JP2020080851A - Methods for preparing rice bran lipase - Google Patents

Abstract

To provide methods for preparing rice bran lipase.SOLUTION: The method of the invention for preparing rice bran lipase comprises an extraction step and a purification step. In the extraction step, rice bran is added to an enzyme extraction solution, stirred, and centrifuged to obtain a rice bran lipase extract. In the purification step, the rice bran lipase extract is added to a solution of ammonium sulfate and an alcohol and stirred to precipitate purified rice bran lipase. The effect of the invention is that enzyme purification carried out using a co-precipitation process with ammonium sulfate and alcohol effectively increases the preparation efficiency of the rice bran lipase and shortens the rice bran lipase production process. Scale-up examination has verified that the invention is applicable for commercial production of rice bran lipase.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for preparing rice bran lipase, including a process for extracting and purifying rice bran lipase to obtain a purified rice bran lipase enzyme.

Currently, most of Taiwan's rice bran is used as a feed for low cost animals, and the price is about 6-10 yuan/kg. Rice bran is an agricultural waste, but it has many nutrients such as dietary fiber, amino acid composition balance, fatty acid composition balance, brown rice oil, abundant vitamin E, abundant vitamin B group and minerals, phytosterols, ferulic acid, and γ-oryzanol. Including the price. Rice bran has various nutritional values, but its utilization rate was not high.

Since rice bran has the drawback of ranciding oils and fats and easily proliferates microorganisms, it is not suitable for storage. The main cause of rancidity of rice bran is that rice bran itself has an enzymatic action. This enzyme is called a lipase. Rice bran lipase is mainly present in rice husks and has no effect unless it comes into contact with fat. However, when lipase comes into contact with oils and fats after crushing rice husks to produce rice bran, the activity of lipase rapidly increases, fats are decomposed to release free fatty acids, and oils and fats are oxidized to rancid, finally. Causes rancid deterioration of rice bran (see Chinese Patent Application No. CN102279166A). Therefore, the presence of lipase is disadvantageous for the storage and subsequent use of rice bran. However, in addition to breaking down ester bonds and releasing fatty acids, lipases also contain many enzymatic activities. For example, transesterification activity has been used in biodiesel processes. Compared with conventional chemical reactions, the reaction conditions of lipase are mild, have good substrate stereoselectivity (used for drug preparation), and are environmentally friendly and cause no pollution. Therefore, lipase has been widely applied to the industrial fields such as food, leather, medicine, feed, and washing.

In terms of the industrial enzyme market, the production value in the global market in 2014 reached $4.2 billion, of which lipase accounts for about 10% of the total market. Analyzing the lipase market, the global market is expected to reach nearly $600 million by 2020, with an annual growth rate of 6.5% between 2015 and 2020. Mainly used in the washing and food industries, Asia is its largest market. Many companies around the world have entered the industrial enzyme market with enzymes and mainly use lipases. Lipase-related products are used in biocatalysts and detergents for oil stains and the enzymes are obtained from microorganisms (fungi) and oil stain decomposers (Bacillus sp).

Conventional patent documents describe crude enzyme extraction and storage methods for rice bran lipase (see, for example, Patent Document 1). According to the above method, n-hexane was uniformly mixed at a ratio of 3 mL per 1 g of rice bran and then shaken (170 to 190 rpm) for 25 to 35 minutes, and then the rice bran granules were taken out from the sufficiently filtered residue. , Stir at room temperature for 4 to 8 hours, stir 3 or 4 times per hour to fully evaporate n-hexane, and degreasing rice bran granules that have been thoroughly dried to 1 g of defatted rice bran After uniformly mixing water at a ratio of 8 mL, shaking (170 to 190 rpm) treatment is performed for 25 to 35 minutes, and centrifugation (2,000 to 4,000 rpm) treatment is performed at low temperature (2 to 6°C) for 25 to 35 minutes. After that, remove the centrifuged supernatant liquid and use it as a crude enzyme extract of rice bran lipase to degrease the rice bran lipase extract, and prepare a freeze-dried powder from the crude enzyme extract, or add 0.5 mM chloride. A step of preserving the rice bran lipase crude enzyme, which is preserved with a phosphate buffer (50 mM, pH 7.0) containing calcium. In this patent, by extracting distilled rice bran lipase using a distilled water shaking method, the crude enzyme activity of the extracted rice bran lipase is higher and the purity is higher than the conventional phosphate buffer pulverization extraction method. The method is easy to operate and requires less time. The crude enzyme obtained is further purified to obtain high-purity lipase.

Patent Document 2 describes a method for purifying rice bran lipase. The method comprises a defatting step of defatting rice bran at a ratio of 10 mL of diethyl ether to 1 g of rice bran, and a defatted rice bran granule containing Tris-HCl buffer solution (10 mM, pH 7.5) containing 1 mM EDTA. After stirring for 12 hours, the mixture was filtered through a multi-layered cotton cloth, and the obtained filtrate was subjected to centrifugation (3,000 xg) for 30 minutes, and then the centrifuged supernatant was treated with rice bran. Defatting step of rice bran lipase extract extracted as crude enzyme extract of lipase, and rice bran lipase crude using an octyl-Sepharose column (preliminarily balanced with 0.01 M Tris-HCl buffer of pH 7.5) Purification of the enzyme extract, linear gradient elution using 10-40% methanol (divided collection volume of eluent is 10 mL), and purified rice bran lipase was present in the eluent eluted in the latter stage A step of purifying the enzyme extract. The final purification rate is 6.8, and the activity recovery rate is 20%. This patent can obtain high-purity rice bran lipase and can perform detailed analysis of enzyme characteristics, but the process is complicated, time-consuming, and operation is dangerous (because of the degreasing of rice bran using diethyl ether, ). Moreover, the content of patents is biased toward academic research purposes, and is not suitable for commercial production of rice bran lipase.

