Method for producing secretory β-galactosidase

Abstract

This invention provides a method for manufacturing secretory β-galactosidase, characterized in that a non-secretory β-galactosidase gene from a basidiomycete yeast is introduced into Aspergillus oryzae to induce the production of secretory β-galactosidase, and galactooligosaccharides are readily manufactured by utilizing the β-galactosidase produced by this method.

Description

Technical Field

[0001] The present invention relates to a method for producing secretory β-galactosidase, which method can be easily used for producing galacto-oligosaccharides. Background Art

[0002] β-Galactosidase is known to catalyze the hydrolysis of β-D-galactosidic bonds of lactose and the like, and also to catalyze the galactosyl transfer reaction, and is used to produce galacto-oligosaccharides that selectively promote the proliferation of bifidobacteria in the intestine.

[0003] The present applicant has reported a technique for producing galacto-oligosaccharides using β-galactosidase derived from a high-titer mutant 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 prepare a cell concentrate containing the cells of Sporobolomyces singularis that produces the enzyme for use in the reaction.

[0005] This bacterial cell concentrate is easily deteriorated because it contains live bacteria. Moreover, since the bacterial cells are simply concentrated, the specific activity is low, and there are also problems such as leakage of bacterial cell contents into the galacto-oligosaccharide reaction solution, which leads to an increase in purification cost.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2006-223268 Summary of the invention

[0009] An object of the present invention is to provide a method for producing β-galactosidase which solves the above-mentioned problems and can be easily used for the production of galacto-oligosaccharides.

[0010] The present inventors have conducted intensive studies to solve the above problems and have found that secretory β-galactosidase can be produced by introducing a non-secretory β-galactosidase gene from basidiomycete yeast into Aspergillus oryzae and that it can be easily used for the production of galacto-oligosaccharides, thereby completing the present invention.

[0011] That is, the present invention is a method for producing secretory β-galactosidase, characterized in that a non-secretory β-galactosidase gene derived from basidiomycete yeast is introduced into Aspergillus oryzae to produce secretory β-galactosidase.

[0012] Furthermore, the present invention provides a non-secretory β-galactosidase gene derived from a basidiomycete yeast, wherein the β-galactosidase gene is a sequence as set forth in SEQ ID NOs: 7, 13, and 19.

[0013] Furthermore, the present invention provides a transformant of Aspergillus oryzae, characterized in that a non-secretory β-galactosidase gene derived from a basidiomycete yeast is introduced into Aspergillus oryzae to thereby produce secretory β-galactosidase.

[0014] Furthermore, the present invention provides a method for producing galacto-oligosaccharides, characterized in that β-galactosidase produced by the above-mentioned method for producing β-galactosidase is allowed to act on a substrate containing at least lactose.

[0015] Furthermore, the present invention provides a secretory β-galactosidase obtained by introducing a non-secretory β-galactosidase gene derived from a basidiomycete yeast into Aspergillus oryzae and then culturing the resulting bacteria.

[0016] The method for producing a secretory β-galactosidase of the present invention can obtain a non-secretory β-galactosidase derived from a basidiomycete yeast by converting it into a secretory one.

[0017] Therefore, the β-galactosidase obtained by the method for producing secretory β-galactosidase of the present invention has high β-galactosidase activity and high thermal stability, and can be easily isolated and purified, and can be easily used for the production of galacto-oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a figure which shows the result of SDS-PAGE and activity measurement using SsGal strain.

[0019] Figure 2 It is a figure which shows the result of SDS-PAGE and activity measurement using SmGal strain.

[0020] Figure 3 It is a figure which shows the result of SDS-PAGE and activity measurement using RmGal strain.

[0021] Figure 4 It is a figure which shows the result of SDS-PAGE and activity measurement using SeGal strain.

[0022] Figure 5 The graphs show the results of SDS-PAGE and activity measurement (heat inactivation) using the SsGal strain.

[0023] Figure 6 The graphs show the results of SDS-PAGE and activity measurement (heat inactivation) using the SmGal strain.

[0024] Figure 7 The graphs show the results of SDS-PAGE and activity measurement (heat inactivation) using the RmGal strain.

[0025] Figure 8 The graphs show the results of SDS-PAGE and activity measurement (heat inactivation) using the SeGal strain.

[0026] Fig. 9 This is a graph showing the results of activity measurement after heat treatment using the SeGal strain and the mother strain.

[0027] Fig.10 This is a graph showing the reaction time and the sugar composition in the solution in the production of galacto-oligosaccharide using the SsGal strain.

[0028] Fig.11 This is a graph showing the reaction time and the sugar composition in the solution in the production of galacto-oligosaccharides using the SmGal strain.

[0029] Fig.12 This is a graph showing the reaction time and the sugar composition in the solution in the production of galacto-oligosaccharides using the SeGal strain.

[0030] Fig.13 Graphs showing the relationship between reaction time and sugar composition in the solution in the production of galacto-oligosaccharides using the SeGal strain ((a): 70°C, (b): 80°C).

[0031] Fig.14 This is a graph showing the relationship between the reaction time and the sugar composition in the solution in the production of galacto-oligosaccharides using the SeGal strain ((c): 90°C). DETAILED DESCRIPTION

[0032] The method for producing secretory β-galactosidase of the present invention (hereinafter referred to as "the production method of the present invention") is to introduce a non-secretory β-galactosidase gene derived from basidiomycete yeast into Aspergillus oryzae to produce secretory β-galactosidase.

[0033] The non-secretory β-galactosidase gene derived from basidiomycete yeast used in the preparation method of the present invention can encode non-secretory β-galactosidase produced by basidiomycete yeast. The non-secretory type here means that it has cell wall binding, which can be confirmed by activity staining or the like.

[0034] In addition, 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 Rodotorula minuta, the genus Sterigmatomyces such as Sterigmatomyces elviae, the genus Cryptococcus such as Cryptococcus laurentii, etc. Among these basidiomycete yeasts, basidiomycete yeasts belonging to the genus Sporobolomyces or the genus Sirobasidium are preferred, and Sporobolomyces singularis or Sterigmatomyces elviae are more preferred.

[0035] Furthermore, as a gene encoding a non-secretory β-galactosidase produced by a basidiomycete yeast, firstly, a gene cloned from a basidiomycete yeast that produces the above-mentioned non-secretory β-galactosidase according to a conventional method such as PCR can be mentioned. It should be noted that this gene is preferably fully synthesized in combination with a host based on the information of the gene obtained in the above manner.

[0036] Specifically, the following genes are mentioned. It should be noted that this gene also contains a signal sequence.

[0037] β-galactosidase gene from Sporobolomyces singularis consisting of the base sequence described in SEQ ID NO: 1 (Sequence Nos. 1 to 57 are signal sequences)

[0038] β-galactosidase gene from the large chain worm consisting of the base sequence described in SEQ ID NO: 7 (numbers 1 to 48 in the sequence are signal sequences)

[0039] β-galactosidase gene from Rhodotorula microtypingii consisting of the base sequence described in SEQ ID NO: 13 (numbers 1 to 57 in the sequence are signal sequences)

[0040] β-galactosidase gene from Sterigmatomyces elviae consisting of the base sequence described in SEQ ID NO: 19 (numbers 1 to 57 in the sequence are signal sequences)

[0041] Furthermore, a preferred gene among the above genes is a gene in which the signal sequence of each of the above basidiomycete yeasts is replaced with the signal sequence of Aspergillus oryzae. Examples of the signal sequence of Aspergillus oryzae include the secretory signal sequence (TAA signal) of α-amylase (Taka-amylase: TAA) of Aspergillus oryzae (Okazaki, F., Aoki, J., Tabuchi, S., Tanaka, T., Ogino, C., and Kondo, A., Efficient heterologous expression and secretion in Aspergillus oryzae of α-amylase variable hea vy-chain antibody fragment V (HH) against EGFR. Appl Microbiol Biotec hnol 96, 81-88 (2012).), and the secretory signal of lipase of Rhizopus oryzae (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 indirecting proteins to secretory pathway of Aspergillus oryzae. Appl Microbiol Biotechnol 79, 1009-1018 (2008).) etc. Such signal sequence substitution can be performed according to conventional methods.

[0042] Among the β-galactosidase genes in which the signal sequence of each basidiomycete yeast is replaced with the signal sequence of Aspergillus oryzae, the following genes are preferred. These sequences consist of a secretion signal (TAA signal) sequence of Aspergillus oryzae and a sequence encoding natural β-galactosidase.

[0043] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 3 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0044] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 9 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0045] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 15 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0046] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 21 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0047] Among the above genes, it is preferred that the codons of the sequence encoding natural β-galactosidase be changed within the range of not changing the amino acid sequence of β-galactosidase. As such β-galactosidase genes, the following genes can be mentioned. These sequences are composed of a secretion signal (TAAsignal) sequence of Aspergillus oryzae and a sequence of codons of the sequence encoding natural β-galactosidase be changed within the range of not changing the amino acid sequence of β-galactosidase.

[0048] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 5 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0049] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 11 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0050] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 17 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0051] β-galactosidase gene consisting of the base sequence described in SEQ ID NO: 23 (Sequence Nos. 1 to 63 are secretion signal sequences)

[0052] Among the above genes, β-galactosidase genes consisting of the base sequences described in SEQ ID NOs: 5, 11, and 23 are preferred.

[0053] The Aspergillus oryzae into which the β-galactosidase gene is introduced used in the preparation method of the present invention is not particularly limited, and examples thereof include ATP sulfurylase gene (sC) - and nitrate reductase gene (niaD) - Aspergillus oryzae NS4 strain (available from the Independent Administrative Institution Sake Research Institute, 739-0046, Kagamiyama 3-7-1, Higashihiroshima City, Hiroshima Prefecture), Aspergillus oryzae niaD300, Aspergillus oryzae RIB40, and Aspergillus oryzae ATCC11488. Of these, Aspergillus oryzae NS4 strain is preferred.

[0054] In the preparation method of the present invention, the method for introducing the above-mentioned gene into Aspergillus oryzae is not particularly limited, for example, as long as the above-mentioned gene is introduced into an expression vector by a conventional method. The type of expression vector is not particularly limited, preferably an expression vector from Aspergillus oryzae, particularly preferably: comprising a modified promoter (by introducing a cis-acting element to improve Aspergillus oryzae enolase promoter, Tsuboi, H. et al., Biosci. Biotec hnol. Biochem., 69, 206-208 (2005)) and a high expression vector (Japanese Patent No. 4413557) of a 5'UTR sequence with high translation efficiency) utilizing a cis-acting element (Region III) related to the expression control of amylase-based genes. Furthermore, in these vectors, in order to select transformants, resistance genes of antibiotics such as ampicillin can also be introduced, or an ATP sulfurylase expression cassette as a marker can be introduced.

[0055] The above-mentioned expression vector can be prepared based on the method described in the above-mentioned literature, or can be prepared by using the protein expression service provided by, for example, Ozeki Co., Ltd. (4-9 Imazu Ideie-cho, Nishinomiya-shi, Hyogo Prefecture, 663-8227).

[0056] After the above-mentioned gene is imported into the expression vector, it is imported into Aspergillus oryzae and transformed. The method that Aspergillus oryzae is transformed is not particularly limited, for example, ordinary methods such as protoplast-PEG method, electroporation can be used to carry out. After transforming, also can suitably clean, select, collect bacteria etc. according to ordinary method.

[0057] In this way, a non-secretory β-galactosidase gene from basidiomycete yeast is introduced into Aspergillus oryzae to obtain an Aspergillus oryzae transformant that produces secretory β-galactosidase. By culturing the transformant appropriately in a DPY medium, CDD medium, etc., secretory β-galactosidase can be produced by Aspergillus oryzae.

[0058] Since the β-galactosidase obtained above is secretory, it can be purified by filtering the culture solution after cultivation and separating it by centrifugation, etc., and only the supernatant can be collected. In addition, the supernatant can also be concentrated using an ultrafiltration membrane, etc. This β-galactosidase has the following characteristics: the activity of β-galactosidase is high, the thermal stability is also high, and there are few impurities.

[0059] Preferred amino acid sequences of such secretory β-galactosidase are shown below, for example.

[0060] β-galactosidase from Sporobolomyces singularis consisting of the amino acid sequence described in SEQ ID NO: 2 (Sequence Nos. 1 to 575) (Sequence Nos. 4 and 6 also have the same amino acid sequence (Sequence Nos. 1 to 575))

[0061] β-galactosidase derived from a large-chain β-galactosidase consisting of the amino acid sequence described in SEQ ID NO: 8 (Nos. 1 to 685 in the sequence) (the amino acid sequences described in SEQ ID NOs. 10 and 12 are also the same (Nos. 1 to 685 in the sequence))

[0062] β-galactosidase from Rhodotorula microtypingensis consisting of the amino acid sequence described in SEQ ID NO: 14 (Sequence Nos. 1 to 581) (Sequence Nos. 16 and 18 also have the same amino acid sequence (Sequence Nos. 1 to 581))

[0063] β-galactosidase from Sterigmatomyces elviae consisting of the amino acid sequence described in SEQ ID NO: 20 (Sequence Nos. 1 to 581) (Sequence Nos. 22 and 24 also have the same amino acid sequence (Sequence Nos. 1 to 581))

[0064] Among the above-mentioned β-galactosidases, preferred are: β-galactosidase derived from Sporobolomyces singularis consisting of the amino acid sequence recorded in SEQ ID NO: 2, β-galactosidase derived from Macrobrachium truncatum consisting of the amino acid sequence recorded in SEQ ID NO: 8, and β-galactosidase derived from Sterigmatomyces elviae consisting of the amino acid sequence recorded in SEQ ID NO: 20.

[0065] In addition to being secreted outside the bacteria, the β-galactosidase also has the properties that the activity of the β-galactosidase does not decrease even after long-term storage, and has good thermal stability and storage properties. It should be noted that the activity of the β-galactosidase can be confirmed by the method described in the examples described below. Generally speaking, in order to effectively produce oligogalactose, a variety of β-galactosidases are used, but the β-galactosidase obtained above can also effectively produce oligogalactose even if used alone.

[0066] The β-galactosidase obtained above can be used, for example, to make the β-galactosidase act on a substrate containing at least lactose to produce galacto-oligosaccharides, similar to the conventionally known β-galactosidase. It should be noted that since the β-galactosidase is secretory, it is not necessary to remove the bacterial cells when producing galacto-oligosaccharides.

[0067] Specifically, in order to make the β-galactosidase obtained above act on a substrate containing at least lactose, it is sufficient to add β-galactosidase to the substrate containing at least lactose and maintain a predetermined temperature. The amount of β-galactosidase added is not particularly limited, for example, it is 1 to 50 U relative to 100 g of lactose, preferably 5 to 10 U. Furthermore, the temperature at which β-galactosidase acts on the substrate is not particularly limited, and is 30 to 90° C., preferably 60 to 90° C., and the maintenance time can be appropriately set. Galactosylated sugars can also be added to the substrate containing at least lactose. Such sugars are not particularly limited, and examples thereof include galactose, mannose, ribose, xylose, arabinose, rhamnose, N-acetylglucosamine, α-methyl mannoside, α-methyl galactoside, α-methyl glucoside, 2-deoxyglucose, 2-deoxygalactose, and the like.

[0068] The galacto-oligosaccharides produced as described above contain a large amount of galacto-oligosaccharides of penta-saccharides or less, particularly galacto-oligosaccharides of tri-saccharides.

[0069] It should be noted that the galacto-oligosaccharide produced as described above can be used directly or separated and purified using a general purification method. The purification method is not particularly limited, and specifically, purification can be performed by applying various chromatography methods such as ion exchange, gel filtration, activated carbon, and affinity chromatography.

[0070] The galacto-oligosaccharide obtained in this manner can be used as a useful food raw material, pharmaceutical raw material, or reagent.

[0071] Example

[0072] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[0073] The accession numbers of the basidiomycete yeasts used in these Examples are as follows.

[0074] ·Sporobolomyces singularis ATCC 24193

[0075] ·Red yeast CBS 319

[0076] ·Sterigmatomyces elviae IFO 1843

[0077] ·Large chain lug CBS 6803

[0078] ATCC: 10801 University Boulevard Manassas, VA 20110 USA

[0079] CBS: Uppsalalaan 8,3584 CT,Utrecht,The Netherlands

[0080] IFO: 〒532-8686 No. 17-85, 2-chome, Jusanhonmachi, Yodogawa-ku, Osaka City

[0081] Example 1

[0082] Acquisition of the β-galactosidase gene from Sporobolomyces singularis:

[0083] 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).), the β-galactosidase gene (SEQ ID NO. 1) of Sporobolomyces singularis was obtained. This gene consists of a signal sequence and a sequence encoding β-galactosidase. A sequence (SEQ ID NO. 3) in which the signal sequence of this gene was replaced with the TAA signal sequence of Aspergillus oryzae was obtained on a computer. Furthermore, for the β-galactosidase gene, a sequence (SEQ ID NO. 5) (SsGal) in which the codons of the sequence encoding the natural β-galactosidase were changed within the range of not changing the amino acid sequence of the β-galactosidase was obtained. The total synthesis of SsGal was entrusted to GenScript.

[0084] Example 2

[0085] Obtaining the β-galactosidase gene from the large-chain spores:

[0086] Degenerate primers (Table 1) (SEQ ID NOs: 25 to 29) were designed from the conserved region, and partial sequences were cloned using RT-PCR in 6 combinations of 2 sense and 3 antisense. 5' RACE and 3' RACE were performed from the partial sequences to obtain full-length cDNA.

[0087] [Table 1]

[0088] Serial Number name Orientation Degenerate primer base sequence 25 F1 justice gccggcgcggctathcargtngarggngcn 26 F2 justice gtcaagacntggttyacnttyaaygarccn 27 R1 Antonym ctcggcccacccraaytcnswraartadat 28 R2 Antonym ccattcccarttrtcnacraanswcca 29 C-R70 Antonym gacgaggccnswrttccaytcraarttrtc

[0089] Based on the above full-length cDNA, the β-galactosidase gene (sequence number 7) from the large-chain β-galactosidase was obtained by analogy with the start codon (ATG) in the upstream region. This gene consists of a signal sequence and a sequence encoding β-galactosidase. A sequence (sequence number 9) in which the signal sequence of this gene is replaced with the TAA signal sequence of Aspergillus oryzae was obtained on a computer. Further, for the β-galactosidase gene, a sequence (sequence number 11) (SmGal) in which the codons of the sequence encoding natural β-galactosidase were changed within the range of not changing the amino acid sequence of β-galactosidase was obtained. This SmGal was entrusted to GenScript Company for total synthesis.

[0090] Example 3

[0091] Acquisition of β-galactosidase gene from Rhodotorula microtypingii:

[0092] The β-galactosidase gene (sequence number 13) of Rhodotorula microphylla was obtained by the same method as the β-galactosidase gene from the large chain worm. This gene consists of a signal sequence and a sequence encoding β-galactosidase. A sequence (sequence number 15) in which the signal sequence of this gene is replaced by the TAA signal sequence of Aspergillus oryzae is obtained on a computer. Further, for the β-galactosidase gene, a sequence (sequence number 17) (RmGal) in which the codons of the sequence encoding the natural β-galactosidase are changed within the range of not changing the amino acid sequence of the β-galactosidase is obtained. This RmGal was entrusted to GenScript Company for total synthesis.

[0093] Example 4

[0094] Acquisition of β-galactosidase gene from Sterigmatomyces elviae:

[0095] The β-galactosidase gene of Sterigmatomyces elviae (sequence number 19) was obtained by the same method as the β-galactosidase gene from the large-chain β-galactosidase. This gene consists of a signal sequence and a sequence encoding β-galactosidase. A sequence (sequence number 21) in which the signal sequence of this gene is replaced by the TAA signal sequence of Aspergillus oryzae was obtained on a computer. Further, for the β-galactosidase gene, a sequence (sequence number 23) (SeGal) in which the codons of the sequence encoding the natural β-galactosidase were changed within the range of not changing the amino acid sequence of the β-galactosidase was obtained. This SeGal was entrusted to GenScript Company for total synthesis.

[0096] Example 5

[0097] Obtaining SsGal transformants:

[0098] The SsGal obtained in Example 1 was sent to Ozeki Co., Ltd. (4-9 Imazu-Ideie-cho, Nishinomiya-shi, Hyogo Prefecture, 663-8227) for protein expression service and introduced into an expression vector.

[0099] As a host for transformation, a nitrate reductase gene (niaD) from Aspergillus oryzae was used. - , ATP sulfurylase gene (sC) - The NS4 strain of the strain (sold by the Independent Administrative Institution Sake Research Institute, Kagamiyama 3-7-1, Higashihiroshima City, Hiroshima Prefecture, 739-0046) was introduced into the expression vector by the general protoplast-PEG method to obtain a transformant (SsGal strain). It should be noted that the selection of the transformant was carried out by mixing with sC - to complement each other.

[0100] Example 6

[0101] Obtaining SmGal transformants:

[0102] The same procedure as in Example 5 was carried out except that SmGal obtained in Example 2 was used to obtain an expression vector and a transformant (SmGal strain) after introduction.

[0103] Example 7

[0104] Obtaining RmGal transformants:

[0105] The same procedure as in Example 5 was carried out except that RmGal obtained in Example 3 was used to obtain an expression vector and a transformant (RmGal strain) after introduction.

[0106] Example 8

[0107] Obtaining SeGal transformants:

[0108] The same procedure as in Example 5 was carried out except that SeGal obtained in Example 4 was used to obtain an expression vector and a transformant (SeGal strain) after introduction.

[0109] Example 9

[0110] Evaluation of β-galactosidase production of transformants:

[0111] (1) Activity assay

[0112] Among the transformants obtained in Examples 5 to 8, the SsGal strain was cultured using 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, 200mM MOPS-NaOH buffer pH 7.0) at 30°C for 144 hours (15mL / 100mL triangular graduated flask). RmGal strain was cultured at 30°C for 144 hours (150mL / 500mL round-bottom graduated flask) using 2×DPY medium (4% dextrin, 2% polypeptone, 2% yeast extract, 1% KH2PO4, 0.1% MgSO4·7H2O). SmGal strain was cultured at 30°C for 168 hours (150mL / 500mL round-bottom graduated flask) using 2×DPY medium. SeGal strain was cultured at 30°C for 168 hours using DPY medium (2% dextrin, 1% polypeptone, 1% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O). The culture supernatant was recovered, mixed with an equal amount of 2× sample buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 0.01% bromophenol blue, 4% SDS, 200 mM DTT), treated at 100° C. for 10 minutes, and subjected to SDS-PAGE (CBB staining).

