable fat, tert-butyl alcohol is added for emulsification, and before the mixture is cooled, methanol and dye are added for emulsification;

[0020] (3) Pour the mixed solution from step (2) onto the solid medium in the culture dish, seal it, and place it at low temperature to cool.

[0021] The lipase transesterification activity test plate of the present invention is used in the rapid and intuitive test of the lipase transesterification activity.

[0022] Furthermore, the application process is: spotting the sample containing lipase on the lipase transesterification activity test plate, placing it in an incubator for reaction for 8-36 hours, and then taking it out for inspection.

[0023] Furthermore, the inspection is to inspect the color difference, refractive index difference or diameter of the transparent circle around the lipase sample spot and the heat-inactivated enzyme solution sample spot to screen the desired transesterification active lipase.

[0024] There is no report on the plate method for detecting the transesterification activity of lipase in the prior art. The existing plate methods are all for detecting the hydrolysis activity of lipase, and the agar used therein is based on a water-based system and cannot be directly used for the transesterification reaction in a non-aqueous phase. With the help of animal fats with a solidifying effect, not only because of its solid form at room temperature, but also because it can be directly used as a substrate for preparing biodiesel, with the aid of solid media such as gauze, methanol is successfully emulsified and fixed in the transesterification activity test plate, so that people can quickly and intuitively check the transesterification activity of lipase.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] The method of the present invention can visually inspect the transesterification activity of lipase. Unlike the operation of ordinary transesterification reaction, it does not need to use liquid method to react, and the operation is simple. The transesterification activity of lipase can also be quantitatively calculated by measuring the diameter of the transparent circle. The present invention only needs to spot the enzyme solution on the test plate, and after standing in the incubator for a period of time, the specific phenomenon of lipase transesterification activity can be directly observed. At present, there is no relevant report on the transesterification activity plate detection method. The method of the present invention is intuitive, easy to observe, greatly saves manpower and time, and provides great convenience for screening specific transesterification activity lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Activity display of lipases with different transesterification activities on the transesterification activity test plate, where (A) shows the activity of lipases with different transesterification activities fixed in two layers of gauze containing a mixture of lard and soybean oil (3:1), tert-butanol, and methanol, and reacted for 18 hours; (B) shows the activity of lipases with different transesterification activities fixed in a layer of gauze containing lard, tert-butanol, and methanol for 24 hours;

[0028] Figure 2 The relationship between the transparent zone diameter measurement of lipases with different transesterification activities on the transesterification activity test plate and its functional relationship with enzyme activity;

[0029] Figure 3 Gas chromatography test results of the substances in the refractive index difference portion of the hydrolysis activity and transesterification activity plates, wherein (A) is a gas chromatogram of the substances in the transparent circle of the hydrolysis activity test plate after extraction with n-hexane; (B) is a gas chromatogram of the substances in the transparent circle of the transesterification activity test plate after extraction with n-hexane;

[0030] Figure 4 The transesterification activity of lipase on transesterification activity test plates with different solid media, wherein (A) is the display of sponge-transesterification activity test plate; (B) is the display of non-woven fabric-transesterification activity test plate; (C) is the display of printing paper-transesterification activity test plate; (D) is the display of gauze-transesterification activity test plate; (E) is the display of facial tissue-transesterification activity test plate; (F) is the display of filter paper-transesterification activity test plate; (G) is the display of no solid medium-transesterification activity test plate;

[0031] Figure 5 The display of transesterification activity of lipase on transesterification activity test plates with different numbers of gauze layers, wherein (A) is the display of the front and back sides of the transesterification activity test plate with one layer of gauze; (B) is the display of the front and back sides of the transesterification activity test plate with two layers of gauze; (C) is the display of the front and back sides of the transesterification activity test plate with three layers of gauze;

[0032] Figure 6 The transesterification activity of lipase on the transesterification activity test plate of mixed oils with different components, wherein (A) is the display on the transesterification activity test plate of pure lard; (B) is the display on the transesterification activity test plate of lard and soybean oil mixed in a ratio of 1:1; (C) is the display on the transesterification activity test plate of lard and soybean oil mixed in a ratio of 2:1; (D) is the display on the transesterification activity test plate of lard and soybean oil mixed in a ratio of 3:1 and one layer of gauze; (E) is the display on the transesterification activity test plate of lard and soybean oil mixed in a ratio of 3:1 and two layers of gauze;