As can be seen by arranging the above patents, Patent Document 1 presents a rice bran lipase extraction method that is easy to operate and does not take much time, but the obtained rice bran lipase is only a crude enzyme and can be further purified. It is not possible to obtain high-purity lipase. Although a detailed rice bran lipase purification method is presented in WO02/101033A1, the process is too complicated, time-consuming, and dangerous (because of diethyl ether), and is not suitable for commercial production of a large amount of rice bran lipase. there were. As described above, the mass production process technology of rice bran lipase cannot be said to be perfect even today, and therefore, a new rice bran lipase preparation process that boasts good enzyme extraction and purification efficiency, simple operation process and low cost It is required to develop. Therefore, the process of extracting and purifying rice bran lipase according to the present invention satisfies the requirements of novelty and inventive step, which are the requirements for patent application, and is practicable.

Chinese Patent Application Publication No. CN103642771B European Patent Application Publication No. WO02/101033A1

Therefore, the present inventor thought that the above-mentioned drawbacks could be ameliorated and, as a result of intensive studies, came to the proposal of the present invention to rationally and effectively improve the problem.

The present invention has been made in view of such conventional problems, and an object thereof is to provide a method for preparing rice bran lipase. In other words, the efficiency of extraction and purification of rice bran lipase can be improved, and the use of rice bran lipase enables the development of enzyme-related products with high added value.

In order to solve the above problems, a method for preparing rice bran lipase according to one aspect of the present invention includes an extraction step and a purification step. In the extraction step, rice bran is added to the enzyme extraction solution and stirred, and a rice bran lipase extract is obtained after centrifugation. In the purification step, a rice bran lipase extract is added to ammonium sulfate and alcohol and stirred to obtain a rice bran lipase purified enzyme precipitate.

In the method for preparing rice bran lipase according to the present invention, the rice bran is undefatted rice bran.

In the method for preparing rice bran lipase according to the present invention, the ratio of rice bran to the enzyme extraction solution in the extraction step is 2:1 or more.

In the method for preparing rice bran lipase according to the present invention, the ratio of rice bran to the enzyme extraction solution in the extraction step is 4:1.

In the method for preparing rice bran lipase according to the present invention, in the extraction step, the enzyme extraction solution is distilled water containing a surfactant.

In the method for preparing rice bran lipase according to the present invention, in the extraction step, the enzyme extraction solution is distilled water containing 0.1% Triton X-100 surfactant.

Further, the purification step of the method for preparing rice bran lipase according to the present invention is performed in two steps. In the first-step purification step, the rice bran lipase extract is added to ammonium sulfate and stirred to obtain a rice-bran lipase solution that has been completely purified in the first step. In the second-step purification step, the rice bran lipase solution obtained by completing the first-step purification is added to alcohol, and the mixture is stirred and then allowed to stand to obtain a rice bran lipase-purified enzyme precipitate.

In the method for preparing rice bran lipase according to the present invention, the alcohol concentration is 20% or more.

In the method for preparing rice bran lipase according to the present invention, the alcohol concentration is 50%.

The method for preparing rice bran lipase according to the present invention includes an extraction step and a purification step. In the extraction step, rice bran is added to an enzyme extraction solution, which is distilled water containing a surfactant, and stirred, and a rice bran lipase extract is obtained after centrifugation. In the purification step, a rice bran lipase extract is added to ammonium sulfate and alcohol and stirred to obtain a rice bran lipase purified enzyme precipitate.

The present invention utilizes the ammonium sulfate and alcohol coprecipitation method to purify the enzyme. Unlike the conventional step-step separation process (ammonium sulfate precipitation step and alcohol precipitation step), it effectively enhances the rice bran lipase preparation efficiency and shortens the rice bran lipase production process. Further, it was proved that the present invention can be applied to the commercial production of rice bran lipase through the scale-up verification.

1 is a schematic view of a flow chart showing a method for preparing rice bran lipase according to one embodiment of the present invention. It is a lipase enzyme activity standard curve of a lipase standard product. It is a protein concentration standard curve of a bovine serum albumin standard product (protein concentration method (1)). It is a protein concentration standard curve of a bovine serum albumin standard product (protein concentration method (2)).

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Needless to say, the present invention is not limited to the following examples and can be arbitrarily changed without departing from the spirit of the present invention.

FIG. 1 is a flow chart showing a preferred embodiment of the method for preparing rice bran lipase of the present invention. It includes an extraction step 1 and a purification step 2.

<Extraction step 1>
Rice bran is added to the enzyme extraction solution and stirred for 30 to 60 minutes, and a rice bran lipase extract is obtained after centrifugation. By the way, the enzyme extraction solution is distilled water containing a surfactant, and the surfactant is 0.1% Triton X-100.