[0113] In addition, the activity assay using ONPG as a substrate was carried out as follows. A solution was prepared by adding 2-nitrophenyl-β-galactoside (ONPG) to 50 mM citrate phosphate buffer (pH 4.0) to 12.5 mM. 0.2 mL of culture supernatant was added to 0.8 mL of the solution, and the supernatant was reacted at 30° C. for 10 minutes (test solution), wherein the culture supernatant was obtained by containing the above-mentioned β-galactosidase diluted in 50 mM citrate phosphate buffer (pH 4.0) with an absorbance of 0.2 to 0.8 at 420 nm. 4 mL of 0.25 M sodium carbonate solution was added to terminate the reaction, and then centrifuged (3000 g, 10 minutes), and the absorbance at 420 nm was measured by a spectrophotometer to quantify the amount of free 2-nitrophenol contained in the supernatant. On the other hand, 50mM citric acid phosphate buffer (pH 4.0) was added to the 2-nitrophenyl-β-galactoside solution, which was used as a blank reagent, and a sodium carbonate solution was added in advance, and the reaction was stopped and color was developed at the same time as the addition and mixing of the culture supernatant containing the above-mentioned β-galactosidase, and this was used as the reaction initial solution (blind test). 1 unit (U) of enzyme activity was set as the amount of enzyme that released 1 micromole of 2-nitrophenol in 1 minute under this condition, and was calculated according to the following formula.

[0114]

[0115] The results of SDS-PAGE and activity assay are shown in Figures 1 to 4 . By CBB staining, special bands not found in the parent strains were detected in the RmGal strain, SmGal strain, and SeGal strain, and they were presumed to be the respective β-galactosidase. Although no special bands were found in the SsGal strain in DPY medium, special bands not found in the parent strain were detected when cultured in CDD (pH 7.0) medium, and they were presumed to be β-galactosidase. The culture conditions that can improve the secretion productivity of each β-galactosidase were studied. As a result, the activity of the SsGal strain reached the maximum under the conditions of CDD (pH 7.0) medium, 30°C, and 144 hours, the RmGal strain under the conditions of 2×DPY medium, 30°C, and 144 hours, the SmGal strain under the conditions of 2×DPY medium, 30°C, and 168 hours, and the SeGal strain under the conditions of DPY medium, 30°C, and 168 hours. Furthermore, based on the concentrations of the SDS-PAGE bands, it was estimated that the productivity of the SsGal strain, the RmGal strain, the SmGal strain, and the SeGal strain were approximately 200 mg / L, approximately 200 mg / L, approximately 200 mg / L, and approximately 1 g / L, respectively.

[0116] (2) Estimation of the number of copies

[0117] Furthermore, the number of expression cassettes introduced into the transformants was estimated by real-time PCR.

[0118] From the PCR results, it can be inferred that the SsGal strain, the RmGal strain, and the SmGal strain are strains into which one replication expression cassette is inserted, and the SeGal strain is a strain into which two replication expression cassettes are inserted.

[0119] (3) Heat inactivation test

[0120] 1 mL of the culture solution of each transformant and the mother strain (NS4 strain) cultured under the culture conditions described in (1) was cultured at 40°C, 50°C, 60°C, 70°C, and 80°C for 1 hour each, and then enzyme activity was measured and SDS-PAGE was performed.

[0121] The results of SDS-PAGE and activity assay are shown in Figures 5 to 8. The SsGal strain can maintain activity until 40°C, but the activity decreases by about 70% after culturing at 50°C for 1 hour, and disappears at 70°C. The activity of the parent strain cultured under the same conditions can be detected in trace amounts until 60°C, but disappears at 70°C. The RmGal strain can maintain activity until 50°C, but the activity disappears after culturing at 60°C for 1 hour. The activity of the parent strain cultured under the same conditions can be detected until 70°C, but disappears at 80°C. The SmGal strain can maintain activity until 50°C, but the activity decreases by about 20% after culturing at 60°C for 1 hour, and disappears at 80°C. The activity of the parent strain cultured under the same conditions can be detected until 70°C, but disappears at 80°C. The SeGal strain can maintain activity until 70°C. The activity decreases by about 97% after culturing at 80°C for 1 hour. The activity of the parent strain cultured under the same conditions can be detected until 40°C, but disappears at 50°C. SeGal was treated at 80°C for less than 1 hour (5 minutes, 10 minutes, and 20 minutes). The results showed that the activity decreased by about 37% after treatment at 80°C for 5 minutes, and decreased by about 98% after treatment for 20 minutes. Furthermore, the results of measuring the activity showed that the activity of the 40°C treatment and the 50°C treatment was higher than that of the mother plant.

[0122] From the above, it can be seen that the SeGal strain can maintain its activity even at high temperatures.

[0123] Example 10

[0124] Removal of impurity enzymes:

[0125] As shown in (3) of Example 9, it was found that the SeGal strain can maintain activity even at high temperatures. On the other hand, the heat inactivation test was carried out in the same manner as in (3) of Example 9 for the hybrid enzyme derived from Aspergillus oryzae, which is the mother strain of the SeGal strain, and 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 ( Fig. 9 ).

[0126] Embodiment 11

[0127] Production of galacto-oligosaccharides (1):

[0128] To 150 mL of a solution containing 66% (w / v) lactose, the culture supernatant of the SsGal strain, SmGal strain, and SeGal strain obtained in Example 9 was added in an amount equivalent to 10 U, respectively, and the mixture was reacted at a specified temperature for a specified time to produce oligogalactose. The sugar composition and amount were determined by high performance liquid chromatography. The reaction time and the sugar composition in the solution are shown in Figures 10-12 ( Fig.10 : SsGal strain, Fig.11 : SmGal strain, Fig.12 : SeGal strain).

[0129] As can be seen from the figure, β-galactosidase produced by the SsGal strain, the SmGal strain, and the SeGal strain can produce galacto-oligosaccharides mainly consisting of trisaccharides from lactose.

[0130] In addition, when β-galactosidase produced by the SsGal strain was used to produce galactoligosaccharides, the galactoligosaccharide content was 56.0%; when β-galactosidase produced by the SmGal strain was used to produce galactoligosaccharides, the galactoligosaccharide content was 66.7%; when β-galactosidase produced by the SeGal strain was used to produce galactoligosaccharides, the galactoligosaccharide content was 68.5%.

[0131] In addition, since the above-mentioned β-galactosidase is of the secretory type, it is not necessary to perform treatment of the bacterial cells after the production of galactoligosaccharides, and galactoligosaccharides can be produced efficiently.

[0132] Example 12

[0133] Production of galacto-oligosaccharides (2):

[0134] To 150 mL of a solution containing 66% (w / v) lactose, the culture supernatant of the SeGal strain obtained in Example 9 was added in an amount equivalent to 1.0 U, and the mixture was reacted at 70°C, 80°C, and 90°C for a specified time to produce galacto-oligosaccharides. The sugar composition and amount were determined by high performance liquid chromatography. The reaction time and the sugar composition in the solution are shown in Fig.13 ((a): 70℃, (b): 80℃), Fig.14 ((c): 90℃).

[0135] β-galactosidase derived from the SeGal strain has high heat resistance and can produce GOS at 70°C to 90°C.