[0033] Figure 7 The transesterification activity of lipase on different animal fat transesterification activity test plates, wherein (A) is the display on the lard-transesterification activity test plate; (B) is the display on the beef tallow-transesterification activity test plate; (C) is the display on the chicken fat-transesterification activity test plate;

[0034] Figure 8 The transesterification activity of lipase on different dye composition-lard-transesterification activity test plates, wherein (A) is the display of the lard transesterification activity test plate without dye addition under fluorescent light and ultraviolet light; (B) the display of the rhodamine B lard transesterification activity test plate under fluorescent light and ultraviolet light; (C) the display of the Nile red lard transesterification activity test plate under fluorescent light and ultraviolet light; (D) the display of the methyl red lard transesterification activity test plate under fluorescent light and ultraviolet light;

[0035] Fig. 9 The transesterification activity of lipase on different dye component-beef oil-transesterification activity test plates, where (A) is the display of the beef oil transesterification activity test plate without dye addition under fluorescent light and ultraviolet light; (B) is the display of the Nile red beef oil transesterification activity test plate under fluorescent light and ultraviolet light; (C) is the display of the methyl red beef oil transesterification activity test plate under fluorescent light and ultraviolet light; (D) is the display of the rhodamine B beef oil transesterification activity test plate under fluorescent light and ultraviolet light.

[0036] Fig.10 The transesterification activity of lipase on the transesterification activity test plate with different tert-butanol addition amounts, where (A) is the display on the transesterification activity test plate when the volume ratio of tert-butanol to lard is 1:3; (B) is the display on the transesterification activity test plate when the volume ratio of tert-butanol to lard is 1:2; (C) is the display on the transesterification activity test plate when the volume ratio of tert-butanol to lard is 1:1.

[0037] Fig.11 The transesterification activity of lipase on the transesterification activity test plate with different methanol addition amounts, wherein (A) is the display on the transesterification activity test plate when the volume ratio of methanol to lard is 1:4.5; (B) is the display on the transesterification activity test plate when the volume ratio of methanol to lard is 1:4; (C) is the display on the transesterification activity test plate when the volume ratio of methanol to lard is 1:3.5; (D) is the display on the transesterification activity test plate when the volume ratio of methanol to lard is 1:3; (E) is the display on the transesterification activity test plate when the volume ratio of methanol to lard is 1:2.5; (F) is the display on the transesterification activity test plate when the volume ratio of methanol to lard is 1:2;

[0038] Fig.12 Illustration of the preparation and use of the rapid visual assay plate for lipase transesterification activity. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0041] The soybean oil, lard, chicken fat and beef tallow in the present invention are all commonly available commercially or homemade.

[0042] The soybean oil in the examples was purchased from JD.com COFCO Group Fortune Flagship Store, product number: 1523698;

[0043] The present invention can also use homemade lard, chicken fat, and beef tallow: the corresponding animal fat tissue is purchased from a vegetable market in Huai'an, Jiangsu, heated and boiled until the amount of oil no longer increases, and the oil residue is filtered out;

[0044] Agar powder, potassium dihydrogen phosphate, disodium hydrogen phosphate: products of Shanghai Sangon Biotechnology Co., Ltd.;

[0045] Nile Red: CAS No. 7385-67-3;

[0046] Polyvinyl alcohol (average molecular weight 1750±50), tert-butyl alcohol, methanol, methyl red, and rhodamine B: products of Sinopharm Chemical Reagent Co., Ltd.;

[0047] Recombinant Escherichia coli producing Candida antarctica lipase B: preserved in the laboratory of the School of Life Science and Food Engineering of Huaiyin Institute of Technology, Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation, DOI:10.1007 / s13205-021-03095-x;

[0048] Antarctic Candida lipase B enzyme solution: Antarctic Candida lipase B recombinant Escherichia coli was obtained according to the lipase expression and purification method described in the literature: Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation, DOI: 10.1007 / s13205-021-03095-x;

[0049] All actual enzyme activity calculation methods in this article refer to the reference: Carbon-based magnetic nano-particle utilizing nano-biochar as core and its immobilizing lipase for biodiesel preparation, DOI: 10.1016 / j.indcrop.2024.119693.

