Method for manufacturing secretory β-galactosidase

Abstract

A method for producing secretory β-galactosidase is characterized by introducing a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae to induce the production of secretory β-galactosidase; and by a method for producing galactooligosaccharides using the β-galactosidase produced by this method, galactooligosaccharides can be readily produced.

Description

Technical Field

[0001] This invention relates to a method for manufacturing a secretory β-galactosidase that is readily available for the production of galactooligosaccharides. Prior Technology

[0002] It is well known that β-galactosidase catalyzes the hydrolysis of β-D-galactosidic bonds in lactose and other substances, and also catalyzes galactose transfer reactions, thus being used to produce galactooligosaccharides that selectively promote the proliferation of Bifidobacterium bifidum in the intestine.

[0003] To date, the applicant has reported a technology for producing galactooligosaccharides using β-galactosidase (Patent Document 1), wherein the β-galactosidase is derived from a unique high-valence mutant strain of *Saccharomyces cerevisiae*, a basidiomycete yeast.

[0004] However, since the β-galactosidase used in this technique is non-secretory (cell wall bound), it needs to be prepared into a cell concentrate containing the unique *Saccharomyces cerevisiae* cells that produce it for use in the reaction.

[0005] This bacterial concentrate is prone to spoilage because it contains live bacteria; moreover, since it only concentrates the bacterial cells, the specific activity is relatively low, and there are also problems such as leakage of bacterial contents into the galactooligosaccharide reaction solution, which increases the purification cost. [Previous Technical Documents] [Patent Literature]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2006-223268 Summary of the Invention

[0007] [The problem that the invention aims to solve]

[0008] The present invention addresses the problem of providing a method for manufacturing β-galactosidase that is readily applicable to the production of galactooligosaccharides, thereby solving the aforementioned problems. [Methods for solving problems]

[0009] The inventors of this invention, through their dedicated research to solve the aforementioned problems, discovered that by introducing a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae, a secretory β-galactosidase can be produced, which can be easily utilized in the production of galactooligosaccharides, thus completing this invention.

[0010] In other words, the present invention is a method for manufacturing secretory β-galactosidase, characterized by introducing a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae to induce it to produce secretory β-galactosidase.

[0011] Furthermore, the present invention relates to a non-secretory β-galactosidase gene derived from basidiomycete yeast, which is the sequence recorded in sequence numbers 7, 13, and 19.

[0012] Furthermore, the present invention is a transformation of Aspergillus oryzae, characterized by the introduction of a non-secretory β-galactosidase gene derived from Basidiomycota yeast into Aspergillus oryzae to produce a secretory β-galactosidase.

[0013] Furthermore, the present invention is a method for manufacturing galactooligosaccharides, characterized in that the β-galactosidase manufactured by the aforementioned method for manufacturing β-galactosidase is acted on a matrix containing at least lactose.

[0014] Furthermore, the present invention relates to a secretory β-galactosidase, which is obtained by introducing a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae and then culturing it. [Effects of the Invention]

[0015] The method for manufacturing secretory β-galactosidase of the present invention can convert non-secretory β-galactosidase derived from basidiomycete yeast into secretory β-galactosidase.

[0016] Therefore, the β-galactosidase obtained by the method for manufacturing secretory β-galactosidase of the present invention has high β-galactosidase activity and high thermal stability, and is easy to separate and purify, and can be easily used for the manufacture of galactooligosaccharides. Simple Explanation of the Diagram

[0017] [Figure 1] shows the results of SDS-PAGE and activity assays using the SsGal strain. [Figure 2] shows the results of SDS-PAGE and activity assays using the SmGal strain. [Figure 3] shows the results of SDS-PAGE and activity assays using the RmGal strain. [Figure 4] shows the results of SDS-PAGE and activity assays using the SeGal strain. [Figure 5] shows the results of SDS-PAGE and activity assay (thermal inactivation) of the SsGal strain. [Figure 6] shows the results of SDS-PAGE and activity assay (thermal inactivation) of the SmGal strain. [Figure 7] shows the results of SDS-PAGE and activity assay (thermal inactivation) of the RmGal strain. [Figure 8] shows the results of SDS-PAGE and activity assay (thermal inactivation) of the SeGal strain. [Figure 9] shows the results of activity determination after heat treatment of the SeGal strain and the mother plant. [Figure 10] is a graph showing the reaction time and sugar composition in the solution when using galactooligosaccharides from the SsGal strain. [Figure 11] is a graph showing the reaction time and sugar composition in the solution when using SmGal strain galactooligosaccharide for production. [Figure 12] is a graph showing the reaction time and sugar composition in the solution when using galactooligosaccharides from the SeGal strain. [Figure 13] is a graph showing the reaction time and sugar composition in solution when using galactooligosaccharides from the SeGal strain ((a): 70℃, (b): 80℃). [Figure 14] is a graph showing the reaction time and sugar composition in solution when using galactooligosaccharides from the SeGal strain ((c): 90°C). Implementation

