WO2011033633A1

WO2011033633A1 - Lactase preparation

Info

Publication number: WO2011033633A1
Application number: PCT/JP2009/066231
Authority: WO
Inventors: 一磨塩田; 博文堀口; 愛伊豫谷; 吉川　潤; 佐藤　智子
Original assignee: 合同酒精株式会社
Priority date: 2009-09-17
Filing date: 2009-09-17
Publication date: 2011-03-24

Abstract

Contaminating arylsulfatase in a lactase preparation causes the generation of unpleasant and offensive taste and odor in a food such as a milk product to which the lactase preparation is added. To overcome this problem, it is required to establish a method for measuring arylsulfatase at a high sensitivity. It is also required therefor to develop a lactase preparation in which the content of contaminating arylsulfatase is minimized or, preferably, a lactase preparation from which arylsulfatase is completely removed. A lactase preparation, which is produced by using, as a starting material, a liquid culture medium of a diploid yeast strain carrying lactase gene and showing regulated expression of arylsulfatase protein, or a genetically-modified microorganism carrying lactase gene of a yeast having been transferred thereinto and showing regulated expression of arylsulfatase protein, which shows an arylsulfatase activity of 0.1% or lower on the basis of the lactase activity when measured by a fluorescence method.

Description

Lactase preparation

The present invention relates to a lactase preparation in which arylsulfatase is not contaminated or very small even when contaminated, a method for producing such a lactase preparation, and a dairy product produced using such a lactase preparation.

Since ancient times, milk has long been used as a nutritious and useful food. Milk contains lactose, a kind of sugar. Lactose is broken down in the intestine by lactase, but in some humans, as the amount of lactase secreted into the intestine decreases as it grows, milk and milk processed products (hereinafter collectively referred to as “dairy products”) ) Causes so-called lactose intolerance such as abdominal pain and diarrhea. This contributed to the widespread consumption of this nutritious food.

In recent years, dairy products in which lactose has been reduced or removed in advance have been provided. Such a dairy product can be consumed without problems even by a human with lactose intolerance.

Lactose is reduced or removed by various methods, but the most common is a method of hydrolyzing lactose by treating a dairy product with a lactase preparation.

By the way, until now, dairy products in which lactose has been decomposed by lactase have been generally distributed through a sterilization process. However, recently, a method has been widely used in which lactase preparation is aseptically added to sterilized milk and lactose is decomposed during distribution. This is believed to reduce the amount of lactase preparation used and contribute to cost reduction.

On the other hand, new problems have arisen by adopting a method of aseptically adding a lactase preparation to sterilized milk. It is a problem caused by causing chemical changes in milk components because contaminant enzymes (proteases and arylsulfatases) contained in trace amounts in lactase preparations are not inactivated. In fact, it is known that proteases cause curdling and bitterness, and Non-Patent Document 1 reports that arylsulfatase causes unpleasant taste and odor.

Patent Document 1 describes a lactase preparation in which the amount of arylsulfatase contamination is reduced and a method for producing the same. However, in the method for measuring arylsulfatase activity described in Patent Document 1, arylsulfatase activity is not measured in a trace amount region of 8 units (arylsulfatase activity per 1 LLU of lactase active substance, the same shall apply hereinafter) or less, and is described as below the detection limit. It has only been done. In Table 1 of Patent Document 1, it is described that if the activity of contaminating arylsulfatase is 19 units or less, no odor is produced in the dairy product. However, this is a judgment based on the results of a short-term test with a reaction period of 2 days. Presuming that the cause of the off-flavor in dairy products is a chemical change in the milk component by the enzyme arylsulfatase, a reaction period of up to 2 to 3 months is assumed when long-life milk is assumed as the application. If so, it cannot be said that if the arylsulfatase activity measured by the method described in Patent Document 1 is 19 units or less, no off-flavor is produced in the dairy product. The fact that more precise control is required regarding the amount of arylsulfatase contamination also means that more accurate methods for measuring arylsulfatase activity are required to control the amount of arylsulfatase contamination. Don't wait.

In order to remove the arylsulfatase contaminating the enzyme preparation to the utmost limit, a general purification method such as adsorption, solvent fractionation, ammonium sulfate fractionation, chromatography, and crystallization may be combined. Or what is necessary is just to select and breed the microorganisms which produce the target enzyme, Comprising: Arylsulfatase production ability is deleted. In addition, a microorganism obtained by transforming a host that originally does not produce arylsulfatase to produce the target enzyme can also be used.

Patent Document 1 describes a method for obtaining a microorganism having reduced ability to produce arylsulfatase by mutation treatment and a microorganism having an arylsulfatase gene deleted by genetic engineering techniques. However, although a method for obtaining a microorganism in which arylsulfatase production ability has been reduced by mutation treatment has been described, there is no description of an actual acquisition example, and only the possibility has been shown. That is, it is unclear whether or not it is possible to produce a microorganism having an arylsulfatase production ability reduced by mutation treatment at an industrial level by the method described in Patent Document 1. In yeast diploid strains useful for industrial production of lactase formulations, it is necessary to obtain double mutants to disrupt the arylsulfatase gene by mutation, and such double mutants Acquisition was considered difficult in practice.

Further, regarding deletion of the arylsulfatase gene by genetic engineering techniques, Patent Document 1 only describes an example using a haploid strain called CBS2359 strain. In other words, in the method described in Patent Document 1, it is difficult to effectively disrupt a gene in a diploid strain of yeast useful for the production of a lactase preparation. Therefore, aryl which is a diploid strain of yeast is used. A sulfatase non-producing strain cannot be produced.

Special table 2009-517061

Even if the amount of arylsulfatase contamination in the lactase preparation is very small, when the lactase preparation is added to milk or dairy products, it may have an unpleasant taste and smell that is uncomfortable depending on the action time and temperature conditions. Can be generated. In order not to cause such inconvenience, it is necessary to produce a lactase preparation in which the amount of contamination of arylsulfatase is extremely reduced, and preferably arylsulfatase is completely removed.