[0050] Gauze: pure cotton medical gauze with a density of 28s*26s and a size of 3m*0.8m;

[0051] Sponge: low density, daily-use sponge with a size of 1m*1m*0.5cm;

[0052] Filter paper: double-circle qualitative filter paper with medium filtration rate produced by General Electric Biotechnology (Hangzhou) Co., Ltd.;

[0053] Printing paper: 70g / m 3 M&G multi-purpose copy and printing paper;

[0054] Non-woven fabric: Polypropylene resin is the main raw material, with a gram weight range of 10-300g / m 2 ;

[0055] Facial tissue: 100% virgin wood pulp, density 0.5g / cm 3 ;

[0056] The above raw materials can be the same type of raw materials available from other companies or manufacturers on the market.

[0057] Cell ultrasonic disruptor SM-650D (power 650W), Nanjing Shunma Instrument Equipment Co., Ltd.

[0058] Example 1

[0059] A method for quickly and visually testing the transesterification activity of lipase, comprising the following steps:

[0060] (1) Preparation of transesterification activity test plate:

[0061] Place a single-layer gauze and a double-layer gauze with a diameter of 8 cm in a culture dish with a diameter of 9 cm respectively;

[0062] Preparation of transesterification activity test mixed solution 1: Heat and melt 5g lard (density 0.91g / mL), add 3mL tert-butyl alcohol, and emulsify by ultrasonic until homogenous. When the mixed solution has not cooled down, add 0.925mL methanol and emulsify by ultrasonic until homogenous, which is mixed solution 1;

[0063] Preparation of transesterification activity test mixed solution 2: Heat 5g of mixed oil, in which the mass ratio of lard to soybean oil is 3:1, add 3.5mL of tert-butyl alcohol after melting, and emulsify by ultrasonic until homogenous. When the mixed solution has not cooled down, add 0.8mL of methanol and emulsify by ultrasonic until homogenous, which is mixed solution 2;

[0064] Quickly and evenly pour the ultrasonically homogenized mixed solution 1 onto the single-layer gauze in the culture dish; quickly and evenly pour the ultrasonically homogenized mixed solution 2 onto the double-layer gauze in the culture dish; seal the plate and place it at 4°C to cool it to form a semi-solid plate.

[0065] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the test transesterification activity plate prepared in step (1), and spot 5 μL of an enzyme solution containing an esterification activity of 535.65 U / mL of Candida antarctica lipase B without dilution (100%), 1-fold (50%), 2-fold (33%), 3-fold (25%), 4-fold (20%), and 5-fold (17%) on the circular filter paper. Use an inactivated enzyme solution with an enzyme activity of 0 U / mL as a control. Place the pure lard plate prepared in step (1) in an incubator at 30°C for 24 h and the mixed oil plate for 18 h, then take them out for inspection.

[0066] (3) Quantitative measurement of lipase transesterification activity: On the transesterification plate where the reaction is completed in step (2), a transparent circle diameter is drawn every 45° with the center of the filter paper as the center, and the average diameter of the transparent circle generated by the lipase transesterification reaction is measured to form a functional relationship with its transesterification activity. The functional relationship can be used to directly calculate the lipase transesterification activity based on the average diameter of the transparent circle.

[0067] Figure 1 The figure shows the activity display of lipases with different transesterification activities on the transesterification activity test plate. Compared with the inactivated enzyme solution, the refractive index difference around the enzyme solution spotted on the circular filter paper was observed to determine the difference in the transesterification activity of the lipase. Figure 1It can be seen that although the reaction time of the mixed oil plate is shorter than that of the single lard plate, its enzyme activity is stronger and the transesterification phenomenon is more obvious, but the transesterification phenomenon of weaker enzyme activity is slightly lower than that of the single lard plate. The mixed oil plate is suitable for the detection of transesterification phenomenon with high enzyme activity in a short time, while the single lard plate is suitable for the detection of transesterification phenomenon with weaker enzyme activity for a long time.

