Thermally stable glucocerebrosidase

Abstract

To provide a protein having glucocerebrosidase activity and also having thermostability.SOLUTION: The aforementioned problem is solved by a protein that is plant-derived, belongs to the glycoside hydrolase family 1 (GH1), and has glucocerebrosidase activity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a protein having glucocerebrosidase activity and thermal stability (hereinafter sometimes referred to as "thermally stable glucocerebrosidase"), an enzyme composition containing this protein, a pharmaceutical composition or food composition, and a method for producing ceramide using this protein. [Background technology]

[0002] Glucocerebrosidase is a well-known enzyme that hydrolyzes glucosylceramide, a type of glycolipid, into ceramide. This glucocerebrosidase is mainly found in animals and plays an important role in producing ceramide from glucosylceramide in the animal body, but its presence in plants is almost unknown.

[0003] In humans, there is a congenital metabolic disorder (Gaucher disease) in which the glucocerebrosidase gene is congenitally deficient, preventing the conversion of glucosylceramide into ceramide in the body. In Gaucher disease, glucosylceramide accumulates abnormally in the body, leading to various symptoms such as enlargement of the liver and spleen, anemia, decreased platelet count, and bone abnormalities (Non-patent documents 1 and 2).

[0004] It is estimated that there are more than 5,000 people worldwide suffering from Gaucher disease. The primary treatment involves intravenously replenishing the deficient glucocerebrosidase in the body and converting accumulated glucosylceramide into ceramide. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Annual Review of Genomics and Human Genetics 4,403-436 (2003) [Non-Patent Document 2] British Journal of Haematology 129,178-188 (2005) [Overview of the project] [Problems that the invention aims to solve]

[0006] Currently, the enzyme preparations used as intravenous medications for Gaucher disease primarily utilize imiglucerase, a human-derived glucocerebrosidase produced in the ovarian cells of Chinese hamsters using genetic engineering technology. However, this imiglucerase is expensive and has poor thermal stability, requiring replenishment every two weeks, which poses a significant financial burden for patients.

[0007] Furthermore, since animal-derived glucocerebrosidases are enzymes that act at or near the body temperature of animals, they all have low thermal stability, similar to the imiglucerases mentioned above. Therefore, if there were a glucocerebrosidase that was thermally stable and required fewer supplementation sessions, its medical value would be immeasurable, but no such glucocerebrosidase has been reported yet.

[0008] On the other hand, ceramides, which are present in the stratum corneum of human skin, are essential components for maintaining skin moisture and are used in cosmetics. However, ceramides used as active ingredients in cosmetics are usually produced through chemical synthesis. It might be conceivable to produce ceramide by breaking down glucosylceramide, which is present in high concentrations in animals and plants and is relatively inexpensive, using glucocerebrosidase, and then using this ceramide in cosmetics and reagents. However, the aforementioned imiglucerase is very expensive and has low thermal stability, so it has not been put into practical use. Furthermore, the ceramide content in animals and plants is very low, unlike glucosylceramide, so directly extracting and purifying this ceramide would incur enormous costs.

[0009] Therefore, the present invention aims to provide a protein that has glucocerebrosidase activity and is also thermally stable. [Means for solving the problem]

[0010] To solve the above problems, the inventors diligently conducted research and discovered a plant-derived protein belonging to the glycoside hydrolase family 1 and possessing glucocerebrosidase activity. Furthermore, they found that this protein is thermally stable, thus completing the present invention. To date, there have been no reports of glucocerebrosidases belonging to GH1 being found in plants.

[0011] In other words, the present invention is as follows <1> ~ <17> That is the case. <1> A plant-derived protein belonging to the glycoside hydrolase family 1 and possessing glucocerebrosidase activity. <2> The aforementioned plant is a seed plant. <1> The protein described above. <3> The aforementioned seed plant is one of the following: Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Solanaceae, Rosaceae, Amaryllidaceae, Fabaceae, or Liliaceae. <2> The protein described above. <4> The proteins shown in (A), (B), or (C) below. (A) A protein consisting of the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38-521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19-503 of Sequence ID No. 2 in the sequence listing. (B) A protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38-521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19-503 of Sequence ID No. 2 in the sequence listing, and which belongs to the glycoside hydrolase family 1 and has glucocerebrosidase activity and thermal stability. (C) A protein having 60% or more homology to the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38-521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19-503 of Sequence ID No. 2 in the sequence listing, belonging to the glycoside hydrolase family 1, and possessing glucocerebrosidase activity and thermal stability. <5> <1> ~ <4> DNA that codes for any one of the proteins listed in either of the following. <6> DNA as shown in (a), (b), or (c) below. (a) DNA encoding a protein belonging to glycoside hydrolase family 1 and possessing glucocelerosidase activity, consisting of the nucleotide sequences shown in Sequence ID No. 3, nucleotide numbers 112-1566 of Sequence ID No. 3, Sequence ID No. 4, or nucleotide numbers 55-1512 of Sequence ID No. 4 in the sequence listing. (b) DNA encoding a protein belonging to glycoside hydrolase family 1 and possessing glucocerebrosidase activity and thermal stability, consisting of a sequence in which one or more bases have been substituted, deleted, inserted, or added in the sequence shown in Sequence ID No. 3, Sequence ID No. 3 with base numbers 112-1566, Sequence ID No. 4, or Sequence ID No. 4 with base numbers 55-1512 of the sequence listing. (c) DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 3, base numbers 112-1566 of Sequence ID No. 3, Sequence ID No. 4, or base numbers 55-1512 of Sequence ID No. 4, and which encodes a protein belonging to the glycoside hydrolase family 1 and possessing glucocerebrosidase activity and thermal stability. <7> <5> or <6> An expression vector for expressing a protein belonging to the glycoside hydrolase family 1, containing the DNA described above, and possessing glucocerebrosidase activity and thermal stability. <8> <7> A transformant into which the expression vector described above has been introduced. <9> The transformed organism is a plant, plant cell, animal cell, Escherichia coli, yeast, or filamentous fungus. <8> The transformed organism described above. A method for producing a protein, comprising the steps of breeding or culturing the transformant according to <10><8> or <9>, and recovering the protein according to any one of <1> to <4> from the transformant or the transformant-containing material obtained by this step. <11>An enzyme composition for glucosylceramide hydrolysis, containing the protein according to any one of <1> to <4>. <12>A pharmaceutical composition containing the protein according to any one of <1> to <4> as an active ingredient. <13>The pharmaceutical composition according to <12>, which is for the prevention and treatment of Gaucher's disease. <14>A food composition containing the protein according to any one of <1> to <4> and glucosylceramide isolated from plants, animals, or microorganisms or chemically synthesized glucosylceramide. <15>A method for producing ceramide, comprising the step of generating ceramide from glucosylceramide isolated from plants, animals, or microorganisms or chemically synthesized glucosylceramide using the protein according to any one of <1> to <4>.

[0012] <16>A method for preventing or treating Gaucher's disease, characterized by administering (such as by intravenous drip) the protein according to any one of <1> to <4> or the pharmaceutical composition according to <12> or <13> to a Gaucher's disease patient. <17>The method for preventing or treating Gaucher's disease according to <16>, wherein the protein or the pharmaceutical composition is administered to a Gaucher's disease patient once every 3 to 10 weeks so that the glucocerebrosidase activity is 10 to 200 U / kg of body weight.

Advantages of the Invention

[0013] According to the present invention, it is possible to provide a protein belonging to glycoside hydrolase family 1 and having glucocerebrosidase activity and thermal stability. And it is also possible to provide an enzyme composition, a pharmaceutical composition, and a food composition containing this protein, and further to provide a method for producing ceramide using this protein.