Preferably, non-defatted rice bran is used as the rice bran to reduce material costs. The ratio of rice bran to the enzyme extraction solution is 2:1 or more, more preferably 4:1.

<Purification step 2>
A rice bran lipase extract is added to ammonium sulfate and alcohol and stirred to obtain a rice bran lipase purified enzyme precipitate.

The purification step 2 is performed in two stages. In the purification step 21 of the first stage, the rice bran lipase extract is added to ammonium sulfate having a saturation concentration of 0 to 60% and stirred for 30 to 60 minutes to obtain a rice bran lipase solution that has been purified in the first stage. In the second purification step 22, the rice bran lipase solution which has been purified in the first step is added to alcohol, stirred for 30 to 60 minutes, allowed to stand for 10 minutes, and centrifuged or precipitated by gravity to purify the rice bran lipase. An enzyme precipitate is obtained.

The final concentration of alcohol is 20% or more, preferably 50%.

The present invention simplifies the extraction step 1 and the purification step 2 for obtaining rice bran lipase, and in order to improve the process efficiency, as a result of a study conducted by the applicant, after performing a simple enzyme extraction step 1 on rice bran, It was found that by carrying out the enzyme purification step 2 using the ammonium sulfate and alcohol coprecipitation method, the efficiency of rice bran lipase preparation can be effectively increased and the process of producing rice bran lipase can be shortened. In addition, scale-up verification of the rice bran lipase preparation process was carried out using equipment at the test factory level.

INDUSTRIAL APPLICABILITY The present invention has the effects of purifying the enzyme using the ammonium sulfate and alcohol coprecipitation method, effectively increasing the efficiency of rice bran lipase preparation, and shortening the process of producing rice bran lipase. In addition, through scale-up verification, it was demonstrated that the present invention can be applied to commercial production of rice bran lipase. Effectively enhancing the extraction and purification efficiency of rice bran lipase, it becomes possible to develop high value-added enzyme-related products using rice bran lipase.

The present invention will be further described based on the following experimental examples. However, it is obvious that these experimental examples are merely examples for explanation and do not limit the practice of the present invention.
<<Experimental Example 1: General experimental method>>
1. Analysis of lipase activity:
In this Experimental Example 1, a lipase standard product (Lipase from wheat germ-Type I, Sigma L3001) having various enzyme activities (0, 1.05, 2.63, 5.25, 7.88, and 10.5 U). And 0.1 mL of each of the rice bran lipase samples to be measured are added to 3.2 mL of a substrate solution (containing 0.55 mM p-nitrophenyl-palmitate substrate in Table 1), and a shaking reaction is performed at 35° C. for 20 minutes. After that, the enzyme reaction is stopped by treating at 100° C. for 5 minutes, and then centrifugation (6,000 rpm) is performed for 10 minutes, the centrifuged supernatant is taken out, and the absorbance at 400 nm is measured.

The absorbance at 400 nm of the enzyme reaction of lipase standard products with various enzyme activities is shown on the Y-axis, and the enzyme activity is shown on the X-axis (the lipase standard product is plotted under the conditions of 15 units/mg protein and Protein=70%), Generate a lipase enzyme activity calibration curve (see Figure 2). The absorbance at 400 nm of the enzyme reaction of the rice bran lipase sample to be measured is substituted into the calibration curve to obtain the lipase activity (U), and the obtained enzyme activity value is divided by 0.1 (0.1 mL taken out from the sample The enzyme activity (U/mL) is obtained per 1 ml of the rice bran lipase sample.

2. Protein concentration analysis method 1:
In Experimental Example 1, protein concentration analysis is performed using the Pierce ^™ Microplate BCA Protein Assay Kit (Thermo#23252), and the analysis is performed according to the analysis flowchart of this kit. Bovine serum albumin (BSA) with different protein concentrations (0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 mg/mL) and measured rice bran Remove 9 μL of each lipase sample, place it in the center of the well of a 96-well microplate, add 4 μL CRS solution (Compatibility Reagent Solution), shake for 1 minute, and then let react for 15 minutes at 37°C, 260 μL After adding a WR reagent (working reagent) and performing a shaking treatment for 1 minute, the mixture was reacted at 37° C. for 30 minutes, treated at room temperature for 5 minutes, and the absorbance at 562 nm was measured.

The absorbance at 562 nm of bovine serum albumin standards of various protein concentrations is shown on the Y-axis, the protein concentration is shown on the X-axis, and a protein concentration calibration curve is prepared (see FIG. 3). The absorbance at 562 nm of the rice bran lipase sample to be measured is substituted into the calibration curve to determine the protein concentration (mg/mL) of the rice bran lipase sample.

3. Protein concentration analysis method 2:
In Experimental Example 1, bovine serum albumin standard (Bovine serum albumin, Sigma A3803) with various protein concentrations (0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 μg/mL) and 20 μL of each sample of rice bran lipase to be measured was added to 200 μL of dye solvent (Bio-Rad protein assay_dye reagent concentrate#500-0006), mixed uniformly, and allowed to react at room temperature for 5 minutes to give an absorbance of 595 nm. The measurement was performed.