[0018] [Forms of Invention Implementation]

[0019] The method for manufacturing secretory β-galactosidase of the present invention (hereinafter referred to as "the method of the present invention") involves introducing a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae to induce it to produce secretory β-galactosidase.

[0020] The non-secretory β-galactosidase gene line used in the method of this invention encodes the non-secretory β-galactosidase produced by Basidiomycota yeast. The term "non-secretory" here refers to a cell wall-bound form, which can be identified by methods such as viability staining.

[0021] Furthermore, the basidiomycete yeasts that can produce non-secretory β-galactosidase are not particularly limited, and examples include basidiomycete yeasts belonging to the genera *Sporobolomyces singularis*, *Sirobasidium magnum*, *Rodotorula minuta*, *Sterigmatomyces elviae*, and *Cryptococcus laurentii*. Among these basidiomycete yeasts, those belonging to the genera *Sporobolomyces* or *Sterigmatomyces* are preferred, and *Sporobolomyces singularis* or *Sterigmatomyces elviae* are even more preferred.

[0022] Furthermore, the gene encoding the non-secretory β-galactosidase produced by basidiomycete yeast can be exemplified by genes selected and propagated from basidiomycete yeasts capable of producing the aforementioned non-secretory β-galactosidase using common methods such as PCR. Moreover, this gene is preferably obtained by total synthesis in conjunction with the host, based on the information from the gene obtained as described above.

[0023] Specifically, the following genes can be cited. Furthermore, this gene also contains a message sequence.

[0024] • The β-galactosidase gene derived from the unique *Saccharomyces cerevisiae*, consisting of the base sequence recorded in sequence number 1 (sequences 1-57 are message sequences). • The β-galactosidase gene derived from large-chain daphnia, consisting of the base sequence recorded in sequence number 7 (sequences 1-48 are message sequences). • The β-galactosidase gene derived from *Rhodotorula pulveratum*, consisting of the base sequence recorded in sequence number 13 (sequences 1-57 are message sequences). • The β-galactosidase gene derived from *Sterigmatomyces elviae*, consisting of the base sequence recorded in sequence number 19 (sequences 1-57 are message sequences).

[0025] Furthermore, a preferred embodiment of the aforementioned gene is one in which the message sequences of the aforementioned basidiomycetes and yeasts are replaced with the message sequences of Aspergillus oryzae. Examples of signaling sequences from Aspergillus oryzae include the secretion signal (TAA) sequence of its α-amylase (TAA) (Okazaki, F., Aoki, J., Tabuchi, S., Tanaka, T., Ogino, C., and Kondo, A., Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR. Appl Microbiol Biotechnol 96, 81-88 (2012)). Another example is the secretion signal of Rhizopus oryzae lipase (Hama, S., Tamalampudi, S., Shindo, N., Numata, T., Yamaji, H., Fukuda, H., and Kondo, A., Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins). (e.g., to the secretory pathway of Aspergillus oryzae. Appl Microbiol Biotechnol 79, 1009-1018 (2008)). Substitution of such message sequences can be carried out using common methods.

[0026] Among the β-galactosidase genes that replace the aforementioned basidiomycete yeast signal sequences with the signal sequences of Aspergillus oryzae, the following genes are preferred examples. These sequences consist of the Aspergillus oryzae secretory signal (TAA signal) sequence and the sequence encoding the natural β-galactosidase.