Brief Description of the Drawings

[0014] [Figure 1] A graph showing the relationship between the incubation time and the relative residual activity of rice-derived glucocerebrosidase (RGC1) and human-derived glucocerebrosidase (imiglucerase) at 37°C (pH 5). [Figure 2] A graph showing the relationship between the incubation time and the relative residual activity of rice-derived glucocerebrosidase (RGC1) and human-derived glucocerebrosidase (imiglucerase) at 37°C (pH 7). [Figure 3] A graph showing the relationship between the incubation time and the relative residual activity of soybean-derived glucocerebrosidase (SGC1) and human-derived glucocerebrosidase (imiglucerase) at 45°C (pH 5). [Figure 4] A graph showing the relationship between the incubation time and the relative residual activity of soybean-derived glucocerebrosidase (SGC1) and human-derived glucocerebrosidase (imiglucerase) at 45°C (pH 7). [Figure 5] The amino acid sequence of rice-derived glucocerebrosidase (RGC1). [Figure 6] The nucleotide sequence of DNA encoding rice-derived glucocerebrosidase (RGC1). [Figure 7] The amino acid sequences of nine peptide fragments obtained by trypsin treatment of rice-derived glucocerebrosidase (RGC1). [Figure 8] The nucleotide sequences of the F-primers and R-primers used for cloning DNA encoding rice-derived glucocerebrosidase (RGC1). [Figure 9] The amino acid sequence of soybean-derived glucocerebrosidase (SGC1). [Figure 10] The nucleotide sequence of DNA encoding soybean-derived glucocerebrosidase (SGC1). [Figure 11]These are the amino acid sequences of seven peptide fragments obtained by trypsin treatment of soybean-derived glucocerebrosidase (SGC1). [Figure 12] These are the base sequences of the F-primer and R-primer used to clone the DNA encoding soybean-derived glucocerebrosidase (SGC1). [Modes for carrying out the invention]

[0015] The present invention will now be described. The present invention relates to a plant-derived protein belonging to the glycoside hydrolase family 1 and possessing glucocerebrosidase activity (hereinafter referred to as "the protein of the present invention" or "the heat-stable glucocerebrosidase of the present invention"), DNA encoding the protein of the present invention and an expression vector containing this DNA, a transformant into which this expression vector has been introduced and a method for producing the protein of the present invention using this transformant, an enzyme composition containing the protein of the present invention, a pharmaceutical composition or food composition, and a method for producing ceramide using the protein of the present invention, etc.

[0016] First, the protein of the present invention will be described in detail. The protein of the present invention is plant-derived, belongs to the glycoside hydrolase family 1 (GH1), and is a protein that possesses glucocerebrosidase activity. Furthermore, this plant-derived GH1-belonging protein with glucocerebrosidase activity is thermally stable.

[0017] Here, "glucocerebrosidase activity" refers to the enzymatic activity of the enzyme (glucocerebrosidase) with EC number (EC3.2.1.45), specifically the activity that catalyzes the hydrolysis of the β-1,4-glycosyl bond between glucose and ceramide in the glycolipid glucosylceramide to produce ceramide. Furthermore, "Glycoside Hydrolase family 1" is one of the families of carbohydrate hydrolases classified into approximately 130 categories by the Carbohydrate Active enzyme database (CAZy database, http: / / www.cazy.org / ), and glucocerebrosidases are known to belong to glycoside hydrolase family 1 (GH1), glycoside hydrolase family 30 (GH30), and glycoside hydrolase family 116 (GH116).

[0018] Furthermore, "plant-derived" means that the protein is expressed from the genes of a plant, and these plant genes can be obtained from, for example, at least one selected from the group consisting of plant leaves, stems, roots, seeds, fruits, petals, pistils, pollen (stamens), rhizoids, and sporangia. Furthermore, "thermal stability" refers to the ability to retain 80% or more of the glucocerebrosidase activity when incubated at 37°C for 30 hours in a solution containing a protein with glucocerebrosidase activity at a level of 0.0002 to 0.0008 U / mL in a 50 mM acetate buffer (pH 5.0) containing 0.1% Triton X-100 (registered trademark, hereinafter the same) and 0.05% sodium cholate (where the relative residual activity is 80% or more, with the glucocerebrosidase activity before incubation set to 100%). Preferably, this thermal stability is achieved when the relative residual activity is 90% or more. Here, the glucocerebrosidase activity was measured as follows. Specifically, a predetermined amount of the sample is added to 50 mM acetate buffer (pH 5.5) containing 100 μM glucosylceramide (cerebroside B, (4E,8E)-ND-2'-hydroxypalmitoyl-1-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine ("E", "N", and "O" are italicized, "D" is a small uppercase letter): The Journal of Antibiotics 41, (1988) 469-480, the same applies hereafter), 0.1% Tween 20, and 0.05% sodium cholate. The mixture is incubated at 37°C for 15-60 minutes. Then, four times the volume of ethanol is added to the resulting enzyme reaction solution and mixed. The mixture is then centrifuged at 15,000 rpm for 20 minutes, and the supernatant is subjected to high-performance liquid chromatography analysis to measure the amount of ceramide produced by the enzymatic reaction. Other conditions necessary for measuring glucocerebrosidase activity will be carried out according to the method described in Example 2 below.

[0019] Furthermore, the protein of the present invention is preferably derived from seed plants. Seed plants are not limited to, but include Malvaceae, Chenopodiaceae, Rubiaceae, Cannabaceae, Hydrangeaceae, Brassicaceae, Iridaceae, Poaceae, Araliaceae, Cucurbitaceae, Anacardiaceae, Cyperaceae, Campanulaceae, Asteraceae, Lauraceae, Moraceae, Papaveraceae, Araceae, Cactaceae, Lamiaceae, Nymphaeaceae, Apiaceae, Polygonaceae, Ericaceae, Specific examples include plants from the Camellia family, Solanaceae family, Caryophyllaceae family, Ulmaceae family, Nelumbonaceae family, Rosaceae family, Nelumbonaceae family, Amaryllidaceae family, Convolvulaceae family, Vitaceae family, Fagaceae family, Paeoniaceae family, Fabaceae family, Rutaceae family, Pontederiaceae family, Oleaceae family, Palm family, Salicaceae family, Liliaceae family, Orchidaceae family, Taxaceae family, Ginkgoaceae family, Cupressaceae family, Cycadaceae family, Cupressaceae family, and Pinaceae family.

[0020] To give more specific examples of seed plants, there are cotton, hibiscus, spinach, goosefoot, beet, madder, gardenia, coffee plant, hemp, hops, hydrangea, Arabidopsis thaliana, rapeseed, radish, Chinese cabbage, cabbage, cauliflower, broccoli, komatsuna, bok choy, wasabi, iris, Japanese iris, Japanese iris, rice, timothy, wheat, corn, sorghum, barley, rye, sugarcane, oats, barnyard millet, foxtail millet, grass, reed, bamboo, dwarf bamboo, and tadpoles. Lani tree, Udo, Fatsia japonica, Cucumber, Bitter melon, Melon, Watermelon, Goya, Pumpkin, Loofah, Winter melon, Gourd, Moonflower, Poison ivy, Japanese wax tree, Star grass, Balloon flower, etc., Coreopsis, Lettuce, Gerbera, Gazania, Thistle, Burdock, Sunflower, Cosmos, Dandelion, Calendula, Butterbur, Ragweed, Camphor tree, Bay laurel, Mulberry, Fig, Poppy, Water lily, Taro, Cactus, Perilla, Salvia, Lavender, Water lily, Carrot, Japanese parsley, Se Loli, buckwheat, knotweed, azalea, blueberry, rhododendron, camellia, tomato, eggplant, tobacco, petunia, chili pepper, potato, carnation, baby's breath, chickweed, zelkova, hackberry, lotus, rose, cherry blossom, almond, apricot, strawberry, plum, apple, pear, loquat, peach, lotus, garlic, leek, onion, bindweed, morning glory, sweet potato, grape, beech, sawtooth oak, chestnut, peony, soybean, broad bean, black bean, wisteria, Japanese wisteria, rupee The list includes eggplant, green beans, peas, alfalfa, peanuts, sweet peas, lotus, Satsuma mandarin, Japanese pepper, summer orange, orange, lime, lemon, grapefruit, trifoliate orange, water hyacinth, olive, jasmine, coconut palm, oil palm, date palm, palm tree, poplar, willow, lily, dwarf lily, tulip, narcissus, phalaenopsis orchid, cattleya, vanilla, yew, Japanese iris, ginkgo, cedar, cycad, cypress, pine, and others.