[0068] Figure 2 The figure shows the relationship between the transparent zone diameter measurement of lipases with different transesterification activities on the transesterification activity test plate and its functional relationship with the enzyme activity. 5 μL of the enzyme solution containing the transesterification activity of Antarctic Candida lipase B of 535.65U / mL and its 1-fold, 2-fold, 3-fold, 4-fold, and 5-fold dilutions were spotted on circular filter paper in parallel, and the inactivated enzyme solution with an enzyme activity of 0U / mL was used as a control; the pure lard plate was placed in a 30°C incubator for 24 hours, and the mixed oil plate (containing lard and soybean oil (3:1)) was placed for 18 hours, then taken out for inspection, and a functional relationship curve was constructed, in which, Figure 2 (A) is the functional relationship between the diameter of the transparent circle (x) and the enzyme activity (y) on the transesterification activity test plate containing lard, tert-butyl alcohol and methanol: y = 4.97208x 2 -72.64395x+380.6642; Figure 2 (B) is a transesterification activity test plate containing a mixture of lard and soybean oil (3:1), tert-butyl alcohol, and methanol. The functional relationship between the diameter of the transparent circle (x) and the enzyme activity (y) is: y = 4.61176x 2 -57.93655x+317.60057;

[0069] The diameters of the transparent circles of the transesterification activity test plates containing lard, tert-butyl alcohol, methanol, a mixture of lard and soybean oil (3:1), tert-butyl alcohol, and methanol were measured respectively. The results are as follows Figure 2 (C) Figure 2 (D) shows the average diameter of the transparent circle. Figure 2 (C) The enzyme solution containing the transesterification activity of Candida antarctica lipase B of 535.65 U / mL was diluted 1-fold and 2-fold, and 5 μL of each solution was spotted on a circular filter paper in parallel, and an inactivated enzyme solution with an enzyme activity of 0 U / mL was used as a control; Figure 2 In (D), 5 μL of the enzyme solution containing the transesterification activity of Antarctic Candida lipase B of 535.65 U / mL was taken without dilution and diluted 1-fold and spotted on the circular filter paper in parallel, and the inactivated enzyme solution with an enzyme activity of 0 U / mL was used as a control; the correctness of the functional relationship was verified as shown in Tables 1 and 2. It can be seen from Tables 1 and 2 that the relationship between the lipase transesterification activity and the transparent zone diameter shown in the functional relationship is basically correct, and can be used for quantitative measurement of lipase transesterification activity.

[0070] 5g soybean oil, 3mL tert-butyl alcohol, 0.925mL methanol, 150μL water and 10mg freeze-dried enzyme powder containing Antarctic Candida lipase B (enzyme activity of 35.71U / mg) were reacted at 30℃ and 200rpm for 30min. The transesterification reaction mixture was centrifuged and the upper layer was taken for quantitative analysis. One transesterification activity unit (U) refers to the amount of enzyme that can produce 1.0μmol / min fatty acid methyl ester under standard test conditions.

[0071] Table 1 Functional relationship test of transesterification activity test plate containing lard, tert-butyl alcohol and methanol

[0072] Ratio of actual enzyme activity to original enzyme activity 0.5 0.5 0.33 0.33 Actual enzyme activity (U / mL) 267.83 267.83 178.50 178.50 x(average diameter of transparent circle / mm) 13.2 12.8 11.803 10.598 y(Calculate enzyme activity U / mL) 288.10 265.45 215.89 169.22

[0073] Table 2 Plate function relationship test of transesterification activity test containing lard and soybean oil (3:1) mixture, tert-butyl alcohol and methanol

[0074] Ratio of actual enzyme activity to original enzyme activity 1.0 1.0 0.5 0.5 Actual enzyme activity (U / mL) 535.65 535.65 267.83 267.83 x(average diameter of transparent circle / mm) 15.691 15.22 11.482 13.028 y(Calculate enzyme activity U / mL) 543.97 504.11 260.37 345.55

[0075] Example 2

[0076] Verifying that the transparent circle shown on the transesterification activity test plate is due to the transesterification activity of lipase includes the following steps:

[0077] (1) Preparation of lard water emulsion plates for testing lipase hydrolysis activity:

[0078] Agar powder, 3% polyvinyl alcohol (PVA), 50 mM KH 2 PO 4 -Na 2 HPO 4 Buffer solution (pH 7.0) was mixed in a volume ratio of 1:5:59 and heated in a microwave oven until it boiled several times. 3% (v / v) lard was added and ultrasonic emulsified until homogeneous, and cooled to form a lard-water emulsified plate.