[0136] Industrial Applicability

[0137] The β-galactosidase obtained by the method for producing secretory β-galactosidase can be easily isolated and purified, and can be used for the production of galacto-oligosaccharides. Sequence Listing <110> Yakult Co., Ltd. Head Office <120> Method for producing secretory β-galactosidase <130> PF-190014-WO <150> JP2018-212757 <151> 2018-11-13 <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 1785 <212> DNA <213> Sporobolomyces singularis <220> <221> CDS <222> (1)..(1785) <223> Inventors: Ishikawa, Eiji; Ikeda, Masakazu; Anbe, Minako; Hatano, Hiroshi <220> <221> Signal peptide <222> (1)..(57) <220> <221> Mature peptide <222> (58)..(1782) <400> 1 atg atg ctg cat gcg gca ctg ctc gtt gcg ctc ccc tgc gtg gtt ctt 48 Met Met Leu His Ala Ala Leu Leu Val Ala Leu Pro Cys Val Val Leu -15 -10 -5 gct cgt ccc gcc ggt gca gtt acc tac ccc ggt gcg att cca ctt agc 96 Ala Arg Pro Ala Gly Ala Val Thr Tyr Pro Gly Ala Ile Pro Leu Ser -1 1 5 10 ttg acc agc aat tac gag acg ccg agt ccg acc gcc atc ccc ctg gag 144 Leu Thr Ser Asn Tyr Glu Thr Pro Ser Pro Thr Ala Ile Pro Leu Glu 15 20 25 ccg acc cca acg gcg acc gga acc gcc gaa ctt gat gcg ctc tgg aat 192 Pro Thr Pro Thr Ala Thr Gly Thr Ala Glu Leu Asp Ala Leu Trp Asn 30 35 40 45 ttg gtg gaa gca cag tac cct gtt cag acg gcg gct gtc acc acc ctg 240 Leu Val Glu Ala Gln Tyr Pro Val Gln Thr Ala Ala Val Thr Thr Leu 50 55 60 gtg acg gtg ccc gac gac tac aag ttt gaa gca gac cct cct tcc tat 288 Val Thr Val Pro Asp Asp Tyr Lys Phe Glu Ala Asp Pro Pro Ser Tyr 65 70 75 gct ctt gct ggc tac gag aca tca gaa att gcc ggc ttg aag ttc ccg 336 Ala Leu Ala Gly Tyr Glu Thr Ser Glu Ile Ala Gly Leu Lys Phe Pro 80 85 90 aag ggg ttc aag ttt ggc gtg gcc ggc gcg gct att caa gtg gaa ggc 384 Lys Gly Phe Lys Phe Gly Val Ala Gly Ala Ala Ile Gln Val Glu Gly 95 100 105 gca gcg aaa gca gag gga cga ggc cca tcc act tgg gat tac ttg tgc 432 Ala Ala Lys Ala Glu Gly Arg Gly Pro Ser Thr Trp Asp Tyr Leu Cys 110 115 120 125 cac cat tac gcg tcc aca cag tgc aac aac tat gat cct gac att acg 480 His His Tyr Ala Ser Thr Gln Cys Asn Asn Tyr Asp Pro Asp Ile Thr 130 135 140 acg aac cat tac tac ctt tac cct ctt gat ttc gcc cgg ctc cag cat 528 Thr Asn His Tyr Tyr Leu Tyr Pro Leu Asp Phe Ala Arg Leu Gln His 145 150 155 cta ggc atc aac acg tat tcg ttt tca atc tcc tgg act cgt ata tac 576 Leu Gly Ile Asn Thr Tyr Ser Phe Ser Ile Ser Trp Thr Arg Ile Tyr 160 165 170 cct ctg ggt gct ggc tac gtt aac gaa gcc ggt ttg gcg cat tac gac 624 Pro Leu Gly Ala Gly Tyr Val Asn Glu Ala Gly Leu Ala His Tyr Asp 175 180 185 gcg gta atc cac tcg gcc aag aag tac ggg ctg gag cct gtc gga aca 672 Ala Val Ile His Ser Ala Lys Lys Tyr Gly Leu Glu Pro Val Gly Thr 190 195 200 205 gta ttt cac tgg gac acc cct ctc agc ctc atg ctc aaa tat ggc gcg 720 Val Phe His Trp Asp Thr Pro Leu Ser Leu Met Leu Lys Tyr Gly Ala 210 215 220 tgg caa gat acc ggc gac cag atc gtt aaa gat ttc gtc aca tac gcc 768 Trp Gln Asp Thr Gly Asp Gln Ile Val Lys Asp Phe Val Thr Tyr Ala 225 230 235 acc acc gtc ttc aaa cga tac ggt aat gaa gtc aag acc tgg ttc acg 816 Thr Thr Val Phe Lys Arg Tyr Gly Asn Glu Val Lys Thr Trp Phe Thr 240 245 250 ttc aat gag cct cgc gtg ttc tgt tct caa aac agt ggc ctt ccc tat 864 Phe Asn Glu Pro Arg Val Phe Cys Ser Gln Asn Ser Gly Leu Pro Tyr 255 260 265 aac ctc acg tat cct gag gga atc aac tca act tca gcc gtc ttc cgg 912 Asn Leu Thr Tyr Pro Glu Gly Ile Asn Ser Thr Ser Ala Val Phe Arg 270 275 280 285 tgt act tat aac gtc ctg aaa gcc cat ggc cac gcg gtt aag gtt tac 960 Cys Thr Tyr Asn Val Leu Lys Ala His Gly His Ala Val Lys Val Tyr 290 295 300 cgg gat ctc gtt gcc agc gga acc att gct gct gga gag atc ggc ttc 1008 Arg Asp Leu Val Ala Ser Gly Thr Ile Ala Ala Gly Glu Ile Gly Phe 305 310 315 aag tcg gac gac aac tac cca atc cca gcg cgg ccc gga aac gcg gac 1056 Lys Ser Asp Asp Asn Tyr Pro Ile Pro Ala Arg Pro Gly Asn Ala Asp 320 325 330 gac gag gaa tcc gcc aaa cgt cac gaa gcg ttc cga atc gga atc ttt 1104 Asp Glu Glu Ser Ala Lys Arg His Glu Ala Phe Arg Ile Gly Ile Phe 335 340 345 gcc cag cca gtt tac gga aac ggc gac tat cct gat gta gta aaa gag 1152 Ala Gln Pro Val Tyr Gly Asn Gly Asp Tyr Pro Asp Val Val Lys Glu 350 355 360 365 acc gtt ggc gac atg ctg ccc gcc ctg acg gat gag gac aag ggc tac 1200 Thr Val Gly Asp Met Leu Pro Ala Leu Thr Asp Glu Asp Lys Gly Tyr 370 375 380 atc aag ggc agc ggc gac atc ttc gcc att gac ggt tac cgg acc gat 1248 Ile Lys Gly Ser Gly Asp Ile Phe Ala Ile Asp Gly Tyr Arg Thr Asp 385 390 395 atc tcg cat gcc gca ctg aat gga atc gcg aat tgc atc aga aac cag 1296 Ile Ser His Ala Ala Leu Asn Gly Ile Ala Asn Cys Ile Arg Asn Gln 400 405 410 tcg gac cct aac tgg cct gtt tgc gag gaa ggg tct gac ccg ttc gcc 1344 Ser Asp Pro Asn Trp Pro Val Cys Glu Glu Gly Ser Asp Pro Phe Ala 415 420 425 cac gta tac ccg tct ggt ttc gcc atc ggc cag tcc gcc gat ccg ctg 1392 His Val Tyr Pro Ser Gly Phe Ala Ile Gly Gln Ser Ala Asp Pro Leu 430 435 440 445 tcg tca tgg ctc gtc aac tcc gcc cca ttt att cgc gac cag ctg aag 1440 Ser Ser Trp Leu Val Asn Ser Ala Pro Phe Ile Arg Asp Gln Leu Lys 450 455 460 ttc ctc act caa acg tac ccg gca aag gga ggt att tac ttc agc gag 1488 Phe Leu Thr Gln Thr Tyr Pro Ala Lys Gly Gly Ile Tyr Phe Ser Glu 465 470 475 ttt ggg tgg gcc gag gat gcg gag tac gac cgc cag ctg ttg tac caa 1536 Phe Gly Trp Ala Glu Asp Ala Glu Tyr Asp Arg Gln Leu Leu Tyr Gln 480 485 490 atc acc tgg gac ggt ctt agg acc cag tat ctc act gac tac ctg tcc 1584 Ile Thr Trp Asp Gly Leu Arg Thr Gln Tyr Leu Thr Asp Tyr Leu Ser 495 500 505 caa ctc ctg ctc gcc gtc cat aag gat ggg att aat ctt cgc ggc gcg 1632 Gln Leu Leu Leu Ala Val His Lys Asp Gly Ile Asn Leu Arg Gly Ala 510 515 520 525 tta acc tgg agt ttc gtc gac aac tgg gaa tgg gga ctg ggg atg caa 1680 Leu Thr Trp Ser Phe Val Asp Asn Trp Glu Trp Gly Leu Gly Met Gln 530 535 540 cag aaa ttc gga ttc cag ttt gtc aat cag tcg gac cca gat ctc acc 1728 Gln Lys Phe Gly Phe Gln Phe Val Asn Gln Ser Asp Pro Asp Leu Thr 545 550 555 agg acc ttc aaa ctc tct gcg cac gct tac gct caa ttt ggt cgc aac 1776 Arg Thr Phe Lys Leu Ser Ala His Ala Tyr Ala Gln Phe Gly Arg Asn 560 565 570 cac ctc tga 1785 His Leu 575 <210> 2 <211> 594 <212> PRT <213> Sporobolomyces singularis <400> 2 Met Met Leu His Ala Ala Leu Leu Val Ala Leu Pro Cys Val Val Leu -15 -10 -5 Ala Arg Pro Ala Gly Ala Val Thr Tyr Pro Gly Ala Ile Pro Leu Ser -1 1 5 10 Leu Thr Ser Asn Tyr Glu Thr Pro Ser Pro Thr Ala Ile Pro Leu Glu 15 20 25 Pro Thr Pro Thr Ala Thr Gly Thr Ala Glu Leu Asp Ala Leu Trp Asn 30 35 40 45 Leu Val Glu Ala Gln Tyr Pro Val Gln Thr Ala Ala Val Thr Thr Leu 50 55 60 Val Thr Val Pro Asp Asp Tyr Lys Phe Glu Ala Asp Pro Pro Ser Tyr 65 70 75 Ala Leu Ala Gly Tyr Glu Thr Ser Glu Ile Ala Gly Leu Lys Phe Pro 80 85 90 Lys Gly Phe Lys Phe Gly Val Ala Gly Ala Ala Ile Gln Val Glu Gly 95 100 105 Ala Ala Lys Ala Glu Gly Arg Gly Pro Ser Thr Trp Asp Tyr Leu Cys 110 115 120 125 His His Tyr Ala Ser Thr Gln Cys Asn Asn Tyr Asp Pro Asp Ile Thr 130 135 140 Thr Asn His Tyr Tyr Leu Tyr Pro Leu Asp Phe Ala Arg Leu Gln His 145 150 155 Leu Gly Ile Asn Thr Tyr Ser Phe Ser Ile Ser Trp Thr Arg Ile Tyr 160 165 170 Pro Leu Gly Ala Gly Tyr Val Asn Glu Ala Gly Leu Ala His Tyr Asp 175 180 185 Ala Val Ile His Ser Ala Lys Lys Tyr Gly Leu Glu Pro Val Gly Thr 190 195 200 205 Val Phe His Trp Asp Thr Pro Leu Ser Leu Met Leu Lys Tyr Gly Ala 210 215 220 Trp Gln Asp Thr Gly Asp Gln Ile Val Lys Asp Phe Val Thr Tyr Ala 225 230 235 Thr Thr Val Phe Lys Arg Tyr Gly Asn Glu Val Lys Thr Trp Phe Thr 240 245 250 Phe Asn Glu Pro Arg Val Phe Cys Ser Gln Asn Ser Gly Leu Pro Tyr 255 260 265 Asn Leu Thr Tyr Pro Glu Gly Ile Asn Ser Thr Ser Ala Val Phe Arg 270 275 280 285 Cys Thr Tyr Asn Val Leu Lys Ala His Gly His Ala Val Lys Val Tyr 290 295 300 Arg Asp Leu Val Ala Ser Gly Thr Ile Ala Ala Gly Glu Ile Gly Phe 305 310 315 Lys Ser Asp Asp Asn Tyr Pro Ile Pro Ala Arg Pro Gly Asn Ala Asp 320 325 330 Asp Glu Glu Ser Ala Lys Arg His Glu Ala Phe Arg Ile Gly Ile Phe 335 340 345 Ala Gln Pro Val Tyr Gly Asn Gly Asp Tyr Pro Asp Val Val Lys Glu 350 355 360 365 Thr Val Gly Asp Met Leu Pro Ala Leu Thr Asp Glu Asp Lys Gly Tyr 370 375 380 Ile Lys Gly Ser Gly Asp Ile Phe Ala Ile Asp Gly Tyr Arg Thr Asp 385 390 395 Ile Ser His Ala Ala Leu Asn Gly Ile Ala Asn Cys Ile Arg Asn Gln 400 405 410 Ser Asp Pro Asn Trp Pro Val Cys Glu Glu Gly Ser Asp Pro Phe Ala 415 420 425 His Val Tyr Pro Ser Gly Phe Ala Ile Gly Gln Ser Ala Asp Pro Leu 430 435 440 445 Ser Ser Trp Leu Val Asn Ser Ala Pro Phe Ile Arg Asp Gln Leu Lys 450 455 460 Phe Leu Thr Gln Thr Tyr Pro Ala Lys Gly Gly Ile Tyr Phe Ser Glu 465 470 475 Phe Gly Trp Ala Glu Asp Ala Glu Tyr Asp Arg Gln Leu Leu Tyr Gln 480 485 490 Ile Thr Trp Asp Gly Leu Arg Thr Gln Tyr Leu Thr Asp Tyr Leu Ser 495 500 505 Gln Leu Leu Leu Ala Val His Lys Asp Gly Ile Asn Leu Arg Gly Ala 510 515 520 525 Leu Thr Trp Ser Phe Val Asp Asn Trp Glu Trp Gly Leu Gly Met Gln 530 535 540 Gln Lys Phe Gly Phe Gln Phe Val Asn Gln Ser Asp Pro Asp Leu Thr 545 550 555 Arg Thr Phe Lys Leu Ser Ala His Ala Tyr Ala Gln Phe Gly Arg Asn 560 565 570 His Leu 575 <210> 3 <211> 1791 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(1791) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(1788) <400> 3 atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct gcc ggt gca gtt acc tac ccc ggt gcg att cca 96 Ala Pro Ala Leu Ala Ala Gly Ala Val Thr Tyr Pro Gly Ala Ile Pro -5 -1 1 5 10 ctt agc ttg acc agc aat tac gag acg ccg agt ccg acc gcc atc ccc 144 Leu Ser Leu Thr Ser Asn Tyr Glu Thr Pro Ser Pro Thr Ala Ile Pro 15 20 25 ctg gag ccg acc cca acg gcg acc gga acc gcc gaa ctt gat gcg ctc 192 Leu Glu Pro Thr Pro Thr Ala Thr Gly Thr Ala Glu Leu Asp Ala Leu 30 35 40 tgg aat ttg gtg gaa gca cag tac cct gtt cag acg gcg gct gtc acc 240 Trp Asn Leu Val Glu Ala Gln Tyr Pro Val Gln Thr Ala Ala Val Thr 45 50 55 acc ctg gtg acg gtg ccc gac gac tac aag ttt gaa gca gac cct cct 288 Thr Leu Val Thr Val Pro Asp Asp Tyr Lys Phe Glu Ala Asp Pro Pro 60 65 70 75 tcc tat gct ctt gct ggc tac gag aca tca gaa att gcc ggc ttg aag 336 Ser Tyr Ala Leu Ala Gly Tyr Glu Thr Ser Glu Ile Ala Gly Leu Lys 80 85 90 ttc ccg aag ggg ttc aag ttt ggc gtg gcc ggc gcg gct att caa gtg 384 Phe Pro Lys Gly Phe Lys Phe Gly Val Ala Gly Ala Ala Ile Gln Val 95 100 105 gaa ggc gca gcg aaa gca gag gga cga ggc cca tcc act tgg gat tac 432 Glu Gly Ala Ala Lys Ala Glu Gly Arg Gly Pro Ser Thr Trp Asp Tyr 110 115 120 ttg tgc cac cat tac gcg tcc aca cag tgc aac aac tat gat cct gac 480 Leu Cys His His Tyr Ala Ser Thr Gln Cys Asn Asn Tyr Asp Pro Asp 125 130 135 att acg acg aac cat tac tac ctt tac cct ctt gat ttc gcc cgg ctc 528 Ile Thr Thr Asn His Tyr Tyr Leu Tyr Pro Leu Asp Phe Ala Arg Leu 140 145 150 155 cag cat cta ggc atc aac acg tat tcg ttt tca atc tcc tgg act cgt 576 Gln His Leu Gly Ile Asn Thr Tyr Ser Phe Ser Ile Ser Trp Thr Arg 160 165 170 ata tac cct ctg ggt gct ggc tac gtt aac gaa gcc ggt ttg gcg cat 624 Ile Tyr Pro Leu Gly Ala Gly Tyr Val Asn Glu Ala Gly Leu Ala His 175 180 185 tac gac gcg gta atc cac tcg gcc aag aag tac ggg ctg gag cct gtc 672 Tyr Asp Ala Val Ile His Ser Ala Lys Lys Tyr Gly Leu Glu Pro Val 190 195 200 gga aca gta ttt cac tgg gac acc cct ctc agc ctc atg ctc aaa tat 720 Gly Thr Val Phe His Trp Asp Thr Pro Leu Ser Leu Met Leu Lys Tyr 205 210 215 ggc gcg tgg caa gat acc ggc gac cag atc gtt aaa gat ttc gtc aca 768 Gly Ala Trp Gln Asp Thr Gly Asp Gln Ile Val Lys Asp Phe Val Thr 220 225 230 235 tac gcc acc acc gtc ttc aaa cga tac ggt aat gaa gtc aag acc tgg 816 Tyr Ala Thr Thr Val Phe Lys Arg Tyr Gly Asn Glu Val Lys Thr Trp 240 245 250 ttc acg ttc aat gag cct cgc gtg ttc tgt tct caa aac agt ggc ctt 864 Phe Thr Phe Asn Glu Pro Arg Val Phe Cys Ser Gln Asn Ser Gly Leu 255 260 265 ccc tat aac ctc acg tat cct gag gga atc aac tca act tca gcc gtc 912 Pro Tyr Asn Leu Thr Tyr Pro Glu Gly Ile Asn Ser Thr Ser Ala Val 270 275 280 ttc cgg tgt act tat aac gtc ctg aaa gcc cat ggc cac gcg gtt aag 960 Phe Arg Cys Thr Tyr Asn Val Leu Lys Ala His Gly His Ala Val Lys 285 290 295 gtt tac cgg gat ctc gtt gcc agc gga acc att gct gct gga gag atc 1008 Val Tyr Arg Asp Leu Val Ala Ser Gly Thr Ile Ala Ala Gly Glu Ile 300 305 310 315 ggc ttc aag tcg gac gac aac tac cca atc cca gcg cgg ccc gga aac 1056 Gly Phe Lys Ser Asp Asp Asn Tyr Pro Ile Pro Ala Arg Pro Gly Asn 320 325 330 gcg gac gac gag gaa tcc gcc aaa cgt cac gaa gcg ttc cga atc gga 1104 Ala Asp Asp Glu Glu Ser Ala Lys Arg His Glu Ala Phe Arg Ile Gly 335 340 345 atc ttt gcc cag cca gtt tac gga aac ggc gac tat cct gat gta gta 1152 Ile Phe Ala Gln Pro Val Tyr Gly Asn Gly Asp Tyr Pro Asp Val Val 350 355 360 aaa gag acc gtt ggc gac atg ctg ccc gcc ctg acg gat gag gac aag 1200 Lys Glu Thr Val Gly Asp Met Leu Pro Ala Leu Thr Asp Glu Asp Lys 365 370 375 ggc tac atc aag ggc agc ggc gac atc ttc gcc att gac ggt tac cgg 1248 Gly Tyr Ile Lys Gly Ser Gly Asp Ile Phe Ala Ile Asp Gly Tyr Arg 380 385 390 395 acc gat atc tcg cat gcc gca ctg aat gga atc gcg aat tgc atc aga 1296 Thr Asp Ile Ser His Ala Ala Leu Asn Gly Ile Ala Asn Cys Ile Arg 400 405 410 aac cag tcg gac cct aac tgg cct gtt tgc gag gaa ggg tct gac ccg 1344 Asn Gln Ser Asp Pro Asn Trp Pro Val Cys Glu Glu Gly Ser Asp Pro 415 420 425 ttc gcc cac gta tac ccg tct ggt ttc gcc atc ggc cag tcc gcc gat 1392 Phe Ala His Val Tyr Pro Ser Gly Phe Ala Ile Gly Gln Ser Ala Asp 430 435 440 ccg ctg tcg tca tgg ctc gtc aac tcc gcc cca ttt att cgc gac cag 1440 Pro Leu Ser Ser Trp Leu Val Asn Ser Ala Pro Phe Ile Arg Asp Gln 445 450 455 ctg aag ttc ctc act caa acg tac ccg gca aag gga ggt att tac ttc 1488 Leu Lys Phe Leu Thr Gln Thr Tyr Pro Ala Lys Gly Gly Ile Tyr Phe 460 465 470 475 agc gag ttt ggg tgg gcc gag gat gcg gag tac gac cgc cag ctg ttg 1536 Ser Glu Phe Gly Trp Ala Glu Asp Ala Glu Tyr Asp Arg Gln Leu Leu 480 485 490 tac caa atc acc tgg gac ggt ctt agg acc cag tat ctc act gac tac 1584 Tyr Gln Ile Thr Trp Asp Gly Leu Arg Thr Gln Tyr Leu Thr Asp Tyr 495 500 505 ctg tcc caa ctc ctg ctc gcc gtc cat aag gat ggg att aat ctt cgc 1632 Leu Ser Gln Leu Leu Leu Ala Val His Lys Asp Gly Ile Asn Leu Arg 510 515 520 ggc gcg tta acc tgg agt ttc gtc gac aac tgg gaa tgg gga ctg ggg 1680 Gly Ala Leu Thr Trp Ser Phe Val Asp Asn Trp Glu Trp Gly Leu Gly 525 530 535 atg caa cag aaa ttc gga ttc cag ttt gtc aat cag tcg gac cca gat 1728 Met Gln Gln Lys Phe Gly Phe Gln Phe Val Asn Gln Ser Asp Pro Asp 540 545 550 555 ctc acc agg acc ttc aaa ctc tct gcg cac gct tac gct caa ttt ggt 1776 Leu Thr Arg Thr Phe Lys Leu Ser Ala His Ala Tyr Ala Gln Phe Gly 560 565 570 cgc aac cac ctc tga 1791 Arg Asn His Leu 575 <210> 4 <211> 596 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 4 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ala Gly Ala Val Thr Tyr Pro Gly Ala Ile Pro -5 -1 1 5 10 Leu Ser Leu Thr Ser Asn Tyr Glu Thr Pro Ser Pro Thr Ala Ile Pro 15 20 25 Leu Glu Pro Thr Pro Thr Ala Thr Gly Thr Ala Glu Leu Asp Ala Leu 30 35 40 Trp Asn Leu Val Glu Ala Gln Tyr Pro Val Gln Thr Ala Ala Val Thr 45 50 55 Thr Leu Val Thr Val Pro Asp Asp Tyr Lys Phe Glu Ala Asp Pro Pro 60 65 70 75 Ser Tyr Ala Leu Ala Gly Tyr Glu Thr Ser Glu Ile Ala Gly Leu Lys 80 85 90 Phe Pro Lys Gly Phe Lys Phe Gly Val Ala Gly Ala Ala Ile Gln Val 95 100 105 Glu Gly Ala Ala Lys Ala Glu Gly Arg Gly Pro Ser Thr Trp Asp Tyr 110 115 120 Leu Cys His His Tyr Ala Ser Thr Gln Cys Asn Asn Tyr Asp Pro Asp 125 130 135 Ile Thr Thr Asn His Tyr Tyr Leu Tyr Pro Leu Asp Phe Ala Arg Leu 140 145 150 155 Gln His Leu Gly Ile Asn Thr Tyr Ser Phe Ser Ile Ser Trp Thr Arg 160 165 170 Ile Tyr Pro Leu Gly Ala Gly Tyr Val Asn Glu Ala Gly Leu Ala His 175 180 185 Tyr Asp Ala Val Ile His Ser Ala Lys Lys Tyr Gly Leu Glu Pro Val 190 195 200 Gly Thr Val Phe His Trp Asp