[0021] Among these, proteins belonging to GH1 derived from any of the following plants—Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Solanaceae, Rosaceae, Amaryllidaceae, Fabaceae, or Liliaceae—and possessing glucocerebrosidase activity are particularly suitable due to their excellent thermal stability. For example, proteins belonging to GH1 derived from rice or soybeans and possessing glucocerebrosidase activity are extremely suitable in terms of both their glucocerebrosidase activity and thermal stability.

[0022] Furthermore, the heat-stable glucocerebrosidase of the present invention, derived from rice, consists of the amino acid sequence shown in Sequence ID No. 1 of the sequence listing, or the amino acid sequence shown from amino acid numbers 38 to 521 in Sequence ID No. 1, which is the amino acid sequence after the signal peptide has been cleaved. In addition, the heat-stable glucocerebrosidase of the present invention, derived from soybeans, consists of the amino acid sequence shown in Sequence ID No. 2 of the sequence listing, or the amino acid sequence shown from amino acid numbers 19 to 503 in Sequence ID No. 2, which is the amino acid sequence after the signal peptide has been cleaved.

[0023] Furthermore, the present invention also includes proteins that belong to GH1 and possess glucocerebrosidase activity and thermal stability, which consist of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added in any of the amino acid sequences shown in Sequence ID No. 1 of Sequence ID No. 1, amino acid numbers 38 to 521 of Sequence ID No. 2 of Sequence ID No. 2, or amino acid numbers 19 to 503 of Sequence ID No. 2. In this case, it is preferable that the protein is of plant origin, but it does not have to be (the "proteins of the present invention" described later may also include those that are not of plant origin). Here, "a few" means 10 or fewer, preferably 6 or fewer, and more preferably 5 or fewer.

[0024] Furthermore, these amino acid substitutions, deletions, insertions, or additions are conservative. That is, they involve substituting, deleting, inserting, or adding one or more amino acid residues in a way that does not substantially alter the properties of the protein. Examples include substituting a hydrophobic amino acid residue with another hydrophobic amino acid residue, or substituting a polar amino acid residue with another polar amino acid residue having the same charge. Specifically, examples of functionally similar amino acids include hydrophobic (nonpolar) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Among polar amino acids, neutral amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Basic amino acids include arginine, histidine, and lysine. Acidic amino acids include aspartic acid and glutamic acid.

[0025] Furthermore, the present invention also includes proteins that have 60% or more, more preferably 62% or more, even more preferably 65% ​​or more, even more preferably 67% or more, even more preferably 70% or more, even more preferably 73% or more, and even more preferably 77% or more homology to the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38-521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19-503 of Sequence ID No. 2 in the sequence listing, belong to GH1, and possess glucocerebrosidase activity and thermal stability. In this case as well, it is preferable that the protein is of plant origin, but it does not have to be plant origin. Here, "homology" refers to a numerical value calculated using the default parameters in the homology search program EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ).

[0026] Next, the DNA encoding the protein of the present invention and a method for producing the protein using this DNA will be described in detail.

[0027] The DNA encoding the protein of the present invention is not limited to naturally occurring DNA or DNA synthesized using a portion of naturally occurring DNA, as long as it is composed of a base sequence capable of expressing the protein. For example, as the DNA encoding the heat-stable glucocerebrosidase of the present invention derived from rice as described above, DNA consisting of the base sequence shown in Sequence ID No. 3 or base numbers 112 to 1566 of Sequence ID No. 3 in the sequence listing is shown. Also, as the DNA encoding the heat-stable glucocerebrosidase of the present invention derived from soybeans as described above, DNA consisting of the base sequence shown in Sequence ID No. 4 or base numbers 55 to 1512 of Sequence ID No. 4 in the sequence listing is shown.

[0028] Furthermore, examples include DNA that encodes a protein belonging to GH1 and possessing glucocerebrosidase activity and thermal stability, consisting of a base sequence in which one or more bases have been substituted, deleted, inserted, or added in any of the base sequences shown in Sequence ID No. 3, base numbers 112-1566 of Sequence ID No. 3, Sequence ID No. 4, or base numbers 55-1512 of Sequence ID No. 4 in the sequence listing. Here, "a few" means 20 or fewer, preferably 10 or fewer, and more preferably 6 or fewer.

[0029] Furthermore, examples include DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to any of the base sequences shown in Sequence ID No. 3, Sequence ID No. 3 (base numbers 112-1566), Sequence ID No. 4, or Sequence ID No. 4 (base numbers 55-1512), and that encodes a protein belonging to GH1 and possessing glucocerebrosidase activity and thermal stability. Here, "stringent conditions" refer to conditions under which so-called specific hybrids are formed, and nonspecific hybrids are not formed. For example, one such condition is when hybridization is performed at 65°C in the presence of 0.7-1.0M sodium chloride, followed by washing 1-3 times at 60°C, preferably 65°C, more preferably 68°C using a 0.1-5×SSC, 0.1% SDS solution (composition of 1×SSC: 150mM sodium chloride, 15mM sodium citrate).

[0030] Furthermore, the present invention also provides an expression vector that is replicable within a host plant, plant cell, animal cell (including insect cell), or microorganism, and that contains the aforementioned DNA in a state capable of expressing the protein it encodes. In other words, an expression vector for expressing a protein belonging to GH1 and possessing glucocerebrosidase activity and thermal stability is also provided. This expression vector can be constructed using, for example, a self-replicating vector that exists independently outside the chromosome of a host cell and whose replication does not depend on the replication of chromosomal DNA, or a vector that is incorporated into the chromosomal DNA of a host cell and replicates together with this chromosomal DNA. Plasmid vectors and viral vectors are shown as preferred examples. The procedure and method for constructing the expression vector can be the same as those commonly used in the field of genetic engineering.

[0031] Preferably, this expression vector contains, in addition to the DNA encoding the protein of the present invention, a nucleotide sequence that controls its expression and a genetic marker for selecting transformants, in order to express the protein of the present invention in a transformant into which the vector has been introduced. Examples of nucleotide sequences that control expression include promoters, terminators, and nucleotide sequences that encode signal peptides other than those mentioned above. The promoter is not particularly limited as long as it exhibits transcriptional activity in the host. The signal peptide is also not particularly limited as long as it contributes to the extracellular secretion of the protein in the host. Furthermore, the genetic marker may be appropriately selected depending on the method of selecting the transformant, but for example, a drug resistance gene or a gene that complements nutritional requirements can be used.

[0032] Furthermore, the present invention also provides transformants in which the above-described expression vector has been introduced into the intracellular and / or chromosomal DNA of a host. This host-vector system is not particularly limited, and for example, systems using plants, plant cells, animal cells, or microorganisms (such as Escherichia coli, yeast, or filamentous fungi), or fusion protein expression systems using these with other proteins can be used. In addition, the introduction of the above-described expression vector into a host, that is, the transformation of the host using the above-described expression vector, can be carried out according to methods commonly used in this field. Here, it is preferable to use any of the following as the host to be transformed: plants, plant cells, animal cells, Escherichia coli, yeast, or filamentous fungi. In other words, it is preferable to use a transformant of any of the following: plants, plant cells, animal cells, Escherichia coli, yeast, or filamentous fungi into which the above-described expression vector has been introduced. For example, the protein of the present invention can be expressed in large quantities in a transformant of plants, plant cells, or Escherichia coli into which the above-described expression vector has been introduced.

[0033] The transformant can then be bred or cultured under conditions that allow it to grow and multiply while maintaining its traits, and the protein of the present invention can be recovered from the resulting transformant (plant body, plant cultured cells, animal cultured cells, etc.) or its contents (culture medium containing cultured cells, solid culture medium, etc.). Therefore, the present invention provides a method for producing the protein of the present invention, comprising the steps of breeding or culturing the transformant, and recovering (crude or purified) the protein of the present invention from the transformant or its contents obtained by this step. The breeding and culture methods and conditions for the transformant are not particularly limited, as long as they allow the transformant to grow and multiply while maintaining its traits, but they may be substantially equivalent to the breeding or culture methods and conditions for the plant body, plant cells, animal cells, or microorganism used as the host. Furthermore, the method for recovering the target protein after breeding or culturing the transformant can also be a crude preparation method or purification method that is commonly used in this field.

