วิธีสำหรับการผลิต เบต้า-กาแลคโตสิเดสที่ปลดปล่อยออก

Abstract

วิธีสำหรับการผลิต(เบตา)-กาแลคโตสิเดสที่ปลดปล่อยออกได้ที่มีลักษณะเฉพาะโดยการแทรกตัวยีน(เบตา)-กาแลคโตสิเดสที่ไม่ได้รับการปลดปล่อยที่อนุพัทธ์จากยีสต์เบซิดิโอมัยซีตัสเข้าในAspergillusoryzaeเพื่อผลิต(เบตา)-กาแลคโตสิเดสที่ปลดปล่อยออกได้และวิธีสำหรับการผลิตกาแลกโตโอลิโกแซคคาไรด์โดยใช้(เบตา)-กาแลกโตสิเดสที่ผลิตโดยวิธีที่เสริมการผลิตของกาแลกโตโอลิโกแซคคาไรด์

Description

Method for producing secretory β-galactosidase

[0001] The present invention relates to a method for producing a secretory β-galactosidase that can be easily used in the production of galactooligosaccharides.

[0002] β-Galactosidase is known to catalyze not only the hydrolysis of β-D-galactoside bonds in lactose and the like but also the galactosyl transfer reaction, and is used to produce galactooligosaccharides that selectively grow bifidobacteria in the intestine.

[0003] The present applicant has previously reported a technique for producing galactooligosaccharides using β-galactosidase derived from a high-titer mutant strain of Sporobolomyces singularis, a basidiomycete yeast (Patent Document 1).

[0004] However, since the β-galactosidase used in this technique is non-secretory (cell wall-bound), it is necessary to use a concentrated cell solution containing cells of Sporobolomyces singularis, which produces this enzyme, in the reaction.

[0005] This concentrated bacterial cell solution is prone to deterioration because it is made from live bacteria, and furthermore, since it is simply a concentrated bacterial cell solution, it has low specific activity and the contents of the bacterial cell leak into the galactooligosaccharide reaction solution, resulting in high purification costs.

[0006] Japanese Patent Application Laid-Open No. 2006-223268

[0007] An object of the present invention is to provide a method for producing β-galactosidase which solves the above problems and can be easily used in the production of galactooligosaccharides.

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that by incorporating a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae, secretory β-galactosidase can be produced and that this can be easily used to produce galactooligosaccharides, thereby completing the present invention.

[0009] That is, the present invention provides a method for producing secretory β-galactosidase, which comprises inserting a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae to produce secretory β-galactosidase.

[0010] The present invention also relates to non-secretory β-galactosidase genes derived from basidiomycete yeast, which have the sequences set forth in SEQ ID NOS: 7, 13 and 19.

[0011] Furthermore, the present invention relates to a transformant of Aspergillus oryzae, which is characterized in that a non-secretory β-galactosidase gene derived from a basidiomycete yeast is integrated into Aspergillus oryzae and produces secretory β-galactosidase.

[0012] Furthermore, the present invention provides a method for producing galactooligosaccharides, which comprises allowing β-galactosidase produced by the above-mentioned method for producing β-galactosidase to act on a substrate containing at least lactose.

[0013] The present invention also relates to a secretory β-galactosidase obtained by inserting a non-secretory β-galactosidase gene derived from a basidiomycete yeast into Aspergillus oryzae and culturing the same.

[0014] The method for producing secretory β-galactosidase of the present invention can obtain non-secretory β-galactosidase derived from basidiomycete yeast in a secretory form.

[0015] Therefore, the β-galactosidase obtained by the method for producing secretory β-galactosidase of the present invention has high β-galactosidase activity and high thermostability, and is easy to separate and purify, making it easy to use in the production of galactooligosaccharides.