Thr Pro Leu Ser Leu Met Leu Lys Tyr 205 210 215 Gly Ala Trp Gln Asp Thr Gly Asp Gln Ile Val Lys Asp Phe Val Thr 220 225 230 235 Tyr Ala Thr Thr Val Phe Lys Arg Tyr Gly Asn Glu Val Lys Thr Trp 240 245 250 Phe Thr Phe Asn Glu Pro Arg Val Phe Cys Ser Gln Asn Ser Gly Leu 255 260 265 Pro Tyr Asn Leu Thr Tyr Pro Glu Gly Ile Asn Ser Thr Ser Ala Val 270 275 280 Phe Arg Cys Thr Tyr Asn Val Leu Lys Ala His Gly His Ala Val Lys 285 290 295 Val Tyr Arg Asp Leu Val Ala Ser Gly Thr Ile Ala Ala Gly Glu Ile 300 305 310 315 Gly Phe Lys Ser Asp Asp Asn Tyr Pro Ile Pro Ala Arg Pro Gly Asn 320 325 330 Ala Asp Asp Glu Glu Ser Ala Lys Arg His Glu Ala Phe Arg Ile Gly 335 340 345 Ile Phe Ala Gln Pro Val Tyr Gly Asn Gly Asp Tyr Pro Asp Val Val 350 355 360 Lys Glu Thr Val Gly Asp Met Leu Pro Ala Leu Thr Asp Glu Asp Lys 365 370 375 Gly Tyr Ile Lys Gly Ser Gly Asp Ile Phe Ala Ile Asp Gly Tyr Arg 380 385 390 395 Thr Asp Ile Ser His Ala Ala Leu Asn Gly Ile Ala Asn Cys Ile Arg 400 405 410 Asn Gln Ser Asp Pro Asn Trp Pro Val Cys Glu Glu Gly Ser Asp Pro 415 420 425 Phe Ala His Val Tyr Pro Ser Gly Phe Ala Ile Gly Gln Ser Ala Asp 430 435 440 Pro Leu Ser Ser Trp Leu Val Asn Ser Ala Pro Phe Ile Arg Asp Gln 445 450 455 Leu Lys Phe Leu Thr Gln Thr Tyr Pro Ala Lys Gly Gly Ile Tyr Phe 460 465 470 475 Ser Glu Phe Gly Trp Ala Glu Asp Ala Glu Tyr Asp Arg Gln Leu Leu 480 485 490 Tyr Gln Ile Thr Trp Asp Gly Leu Arg Thr Gln Tyr Leu Thr Asp Tyr 495 500 505 Leu Ser Gln Leu Leu Leu Ala Val His Lys Asp Gly Ile Asn Leu Arg 510 515 520 Gly Ala Leu Thr Trp Ser Phe Val Asp Asn Trp Glu Trp Gly Leu Gly 525 530 535 Met Gln Gln Lys Phe Gly Phe Gln Phe Val Asn Gln Ser Asp Pro Asp 540 545 550 555 Leu Thr Arg Thr Phe Lys Leu Ser Ala His Ala Tyr Ala Gln Phe Gly 560 565 570 Arg Asn His Leu 575 <210> 5 <211> 1791 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(1791) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(1788) <400> 5 atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct gct ggc gcc gtc aca tac ccc gga gct att cca 96 Ala Pro Ala Leu Ala Ala Gly Ala Val Thr Tyr Pro Gly Ala Ile Pro -5 -1 1 5 10 ctg tcc ctc aca agc aac tac gag aca cct tct cct acc gcc att cct 144 Leu Ser Leu Thr Ser Asn Tyr Glu Thr Pro Ser Pro Thr Ala Ile Pro 15 20 25 ttg gag cca act ccg act gca act gga acc gca gaa ctg gat gcg ctc 192 Leu Glu Pro Thr Pro Thr Ala Thr Gly Thr Ala Glu Leu Asp Ala Leu 30 35 40 tgg aac ctt gtc gaa gct cag tat ccc gtc caa acg gcc gca gtc acc 240 Trp Asn Leu Val Glu Ala Gln Tyr Pro Val Gln Thr Ala Ala Val Thr 45 50 55 acc ctc gtc acc gtc cct gac gac tac aag ttc gag gcc gat cct cct 288 Thr Leu Val Thr Val Pro Asp Asp Tyr Lys Phe Glu Ala Asp Pro Pro 60 65 70 75 agc tat gca ctc gca ggt tac gag act tcc gag att gcg gga ctg aag 336 Ser Tyr Ala Leu Ala Gly Tyr Glu Thr Ser Glu Ile Ala Gly Leu Lys 80 85 90 ttc ccc aaa ggt ttc aag ttc ggc gtt gct ggg gcc gcg att cag gtc 384 Phe Pro Lys Gly Phe Lys Phe Gly Val Ala Gly Ala Ala Ile Gln Val 95 100 105 gaa ggc gct gct aaa gcg gaa gga cgt ggg ccc tct aca tgg gat tac 432 Glu Gly Ala Ala Lys Ala Glu Gly Arg Gly Pro Ser Thr Trp Asp Tyr 110 115 120 ctc tgt cat cac tat gcc agc act cag tgc aac aac tac gat ccc gat 480 Leu Cys His His Tyr Ala Ser Thr Gln Cys Asn Asn Tyr Asp Pro Asp 125 130 135 atc acc acc aac cat tac tac ctc tac ccc ctc gat ttc gcg cgt ctt 528 Ile Thr Thr Asn His Tyr Tyr Leu Tyr Pro Leu Asp Phe Ala Arg Leu 140 145 150 155 caa cat ctg ggc atc aac acc tac tcc ttt tcc att tcc tgg acc cga 576 Gln His Leu Gly Ile Asn Thr Tyr Ser Phe Ser Ile Ser Trp Thr Arg 160 165 170 atc tac cct ctc ggc gcc ggt tac gtc aac gag gcc ggc ttg gca cac 624 Ile Tyr Pro Leu Gly Ala Gly Tyr Val Asn Glu Ala Gly Leu Ala His 175 180 185 tac gat gct gtc att cac tcc gcc aag aag tac gga ttg gag cca gtt 672 Tyr Asp Ala Val Ile His Ser Ala Lys Lys Tyr Gly Leu Glu Pro Val 190 195 200 ggc acg gtc ttt cac tgg gac act cct ctg tcg ctc atg ctt aag tac 720 Gly Thr Val Phe His Trp Asp Thr Pro Leu Ser Leu Met Leu Lys Tyr 205 210 215 ggg gcg tgg cag gat act ggt gat cag atc gtc aag gac ttt gtg acg 768 Gly Ala Trp Gln Asp Thr Gly Asp Gln Ile Val Lys Asp Phe Val Thr 220 225 230 235 tat gcc acg acc gtt ttc aag cgc tat ggt aac gag gtc aag aca tgg 816 Tyr Ala Thr Thr Val Phe Lys Arg Tyr Gly Asn Glu Val Lys Thr Trp 240 245 250 ttc aca ttc aac gag cca cgt gtc ttc tgc tcc cag aat tcc ggg ctt 864 Phe Thr Phe Asn Glu Pro Arg Val Phe Cys Ser Gln Asn Ser Gly Leu 255 260 265 ccg tac aac ctg acc tat cct gaa ggc atc aac tct act tct gcg gtg 912 Pro Tyr Asn Leu Thr Tyr Pro Glu Gly Ile Asn Ser Thr Ser Ala Val 270 275 280 ttc cgt tgc acg tac aac gtg ctt aag gct cat ggt cat gct gtc aaa 960 Phe Arg Cys Thr Tyr Asn Val Leu Lys Ala His Gly His Ala Val Lys 285 290 295 gtg tat cga gat ctt gtg gca tcg ggt aca atc gct gcc ggc gag atc 1008 Val Tyr Arg Asp Leu Val Ala Ser Gly Thr Ile Ala Ala Gly Glu Ile 300 305 310 315 ggc ttc aag agc gac gac aac tac ccg atc ccg gct cgg cct ggt aat 1056 Gly Phe Lys Ser Asp Asp Asn Tyr Pro Ile Pro Ala Arg Pro Gly Asn 320 325 330 gcc gac gac gag gag tcg gcc aag cgc cac gaa gca ttt cga atc ggc 1104 Ala Asp Asp Glu Glu Ser Ala Lys Arg His Glu Ala Phe Arg Ile Gly 335 340 345 atc ttc gcc cag cct gtg tat ggg aat ggt gac tat ccc gat gtg gtg 1152 Ile Phe Ala Gln Pro Val Tyr Gly Asn Gly Asp Tyr Pro Asp Val Val 350 355 360 aag gag acc gtg ggc gac atg ctc ccc gcc ctt acc gat gag gac aaa 1200 Lys Glu Thr Val Gly Asp Met Leu Pro Ala Leu Thr Asp Glu Asp Lys 365 370 375 ggt tac atc aag ggc tcg ggc gac atc ttc gcg att gac ggc tat cgg 1248 Gly Tyr Ile Lys Gly Ser Gly Asp Ile Phe Ala Ile Asp Gly Tyr Arg 380 385 390 395 act gac atc tcg cac gcg gct ctg aat ggc atc gca aac tgc att cgc 1296 Thr Asp Ile Ser His Ala Ala Leu Asn Gly Ile Ala Asn Cys Ile Arg 400 405 410 aat cag agc gac cct aac tgg ccg gtg tgt gaa gaa gga agc gat ccg 1344 Asn Gln Ser Asp Pro Asn Trp Pro Val Cys Glu Glu Gly Ser Asp Pro 415 420 425 ttt gcc cat gtg tat ccc tct ggc ttt gca att gga caa agc gct gat 1392 Phe Ala His Val Tyr Pro Ser Gly Phe Ala Ile Gly Gln Ser Ala Asp 430 435 440 cct ctg tct tct tgg ctc gtt aac tcc gct ccc ttc atc cga gat caa 1440 Pro Leu Ser Ser Trp Leu Val Asn Ser Ala Pro Phe Ile Arg Asp Gln 445 450 455 ctg aag ttc ctg acg caa acc tat cct gca aaa ggc ggc atc tac ttc 1488 Leu Lys Phe Leu Thr Gln Thr Tyr Pro Ala Lys Gly Gly Ile Tyr Phe 460 465 470 475 tcc gag ttt gga tgg gca gag gat gcg gaa tac gat cgg caa ctc ctt 1536 Ser Glu Phe Gly Trp Ala Glu Asp Ala Glu Tyr Asp Arg Gln Leu Leu 480 485 490 tac cag atc acc tgg gac ggg ctt cgc act cag tat ctc acc gat tac 1584 Tyr Gln Ile Thr Trp Asp Gly Leu Arg Thr Gln Tyr Leu Thr Asp Tyr 495 500 505 ctc tcc cag ctg ctg ttg gct gtg cat aag gat ggg atc aac ctt cgg 1632 Leu Ser Gln Leu Leu Leu Ala Val His Lys Asp Gly Ile Asn Leu Arg 510 515 520 ggc gca ttg acc tgg tct ttc gtg gac aac tgg gaa tgg ggt ctg ggc 1680 Gly Ala Leu Thr Trp Ser Phe Val Asp Asn Trp Glu Trp Gly Leu Gly 525 530 535 atg cag cag aag ttc gga ttc caa ttc gtc aat cag agc gat cca gac 1728 Met Gln Gln Lys Phe Gly Phe Gln Phe Val Asn Gln Ser Asp Pro Asp 540 545 550 555 ttg acc cgc aca ttc aag ctc agc gct cat gcg tat gcc cag ttc ggg 1776 Leu Thr Arg Thr Phe Lys Leu Ser Ala His Ala Tyr Ala Gln Phe Gly 560 565 570 cgc aat cac ctg taa 1791 Arg Asn His Leu 575 <210> 6 <211> 596 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <400> 6 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ala Gly Ala Val Thr Tyr Pro Gly Ala Ile Pro -5 -1 1 5 10 Leu Ser Leu Thr Ser Asn Tyr Glu Thr Pro Ser Pro Thr Ala Ile Pro 15 20 25 Leu Glu Pro Thr Pro Thr Ala Thr Gly Thr Ala Glu Leu Asp Ala Leu 30 35 40 Trp Asn Leu Val Glu Ala Gln Tyr Pro Val Gln Thr Ala Ala Val Thr 45 50 55 Thr Leu Val Thr Val Pro Asp Asp Tyr Lys Phe Glu Ala Asp Pro Pro 60 65 70 75 Ser Tyr Ala Leu Ala Gly Tyr Glu Thr Ser Glu Ile Ala Gly Leu Lys 80 85 90 Phe Pro Lys Gly Phe Lys Phe Gly Val Ala Gly Ala Ala Ile Gln Val 95 100 105 Glu Gly Ala Ala Lys Ala Glu Gly Arg Gly Pro Ser Thr Trp Asp Tyr 110 115 120 Leu Cys His His Tyr Ala Ser Thr Gln Cys Asn Asn Tyr Asp Pro Asp 125 130 135 Ile Thr Thr Asn His Tyr Tyr Leu Tyr Pro Leu Asp Phe Ala Arg Leu 140 145 150 155 Gln His Leu Gly Ile Asn Thr Tyr Ser Phe Ser Ile Ser Trp Thr Arg 160 165 170 Ile Tyr Pro Leu Gly Ala Gly Tyr Val Asn Glu Ala Gly Leu Ala His 175 180 185 Tyr Asp Ala Val Ile His Ser Ala Lys Lys Tyr Gly Leu Glu Pro Val 190 195 200 Gly Thr Val Phe His Trp Asp Thr Pro Leu Ser Leu Met Leu Lys Tyr 205 210 215 Gly Ala Trp Gln Asp Thr Gly Asp Gln Ile Val Lys Asp Phe Val Thr 220 225 230 235 Tyr Ala Thr Thr Val Phe Lys Arg Tyr Gly Asn Glu Val Lys Thr Trp 240 245 250 Phe Thr Phe Asn Glu Pro Arg Val Phe Cys Ser Gln Asn Ser Gly Leu 255 260 265 Pro Tyr Asn Leu Thr Tyr Pro Glu Gly Ile Asn Ser Thr Ser Ala Val 270 275 280 Phe Arg Cys Thr Tyr Asn Val Leu Lys Ala His Gly His Ala Val Lys 285 290 295 Val Tyr Arg Asp Leu Val Ala Ser Gly Thr Ile Ala Ala Gly Glu Ile 300 305 310 315 Gly Phe Lys Ser Asp Asp Asn Tyr Pro Ile Pro Ala Arg Pro Gly Asn 320 325 330 Ala Asp Asp Glu Glu Ser Ala Lys Arg His Glu Ala Phe Arg Ile Gly 335 340 345 Ile Phe Ala Gln Pro Val Tyr Gly Asn Gly Asp Tyr Pro Asp Val Val 350 355 360 Lys Glu Thr Val Gly Asp Met Leu Pro Ala Leu Thr Asp Glu Asp Lys 365 370 375 Gly Tyr Ile Lys Gly Ser Gly Asp Ile Phe Ala Ile Asp Gly Tyr Arg 380 385 390 395 Thr Asp Ile Ser His Ala Ala Leu Asn Gly Ile Ala Asn Cys Ile Arg 400 405 410 Asn Gln Ser Asp Pro Asn Trp Pro Val Cys Glu Glu Gly Ser Asp Pro 415 420 425 Phe Ala His Val Tyr Pro Ser Gly Phe Ala Ile Gly Gln Ser Ala Asp 430 435 440 Pro Leu Ser Ser Trp Leu Val Asn Ser Ala Pro Phe Ile Arg Asp Gln 445 450 455 Leu Lys Phe Leu Thr Gln Thr Tyr Pro Ala Lys Gly Gly Ile Tyr Phe 460 465 470 475 Ser Glu Phe Gly Trp Ala Glu Asp Ala Glu Tyr Asp Arg Gln Leu Leu 480 485 490 Tyr Gln Ile Thr Trp Asp Gly Leu Arg Thr Gln Tyr Leu Thr Asp Tyr 495 500 505 Leu Ser Gln Leu Leu Leu Ala Val His Lys Asp Gly Ile Asn Leu Arg 510 515 520 Gly Ala Leu Thr Trp Ser Phe Val Asp Asn Trp Glu Trp Gly Leu Gly 525 530 535 Met Gln Gln Lys Phe Gly Phe Gln Phe Val Asn Gln Ser Asp Pro Asp 540 545 550 555 Leu Thr Arg Thr Phe Lys Leu Ser Ala His Ala Tyr Ala Gln Phe Gly 560 565 570 Arg Asn His Leu 575 <210> 7 <211> 2106 <212> DNA <213> Tremella magnata <220> <221> CDS <222> (1)..(2106) <220> <221> Signal peptide <222> (1)..(48) <220> <221> Mature peptide <222> (49)..(2103) <400> 7 atg ttc aag ctt acc tct gtg ctg ttg ctg ctc ggt gca gct caa gca 48 Met Phe Lys Leu Thr Ser Val Leu Leu Leu Leu Gly Ala Ala Gln Ala -15 -10 -5 -1 gct gtt cta aac cct cgt caa gct ggc agc ggt aat tcc act gcc agc 96 Ala Val Leu Asn Pro Arg Gln Ala Gly Ser Gly Asn Ser Thr Ala Ser 1 5 10 15 ggc tcg ata gcc ggc gat tcc act aga cca gcc acg aca tcc tcg gtc 144 Gly Ser Ile Ala Gly Asp Ser Thr Arg Pro Ala Thr Thr Ser Ser Val 20 25 30 gtc tca ccc tct gca gcc aga aac tcc act gcc gca gct act ggt aat 192 Val Ser Pro Ser Ala Ala Arg Asn Ser Thr Ala Ala Ala Thr Gly Asn 35 40 45 gct tct cgc aat gct act gcg aca ggt act gcc gtc gct aca gcc act 240 Ala Ser Arg Asn Ala Thr Ala Thr Gly Thr Ala Val Ala Thr Ala Thr 50 55 60 ggc ggg gtt aca gca gcc acg tcc act gga atg gcg gtg act tcc cct 288 Gly Gly Val Thr Ala Ala Thr Ser Thr Gly Met Ala Val Thr Ser Pro 65 70 75 80 gcc cag gga gcc ggt acc gga gtc ggt acc gca gcc gct gct acg acg 336 Ala Gln Gly Ala Gly Thr Gly Val Gly Thr Ala Ala Ala Ala Thr Thr 85 90 95 act acc gcc acg cct agc caa tcc gac ttt gat aat tgg gtc ctc acc 384 Thr Thr Ala Thr Pro Ser Gln Ser Asp Phe Asp Asn Trp Val Leu Thr 100 105 110 agt gga ttg cct acc atc acc act tca ttg atc agt acc aat ccc gat 432 Ser Gly Leu Pro Thr Ile Thr Thr Ser Leu Ile Ser Thr Asn Pro Asp 115 120 125 gcc att act ccg act gcc agt act tca gga ccg aag cct acg gtc acg 480 Ala Ile Thr Pro Thr Ala Ser Thr Ser Gly Pro Lys Pro Thr Val Thr 130 135 140 ttc agc tcg tac tcg gac caa gag ctg gag aat ctc tgg gac gac ttt 528 Phe Ser Ser Tyr Ser Asp Gln Glu Leu Glu Asn Leu Trp Asp Asp Phe 145 150 155 160 gtg gga caa gta caa caa cct cca ttc agc tat gtt cca gaa ccc caa 576 Val Gly Gln Val Gln Gln Pro Pro Phe Ser Tyr Val Pro Glu Pro Gln 165 170 175 aac ccc tat cct ctg cca aac acc cca cca tcc ctc tat cca gac tgg 624 Asn Pro Tyr Pro Leu Pro Asn Thr Pro Pro Ser Leu Tyr Pro Asp Trp 180 185 190 tac gtc aat tgc cct aca aag agt cta ccg ggg tac aaa ttc ccc aga 672 Tyr Val Asn Cys Pro Thr Lys Ser Leu Pro Gly Tyr Lys Phe Pro Arg 195 200 205 gga ttc ctg ttc ggc tgg gct aca gct gcg caa cag tgg gaa ggg gct 720 Gly Phe Leu Phe Gly Trp Ala Thr Ala Ala Gln Gln Trp Glu Gly Ala 210 215 220 gtc aag gcg gat ggt aag ggt cct agt atc tgg gac tgg gca agt aga 768 Val Lys Ala Asp Gly Lys Gly Pro Ser Ile Trp Asp Trp Ala Ser Arg 225 230 235 240 tac ccc ggc ttc atc gcg gac aac act tct gat gtg gga gat ctg 816 Tyr Pro Gly Phe Ile Ala Asp Asn Thr Thr Ser Asp Val Gly Asp Leu 245 250 255 gga tat tac cta tac aaa gaa gat atg gca cgc ctc gct gcg ttg gga 864 Gly Tyr Tyr Leu Tyr Lys Glu Asp Met Ala Arg Leu Ala Ala Leu Gly 260 265 270 gga aac gtc tac tct tc tc tc tc tgg act cgt atc ctc ccc ttt 912 Gly Asn Val Tyr Ser Phe Ser Ile Phe Trp Thr Arg Ile Leu Pro Phe 275 280 285 gcg gtc caa gga tcc ccc gtg aac caa aag gga gta gac ttt cgg 960 Ala Val Gln Gly Ser Pro Val Asn Gln Lys Gly Val Asp Phe Tyr Arg 290,295,300 gac ttg atc gat tat tgc tgg agt ttg ggt atc gag cct gtc gtg aca 1008 Asp With Asp Tyr Cys Trp Ser Leu Gly With Glu Pro Val Val Thr 305 310 315 320 ctg ttc cac tgg gat aca cct tta gcg gtg caa ctc ctc tat gga gga 1056 Leu Phe His Trp Asp Thr Pro Leu Ala Val Gln Leu Leu Tyr Gly Gly 325 330 335 ttc gca agt gac aag atc att gat gat tat gtc aat tat gcc gaa acg 1104 Phe Ala Served Asp Lys Ile Ile Asp Asp Tyr Val Asn Tyr Ala Glu Thr 340 345 350 gtg ttc act gcc tat aat ggc tcg gtt cac aaa tgg atc acc ttc aac 1152 Val Phe Thr Ala Tyr Asn Gly Ser Val His Lys Trp Ile Thr Phe Asn 355 360 365 gaa cca gta gta ttc tgc agc cag atg gct tct cct gtg aat tca aca 1200 Glu Pro Val Val Phe Cys Ser Gln Met Ala Ser Pro Val Asn Ser Thr 370 375 380 ctg ccc gaa ggg ttg aac agc acc aca tac cca tac aca tgt agc tac 1248 Leu Pro Glu Gly Leu Asn Ser Thr Thr Tyr Pro Tyr Thr Cys Ser Tyr 385 390 395 400 cat ctc acc ctg gct cac gcc aag acc gtc caa cga ttc aga gag ctc 1296 His Leu Thr Leu Ala His Ala Lys Thr Val Gln Arg Phe Arg Glu Leu 405 410 415 aac atc cag gga gag att gcg ctc aag tcg gac aac ttt aat ggt atc 1344 Asn Ile Gln Gly Glu Ile Ala Leu Lys Ser Asp Asn Phe Asn Gly Ile 420 425 430 cct tgg agg gaa ggg aat ccc gac gat gaa gaa gcc gtt gct agg cat 1392 Pro Trp Arg Glu Gly Asn Pro Asp Asp Glu Glu Ala Val Ala Arg His 435 440 445 tct gca tac cag att ggc atc ttt gcg gaa ccg ata tac aac act ggc 1440 Ser Ala Tyr Gln Ile Gly Ile Phe Ala Glu Pro Ile Tyr Asn Thr Gly 450 455 460 gac tgg cca gaa ctg atc aag aac gat ctt gga ccc gac atc ttg ccc 1488 Asp Trp Pro Glu Leu Ile Lys Asn Asp Leu Gly Pro Asp Ile Leu Pro 465 470 475 480 cga ttc acc gat gag cag atc cag atg atc aag ggt act gcc gac ttc 1536 Arg Phe Thr Asp Glu Gln Ile Gln Met Ile Lys Gly Thr Ala Asp Phe 485 490 495 ttt gcc att gat ggg tat cga gat ggc tgg gtc act gcc cca cct gct 1584 Phe Ala Ile Asp Gly Tyr Arg Asp Gly Trp Val Thr Ala Pro Pro Ala 500 505 510 gga gtg cag gct tgc gtg gcc aat atc agt gat ccc ctc tgg cct gtg 1632 Gly Val Gln Ala Cys Val Ala Asn Ile Ser Asp Pro Leu Trp Pro Val 515 520 525 tgc aat caa gtc aac ttc tac gac tct tct ccc gca ggt tgg gga atc 1680 Cys Asn Gln Val Asn Phe Tyr Asp Ser Ser Pro Ala Gly Trp Gly Ile 530 535 540 gga gcg ttt ggt aat tgg cct acc act ccc tgg ctg caa aac act tgg 1728 Gly Ala Phe Gly Asn Trp Pro Thr Thr Pro Trp Leu Gln Asn Thr Trp 545 550 555 560 caa ttt gtc cgg cca ttt ttg aaa gaa ttg act cag cag tac ccc acc 1776 Gln