[0034] As an example of a preferred embodiment of the method for producing the protein of the present invention, a method using a plant body, plant cell, or transformed animal cell is mentioned. Examples of animal cells include Chinese hamsters and humans. Examples of plant bodies and plant cells include Malvaceae, Chenopodiaceae, Rubiaceae, Cannabaceae, Hydrangeaceae, Brassicaceae, Iridaceae, Poaceae, Araliaceae, Cucurbitaceae, Anacardiaceae, Cyperaceae, Campanulaceae, Asteraceae, Lauraceae, Moraceae, Papaveraceae, Araceae, Cactaceae, Lamiaceae, Nymphaeaceae, Apiaceae, Polygonaceae, Ericaceae, and Viburnum. Examples include plants of the Pitraceae family, Solanaceae family, Caryophyllaceae family, Ulmaceae family, Nelumbonaceae family, Rosaceae family, Nelumbonaceae family, Amaryllidaceae family, Convolvulaceae family, Vitaceae family, Fagaceae family, Paeoniaceae family, Fabaceae family, Rutaceae family, Pontederiaceae family, Oleaceae family, Palm family, Salicaceae family, Liliaceae family, Orchidaceae family, Taxaceae family, Ginkgoaceae family, Cupressaceae family, Cycadaceae family, Cupressaceae family, and Pinaceae family.More specifically, cotton, hibiscus, spinach, goosefoot, beet, madder, gardenia, coffee plant, hemp, hops, hydrangea, white marbled thrush, rapeseed, radish, Chinese cabbage, cabbage, cauliflower, broccoli, komatsuna, bok choy, wasabi, iris, Japanese iris, Japanese iris, rice, timothy, wheat, corn, sorghum, barley, rye, sugarcane, oats, barnyard millet, foxtail millet, grass, reed, bamboo, dwarf bamboo, Japanese angelica tree, Japanese angelica tree, Fatsia japonica, cucumber, bitter melon, melon, watermelon, bitter gourd, pumpkin, loofah, winter melon, gourd, bottle gourd, lacquer tree, sumac, star grass, balloon flower, etc., coreopsis, lettuce, gerbera, gazania, thistle, burdock, sunflower, cosmos, dandelion, calendula, butterbur, ragweed, camphor tree, bay laurel, mulberry, fig, poppy, water lily, taro, cactus, perilla, salvia, lavender, water lily, carrot, water dropwort, celery, buckwheat , knotweed, azalea, blueberry, rhododendron, camellia, tomato, eggplant, tobacco, petunia, chili pepper, potato, carnation, baby's breath, chickweed, zelkova, hackberry, lotus, rose, cherry blossom, almond, apricot, strawberry, plum, apple, pear, loquat, peach, lotus, garlic, leek, onion, bindweed, morning glory, sweet potato, grape, beech, sawtooth oak, chestnut, peony, soybean, broad bean, black bean, wisteria, Japanese wisteria, lupine, Examples include green beans, peas, alfalfa, peanuts, sweet peas, lotus, Satsuma mandarins, Japanese pepper, summer oranges, oranges, limes, lemons, grapefruits, trifoliate oranges, water hyacinths, olives, jasmine, coconut palms, oil palms, date palms, palm trees, poplars, willows, lilies, dwarf lilies, tulips, daffodils, phalaenopsis orchids, cattleyas, vanilla, yew, Japanese irises, ginkgo trees, cedars, cycads, cypress trees, and pine trees.

[0035] Furthermore, other examples of preferred embodiments of the method for producing the protein of the present invention include methods using transformed cells of Escherichia coli, yeast, or filamentous fungi. Examples of yeast cells include microorganisms belonging to the genera Saccharomyces, Hansenula, or Pichia, such as Saccharomyces cerevisiae. Examples of filamentous fungi include those belonging to the genera Humicola, Trichoderma, Staphylotrichum, Aspergillus, Fusarium, or Acremonium (all scientific names in parentheses are italicized).

[0036] Next, the enzyme composition, pharmaceutical composition, and food composition containing the protein of the present invention will be described in detail.

[0037] The present invention provides an enzyme composition containing the protein of the present invention as described above. This enzyme composition contains the protein of the present invention as an active ingredient (the active ingredient of the glucocerebrosidase activity possessed by the enzyme composition) and can be suitably used for glucosylceramide hydrolysis (for the hydrolysis of glucose in the glucosylceramide molecule), that is, for the conversion of glucosylceramide to ceramide.

[0038] Furthermore, by incorporating the protein of the present invention as an active ingredient, a pharmaceutical composition can be provided. This pharmaceutical composition can be suitably used for the prevention and treatment of Gaucher disease. While it is preferably administered as an intravenous drug, it may also be administered orally (tablets, powders, syrups, etc.).

[0039] In other words, the present invention provides a method for preventing or treating Gaucher disease, which involves administering (e.g., intravenously) a pharmaceutical composition containing the protein of the present invention as an active ingredient to a patient with Gaucher disease. The preferred method of use and dosage is to administer the protein of the present invention or a pharmaceutical composition containing the protein of the present invention as an active ingredient (a preventive and therapeutic agent for Gaucher disease) to a patient with Gaucher disease once every 3 to 10 weeks, more preferably once every 4 to 8 weeks, so that the glucocerebrosidase activity is 10 to 200 U / kg of body weight. In this case, "U (unit)" refers to the unit of decomposition of the synthetic substrate p-nitrophenyl-β-D-glucopyranoside ("p" is italicized, "D" is a small uppercase letter) at a rate of 1 μmol per minute at 37°C.

[0040] Furthermore, by incorporating the protein of the present invention with glucosylceramide isolated from plants, animals, or microorganisms (e.g., basidiomycetes), or chemically synthesized glucosylceramide, it is possible to provide a food composition in which ceramide can be easily released from glucosylceramide in the composition. By incorporating the protein of the present invention and the above-mentioned glucosylceramide as active ingredients, it is also possible to provide a functional food composition in which this released ceramide acts as a functional material, for one or more uses selected from those for improving skin moisture, preventing skin damage caused by ultraviolet rays, preventing inflammatory bowel disease, and preventing colorectal cancer. Furthermore, it is also possible to provide a food composition for preventing lifestyle-related diseases (e.g., diabetes, heart disease, hypertension, hyperlipidemia, etc.).

[0041] Furthermore, by incorporating the heat-stable glucocerebrosidase of the present invention as an active ingredient, the present invention can also provide a functional food composition for the prevention or treatment of Gaucher disease, or a ceramide supplementation food composition to be taken together with a food composition containing glucosylceramide. The usage and dosage of the above-mentioned food composition for the prevention or treatment of Gaucher disease may be the same as those of the pharmaceutical composition for the prevention and treatment of Gaucher disease described above.

[0042] Next, a method for producing ceramide using the protein of the present invention will be described in detail. The method for producing ceramide using the protein of the present invention includes the step of producing ceramide from glucosylceramide isolated from plants, animals, or microorganisms (e.g., basidiomycetes) or from chemically synthesized glucosylceramide using the protein of the present invention. This production method may include any other steps not mentioned above, as long as they do not significantly affect the effects of the present invention.

[0043] Glucosylceramide is found in relatively large quantities in living organisms such as plants and animals compared to ceramide. By using the heat-stable glucocerebrosidase of the present invention, which can be obtained inexpensively by expressing such glucosylceramide in large quantities using the aforementioned transformant, ceramide can be produced at low cost. Furthermore, using the ceramide obtained in this way, cosmetics or functional foods aimed at improving skin hydration, preventing skin damage caused by ultraviolet rays, and preventing colorectal cancer can be provided at low cost. In addition, ceramide can also be provided inexpensively as a research reagent or pharmaceutical.

[0044] The embodiments described above are merely examples to facilitate understanding of the present invention and do not limit it.