[0016] FIG. 1 shows the results of SDS-PAGE and activity measurement using the SsGal strain. FIG. 2 shows the results of SDS-PAGE and activity measurement using the SmGal strain. FIG. 3 shows the results of SDS-PAGE and activity measurement using the RmGal strain. FIG. 4 shows the results of SDS-PAGE and activity measurement using the SeGal strain. FIG. 5 shows the results of SDS-PAGE and activity measurement (heat inactivation) using the SsGal strain. FIG. 6 shows the results of SDS-PAGE and activity measurement (heat inactivation) using the SmGal strain. FIG. 7 shows the results of SDS-PAGE and activity measurement (heat inactivation) using the RmGal strain. FIG. 8 shows the results of SDS-PAGE and activity measurement (heat inactivation) using the SeGal strain. FIG. 9 shows the results of activity measurement after heat treatment using the SeGal strain and the parent strain. FIG. 10 shows the reaction time and sugar composition in solution during galactooligosaccharide production using the SsGal strain. FIG. 11 shows the reaction time and sugar composition in solution during galactooligosaccharide production using the SmGal strain. FIG. 12 shows the reaction time and sugar composition in solution during galactooligosaccharide production using the SeGal strain. 1 shows the reaction time and sugar composition in the solution during galactooligosaccharide production using the SeGal strain ((a): 70°C, (b): 80°C). 2 shows the reaction time and sugar composition in the solution during galactooligosaccharide production using the SeGal strain ((c): 90°C).

[0017] The method for producing secretory β-galactosidase of the present invention (hereinafter referred to as "the method of the present invention") involves inserting a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae to produce secretory β-galactosidase.

[0018] The non-secretory β-galactosidase gene derived from basidiomycete yeast used in the production method of the present invention encodes a non-secretory β-galactosidase produced by basidiomycete yeast. Here, the term "non-secretory" refers to a gene that has cell wall-binding ability, which can be confirmed by activity staining or the like.

[0019] Furthermore, the basidiomycete yeast that produces non-secretory β-galactosidase is not particularly limited, and examples thereof include basidiomycete yeasts belonging to the genus Sporobolomyces such as Sporobolomyces singularis, the genus Sirobasidium such as Sirobasidium magnum, the genus Rhodotorula such as Rhodotorula minuta, the genus Sterigmatomyces such as Sterigmatomyces elviae, and the genus Cryptococcus such as Cryptococcus laurentii. Among these basidiomycete yeasts, those belonging to the genus Sporobolomyces or Sterigmatomyces are preferred, with Sporobolomyces singularis or Sterigmatomyces elviae being more preferred.

[0020] Furthermore, examples of genes encoding non-secretory β-galactosidase produced by basidiomycete yeast include genes cloned from the above-mentioned basidiomycete yeast that produces non-secretory β-galactosidase by standard methods such as PCR. Preferably, this gene is totally synthesized based on the gene information obtained as described above, in accordance with the host.

[0021] Specifically, the following genes are included: These genes also contain signal sequences.

[0022] - A β-galactosidase gene derived from Sporobolomyces singularis consisting of the base sequence set forth in SEQ ID NO: 1 (positions 1 to 57 in the sequence are a signal sequence) - A β-galactosidase gene derived from Silobacidium magnum consisting of the base sequence set forth in SEQ ID NO: 7 (positions 1 to 48 in the sequence are a signal sequence) - A β-galactosidase gene derived from Rhodotorula minuta consisting of the base sequence set forth in SEQ ID NO: 13 (positions 1 to 57 in the sequence are a signal sequence) - A β-galactosidase gene derived from Sterigmatomyces erbiae consisting of the base sequence set forth in SEQ ID NO: 19 (positions 1 to 57 in the sequence are a signal sequence)

[0023] Furthermore, preferred genes are those in which the signal sequence of each of the basidiomycete yeasts is replaced with the signal sequence of Aspergillus oryzae. Examples of signal sequences from Aspergillus oryzae include the secretion signal (TAA signal) sequence of Aspergillus oryzae 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)), and the secretion signal sequence 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 to (Secretory pathway of Aspergillus oryzae. Appl Microbiol Biotechnol 79, 1009-1018 (2008)). Such substitution of a signal sequence can be carried out by a conventional method.

[0024] Among the β-galactosidase genes in which the signal sequences of the above-mentioned basidiomycete yeasts have been replaced with the signal sequence of Aspergillus oryzae, the following genes are preferred: These sequences consist of the secretion signal (TAA signal) sequence of Aspergillus oryzae and a sequence encoding native β-galactosidase.