Phe Val Arg Pro Phe Leu Lys Glu Leu Thr Gln Gln Tyr Pro Thr 565 570 575 aaa ggt ggt atc tac ctc tcg gaa ttt ggc ttc tcc gaa cca ttc gag 1824 Lys Gly Gly Ile Tyr Leu Ser Glu Phe Gly Phe Ser Glu Pro Phe Glu 580 585 590 aac gag aaa aac ttc atc tac cag atc acg act gac ccg gga cgg gtg 1872 Asn Glu Lys Asn Phe Ile Tyr Gln Ile Thr Thr Asp Pro Gly Arg Val 595 600 605 gca tac ttt aac agt tac ctc ggt gaa gtg ctc ttg gcg atc aac gag 1920 Ala Tyr Phe Asn Ser Tyr Leu Gly Glu Val Leu Leu Ala Ile Asn Glu 610 615 620 gat gaa aca gat gtg aga ggg act ttt gga tgg agt ctt ttg gac aac 1968 Asp Glu Thr Asp Val Arg Gly Thr Phe Gly Trp Ser Leu Leu Asp Asn 625 630 635 640 ttt gag tgg aac tcg ggg ttg tcg act cgg ttc ggt gtc caa tat gtc 2016 Phe Glu Trp Asn Ser Gly Leu Ser Thr Arg Phe Gly Val Gln Tyr Val 645 650 655 gat tac aac agt cct acg ctc gaa agg acg ttc aag cgc tct gcg atc 2064 Asp Tyr Asn Ser Pro Thr Leu Glu Arg Thr Phe Lys Arg Ser Ala Ile 660 665 670 gag atg agc cag ttc tgg aac act cat cgt tgc gag gac tag 2106 Glu Met Ser Gln Phe Trp Asn Thr His Arg Cys Glu Asp 675 680 685 <210> 8 <211> 701 <212> PRT <213> Auricularia magna <400> 8 Met Phe Lys Leu Thr Ser Val Leu Leu Leu Leu Gly Ala Ala Gln Ala -15 -10 -5 -1 Ala Val Leu Asn Pro Arg Gln Ala Gly Ser Gly Asn Ser Thr Ala Ser 1 5 10 15 Gly Ser Ile Ala Gly Asp Ser Thr Arg Pro Ala Thr Thr Ser Ser Val 20 25 30 Val Ser Pro Ser Ala Ala Arg Asn Ser Thr Ala Ala Ala Thr Gly Asn 35 40 45 Ala Ser Arg Asn Ala Thr Ala Thr Gly Thr Ala Val Ala Thr Ala Thr 50 55 60 Gly Gly Val Thr Ala Ala Thr Ser Thr Gly Met Ala Val Thr Ser Pro 65 70 75 80 Ala Gln Gly Ala Gly Thr Gly Val Gly Thr Ala Ala Ala Ala Thr Thr 85 90 95 Thr Thr Ala Thr Pro Ser Gln Ser Asp Phe Asp Asn Trp Val Leu Thr 100 105 110 Ser Gly Leu Pro Thr Ile Thr Thr Ser Leu Ile Ser Thr Asn Pro Asp 115 120 125 Ala Ile Thr Pro Thr Ala Ser Thr Ser Gly Pro Lys Pro Thr Val Thr 130 135 140 Phe Ser Ser Tyr Ser Asp Gln Glu Leu Glu Asn Leu Trp Asp Asp Phe 145 150 155 160 Val Gly Gln Val Gln Gln Pro Pro Phe Ser Tyr Val Pro Glu Pro Gln 165 170 175 Asn Pro Tyr Pro Leu Pro Asn Thr Pro Pro Ser Leu Tyr Pro Asp Trp 180 185 190 Tyr Val Asn Cys Pro Thr Lys Ser Leu Pro Gly Tyr Lys Phe Pro Arg 195 200 205 Gly Phe Leu Phe Gly Trp Ala Thr Ala Ala Gln Gln Trp Glu Gly Ala 210 215 220 Val Lys Ala Asp Gly Lys Gly Pro Ser Ile Trp Asp Trp Ala Ser Arg 225 230 235 240 Tyr Pro Gly Phe Ile Ala Asp Asn Thr Thr Ser Asp Val Gly Asp Leu 245 250 255 Gly Tyr Tyr Leu Tyr Lys Glu Asp Met Ala Arg Leu Ala Ala Leu Gly 260 265 270 Gly Asn Val Tyr Ser Phe Ser Ile Phe Trp Thr Arg Ile Leu Pro Phe 275 280 285 Ala Val Gln Gly Ser Pro Val Asn Gln Lys Gly Val Asp Phe Tyr Arg 290 295 300 Asp Leu Ile Asp Tyr Cys Trp Ser Leu Gly Ile Glu Pro Val Val Thr 305 310 315 320 Leu Phe His Trp Asp Thr Pro Leu Ala Val Gln Leu Leu Tyr Gly Gly 325 330 335 Phe Ala Ser Asp Lys Ile Ile Asp Asp Tyr Val Asn Tyr Ala Glu Thr 340 345 350 Val Phe Thr Ala Tyr Asn Gly Ser Val His Lys Trp Ile Thr Phe Asn 355 360 365 Glu Pro Val Val Phe Cys Ser Gln Met Ala Ser Pro Val Asn Ser Thr 370 375 380 Leu Pro Glu Gly Leu Asn Ser Thr Thr Tyr Pro Tyr Thr Cys Ser Tyr 385 390 395 400 His Leu Thr Leu Ala His Ala Lys Thr Val Gln Arg Phe Arg Glu Leu 405 410 415 Asn Ile Gln Gly Glu Ile Ala Leu Lys Ser Asp Asn Phe Asn Gly Ile 420 425 430 Pro Trp Arg Glu Gly Asn Pro Asp Asp Glu Glu Ala Val Ala Arg His 435 440 445 Ser Ala Tyr Gln Ile Gly Ile Phe Ala Glu Pro Ile Tyr Asn Thr Gly 450 455 460 Asp Trp Pro Glu Leu Ile Lys Asn Asp Leu Gly Pro Asp Ile Leu Pro 465 470 475 480 Arg Phe Thr Asp Glu Gln Ile Gln Met Ile Lys Gly Thr Ala Asp Phe 485 490 495 Phe Ala Ile Asp Gly Tyr Arg Asp Gly Trp Val Thr Ala Pro Pro Ala 500 505 510 Gly Val Gln Ala Cys Val Ala Asn Ile Ser Asp Pro Leu Trp Pro Val 515 520 525 Cys Asn Gln Val Asn Phe Tyr Asp Ser Ser Pro Ala Gly Trp Gly Ile 530 535 540 Gly Ala Phe Gly Asn Trp Pro Thr Thr Pro Trp Leu Gln Asn Thr Trp 545 550 555 560 Gln Phe Val Arg Pro Phe Leu Lys Glu Leu Thr Gln Gln Tyr Pro Thr 565 570 575 Lys Gly Gly Ile Tyr Leu Ser Glu Phe Gly Phe Ser Glu Pro Phe Glu 580 585 590 Asn Glu Lys Asn Phe Ile Tyr Gln Ile Thr Thr Asp Pro Gly Arg Val 595 600 605 Ala Tyr Phe Asn Ser Tyr Leu Gly Glu Val Leu Leu Ala Ile Asn Glu 610 615 620 Asp Glu Thr Asp Val Arg Gly Thr Phe Gly Trp Ser Leu Leu Asp Asn 625 630 635 640 Phe Glu Trp Asn Ser Gly Leu Ser Thr Arg Phe Gly Val Gln Tyr Val 645 650 655 Asp Tyr Asn Ser Pro Thr Leu Glu Arg Thr Phe Lys Arg Ser Ala Ile 660 665 670 Glu Met Ser Gln Phe Trp Asn Thr His Arg Cys Glu Asp 675 680 685 <210> 9 <211> 2121 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(2121) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(2118) <400> 9 atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct gct gtt cta aac cct cgt caa gct ggc agc ggt 96 Ala Pro Ala Leu Ala Ala Val Leu Asn Pro Arg Gln Ala Gly Ser Gly -5 -1 1 5 10 aat tcc act gcc agc ggc tcg ata gcc ggc gat tcc act aga cca gcc 144 Asn Ser Thr Ala Ser Gly Ser Ile Ala Gly Asp Ser Thr Arg Pro Ala 15 20 25 acg aca tcc tcg gtc gtc tca ccc tct gca gcc aga aac tcc act gcc 192 Thr Thr Ser Ser Val Val Ser Pro Ser Ala Ala Arg Asn Ser Thr Ala 30 35 40 gca gct act ggt aat gct tct cgc aat gct act gcg aca ggt act gcc 240 Ala Ala Thr Gly Asn Ala Ser Arg Asn Ala Thr Ala Thr Gly Thr Ala 45 50 55 gtc gct aca gcc act ggc ggg gtt aca gca gcc acg tcc act gga atg 288 Val Ala Thr Ala Thr Gly Gly Val Thr Ala Ala Thr Ser Thr Gly Met 60 65 70 75 gcg gtg act tcc cct gcc cag gga gcc ggt acc gga gtc ggt acc gca 336 Ala Val Thr Ser Pro Ala Gln Gly Ala Gly Thr Gly Val Gly Thr Ala 80 85 90 gcc gct gct acg acg act acc gcc acg cct agc caa tcc gac ttt gat 384 Ala Ala Ala Thr Thr Thr Thr Ala Thr Pro Ser Gln Ser Asp Phe Asp 95 100 105 aat tgg gtc ctc acc agt gga ttg cct acc atc acc act tca ttg atc 432 Asn Trp Val Leu Thr Ser Gly Leu Pro Thr Ile Thr Thr Ser Leu Ile 110 115 120 agt acc aat ccc gat gcc att act ccg act gcc agt act tca gga ccg 480 Ser Thr Asn Pro Asp Ala Ile Thr Pro Thr Ala Ser Thr Ser Gly Pro 125 130 135 aag cct acg gtc acg ttc agc tcg tac tcg gac caa gag ctg gag aat 528 Lys Pro Thr Val Thr Phe Ser Ser Tyr Ser Asp Gln Glu Leu Glu Asn 140 145 150 155 ctc tgg gac gac ttt gtg gga caa gta caa caa cct cca ttc agc tat 576 Leu Trp Asp Asp Phe Val Gly Gln Val Gln Gln Pro Pro Phe Ser Tyr 160 165 170 gtt cca gaa ccc caa aac ccc tat cct ctg cca aac acc cca cca tcc 624 Val Pro Glu Pro Gln Asn Pro Tyr Pro Leu Pro Asn Thr Pro Pro Ser 175 180 185 ctc tat cca gac tgg tac gtc aat tgc cct aca aag agt cta ccg ggg 672 Leu Tyr Pro Asp Trp Tyr Val Asn Cys Pro Thr Lys Ser Leu Pro Gly 190 195 200 tac aaa ttc ccc aga gga ttc ctg ttc ggc tgg tgg aca gct gcg caa 720 Tyr Lys Phe Pro Arg Gly Phe Leu Phe Gly Trp Ala Thr Ala Ala Gln 205 210 215 cag tgg gaa ggg gct gtc aag gcg gat ggt aag ggt cct agt atc tgg 768 Gln Trp Glu Gly Ala Val Lys Ala Asp Gly Lys Gly Pro Ser Ile Trp 220 225 230 235 gac tgg gca agt aga tac ccc ggc ttc atc gcg gac aac act tct 816 Asp Trp Ala Ser Arg Tyr Pro Gly Phe Ile Ala Asp Asn Thr Thr Ser 240 245 250 gat gtg gga gat ctg gga tat tac cta tac aaa gaa gat atg gca cgc 864 Asp Val Gly Asp Leu Gly Tyr Tyr Leu Tyr Lys Glu Asp Met Ala Arg 255 260 265 ctc gct gcg tg gga gga aac gtc tct ttc tcc atc ttc tgg act 912 Leu Ala Ala Leu Gly Gly Asn Val Tyr Ser Phe Ser Ile Phe Trp Thr 270 275 280 cgt atc ctc ccc ttt gcg gtc CA gga tcc ccc gtg aac CA aag gga 960 Arg Ile Leu Pro Phe Ala Val Gln Gly Ser Pro Val Asn Gln Lys Gly 285,290,295 gta gac ttt tat cgg gac ttg atc gat tat tgc tgg agt ttg ggt atc 1008 Val Asp Phe Tyr Arg Asp Leu Ile Asp Tyr Cys Trp Ser Leu Gly Ile 300 305 310 315 gag cct gtc gtg aca ctg ttc cac tgg gat aca cct tta gcg gtg caa 1056 Glu Pro Val Val Thr Leu Phe His Trp Asp Thr Pro Leu Ala Val Gln 320 325 330 ctg ctc tat gga gga ttc gca agt gac aag atc att gat gat tat gtc 1104 Leu Leu Tyr Gly Gly Phe Ala Served Asp Lys Ile Ile Asp Asp Tyr Val 335 340 345 aat tat gcc gaa acg gtg ttc act gcc tat aat ggc tcg gtt cac aaa 1152 Asn Tyr Ala Glu Thr Val Phe Thr Ala Tyr Asn Gly Ser Val His Lys 350 355 360 tgg atc acc ttc aac gaa cca gta gta ttc tgc agc cag atg gct tct 1200 Trp Ile Thr Phe Asn Glu Pro Val Val Phe Cys Ser Gln Met Ala Ser 365 370 375 cct gtg aat tca aca ctg ccc gaa ggg ttg aac agc acc aca tac cca 1248 Pro Val Asn Ser Thr Leu Pro Glu Gly Leu Asn Ser Thr Thr Tyr Pro 380 385 390 395 tac aca tgt agc tac cat ctc acc ctg gct cac gcc aag acc gtc caa 1296 Tyr Thr Cys Ser Tyr His Leu Thr Leu Ala His Ala Lys Thr Val Gln 400 405 410 cga ttc aga gag ctc aac atc cag gga gag att gcg ctc aag tcg gac 1344 Arg Phe Arg Glu Leu Asn Ile Gln Gly Glu Ile Ala Leu Lys Ser Asp 415 420 425 aac ttt aat ggt atc cct tgg agg gaa ggg aat ccc gac gat gaa gaa 1392 Asn Phe Asn Gly Ile Pro Trp Arg Glu Gly Asn Pro Asp Asp Glu Glu 430 435 440 gcc gtt gct agg cat tct gca tac cag att ggc atc ttt gcg gaa ccg 1440 Ala Val Ala Arg His Ser Ala Tyr Gln Ile Gly Ile Phe Ala Glu Pro 445 450 455 ata tac aac act ggc gac tgg cca gaa ctg atc aag aac gat ctt gga 1488 Ile Tyr Asn Thr Gly Asp Trp Pro Glu Leu Ile Lys Asn Asp Leu Gly 460 465 470 475 ccc gac atc ttg ccc cga ttc acc gat gag cag atc cag atg atc aag 1536 Pro Asp Ile Leu Pro Arg Phe Thr Asp Glu Gln Ile Gln Met Ile Lys 480 485 490 ggt act gcc gac ttc ttt gcc att gat ggg tat cga gat ggc tgg gtc 1584 Gly Thr Ala Asp Phe Phe Ala Ile Asp Gly Tyr Arg Asp Gly Trp Val 495 500 505 act gcc cca cct gct gga gtg cag gct tgc gtg gcc aat atc agt gat 1632 Thr Ala Pro Pro Ala Gly Val Gln Ala Cys Val Ala Asn Ile Ser Asp 510 515 520 ccc ctc tgg cct gtg tgc aat caa gtc aac ttc tac gac tct tct ccc 1680 Pro Leu Trp Pro Val Cys Asn Gln Val Asn Phe Tyr Asp Ser Ser Pro 525 530 535 gca ggt tgg gga atc gga gcg ttt ggt aat tgg cct acc act ccc tgg 1728 Ala Gly Trp Gly Ile Gly Ala Phe Gly Asn Trp Pro Thr Thr Pro Trp 540 545 550 555 ctg caa aac act tgg caa ttt gtc cgg cca ttt ttg aaa gaa ttg act 1776 Leu Gln Asn Thr Trp Gln Phe Val Arg Pro Phe Leu Lys Glu Leu Thr 560 565 570 cag cag tac ccc acc aaa ggt ggt atc tac ctc tcg gaa ttt ggc ttc 1824 Gln Gln Tyr Pro Thr Lys Gly Gly Ile Tyr Leu Ser Glu Phe Gly Phe 575 580 585 tcc gaa cca ttc gag aac gag aaa aac ttc atc tac cag atc acg act 1872 Ser Glu Pro Phe Glu Asn Glu Lys Asn Phe Ile Tyr Gln Ile Thr Thr 590 595 600 gac ccg gga cgg gtg gca tac ttt aac agt tac ctc ggt gaa gtg ctc 1920 Asp Pro Gly Arg Val Ala Tyr Phe Asn Ser Tyr Leu Gly Glu Val Leu 605 610 615 ttg gcg atc aac gag gat gaa aca gat gtg aga ggg act ttt gga tgg 1968 Leu Ala Ile Asn Glu Asp Glu Thr Asp Val Arg Gly Thr Phe Gly Trp 620 625 630 635 agt ctt ttg gac aac ttt gag tgg aac tcg ggg ttg tcg act cgg ttc 2016 Ser Leu Leu Asp Asn Phe Glu Trp Asn Ser Gly Leu Ser Thr Arg Phe 640 645 650 ggt gtc caa tat gtc gat tac aac agt cct acg ctc gaa agg acg ttc 2064 Gly Val Gln Tyr Val Asp Tyr Asn Ser Pro Thr Leu Glu Arg Thr Phe 655 660 665 aag cgc tct gcg atc gag atg agc cag ttc tgg aac act cat cgt tgc 2112 Lys Arg Ser Ala Ile Glu Met Ser Gln Phe Trp Asn Thr His Arg Cys 670 675 680 gag gac tag 2121 Glu Asp 685 <210> 10 <211> 706 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ala Val Leu Asn Pro Arg Gln Ala Gly Ser Gly -5 -1 1 5 10 Asn Ser Thr Ala Ser Gly Ser Ile Ala Gly Asp Ser Thr Arg Pro Ala 15 20 25 Thr Thr Ser Ser Val Val Ser Pro Ser Ala Ala Arg Asn Ser Thr Ala 30 35 40 Ala Ala Thr Gly Asn Ala Ser Arg Asn Ala Thr Ala Thr Gly Thr Ala 45 50 55 Val Ala Thr Ala Thr Gly Gly Val Thr Ala Ala Thr Ser Thr Gly Met 60 65 70 75 Ala Val Thr Ser Pro Ala Gln Gly Ala Gly Thr Gly Val Gly Thr Ala 80 85 90 Ala Ala Ala Thr Thr Thr Thr Ala Thr Pro Ser Gln Ser Asp Phe Asp 95 100 105 Asn Trp Val Leu Thr Ser Gly Leu Pro Thr Ile Thr Thr Ser Leu Ile 110 115 120 Ser Thr Asn Pro Asp Ala Ile Thr Pro Thr Ala Ser Thr Ser Gly Pro 125 130 135 Lys Pro Thr Val Thr Phe Ser Ser Tyr Ser Asp Gln Glu Leu Glu Asn 140 145 150 155 Leu Trp Asp Asp Phe Val Gly Gln Val Gln Gln Pro Pro Phe Ser Tyr 160 165 170 Val Pro Glu Pro Gln Asn Pro Tyr Pro Leu Pro Asn Thr Pro Pro Ser 175 180 185 Leu Tyr Pro Asp Trp Tyr Val Asn Cys Pro Thr Lys Ser Leu Pro Gly 190 195 200 Tyr Lys Phe Pro Arg Gly Phe Leu Phe Gly Trp Ala Thr Ala Ala Gln 205 210 215 Gln Trp Glu Gly Ala Val Lys Ala Asp Gly Lys Gly Pro Ser Ile Trp 220 225 230 235 Asp Trp Ala Ser Arg Tyr Pro Gly Phe Ile Ala Asp Asn Thr Thr Ser 240 245 250 Asp Val Gly Asp Leu Gly Tyr Tyr Leu Tyr Lys Glu Asp Met Ala Arg 255 260 265 Leu Ala Ala Leu Gly Gly Asn Val Tyr Ser Phe Ser Ile Phe Trp Thr 270 275 280 Arg Ile Leu Pro Phe Ala Val Gln Gly Ser Pro Val Asn Gln Lys Gly 285 290 295 Val Asp Phe Tyr Arg Asp Leu Ile Asp Tyr Cys Trp Ser Leu Gly Ile 300 305 310 315 Glu Pro Val Val Thr Leu Phe His Trp Asp Thr Pro Leu Ala Val Gln 320 325 330 Leu Leu Tyr Gly Gly Phe Ala Ser Asp Lys Ile Ile Asp Asp Tyr Val 335 340 345 Asn Tyr Ala Glu Thr Val Phe Thr Ala Tyr Asn Gly Ser Val His Lys 350 355 360 Trp Ile Thr Phe Asn Glu Pro Val Val Phe Cys Ser Gln Met Ala Ser 365 370 375 Pro Val Asn Ser Thr Leu Pro Glu Gly Leu Asn Ser Thr Thr Tyr Pro 380 385 390 395 Tyr Thr Cys Ser Tyr His Leu Thr Leu Ala His Ala Lys Thr Val Gln 400 405 410 Arg Phe Arg Glu Leu Asn Ile Gln Gly Glu Ile Ala Leu Lys Ser Asp 415 420 425 Asn Phe Asn Gly Ile Pro Trp Arg Glu Gly Asn Pro Asp Asp Glu Glu 430 435 440 Ala Val Ala Arg His Ser Ala Tyr Gln Ile Gly Ile Phe Ala Glu Pro 445 450 455 Ile Tyr Asn Thr Gly Asp Trp Pro Glu Leu Ile Lys Asn Asp Leu Gly 460 465 470 475 Pro Asp Ile Leu Pro Arg Phe Thr Asp Glu Gln Ile Gln Met Ile Lys 480 485 490 Gly Thr Ala Asp Phe Phe Ala Ile Asp Gly Tyr Arg Asp Gly Trp Val 495 500 505 Thr Ala Pro Pro Ala Gly Val Gln Ala Cys Val Ala Asn Ile Ser Asp 510 515 520 Pro Leu Trp Pro Val Cys Asn Gln Val Asn Phe Tyr Asp Ser Ser Pro 525 530 535 Ala Gly Trp Gly Ile Gly Ala Phe Gly Asn Trp Pro Thr Thr Pro Trp 540 545 550 555 Leu Gln Asn Thr Trp Gln Phe Val Arg Pro Phe Leu Lys Glu Leu Thr 560 565 570 Gln Gln Tyr Pro Thr Lys Gly Gly Ile Tyr Leu Ser Glu Phe Gly Phe 575 580 585 Ser Glu Pro Phe Glu Asn Glu Lys Asn Phe Ile Tyr Gln Ile Thr Thr 590 595 600 Asp Pro Gly Arg Val Ala Tyr Phe Asn Ser Tyr Leu Gly Glu Val Leu 605 610 615 Leu Ala Ile Asn Glu Asp Glu Thr Asp Val Arg Gly Thr Phe Gly Trp 620 625 630 635 Ser Leu Leu Asp Asn Phe Glu Trp Asn Ser Gly Leu Ser Thr Arg Phe 640 645 650 Gly Val Gln Tyr Val Asp Tyr Asn Ser Pro Thr Leu Glu Arg Thr Phe 655 660 665 Lys Arg Ser Ala Ile Glu Met Ser Gln Phe Trp Asn Thr His Arg Cys 670 675 680 Glu Asp 685 <210> 11 <211> 2121 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(2121) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(2118) <400> 11 atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct gcg gtg ctc aac cca cgc cag gct ggc tct gga 96 Ala Pro Ala Leu Ala Ala Val Leu Asn Pro Arg Gln Ala Gly Ser Gly -5 -1 1 5 10 aac tct act gcc tct ggg tcc att gct ggc gac tcg aca cgc ccg gct 144 Asn Ser Thr Ala Ser Gly Ser Ile Ala Gly Asp Ser Thr Arg Pro Ala 15 20 25 acg acc tct tcg gtt gtc tcc cct tcc gca gcc cga aac tcc act gcc 192 Thr Thr Ser Ser Val Val Ser Pro Ser Ala Ala Arg Asn Ser Thr Ala 30 35 40 gcc gcc acg ggt aat gct tcc cgc aac gct acg gcc acc gga aca gcc 240 Ala Ala Thr Gly Asn Ala Ser Arg Asn Ala Thr Ala Thr Gly Thr Ala 45 50 55 gtt gcc act gcc acg ggc ggc gtt acg gcg gca acg tcc acg ggc atg 288 Val Ala Thr Ala Thr Gly Gly Val Thr Ala Ala Thr Ser Thr Gly Met 60 65 70 75 gct gtt acg tct ccc gct cag ggt gcc gga acg ggt gtt ggc acc gcc 336 Ala Val Thr Ser Pro Ala Gln Gly Ala Gly Thr Gly Val Gly Thr Ala 80 85 90 gca gcc gca aca aca acg acg gca acg ccc tct caa tcc gac ttc gac 384 Ala Ala Ala Thr Thr Thr Thr Ala Thr Pro Ser Gln Ser Asp Phe Asp 95 100 105 aac tgg gtg ctc acg tcg ggg ctc ccc aca atc aca act agc ctg atc 432 Asn Trp Val Leu Thr Ser