[0027] • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 3 (sequences 1-63 are secretion signal sequences). • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 9 (sequences 1-63 are secretion signal sequences). • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 15 (sequences 1-63 are secretion signal sequences). • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 21 (sequences 1-63 are secretion signal sequences).

[0028] Of the aforementioned genes, those that modify the codons encoding the natural β-galactosidase sequence without altering the amino acid sequence of the β-galactosidase are preferred. Examples of such β-galactosidase genes include the following. These sequences consist of the secretory signal (TAA) sequence of *Aspergillus oryzae* and a sequence modifying the codons encoding the natural β-galactosidase sequence without altering the amino acid sequence of the β-galactosidase.

[0029] • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 5 (sequences 1-63 are secretion signal sequences). • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 11 (sequences 1-63 are secretion signal sequences). • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 17 (sequences 1-63 are secretion signal sequences). • The β-galactosidase gene, consisting of the base sequence recorded in sequence number 23 (sequences 1-63 are secretion signal sequences).

[0030] Among the above-mentioned genes, the β-galactosidase gene, which is composed of the base sequences recorded in sequence numbers 5, 11, and 23, is preferred.

[0031] The *Kojirae* strain used in the preparation method of this invention to which the above-mentioned β-galactosidase gene is to be introduced is not particularly limited. Examples include *Kojirae* strain NS4 (available from the National Institute of Alcoholic Drinks, Hiroshima City, Higashi-Hiroshima Prefecture 3-7-1, Postal Code 739-0046), *Kojirae* strain niaD300, *Kojirae* strain RIB40, and *Kojirae* strain ATCC11488. Among these, *Kojirae* strain NS4 is preferred.

[0032] In the preparation method of this invention, the method of introducing the above-mentioned gene into *Aspergillus oryzae* is not particularly limited. For example, the gene can be introduced into the expression vector using common methods. The type of expression vector is not particularly limited, but it is preferably an expression vector derived from *Aspergillus oryzae*, especially using a modified promoter of a cis-acting element (Region III) related to the expression control of amylase system genes (by introducing a modified Aspergillus oryzae enolase promoter with a cis-acting element, Tsuboi, H. et al., Biosci. Biotechnol. Biochem., 69, 206-208 (2005)) and a high-expression vector containing a 5'UTR sequence with high translation efficiency (Japanese Patent No. 4413557). Furthermore, in order to select transfectants, antibiotic resistance genes such as ampicillin or a marker adenosine triphosphate sulfatase expression capsule can also be introduced into such vectors.

[0033] The aforementioned expression vectors can be prepared using the methods described in the aforementioned literature, or they can be commissioned to protein expression services such as those provided by Ozeki Co., Ltd. (Postal code 663-8227, 4-9 Imazudeya-cho, Nishinomiya City, Hyogo Prefecture).

[0034] After introducing the aforementioned genes into the expression vector, it is then introduced into *Aspergillus oryzae* to induce transformation. The method for transforming *Aspergillus oryzae* is not particularly limited; common methods such as protoplast-PEG or electroporation are acceptable. After transformation, appropriate washing, selection, and collection of bacteria can be performed using common methods.

[0035] By introducing a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae, a transgenic Aspergillus oryzae capable of producing secretory β-galactosidase can be obtained. By appropriately culturing this transgenic strain in DPY or CDD media, Aspergillus oryzae can produce secretory β-galactosidase.

[0036] The β-galactosidase obtained above is a secretory form, and purification can be achieved by simply filtering and centrifuging the culture medium after cultivation, and collecting the supernatant. Alternatively, ultrafiltration membranes can be used to concentrate the supernatant. This β-galactosidase is characterized by high β-galactosidase activity, high thermal stability, and fewer impurities.

[0037] The preferred amino acid sequence of this type of secretory β-galactosidase is shown below. • A β-galactosidase derived from the unique *Saccharomyces cerevisiae*, consisting of the amino acid sequence described in sequence number 2 (sequences 1-575 in sequence) (the amino acid sequences described in sequences 4 and 6 are also the same (sequences 1-575 in sequence)). • A β-galactosidase derived from large-chain baicale, consisting of the amino acid sequence described in sequence number 8 (sequences 1-685) (the amino acid sequences described in sequences 10 and 12 are also the same (sequences 1-685)). • A β-galactosidase derived from *Rhodotorula pulveratum* consisting of the amino acid sequence described in sequence number 14 (sequences 1-581 in sequence) (the amino acid sequences described in sequences 16 and 18 are also the same (sequences 1-581 in sequence)). • The β-galactosidase derived from *Sterigmatomyces elviae* (sequence numbers 1-581) consisting of the amino acid sequence described in sequence number 20 (the amino acid sequences described in sequences 22 and 24 are also the same (sequence numbers 1-581)).