[0025] - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 3 (in the sequence, positions 1 to 63 are a secretory signal sequence) - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 9 (in the sequence, positions 1 to 63 are a secretory signal sequence) - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 15 (in the sequence, positions 1 to 63 are a secretory signal sequence) - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 21 (in the sequence, positions 1 to 63 are a secretory signal sequence)

[0026] Among the above genes, those in which the codons of the sequence encoding native β-galactosidase are altered without changing the amino acid sequence of β-galactosidase are preferred. Examples of such β-galactosidase genes include the following genes. These sequences consist of the secretion signal (TAA signal) sequence of Aspergillus oryzae and sequences in which the codons of the sequence encoding native β-galactosidase are altered without changing the amino acid sequence of β-galactosidase.

[0027] - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 5 (positions 1 to 63 in the sequence are a secretory signal sequence) - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 11 (positions 1 to 63 in the sequence are a secretory signal sequence) - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 17 (positions 1 to 63 in the sequence are a secretory signal sequence) - A β-galactosidase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 23 (positions 1 to 63 in the sequence are a secretory signal sequence)

[0028] Among the above genes, the β-galactosidase genes consisting of the base sequences set forth in SEQ ID NOs: 5, 11, and 23 are preferred.

[0029] The Aspergillus oryzae into which the β-galactosidase gene is to be inserted in the production method of the present invention is not particularly limited. For example, the Aspergillus oryzae may contain the ATP sulfurylase gene (sC). － and the nitrate reductase gene (niaD) －Examples of suitable strains include Aspergillus oryzae NS4 strain (available from the National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima City, Hiroshima Prefecture, 739-0046), Aspergillus oryzae nia D300, Aspergillus oryzae RIB40, and Aspergillus oryzae ATCC 11488. Among these, Aspergillus oryzae NS4 strain is preferred.

[0030] In the production method of the present invention, the above-mentioned gene can be incorporated into Aspergillus oryzae by any method, including, for example, incorporating the gene into an expression vector by standard methods. The type of expression vector is also not particularly limited, but an expression vector derived from Aspergillus oryzae is preferred. In particular, an improved promoter utilizing a cis-element (Region III) involved in the expression control of amylase-related genes (Improvement of the Aspergillus oryzae enolase promoter by introduction of a cis-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) are preferred. Furthermore, these vectors may incorporate a resistance gene for antibiotics such as ampicillin for the selection of transformants, or an ATP sulfurylase expression cassette or other marker.

[0031] The expression vector may be prepared based on the method described in the above-mentioned literature, or may be prepared by, for example, the protein expression service of Ozeki Co., Ltd. (4-9, Imazu Dezai-cho, Nishinomiya-shi, Hyogo 663-8227).

[0032] After the gene is incorporated into an expression vector, it is then incorporated into Aspergillus oryzae to transform it. The method for transforming Aspergillus oryzae is not particularly limited, and may be performed by a conventional method such as the protoplast-PEG method or electroporation. After transformation, washing, selection, collection of bacteria, etc. may be performed as appropriate according to conventional methods.

[0033] In this way, a non-secretory β-galactosidase gene derived from basidiomycete yeast can be inserted into Aspergillus oryzae to obtain a transformant of Aspergillus oryzae that produces secretory β-galactosidase. By culturing this transformant in an appropriate medium such as DPY medium or CDD medium, secretory β-galactosidase is produced from Aspergillus oryzae.

[0034] Since the β-galactosidase obtained above is a secretory type, purification can be achieved by, for example, separating the culture medium after cultivation by filtration, centrifugation, or the like, and collecting the supernatant. The supernatant can also be concentrated using an ultrafiltration membrane or the like. This β-galactosidase has the advantages of high β-galactosidase activity, high thermostability, and few impurities.