Prior to solving the above problems, the present inventors selected a highly sensitive fluorescence method as a method for measuring arylsulfatase activity, examined appropriate measurement conditions, and established a measurement method. And by the method for measuring the activity of arylsulfatase, arylsulfatase is used for measuring the activity of arylsulfatase in a region that has been conventionally below the detection limit, so as not to generate an unpleasant taste or smell in milk or dairy products. The amount of contamination was identified and the present invention was completed.

That is, the present invention relates to a yeast diploid strain having a lactase gene and restricted expression of arylsulfatase protein, or a recombinant microorganism into which a yeast lactase gene has been introduced and expression of arylsulfatase protein is restricted. A lactase preparation produced from the above culture broth as a raw material, and the arylsulfatase activity (unit: U / g) measured and calculated by the following method based on the lactase activity (unit: NLU / g) by the FCC IV method. ) For lactase preparations characterized in that they are 0.1% or less:
(Measurement method of arylsulfatase activity)
(1) A specimen in which the presence of arylsulfatase is predicted is appropriately diluted with a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5 N potassium chloride to prepare a sample.
(2) An aqueous solution containing 2-methylumbelliferone sulfate potassium at a concentration of 2 mM is prepared.
(3) The sample and a 4-methylumbelliferone sulfate aqueous solution are mixed at 1: 1 (volume basis) and reacted at 37 ° C. for 3 hours.
(4) To the reaction solution, add the same amount (volume basis) of 0.1N sodium hydroxide aqueous solution as the reaction solution to stop the reaction and prepare a measurement sample.
(5) The fluorescence intensity is measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.
(6) 4-Methylumbelliferone is dissolved in a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5N potassium chloride to obtain a solution with an appropriate concentration. An aqueous sodium oxide solution is added, and the fluorescence intensity is measured under the same conditions as in (5).
(7) A calibration curve is created from (6).
(8) From the fluorescence intensity measured in (5) and the calibration curve created in (7), the 4-methylumbelliferone concentration of the measurement sample is calculated, divided by 3, and the reaction time is 1 hour. If so, determine the 4-methylumbelliferone concentration. Further, the amount of 4-methylumbelliferone generated during the reaction for 1 hour is calculated from the volume of the reaction solution.
(9) Since the amount of 4-methylumbelliferone calculated in this way is based on the amount of sample contained in the sample prepared in (1), it is converted to the amount of 4-methylumbelliferone per gram of sample. To do.
(10) The case where the amount of 4-methylumbelliferone produced per 1 hour of the reaction time between the substrate and the enzyme is 1 nmole is defined as 1 unit (U), and the unit is the unit amount per 1 g of the specimen, that is, the enzyme preparation, That is, it is represented by “unit (U) / g”.

In step (10), “substrate” is potassium 4-methylumbelliferone sulfate, and “enzyme” is arylsulfatase.

In the present invention described above, “expression of arylsulfatase protein is restricted” means, for example, an expression in which the arylsulfatase gene (structural gene) is disrupted or the arylsulfatase gene functions to express the arylsulfatase protein It means that the arylsulfatase protein is not produced or the production amount thereof is reduced because the regulatory gene is disrupted or the arylsulfatase gene and / or the arylsulfatase protein expression regulatory gene is not present. It is preferable that the arylsulfatase protein is not expressed, that is, the production amount is zero, but the arylsulfatase activity (unit: U / g) / lactase activity (unit: NLU / g) is 0.1% or less. However, the expression of the arylsulfatase protein may be limited so as to be preferably 0.02% or less.

The lactase preparation according to the present invention preferably has a lactase activity of 1,000 NLU / g or more, more preferably 2,000 NLU / g or more, and further preferably 3,000 NLU / g or more. More preferably, it is more preferably 4,000 NLU / g or more, and most preferably 5,000 NLU / g or more.

The yeast diploid strain having a lactase gene and restricted expression of arylsulfatase protein may be a mutant strain obtained by a mutation treatment of a yeast diploid strain or a yeast diploid strain. It may be a mutant obtained by subjecting a body strain to deletion of an arylsulfatase gene or an arylsulfatase protein expression regulatory gene. A mutant strain whose parent strain is a diploid strain of yeast having a large production amount of lactase protein is preferred.

The diploid strain of yeast is preferably a diploid strain of Kluyveromyces lactis or its closely related species, Kluyveromyces marxianus. In addition, the genetically modified microorganism into which the yeast lactase gene is introduced and the expression of the arylsulfatase protein is restricted is preferably a genetically modified microorganism into which the Klaveromyces lactis or Kriveromyces marixianus lactase gene is introduced.

The present invention relates to a yeast diploid strain having a lactase gene and restricted expression of an arylsulfatase protein, or a genetically modified microorganism into which a yeast lactase gene has been introduced and the expression of an arylsulfatase protein is restricted. The present invention also relates to a method for producing a lactase preparation, which comprises culturing, recovering a culture solution with or without destroying cell walls, and preparing a lactase preparation. The case where the culture solution is collected without destroying the cell wall is a case where the genetically modified microorganism secretes lactase.

The preparation process of the lactase preparation may include purification steps performed in the art, such as concentration of lactase protein and removal of contaminants. The produced lactase preparation has, for example, an arylsulfatase activity (unit: U / g) according to the activity measuring method according to the present invention described above / a lactase activity (unit: NLU / g) by the FCC IV method of 0.1%. It is as follows.

Furthermore, the present invention relates to a dairy product produced using the lactase preparation according to the present invention.

Since a highly sensitive method for measuring arylsulfatase activity has been established, it has become possible to accurately grasp the amount of arylsulfatase in a lactase preparation using the activity as an index. In addition, a diploid strain of yeast having a lactase gene and restricted expression of arylsulfatase protein, or cultivation of a genetically modified microorganism into which yeast lactase gene is introduced and expression of arylsulfatase gene protein is restricted By using the liquid as a raw material, it has become possible to provide a lactase preparation containing a very small amount of arylsulfatase contamination or no arylsulfatase. If the lactase preparation of the present invention is used, the effect of suppressing the occurrence of an unpleasant taste and unpleasant taste in long life milk and the like can be obtained.