[0045] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments, and various modifications are possible within the technical concept of the present invention. [Examples]

[0046] (Example 1) Ten g of young leaves of rice (Oryza sativa L. cv. Nipponbare) were collected and homogenized in 50 mM acetate buffer (pH 5.5) containing 0.05% sodium cholate and 0.3% Triton X-100. The homogenized solution was centrifuged at 15,000 rpm at 4°C for 20 minutes, and the supernatant was dialyzed with 1000 times the volume of 40 mM sodium acetate buffer (pH 5.5) containing 0.05% Tween 20 (registered trademark, hereinafter the same) and 0.025% sodium cholate to obtain an enzyme extract. To 50 mL of this enzyme extract, 10 mM glucosylceramide (cerebroside C, (4E,8E)-ND-2´-hydroxy-(E)-3´-octadecenoyl-1-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine ("E", "N", and "O" are italicized, "D" is a small uppercase letter): The Journal of Antibiotics 41, (1988) 469-480, the same applies hereafter) dissolved in 1 mL of ethanol and 49 mL of 0.4% sodium cholate were added and the mixture was reacted at 45°C for 5 hours. The resulting reaction solution was adjusted to pH 11.5 with sodium carbonate solution and sodium hydroxide solution, then ethyl acetate solution was added and mixed, and the mixture was centrifuged at 3000 rpm at 4°C for 20 minutes. After centrifugation, the resulting ethyl acetate layer was allowed to dry, and this dried material was dissolved in 80% ethanol solution and subjected to high-performance liquid chromatography analysis. Specifically, the sample was injected into a TSKgel ODS-120T column (4.6 mm × 30 cm, manufactured by Tosoh Corporation, registered trademark (hereinafter the same)), and a solvent with an 81% ethanol concentration was flowed through it at a flow rate of 1.0 mL / min. The newly generated substance from the enzymatic reaction was then isolated by detection using a UV detector (ultraviolet absorption wavelength 215 nm). Then, the IR (FTS-135, manufactured by Bio-Rad) of the separated substance, 1 1H NMR, 1313C NMR (Varian UNITY plus 500 spectrometer, Agilent) and ESI-MS analysis (Agilent 6460, Agilent) were performed. The results are shown in Table 1 below.

[0047] [Table 1]

[0048] These results revealed that the substance produced from cerebroside C by the rice-derived enzyme described above is ceramide derived from cerebroside C. Furthermore, this substance is the same as the ceramide produced from cerebroside C by imiglucerase (manufactured by Sanofi), a human-derived glucocerebrosidase, and the above-mentioned IR. 1 1H NMR, 13 The 13C NMR and ESI-MS analysis results were in complete agreement. These results clearly indicate that this rice-derived enzyme possesses glucocerebrosidase activity.

[0049] (Example 2) 110 g of rice (Nipponbare) stems were collected, finely chopped, and then homogenized in 20 mM acetate buffer (pH 5.5) containing 0.05% sodium cholate. The homogenized mixture was centrifuged at 10,000 rpm at 4°C for 60 minutes, and the supernatant was removed. Further, 20 mM acetate buffer (pH 5.5) containing 0.05% sodium cholate was added to this precipitate, stirred for 5 minutes, and then centrifuged at 10,000 rpm at 4°C for 60 minutes, and the supernatant was removed. Finally, 20 mM acetate buffer (pH 5.5) containing 0.05% sodium cholate was added to this precipitate, and a sufficient amount of Triton X-100 for glucocerebrosidase elution was added. After stirring for 30 minutes, the mixture was centrifuged at 18,000 rpm at 4°C for 60 minutes. The obtained supernatant was filtered through a Durapore membrane filter 0.45 μm HV (Merck Millipore, registered trademark (hereinafter the same)), then desalted and concentrated to obtain an enzyme extract.

[0050] This enzyme extract was applied to a HiTrap Q HP column (manufactured by Amersham Biosciences, registered trademark (hereinafter the same)) equilibrated with 20 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate and 0.1% Triton X-100. Then, the extract was eluted by gradient elution from 20 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate and 0.1% Triton X-100 to a buffer containing 1 M sodium chloride in 20 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate and 0.1% Triton X-100. Next, the fraction showing strong glucocerebrosidase activity was pooled and desalted and concentrated by ultrafiltration. This desalted concentrate was applied to a HiTrap SP HP column (Amersham Biosciences) equilibrated with 20 mM acetate buffer (pH 5.5) containing 0.05% sodium cholate and 0.1% Triton X-100. Then, the enzyme was eluted by gradient elution from 20 mM acetate buffer (pH 5.5) containing 0.05% sodium cholate and 0.1% Triton X-100 to a buffer containing 1 M sodium chloride in 20 mM acetate buffer (pH 5.78) containing 0.05% sodium cholate and 0.1% Triton X-100, and fractionated. The enzyme fraction showing strong glucocerebrosidase activity was pooled, and the desalted and concentrated solution was obtained by ultrafiltration as rice-derived glucocerebrosidase (RGC1).

[0051] The glucocerebrosidase activity of the fraction was measured as follows. First, a predetermined amount of sample was added to 50 mM acetate buffer (pH 5.5) containing 100 μM glucosylceramide (cerebroside B), 0.1% Tween 20, and 0.05% sodium cholate, and incubated at 37°C for 15 minutes. Next, four times the volume of ethanol was added to the resulting reaction mixture and mixed. The mixture was then centrifuged at 15,000 rpm for 20 minutes, and the supernatant was subjected to high-performance liquid chromatography analysis. In high-performance liquid chromatography analysis, the sample was injected into a TSKgel ODS-120T column (4.6 mm × 30 cm), and an 83% ethanol solvent was flowed through it at a flow rate of 0.8 mL / min. The amount of ceramide newly generated by the enzymatic reaction was measured by detection with a UV detector (ultraviolet absorption wavelength 215 nm). The amount of ceramide was determined using ceramide produced from cerebroside B by imiglucerase (Sanofi), a human-derived glucocerebrosidase, as a standard. Furthermore, the molecular weight of the produced ceramide was confirmed to be ceramide produced from cerebroside B by examining it using negative LC-MS (Agilent). (ESI-MS m / z: 564.5 [MH]) - The amount of enzyme that produces 1 μmol of ceramide per minute in the enzyme reaction solution was defined as 1 U (unit), and the glucocerebrosidase activity per 1 mL of enzyme solution was calculated.

[0052] Furthermore, the protein concentration of purified RGC1 was determined using a protein assay kit (Bio-Rad Laboratory) with bovine serum albumin as the standard and imiglucerase, whose protein concentration had been measured in advance, as the reference sample. Specifically, the purified RGC1 solution and imiglucerase solution were injected into a TSKgel Octyl-80Ts column (4.6 mm × 15 cm, Tosoh Corporation), flowed at a flow rate of 0.8 mL / min in gradient mode by increasing the ratio of acetonitrile solution in 0.05% trifluoroacetic acid, and detected with a UV detector (ultraviolet absorption wavelength 280 nm). The peak area generated was determined and compared with that of imiglucerase to determine the protein concentration of purified RGC1.

[0053] The fraction containing RGC1 showed a single band on SDS-PAGE, with an average molecular weight (MW) of approximately 62 kD. SDS-PAGE was performed using AE-6000 electrophoresis (ATTO Corporation) and precast minigel e-PAGEL (E-R10L / gel concentration 10% / 18 samples, ATTO Corporation), and silver staining was performed using Silver Stain MS Kit (Wako Pure Chemical Industries, Ltd.). The molecular weight marker used was SDS-PAGE Molecular Weight Standards Low Range (Bio-Rad Laboratory).

[0054] (Example 3) The RGC1 obtained in Example 2 was added to 50 mM acetate buffer (pH 5.5) containing 12.5 μM glucosylceramide (one of the 12 types listed in Table 2 below, derived from animals, plants, or filamentous fungi) or synthetic β-glucoside, 0.1% Tween 20, and 0.05% sodium cholate, and incubated at 37°C for 15 minutes. Next, four times the volume of ethanol was added to the resulting reaction mixture and mixed. The mixture was then centrifuged at 15,000 rpm for 20 minutes, and the amount of ceramide in the supernatant was measured using various reversed-phase columns by high-performance liquid chromatography to determine the glucocerebrosidase activity. The amount of ceramide was determined using the ceramide produced from the above glucosylceramide by imiglucerase as a standard. The glucocerebrosidase activity was determined as a relative activity, with the activity using the animal-derived glucosylceramide d18:1(4E)-C8:0-GluCer ("E" is italicized) as the substrate set to 100 (far right column of Table 2 below). This test was performed five times, and the average value of the relative activity was calculated. The results are shown in Table 2 below.