[0079] (2) Transesterification and hydrolysis reaction: A filter paper with a diameter of 5 mm was placed on the lard water emulsification plate prepared in step (1) of this example and the single lard transesterification activity plate prepared in Example 1, and 5 μL of the enzyme solution containing Antarctic Candida lipase B (enzyme activity of 535.65 U / mL) was spotted on the filter paper, and placed in a 30°C incubator for 24 hours. The transparent circle substances on the two plates were respectively scooped out, and the extracts were analyzed by gas chromatography using n-hexane as the solvent.

[0080] Figure 3The figures show the gas chromatographic peaks of the hydrolysis activity lard water emulsion plate and the transesterification activity test plate transparent circle material after n-hexane extraction. According to the different peak elution times, it is determined that the reaction products produced by the transesterification activity test plate and the lard water emulsion plate are different, that is, the activities of lipase on the two plates are different, and the transesterification activity test plate shows transesterification activity. As can be seen from the figure, the gas chromatogram of the hydrolysis activity test transparent circle material only has the solvent peak of n-hexane, while the gas chromatogram of the transesterification activity test transparent circle material has four methyl ester peaks representing the biodiesel generated by transesterification, namely: peak 1 palmitic acid methyl ester, peak 2 linoleic acid methyl ester, peak 3 oleic acid methyl ester, and peak 4 stearic acid methyl ester. This shows that the transparent circle formed in the lipase transesterification activity test plate is the transesterification product of lipase, not the hydrolysis transparent circle of lipase.

[0081] in Figure 3 The specific detected components in (A) are shown in Table 3. Figure 3 The specific detected components in (B) are shown in Table 4.

[0082] Table 3 Hydrolysis activity test plate transparent circle n-hexane extraction material gas chromatogram detected components

[0083]

[0084]

[0085] Table 4 Gas chromatogram of substances extracted with n-hexane from the transparent circle of the transesterification activity test plate Detected components

[0086] serial number Peak time area high area% concentration Compound Name 0 1.371 510846286 282568851 99.970 99.970 n-Hexane 1 4.107 20044 6651 0.004 0.004 Methyl palmitate 2 5.639 28997 8866 0.006 0.006 Methyl linoleate 3 5.723 80574 18982 0.016 0.016 Methyl oleate 4 6.013 23150 6096 0.005 0.005 Methyl stearate total - 510999051 282609446 100 - -

[0087] Example 3

[0088] The solid medium optimization of the transesterification activity test plate specifically includes the following steps:

[0089] (1) Preparation of transesterification activity test plate:

[0090] Place single-layer gauze, double-layer gauze, triple-layer gauze, sponge, non-woven fabric, printing paper, facial tissue, and filter paper with a diameter of 8 cm that matches the size of the culture dishes in multiple culture dishes with a diameter of 9 cm respectively;

[0091] Preparation of transesterification activity test mixture: Heat and melt 5g lard, add 3mL tert-butyl alcohol, and emulsify by ultrasonic until homogenous. When the mixture has not cooled down, add 0.925mL methanol and emulsify by ultrasonic until homogenous;

[0092] The homogenized mixed solution after ultrasonication was quickly and evenly poured onto the solid medium in each culture dish. After the plate was sealed, it was placed at 4°C to cool to form a semi-solid plate.

[0093] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the transesterification activity test plate prepared in step (1), take 5 μL of the enzyme solution containing Antarctic Candida antarctica lipase B (enzyme activity of 535.65 U / mL) and spot it on the filter paper, use the inactivated enzyme solution as a control, place in a 30°C incubator for 24 hours, then take out and inspect.

[0094] Figure 4 The figure shows the transesterification activity of lipase on the transesterification activity plate of different solid media. It can be concluded that sponge, non-woven fabric, printing paper, and no solid medium (i.e., pouring the homogenized mixed liquid into the culture dish) are not suitable as supporting media, and the transesterification phenomenon cannot be clearly seen; gauze (two layers), facial tissue, and filter paper show different transesterification activity phenomena, and it is found that the transesterification phenomenon of two layers of gauze is more obvious than that of facial tissue and filter paper. Figure 5 The figure shows the transesterification activity of lipase on the transesterification activity plate tested with different layers of gauze. Among them, the test effect of single-layer gauze is the best.