It is a graph which shows the measurement result of the aryl sulfatase activity by a colorimetric method. It is a graph which shows the measurement result of the arylsulfatase activity by a fluorescence method. It is a schematic diagram which shows the construction method of the arylsulfatase gene disruption vector pdSuC1. It is a schematic diagram which shows the construction method of the arylsulfatase gene disruption vector pdSuCM6. It is a schematic diagram showing a method for introducing two arylsulfatase gene disruption vectors. It is a photograph showing the results of Southern blotting of a strain containing two arylsulfatase genes, a strain in which one arylsulfatase gene was disrupted, and a strain in which two arylsulfatase genes were disrupted.

First, a method for measuring the activity of arylsulfatase will be described.

A conventionally known method for measuring the activity of arylsulfatase is a colorimetric method using a compound in which a sulfate group is bound to a chromophore such as p-nitrophenol as a substrate. In this method, the amount of chromophore released by the removal of sulfate groups from the substrate due to the reaction between the substrate and arylsulfatase is measured by absorbance. However, when the amount of liberated chromophore such as p-nitrophenol is small, the change in absorbance is small and it is difficult to obtain a clear measured value.

As a method for measuring the activity of arylsulfatase, a fluorescence method using a compound in which a sulfate group is bound to a fluorophore, such as 4-methylumbelliferyl sulfate, as a substrate is also known. The sensitivity of the fluorescence method is generally said to be at least 100 times that of the colorimetric method. Therefore, the present inventors have examined the reaction conditions and measurement conditions between the substrate and the enzyme and established a highly sensitive measurement method for the fluorescence method for measuring arylsulfatase activity. By carrying out this highly sensitive activity measurement method, the amount of arylsulfatase contaminated in the lactase preparation can be accurately grasped. As a result, it has become possible to provide lactase preparations having zero or very little arylsulfatase content.

The method for measuring the activity of arylsulfatase according to the present invention is as follows.

(1) A specimen in which the presence of arylsulfatase is predicted is appropriately diluted with a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5 N potassium chloride to prepare a sample.
(2) An aqueous solution containing 2-methylumbelliferone sulfate potassium at a concentration of 2 mM is prepared.
(3) The sample and a 4-methylumbelliferone sulfate aqueous solution are mixed at 1: 1 (volume basis) and reacted at 37 ° C. for 3 hours.
(4) To the reaction solution, add the same amount (volume basis) of 0.1N sodium hydroxide aqueous solution as the reaction solution to stop the reaction and prepare a measurement sample.
(5) The fluorescence intensity is measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.
(6) 4-Methylumbelliferone is dissolved in a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5N potassium chloride to obtain a solution with an appropriate concentration. An aqueous sodium oxide solution is added, and the fluorescence intensity is measured under the same conditions as in (5).
(7) A calibration curve is created from (6).
(8) From the fluorescence intensity measured in (5) and the calibration curve created in (7), the 4-methylumbelliferone concentration of the measurement sample is calculated, divided by 3, and the reaction time is 1 hour. If so, determine the 4-methylumbelliferone concentration. Further, the amount of 4-methylumbelliferone generated during the reaction for 1 hour is calculated from the volume of the reaction solution.
(9) Since the amount of 4-methylumbelliferone calculated in this way is based on the amount of sample contained in the sample prepared in (1), it is converted to the amount of 4-methylumbelliferone per gram of sample. To do.
(10) The case where the amount of 4-methylumbelliferone produced per 1 hour of the reaction time between the substrate and the enzyme is 1 nmole is defined as 1 unit (U), and the unit is the unit amount per 1 g of the specimen, that is, the enzyme preparation, That is, it is represented by “unit (U) / g”.

The lactase preparation according to the present invention was measured by the above method, and the calculated arylsulfatase activity was determined by the FCC IV method (Food Chemicals Codex Fourth Edition, Effective Jury 1, 1996, Committee on Food 2 Chemical 80. Chemp. 80 ) Based on lactase activity (unit: NLU / g), it is 0.1% or less, preferably 0.02% or less.

In the production of the lactase preparation of the present invention, a diploid strain of yeast that produces lactase protein, has a lactase gene, and is restricted in the expression of arylsulfatase protein is used. Such a yeast diploid strain is, for example, a mutant strain obtained by microbial mutation treatment. For example, such mutant strains can be treated by exposing yeast to UV irradiation or chemical mutagens to destroy the arylsulfatase gene or the arylsulfatase protein expression regulatory gene for both diploid genes. Alternatively, it can be obtained by a deletion method, a method of deleting an arylsulfatase gene or an expression regulator gene of an arylsulfatase protein by genetic engineering techniques, etc. for both diploid genes. Whether or not a desired mutant strain has been obtained may be determined by measuring the arylsulfatase activity of the cultivated yeast culture solution by the method for measuring arylsulfatase activity (fluorescence method) according to the present invention.

Induction of mutation by ultraviolet rays is performed, for example, by irradiating a suspension of diploid yeast with ultraviolet rays. In addition, chemical mutagenesis is performed by adding a chemical mutagen to a suspension of diploid yeast, for example. Examples of chemical mutagens include 5-bromouracil, 2-aminopurine, nitrous acid, hydroxylamine, acriflavine, methanesulfonic acid compound, nitrosoguanidine and the like.

In order to delete an arylsulfatase gene or an expression regulator gene of an arylsulfatase protein by genetic engineering techniques, a gene fragment having a homologous sequence of the gene to be deleted is obtained, and the fragment is subcloned into a vector and deleted. Ordinary genetic engineering techniques, such as constructing a disruption vector for a power gene and transforming a diploid strain of yeast using the vector, may be applied.

In the production of the lactase preparation of the present invention, a recombinant microorganism into which a yeast lactase gene is introduced so as to express a lactase protein and the expression of an arylsulfatase protein is restricted can also be used. As described above, “expression of arylsulfatase protein is restricted” means that, for example, a gene involved in the production of arylsulfatase protein is restricted, and more specifically, arylsulfatase gene and / or Because there is no aryl sulfatase protein expression regulatory gene, or because the aryl sulfatase gene (structural gene) is disrupted or the expression regulatory gene that causes the aryl sulfatase gene to express the aryl sulfatase protein is disrupted It means that arylsulfatase protein is not produced or its production amount is reduced.