[0055] [Table 2]

[0056] These results show that RGC1 hardly reacts to lactosylceramides such as d18:1(4E)-C18:0-GM3 and d18:1(4E)-C8:0-LacCer, and galactosylceramides such as d18:1(4E)-C8:0-GalCer (all with italicized "E"), indicating that it specifically recognizes and reacts to the glucose structure of glucosylceramide. Furthermore, it hardly reacts to pNP-β-glucoside (with italicized "p"), demonstrating that it also specifically recognizes and reacts to the ceramide structure of glucosylceramide. These results reveal that RGC1 is a glucocerebrosidase. In addition, it was shown that RGC1 reacts to glucosylceramide substrates of all types, including those derived from plants, animals, and filamentous fungi.

[0057] (Example 4) Four types of animal-derived glucocerebrosidases are known: glucocerebrosidase 1 (GBA1; imiglucerase, belonging to GH30), glucocerebrosidase 2 (GBA2, belonging to GH116), glucocerebrosidase 3 (GBA3, belonging to GH1), and lactase-phlorizin hydrolase (belonging to GH1). In addition, a plant-derived glucocerebrosidase, glucocerebrosidase belonging to GH116 (AtGCD3), has been isolated from Arabidopsis thaliana. Therefore, using the synthetic glucosylceramide substrate N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-glucosyl-β1-1´-sphingosine (C6-NBD glucosylceramide: "N" is italicized, "D" is a small uppercase letter), the K of RGC1 and imiglucerase obtained in Example 2 was used. cat / K mThe value (the value obtained by dividing the number of substrate molecules that can be converted by 1 molecule of enzyme per second by the substrate concentration that gives a reaction rate of 50% of the maximum reaction rate of the enzyme: "K" is in italics) was determined using the Hanes-Woolf plot according to the reaction method of Example 2. Also, the K cat / K m values were cited from the literature values (Journal of Biological Chemistry 282, (2007) 30889-30900, and Journal of Biological Chemistry 295, (2020) 717-728). The results are shown in Table 3 below. From these results, RGC1 clearly has a higher K cat / K m value compared to other glucocerebrosidases, indicating that it is an excellent glucocerebrosidase.

[0058]

Table 3

[0059] (Example 5) RGC1 and imiglucerase obtained in Example 2 were added to 50 mM acetate buffer (pH 5.5) containing 100 μM glucosylceramide (cerebroside B), 0.1% Tween 20, and 0.05% sodium cholate, and incubated at each temperature for 15 minutes. Then, 4 volumes of ethanol were added to the resulting reaction solution and mixed, followed by centrifugation at 15,000 rpm for 20 minutes, and the supernatant was subjected to high performance liquid chromatography analysis to measure the amount of ceramide produced by the enzyme reaction. The high performance liquid chromatography analysis was carried out in the same manner as in Example 2. Also, the calculation of the amount of ceramide and enzyme activity was carried out in the same manner as in Example 2. And the temperature with the highest glucocerebrosidase activity was taken as the optimum temperature. This test was performed 3 times, and the average value was determined. The results are shown in Table 4 below. These results suggest that while the optimal temperature for imiglucerase, a human-derived glucocerebrosidase, was 42.5°C, the optimal temperature for RGC1, a rice-derived glucocerebrosidase, was a higher 54.0°C, indicating that RGC1 is highly stable in vivo.

[0060] [Table 4]

[0061] (Example 6) In general, enzymes are known to exhibit better thermal stability the higher their optimal temperature. From Example 5, RGC1 was expected to have thermal stability because its optimal temperature for glucocerebrosidase activity is higher than that of imiglucerase. Therefore, the following tests were conducted to confirm the thermal stability of glucocerebrosidase in lysosomes (pH 5) and cytoplasm (pH 7), where it is mainly present and acts in the human body. The RGC1 and imiglucerase obtained in Example 2 were incubated at 37°C for 6 to 106 hours in pH 5 (50 mM acetate buffer containing 0.1% Triton X-100 and 0.05% sodium cholate (pH 5.0)), simulating lysosomes, and at 37°C for 4 to 48 hours in pH 7 (50 mM potassium phosphate buffer containing 0.1% Triton X-100 and 0.05% sodium cholate (pH 7.0)), simulating cytoplasm. Glucocerebrosidase activity after each incubation period was measured according to the method of Example 2. Residual activity was calculated as a relative activity value with the pre-incubation activity set to 100. This test was performed three times, and the average value was calculated. The results are shown in Figures 1 and 2. In these figures, an asterisk (*) is used to indicate cases where the glucocerebrosidase activity of RGC1 after each reaction time was significantly higher than that of imiglucerase (1% significance). Figures 1 and 2 clearly show that RGC1 is more stable and has better thermal stability than imiglucerase at both pH 5 and pH 7.

[0062] (Example 7) After cleaving the RGC1 band obtained by SDS-PAGE in Example 2, trypsin treatment was performed, and the molecular mass of the resulting peptide fragments was analyzed by MALDI-TOFMS (Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometer: Microflex LRF 20, Bruker Daltonics). The amino acid sequences of the nine peptide fragments shown in Sequence IDs 5-13 of the sequence listing and Figure 7 were determined. The precise molecular weights of these nine peptide fragments of RGC1 perfectly matched those of nine peptides derived from a protein named Os3BGlu6, derived from rice (Nipponbare), whose function is yet to be identified. This suggests that RGC1 may be the same protein as Os3BGlu6. To date, there are no reports that Os3BGlu6 is a glucocerebrosidase.

[0063] (Example 8) Total RNA was extracted from rice (Nipponbare) callus using the RNeasy Plant Mini Kit (Qiagen, registered trademark (hereinafter the same)), and mRNA was purified from the total RNA using the Absolutely mRNA Purification Kit (Agilent). cDNA synthesis from mRNA and subsequent RACE analysis were performed using SuperScript III Reverse Transcriptase (Thermo Fisher, registered trademark). Os3BGlu6 cDNA was obtained by PCR amplification using sequence numbers 14 and 15 of the sequence listing prepared from the open reading frame estimated from the whole genome sequence of rice (Nipponbare), and two primers (F-primer (RGC1-CN) and R-primer (RGC1-CC)) shown in Figure 8. The specific PCR conditions involved adding KOD-Plus-Neo (Toyobo Co., Ltd.) to the above-mentioned rice callus cDNA and the two primers, and amplifying the DNA by repeating the reaction conditions of 94°C for 2 minutes, 60°C for 0.5 minutes, and 68°C for 1 minute 35 times. The amplified fragment was then subcloned into the plasmid vector pUC19. Furthermore, using a similar method, a longer DNA region including the DNA region outside the amplified fragment was amplified, and the complete cDNA sequence of the Os3BGlu6 gene was determined by analyzing the base sequence of that fragment using a standard method. This base sequence is shown in Sequence ID No. 3 of the sequence listing and in Figure 6. The amino acid sequence translated from this base sequence is shown in Sequence ID No. 1 of the sequence listing and in Figure 5. From this amino acid sequence, it was revealed that Os3BGlu6 belongs to GH1.

[0064] (Example 9) To determine that the isolated Os3BGlu6 gene is a glucocerebrosidase gene (the gene encoding RGC1), we investigated whether Os3BGlu6 produced in E. coli had glucocerebrosidase activity. Using pUC19, which was obtained by subcloning the Os3BGlu6 gene in Example 8, E. coli (DH5α) was transformed by introducing this gene. Next, these transformants were cultured in LB liquid medium (1.0% tryptone, 0.5% yeast extract, 1.0% sodium chloride, 50 μg / mL ampyricillin) at 37°C for 24 hours, and then centrifuged at 15000 rpm for 10 minutes to collect the cells. The obtained cells were washed twice with 50 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate. Subsequently, the cells were collected by centrifugation at 15,000 rpm for 10 minutes, suspended in 50 mM acetate buffer (pH 6.0) containing 0.3% Triton X-100 and 0.05% sodium cholate, and then sonicated. This sonicated solution was centrifuged at 18,000 rpm at 4°C for 60 minutes. The supernatant was then filtered through a Durapore membrane filter 0.45 μm HV (Merck Millipore), desalted and concentrated to obtain an enzyme extract. This enzyme extract was added to 50 mM sodium acetate buffer (pH 5.5) containing 100 μM glucosylceramide (cerebroside C), 0.1% Tween 20, and 0.05% sodium cholate, and incubated at 37°C for 13 minutes. Next, four times the volume of ethanol was added to the resulting reaction solution and mixed. The mixture was then centrifuged at 15,000 rpm for 20 minutes, and the supernatant was subjected to high-performance liquid chromatography analysis to measure the amount of ceramide produced by the enzymatic reaction. The high-performance liquid chromatography analysis was performed in the same manner as in Example 1. The molecular weight of the produced ceramide was determined by negative LC-MS (Agilent), confirming that it was ceramide produced from cerebroside C (ESI-MS m / z: 590.5 [MH]). - Furthermore, the amount of enzyme that produces 1 μmol of ceramide per minute in the enzyme reaction solution was defined as 1 U (unit), and the glucocerebrosidase activity per 1 mL of enzyme extract was calculated. The results are shown in Table 5 below.