[0035] Preferred examples of the amino acid sequence of such secretory β-galactosidase are as follows: - β-galactosidase derived from Sporobolomyces singularis consisting of the amino acid sequence set forth in SEQ ID NO: 2 (positions 1 to 575 in the sequence) (the same as the amino acid sequences set forth in SEQ ID NOs: 4 and 6 (positions 1 to 575 in the sequence)) - β-galactosidase derived from Silobasidium magnum consisting of the amino acid sequence set forth in SEQ ID NO: 8 (positions 1 to 685 in the sequence) (the same as the amino acid sequences set forth in SEQ ID NOs: 10 and 12 (positions 1 to 685 in the sequence)) - β-galactosidase derived from Rhodotorula minuta consisting of the amino acid sequence set forth in SEQ ID NO: 14 (positions 1 to 581 in the sequence) (the same as the amino acid sequences set forth in SEQ ID NOs: 16 and 18 (positions 1 to 581 in the sequence)) - β-galactosidase derived from Sterigmatomyces erbiae consisting of the amino acid sequence set forth in SEQ ID NO: 20 (positions 1 to 581 in the sequence) (the same as the amino acid sequences set forth in SEQ ID NOs: 22 and 24 (positions 1 to 581 in the sequence))

[0036] Among the above-mentioned β-galactosidases, β-galactosidase derived from Sporobolomyces singularis and consisting of the amino acid sequence set forth in SEQ ID NO: 2, β-galactosidase derived from Syllobasidium magnum and consisting of the amino acid sequence set forth in SEQ ID NO: 8, and β-galactosidase derived from Sterigmatomyces erbiae and consisting of the amino acid sequence set forth in SEQ ID NO: 20 are preferred.

[0037] This β-galactosidase is secreted outside the bacterial cells, and its activity does not decrease even after long-term storage, and it has good thermostability and storage stability. The activity of β-galactosidase can be confirmed by the method described in the Examples below. While multiple β-galactosidases are usually used to efficiently produce galactooligosaccharides, the β-galactosidase obtained above can be used alone to efficiently produce galactooligosaccharides.

[0038] The β-galactosidase obtained above can be used, like conventionally known β-galactosidases, to produce galactooligosaccharides by allowing the β-galactosidase to act on a substrate containing at least lactose. Since this β-galactosidase is secretory, there is no need to remove the bacterial cells during the production of galactooligosaccharides.

[0039] Specifically, to allow the β-galactosidase obtained above to act on a substrate containing at least lactose, β-galactosidase is added to the substrate containing at least lactose and maintained at a predetermined temperature. The amount of β-galactosidase added is not particularly limited, but may be, for example, 1 to 50 U, preferably 5 to 10 U, per 100 g of lactose. Furthermore, the temperature at which the β-galactosidase is allowed to act on the substrate is not particularly limited, but may be 30 to 90°C, preferably 60 to 90°C, and the maintenance time may be set appropriately. A sugar to be galactosylated may be added to the substrate containing at least lactose. Examples of such sugars include, but are not limited to, galactose, mannose, ribose, xylose, arabinose, rhamnose, N-acetylglucosamine, α-methylmannoside, α-methylgalactoside, α-methylglucoside, 2-deoxyglucose, and 2-deoxygalactose.

[0040] The galactooligosaccharides produced as described above contain a large amount of galactooligosaccharides of 5 or less sugars, particularly galactooligosaccharides of 3 sugars.

[0041] The galactooligosaccharides produced as described above may be used as they are, or may be separated and purified using a common purification method, which is not particularly limited, but may be purified by various types of chromatography such as ion exchange, gel filtration, activated carbon, and affinity chromatography.

[0042] The galactooligosaccharides thus obtained can be used as useful food materials, pharmaceutical raw materials, and reagents.