Gly Leu Pro Thr Ile Thr Thr Ser Leu Ile 110 115 120 tcg aca aac cct gac gcc att acc cca acc gca tct acc tcg gga ccc 480 Ser Thr Asn Pro Asp Ala Ile Thr Pro Thr Ala Ser Thr Ser Gly Pro 125 130 135 aaa cca acg gtt acc ttc tcc tcc tac tct gat cag gag ttg gag aat 528 Lys Pro Thr Val Thr Phe Ser Ser Tyr Ser Asp Gln Glu Leu Glu Asn 140 145 150 155 ctg tgg gac gac ttt gtc ggg cag gtt cag cag cct ccg ttc tcg tat 576 Leu Trp Asp Asp Phe Val Gly Gln Val Gln Gln Pro Pro Phe Ser Tyr 160 165 170 gtt cca gaa ccc caa aac ccg tac cca ttg ccc aac acc cca ccg tct 624 Val Pro Glu Pro Gln Asn Pro Tyr Pro Leu Pro Asn Thr Pro Pro Ser 175 180 185 ctc tac ccc gac tgg tac gtc aat tgt ccc acc aag agc ttg cct ggt 672 Leu Tyr Pro Asp Trp Tyr Val Asn Cys Pro Thr Lys Ser Leu Pro Gly 190 195 200 tac aag ttc cct cga ggc ttc ctc ttc ggt tgg gcg acg gct gcg caa 720 Tyr Lys Phe Pro Arg Gly Phe Leu Phe Gly Trp Ala Thr Ala Ala Gln 205 210 215 cag tgg gag ggg gct gtg aaa gct gac ggc aaa gga cca tcc atc tgg 768 Gln Trp Glu Gly Ala Val Lys Ala Asp Gly Lys Gly Pro Ser Ile Trp 220 225 230 235 gac tgg gcc tcc cga tat ccg ggc ttc atc gcc gat aac acc aca tcg 816 Asp Trp Ala Ser Arg Tyr Pro Gly Phe Ile Ala Asp Asn Thr Thr Ser 240 245 250 gac gtg ggc gac ttg ggc tac tac ttg tac aag gag gat atg gct cgt 864 Asp Val Gly Asp Leu Gly Tyr Tyr Leu Tyr Lys Glu Asp Met Ala Arg 255 260 265 ctc gcc gcc ctc ggc ggc aat gtg tat tcc ttt tcc atc ttc tgg acg 912 Leu Ala Ala Leu Gly Gly Asn Val Tyr Ser Phe Ser Ile Phe Trp Thr 270 275 280 cga att ctg ccg ttc gca gtg caa ggt tct ccg gtc aat cag aag ggt 960 Arg Ile Leu Pro Phe Ala Val Gln Gly Ser Pro Val Asn Gln Lys Gly 285 290 295 gtt gac ttc tac cgg gac ctg atc gat tac tgt tgg agc ctt ggt atc 1008 Val Asp Phe Tyr Arg Asp Leu Ile Asp Tyr Cys Trp Ser Leu Gly Ile 300 305 310 315 gaa ccg gtc gtc aca ttg ttc cac tgg gat act ccg ttg gca gtg caa 1056 Glu Pro Val Val Thr Leu Phe His Trp Asp Thr Pro Leu Ala Val Gln 320 325 330 ctg ctc tat gga ggt ttc gcc tcg gac aag atc atc gac gac tac gtc 1104 Leu Leu Tyr Gly Gly Phe Ala Ser Asp Lys Ile Ile Asp Asp Tyr Val 335 340 345 aac tac gcc gaa acc gtg ttc aca gcg tac aac ggt tcg gtc cat aag 1152 Asn Tyr Ala Glu Thr Val Phe Thr Ala Tyr Asn Gly Ser Val His Lys 350 355 360 tgg atc acc ttc aac gag cca gtt gtg ttc tgt tcc caa atg gca tcg 1200 Trp Ile Thr Phe Asn Glu Pro Val Val Phe Cys Ser Gln Met Ala Ser 365 370 375 cca gtg aat agc acg ctg cca gag ggg ttg aac tcg acc acc tac ccc 1248 Pro Val Asn Ser Thr Leu Pro Glu Gly Leu Asn Ser Thr Thr Tyr Pro 380 385 390 395 tat acg tgc tcg tac cat ctg acc ctt gcg cac gcg aag acc gtg caa 1296 Tyr Thr Cys Ser Tyr His Leu Thr Leu Ala His Ala Lys Thr Val Gln 400 405 410 cgg ttc cgt gag ctc aac atc cag ggg gag att gcc ttg aag agc gat 1344 Arg Phe Arg Glu Leu Asn Ile Gln Gly Glu Ile Ala Leu Lys Ser Asp 415 420 425 aac ttc aac ggc att ccg tgg cgg gag gga aac cct gat gat gaa gag 1392 Asn Phe Asn Gly Ile Pro Trp Arg Glu Gly Asn Pro Asp Asp Glu Glu 430 435 440 gct gtc gcc cga cat tct gcc tat cag atc gga atc ttc gca gaa cct 1440 Ala Val Ala Arg His Ser Ala Tyr Gln Ile Gly Ile Phe Ala Glu Pro 445 450 455 atc tac aac act ggt gat tgg ccg gag ctg atc aag aac gat ctg ggg 1488 Ile Tyr Asn Thr Gly Asp Trp Pro Glu Leu Ile Lys Asn Asp Leu Gly 460 465 470 475 ccg gat atc ttg ccc cgc ttc act gat gaa cag atc cag atg atc aag 1536 Pro Asp Ile Leu Pro Arg Phe Thr Asp Glu Gln Ile Gln Met Ile Lys 480 485 490 ggg acc gct gat ttc ttc gcg atc gat ggt tac cgt gac ggc tgg gtc 1584 Gly Thr Ala Asp Phe Phe Ala Ile Asp Gly Tyr Arg Asp Gly Trp Val 495 500 505 aca gca cca cct gct ggt gtc caa gct tgc gtg gcg aac atc tct gat 1632 Thr Ala Pro Pro Ala Gly Val Gln Ala Cys Val Ala Asn Ile Ser Asp 510 515 520 ccg ttg tgg ccc gtg tgt aac cag gtc aac ttc tac gat tcg agc ccg 1680 Pro Leu Trp Pro Val Cys Asn Gln Val Asn Phe Tyr Asp Ser Ser Pro 525 530 535 gcc ggt tgg gga att ggc gca ttt ggt aac tgg cca act act ccg tgg 1728 Ala Gly Trp Gly Ile Gly Ala Phe Gly Asn Trp Pro Thr Thr Pro Trp 540 545 550 555 ctt cag aac aca tgg cag ttt gtc cgc ccg ttt ctc aag gag ctt act 1776 Leu Gln Asn Thr Trp Gln Phe Val Arg Pro Phe Leu Lys Glu Leu Thr 560 565 570 cag caa tac cca acg aag ggg ggt atc tac ctc tcc gaa ttc ggt ttc 1824 Gln Gln Tyr Pro Thr Lys Gly Gly Ile Tyr Leu Ser Glu Phe Gly Phe 575 580 585 tcg gag ccc ttt gag aac gaa aag aac ttc atc tac caa atc aca aca 1872 Ser Glu Pro Phe Glu Asn Glu Lys Asn Phe Ile Tyr Gln Ile Thr Thr 590 595 600 gac ccc ggc cgg gtg gcc tac ttc aac agc tat ctg ggg gaa gtg ctc 1920 Asp Pro Gly Arg Val Ala Tyr Phe Asn Ser Tyr Leu Gly Glu Val Leu 605 610 615 ttg gcc atc aac gaa gat gag acg gac gtc cgc ggt act ttc gga tgg 1968 Leu Ala Ile Asn Glu Asp Glu Thr Asp Val Arg Gly Thr Phe Gly Trp 620 625 630 635 tct ctt ctc gac aac ttc gag tgg aac tct gga ctg agc aca cga ttc 2016 Ser Leu Leu Asp Asn Phe Glu Trp Asn Ser Gly Leu Ser Thr Arg Phe 640 645 650 ggg gtc cag tat gtg gac tac aac tcc cct acg ctt gaa cgg acc ttc 2064 Gly Val Gln Tyr Val Asp Tyr Asn Ser Pro Thr Leu Glu Arg Thr Phe 655 660 665 aag cgc tct gcg atc gaa atg tcc cag ttc tgg aac acg cat cgt tgc 2112 Lys Arg Ser Ala Ile Glu Met Ser Gln Phe Trp Asn Thr His Arg Cys 670 675 680 gag gac taa 2121 Glu Asp 685 <210> 12 <211> 706 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <400> 12 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ala Val Leu Asn Pro Arg Gln Ala Gly Ser Gly -5 -1 1 5 10 Asn Ser Thr Ala Ser Gly Ser Ile Ala Gly Asp Ser Thr Arg Pro Ala 15 20 25 Thr Thr Ser Ser Val Val Ser Pro Ser Ala Ala Arg Asn Ser Thr Ala 30 35 40 Ala Ala Thr Gly Asn Ala Ser Arg Asn Ala Thr Ala Thr Gly Thr Ala 45 50 55 Val Ala Thr Ala Thr Gly Gly Val Thr Ala Ala Thr Ser Thr Gly Met 60 65 70 75 Ala Val Thr Ser Pro Ala Gln Gly Ala Gly Thr Gly Val Gly Thr Ala 80 85 90 Ala Ala Ala Thr Thr Thr Thr Ala Thr Pro Ser Gln Ser Asp Phe Asp 95 100 105 Asn Trp Val Leu Thr Ser Gly Leu Pro Thr Ile Thr Thr Ser Leu Ile 110 115 120 Ser Thr Asn Pro Asp Ala Ile Thr Pro Thr Ala Ser Thr Ser Gly Pro 125 130 135 Lys Pro Thr Val Thr Phe Ser Ser Tyr Ser Asp Gln Glu Leu Glu Asn 140 145 150 155 Leu Trp Asp Asp Phe Val Gly Gln Val Gln Gln Pro Pro Phe Ser Tyr 160 165 170 Val Pro Glu Pro Gln Asn Pro Tyr Pro Leu Pro Asn Thr Pro Pro Ser 175 180 185 Leu Tyr Pro Asp Trp Tyr Val Asn Cys Pro Thr Lys Ser Leu Pro Gly 190 195 200 Tyr Lys Phe Pro Arg Gly Phe Leu Phe Gly Trp Ala Thr Ala Ala Gln 205 210 215 Gln Trp Glu Gly Ala Val Lys Ala Asp Gly Lys Gly Pro Ser Ile Trp 220 225 230 235 Asp Trp Ala Ser Arg Tyr Pro Gly Phe Ile Ala Asp Asn Thr Thr Ser 240 245 250 Asp Val Gly Asp Leu Gly Tyr Tyr Leu Tyr Lys Glu Asp Met Ala Arg 255 260 265 Leu Ala Ala Leu Gly Gly Asn Val Tyr Ser Phe Ser Ile Phe Trp Thr 270 275 280 Arg Ile Leu Pro Phe Ala Val Gln Gly Ser Pro Val Asn Gln Lys Gly 285 290 295 Val Asp Phe Tyr Arg Asp Leu Ile Asp Tyr Cys Trp Ser Leu Gly Ile 300 305 310 315 Glu Pro Val Val Thr Leu Phe His Trp Asp Thr Pro Leu Ala Val Gln 320 325 330 Leu Leu Tyr Gly Gly Phe Ala Ser Asp Lys Ile Ile Asp Asp Tyr Val 335 340 345 Asn Tyr Ala Glu Thr Val Phe Thr Ala Tyr Asn Gly Ser Val His Lys 350 355 360 Trp Ile Thr Phe Asn Glu Pro Val Val Phe Cys Ser Gln Met Ala Ser 365 370 375 Pro Val Asn Ser Thr Leu Pro Glu Gly Leu Asn Ser Thr Thr Tyr Pro 380 385 390 395 Tyr Thr Cys Ser Tyr His Leu Thr Leu Ala His Ala Lys Thr Val Gln 400 405 410 Arg Phe Arg Glu Leu Asn Ile Gln Gly Glu Ile Ala Leu Lys Ser Asp 415 420 425 Asn Phe Asn Gly Ile Pro Trp Arg Glu Gly Asn Pro Asp Asp Glu Glu 430 435 440 Ala Val Ala Arg His Ser Ala Tyr Gln Ile Gly Ile Phe Ala Glu Pro 445 450 455 Ile Tyr Asn Thr Gly Asp Trp Pro Glu Leu Ile Lys Asn Asp Leu Gly 460 465 470 475 Pro Asp Ile Leu Pro Arg Phe Thr Asp Glu Gln Ile Gln Met Ile Lys 480 485 490 Gly Thr Ala Asp Phe Phe Ala Ile Asp Gly Tyr Arg Asp Gly Trp Val 495 500 505 Thr Ala Pro Pro Ala Gly Val Gln Ala Cys Val Ala Asn Ile Ser Asp 510 515 520 Pro Leu Trp Pro Val Cys Asn Gln Val Asn Phe Tyr Asp Ser Ser Pro 525 530 535 Ala Gly Trp Gly Ile Gly Ala Phe Gly Asn Trp Pro Thr Thr Pro Trp 540 545 550 555 Leu Gln Asn Thr Trp Gln Phe Val Arg Pro Phe Leu Lys Glu Leu Thr 560 565 570 Gln Gln Tyr Pro Thr Lys Gly Gly Ile Tyr Leu Ser Glu Phe Gly Phe 575 580 585 Ser Glu Pro Phe Glu Asn Glu Lys Asn Phe Ile Tyr Gln Ile Thr Thr 590 595 600 Asp Pro Gly Arg Val Ala Tyr Phe Asn Ser Tyr Leu Gly Glu Val Leu 605 610 615 Leu Ala Ile Asn Glu Asp Glu Thr Asp Val Arg Gly Thr Phe Gly Trp 620 625 630 635 Ser Leu Leu Asp Asn Phe Glu Trp Asn Ser Gly Leu Ser Thr Arg Phe 640 645 650 Gly Val Gln Tyr Val Asp Tyr Asn Ser Pro Thr Leu Glu Arg Thr Phe 655 660 665 Lys Arg Ser Ala Ile Glu Met Ser Gln Phe Trp Asn Thr His Arg Cys 670 675 680 Glu Asp 685 <210> 13 <211> 1803 <212> DNA <213> Rhodotorula <220> <221> CDS <222> (1)..(1803) <220> <221> Signal peptide <222> (1)..(57) <220> <221> Mature peptide <222> (58)..(1800) <400> 13 atg gtc atc ctg cgc cac acg gta cta gcg gca gcg gtc gtc cag atc 48 Met Val Ile Leu Arg His Thr Val Leu Ala Ala Ala Val Val Gln Ile -15 -10 -5 gcc ctc ggg gct ccg cag ttt agc ccc aca gtc aat acg gaa ggt atc 96 Ala Leu Gly Ala Pro Gln Phe Ser Pro Thr Val Asn Thr Glu Gly Ile -1 1 5 10 att cca att tcg gag cgc cct tcg cct aca caa tct tcg tcg tcc agc 144 Ile Pro Ile Ser Glu Arg Pro Ser Pro Thr Gln Ser Ser Ser Ser Ser 15 20 25 gga ccc gtg to cca gct ggc tca tat gtc tcc gac ttt gat gcc agc 192 Gly Pro Val Ile Pro Ala Gly Ser Tyr Val Ser Asp Phe Asp Ala Ser 30 35 40 45 gga ctt gca aac tta tgg agc caa gtc gaa gta gac ata cct gtc gag 240 Gly Leu Ala Asn Leu Trp Ser Gln Val Glu Val Asp Ile Pro Val Glu 50 55 60 tct cca agg to tct gcc gta cct cct ctc aac aaa acg ttc agt gtt 288 Ser Pro Arg Ile Ser Ala Val Pro Pro Leu Asn Lys Thr Phe Ser Val 65 70 75 ccg aaa act cca gtc ctg ccc aac tcc ctg cag gat cac ctt cca aaa 336 Pro Lys Thr Pro Val Leu Pro Asn Ser Leu Gln Asp His Leu Pro Lys 80 85 90 gat gtc aaa gca ccc gaa ggc ttt gca tgg ggc gtg gcc tcc gtt gct 384 Asp Val Lys Ala Pro Glu Gly Phe Ala Trp Gly Val Ala Ser Val Ala 95 100 105 cag cag tac gag ggt gcc gtc aaa gca gat gga cga gga cca tct cat 432 Gln Gln Tyr Glu Gly Ala Val Lys Ala Asp Gly Arg Gly Pro Ser His 110 115 120 125 tgg gat ttc ctt tgc cat aga aac cct tcc agc tgc aca aac tac acc 480 Trp Asp Phe Leu Cys His Arg Asn Pro Ser Ser Cys Thr Asn Tyr Thr 130 135 140 agt gat atc act gat ctt ggc cgt tac tac tat aag aat gac ttg gca 528 Ser Asp Ile Thr Asp Leu Gly Arg Tyr Tyr Tyr Lys Asn Asp Leu Ala 145 150 155 cga atg gct gcc atg ggt ata act cac tac tca ttt tca gtg agc tgg 576 Arg Met Ala Ala Met Gly Ile Thr His Tyr Ser Phe Ser Val Ser Trp 160 165 170 acc aga gtt gtg cca ttc ggc aag aag ggc agt cca gtc agc aac gaa 624 Thr Arg Val Val Pro Phe Gly Lys Gly Ser Pro Val Ser Asn Glu 175 180 185 ggc ctc gac tat tac gaa gat atc tgc aag gct ttg agc ttt gga 672 Gly Leu Asp Tyr Tyr Glu Asp With Cys Thr Ala Leu Ser Phe Gly 190 195 200 205 atc aag cct gtc att act tta ttc cac tgg gat act cct gcc aac tta 720 Ile Lys Pro Val Ile Thr Leu Phe His Trp Asp Thr Pro Ala Asn Leu 210 215 220 ctc ttc gaa tat gga ggt ttc ctc aac ggg aca atc ​​gta gat gac tac 768 Leu Phe Glu Tyr Gly Gly Phe Leu Asn Gly Thr Ile Val Asp Tyr 225 230 235 tac tat tat gca gat ata gta ttc aga aga cta ggc aaa tat gcc gaa Tyr Tyr Tyr Ala Asp Ile Val Phe Arg Arg Leu Gly Lys Tyr Ala Glu 240 245 250 acc ttc ttc acc ttc aat gag cct cgt gta tac tgc agc gag tat act 864 Thr Phe Phe Thr Phe Asn Glu Pro Arg Val Tyr Cys Ser Glu Tyr Thr 255 260 265 ggt cct ccg ttc gat gcc tat tac gaa cgc tat ggg ctg aat tct agc 912 Gly Pro Pro Phe Asp Ala Tyr Tyr Glu Arg Tyr Gly Leu Asn Ser Ser 270 275 280 285 act gca cca tat ccc tgc tcg tac aat ctc cga gcc cat ggt gcc 960 Thr Ala Pro Tyr Pro Cys Ser Tyr Asn Leu Leu Arg Ala His Gly Ala 290,295,300 gca gtc ggc agg tat cgt gcc ctg gtc aaa gaa ggc agc atc aaa tct 1008 Ala Val Gly Arg Tyr Arg Ala Leu Val Lys Glu Gly Ser Ile Lys Ser 305 310 315 ggt gaa atc ​​gca ttc aaa aac gac gat agt tac cag cta cct caa aat 1056 Gly Glu Ile Ala Phe Lys Asn Asp Asp Ser Tyr Gln Leu Pro Gln Asn 320 325 330 cca gac tct gac gcc gac aag cga gct gca aaa cgc cac ttc gac ttc 1104 Pro Asp Ser Asp Ala Asp Lys Arg Ala Ala Lys Arg His Phe Asp Phe 335 340 345 tac atc ggc ata ttc tcg caa ccc gta tac ggc aac ggc tac tat ccc 1152 Tyr Ile Gly Ile Phe Ser Gln Pro Val Tyr Gly Asn Gly Tyr Tyr Pro 350 355 360 365 gaa acc gtc cgg aac aca att tca gag cgc ttc ctc cca gag ttc acc 1200 Glu Thr Val Arg Asn Thr Ile Ser Glu Arg Phe Leu Pro Glu Phe Thr 370 375 380 gca gcc gag cgc gaa caa att cag gga tca gcg gac ttc tac gcc atc 1248 Ala Ala Glu Arg Glu Gln Ile Gln Gly Ser Ala Asp Phe Tyr Ala Ile 385 390 395 gac ggt tat agg acg aat atc gca agc gct gct ccc aat ggt att gac 1296 Asp Gly Tyr Arg Thr Asn Ile Ala Ser Ala Ala Pro Asn Gly Ile Asp 400 405 410 gcg tgc ttg aga aac gct agt gat ccc aac tgg cca gtt tgc cag gac 1344 Ala Cys Leu Arg Asn Ala Ser Asp Pro Asn Trp Pro Val Cys Gln Asp 415 420 425 aac agt aac aca ggc caa tac gct act ctt gag gga ttc gca ttg gga 1392 Asn Ser Asn Thr Gly Gln Tyr Ala Thr Leu Glu Gly Phe Ala Leu Gly 430 435 440 445 cct cct gca gat ccc aac gcg aat tgg cta tac aac act gca cct tac 1440 Pro Pro Ala Asp Pro Asn Ala Asn Trp Leu Tyr Asn Thr Ala Pro Tyr 450 455 460 ctg cgt tac caa ttc aaa gtt ctg aaa gag aac ttc at tac aag aag 1488 Leu Arg Tyr Gln Phe Lys Val Leu Lys Glu Asn Phe Asn Tyr Lys Lys 465 470 475 atc tac ttg acg gaa ttc ggc ttt gca gaa cct ttt agc tat ttg cga 1536 Ile Tyr Leu Thr Glu Phe Gly Phe Ala Glu Pro Phe Ser Tyr Leu Arg 480 485 490 cag gat ctg tat gca ttg ctg tat gac act gat cgc act gca tat tat 1584 Gln Asp Leu Tyr With Leu Tyr Asp Thr Asp Arg Thr With Tyr 495,500,505 CA Gac Tat CTA GCG CAG TGC ATG CTG GCT ATCA AAA GAGA GAT GCC ATC 1632 Gln Asp Tyr Ile Gln Cys Met Leu Ile Lys Glu Asp Gly Ile 510 515 520 525 cct ctt gct ggt gtt ttc gca tgg tca ttt gtt gat aac ttc gaa tgg 1680 Pro Leu Ala Gly Val Phe Ala Trp Ser Phe Val Asp Asn Phe Glu Trp 530 535 540 ggt tcc ggt ctt gag cag aga ttt gga atg caa tat gtc aac tac acc 1728 Gly Ser Gly Leu Glu Gln Arg Phe Gly Met Gln Tyr Val Asn Tyr Thr 545 550 555 gat ccg gat ctc cca cga acc ttc aag ctt tct ttc ctg gca tat cgt 1776 Asp Pro Asp Leu Pro Arg Thr Phe Lys Leu Ser Phe Leu Ala Tyr Arg 560 565 570 gat ttc atc aaa aac cac aaa aag tga 1803 Asp Phe Ile Lys Asn His Lys Lys 575 580 <210> 14 <211> 600 <212> PRT <213> Rhodotorula minuta <400> 14 Met Val Ile Leu Arg His Thr Val Leu Ala Ala Ala Val Val Gln Ile -15 -10 -5 Ala Leu Gly Ala Pro Gln Phe Ser Pro Thr Val Asn Thr Glu Gly Ile -1 1 5 10 Ile Pro Ile Ser Glu Arg Pro Ser Pro Thr Gln Ser Ser Ser Ser Ser 15 20 25 Gly Pro Val Ile Pro Ala Gly Ser Tyr Val Ser Asp Phe Asp Ala Ser 30 35 40 45 Gly Leu Ala Asn Leu Trp Ser Gln Val Glu Val Asp Ile Pro Val Glu 50 55 60 Ser Pro Arg Ile Ser Ala Val Pro Pro Leu Asn Lys Thr Phe Ser Val 65 70 75 Pro Lys Thr Pro Val Leu Pro Asn Ser Leu Gln Asp His Leu Pro Lys 80 85 90 Asp Val Lys Ala Pro Glu Gly Phe Ala Trp Gly Val Ala Ser Val Ala 95 100 105 Gln Gln Tyr Glu Gly Ala Val Lys Ala Asp Gly Arg Gly Pro Ser His 110 115 120 125 Trp Asp Phe Leu Cys His Arg Asn Pro Ser Ser Cys Thr Asn Tyr Thr 130 135 140 Ser Asp Ile Thr Asp Leu Gly Arg Tyr Tyr Tyr Lys Asn Asp Leu Ala 145 150 155 Arg Met Ala Ala Met Gly Ile Thr His Tyr Ser Phe Ser Val Ser Trp 160 165 170 Thr Arg Val Val Pro Phe Gly Lys Lys Gly Ser Pro Val Ser Asn Glu 175 180 185 Gly Leu Asp Tyr Tyr Glu Asp Ile Cys Lys Thr Ala Leu Ser Phe Gly 190 195 200 205 Ile Lys Pro Val Ile Thr Leu Phe His Trp Asp Thr Pro Ala Asn Leu 210 215 220 Leu Phe Glu Tyr Gly Gly Phe Leu Asn Gly Thr Ile Val Asp Asp Tyr 225 230 235 Tyr Tyr Tyr Ala Asp Ile Val Phe Arg Arg Leu Gly Lys Tyr Ala Glu 240 245 250 Thr Phe Phe Thr Phe Asn Glu Pro