[0065] [Table 5]

[0066] These results clearly demonstrated that the enzyme extract obtained from E. coli transformed with the Os3BGlu6 gene possessed glucocerebrosidase activity. Therefore, it was revealed that the isolated RGC1 is Os3BGlu6 and a glucocerebrosidase belonging to the GH1 group. To date, there have been no reports of glucocerebrosidases belonging to the GH1 group being found in plants.

[0067] (Example 10) The homology (identity) of the amino acid sequence of RGC1 with animal-derived glucocerebrosidase and the Arabidopsis thaliana-derived glucocerebrosidase AtGCD3 was investigated using the EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The results showed homology with GBA1 (14.9%), GBA2 (10.1%), GBA3 (35.8%), Lactase-phlorizin hydrolase (11.3%), and AtGCD3 (3.3%). From these results, it became clear that RGC1 is a glucocerebrosidase that is completely different from any glucocerebrosidase isolated to date, even in terms of its amino acid sequence.

[0068] (Example 11) 102 g of soybean (Glycine max (L.) Merr. Enrei) stems were harvested, finely chopped, and then homogenized in 20 mM acetate buffer (pH 5.5) containing 0.01% sodium cholate. The resulting mixture was centrifuged at 10,000 rpm at 4°C for 60 minutes, and the supernatant was removed. Further, 20 mM acetate buffer (pH 5.5) containing 0.01% sodium cholate was added to the centrifugation precipitate, stirred for 5 minutes, and then centrifuged at 10,000 rpm at 4°C for 60 minutes, and the supernatant was removed. An enzyme extract was then obtained from the resulting centrifugation precipitate using the same method as in Example 2.

[0069] The enzyme extract was applied to a HiTrap Q HP column (Amersham Biosciences) equilibrated with 0.5 mM phosphate buffer (pH 6.7) containing 0.05% sodium cholate and 0.05% Triton X-100. Then, the extract was eluted by gradient elution from 0.5 mM phosphate buffer (pH 6.7) containing 0.05% sodium cholate and 0.05% Triton X-100 to a solution containing 1 M sodium chloride in 20 mM phosphate buffer (pH 6.7) containing 0.05% sodium cholate and 0.1% Triton X-100. Next, fractions showing strong glucocerebrosidase activity were pooled and desalted and concentrated by ultrafiltration. This desalted concentrate was applied to a HiTrap SP HP column (Amersham Biosciences) equilibrated with 0.5 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate and 0.05% Triton X-100. Then, the enzyme was eluted by gradient elution from 0.5 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate and 0.05% Triton X-100 to a solution containing 1 M sodium chloride in 0.5 mM acetate buffer (pH 6.0) containing 0.05% sodium cholate and 0.05% Triton X-100, and fractionated. As a result, the fraction showing strong glucocerebrosidase activity was pooled, and the enzyme solution was desalted and concentrated by ultrafiltration to obtain soybean-derived glucocerebrosidase (SGC1).

[0070] The glucocerebrosidase activity and SGC1 protein concentration of the fraction were analyzed using the same method as in Example 2. This SGC1 fraction showed a single band on SDS-PAGE, and its average molecular weight (MW) was approximately 62.0 kD. SDS-PAGE was also performed using the same method as in Example 2.

[0071] (Example 12) The optimal temperature for the glucoserebrosidase activity of SGC1 and imiglucerase obtained in Example 11 was confirmed using the same method as in Example 5. The results are shown in Table 6 below. These results suggest that SGC1 may be highly stable in vivo, as its optimal temperature was 64.0°C, compared to 42.5°C for imiglucerase.

[0072] [Table 6]

[0073] (Example 13) As mentioned above, it is known that the higher the optimal temperature of an enzyme, the better its thermal stability. From Example 12, SGC1 has a higher optimal temperature for glucocerebrosidase activity than imiglucerase, so it was expected to have thermal stability. Therefore, the following tests were conducted to confirm its thermal stability in lysosomes (pH 5) and cytoplasm (pH 7). The two glucocerebrosidases obtained in Example 11, SGC1 and imiglucerase, were incubated at 45°C for 1 to 31 hours in pH 5 (50 mM acetate buffer containing 0.1% Triton X-100 and 0.05% sodium cholate (pH 5.0)), simulating lysosomes, and at 45°C for 0.3 to 5 hours in pH 7 (50 mM potassium phosphate buffer containing 0.1% Triton X-100 and 0.05% sodium cholate (pH 7.0)), simulating cytoplasm. The glucocerebrosidase activity after each reaction time was measured according to the method of Example 2. The residual activity was calculated as a relative activity value with the activity before incubation set to 100. This test was performed three times, and the average value was calculated. The results are shown in Figures 3 and 4. In these figures, an asterisk (*) is used to indicate cases where the glucocerebrosidase activity of SGC1 after each reaction time was significantly higher than that of imiglucerase (1% significance). Figures 3 and 4 clearly show that SGC1 is far more stable and thermally stable than imiglucerase at both pH 5 and pH 7.

[0074] (Example 14) After cleaving the SGC1 band obtained by SDS-PAGE in Example 11, trypsin treatment was performed, and the molecular mass of the resulting peptide fragments was analyzed by MALDI-TOFMS (Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometer: Microflex LRF 20, Bruker Daltonics). The amino acid sequences of the seven peptide fragments shown in Sequence IDs 16-22 of the sequence listing and Figure 11 were determined. The precise molecular weights of these seven peptide fragments derived from SGC1 perfectly matched those of seven peptides derived from a protein named β-glucosidase 40 from a US soybean variety (Glycine max (L.) Merr. Williams 82 ("Glycine max" is italicized)) whose function is unidentified. This suggests that SGC1 may be the same protein as β-glucosidase 40. It should be noted that β-glucosidase 40 was simply named based on homology searches with the genome sequence, and its enzymatic activity as a β-glucosidase has not been confirmed, nor have there been any reports to date that it is a glucocerebrosidase.

[0075] (Example 15) Total RNA was extracted from soybean (Enrei) leaves using the RNeasy Plant Mini Kit (Qiagen), and cDNA was synthesized from this total RNA using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara Co., Ltd.). The cDNA of soybean (Enrei)-derived β-glucosidase 40 was obtained by PCR amplification using sequence numbers 23 and 24 of the sequence listing prepared from the open reading frame estimated from the whole genome sequence of US soybean (Williams 82), and two primers (F-primer (SGC1-CN) and R-primer (SGC1-CC)) shown in Figure 12. The specific PCR conditions involved adding KOD-Plus-Neo (manufactured by Toyobo Co., Ltd.) to the cDNA from the leaves of the soybean (Enrei) mentioned above and the two primers mentioned above, and amplifying the DNA by repeating the reaction conditions of 94°C for 2 minutes, 60°C for 0.5 minutes, and 68°C for 1 minute 40 times. The amplified fragment was then subcloned into the plasmid vector pUC19. Furthermore, a longer DNA region, including the DNA region outside the amplified fragment, was amplified using a similar method, and the complete cDNA sequence of the soybean (Enrei)-derived β-glucosidase 40 gene was determined by analyzing the base sequence of that fragment using a standard method. This base sequence is shown in Sequence ID No. 4 of the sequence listing and in Figure 10. The amino acid sequence translated from this base sequence is shown in Sequence ID No. 2 of the sequence listing and in Figure 9. From this amino acid sequence, it was revealed that soybean (Enrei)-derived β-glucosidase 40 belongs to GH1.