The absorbance at 595 nm of bovine serum albumin standards of various protein concentrations is shown on the Y-axis, the protein concentration is shown on the X-axis, and a protein concentration calibration curve is prepared (see FIG. 4). The absorbance at 595 nm of the rice bran lipase sample to be measured is substituted into the calibration curve to determine the protein concentration (μg/mL) of the rice bran lipase sample.

<<Experimental example 2. Comparison of extraction ability of rice bran lipase with different enzyme extraction solutions>>
In this Experimental Example 2, different enzyme extraction solutions are used. This includes the rice bran lipase extraction solution according to the present invention, the above-mentioned Patent Document 1 (Chinese Patent Application Publication No. CN103642771B), and the conventional extraction solution, and the extraction of lipase for each rice bran is performed. Conduct and compare the extraction ability of rice bran lipase of different enzyme extraction solutions.

Experimental materials:
1. Preparation of undefatted rice bran and defatted rice bran:
The non-defatted rice bran used in this Experimental Example 2 was purchased from the Lianhe rice mill (Tainan, Taiwan). Regarding the preparation of defatted rice bran, first, the non-defatted rice bran purchased from the Unigen milling plant was added to n-hexane at a ratio of 25% (v/v) to carry out a defatting reaction, stirred at room temperature for 30 minutes, and the supernatant was added. After removing the liquid, the defatted rice bran is placed on filter paper and dried in a draft chamber for 60 minutes, and the rice bran thus obtained is used as defatted rice bran.
2. Enzyme extract solution:
The enzyme extraction solution used in this Experimental Example 2 was distilled water (0.1 to 1% Triton X-100, SDS (sodium lauryl sulfate), or rice bran lipase extraction of the present invention containing a surfactant such as Tween 20). Solution), distilled water (rice bran lipase extraction solution of Patent Document 1 (Chinese Patent Application Publication No. CN103642771B) mentioned above), or a conventional rice bran containing 50 mM phosphate buffer (pH 7, 0.5 mM CaCl _{2 ).} Lipase extraction solution).

experimental method:
Extraction of lipase from undefatted rice bran or defatted rice bran is performed with reference to the experimental method described in Patent Document 1 (Chinese Patent Application Publication No. CN103642771B) (In the above-mentioned Patent Document 1, lipase extraction is not performed on undefatted rice bran. ). After uniformly mixing the enzyme extraction solution at a ratio of 8 mL to 1 g of dried defatted rice bran or undefatted rice bran granules, shake (180 rpm) treatment for 30 to 60 minutes, and centrifuge (3,000 xg) treatment for 30 minutes After the above, the centrifuged supernatant is taken out to make an enzyme extract of rice bran lipase, and the lipase activity of each enzyme extract is analyzed.

Experimental result:
Table 2 shows a comparison of the results of the lipase activity and the specific activity per milliliter of the protein concentration measured by the enzyme extraction solution different from the defatted rice bran and the non-defatted rice bran in this experiment.

Protein concentration, lipase activity and specific activity per milliliter measured by enzyme extraction solutions different from defatted rice bran and non-defatted rice bran.
*50 mM phosphate buffer (pH ₇ , containing 0.5 mM CaCl ₂ )
**Perform protein concentration analysis using protein concentration analysis method 1.
Note: Experimental data are the average of three independent analysis data.

As can be seen from the results in Table 2, when rice bran was used as the defatted rice bran, the lipase activity and the specific activity per milliliter of the rice bran lipase extract obtained by the three different enzyme extraction solutions were compared, and as a result, the present invention was obtained. When the rice bran lipase extraction solution (distilled water (containing 0.1% Triton X-100)) according to Example 1 was subjected to rice bran lipase extraction, the highest lipase activity and specific activity per milliliter were obtained. The lipase activity per milliliter of the rice bran lipase extract obtained by the enzyme extract solution of the present invention is higher than that of the other two enzyme extract solutions by the rice bran lipase extract obtained by the distilled water and the phosphate buffer extract solution. The solutions were 1.6 times and 1.7 times, respectively, and the specific activities were 1.2 times and 1.5 times, respectively.

When rice bran is used as the non-defatted rice bran, the result of comparing the lipase activity and the specific activity per milliliter of the rice bran lipase extract obtained by the three different enzyme extraction solutions, and as a result, the formulation of the rice bran lipase extract solution according to the present invention When rice bran lipase extraction was carried out, the highest lipase activity and specific activity per milliliter were obtained. The lipase activity per milliliter of the rice bran lipase extract obtained by the enzyme extract solution of the present invention is higher than that of the other two enzyme extract solutions by the rice bran lipase extract obtained by the distilled water and the phosphate buffer extract solution. The solution was 1.6 times and 1.8 times, respectively, and the specific activities were 1.3 times and 1.5 times, respectively.

As a result of comparing the lipase specific activity of the rice bran lipase extract obtained by the experiment of defatted rice bran and non-defatted rice bran, when the rice bran lipase extraction was performed by the rice bran lipase extraction solution according to the present invention, irrespective of whether the rice bran was defatting A rice bran lipase extract having the same specific activity was obtained. On the other hand, in the other two enzyme extraction solutions, when rice bran that had not been defatted was used, the specific activity of the extracted rice bran lipase extract remarkably or slightly decreased.