[0043] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0044] The deposit numbers of the basidiomycete yeasts used in these examples are as follows: Sporobolomyces singularis ATCC 24193 Rhodotorula minuta CBS 319 Sterigmatomyces erbiae IFO 1843 Sillobacidium magnum CBS 6803 ATCC: 10801 University Boulevard, Manassas, VA 20110 USA CBS: Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands IFO: 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686

[0045] Example 1: Obtaining a β-galactosidase gene from Sporobolomyces singularis: The β-galactosidase gene from Sporobolomyces singularis was obtained (SEQ ID NO: 1) based on the literature (Ishikawa, E., Sakai, T., Ikemura, H., Matsumoto, K., and Abe, H., Identification, cloning, and characterization of a Sporobolomyces singularis β-galactosidase-like enzyme involved in galacto-oligosaccharide production. J Biosci Bioeng 99, 331-339 (2005).). This gene consists of a signal sequence and a sequence encoding β-galactosidase. The signal sequence of this gene was replaced with the TAA signal sequence of Aspergillus oryzae to obtain a sequence (SEQ ID NO: 3) on a computer, and the β-galactosidase gene was further modified by changing the codons in the sequence encoding native β-galactosidase without altering the amino acid sequence of β-galactosidase to obtain a sequence (SEQ ID NO: 5) (SsGal). This SsGal was totally synthesized by GenScript.

[0046] Example 2: Isolation of the β-galactosidase gene from Psilobasidium magnum: Degenerate primers (Table 1) (SEQ ID NOs: 25-29) were designed from the conserved region, and partial sequences were cloned by RT-PCR using six combinations (two forward primers and three reverse primers). 5' RACE and 3' RACE were performed from the partial sequences to obtain full-length cDNA.

[0047]

[0048] Based on the full-length cDNA described above, a β-galactosidase gene derived from Psilocytoma magnum was obtained by analogy with the initiation codon (ATG) in the upstream region (SEQ ID NO: 7). This gene consists of a signal sequence and a sequence encoding β-galactosidase. The signal sequence of this gene was replaced with the TAA signal sequence of Aspergillus oryzae to obtain a sequence (SEQ ID NO: 9) on a computer. Furthermore, the β-galactosidase gene was modified by altering the codons of the native β-galactosidase-encoding sequence without altering the amino acid sequence of β-galactosidase (SEQ ID NO: 11) (SmGal). GenScript, Inc., conducted the total synthesis of this SmGal.

[0049] Example 3: Obtaining the β-galactosidase gene from Rhodotorula minuta: The β-galactosidase gene from Rhodotorula minuta was obtained by a method similar to that for the β-galactosidase gene from Xylobasidium magnum (SEQ ID NO: 13). This gene consists of a signal sequence and a sequence encoding β-galactosidase. The signal sequence of this gene was replaced with the TAA signal sequence from Aspergillus oryzae to obtain a sequence (SEQ ID NO: 15) on a computer. Furthermore, the β-galactosidase gene was modified by altering the codons of the sequence encoding native β-galactosidase without altering the amino acid sequence of β-galactosidase (SEQ ID NO: 17) (RmGal). This RmGal was totally synthesized by GenScript, Inc.

[0050] Example 4: Obtaining a β-galactosidase gene from Sterigmatomyces erbiae: The β-galactosidase gene from Sterigmatomyces erbiae was obtained by a method similar to that for the β-galactosidase gene from Sillobacillus magnum (SEQ ID NO: 19). This gene consists of a signal sequence and a sequence encoding β-galactosidase. The signal sequence of this gene was replaced with the TAA signal sequence of Aspergillus oryzae to obtain a sequence (SEQ ID NO: 21) on a computer. Furthermore, the β-galactosidase gene was modified by altering the codons of the sequence encoding native β-galactosidase without altering the amino acid sequence of β-galactosidase (SEQ ID NO: 23) (SeGal). This SeGal was totally synthesized by GenScript, Inc.

[0051] Example 5 Obtaining SsGal transformant: The SsGal obtained in Example 1 was sent to the protein expression service of Ozeki Co., Ltd. (4-9, Imazu Dezaike-cho, Nishinomiya-shi, Hyogo 663-8227) and inserted into an expression vector.

[0052] The host for transformation contained the nitrate reductase gene (niaD) from Aspergillus oryzae. － , ATP sulfurylase gene (sC) － The NS4 strain (provided by the National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima Prefecture, 739-0046) was used, and an expression vector was introduced into it by the conventional protoplast-PEG method to obtain a transformant (SsGal strain). － This was done by complementation of the trait.

[0053] Example 6 Obtaining SmGal transformants: An expression vector and a transformant (SmGal strain) were obtained in the same manner as in Example 5, except that the SmGal obtained in Example 2 was used.