[0095] Example 4

[0096] The optimization of different mixed oil components of the transesterification activity test plate specifically includes the following steps:

[0097] (1) Preparation of transesterification activity test plate:

[0098] Place a single-layer gauze of 8 cm in diameter, a double-layer gauze of 8 cm in diameter, and a triple-layer gauze of 8 cm in diameter in the culture dish respectively;

[0099] Preparation of transesterification activity test mixture: 5g of mixed oil, lard: soybean oil in a mass ratio of 1:0, 1:1, 2:1, 3:1, respectively, was mixed and melted by microwave heating, and then 2mL of tert-butyl alcohol was added and ultrasonic emulsified until homogenous. When the mixed solution has not cooled down, 0.9mL of methanol was added and ultrasonic emulsified until homogenous;

[0100] Quickly pour the various mixed solutions (lard alone, lard and soybean oil in different proportions) after ultrasonic homogenization evenly onto the gauze in each culture dish, seal the plate and place it at 4°C to cool it to form a semi-solid plate.

[0101] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the transesterification activity test plate prepared in step (1), take 5 μL of the enzyme solution containing Antarctic Candida lipase B and spot it on the filter paper, use the inactivated enzyme solution as a control, place it in a 30°C incubator for 24 hours, then take it out for inspection.

[0102] Figure 6 The figure shows the transesterification activity of lipase on the transesterification activity test plate of different mixed oil components. It can be concluded that although the addition of soybean oil can improve the transesterification effect during the plate method test, the increase of soybean oil will lead to the enhancement of the fluidity of the semi-solid object on the plate. At this time, the fluidity of the semi-solid object can be reduced by increasing the number of gauze layers. When the number of gauze layers in the mixed oil is one layer, the fluidity of the plate is strong, so that the interface cannot be fixed, and the specific transesterification phenomenon on the plate cannot be directly observed well; when the number of gauze layers is three layers, the fluidity of the plate is weakened, but the light transmittance of the plate is weakened, which hinders the diffusion of the enzyme, making the phenomenon not obvious, and the specific transesterification phenomenon on the plate cannot be directly observed well. When the mass ratio of lard to soybean oil reaches 3:1 and the number of gauze layers is two layers, the test plate shows the most stable and good transesterification activity of lipase. Therefore, it is concluded that the mixed oil plate is suitable for the test of transesterification phenomenon with high enzyme activity for a short time, while the single lard plate is suitable for the test of transesterification phenomenon with weak enzyme activity for a long time.

[0103] Example 5

[0104] The optimization of the lipid composition of the transesterification activity test plate includes the following steps:

[0105] (1) Preparation of transesterification activity test plate:

[0106] Place a single layer of gauze with a diameter of 8 cm that matches the size of the culture dishes in each of the culture dishes with a diameter of 9 cm;

[0107] Preparation of transesterification activity test mixture: Heat and melt 5g lard (density 0.91g / mL), chicken fat (density 0.80g / mL), and beef tallow (density 0.93g / mL), add 4mL tert-butyl alcohol, and emulsify by ultrasonic until homogenous. When the mixture has not cooled down, add 1mL methanol and emulsify by ultrasonic until homogenous;

[0108] Quickly pour the ultrasonically homogenized mixture evenly onto the single layer of gauze in each culture dish, seal the plate and place it at 4°C to cool it down to form a semi-solid plate.

[0109] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the transesterification activity test plate prepared in step (1), take 5 μL of the enzyme solution containing Antarctic Candida lipase B and spot it on the filter paper, use the inactivated enzyme solution as a control, place it in a 30°C incubator for 24 hours, then take it out for inspection.

[0110] Figure 7 The figure shows the transesterification activity display of lipase on transesterification activity test plates with different animal fat components. It can be concluded that although the beef tallow-transesterification activity test plate is more fixed and less likely to flow than the lard-transesterification activity test plate, the actual transesterification display effect is basically the same, but the chicken fat-transesterification activity test plate is more difficult to fix and the transesterification display effect is poor; and compared with the lard-transesterification activity test plate, the beef tallow-transesterification activity test plate is weaker in dye transmittance, and lard is cheaper and easier to obtain. Therefore, lard was selected as the main animal fat for the transesterification activity rapid and intuitive test plate.