[0076] (Example 16) To clarify that the soybean (Enrei)-derived β-glucosidase 40 gene is a glucocerebrosidase gene, we investigated whether the soybean-derived β-glucosidase 40 gene produced in E. coli had glucocerebrosidase activity. Using pUC19, which was obtained by subcloning the soybean-derived β-glucosidase 40 gene obtained in Example 15, E. coli (DH5α) was transformed by introducing this gene. Using these transformants, the glucocerebrosidase activity per 1 mL of enzyme extract was calculated using the same method as in Example 9, except that the enzyme reaction was incubated at 60°C for 30 minutes. The results are shown in Table 7 below.

[0077] [Table 7]

[0078] These results clearly demonstrated that the enzyme extract obtained from Escherichia coli transformed with the soybean (Enrei)-derived β-glucosidase 40 gene possessed glucocerebrosidase activity. Therefore, it was revealed that the isolated soybean-derived SGC1 is soybean-derived β-glucosidase 40 and a glucocerebrosidase belonging to the GH1 group. As mentioned earlier, to date, there have been no reports of glucocerebrosidases belonging to the GH1 group being found in plants.

[0079] (Example 17) The homology (identity) of the amino acid sequence of SGC1 with animal-derived glucocerebrosidase and the Arabidopsis thaliana-derived glucocerebrosidase AtGCD3 was investigated using the EMBOSS Needle described above. The results showed homology with GBA1 of 14.9%, GBA2 of 10.0%, GBA3 of 35.9%, Lactase-phlorizin hydrolase of 10.4%, and AtGCD3 of 7.6%. From these results, it became clear that SGC1 is a glucocerebrosidase that is completely different from any glucocerebrosidase isolated to date, even in terms of its amino acid sequence.

[0080] (Example 18) By using BLASTp search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), we searched for proteins with high homology to the amino acid sequence of RGC1 and found that a wide range of highly homologous proteins exist in seed plants. Homology (identity) was investigated using the EMBOSS Needle described above. The results are shown in Table 8 below.

[0081] [Table 8]

[0082] Table 8 shows that the amino acid sequence of RGC1 showed 73.8–84.9% homology with β-glucosidase 6 and β-glucosidase 34 from monocotyledonous plants, and 62.6–70.0% homology with β-glucosidase 6, β-glucosidase 40, and β-glucosidase 34 from dicotyledonous plants. It also showed 46.6–53.2% homology with β-glucosidase 6 and β-glucosidase 40 from ferns and mosses. Furthermore, all of these amino acid sequences possessed the conserved region required for GH1 and belonged to GH1. Based on these results, β-glucosidase 6, β-glucosidase 40, and β-glucosidase 34 were considered to be glucocerebrosidases.

[0083] (Example 19) To investigate whether other plants besides rice also exhibit similar glucocerebrosidase activity, the following tests were conducted. 0.01 to 1 g of each part of various plants—leaves, roots, stems, petals, pistils, pollen, fruits, rhizoids, and sporangia—were collected and homogenized in 50 mM acetate buffer (pH 5.0) containing 0.05% sodium cholate and 0.3% Triton X-100. The resulting mixture was centrifuged at 15,000 rpm at 4°C for 20 minutes, and the supernatant was used as the enzyme extract. The enzyme extract was then incubated at 45°C for 60 minutes in 50 mM acetate buffer (pH 5.0) containing 100 μM glucosylceramide (cerebroside B), 0.05% sodium cholate, and 0.2% Triton X-100. Next, four times the volume of ethanol was added to the resulting reaction solution and mixed. The mixture was then centrifuged at 15,000 rpm for 20 minutes, and the supernatant was subjected to high-performance liquid chromatography analysis to measure the amount of ceramide produced by the enzymatic reaction. The high-performance liquid chromatography analysis was performed in the same manner as in Example 2. The amount of ceramide and enzyme activity were also calculated in the same manner as in Example 2, and the number of glucocerebrosidase activity units per gram of plant fresh weight was calculated. This test was performed three times, and the average value was calculated. The results are shown in Table 9 below.

[0084] [Table 9]

[0085] These results indicate that glucocerebrosidase activity was detected in the extracts of all plants measured. Considering the results in Tables 8 and 9, it was shown that glucocerebrosidase is present not only in rice and soybeans, but in plants in general. Furthermore, the fact that RGC1 derived from monocots and SGC1 derived from dicots are glucocerebrosidases belonging to GH1 (Examples 9 and 16), that enzymes belonging to GH1 with high homology to the amino acid sequence of RGC1 are present in many seed plants (Example 18), and that high glucocerebrosidase activity was detected in extracts of many seed plants as shown in Table 9 above, indicates that seed plants possess glucocerebrosidases belonging to GH1. In addition, the presence of β-glucosidase 6 and β-glucosidase 40 in ferns and mosses (Table 8 above), and the detection of glucocerebrosidase activity in extracts of ferns and mosses, strongly suggests that ferns and mosses also possess glucocerebrosidases belonging to GH1. Next, considering homology, the amino acid sequence homology between RGC1 from monocots and SGC1 from dicots, which have been proven to be GH1-belonging glucocerebrosidases in Examples 9 and 16, is 67.6%. Therefore, enzymes with at least 67.5% homology to the amino acid sequences of RGC1 and SGC1 are definitely glucocerebrosidases belonging to GH1. Furthermore, as shown in Table 9 above, seed plants have been found to possess glucocerebrosidases. Therefore, at least the enzymes with the lowest homology to the amino acid sequence of RGC1 in seed plants, shown in Table 8 above (62.6% or higher in Rosa chinensis), are also considered to be glucocerebrosidases belonging to GH1.

[0086] (Example 20) Enzyme extracts from various plants, extracted according to Example 19, as well as the aforementioned purified rice-derived RGC1 and purified soybean-derived SGC1, were added to a 50 mM sodium acetate buffer (pH 5.5) containing 100 μM glucosylceramide (cerebroside B), 0.1% Tween 20, and 0.05% sodium cholate, and incubated at each temperature for 15 minutes. Then, four times the volume of ethanol was added to the resulting reaction solution and mixed. The mixture was then centrifuged at 15,000 rpm for 20 minutes, and the supernatant was subjected to high-performance liquid chromatography analysis to measure the amount of ceramide produced by the enzymatic reaction. The high-performance liquid chromatography analysis was performed in the same manner as in Example 5. The amount of ceramide and enzyme activity were also calculated in the same manner as in Example 5. The temperature at which the glucocerebrosidase activity was highest was determined to be the optimal temperature. This test was performed three times, and the average value was calculated. The results are shown in Table 10 below.

[0087] [Table 10]

[0088] These results show that glucocerebrosidases derived from many seed plants, including rice and soybeans, have higher optimal temperatures than imiglacerase, a human-derived glucocerebrosidase. As shown in Examples 6 and 13, it is generally known that enzymes with higher optimal temperatures have better thermal stability. Therefore, it can be said that glucocerebrosidases belonging to GH1 derived from seed plants have better thermal stability than imiglacerase, a human-derived glucocerebrosidase, meaning they possess thermal stability.

Claims

1. An enzyme composition for hydrolyzing glucosylceramide, comprising the protein shown in (A) or (B) below as an active ingredient. (A) A protein consisting of the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38-521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19-503 of Sequence ID No. 2 in the sequence listing. (B) A protein having glycoside hydrolase family 1 and possessing glucocerebrosidase activity and thermal stability, comprising an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38 to 521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19 to 503 of Sequence ID No. 2 in the sequence listing.

2. A method for producing ceramide, comprising the step of producing ceramide from glucosylceramide isolated from plants, animals, or microorganisms, or from chemically synthesized glucosylceramide, using the protein shown in (A) or (B) below as an active ingredient. (A) A protein consisting of the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38-521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19-503 of Sequence ID No. 2 in the sequence listing. (B) A protein having glycoside hydrolase family 1 and possessing glucocerebrosidase activity and thermal stability, comprising an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in Sequence ID No. 1, amino acid numbers 38 to 521 of Sequence ID No. 1, Sequence ID No. 2, or amino acid numbers 19 to 503 of Sequence ID No. 2 in the sequence listing.

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