[0054] Example 7 Obtaining RmGal transformants: An expression vector and a transformant (RmGal strain) were obtained in the same manner as in Example 5, except that the RmGal obtained in Example 3 was used.

[0055] Example 8 Obtaining SeGal transformants: An expression vector and a transformant (SeGal strain) were obtained in the same manner as in Example 5, except that SeGal obtained in Example 4 was used.

[0056] Example 9 Evaluation of β-galactosidase production by transformants: (1) Activity measurement Of the transformants obtained in Examples 5 to 8, the SsGal strain was grown 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 The RmGal strain was cultured in 2xDPY medium (4% dextrin, 2% hippocampal saccharin, 2% yeast extract, 1% KH2PO4, 0.1% MgSO4·7H2O) at 30°C for 144 hours (15 mL / 100 mL Erlenmeyer flask scale). The SmGal strain was cultured in 2xDPY medium (4% dextrin, 2% hippocampal saccharin, 2% yeast extract, 1% KH2PO4, 0.1% MgSO4·7H2O) at 30°C for 144 hours (150 mL / 500 mL Sakaguchi flask scale). The SeGal strain was cultured in DPY medium (2% dextrin, 1% hippocampal saccharin, 1% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O) at 30°C for 168 hours. The culture supernatant was collected and mixed with an equal volume of 2x sample buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 0.01% bromophenol blue, 4% SDS, 200 mM DTT), incubated at 100°C for 10 minutes, and then subjected to SDS-PAGE (CBB staining).

[0057] Additionally, activity measurements using ONPG as a substrate were performed according to the following method. A solution of 2-nitrophenyl-β-galactoside (ONPG) was prepared by adding 12.5 mM ONPG to 50 mM citrate phosphate buffer (pH 4.0). 0.2 mL of the above-mentioned β-galactosidase-containing culture supernatant, diluted with 50 mM citrate phosphate buffer (pH 4.0) to an absorbance of 0.2-0.8 at 420 nm, was added to 0.8 mL of this solution and allowed to react for 10 minutes at 30°C (test solution). The reaction was stopped by adding 4 mL of 0.25 M sodium carbonate solution, followed by centrifugation (3,000 g, 10 minutes). The amount of liberated 2-nitrophenol in the supernatant was quantified by measuring the absorbance at 420 nm using a spectrophotometer. Separately, a 2-nitrophenyl-β-galactoside solution to which 50 mM citrate phosphate buffer (pH 4.0) was added was used as a reagent blank, and a starting reaction solution (blank) was prepared by adding sodium carbonate solution to the solution, and then adding and mixing the culture supernatant containing β-galactosidase to the solution, which simultaneously stopped the reaction and developed color. One unit (U) of enzyme activity was defined as the amount of enzyme that liberates 1 μmole of 2-nitrophenol per minute under these conditions, and was calculated using the following formula:

[0058]

[0059] 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 that were not present in the parent strains, which were presumed to represent the respective β-galactosidases. The SsGal strain did not exhibit a specific band in DPY medium, but when cultured in CDD (pH 7.0) medium, a specific band was detected that was not present in the parent strain, presumably representing β-galactosidase. Culture conditions that maximized the secretion productivity of each β-galactosidase were examined. The SsGal strain exhibited maximum activity in CDD (pH 7.0) medium at 30°C for 144 hours, the RmGal strain in 2xDPY medium at 30°C for 144 hours, the SmGal strain in 2xDPY medium at 30°C for 168 hours, and the SeGal strain in DPY medium at 30°C for 168 hours. Furthermore, the productivities of the SsGal strain, RmGal strain, SmGal strain, and SeGal strain were estimated to be approximately 200 mg / L, approximately 200 mg / L, approximately 200 mg / L, and approximately 1 g / L, respectively, based on the concentration of the SDS-PAGE bands.

[0060] (2) Copy number estimation The number of expression cassettes integrated into the transformants was also estimated by real-time PCR.

[0061] From the PCR results, it was estimated that the SsGal, RmGal, and SmGal strains contained one copy of the expression cassette, while the SeGal strain contained two copies.