As can be seen from the above experimental results, the rice bran lipase extraction ability of the rice bran lipase extraction solution according to the present invention is clearer than that of the above-mentioned Patent Document 1 (Chinese Patent Application Publication No. CN103642771B) and the conventional extraction solution. Very expensive. In addition, the rice bran lipase extraction solution according to the present invention all achieved the same rice bran lipase extraction ability regardless of whether or not the rice bran was degreased.

<<Experimental Example 3. Examination of solid-liquid ratio of rice bran lipase extraction process: >>
In this Experimental Example 3, the rice bran and the enzyme extraction solution having different solid-liquid ratios are used, lipase extraction is performed on the rice bran, and the extraction ability of the rice bran lipase at different solid-liquid ratios is compared. The main purpose is to reduce the amount of enzyme extraction solution used by increasing the solid-liquid ratio and to reduce the operation volume of the rice bran lipase extraction process.

Experimental materials:
1. Undefatted rice bran:
The non-defatted rice bran used in this example was purchased from the Unigen rice mill.
2. Enzyme extract solution:
The enzyme extract solution used in this example is distilled water (rice bran lipase extract solution according to the invention containing 0.1% Triton X-100).

experimental method:
First, lipase extraction is performed on undefatted rice bran. Based on the different solid-liquid ratios of rice bran and enzyme extraction solution, each solid-liquid ratio (rice bran:enzyme extraction solution) was set to 1:1, 2:1, 4:1, and 1:8, 96g, 48g, 24g. , And 12 g of rice bran granules were respectively taken out and added to 96 mL of the enzyme extraction solution, uniformly mixed, shaken (180 rpm) for 30 to 60 minutes, and then centrifuged (3,000 xg) for 30 minutes. , Take out the centrifuged supernatant and use it as an enzyme extract of rice bran lipase.

Experimental result:
In this experiment, Table 3 shows a comparison of the results of protein concentration, lipase activity per milliliter, specific activity and total activity measured by the rice bran lipase extract at each solid-liquid ratio.

The protein concentration measured by the rice bran lipase extract at each solid-liquid ratio, lipase activity per milliliter, specific activity and total activity.
*The solid-liquid ratio is rice bran: enzyme extraction solution.
** Perform protein concentration analysis using Protein Concentration Analysis Method 2.
Note: Experimental data are the average of three independent analysis data.

As can be seen from the results in Table 3, if the solid-liquid ratio is too high (solid:liquid=1:1), the re-dissolved liquid of rice bran becomes too viscous and becomes semi-coagulated, which facilitates the rice bran enzyme extraction process. I can't do it. As a result of comparing the lipase activity, the specific activity and the total activity per milliliter of the rice bran lipase extract obtained by the other three different solid-liquid ratios, the solid-liquid ratio was compared with the original solid-liquid ratio (1:8). When the ratio is increased to 1:4, the lipase activity per milliliter is increased 2.2-fold, the specific activity is increased 1.4-fold, and the total activity is increased 2-fold. When the solid-liquid ratio increases to 1:2, the lipase activity per milliliter increases 3.4 times, the specific activity increases 1.6 times, and the total activity increases 2.8 times. Judging from the results of the amount of rice bran used and the total activity, when the solid-liquid ratio was increased to 1:4, the rice bran lipase extraction capacity was similar to the original process (solid-liquid ratio=1:8). However, when the solid-liquid ratio is increased to 1:2, the rice bran lipase extraction ability is inferior to the original process (solid-liquid ratio=1:8).

As can be seen from the above experimental results, different rice bran and enzyme extraction solution solid-liquid ratios definitely affect the rice bran lipase extraction efficiency. When the solid-liquid ratio used in the process is increased to 1:4, the rice bran lipase extraction capacity is similar to the original process (solid-liquid ratio = 1:8), and the amount of enzyme extraction solution used is effectively reduced. The operating volume of the lipase extraction process is reduced.

<<Experimental Example 4. Examination of rice bran lipase purification process>>
In Experimental Example 4, two different enzyme purification methods are used to perform lipase purification on a rice bran lipase extract. The two enzyme purification methods are the ammonium sulfate and alcohol purification methods of rice bran lipase according to the present invention and the conventional ammonium sulfate purification method, respectively, and the purification efficiency of rice bran lipase by the two enzyme purification methods will be compared.

Experimental materials:
1. Undefatted rice bran:
The non-defatted rice bran used in this Experimental Example 4 was purchased from the United States rice mill.
2. Enzyme extract solution:
The enzyme extraction solution used in this Experimental Example 4 is distilled water (compound of the rice bran lipase extraction solution of the present invention containing 0.1% Triton X-100).
3. Enzyme Precipitate Redissolved Solution The enzyme precipitate reconstituted solution used in this Experimental Example 4 is a 50 to 100 mM phosphate buffer solution (pH 8.0).

experimental method:
First, lipase extraction is performed on undefatted rice bran using two types of solid-liquid ratio (1:4 and 1:8). When the solid-liquid ratio is 1:4, 7.5 g of rice bran granules are taken out and added to 30 mL of enzyme extraction solution to perform enzyme extraction. When the solid-liquid ratio is 1:8, 3.75 g of rice bran granules are taken out and added to 30 mL of enzyme extraction solution to perform enzyme extraction. After uniformly mixing the rice bran granules and the enzyme extraction solution, shake (180 rpm) treatment for 30 to 60 minutes, centrifuge (3,000 xg) treatment at room temperature for 30 minutes, and then centrifuge the supernatant. Take it out and use it as an enzyme extract of rice bran lipase.