[0111] Example 6

[0112] The dye addition optimization of the transesterification activity test plate includes the following steps:

[0113] (1) Preparation of transesterification activity test plate:

[0114] Place a single layer of gauze with a diameter of 8 cm that matches the size of the culture dishes in each of the culture dishes with a diameter of 9 cm;

[0115] Preparation of transesterification activity test mixture: Heat and melt 5g lard and 5g beef tallow respectively, add 4mL tert-butyl alcohol, and emulsify by ultrasonic until homogenous. When the mixture has not cooled down, add 1mL methanol and 20μL 1mg / mL Nile red, methyl red and rhodamine B in anhydrous ethanol as solvent respectively, and emulsify by ultrasonic until homogenous;

[0116] Quickly pour the ultrasonically homogenized mixed solution evenly onto the solid medium in each culture dish, seal the plate and place it at 4°C to cool it to form a semi-solid plate.

[0117] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the transesterification activity test plate prepared in step (1), take 5 μL of the enzyme solution containing Antarctic Candida lipase B and spot it on the filter paper, use the inactivated enzyme solution as a control, place it in a 30°C incubator for 24 hours, then take it out for inspection.

[0118] Figure 8 and Fig. 9The figure shows the transesterification activity of lipase on transesterification activity test plates with different dye components. It can be concluded that the observation of the lard transesterification activity test plate with rhodamine B as the dye is most obvious under ultraviolet light. The beef tallow transesterification activity test plate is not as obvious as the lard transesterification activity test plate under ultraviolet light because the beef tallow is thicker. Compared with the lard transesterification activity test plate without dye, the lard transesterification test plate with dye added is more obvious under ultraviolet light and easier to observe.

[0119] Example 7

[0120] The optimization of the amount of tert-butyl alcohol added to the transesterification activity test plate specifically includes the following steps:

[0121] (1) Preparation of transesterification activity test plate:

[0122] Place a single layer of gauze with a diameter of 8 cm that matches the size of the culture dishes in each of the culture dishes with a diameter of 9 cm;

[0123] Preparation of transesterification activity test mixture: After heating and melting 5g of lard, add tert-butyl alcohol in a volume ratio of 1:1, 1:2 and 1:3 to lard, respectively, and emulsify by ultrasonic until homogenous. When the mixture has not cooled down, add 1mL of methanol and emulsify by ultrasonic until homogenous;

[0124] Quickly pour the ultrasonically homogenized mixture evenly onto the single layer of gauze in each culture dish, seal the plate and place it at 4°C to cool it down to form a semi-solid plate.

[0125] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the transesterification activity test plate prepared in step (1), take 5 μL of the enzyme solution containing Antarctic Candida lipase B and spot it on the filter paper, use the inactivated enzyme solution as a control, place it in a 30°C incubator for 24 hours, then take it out for inspection.

[0126] Fig.10 The figure shows the transesterification activity of lipase on the transesterification activity test plate with different tert-butanol addition amounts. It can be concluded that although the transesterification phenomenon of lipase is more obvious with the addition of more tert-butanol, the fluidity of the plate gradually increases with the increase of the amount of tert-butanol, affecting the observation of the transesterification phenomenon; when the ratio of tert-butanol to lard is 1:2, the transesterification activity test plate is optimal.

[0127] Example 8

[0128] The optimization of the amount of methanol added to the transesterification activity test plate specifically includes the following steps:

[0129] (1) Preparation of transesterification activity test plate:

[0130] Place a single layer of gauze with a diameter of 8 cm that matches the size of the culture dishes in each of the culture dishes with a diameter of 9 cm;

[0131] Preparation of transesterification activity test mixture: After heating and melting 5g lard, add 3mL tert-butyl alcohol and emulsify by ultrasonic until homogenous. When the mixture has not cooled down, add methanol and lard in volume ratios of 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5 and 1:2 respectively, and emulsify by ultrasonic until homogenous;

[0132] Quickly pour the ultrasonically homogenized mixed solution evenly onto the solid medium in each culture dish, seal the plate and place it at 4°C to cool it to form a semi-solid plate.

[0133] (2) Rapid test of lipase transesterification activity: Place a circular filter paper with a diameter of 5 mm on the transesterification activity test plate prepared in step (1), take 5 μL of the enzyme solution containing Antarctic Candida lipase B and spot it on the filter paper, use the inactivated enzyme solution as a control, place it in a 30°C incubator for 24 hours, then take it out for inspection.

[0134] Fig.11 The figure shows the transesterification activity of lipase on the transesterification activity test plate with different methanol addition amounts. It can be concluded that the transesterification phenomenon is most obvious when the methanol addition amount is 1.82 mL, and the transesterification activity test plate is the best.