[0038] Among the aforementioned β-galactosidases, the preferred ones are the β-galactosidase derived from *Sterigmatomyces elviae* whose amino acid sequence is described in sequence number 2, the β-galactosidase derived from *Bacillus macrocarpa* whose amino acid sequence is described in sequence number 8, and the β-galactosidase derived from *Sterigmatomyces elviae* whose amino acid sequence is described in sequence number 20.

[0039] This β-galactosidase, in addition to being secreted by bacteria, also exhibits good thermal stability and shelf life, with its activity remaining constant even after long-term storage. Furthermore, the activity of the β-galactosidase can be confirmed using the methods described in the examples below. Generally, multiple β-galactosidases are sometimes used to efficiently produce galactooligosaccharides; however, the β-galactosidase obtained above can also efficiently produce galactooligosaccharides when used alone.

[0040] The β-galactosidase obtained above can be used in the same way as known β-galactosidases, for example, to act on a matrix containing at least lactose to produce galactooligosaccharides. Furthermore, since this β-galactosidase is secreted, no special cell removal is required during the production of galactooligosaccharides.

[0041] Specifically, to enable the β-galactosidase obtained above to act on a matrix containing at least lactose, simply add β-galactosidase to the matrix containing at least lactose and maintain a predetermined temperature. The amount of β-galactosidase added is not particularly limited; for example, 1-50 U relative to 100g of lactose, preferably 5-10 U. Furthermore, the temperature at which the β-galactosidase acts on the matrix is ​​not particularly limited; it is 30-90°C, preferably 60-90°C, and the maintenance time can be set appropriately. Galactosylated sugars can also be added to the matrix containing at least lactose; such sugars are not particularly limited and examples include galactose, mannose, ribose, xylose, arabinose, rhamnose, N-acetylglucosamine, α-methylmannoside, α-methylgalactoside, α-methylglucoside, 2-deoxyglucose, and 2-deoxygalactose.

[0042] The galactooligosaccharides produced as described above are rich in galactooligosaccharides with 5 sugars or less, especially galactooligosaccharides with 3 sugars.

[0043] Furthermore, the galactooligosaccharides produced as described above can be used directly, but they can also be separated and purified using common purification methods. There are no special limitations on the purification methods; specifically, they can be purified using various chromatographic methods such as ion exchange, gel filtration, activated carbon, and affinity chromatography.

[0044] The resulting galactooligosaccharides can be used as useful food ingredients, pharmaceutical raw materials, or reagents. [Example]

[0045] The following examples illustrate the invention in detail, but the invention is not limited to these examples.

[0046] The basidiomycete yeasts used in these embodiments have the following registration numbers. · Unique Sporozoan ATCC 24193 • Little Red Yeast CBS 319 ·Sterigmatomyces elviae IFO 1843 ·Large chain lug CBS 6803 ATCC:10801 University Boulevard Manassas, VA 20110 USA CBS:Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands IFO: Postal code 532-8686 No. 17-85, 2-chome, Jusanhonmachi, Yodogawa-ku, Osaka City

[0047] Example 1 Obtaining the β-galactosidase gene from the unique *Saccharomyces cerevisiae*: Based on the literature (Ishikawa, E., Sakai, T., Ikemura, H., Matsumoto, K., and Abe, H., Identification, cloning, and characterization of a Sporobolomyces singularis beta-galactosidase-like enzyme involved in galacto-oligosaccharide production. J Biosci Bioeng 99, 331-339 (2005).), a unique Sporobolomyces β-galactosidase gene (sequence number 1) was obtained. This gene contains a message sequence and a sequence encoding β-galactosidase. The message sequence of this gene was replaced with the TAA signal sequence of Aspergillus oryzae (sequence number 3) obtained by computer. Furthermore, the β-galactosidase gene was modified to obtain a sequence (sequence number 5) (SsGal) in which the codons encoding the natural β-galactosidase sequence were changed without altering the amino acid sequence of β-galactosidase. GenScript was commissioned to fully synthesize this SsGal.