The enzyme extract is purified with lipase and divided into an experimental group and a control group. In the control group, the conventional ammonium sulfate purification method was used, and in the experimental step, 25 mL of the enzyme extract solution was added to 9.025 g of ammonium sulfate (ammonium sulfate having a saturation concentration of 0 to 60%) and mixed uniformly for 30 to 60 minutes. After stirring for 15 minutes and performing high-speed centrifugation (12,000×g) for 15 minutes, the centrifugation precipitate is taken out to be a purified enzyme precipitate of a control group of rice bran lipase. The purified enzyme precipitate of the control group is redissolved with the enzyme precipitate re-dissolution solution, and after the redissolution, it is used as the purified enzyme solution of the control group.

In the experimental group, the ammonium sulfate and alcohol purification method according to the present patent was used, and in the experimental step of the experimental group, 25 mL of the enzyme extract was added to 9.025 g of ammonium sulfate (ammonium sulfate having a saturated concentration of 0 to 60%) to homogenize. Stir for 3 minutes to mix (total volume 30 mL) and add 8 mL, 13.8 mL, 21.8 mL, and 33.3 mL of 95% industrial alcohol respectively (final alcohol concentration 20%, 30% respectively). , 40%, and 50%), and after stirring for 30 minutes so that they are uniformly mixed at room temperature, the mixture is allowed to stand for 10 minutes, then subjected to low-speed centrifugation (3,000×g) for 10 minutes, and then centrifuged. The separated precipitate is taken out and used as a purified enzyme precipitate of the rice bran lipase experimental group. The purified enzyme precipitate of the experimental group is redissolved with the enzyme precipitate re-dissolved solution, and after the redissolved, it is used as the purified enzyme solution of the experimental group.

Experimental result:
Table 4 shows a comparison of the results of the purified enzyme solutions produced by the two types of enzyme purification methods according to the present experiment, their protein concentrations, lipase activity per milliliter, specific activity and total activity.

The protein concentration of the purified enzyme solution is lipase activity, specific activity and total activity per milliliter.
*Alcohol concentrations of 20%, 30%, 40%, and 50% are indicated as experimental groups, and after the enzyme extract has been treated with ammonium sulfate at a saturation concentration of 0 to 60%, alcohol is added to give the final alcohol concentration. Are 20%, 30%, 40%, and 50%, respectively. In the control group, the enzyme extract was only treated with ammonium sulfate having a saturated concentration of 0 to 60%, and was not treated with alcohol.
** Perform protein concentration analysis using Protein Concentration Analysis Method 2.
***The enzyme precipitate of the control group is obtained after high-speed centrifugation (12,000×g) for 15 minutes. The enzyme precipitate of the experimental group is obtained after low-speed centrifugation (3,000×g) treatment for 10 minutes.
Note: Experimental data are the average of three independent analysis data.

As can be seen from the results of Table 4, when the solid-liquid ratio was 1:8, the lipase per milliliter of the experimental groups with final alcohol concentrations of 20%, 30%, 40%, and 50% was compared to the control group. The activities were 0.46 times, 0.45 times, 0.44 times, and 1.05 times that of the control group, and the specific activities were 5.67 times, 5.55 times, 5.45 times that of the control group, respectively. And 8.23 times, and the total activity is 0.48 times, 0.73 times, 0.68 times, and 3.96 times that of the control group, respectively. When the solid-liquid ratio was 1:4, the lipase activity per milliliter of the experimental groups with final alcohol concentrations of 20%, 30%, 40%, and 50% was 0.48 of that of the control group, respectively, when compared with the control group. Fold, 0.47 fold, 0.51 fold, and 1.01 fold, and the specific activities were 6.35 fold, 5.72 fold, 6.28 fold, and 10.07 fold of the control group, respectively, and the total activity was Are 0.54 times, 0.58 times, 0.71 times, and 3.67 times that of the control group, respectively.

Based on the result of total lipase activity, the extraction and purification efficiency of rice bran lipase with different solid-liquid ratios is determined. Regarding the control group, the total lipase activity of the control group with a solid-liquid ratio of 1:4 was 1.94 times that of the control group with a solid-liquid ratio of 1:8, and the solid-liquid ratio increased to 1:4. , And the extraction and purification efficiency of the rice bran lipase is clearly similar to the original process (solid-liquid ratio=1:8).