[0062] (3) Thermal inactivation test One mL of the culture medium of each transformant and the parent strain (NS4 strain) cultured under the culture conditions described in (1) was incubated at 40°C, 50°C, 60°C, 70°C, and 80°C for 1 hour each, and enzyme activity was measured and SDS-PAGE was performed.

[0063] The results of SDS-PAGE and activity measurements are shown in Figures 5 to 8. The SsGal strain retained its activity up to 40°C, but after 1 hour of incubation at 50°C, its activity decreased by approximately 70% and disappeared at 70°C. The parent strain cultured under the same conditions had a trace amount of activity up to 60°C and disappeared at 70°C. The RmGal strain maintained its activity up to 50°C, but its activity disappeared after 1 hour of incubation at 60°C. The parent strain cultured under the same conditions had detectable activity up to 70°C and disappeared at 80°C. The SmGal strain maintained its activity up to 50°C, but its activity decreased by approximately 20% after 1 hour of incubation at 60°C and disappeared at 80°C. The parent strain cultured under the same conditions had detectable activity up to 70°C and disappeared at 80°C. The SeGal strain maintained its activity up to 70°C, but its activity decreased by approximately 97% after 1 hour of incubation at 80°C. The activity of the parent 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 times than 1 hour (5, 10, or 20 minutes). As a result, the activity decreased by approximately 37% after 5 minutes of treatment at 80°C, and by approximately 98% after 20 minutes of treatment. Furthermore, activity measurements showed that both treatments at 40°C and 50°C showed higher activity than the parent strain.

[0064] These results indicate that the SeGal strain can retain its activity even at high temperatures.

[0065] Example 10 Removal of contaminating enzymes: As shown in Example 9(3), it was found that the SeGal strain can retain its activity even at high temperatures. On the other hand, when a heat inactivation test was performed on a contaminating enzyme derived from Aspergillus oryzae, the parent strain of the SeGal strain, in the same manner as in Example 9(3), it was found that it could be inactivated by heat treatment at 70°C. Therefore, it was found that the β-galactosidase produced by the SeGal strain can be purified by heat treatment (Figure 9).

[0066] Example 11 Production of Galactooligosaccharides (1): 10 U of each of the culture supernatants from the SsGal, SmGal, and SeGal strains obtained in Example 9 was added to 150 mL of a solution containing 66% (w / v) lactose, and the mixture was allowed to react at a predetermined temperature for a predetermined time to produce galactooligosaccharides. The sugar composition and amount were measured by high-performance liquid chromatography. The reaction time and sugar composition in the solution are shown in Figures 10 to 12 (Figure 10: SsGal strain, Figure 11: SmGal strain, Figure 12: SeGal strain).

[0067] The figure shows that β-galactosidase produced by the SsGal, SmGal, and SeGal strains can produce mainly trisaccharide galactooligosaccharides from lactose.

[0068] Furthermore, when galactooligosaccharides were produced using β-galactosidase produced by the SsGal strain, the galactooligosaccharide content was 56.0%, when galactooligosaccharides were produced using β-galactosidase produced by the SmGal strain, the galactooligosaccharide content was 66.7%, and when galactooligosaccharides were produced using β-galactosidase produced by the SeGal strain, the galactooligosaccharide content was 68.5%.

[0069] Since the β-galactosidase is secretory, there is no need to treat the cells after producing galactooligosaccharides, and galactooligosaccharides can be produced efficiently.

[0070] Example 12 Production of Galactooligosaccharides (2): 1.0 U of the culture supernatant of the SeGal strain obtained in Example 9 was added to 150 mL of a solution containing 66% (w / v) lactose, and the mixture was reacted at 70°C, 80°C, and 90°C for a predetermined time to produce galactooligosaccharides. The sugar composition and amount were measured 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).

[0071] The β-galactosidase derived from the SeGal strain was highly thermostable and enabled GOS production at temperatures between 70°C and 90°C.

[0072] The β-galactosidase obtained by the method for producing secretory β-galactosidase can be easily separated and purified, and can be used to produce galactooligosaccharides.

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