[0048] Example 2 Obtaining the β-galactosidase gene from large-chain basidiomycetes: Degenerate primers (Table 1) (sequence numbers 25-29) were designed from the preserved regions, and partial sequences were selected by RT-PCR using 6 combinations of forward (2 types) × reverse (3 types). Full-length cDNA was obtained from these partial sequences through 5' and 3' RACE.

[0049]

[0050] Based on the full-length cDNA described above, the β-galactosidase gene (sequence number 7) derived from *Aspergillus oryzae* was obtained by analogy from the start codon (ATG) in the upstream region. This gene contains a signal sequence and a sequence encoding β-galactosidase. The sequence (sequence number 9) of which the signal sequence of this gene was replaced with the TAA signal sequence of *Aspergillus oryzae* was obtained on a computer. Furthermore, the β-galactosidase gene was modified to obtain a sequence (sequence number 11) (SmGal) in which the codons encoding the natural β-galactosidase sequence were changed without altering the amino acid sequence of β-galactosidase. GenScript was commissioned to perform the total synthesis of this SmGal.

[0051] Example 3 Obtaining the β-galactosidase gene from Rhodotorula glutinis: The β-galactosidase gene (sequence number 13) of *Rhodotorula glutinis* was obtained using the same method as the β-galactosidase gene derived from *Bacillus oryzae*. This gene contains a message sequence and a sequence encoding β-galactosidase. The message sequence of this gene was replaced with the TAA signal sequence of *Aspergillus oryzae* (sequence number 15) on a computer. Furthermore, the β-galactosidase gene was modified to obtain a sequence (sequence number 17) (RmGal) in which the codons encoding the natural β-galactosidase sequence were changed without altering the amino acid sequence of β-galactosidase. GenScript was commissioned to perform the total synthesis of this RmGal.

[0052] Example 4 Obtaining the β-galactosidase gene from *Sterigmatomyces elviae*: The β-galactosidase gene of *Sterigmatomyces elviae* (sequence number 19) was obtained using the same method as the β-galactosidase gene derived from *Bacillus oryzae*. This gene contains a message sequence and a sequence encoding β-galactosidase. The message sequence of this gene was replaced with the TAA signal sequence of *Aspergillus oryzae* (sequence number 21) on a computer. Furthermore, the β-galactosidase gene was modified to obtain a sequence (sequence number 23) (SeGal) in which the codons encoding the natural β-galactosidase sequence were changed without altering the amino acid sequence of β-galactosidase. This SeGal was commissioned to GenScript for total synthesis.

[0053] Example 5 Obtaining the SsGal transform: The SsGal obtained in Example 1 was sent to the protein expression service of Ozeki Co., Ltd. (Postal code 663-8227, 4-9 Imazudeya-cho, Nishinomiya City, Hyogo Prefecture) and imported into the expression vector.

[0054] The host cell used for the transgenic strain was NS4, derived from the nitrate reductase gene (niaD)- and the adenosine triphosphate sulfatase gene (sC)- of *Fragaria oryzae* (distributed by the National Institute of Alcoholics, Sake Research, 3-7-1 Kageyama, Higashi-Hiroshima City, Hiroshima Prefecture, 739-0046, Japan). The transgenic strain (SsGal strain) was introduced into the expression vector using the standard protoplast-PEG method. Furthermore, selection of the transgenic strain was performed through morpho-plasmo-complementation of sC-.

[0055] Example 6 Obtaining the SmGal transform: Except for the SmGal obtained in Example 2, the introduced expression vector and transgenic strain (SmGal strain) were obtained in the same manner as in Example 5.

[0056] Example 7 Obtaining the RmGal transform: Except for the RmGal obtained in Example 3, the introduced expression vector and transgenic strain (RmGal strain) were obtained in the same manner as in Example 5.

[0057] Example 8 Obtaining the SeGal transform: Except for the SeGal obtained in Example 4, the introduced expression vector and transgenic strain (SeGal strain) were obtained in the same manner as in Example 5.