Based on the result of total lipase activity, the extraction and purification efficiency of rice bran lipase by different enzyme purification methods is judged. Compared with the conventional ammonium sulfate purification method (control group), the extraction and purification efficiency of ammonium sulfate of rice bran lipase according to the present invention and rice bran lipase of the alcohol purification method (experimental group with an alcohol concentration of 50%) were clearly higher, and the solid-liquid ratio was 1 In the case of :8 and 1:4, the total activity of rice bran lipase in the experimental group having an alcohol concentration of 50% was 3.96 times and 3.67 times that of the rice bran lipase in the control group, respectively. However, when the alcohol concentration was 50% or less (experimental groups with alcohol concentrations of 20%, 30%, and 40%), the total activity of the rice bran lipase was lower than that of the control group. In addition, when obtaining the enzyme precipitates of the experimental group with the alcohol concentration of 50%, the low-speed centrifugation (3,000 xg) treatment was performed for 10 minutes, as compared with the control group and the experimental group with the alcohol concentrations of 20%, 30%, and 40%. After the above, the rice bran lipase precipitate can be obtained very easily, and an effect similar to the precipitation can be achieved by using gravity precipitation. On the other hand, it is difficult to obtain a rice bran lipase precipitate in the control group and the experimental groups having alcohol concentrations of 20%, 30% and 40%.

As can be seen from the above experimental results, the enzyme purification capacity of the rice bran lipase according to the present invention in the ammonium sulfate and alcohol purification method is clearly superior to the conventional ammonium sulfate purification method. Further, the enzyme purification process developed by the present invention is easy to operate, and the purified enzyme precipitate can be smoothly obtained without using high-speed centrifugation equipment, which is advantageous for scale-up of the process.

<<Experimental Example 5. Scale-up verification of rice bran lipase preparation process>>
In this Experimental Example 5, equipment at a test factory level is used, and scale-up verification is performed on the rice bran lipase preparation process of the present invention, the rice bran lipase preparation efficiency is effectively increased, and the rice bran lipase production process is shortened.

Experimental materials:
1. Undefatted Rice Bran The undefatted rice bran used in this Experimental Example 5 was purchased from the Uniha rice mill.
2. Enzyme Extraction Solution The enzyme extraction solution used in this Experimental Example 5 is distilled water (compound of rice bran lipase extraction solution according to the present invention containing 0.1% Triton X-100).
3. Enzyme precipitate re-dissolution solution The enzyme precipitate re-dissolution solution used in this Experimental Example 5 is a 50 to 100 mM phosphate buffer solution (pH 8.0).
4. Test factory level equipment The test factory level equipment is a 500 liter fermentation tank (Biotop BTF-C500L, Chosei Business Co., Ltd., Taiwan) and a horizontal continuous centrifuge (Lw360×1600, Liaoning Fuichi Machinery Co., Ltd., China. ), system tandem pipeline equipment (Shosei Industrial Co., Ltd., Taiwan) and continuous high-speed centrifuge (S-VT No.6, Quan Tai Iron Factory, Taiwan).

experimental method:
First, lipase extraction is performed on undefatted rice bran, and based on a solid-liquid ratio of rice bran and enzyme extraction solution of 1:4, rice bran granules 35 kg and enzyme extraction solution 140 L are added to a 500-liter fermentation tank for 60 minutes. After stirring (120 rpm) and performing continuous centrifugation for 30 minutes by a horizontal continuous centrifuge, the collected supernatant obtained by centrifugation is used as an enzyme extract of rice bran lipase.

The enzyme extract is purified for lipase and divided into a control group and an experimental group. In the control group, the conventional ammonium sulfate purification method was used, and in the experimental step, the enzyme extract (volume 160 L) prepared above was added to a 500-liter fermentation tank, and 60 kg of ammonium sulfate (saturated ammonium sulfate of 0 to 60%) was added. Was added, and the mixture was stirred (120 rpm) for 60 minutes so as to be uniformly mixed, and then continuously centrifuged by a horizontal continuous centrifuge or a continuous high-speed centrifuge (16,000×g). Couldn't get

In the experimental group, the ammonium sulfate and alcohol purification method according to the present patent is used, and in the experimental step of the experimental group, the enzyme extract (volume 160 L) prepared above is added to a fermentation tank of 500 liters, and 60 kg of ammonium sulfate (0 to 60) is added. % Saturated ammonium sulfate) was added, and stirring (120 rpm) was performed for 60 minutes so as to uniformly mix. At this time, the total volume of the solution was 200 L, and 200 L of 95% industrial alcohol was added and stirred for 60 minutes (120 rpm) so as to mix uniformly, and allowed to stand for 10 minutes, and then wait for gravity precipitation to wait for the rice bran lipase. The purified enzyme precipitate (about 60 kg) of the experimental group of is obtained. The purified enzyme precipitate of the experimental group is redissolved with a redissolved solution of the enzyme precipitate, and after the redissolved, the purified enzyme solution of the experimental group is used.

Experimental result:
In this experiment, rice bran lipase was purified based on the conventional ammonium sulfate purification method using equipment at a test factory level, but an enzyme precipitate (control group) could not be obtained.

In this experiment, the ammonium sulfate and alcohol purification method according to the present patent was used, rice bran lipase was purified using equipment at a test factory level, and an enzyme precipitate (experimental group) could be obtained smoothly. 5 g of the experimental group purified enzyme precipitate was redissolved with 3 mL of the enzyme precipitate re-dissolved solution, and after the redissolution, a purified enzyme solution of the experimental group with a total volume of 6.5 mL was obtained. The protein concentration, lipase activity per milliliter, specific activity and total activity are shown in Table 5.