A genetically modified microorganism into which a yeast lactase gene is introduced and the expression of arylsulfatase protein is restricted can be prepared by a known method. For example, a lactase gene is incorporated into a plasmid resistant to drug A and, if necessary, an expression control gene for the lactase gene is incorporated at the same time. The lactase expression plasmid thus prepared is used to transform a host microorganism. The transformed microorganism is cultured in a medium containing the drug A, and the appearing colonies are selected.

E. coli, yeast, Bacillus subtilis, etc. can be used as a host for obtaining a genetically modified microorganism into which a yeast lactase gene has been introduced and the expression of arylsulfatase protein is restricted.

As a host for obtaining a recombinant microorganism into which a yeast lactase gene has been introduced and the expression of arylsulfatase protein is restricted, a host that does not have an arylsulfatase gene or an arylsulfatase protein expression regulatory gene, or an arylsulfatase A host in which a gene or an arylsulfatase protein expression regulatory gene is disrupted or deleted is desirable.

A lactase for a lactase preparation is a yeast diploid strain that has a lactase gene and the expression of an arylsulfatase protein is restricted, or a gene set in which a yeast lactase gene is introduced and the expression of an arylsulfatase protein is restricted Purify from the culture solution of the replacement microorganism. Note that the concept of “culture solution” includes a culture supernatant and an extract from recovered cells.

Incubation of microorganisms such as yeast can be performed using an incubator such as a flask, miniger, tank, etc. As culture conditions, a temperature, pH, number of stirring, etc. suitable for enzyme production by the microorganisms are selected.

After completion of the culture, a culture solution in which lactase is dissolved is usually obtained by breaking the cell wall. In addition, when the microorganisms used for culture secrete lactase, the cell wall need not be destroyed.

The culture solution is usually separated into a culture supernatant and a residue by an appropriate method usually used in this technical field, such as centrifugation, filtration, or the like. The culture supernatant may be used as it is as an enzyme solution, or may be used as an enzyme solution after being concentrated using an ultrafiltration membrane or the like. Alternatively, a culture solution or culture supernatant that has been purified by subjecting it to one or more purification operations such as adsorption, solvent fractionation, ammonium sulfate fractionation, chromatography, crystallization, etc. may be used as an enzyme solution. Good. The enzyme solution may be pulverized by a method such as spray drying or freeze drying. The enzyme solution itself thus obtained can also be used as a lactase preparation.

The essential component of the lactase preparation according to the present invention is lactase. The lactase preparation contains other components as long as it is a substance that does not inhibit the activity of the lactase and does not inhibit the activity, or unless it has an undesirable effect on the intended use of the lactase preparation. May be present. Examples of substances that may be present include starches, dextrin, and buffering agents that are excipients for ease of use, such as metal salts that contribute to the stabilization of lactase, various sugars, ascorbic acid, glycerin, etc. Inorganic salts and the like. The properties of the lactase preparation are not particularly limited, and may be, for example, powder, granules, solutions and the like.

The present invention also relates to a dairy product produced using the lactase preparation according to the present invention. Dairy products refer to milk such as long life milk, yogurt, fresh cream, sour cream, cheese and the like. The lactase preparation is used in a usual manner or use amount in this technical field.

Hereinafter, the present invention will be described specifically by way of examples.

[Example 1] Measurement of arylsulfatase activity by a conventionally known colorimetric method GODO-YNL2 (Lactase preparation manufactured by Godo Shusei Co., Ltd.) is dissolved in distilled water to obtain a 1% (w / v) solution. It was. This 1% solution was diluted with 100 mM potassium phosphate buffer (pH 6.5) containing 0.5 N potassium chloride, 0.8% (w / v), 0.6% (w / v), 0.4% (w / v) and 0.2% (w / v) solutions were obtained. 0.5 mL of a 20 mM p-nitrophenyl sulfate solution in 100 mM potassium phosphate buffer (pH 6.5) was added to 0.5 mL of each solution and reacted at 37 ° C. for 3 hours. The reaction was stopped by adding 1.5 mL of a 1.5N aqueous sodium hydroxide solution, and the absorbance at 410 nm was measured.

The results are shown in FIG. In the colorimetric method, arylsulfatase activity could not be measured at all in a 1% (w / v) solution of the enzyme preparation.

Example 2 Measurement of Aryl Sulfatase Activity by Fluorescence Method GODO-YNL2 (Lactase preparation manufactured by Godo Sakesei Co., Ltd.) was dissolved in 100 mM potassium phosphate buffer (pH 6.5) containing 0.5 N potassium chloride. % (W / v) solution was obtained. This 1% solution was diluted with the same buffer and 0.8% (w / v), 0.6% (w / v), 0.4% (w / v) and 0.2% ( w / v) A solution was obtained.

0.5 mL of 2 mM 4-methylumbelliferyl sulfate aqueous solution was added to 0.5 mL of each solution and reacted at 37 ° C. for 3 hours. The reaction was stopped by adding 1 mL of a 0.1N sodium hydroxide aqueous solution, and the fluorescence intensity was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.

The results are shown in FIG. Unlike the colorimetric method, the fluorescence method showed good quantitativeness even with an enzyme preparation solution having a concentration of 1% (w / v) or less.

[Example 3] Comparison between colorimetric method and fluorescence method in measuring arylsulfatase activity The difference in sensitivity between the colorimetric method and fluorescence method in measuring arylsulfatase activity was examined. First, purified lactase used in this experiment was prepared.