[0058] Example 9 Assessment of β-galactosidase production in transformed organisms: (1) Activity assay Among the transfectants obtained in Examples 5-8, the SsGal strain was cultured in CDD medium (2% dextrin, 0.2% glucose, 0.2% NH4Cl, 0.002% KCl, 0.001% K2HPO4, 0.0005% MgSO4·7H2O, 2×10-5% CuSO4·5H2O, 1×10-5% FeSO4·7H2O, 1×10-6% ZnSO4·7H2O, 1×10-6% MnSO4·5H2O, 1×10-6% AlCl3, 200 mM MOPS-NaOH buffer pH 7.0) at 30°C for 144 hours (15 mL / 100 mL Erlenmeyer flask). The RmGal strain was cultured in 2×DPY medium (4% dextrin, 2% hipolypepton, 2% yeast extract, 1% KH2PO4, 0.1% MgSO4·7H2O) at 30℃ for 144 hours (150 mL / 500 mL shake flask). The SmGal strain was cultured in 2×DPY medium at 30℃ for 168 hours (150 mL / 500 mL shake flask). The SeGal strain was cultured in DPY medium (2% dextrin, 1% hipolypepton, 1% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O) at 30℃ for 168 hours. The culture supernatant was recovered and mixed with an equal volume of 2× sample buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 0.01% bromophenol blue, 4% SDS, 200 mM DTT), and treated at 100℃ for 10 minutes before being used for SDS-PAGE (CBB staining).

[0059] Furthermore, the activity assay using ONPG as a matrix was performed as follows: A 12.5 mM solution was prepared by adding 2-nitrophenyl-β-galactoside (ONPG) to 50 mM citrate-phosphate buffer (pH 4.0). 0.2 mL of the culture supernatant containing the above β-galactosidase, diluted to 0.2–0.8 with an absorbance of 420 nm in 50 mM citrate-phosphate buffer (pH 4.0), was added to this solution, and the mixture was incubated at 30°C for 10 minutes (test solution). The reaction was stopped by adding 4 mL of 0.25 M sodium carbonate solution, followed by centrifugation (3,000 g, 10 min). The amount of free 2-nitrophenol in the supernatant was quantified by measuring the absorbance at 420 nm using a spectrophotometer. On the other hand, a blank reagent was prepared by adding 50 mM citrate-phosphate buffer (pH 4.0) to a 2-nitrophenyl-β-galactosidase solution. Sodium carbonate solution was added beforehand, and the mixture was simultaneously reacted with culture supernatant containing the aforementioned β-galactosidase. After the reaction was stopped and color developed, this solution was used as the starting solution (blind test). One unit (U) of enzyme activity was defined as the amount of 2-nitrophenol enzyme that releases 1 micromolar per minute under these conditions, calculated using the following formula.

[0060]

Number 1

[0061] The results of SDS-PAGE and activity assays are shown in Figures 1-4. CBB staining revealed specific bands in the RmGal, SmGal, and SeGal strains not found in their parent plants, leading to their identification as β-galactosidases. While no specific band was found in the SsGal strain in DPY medium, a specific band not found in the parent plant was detected in CDD (pH 7.0) medium, leading to its identification as a β-galactosidase. The culture conditions that could enhance the secretory productivity of each β-galactosidase were investigated. The results showed that the SsGal strain achieved maximum activity under the following conditions: CDD (pH 7.0) medium, 30°C, 144 hours; the RmGal strain under 2×DPY medium, 30°C, 144 hours; the SmGal strain under 2×DPY medium, 30°C, 168 hours; and the SeGal strain under DPY medium, 30°C, 168 hours. Furthermore, based on the concentration of the bands in the SDS-PAGE spectrum, the productivity of the SsGal strain, RmGal strain, SmGal strain, and SeGal strain is estimated to be approximately 200 mg / L, approximately 200 mg / L, approximately 200 mg / L, and approximately 1 g / L, respectively.

[0062] (2) Copy number estimation Furthermore, the number of transfectants introduced into the transfector was estimated using real-time PCR.

[0063] Based on the PCR results, it is inferred that the SsGal, RmGal, and SmGal strains have 1 copy of the insertion, while the SeGal strain has 2 copies of the insertion into the cassette.