Protein concentration, lipase activity per milliliter, specific activity and total activity measured by a scaled-up rice bran lipase purified enzyme solution (5 g of purified enzyme precipitate).
* Perform protein concentration analysis using protein concentration analysis method 2.
Note: Experimental data are the average of three independent analysis data.

As can be seen from the results in Table 5, in the rice bran lipase preparation process of the present invention, scale-up verification was smoothly carried out using equipment at a test factory level, and the rice bran lipase purified enzyme solution (3 mL obtained by scale-up verification was used. 5 g of the purified enzyme precipitate was redissolved with the enzyme precipitate re-dissolving solution, and the total volume after re-dissolution was 6.5 mL), the protein concentration was 0.64 mg/mL, and lipase activity per milliliter Is 400 U/mL, the specific activity is 623 U/mg and the total activity is 2598 U.

In the conventional ammonium sulfate purification method, it was not possible to smoothly perform scale-up verification using equipment at a test factory level, but in the rice bran lipase preparation process of the present invention, a novel ammonium sulfate and alcohol purification method was used, and a facility at a test factory level was used. Was used for successful scale-up verification. In addition, the enzyme precipitation process does not require a centrifuge facility or a solid-liquid separation facility, and a purified enzyme precipitate can be obtained smoothly only by using the gravity precipitation method, and the enzyme extraction and purification process of the present invention enables the efficiency of rice bran lipase preparation. It shows that the rice bran lipase production cost can be suppressed effectively.

To make the purpose of this specification clearly understandable, the meaning of the word “comprising” has the meaning corresponding to the words “including but not limited to” and “comprises”.

Incidentally, when a prior art publication is cited herein, said prior art publication does not constitute an admission as follows. In Taiwan or any other nation, the prior art publications only form part of the common general knowledge of the technology.

Unless otherwise defined, all technical and scientific terms used in the text can be understood by those skilled in the technical field to which the present invention belongs. Those skilled in the art will appreciate that many of the examples described herein are methods and materials that have similar or equivalent effects and have been applied in the practice of the invention. Of course, the present invention is not limited to the methods and materials described.

In addition, the official name of the term "lipase" used in this specification is called triacylglycerol lipase (EC3.1.1.3), which is an enzyme having the ability to hydrolyze the ester bond of fat, and has a hydrolytic action on fats and oils. To generate fatty acids and alcohols.

The term "extraction" as used herein refers to the extraction of an enzyme extract containing rice bran lipase by causing an enzyme extraction reaction on rice bran using water, a solvent, a buffer, or the like. The selection of these operating conditions is routinely determined by a person skilled in the art according to a routine.

As used herein, the term "purification" refers to the use of chemicals, solvents, colloids, etc. to cause an enzyme purification reaction on an enzyme extract containing rice bran lipase to obtain a rice bran lipase purified enzyme having enzymatic activity. It means to do. The selection of these operating conditions is routinely determined by a person skilled in the art according to a routine.

As used herein, the term "test plant level equipment" refers to the use of system equipment with an operating volume of 100 liters or greater to perform scale-up verification for the rice bran lipase preparation process. The selection of these operating conditions is routinely determined by a person skilled in the art according to a routine.

In accordance with the present invention, the enzyme extraction and purification process is performed using conventional techniques familiar to those skilled in the art.

As can be seen, the operating conditions for enzyme extraction and purification vary depending on factors such as the solvent and chemicals used, and the best enzyme extraction and purification effect is achieved. The selection of these operating conditions is routinely determined by a person skilled in the art according to a routine.

The term “activity” as used herein refers to lipase activity, and the activity unit of lipase standard (Lipase from wheat germ-Type I, 5-15 units/mg protein, Protein: ≧70%, Sigma L3001) is indicated by U. .. One activity unit (1 U) of the lipase standard product is hydrolyzed by the action of the lipase standard product on the substrate triacylglycerol every hour under the environment of pH value 7.4 (pH 7.4) and 37°C. Is defined as generating 1 microequivalent fatty acid.

The term "specific activity" as used herein refers to the lipase activity contained per milligram of protein and is expressed in U/mg.

As used herein, the term "total activity" refers to the total lipase activity contained in the enzyme solution, the total activity being the activity of the rice bran lipase solution per milliliter (U/mL) and the volume of the rice bran lipase solution (mL). ), denoted by U.

The term "protein concentration" as used herein refers to the total amount of protein contained per milliliter of enzyme solution and is given in mg/mL.

All patents and literature references cited in the present specification are used as reference materials for the present invention. In case of conflict, the detailed description of the proposal, including those defined therein, will control.

Although the embodiments of the present invention have been described in detail above with reference to the drawings, the specific configuration is not limited to these embodiments, and includes design changes and the like within the scope not departing from the spirit of the present invention.

1 Extraction Step 2 Purification Step 21 First Stage of Purification Step 22 Second Stage of Purification Step

Claims (1)

An extraction step of adding rice bran to the enzyme extraction solution and stirring, and obtaining a rice bran lipase extract after centrifugation,
A step of adding a rice bran lipase extract in ammonium sulfate and alcohol and stirring, to obtain a rice bran lipase purified enzyme precipitate,
A method for preparing rice bran lipase, wherein the rice bran is non-defatted rice bran, and the ratio of rice bran to the enzyme extraction solution is 2:1 or more in the extraction step.