(Preparation of purified lactase)
50 kg of GODO-YNL2 (Lactase preparation manufactured by Godo Seisei Co., Ltd.) was hydrolyzed and desalted using an ultrafiltration membrane (ACP membrane manufactured by Asahi Kasei Co., Ltd.) until the conductivity was 3 mSv or less. Water was added to make a total volume of 125L. Next, it was adsorbed on an ion exchange resin (DEAE Toyopearl 650M, 40 cmφ, 50 L, manufactured by Tosoh Corporation) that had been equilibrated in advance with 10 mM potassium phosphate buffer (pH 7). The plate was washed with 40 L of 10 mM potassium phosphate buffer (pH 7) containing 50 mM sodium chloride, and then lactase was eluted with 200 L of 10 mM potassium phosphate buffer (pH 7) containing 100 mM sodium chloride. At this time, the fraction was fractionated into 20 L portions. Lactase activity of each fraction (according to FCC IV method; Food Chemicals Codex Fourth Edition, Effective July 1, 1996, Committee on Food Chemicals p.801-802) and aryl sulfatase activity as described in detail below; ) Was collected, and the fraction in which arylsulfatase was reduced was collected, mixed, and concentrated using an ultrafiltration membrane (ACP membrane manufactured by Asahi Kasei Corporation) to obtain a lactase concentrate. Glycerin was added to this concentrated solution to 50% (w / w) to obtain a purified lactase preparation.

With regard to GODO-YNL2 and purified lactase preparations prepared as described above, both of which have lactase activity of 5000 to 5500 NLU / g by the FCC IV method, arylsulfatase activity (fluorescence method; details are as follows) ), The arylsulfatase activity of the purified lactase preparation was 1/840 before purification (see Table 1 below).

(Preparation of enzyme preparations with various arylsulfatase contamination rates)
The purified lactase preparation prepared as described above and GODO-YNL2 are appropriately mixed to prepare lactase preparations with various arylsulfatase contamination rates, and lactase activity (by FCC IV method) and arylsulfatase activity (colorimetric method) And by fluorescence method).

(Measurement of arylsulfatase activity by colorimetric method)
0.5 mL of a 20 mM p-nitrophenyl sulfate solution in 100 mM potassium phosphate buffer (pH 6.5) was added to 0.5 mL of the lactase preparation and reacted at 37 ° C. for 3 hours. The reaction was stopped by adding 1.5 mL of a 1.5N aqueous sodium hydroxide solution, and the absorbance at 410 nm was measured.

Separately, an aqueous solution containing p-nitrophenol at a concentration of 0 to 0.5 mM was prepared. 0.5 mL of a 100 mM potassium phosphate buffer solution (pH 6.5) was added to 0.5 mL of each of these solutions, and 1.5 mL of a 1.5N sodium hydroxide aqueous solution was further added to obtain a measurement sample. Absorbance at 410 nm was measured to prepare a calibration curve.

From the calibration curve, determine the concentration of p-nitrophenol contained in 1 mL of the reaction solution, and further divide by 3 (because the reaction time is 3 hours) to obtain p-nitrophenol when the reaction time is 1 hour. The concentration of was calculated. Next, from this concentration, the amount of p-nitrophenol contained in 1 mL of the reaction solution was calculated (unit: nmole), and further doubled (the amount of lactase preparation used was 0.5 g, and this amount per 1 g) The aryl sulfatase activity was calculated by the calculation. 1 U is an activity that generates 1 nmole of p-nitrophenol per hour, and arylsulfatase activity is expressed in units of “U / g-enzyme preparation”.

(Measurement of arylsulfatase activity by fluorescence method)
The lactase preparation was diluted with a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5N potassium chloride to obtain a 1% (w / v) solution. 0.5 mL of 2 mM 4-methylumbelliferyl sulfate aqueous solution was added to 0.5 mL of this 1% solution and reacted at 37 ° C. for 3 hours. The reaction was stopped by adding 1 mL of a 0.1N sodium hydroxide aqueous solution, and the fluorescence intensity was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.

Separately, a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5N potassium chloride containing 4-methylumbelliferone at a concentration of 0 to 4 μM was prepared. A sample for measurement was obtained by adding 1 mL of a 0.1 N aqueous sodium hydroxide solution to 1.0 mL of each of these solutions. The fluorescence intensity was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm to prepare a calibration curve.

From the calibration curve, determine the concentration of 4-methylumbelliferone contained in 1 mL of the reaction solution, and further divide by 3 (because the reaction time is 3 hours). -The absolute amount of methylumbelliferone was calculated. Furthermore, the arylsulfatase activity was calculated by 200 times (the amount of lactase preparation used was 0.5 × 0.01 = 0.005 g, which was converted to 1 g of the sample (lactase preparation)). 1 U is an activity that produces 1 nmole of 4-methylumbelliferone per hour, and arylsulfatase activity is expressed in units of “U / g-enzyme preparation”.

(result)
The results are shown in Table 1. In the colorimetric method, measurement could not be performed when the content of arylsulfatase decreased, but in the fluorescence method, it was possible to accurately measure to a concentration range of about 1/100 of the measurement limit of the colorimetric method. .

[Example 4] Comparison of fluorescence method for measuring arylsulfatase activity with the method described in WO07 / 060247 (Special Table 2009-517061) and taste sensory test (Part 1)
WO07 / 060247 states that a lactase preparation having 19 or less contaminating arylsulfatases (units defined in WO07 / 060247) does not produce off-flavors when added to decompose lactose after sterilization of milk. Are listed. However, this is a view based on the results of a test conducted by limiting the reaction days of lactase to 2 days. On the other hand, when a lactase preparation is added to actual long life milk, it is considered that the enzyme reaction proceeds over a long period of one month or longer. Therefore, lactase preparations containing arylsulfatase at various contamination rates were prepared, added to milk, and the flavor was confirmed after a predetermined number of days.

(Preparation of lactase preparations with various arylsulfatase contamination rates)
Lactase preparations A to E containing various contaminating amounts of arylsulfatase include the purified lactase preparation prepared in Example 3 and GODO-YNL2 containing 100 units of the selected arylsulfatase activity (units described in WO07 / 060247). Were mixed as appropriate.