[0064] (3) Thermal deactivation test One mL of culture medium of each transgenic strain and the parent strain (NS4 strain) cultured under the culture conditions described in (1) was cultured at 40℃, 50℃, 60℃, 70℃ and 80℃ for 1 hour each, and enzyme activity was measured and SDS-PAGE was performed.

[0065] The results of SDS-PAGE and activity assays are shown in Figures 5-8. The SsGal strain maintained activity up to 40°C, but activity decreased by approximately 70% after 1 hour of incubation at 50°C, and disappeared at 70°C. The activity of the mother strain cultured under the same conditions was detectable in trace amounts up to 60°C and disappeared at 70°C. The RmGal strain maintained activity up to 50°C, but activity disappeared after 1 hour of incubation at 60°C. The activity of the mother strain cultured under the same conditions was detectable up to 70°C and disappeared at 80°C. The SmGal strain maintained activity up to 50°C, but activity decreased by approximately 20% after 1 hour of incubation at 60°C, and disappeared at 80°C. The activity of the mother strain cultured under the same conditions was detectable up to 70°C and disappeared at 80°C. The SeGal strain maintained activity up to 70°C. Activity decreased by approximately 97% after 1 hour of incubation at 80°C. The activity of the mother strain cultured under the same conditions was detectable up to 40°C and disappeared at 50°C. Furthermore, SeGal was treated at 80°C for shorter periods (5 minutes, 10 minutes, and 20 minutes) than one hour. The results showed that treatment at 80°C for 5 minutes reduced activity by approximately 37%, while treatment for 20 minutes reduced it by approximately 98%. Moreover, activity measurements showed that treatments at 40°C and 50°C exhibited higher activity than the parent plant.

[0066] As can be seen from the above, the SeGal strain can maintain its activity even at high temperatures.

[0067] Example 10 Removal of impurities: As shown in Example 9(3), it can be seen that the SeGal strain can maintain its activity at high temperatures. On the other hand, the mixed enzyme derived from Aspergillus oryzae of the SeGal strain parent strain was subjected to a heat inactivation test in the same manner as in Example 9(3), and it could be inactivated by heat treatment at 70°C. Therefore, it can be seen that the β-galactosidase produced by the SeGal strain can be purified by heat treatment (Figure 9).

[0068] Example 11 Production of galactooligosaccharides (1): A solution containing 66% (w / v) lactose was added to 150 mL of the culture supernatant of the SsGal, SmGal, and SeGal strains obtained in Example 9, each in an amount equivalent to 10 U. The mixture was reacted at a predetermined temperature for a predetermined time to prepare galactooligosaccharides. The sugar composition and amount were determined by high-performance liquid chromatography. The reaction time and sugar composition in the solution are shown in Figures 10-12 (Figure 10: SsGal strain, Figure 11: SmGal strain, Figure 12: SeGal strain).

[0069] As shown in the figure, β-galactosidase produced by the SsGal, SmGal and SeGal strains can produce galactooligosaccharides, which are mainly trisaccharides, from lactose.

[0070] Furthermore, when using β-galactosidase produced by the SsGal strain to produce galactooligosaccharides, the galactooligosaccharide content was 56.0%; when using β-galactosidase produced by the SmGal strain, the galactooligosaccharide content was 66.7%; and when using β-galactosidase produced by the SeGal strain, the galactooligosaccharide content was 68.5%.

[0071] Furthermore, since the aforementioned β-galactosidase is secretory, it can efficiently produce galactooligosaccharides without the need for bacterial cell treatment after production.

[0072] Example 12 Production of galactooligosaccharides (2): A solution containing 66% (w / v) lactose was added to 150 mL of the culture supernatant of the SeGal strain obtained in Example 9, in an amount equivalent to 1.0 U. The mixture was reacted at 70°C, 80°C, and 90°C for predetermined times to prepare galactooligosaccharides. The sugar composition and amount were determined by high-performance liquid chromatography. The reaction time and sugar composition in the solution are shown in Figure 13 ((a): 70°C, (b): 80°C) and Figure 14 ((c): 90°C).

[0073] The β-galactosidase derived from the SeGal strain has high heat resistance and can produce GOS at 70℃~90℃. [Industry-level availability]

[0074] β-galactosidase obtained by the method of producing secretory β-galactosidase is easy to isolate and purify, and can be used for the production of galactooligosaccharides.