Arg Val Tyr Cys Ser Glu Tyr Thr 255 260 265 Gly Pro Pro Phe Asp Ala Tyr Tyr Glu Arg Tyr Gly Leu Asn Ser Ser 270 275 280 285 Thr Ala Pro Tyr Pro Cys Ser Tyr Asn Leu Leu Arg Ala His Gly Ala 290 295 300 Ala Val Gly Arg Tyr Arg Ala Leu Val Lys Glu Gly Ser Ile Lys Ser 305 310 315 Gly Glu Ile Ala Phe Lys Asn Asp Asp Ser Tyr Gln Leu Pro Gln Asn 320 325 330 Pro Asp Ser Asp Ala Asp Lys Arg Ala Ala Lys Arg His Phe Asp Phe 335 340 345 Tyr Ile Gly Ile Phe Ser Gln Pro Val Tyr Gly Asn Gly Tyr Tyr Pro 350 355 360 365 Glu Thr Val Arg Asn Thr Ile Ser Glu Arg Phe Leu Pro Glu Phe Thr 370 375 380 Ala Ala Glu Arg Glu Gln Ile Gln Gly Ser Ala Asp Phe Tyr Ala Ile 385 390 395 Asp Gly Tyr Arg Thr Asn Ile Ala Ser Ala Ala Pro Asn Gly Ile Asp 400 405 410 Ala Cys Leu Arg Asn Ala Ser Asp Pro Asn Trp Pro Val Cys Gln Asp 415 420 425 Asn Ser Asn Thr Gly Gln Tyr Ala Thr Leu Glu Gly Phe Ala Leu Gly 430 435 440 445 Pro Pro Ala Asp Pro Asn Ala Asn Trp Leu Tyr Asn Thr Ala Pro Tyr 450 455 460 Leu Arg Tyr Gln Phe Lys Val Leu Lys Glu Asn Phe Asn Tyr Lys Lys 465 470 475 Ile Tyr Leu Thr Glu Phe Gly Phe Ala Glu Pro Phe Ser Tyr Leu Arg 480 485 490 Gln Asp Leu Tyr Ala Leu Leu Tyr Asp Thr Asp Arg Thr Ala Tyr Tyr 495 500 505 Gln Asp Tyr Leu Ala Gln Cys Met Leu Ala Ile Lys Glu Asp Gly Ile 510 515 520 525 Pro Leu Ala Gly Val Phe Ala Trp Ser Phe Val Asp Asn Phe Glu Trp 530 535 540 Gly Ser Gly Leu Glu Gln Arg Phe Gly Met Gln Tyr Val Asn Tyr Thr 545 550 555 Asp Pro Asp Leu Pro Arg Thr Phe Lys Leu Ser Phe Leu Ala Tyr Arg 560 565 570 Asp Phe Ile Lys Asn His Lys Lys 575 580 <210> 15 <211> 1809 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(1809) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(1806) <400> 15 atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct gct ccg cag ttt agc ccc aca gtc aat acg gaa 96 Ala Pro Ala Leu Ala Ala Pro Gln Phe Ser Pro Thr Val Asn Thr Glu -5 -1 1 5 10 ggt atc att cca att tcg gag cgc cct tcg cct aca caa tct tcg tcg 144 Gly Ile Ile Pro Ile Ser Glu Arg Pro Ser Pro Thr Gln Ser Ser Ser 15 20 25 tcc agc gga ccc gtg att cca gct ggc tca tat gtc tcc gac ttt gat 192 Ser Ser Gly Pro Val Ile Pro Ala Gly Ser Tyr Val Ser Asp Phe Asp 30 35 40 gcc agc gga ctt gca aac tta tgg agc caa gtc gaa gta gac ata cct 240 Ala Ser Gly Leu Ala Asn Leu Trp Ser Gln Val Glu Val Asp Ile Pro 45 50 55 gtc gag tct cca grudge to tct gcc gta cct cct ctc aac aaa acg ttc 288 Val Glu Ser Pro Arg Ile Ser Ala Val Pro Pro Leu Asn Lys Thr Phe 60 65 70 75 agt gtt ccg aaa act cca gtc ctg ccc aac tcc ctg cag gat cac ctt 336 Ser Val Pro Lys Thr Pro Val Leu Pro Asn Ser Leu Gln Asp His Leu 80 85 90 cca aaa gat gtc aaa gca ccc gaa ggc ttt gca tgg ggc gtg gcc tcc 384 Pro Lys Asp Val Lys Ala Pro Glu Gly Phe Ala Trp Gly Val Ala Ser 95 100 105 gtt gct cag cag tac gag ggt gcc gtc aaa gca gat gga cga gga cca 432 Val Ala Gln Gln Tyr Glu Gly Ala Val Lys Ala Asp Gly Arg Gly Pro 110 115 120 tct cat tgg gat ttc ctt tgc cat aga aac cct tcc agc tgc aca aac 480 Ser His Trp Asp Phe Leu Cys His Arg Asn Pro Ser Ser Cys Thr Asn 125 130 135 tac acc agt gat atc act gat ctt ggc cgt tac tac tat aag aat gac 528 Tyr Thr Ser Asp Ile Thr Asp Leu Gly Arg Tyr Tyr Tyr Lys Asn Asp 140 145 150 155 ttg gca cga atg gct gcc atg ggt ata act cac tac tca ttt tca gtg 576 Leu Ala Arg Met Ala Ala Met Gly Ile Thr His Tyr Ser Phe Ser Val 160 165 170 agc tgg acc aga gtt gtg cca ttc ggc aag aag ggc agt cca gtc agc 624 Ser Trp Thr Arg Val Val Pro Phe Gly Lys Gly Ser Pro Val Ser 175 180 185 aac gaa ggc ctc gac tat tac gaa gat atc tgc aag acg gct ttg agc 672 Asn Glu Gly Leu Asp Tyr Tyr Glu Asp With Cys Lys Thr Ala Leu Ser 190 195 200 ttt gga atc ​​aag cct gtc att act tta ttc cac tgg gat act cct gcc 720 Phe Gly Ile Lys Pro Val Ile Thr Leu Phe His Trp Asp Thr Pro Ala 205 210 215 aac tta ctc ttc gaa tat gga ggt ttc ctc aac ggg aca atc ​​gta gat 768 Asn Leu Leu Phe Glu Tyr Gly Gly Phe Leu Asn Gly Thr Ile Val Asp 220 225 230 235 gac tac tac tat gca gat ata gta ttc aga aga cta ggc aaa tat Asp Tyr Tyr Tyr Tyr Ala Asp Ile Val Phe Arg Arg Leu Gly Lys Tyr 240 245 250 gcc gaa acc ttc ttc acc ttc aat gag cct cgt gta tac tgc agc gag 864 Ala Glu Thr Phe Phe Thr Phe Asn Glu Pro Arg Val Tyr Cys Ser Glu 255 260 265 tat act ggt cct ccg ttc gat gcc tat tac gaa cgc tat ggg ctg aat 912 Tyr Thr Gly Pro Pro Phe Asp Ala Tyr Tyr Glu Arg Tyr Gly Leu Asn 270 275 280 tct agc act gca cca tat ccc tgc tcg tac aat ctc ctc cga gcc cat 960 Ser Ser Thr Ala Pro Tyr Pro Cys Ser Tyr Asn Leu Leu Arg Ala His 285,290,295 ggt gcc gc gtc ggc agg tat cgt gcc ctc gtc aaa gaa ggc agc atc 1008 Gly Ala Ala Val Gly Arg Tyr Arg Ala Leu Val Lys Glu Gly Ser Ile 300 305 310 315 aaa tct ggt gaa atc ​​gca ttc aaa aac gac gat agt tac cag cta cct 1056 Lys Ser Gly Glu Ile Ala Phe Lys Asn Asp Asp Ser Tyr Gln Leu Pro 320 325 330 caa aat cca gac tct gac gcc gac aag cga gct gca aaa cgc cac ttc 1104 Gln Asn Pro Asp Ser Asp Ala Asp Lys Arg Ala Ala Lys Arg His Phe 335 340 345 gac ttc tac atc ggc ata ttc tcg caa ccc gta tac ggc aac ggc tac 1152 Asp Phe Tyr Ile Gly Ile Phe Ser Gln Pro Val Tyr Gly Asn Gly Tyr 350 355 360 tat ccc gaa acc gtc cgg aac aca att tca gag cgc ttc ctc cca gag 1200 Tyr Pro Glu Thr Val Arg Asn Thr Ile Ser Glu Arg Phe Leu Pro Glu 365 370 375 ttc acc gc gcc gag cgc gaa caa att cag gga tca gcg gac ttc tac 1248 Phe Thr Ala Glu Arg Glu Gln Ile Gln Gly Ser Ala Asp Phe Tyr 380 385 390 395 gcc atc gac ggt tat agg acg aat atc gca agc gct gct ccc aat ggt 1296 Only With Asp Gly Tyr Arg Thr Asn With Only Ser Only Only Pro Asn Gly 400 405 410 att gac gcg tgc ttg aga aac gct agt gat ccc aac tgg cca gtt tgc 1344 Ile Asp Ala Cys With Arg Asn Ala Ser Asp Pro Asn Trp Pro Val Cys 415 420 425 cag gac aac agt aac aca ggc caa tac gct act ctt gag gga ttc gca 1392 Gln Asp Asn Ser Asn Thr Gly Gln Tyr Ala Thr Leu Glu Gly Phe Ala 430 435 440 ttg gga cct cct gca gat ccc aac gcg aat tgg cta tac aac act gca 1440 Leu Gly Pro Pro Ala Asp Pro Asn Ala Asn Trp Leu Tyr Asn Thr Ala 445 450 455 cct tac ctg cgt tac caa ttc aaa gtt ctg aaa gag aac ttc aat tac 1488 Pro Tyr Leu Arg Tyr Gln Phe Lys Val Leu Lys Glu Asn Phe Asn Tyr 460 465 470 475 aag aag atc tac ttg acg gaa ttc ggc ttt gca gaa cct ttt agc tat 1536 Lys Lys Ile Tyr Leu Thr Glu Phe Gly Phe Ala Glu Pro Phe Ser Tyr 480 485 490 ttg cga cag gat ctg tat gca ttg ctg tat gac act gat cgc act gca 1584 Two Arg Gln Asp Two Tyr Alas Two Tyr Asp Thr Asp Arg Thr Ala 495,500,505 tat tat caa gac tat cta gcg cag tgc atg ctg gct atc aaa gaa gat 1632 Tyr Tyr Gln Asp Tyr Leu Ala Gln Cys Met Leu Ala Ile Lys Glu Asp 510 515 520 ggc atc cct ctt gct ggt gtt ttc gca tgg tca ttt gtt gat aac ttc 1680 Gly Ile Pro Leu Ala Gly Val Phe Ala Trp Ser Phe Val Asp Asn Phe 525 530 535 gaa tgg ggt tcc ggt ctt gag cag aga ttt gga atg caa tat gtc aac 1728 Glu Trp Gly Ser Gly Leu Glu Gln Arg Phe Gly Met Gln Tyr Val Asn 540 545 550 555 tac acc gat ccg gat ctc cca cga acc ttc aag ctt tct ttc ctg gca 1776 Tyr Thr Asp Pro Asp Leu Pro Arg Thr Phe Lys Leu Ser Phe Leu Ala 560 565 570 tat cgt gat ttc atc aaa aac cac aaa aag tga 1809 Tyr Arg Asp Phe Ile Lys Asn His Lys Lys 575 580 <210> 16 <211> 602 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 16 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ala Pro Gln Phe Ser Pro Thr Val Asn Thr Glu -5 -1 1 5 10 Gly Ile Ile Pro Ile Ser Glu Arg Pro Ser Pro Thr Gln Ser Ser Ser 15 20 25 Ser Ser Gly Pro Val Ile Pro Ala Gly Ser Tyr Val Ser Asp Phe Asp 30 35 40 Ala Ser Gly Leu Ala Asn Leu Trp Ser Gln Val Glu Val Asp Ile Pro 45 50 55 Val Glu Ser Pro Arg Ile Ser Ala Val Pro Pro Leu Asn Lys Thr Phe 60 65 70 75 Ser Val Pro Lys Thr Pro Val Leu Pro Asn Ser Leu Gln Asp His Leu 80 85 90 Pro Lys Asp Val Lys Ala Pro Glu Gly Phe Ala Trp Gly Val Ala Ser 95 100 105 Val Ala Gln Gln Tyr Glu Gly Ala Val Lys Ala Asp Gly Arg Gly Pro 110 115 120 Ser His Trp Asp Phe Leu Cys His Arg Asn Pro Ser Ser Cys Thr Asn 125 130 135 Tyr Thr Ser Asp Ile Thr Asp Leu Gly Arg Tyr Tyr Tyr Lys Asn Asp 140 145 150 155 Leu Ala Arg Met Ala Ala Met Gly Ile Thr His Tyr Ser Phe Ser Val 160 165 170 Ser Trp Thr Arg Val Val Pro Phe Gly Lys Lys Gly Ser Pro Val Ser 175 180 185 Asn Glu Gly Leu Asp Tyr Tyr Glu Asp Ile Cys Lys Thr Ala Leu Ser 190 195 200 Phe Gly Ile Lys Pro Val Ile Thr Leu Phe His Trp Asp Thr Pro Ala 205 210 215 Asn Leu Leu Phe Glu Tyr Gly Gly Phe Leu Asn Gly Thr Ile Val Asp 220 225 230 235 Asp Tyr Tyr Tyr Tyr Ala Asp Ile Val Phe Arg Arg Leu Gly Lys Tyr 240 245 250 Ala Glu Thr Phe Phe Thr Phe Asn Glu Pro Arg Val Tyr Cys Ser Glu 255 260 265 Tyr Thr Gly Pro Pro Phe Asp Ala Tyr Tyr Glu Arg Tyr Gly Leu Asn 270 275 280 Ser Ser Thr Ala Pro Tyr Pro Cys Ser Tyr Asn Leu Leu Arg Ala His 285 290 295 Gly Ala Ala Val Gly Arg Tyr Arg Ala Leu Val Lys Glu Gly Ser Ile 300 305 310 315 Lys Ser Gly Glu Ile Ala Phe Lys Asn Asp Asp Ser Tyr Gln Leu Pro 320 325 330 Gln Asn Pro Asp Ser Asp Ala Asp Lys Arg Ala Ala Lys Arg His Phe 335 340 345 Asp Phe Tyr Ile Gly Ile Phe Ser Gln Pro Val Tyr Gly Asn Gly Tyr 350 355 360 Tyr Pro Glu Thr Val Arg Asn Thr Ile Ser Glu Arg Phe Leu Pro Glu 365 370 375 Phe Thr Ala Ala Glu Arg Glu Gln Ile Gln Gly Ser Ala Asp Phe Tyr 380 385 390 395 Ala Ile Asp Gly Tyr Arg Thr Asn Ile Ala Ser Ala Ala Pro Asn Gly 400 405 410 Ile Asp Ala Cys Leu Arg Asn Ala Ser Asp Pro Asn Trp Pro Val Cys 415 420 425 Gln Asp Asn Ser Asn Thr Gly Gln Tyr Ala Thr Leu Glu Gly Phe Ala 430 435 440 Leu Gly Pro Pro Ala Asp Pro Asn Ala Asn Trp Leu Tyr Asn Thr Ala 445 450 455 Pro Tyr Leu Arg Tyr Gln Phe Lys Val Leu Lys Glu Asn Phe Asn Tyr 460 465 470 475 Lys Lys Ile Tyr Leu Thr Glu Phe Gly Phe Ala Glu Pro Phe Ser Tyr 480 485 490 Leu Arg Gln Asp Leu Tyr Ala Leu Leu Tyr Asp Thr Asp Arg Thr Ala 495 500 505 Tyr Tyr Gln Asp Tyr Leu Ala Gln Cys Met Leu Ala Ile Lys Glu Asp 510 515 520 Gly Ile Pro Leu Ala Gly Val Phe Ala Trp Ser Phe Val Asp Asn Phe 525 530 535 Glu Trp Gly Ser Gly Leu Glu Gln Arg Phe Gly Met Gln Tyr Val Asn 540 545 550 555 Tyr Thr Asp Pro Asp Leu Pro Arg Thr Phe Lys Leu Ser Phe Leu Ala 560 565 570 Tyr Arg Asp Phe Ile Lys Asn His Lys Lys 575 580 <210> 17 <211> 1809 <212> DNA <213> Artificial Sequence <220> <223> β - Galactosidase <220> <221> CDS <222> (1)..(1809) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(1806) <400> 17 atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct gcg cca cag ttc tcg cca aca gtc aac acc gaa 96 Ala Pro Ala Leu Ala Ala Pro Gln Phe Ser Pro Thr Val Asn Thr Glu -5 -1 1 5 10 ggc atc atc ccc atc agc gag cgg cca agc ccg aca cag agc tcc tct 144 Gly Ile Ile Pro Ile Ser Glu Arg Pro Ser Pro Thr Gln Ser Ser Ser 15 20 25 tct tct ggg ccc gtg atc cca gcg ggt tcc tat gtc agc gac ttc gat 192 Ser Ser Gly Pro Val Ile Pro Ala Gly Ser Tyr Val Ser Asp Phe Asp 30 35 40 gcc agc ggg ttg gcg aac ctt tgg tcg cag gtt gaa gtg gac att ccg 240 Ala Ser Gly Leu Ala Asn Leu Trp Ser Gln Val Glu Val Asp Ile Pro 45 50 55 gtt gag tct cca cgc att tcg gcc gtt cct ccg ctg aac aag acc ttc 288 Val Glu Ser Pro Arg Ile Ser Ala Val Pro Pro Leu Asn Lys Thr Phe 60 65 70 75 tcg gtt ccg aag act cca gtt ctc ccc aat tcg ctt caa gac cat ctg 336 Ser Val Pro Lys Thr Pro Val Leu Pro Asn Ser Leu Gln Asp His Leu 80 85 90 ccc aag gat gtg aaa gct cct gag ggc ttt gcc tgg gga gtg gcg tcc 384 Pro Lys Asp Val Lys Ala Pro Glu Gly Phe Ala Trp Gly Val Ala Ser 95 100 105 gtg gca cag caa tac gag ggc gca gtc aaa gca gac gga cga ggc cct 432 Val Ala Gln Gln Tyr Glu Gly Ala Val Lys Ala Asp Gly Arg Gly Pro 110 115 120 tct cac tgg gac ttc ctg tgc cat cgc aac ccc tcc tct tgt acc aac 480 Ser His Trp Asp Phe Leu Cys His Arg Asn Pro Ser Ser Cys Thr Asn 125 130 135 tac acc tct gac atc act gat ctg ggc cgc tac tac tac aag aac gat 528 Tyr Thr Ser Asp Ile Thr Asp Leu Gly Arg Tyr Tyr Tyr Lys Asn Asp 140 145 150 155 ctc gcc cgt atg gcc gca atg ggg atc act cac tac tcc ttt tcg gtg 576 Leu Ala Arg Met Ala Ala Met Gly Ile Thr His Tyr Ser Phe Ser Val 160 165 170 tct tgg acc cga gtt gtc ccc ttc ggc aag aag ggt tcg cct gtt tcc 624 Ser Trp Thr Arg Val Val Pro Phe Gly Lys Lys Gly Ser Pro Val Ser 175 180 185 aac gag gga ttg gac tac tac gag gac atc tgc aag act gcc ctc tct 672 Asn Glu Gly Leu Asp Tyr Tyr Glu Asp Ile Cys Lys Thr Ala Leu Ser 190 195 200 ttc ggc atc aag ccc gtc atc acg ctg ttc cac tgg gat acc ccg gca 720 Phe Gly Ile Lys Pro Val Ile Thr Leu Phe His Trp Asp Thr Pro Ala 205 210 215 aac ctc ctg ttt gag tat gga ggt ttc ctc aat ggc acg atc gtc gat 768 Asn Leu Leu Phe Glu Tyr Gly Gly Phe Leu Asn Gly Thr Ile Val Asp 220 225 230 235 gac tac tac tac tac gcc gac att gtc ttc cgg cgg ttg ggc aag tac 816 Asp Tyr Tyr Tyr Tyr Ala Asp Ile Val Phe Arg Arg Leu Gly Lys Tyr 240 245 250 gcg gag acg ttc ttc acg ttc aac gag cca cgc gtc tac tgc agc gag 864 Ala Glu Thr Phe Phe Thr Phe Asn Glu Pro Arg Val Tyr Cys Ser Glu 255 260 265 tac act ggt cct ccc tc gat gcc tac tac gag cga tac ggc ctc aac 912 Tyr Thr Gly Pro Pro Phe Asp Ala Tyr Tyr Glu Arg Tyr Gly Leu Asn 270 275 280 tcc tcg act gcc cca tac cct tgt tcg tac aac ctt ttg cgc gct cat 960 Ser Ser Thr Ala Pro Tyr Pro Cys Ser Tyr Asn Leu Leu Arg Ala His 285,290,295 ggc gcc gct gtc ggg cga tac cgc gcc ctt gtc aag gag ggc tcc atc 1008 Gly Ala Ala Val Gly Arg Tyr Arg Ala Leu Val Lys Glu Gly Ser Ile 300 305 310 315 aag tcc ggt gag att gcg ttc aag aac gat gac agc tat cag ctt cct 1056 Lys Ser Gly Glu Ile Ala Phe Lys Asn Asp Asp Ser Tyr Gln Leu Pro 320 325 330 cag aat cct gat tcc gat gca gac aaa cgg gca gca aaa cgt cac ttt 1104 Gln Asn Pro Asp Ser Asp Ala Asp Lys Arg Ala Ala Lys Arg His Phe 335 340 345 gac ttc tac atc ggg atc tc tcc caa ccg gtt tac gga aat ggg tac 1152 Asp Phe Tyr Ile Gly Ile Phe Ser Gln Pro Val Tyr Gly Asn Gly Tyr 350 355 360 tac ccg gaaca gtg cgt aac aca atc ​​tcc gaa cgc ttc ctg cct gag 1200 Tyr Pro Glu Thr Val Arg Asn Thr Ile Ser Glu Arg Phe Leu Pro Glu 365 370 375 ttc acc gct gct gaa cgc gag cag att cag ggt agc gcc gac ttc tac 1248 Phe Thr Ala Glu Arg Glu Gln Ile Gln Gly Ser Ala Asp Phe Tyr 380 385 390 395 gcg atc gat gga tat cgg acc aac atc gca tct gca gcc ccg aat ggg 1296 Only With Asp Gly Tyr Arg Thr Asn With Only Ser Only Only Pro Asn Gly 400 405 410 att gac gct tgc ttg cgt aac gct agc gac cct aac tgg gtg tgc 1344 Ile Asp Ala Cys With Arg Asn Ala Ser Asp Pro Asn Trp Pro Val Cys 415 420 425 CA gac aac tcg aac aca ggc cag tat gcc acc ctg gaa ggt ttt gca 1392 Gln Asp Asn Ser Asn Thr Gly Gln Tyr Ala Thr Leu Glu Gly Phe Ala 430 435 440 ttg ggg cct ccc gca gac cca aat gcc aac tgg ctg tac aac acg gcg 1440 Leu Gly Pro Pro Ala Asp Pro Asn Ala Asn Trp Leu Tyr Asn Thr Ala 445 450 455 ccg tat ctc cga tac caa ttc aag gtc ttg aag gag aac ttc aac tac 1488 Pro Tyr Leu Arg Tyr Gln Phe Lys Val Leu Lys Glu Asn Phe Asn Tyr 460 465 470 475 aag aag atc tac ctg acc gaa ttc ggg tt gct gag cct ttc tcc tat 1536 Lys Lys Ile Tyr Leu Thr Glu Phe Gly Phe Ala Glu Pro Phe Ser Tyr 480 485 490 ctg cgc caa gac ctg tat gcg ctt ctc tac gat act gat cga acc gct 1584 Leu Arg Gln Asp Leu Tyr Ala Leu Leu Tyr Asp Thr Asp Arg Thr Ala 495 500 505 tac tac cag gat tac ctg gcg cag tgc atg ctg gcc atc aag gag gac 1632 Tyr Tyr Gln Asp Tyr Leu Ala Gln Cys Met Leu Ala Ile Lys Glu Asp 510 515 520 ggt atc cca ttg gcc ggc gtc ttt gct tgg tcc ttc gtt gac aac ttc 1680 Gly Ile Pro Leu Ala Gly Val Phe Ala Trp Ser Phe Val Asp Asn Phe 525 530 535 gag tgg ggt tcg gga ttg gaa cag cgc ttt ggc atg cag tat gtg aac 1728 Glu Trp Gly Ser Gly Leu Glu Gln Arg Phe Gly Met Gln Tyr Val Asn 540 545 550 555 tac acc gac ccc gac ttg ccc cgc aca ttc aag ctc tcc ttt ctc gcc 1776 Tyr Thr Asp Pro Asp Leu Pro Arg Thr Phe Lys Leu Ser Phe Leu Ala 560 565 570 tat cgg gac ttc atc aag aac cac aag aag taa 1809 Tyr Arg Asp Phe Ile Lys Asn His Lys Lys 575 580 <210> 18 <211> 602 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 