The arylsulfatase activities of the prepared lactase preparations A to E were measured by the method described in WO07 / 060247 and the fluorescence method shown in Example 3. Moreover, the lactase activity was measured by FCC IV method. The results are shown in Table 2. Here, the unit described in WO07 / 060247 is ΔOD ₄₁₀ × 10 ⁶ / hour / NLU.

(Flavor sensory test)
Referring to Example 4 of WO07 / 060247, each of lactase preparations A to E was added to commercially available milk (heat pasteurized; pasteurized conditions: 130 ° C. for 2 seconds) so that lactase was 20000 NLU / L-milk. And stored at 30 ° C. After 2 days of storage, after 1 month, and after 3 months, a flavor sensory test was conducted on milk not added with lactase preparation and milk added with lactase preparation.

The flavor sensory test was conducted as a blind test. Eleven to thirteen panelists sniffed the milk after storage for a certain period of time, and also judged whether it had a smell that was uncomfortable and contained in the mouth. The evaluation was made with a score of 0 (-) for those that did not feel odor, 1 (+) for those that felt odor, and 2 (++) for those that felt strong. The summarized results are shown in Table 3.

In the reaction period of the lactase preparation (that is, the storage period of milk) for 2 days, only preparation E showed a clear off-flavor. This was consistent with the description in WO07 / 060247. However, when the reaction period was 1 month or longer, a bad odor was also detected in preparations C and D. This indicates that when considering the use of a lactase preparation in long-life milk or the like, even if the arylsulfatase activity is the unit described in WO07 / 060247 and the detection limit is 8 units, the taste and odor cannot be sufficiently controlled. It is.

On the other hand, in preparations A and B, no off-flavor was detected even when the reaction period was 1 month or 3 months. When considering the use of lactase preparations in long-life milk, it is necessary to assume a reaction period of one month or longer. Therefore, in such applications, it is preferable to use lactase preparation A or B (that is, those having arylsulfatase activity by the method of the present invention / actase activity by the FCC IV method of 0.02% or less), and lactase preparation A It became clear that the use of was more preferable. Moreover, it became clear that the arylsulfatase activity value comparable to lactase preparations A and B needs to be measured by the fluorescence analysis method described in this specification or an analysis method showing sensitivity equal to or higher than that. .

[Example 5] Acquisition of mutant strain with reduced arylsulfatase production ability Cliveromyces lactis G14-427 strain, a diploid strain, was added to 10 mL of YPD medium (1% yeast extract, 1% glucose, 2% peptone). 1 platinum ear was inoculated, and this bacterial suspension was stored at 30 ° C. and cultured. When the logarithmic growth phase was reached, the medium was centrifuged to recover the bacterial cells. The collected cells were dispersed in sterilized water so that the absorbance at 600 nm was 0.5. The bacterial suspension was irradiated with ultraviolet rays for 15 seconds with a UV lamp. The cells were collected by centrifugation and mixed and dispersed in the YPD medium. An appropriate amount was taken from the YPD medium containing the bacterial cells and applied to the YPD agar plate medium. Static culture was performed at 37 ° C. for 7 days. A small amount of the grown colonies were scraped, and this was mixed with 1 mL of a solution containing zymolyce (manufactured by Seikagaku Biobusiness Co., Ltd.) at 1 mg / mL. The reaction was carried out at 30 ° C. for 2 hours to destroy the cell wall. Thereafter, centrifugation was performed and the supernatant was collected.

The lactase activity (FCC IV method) and arylsulfatase activity (by the fluorescence method described in Example 3) of the supernatant were measured. The arylsulfatase activity / lactase activity was calculated, and the one with a small value was selected.

Mutations in which one of the two arylsulfatase genes present in the diploid strain Kriveromyces lactis G14-427 is dysfunctional by repeating the above mutation treatment and selection for the selected strain. (SM1182 strain). The determination that “one of the two arylsulfatase genes became dysfunctional” is that the arylsulfatase activity value of the SM1182 culture supernatant is the aryl of the culture supernatant of the parent strain G14-427. This is because it was about ½ of the sulfatase activity value.

Further, the obtained mutant strain (SM1182 strain) was subjected to mutation treatment as a parent strain, and the other arylsulfatase gene also became dysfunctional, that is, a mutant strain having an arylsulfatase activity of 0 was obtained (SF-81 strain) ).

The parent strain Kriveromyces lactis G14-427 and two mutant strains obtained as described above were each cultured with shaking in a YPD medium (70 mL / flask) at 26 ° C. for 4 days.

Thereafter, Zymoly Ace (manufactured by Seikagaku Biobusiness Co., Ltd.) was added to the culture solution so as to be 2 mg / mL, and reacted at 30 ° C. for 2 hours to destroy the cell wall. The supernatant was collected by centrifugation, and lactase activity was measured by the FCC IV method, and arylsulfatase activity was measured by the method described in Example 3. The results are shown in Table 4.

[Example 6] Acquisition of host strain The diploid strain Kriveromyces lactis G14-427 was inoculated with 1 platinum ear in 10 mL of YPD medium and grown at 30 ° C until the logarithmic growth phase. The medium was centrifuged and the cells were collected. The collected cells were dispersed in sterilized water so that the absorbance at 600 nm was 0.5. The bacterial suspension was irradiated with ultraviolet rays for 15 seconds with a UV lamp. The cells were collected by centrifugation and mixed and dispersed in the YPD medium. An appropriate amount was taken from the YPD medium containing the bacterial cells and applied to the YPD agar plate medium. Static culture was performed at 37 ° C. for 4 days. The grown colonies were replicated in SD medium (east nitrogen base containing no 0.67% amino acid, 2% glucose, 2% agar), and those that did not grow were selected.

Those strains that do not grow on the SD medium are applied to an SD medium containing 20 mg / L L-methionine from the original YPD agar plate medium, and those that grow are referred to as L-methionine-requiring strains (7-19 strains). did.