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Claims

1. A method for manufacturing a secretory β-galactosidase, characterized by introducing a non-secretory β-galactosidase gene derived from the basidiomycete yeast into Aspergillus oryzae. The aforementioned basidiomycete yeast (oryzae) produces secretory β-galactosidase, which belongs to the genera *Sterigmatomyces*, *Streptococcus*, *Rhodotorula*, or *Sterigmatomyces*. The aforementioned non-secretory β-galactosidase gene derived from basidiomycete yeast contains the message sequence of *Eriocheir oryzae* and the sequence encoding β-galactosidase. The aforementioned message sequence and the sequence encoding β-galactosidase are selected from the sequences recorded in sequence numbers 3, 9, 15, and 21, and the sequence encoding β-galactosidase in the aforementioned sequences recorded in sequence numbers 3, 9, 15, and 21 is a sequence in which at least one of the sequences encoding β-galactosidase has been modified within the range of not changing the amino acid sequence of β-galactosidase, and the codon encoding the sequence of natural β-galactosidase has been changed.

2. The method for manufacturing β-galactosidase as claimed in claim 1, wherein the sequence encoding β-galactosidase in the sequences recorded in the aforementioned sequence numbers 3, 9, 15, and 21 is the sequence of sequence numbers 5, 11, 17, and 23, wherein the codons encoding the sequence of natural β-galactosidase are changed within the range of not changing the amino acid sequence of β-galactosidase.

3. A transformation of *Aspergillus oryzae*, characterized by the introduction of a non-secretory β-galactosidase gene derived from a basidiomycete yeast into *Aspergillus oryzae*, thereby producing a secretory β-galactosidase, wherein the basidiomycete yeast belongs to the genera *Sterigmatomyces*, *Streptococcus*, *Rhodotorula*, or *Sterigmatomyces*, wherein the non-secretory β-galactosidase gene derived from the basidiomycete yeast comprises a message sequence of *Aspergillus oryzae* and a sequence encoding β-galactosidase, wherein the message sequence and the sequence encoding β-galactosidase are selected from the sequences recorded in sequence numbers 3, 9, 15, and 21, and wherein the sequence encoding β-galactosidase in the sequences recorded in sequence numbers 3, 9, 15, and 21 is a sequence in which at least one of the sequences encoding β-galactosidase is modified within the range of not changing the amino acid sequence of β-galactosidase, thereby changing the codon encoding the sequence of naturally occurring β-galactosidase.

4. The transform of Aspergillus oryzae as claimed in claim 3, wherein the sequence encoding β-galactosidase in the sequences described in the aforementioned sequence numbers 3, 9, 15, and 21 is the sequence described in sequence numbers 5, 11, 17, and 23, wherein the codons encoding the sequence of natural β-galactosidase are changed within the range of not changing the amino acid sequence of β-galactosidase.

5. A secretory β-galactosidase obtained by introducing a non-secretory β-galactosidase gene derived from a basidiomycete yeast into *Aspergillus oryzae* and then culturing it, wherein the basidiomycete yeast belongs to the genera *Sterigmatomyces*, *Streptococcus*, *Rhodotorula*, or *Sterigmatomyces*, wherein the non-secretory β-galactosidase gene derived from the basidiomycete yeast contains a message sequence of *Aspergillus oryzae* and a sequence encoding β-galactosidase, wherein the message sequence and the sequence encoding β-galactosidase are selected from the sequences recorded in sequence numbers 3, 9, 15, and 21, and wherein the sequence encoding β-galactosidase in the sequences recorded in sequence numbers 3, 9, 15, and 21 is a sequence in which at least one of the sequences encoding β-galactosidase has been modified within the range of not changing the amino acid sequence of β-galactosidase, and the codon encoding the sequence of naturally occurring β-galactosidase has been changed.

6. The secretory β-galactosidase as described in claim 5, wherein the sequence encoding β-galactosidase in the sequences described in the aforementioned sequence numbers 3, 9, 15, and 21 is the sequence described in sequence numbers 5, 11, 17, and 23, wherein the codons encoding the sequence of natural β-galactosidase are changed within the range of not changing the amino acid sequence of β-galactosidase.

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