[0135] In summary, the present invention has optimized the transesterification activity test plate in many aspects, and the overall process and verification are as follows Fig.12 As shown, it is concluded from Examples 1-8 that a single lard transesterification test plate is used in combination with a layer of gauze. When the lard is 5 g, the volume ratio of tert-butanol to lard is 1:2, and the volume ratio of methanol to lard is 1:3, which is the optimal combination for transesterification of the present invention and is suitable for long-term stable transesterification activity testing of various enzyme activities; a lard and soybean oil (3:1) mixed liquid transesterification test plate is used in combination with a double layer of gauze. When the mixed oil is 5 g, 2 mL of tert-butanol and 0.9 mL of methanol are added, which is suitable for short-term high enzyme activity transesterification activity testing; and in the optimal single lard transesterification test plate using rhodamine B as a dye, it is irradiated with ultraviolet light. The color development and inspection effect are easy to see for the single lard transesterification detection plate without adding dye. In the pursuit of simpler operation, the transesterification effect detection can be carried out without adding dye. If a clearer and more intuitive phenomenon analysis is required, dye can be added to it for effect detection. When transparent circles appear on the transesterification test plate, their shapes are mostly circular or elliptical, and there is also the possibility of irregular shapes. For these shapes, multiple transparent circle diameters can be drawn at certain angles, and the average diameter of the transparent circles generated by the lipase transesterification reaction can be measured, and then the functional relationship between the enzyme activity and the transparent circle can be calculated, so as to quantitatively analyze the lipase of unknown enzyme activity.

Claims

1. A lipase transesterification activity test plate, characterized in that: The test plate comprises animal fat or animal and vegetable fat, tert-butyl alcohol, methanol and a solid medium, wherein the volume ratio of tert-butyl alcohol to animal fat or animal and vegetable fat is 1:1-1:5, the volume ratio of animal fat to vegetable fat is 1:0-5:1, and the volume ratio of methanol to animal fat or animal and vegetable fat is 1:2-1:

5.

2. A lipase transesterification activity test plate, characterized in that: The test plate comprises animal fat or animal and vegetable fat, tert-butyl alcohol, methanol, dye and solid medium, wherein the volume ratio of tert-butyl alcohol to animal fat or animal and vegetable fat is 1:1-1:5, the volume ratio of animal fat to vegetable fat is 1:0-5:1, and the volume ratio of methanol to animal fat or animal and vegetable fat is 1:2-1:

5.

3. The lipase transesterification activity test plate according to claim 1 or 2, characterized in that: The animal fat includes any one of lard, beef tallow or chicken fat, and the vegetable fat includes any one or more of soybean oil, peanut oil or rapeseed oil.

4. The lipase transesterification activity test plate according to claim 1 or 2, characterized in that: The solid medium comprises any one of gauze, sponge, non-woven fabric, printing paper, facial tissue or filter paper.

5. The lipase transesterification activity test plate according to claim 4, characterized in that: The gauze includes any one of single-layer gauze, double-layer gauze or triple-layer gauze.

6. A method for preparing the lipase transesterification activity test plate according to claim 1, characterized in that: The steps include: (1) Place solid medium in a culture dish; (2) After heating animal fat or animal and vegetable fat, tert-butyl alcohol is added for emulsification, and before the mixture is cooled, methanol is added for emulsification; (3) Pour the mixed solution from step (2) onto the solid medium in the culture dish, seal it, and place it at low temperature to cool.

7. The preparation method according to claim 6, characterized in that: In step (2), when the mixed solution has not yet cooled, methanol and dye are added for emulsification, wherein the dye includes any one of rhodamine B, methyl red or Nile red.

8. Use of the lipase transesterification activity test plate according to claim 1 or 2 in rapid and visual testing of lipase transesterification activity.

9. The use according to claim 8, characterized in that: The application process is: spot the sample containing lipase on the lipase transesterification activity test plate, place it in an incubator for reaction for 8-36 hours, and then take it out for inspection.

10. The use according to claim 8, characterized in that: The inspection is to inspect the color difference, refractive index difference or transparent circle diameter around the lipase sample spot and the heat-inactivated enzyme solution sample spot, and then screen the desired transesterification active lipase.

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