7-19 strains were inoculated with 1 platinum ear in 10 mL of YPD medium and grown at 30 ° C. until the logarithmic growth phase. The medium was centrifuged and the cells were collected. The collected cells were dispersed in sterilized water so that the absorbance at 600 nm was 0.5. The bacterial suspension was irradiated with ultraviolet rays for 15 seconds with a UV lamp. The cells were collected by centrifugation and mixed and dispersed in the YPD medium. An appropriate amount was taken from the YPD medium containing the bacterial cells and applied to the YPD agar plate medium. Static culture was performed at 37 ° C. for 4 days. The grown colonies were replicated in an SD medium containing 20 mg / L L-methionine, and those that did not grow were selected.

Those strains that do not grow on the SD medium containing L-methionine are applied to the SD medium containing 20 mg / L L-histidine and 20 mg / L L-methionine from the original YPD agar plate medium and grow. Was L-methionine and L-histidine double auxotrophic strain (8-23 strain).

Table 5 shows the growth of the acquired auxotrophic mutants.

[Example 7] Acquisition of gene double disruption strain that does not produce arylsulfatase (acquisition of host strain)
For the 8-23 strain, which is the L-histidine and L-methionine double auxotrophic mutant strain obtained in Example 6, the respective auxotrophy is complemented by the HIS4 gene and the MET6 gene introduced by the lithium acetate method. I was sure that.

(Acquisition of genomic DNA)
The diploid strain Kriveromyces lactis G14-427 was cultured in YPD medium. Genomic DNA was prepared from the obtained culture solution using Gen Torukun ^TM (for yeast) (Takara Bio Inc.). The operation was performed according to the description of Gen Torukun ^TM (for yeast).

(Construction of arylsulfatase gene disruption vector)
Primers SuC-F and SuC-R were designed so that a fragment containing the open reading frame of the arylsulfatase gene was obtained. The primer sequences used, including these, were as shown in Table 6.

Using the genomic DNA prepared previously as a template, PCR was performed using the above primers under the following conditions to obtain a DNA fragment. In addition, Takara Ex Taq (registered trademark; manufactured by Takara Bio Inc.) was used as the polymerase, and the operation was performed according to the attached document.

(PCR conditions)
Stage 1 (1 cycle) 94 ° C. 3 minutes Stage 2 (30 cycles) 94 ° C. 1 minute 54 ° C. 1 minute 72 ° C. 3 minutes Stage 3 (1 cycle) 72 ° C. 10 minutes Hold at 4 ° C.

The obtained fragment was purified by MagExtractor ^™ -PCR & Gel Clean up- (manufactured by Toyobo Co., Ltd.), and then ligated with a pGEM (registered trademark) -T vector (manufactured by Promega). For the ligation reaction, DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio Inc.) was used. Usage was in accordance with each package insert. Thereafter, E. coli prepared by the Hanahan method (Hanahan, D., J. Mol. Biol., 166, 557 (1983)). A competent cell of ColiDH5α strain was transformed, and a plasmid was extracted from the obtained transformant culture solution using MagExtractor ^™ -Plasmid- (manufactured by Toyobo Co., Ltd.). Usage followed the package insert. As a result, a plasmid pGSuC in which the target fragment was subcloned was obtained.

Furthermore, PCR was performed using primers BGLHIS4-F and HIS4-R using genomic DNA as a template to obtain a fragment containing the HIS4 gene. For the PCR reaction, the method described above was followed. As shown in FIG. 3, the obtained fragment containing the HIS4 gene was treated with Bgl II and EcoRI and inserted into the Bgl II-EcoRI site of pGSuC. The plasmid constructed in this way was named pdSuC1. Various methods at the time of construction were the same as those described above.

In addition, in order to construct an arylsulfatase gene disruption vector using the MET6 gene as a marker, primers SuCd-M6F and SuCd-M6R were designed by adding 40 bases of the homologous sequence of the arylsulfatase gene to the 5 ′ side. The homologous sequence of the arylsulfatase gene was in the vicinity of the restriction enzyme ClaI site present in two places on the open reading frame. As shown in FIG. 4, using these primers, a fragment having an arylsulfatase homologous sequence at both ends of the MET6 gene was obtained using genomic DNA as a template. The obtained fragment was subcloned into pGEM (registered trademark) -T vector (Promega) to obtain pdSuCM6. Various methods at the time of construction were the same as those described above.

(Transformation of L-methionine and L-histidine double auxotrophic strain 8-23 using an arylsulfatase gene disruption vector)
FIG. 5 shows a schematic diagram of the construction of the transformant. The plasmid pdSuC1 is linearized by treatment with NcoI and AatII, and the strain 8-23 is transformed by the lithium acetate method using this, and the transformant SuCD grows in SD medium supplemented with 20 μg / ml methionine. Acquired shares.

Next, the plasmid pdSuCM6 was treated with ClaI to form a linear form, and the SuCD strain was transformed by the lithium acetate method using it to obtain a transformant SuCDD5-2 strain that grew on the SD medium.

(Southern blotting of parental and transformed DNA)
Each of the obtained transformants was cultured in a YPD medium, and genomic DNA was prepared from the culture solution using Gentorikun ^TM (for yeast) (manufactured by Takara Bio Inc.). The obtained genomic DNA was digested with BamHI and then subjected to Southern analysis. The probe used was an AatII-EcoRI fragment of an arylsulfatase gene, and for labeling and detection of nucleic acids, AlkPhos Direct Labeling and Detection System with CDP-Star (manufactured by GE Healthcare Biosciences) was used. I followed.

The result of Southern blotting is shown in FIG. Lane 1 is the parent strain G14-427, Lane 2 is the transformant SuCDD5-2 strain, and Lane 3 is the transformant SuCD strain. In lane 3, a band (12.1 kb) of the fragment containing the arylsulfatase gene and the HIS4 gene was detected at the same position (7.8 kb) as in lane 1, and only one of the arylsulfatase genes was destroyed. It was confirmed that On the other hand, in lane 2, the 7.8 kb band was shifted to 5.3 kb, confirming that it was an arylsulfatase gene double disruption strain.

(Detection of arylsulfatase activity in an arylsulfatase gene double disruption strain)
The parent diploid strain Kriveromyces lactis G14-427 and the SuCDD5-2 strain constructed as described above were inoculated in a YPD medium and cultured with shaking at 30 ° C. and 210 rpm for 72 hours. Zymoly ace (manufactured by Seikagaku Biobusiness Co., Ltd.) was added to the culture solution so as to be 2 mg / ml, and reacted at 30 ° C. for 2 hours to destroy the cell wall. The supernatant was collected by centrifugation, and lactase activity was measured by the FCC IV method, and arylsulfatase activity was measured by the method described in Example 3. The results are shown in Table 7.

The culture medium of the SuCDD5-2 strain contains lactase, but does not contain arylsulfatase, or even if it is contained, the amount of the activity may not be detected by fluorescence measurement. It became clear. That is, it was confirmed that the SuCDD5-2 strain maintained lactase productivity but had no or almost no arylsulfatase productivity.

[Example 8] Preparation of enzyme preparation The strain SF-81 obtained in Example 5 was inoculated into a lactase production medium containing 7% corn steep liquor and 2% lactose, and the mixture was incubated at 30 ° C and 210 rpm for 96 hours. After shaking culture, the cells were collected by centrifugation. Sterile purified water was added to the cells, the cell walls of the collected cells were broken with glass beads and ultrasonic waves, and the supernatant was collected by centrifugation. The supernatant was fractionated with ammonium sulfate and concentrated with an ultrafiltration membrane to obtain a lactase preparation having a lactase activity of 5000 NLU / g. The arylsulfatase activity of this lactase preparation was 1 U / g or less according to the measurement method (fluorescence method) of the present invention.

[Example 9] Sensory sensory test (2)
(Preparation of lactase preparations with various arylsulfatase contamination rates)
A lactase preparation itself produced from the SF-81 strain by the method described in Example 8 and an appropriate mixture of GODO-YNL2 and an arylsulfatase activity measured by the fluorescence method described in Example 3 are 1 to 20 U / 5 lactase preparations were prepared. Moreover, the lactase activity of each lactase preparation was measured by FCC IV method.

(Flavor sensory test)
As in Example 4, each of the above 5 lactase preparations was added to commercially available milk so that the lactase was 20000 NLU / L-milk and stored at 30 ° C. After 1 month of storage, a flavor sensory test was performed in the same manner as in Example 4 to compare milk without a lactase preparation and milk with a lactase preparation. The results are shown in Table 8.

From the results shown in Table 8, it was found that the arylsulfatase activity measured by the method according to the present invention is preferably 5 U / g or less. The ratio of arylsulfatase activity (unit: U / g) measured by the method according to the present invention based on lactase activity (unit: NLU / g) by FCC IV method may be 0.1% or less. It became clear that it was preferable.

Claims

A yeast diploid strain that has a lactase gene and expression of arylsulfatase protein is restricted, or a culture solution of a genetically modified microorganism into which yeast lactase gene is introduced and expression of arylsulfatase protein is restricted A lactase preparation produced as above, wherein the calculated arylsulfatase activity (unit: U / g) measured by the following method based on the lactase activity (unit: NLU / g) by the FCC IV method was 0.1 % Lactase preparation characterized by:
(Measurement method of arylsulfatase activity)
(1) A specimen in which the presence of arylsulfatase is predicted is appropriately diluted with a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5 N potassium chloride to prepare a sample.
(2) An aqueous solution containing 2-methylumbelliferone sulfate potassium at a concentration of 2 mM is prepared.
(3) The sample and a 4-methylumbelliferone sulfate aqueous solution are mixed at 1: 1 (volume basis) and reacted at 37 ° C. for 3 hours.
(4) To the reaction solution, add the same amount (volume basis) of 0.1N sodium hydroxide aqueous solution as the reaction solution to stop the reaction and prepare a measurement sample.
(5) The fluorescence intensity is measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.
(6) 4-Methylumbelliferone is dissolved in a 100 mM potassium phosphate buffer (pH 6.5) containing 0.5N potassium chloride to obtain a solution with an appropriate concentration. An aqueous sodium oxide solution is added, and the fluorescence intensity is measured under the same conditions as in (5).
(7) A calibration curve is created from (6).
(8) From the fluorescence intensity measured in (5) and the calibration curve created in (7), the 4-methylumbelliferone concentration of the measurement sample is calculated, divided by 3, and the reaction time is 1 hour. If so, determine the 4-methylumbelliferone concentration. Further, the amount of 4-methylumbelliferone generated during the reaction for 1 hour is calculated from the volume of the reaction solution.
(9) Since the amount of 4-methylumbelliferone calculated in this way is based on the amount of sample contained in the sample prepared in (1), it is converted to the amount of 4-methylumbelliferone per gram of sample. To do.
(10) The case where the amount of 4-methylumbelliferone produced per 1 hour of the reaction time between the substrate and the enzyme is 1 nmole is defined as 1 unit (U), and the unit is the unit amount per 1 g of the specimen, that is, the enzyme preparation, That is, it is represented by “unit (U) / g”.
The lactase preparation according to claim 1, wherein the yeast is Krivellomyces lactis or Krivellomyces marixianus.
The lactase preparation according to claim 1 or 2, wherein an arylsulfatase activity (unit: U / g) based on lactase activity (unit: NLU / g) by FCC IV method is 0.02% or less.
The diploid strain of yeast having a lactase gene and restricted expression of arylsulfatase protein is a mutant obtained by mutation treatment of a yeast diploid strain having a lactase gene. The lactase preparation according to any one of 3 above.
Deletion of an aryl sulfatase gene or an aryl sulfatase protein expression regulatory gene of a yeast diploid strain having a lactase gene and restricted expression of an aryl sulfatase protein. The lactase preparation according to any one of claims 1 to 3, which is a mutant obtained by the method.
Culturing a diploid strain of yeast having a lactase gene and restricted expression of arylsulfatase protein or a genetically modified microorganism introduced with a yeast lactase gene and restricted in expression of arylsulfatase protein. A method for producing a lactase preparation, comprising: recovering a culture solution without destroying or destroying the preparation to prepare a lactase preparation.
A dairy product produced using the lactase preparation according to any one of claims 1 to 5.