18 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ala Pro Gln Phe Ser Pro Thr Val Asn Thr Glu -5 -1 1 5 10 Gly Ile Ile Pro Ile Ser Glu Arg Pro Ser Pro Thr Gln Ser Ser Ser 15 20 25 Ser Ser Gly Pro Val Ile Pro Ala Gly Ser Tyr Val Ser Asp Phe Asp 30 35 40 Ala Ser Gly Leu Ala Asn Leu Trp Ser Gln Val Glu Val Asp Ile Pro 45 50 55 Val Glu Ser Pro Arg Ile Ser Ala Val Pro Pro Leu Asn Lys Thr Phe 60 65 70 75 Ser Val Pro Lys Thr Pro Val Leu Pro Asn Ser Leu Gln Asp His Leu 80 85 90 Pro Lys Asp Val Lys Ala Pro Glu Gly Phe Ala Trp Gly Val Ala Ser 95 100 105 Val Ala Gln Gln Tyr Glu Gly Ala Val Lys Ala Asp Gly Arg Gly Pro 110 115 120 Ser His Trp Asp Phe Leu Cys His Arg Asn Pro Ser Ser Cys Thr Asn 125 130 135 Tyr Thr Ser Asp Ile Thr Asp Leu Gly Arg Tyr Tyr Tyr Lys Asn Asp 140 145 150 155 Leu Ala Arg Met Ala Ala Met Gly Ile Thr His Tyr Ser Phe Ser Val 160 165 170 Ser Trp Thr Arg Val Val Pro Phe Gly Lys Lys Gly Ser Pro Val Ser 175 180 185 Asn Glu Gly Leu Asp Tyr Tyr Glu Asp Ile Cys Lys Thr Ala Leu Ser 190 195 200 Phe Gly Ile Lys Pro Val Ile Thr Leu Phe His Trp Asp Thr Pro Ala 205 210 215 Asn Leu Leu Phe Glu Tyr Gly Gly Phe Leu Asn Gly Thr Ile Val Asp 220 225 230 235 Asp Tyr Tyr Tyr Tyr Ala Asp Ile Val Phe Arg Arg Leu Gly Lys Tyr 240 245 250 Ala Glu Thr Phe Phe Thr Phe Asn Glu Pro Arg Val Tyr Cys Ser Glu 255 260 265 Tyr Thr Gly Pro Pro Phe Asp Ala Tyr Tyr Glu Arg Tyr Gly Leu Asn 270 275 280 Ser Ser Thr Ala Pro Tyr Pro Cys Ser Tyr Asn Leu Leu Arg Ala His 285 290 295 Gly Ala Ala Val Gly Arg Tyr Arg Ala Leu Val Lys Glu Gly Ser Ile 300 305 310 315 Lys Ser Gly Glu Ile Ala Phe Lys Asn Asp Asp Ser Tyr Gln Leu Pro 320 325 330 Gln Asn Pro Asp Ser Asp Ala Asp Lys Arg Ala Ala Lys Arg His Phe 335 340 345 Asp Phe Tyr Ile Gly Ile Phe Ser Gln Pro Val Tyr Gly Asn Gly Tyr 350 355 360 Tyr Pro Glu Thr Val Arg Asn Thr Ile Ser Glu Arg Phe Leu Pro Glu 365 370 375 Phe Thr Ala Ala Glu Arg Glu Gln Ile Gln Gly Ser Ala Asp Phe Tyr 380 385 390 395 Ala Ile Asp Gly Tyr Arg Thr Asn Ile Ala Ser Ala Ala Pro Asn Gly 400 405 410 Ile Asp Ala Cys Leu Arg Asn Ala Ser Asp Pro Asn Trp Pro Val Cys 415 420 425 Gln Asp Asn Ser Asn Thr Gly Gln Tyr Ala Thr Leu Glu Gly Phe Ala 430 435 440 Leu Gly Pro Pro Ala Asp Pro Asn Ala Asn Trp Leu Tyr Asn Thr Ala 445 450 455 Pro Tyr Leu Arg Tyr Gln Phe Lys Val Leu Lys Glu Asn Phe Asn Tyr 460 465 470 475 Lys Lys Ile Tyr Leu Thr Glu Phe Gly Phe Ala Glu Pro Phe Ser Tyr 480 485 490 Leu Arg Gln Asp Leu Tyr Ala Leu Leu Tyr Asp Thr Asp Arg Thr Ala 495 500 505 Tyr Tyr Gln Asp Tyr Leu Ala Gln Cys Met Leu Ala Ile Lys Glu Asp 510 515 520 Gly Ile Pro Leu Ala Gly Val Phe Ala Trp Ser Phe Val Asp Asn Phe 525 530 535 Glu Trp Gly Ser Gly Leu Glu Gln Arg Phe Gly Met Gln Tyr Val Asn 540 545 550 555 Tyr Thr Asp Pro Asp Leu Pro Arg Thr Phe Lys Leu Ser Phe Leu Ala 560 565 570 Tyr Arg Asp Phe Ile Lys Asn His Lys Lys 575 580 <210> 19 <211> 1785 <212> DNA <213> Sterigmatomyces elviae <220> <221> CDS <222> (1)..(1785) <220> <221> Signal peptide <222> (1)..(57) <220> <221> Mature peptide <222> (58)..(1782) <400> 19 atg ctt gtc gga ctt gct ttg act gct ctg tta ggt gcc act cgt tat 48 Met Leu Val Gly Leu Ala Leu Thr Ala Leu Leu Gly Ala Thr Arg Tyr -15 -10 -5 gtt ggc gca atc cct gct ttc cca atc act cca gat ttg gct ggt ggg 96 Val Gly Ala Ile Pro Ala Phe Pro Ile Thr Pro Asp Leu Ala Gly Gly -1 1 5 10 ctg gag tct gtg acc aac act cag acc tcg ctc cct tca gcg agc gct 144 Leu Glu Ser Val Thr Asn Thr Gln Thr Ser Leu Pro Ser Ala Ser Ala 15 20 25 gtg tcg tcg ccc tat aat caa gat gca ctc gac aag ctg tgg gct gag 192 Val Ser Ser Pro Tyr Asn Gln Asp To Leu Asp Lys Leu Trp To Glu 30 35 40 45 gtc gaa aaa gac att cca gtc gag aca cca agc atc tcc agc gtt gtt 240 Val Glu Lys Asp Ile Pro Val Glu Thr Pro Ser Ile Ser Ser Val Val 50 55 60 cca gta aac aac agc ttt gcg gtc ccc aaa acc act act ctg ccc cga 288 Pro Val Asn Asn Ser Phe Ala Val Pro Lys Thr Pro Thr Leu Pro Arg 65 70 75 tct ctt cag gat cat gct acc agt ggc cgc aaa ttc ccc aaa ggc ttc 336 Ser Leu Gln Asp His Ala Thr Ser Gly Arg Lys Phe Pro Lys Gly Phe 80 85 90 aag ttt ggt gtc gcc acc gcc gat cag cag tat gaa ggt gcc gtc aag 384 Lys Phe Gly Val Ala Thr Ala Asp Gln Gln Tyr Glu Gly Ala Val Lys 95 100 105 gct gat ggc cgt ggc ccc tct cac tgg gat tac ctt tgc cat cgt ctc 432 Ala Asp Gly Arg Gly Pro Ser His Trp Asp Tyr Leu Cys His Arg Leu 110 115 120 125 cca cag caa tgc aac aac tac acc tca gac atc act gac ctt ggt cgc 480 Pro Gln Gln Cys Asn Asn Tyr Thr Ser Asp Ile Thr Asp Leu Gly Arg 130 135 140 tac tat tat aag caa gat atc gca cga atc aag gcc atg gga gta aac 528 Tyr Tyr Tyr Lys Gln Asp Ile Ala Arg Ile Lys Ala Met Gly Val Asn 145 150 155 act gta tca ctc acc ctt tct tgg tca cgt atc aag ccc ttc ggc acg 576 Thr Val Ser Leu Thr Leu Ser Trp Ser Arg Ile Lys Pro Phe Gly Thr 160 165 170 gcc gat agt cct gtc agc aaa gaa gga ctc caa ttt tac gat gac ttt 624 Ala Asp Ser Pro Val Ser Lys Glu Gly Leu Gln Phe Tyr Asp Asp Phe 175 180 185 atc aac gag ctc atc gat aat ggc atc gaa cca gtc gtc acc ctg ttc 672 Ile Asn Glu Leu Ile Asp Asn Gly Ile Glu Pro Val Val Thr Leu Phe 190 195 200 205 cat tgg agt aca cca ctc aat ctg gtg ttc gaa tac ggg gcc ttc ctt 720 His Trp Ser Thr Pro Leu Asn Leu Val Phe Glu Tyr Gly Ala Phe Leu 210 215 220 aat ggc agc tcg gtt gaa gat ttc gct agc tat gct aag ctt gtt ttt 768 Asn Gly Ser Ser Val Glu Asp Phe Ala Ser Tyr Ala Lys Leu Val Phe 225 230 235 gag cat ttc ggt gac aga gta acc aca ttc ctt act ttc aac gag cct 816 Glu His Phe Gly Asp Arg Val Thr Thr Phe Leu Thr Phe Asn Glu Pro 240 245 250 cgt gta tac tgc tcc gaa tac act ggc gag cct ttt aat gat tat tgg 864 Arg Val Tyr Cys Ser Glu Tyr Thr Gly Glu Pro Phe Asn Asp Tyr Trp 255 260 265 cac ttt gga gtt ccc aac atc aat gcc acg acc gct ccc tat cct tgc 912 His Phe Gly Gly Pro Asn Ile Asn Ala Thr Thr Ala Pro Tyr Pro Cys 270 275 280 285 acc tat aac att tta aaa gca cac gga cgt gcc gtt caa gaa tac aga 960 Thr Tyr Asn With Lys Alpha His Gly Arg Allocation Val Gln Glu Tyr Arg 290,295,300 gca ctg gtc aac agt gga aag atc aag aaa ggt gaa gtc gca att aaa 1008 Only Leu Will Asn Be Gly Lys Ile Lys Gly Glu Only Only Ile Lys 305 310 315 aac gac gat agt tat ccc gtg cca gtc aac cca gac tcc gaa gcc gat 1056 Asn Asp Asp Ser Tyr Pro Val Pro Val Asn Pro Asp Ser Glu Ala Asp 320 325 330 gta gaa gcg gcc aag cga cac ttc gat ttc tac att ggc att ttc agt 1104 Val Glu Ala Ala Lys Arg His Phe Asp Phe Tyr Ile Gly Ile Phe Ser 335 340 345 cag cct att tac gtt gat gga aag ttc cca gac acc gtt aga aac acc 1152 Gln Pro Ile Tyr Gly Asp Gly Lys Phe Pro Asp Thr Val Arg Asn Thr 350 355 360 365 atc tcc act gaa ttc ctg cca tac ctc acc gat gat gag aaa gcc atg 1200 Served Thr Glu Phe Leu Pro Tyr Leu Thr Asp Asp Glu Lys Ala Met 370 375 380 att aaa gga agt ggt gac tttc gcc ata gac gca tat cga acc aac 1248 Ile Lys Gly Ser Gly Asp Phe Phe Ala Ile Asp Ala Tyr Arg Thr Asn 385,390,395 ctt gca aga gct gcg ccc aat gcc atc caa gcc tgc gta gct aac atc 1296 Leu Wing Arg Wing Pro Asn Gly Ile Gln Wing Cys Val Wing Asn Ile 400 405 410 tcg gat ccc aat tgg cct gta tgc caa gac ac agc cct gaa gga CA 1344 Ser Asp Pro Asn Trp Pro Val Cys Gln Asp Asn Ser Pro Glu Gly Gln 415 420 425 tac caa acc atg gat ggc ttt gct ttc ggt ccc ccg gca gac ccc aac 1392 Tyr Gln Thr Met Asp Gly Phe Ala Phe Gly Pro Pro Ala Asp Pro Asn 430 435 440 445 gct gca tgg cta tat gac acc agc ttc aag ttg cgc tac cag ctt aag 1440 Ala Ala Trp and Tyr Asp Thr Ser Phe Lys and Arg Tyr Gln and Lys 450 455 460 aca ctc aaa gag gca ttc aac tat gac aag atc tac atc tca gag ttt 1488 Thr Leu Lys Glu Ala Phe Asn Tyr Asp Lys Ile Tyr Ile Ser Glu Phe 465 470 475 gga ttt gct cgg cct tac gaa tac ctc tac cct tac ggc ttc gac gtc 1536 Gly Phe Ala Arg Pro Tyr Glu Tyr Leu Tyr Pro Tyr Gly Phe Asp Val 480 485 490 ctg tac gac aca gac cgt gcc att tac caa gac tac atg gct gag 1584 Leu Tyr Asp Thr Asp Arg Ala Ile Tyr Gln Asp Tyr Met Ala Glu 495,500,505 gcc ttg gat gcc att cat gac gac ggc att cct ctg gct ggt gtc ttt 1632 Ala Leu Asp Ala Ile His Asp Asp Gly Ile Pro Leu Ala Gly Val Phe 510 515 520 525 gct tgg tcc ttc gtt gac aat ttc gaa tgg gct tcc gtt ctt gaa cag 1680 Ala Trp Ser Phe Val Asp Asn Phe Glu Trp Ala Ser Gly Leu Glu Gln 530 535 540 cga ttc ggc atg cag ttc gtg aac tac acg aca ctg gaa aga gag tac 1728 Arg Phe Gly Met Gln Phe Val Asn Tyr Thr Leu Glu Arg Glu Tyr 545 550 555 aag ctc tcc ttc ctg ctt tat cgt gac ttc att gaa aac cac agt tgc 1776 Lys Leu Ser Phe Leu Leu Tyr Arg Asp Phe Ile Glu Asn His Ser Cys 560 565 570 your neck 1785 Glu Asp 575 <210> 20 <211> 594 <212> PRT <213> Sterigmatomyces elviae <400> 20 Met Leu Val Gly Leu Ala Leu Thr Ala Leu Leu Gly Ala Thr Arg Tyr -15 -10 -5 Val Gly Ala Ile Pro Ala Phe Pro Ile Thr Pro Asp Leu Ala Gly Gly -1 1 5 10 Leu Glu Ser Val Thr Asn Thr Gln Thr Ser Leu Pro Ser Ala Ser Ala 15 20 25 Val Ser Ser Pro Tyr Asn Gln Asp Ala Leu Asp Lys Leu Trp Ala Glu 30 35 40 45 Val Glu Lys Asp Ile Pro Val Glu Thr Pro Ser Ile Ser Ser Val Val 50 55 60 Pro Val Asn Asn Ser Phe Ala Val Pro Lys Thr Pro Thr Leu Pro Arg 65 70 75 Ser Leu Gln Asp His Ala Thr Ser Gly Arg Lys Phe Pro Lys Gly Phe 80 85 90 Lys Phe Gly Val Ala Thr Ala Asp Gln Gln Tyr Glu Gly Ala Val Lys 95 100 105 Ala Asp Gly Arg Gly Pro Ser His Trp Asp Tyr Leu Cys His Arg Leu 110 115 120 125 Pro Gln Gln Cys Asn Asn Tyr Thr Ser Asp Ile Thr Asp Leu Gly Arg 130 135 140 Tyr Tyr Tyr Lys Gln Asp Ile Ala Arg Ile Lys Ala Met Gly Val Asn 145 150 155 Thr Val Ser Leu Thr Leu Ser Trp Ser Arg Ile Lys Pro Phe Gly Thr 160 165 170 Ala Asp Ser Pro Val Ser Lys Glu Gly Leu Gln Phe Tyr Asp Asp Phe 175 180 185 Ile Asn Glu Leu Ile Asp Asn Gly Ile Glu Pro Val Val Thr Leu Phe 190 195 200 205 His Trp Ser Thr Pro Leu Asn Leu Val Phe Glu Tyr Gly Ala Phe Leu 210 215 220 Asn Gly Ser Ser Val Glu Asp Phe Ala Ser Tyr Ala Lys Leu Val Phe 225 230 235 Glu His Phe Gly Asp Arg Val Thr Thr Phe Leu Thr Phe Asn Glu Pro 240 245 250 Arg Val Tyr Cys Ser Glu Tyr Thr Gly Glu Pro Phe Asn Asp Tyr Trp 255 260 265 His Phe Gly Gly Pro Asn Ile Asn Ala Thr Thr Ala Pro Tyr Pro Cys 270 275 280 285 Thr Tyr Asn Ile Leu Lys Ala His Gly Arg Ala Val Gln Glu Tyr Arg 290 295 300 Ala Leu Val Asn Ser Gly Lys Ile Lys Lys Gly Glu Val Ala Ile Lys 305 310 315 Asn Asp Asp Ser Tyr Pro Val Pro Val Asn Pro Asp Ser Glu Ala Asp 320 325 330 Val Glu Ala Ala Lys Arg His Phe Asp Phe Tyr Ile Gly Ile Phe Ser 335 340 345 Gln Pro Ile Tyr Gly Asp Gly Lys Phe Pro Asp Thr Val Arg Asn Thr 350 355 360 365 Ile Ser Thr Glu Phe Leu Pro Tyr Leu Thr Asp Asp Glu Lys Ala Met 370 375 380 Ile Lys Gly Ser Gly Asp Phe Phe Ala Ile Asp Ala Tyr Arg Thr Asn 385 390 395 Leu Ala Arg Ala Ala Pro Asn Gly Ile Gln Ala Cys Val Ala Asn Ile 400 405 410 Ser Asp Pro Asn Trp Pro Val Cys Gln Asp Asn Ser Pro Glu Gly Gln 415 420 425 Tyr Gln Thr Met Asp Gly Phe Ala Phe Gly Pro Pro Ala Asp Pro Asn 430 435 440 445 Ala Ala Trp Leu Tyr Asp Thr Ser Phe Lys Leu Arg Tyr Gln Leu Lys 450 455 460 Thr Leu Lys Glu Ala Phe Asn Tyr Asp Lys Ile Tyr Ile Ser Glu Phe 465 470 475 Gly Phe Ala Arg Pro Tyr Glu Tyr Leu Tyr Pro Tyr Gly Phe Asp Val 480 485 490 Leu Tyr Asp Thr Asp Arg Ala Ile Tyr Tyr Gln Asp Tyr Met Ala Glu 495 500 505 Ala Leu Asp Ala Ile His Asp Asp Gly Ile Pro Leu Ala Gly Val Phe 510 515 520 525 Ala Trp Ser Phe Val Asp Asn Phe Glu Trp Ala Ser Gly Leu Glu Gln 530 535 540 Arg Phe Gly Met Gln Phe Val Asn Tyr Thr Thr Leu Glu Arg Glu Tyr 545 550 555 Lys Leu Ser Phe Leu Leu Tyr Arg Asp Phe Ile Glu Asn His Ser Cys 560 565 570 Glu Asp 575 <210> twenty one <211> 1791 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(1791) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(1788) <400> twenty one atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct atc cct gct ttc cca atc ​​act cca gat ttg gct 96 Ala Pro Ala Leu Ala Ile Pro Ala Phe Pro Ile Thr Pro Asp Leu Ala -5 -1 1 5 10 ggt ggg ctg gag tct gtg acc aac act cag acc tcg ctc cct tca gcg 144 Gly Gly Leu Glu Ser Val Thr Asn Thr Gln Thr Ser Leu Pro Ser Ala 15 20 25 agc gct gtg tcg tcg ccc tat aat caa gat gca ctc gac aag ctg tgg 192 Ser Ala Val Ser Ser Pro Tyr Asn Gln Asp Ala Leu Asp Lys Leu Trp 30 35 40 gct gag gtc gaaaaa gac att cca gtc gag aca cca agc atc tcc agc 240 Ala Glu Val Glu Lys Asp Ile Pro Val Glu Thr Pro Ser Ile Ser Ser 45 50 55 gtt gtt cca gta aac aac agc ttt gcg gtc ccc aaa acc cct act ctg 288 Val Val Pro Val Asn Asn Ser Phe Ala Val Pro Thr Pro Thr Leu 60 65 70 75 ccc cga tct ctt cag gat cat gct acc agt ggc cgc aaa ttc ccc aaa 336 Pro Arg Ser Leu Gln Asp His Ala Thr Ser Gly Arg Lys Phe Pro Lys 80 85 90 ggc ttc aag ttt ggt gtc gcc acc gcc gat cag cag tat gaa ggt gcc 384 Gly Phe Lys Phe Gly Val Ala Thr Ala Asp Gln Gln Tyr Glu Gly Ala 95 100 105 gtc aag gct gat ggc cgt ggc ccc tct cac tgg gat tac ctt tgc cat 432 Val Lys Ala Asp Gly Arg Gly Pro Ser His Trp Asp Tyr Leu Cys His 110 115 120 cgt ctc cca cag caa tgc aac aac tac acc tca gac atc act gac ctt 480 Arg Leu Pro Gln Gln Cys Asn Asn Tyr Thr Ser Asp Ile Thr Asp Leu 125 130 135 ggt cgc tac tat tat aag caa gat atc gca cga atc aag gcc atg gga 528 Gly Arg Tyr Tyr Tyr Lys Gln Asp Ile Ala Arg Ile Lys Ala Met Gly 140 145 150 155 gta aac act gta tca ctc acc ctt tct tgg tca cgt atc aag ccc ttc 576 Val Asn Thr Val Ser Leu Thr Leu Ser Trp Ser Arg Ile Lys Pro Phe 160 165 170 ggc acg gcc gat agt cct gtc agc aaa gaa gga ctc caa ttt tac gat 624 Gly Thr Ala Asp Ser Pro Val Ser Lys Glu Gly Leu Gln Phe Tyr Asp 175 180 185 gac ttt atc aac gag ctc atc gat aat ggc atc gaa cca gtc gtc acc 672 Asp Phe Ile Asn Glu Leu Ile Asp Asn Gly Ile Glu Pro Val Val Thr 190 195 200 ctg ttc cat tgg agt aca cca ctc aat ctg gtg ttc gaa tac ggg gcc 720 Leu Phe His Trp Ser Thr Pro Leu Asn Leu Val Phe Glu Tyr Gly Ala 205 210 215 ttc ctt aat ggc agc tcg gtt gaa gat ttc gct agc tat gct aag ctt 768 Phe Leu Asn Gly Ser Ser Val Glu Asp Phe Ala Ser Tyr Ala Lys Leu 220 225 230 235 gtt ttt gag cat ttc ggt gac aga gta acc aca ttc ctt act ttc aac 816 Val Phe Glu His Phe Gly Asp Arg Val Thr Thr Phe Leu Thr Phe Asn 240 245 250 gag cct cgt gta tac tgc tcc gaa tac act ggc gag cct ttt aat gat 864 Glu Pro Arg Val Tyr Cys Ser Glu Tyr Thr Gly Glu Pro Phe Asn Asp 255 260 265 tat tgg cac ttt gga ggt ccc aac atc aat gcc acg acc gct ccc tat 912 Tyr Trp His Phe Gly Gly Pro Asn Ile Asn Ala Thr Thr Ala Pro Tyr 270 275 280 cct tgc acc tat aac att tta aaa gca cac gga cgt gcc gtt caa gaa 960 Pro Cys Thr Tyr Asn Ile Lys Ala His Gly Arg Ala Val Gln Glu 285,290,295 tac aga gca ctg gtc aac agt gga aag atc aag aaa ggt gaa gtc gca 1008 Tyr Arg Ala Leu Val Asn Ser Gly Lys Ile Lys Gly Glu Val Ala 300 305 310 315 att aaa aac gac gat agt tat ccc gtg cca gtc aac cca gac tcc gaa 1056 Ile Lys Asn Asp Asp Ser Tyr Pro Val Pro Val Asn Pro Asp Ser Glu 320 325 330 gcc gat gta gaa gcg gcc aag cga cac ttc gat ttc tac att ggc att 1104 Ala Asp Val Glu Ala Ala Lys Arg His Phe Asp Phe Tyr Ile Gly Ile 335 340 345 ttc agt cag cct att tac ggt gat gga aag ttc cca gac acc gtt aga 1152 Phe Ser Gln Pro Ile Tyr Gly Asp Gly Lys Phe Pro Asp Thr Val Arg 350 355 360 aac acc atc tcc act gaa ttc ctg cca tac ctc acc gat gat gag aaa 1200 Asn Thr Ile Thr Glu Phe Leu Pro Tyr Leu Thr Asp Asp Glu Lys 365 370 375 gcc atg att aaa gga agt ggt gac ttt ttc gcc ata gac gca tat cga 1248 Ala Met Ile Lys Gly Ser Gly Asp Phe Phe Ala Ile Asp Ala Tyr Arg 380 385 390 395 acc aac ctt gca aga gct gcg ccc aat ggc atc caa gcc tgc gta gct 1296 Thr Asn Leu Ala Arg Ala Ala Pro Asn Gly Ile Gln Ala Cys Val Ala 400 405 410 aac atc tcg gat ccc aat tgg cct gta tgc caa gac aac agc cct gaa 1344 Asn Ile Ser Asp Pro Asn Trp Pro Val Cys Gln Asp Asn Ser Pro Glu 415 420 425 gga caa tac caa acc atg gat ggc ttt gct ttc ggt ccc ccg gca gac 1392 Gly Gln Tyr Gln Thr Met Asp Gly Phe Ala Phe Gly Pro Pro Ala Asp 430 435 440 ccc aac gct gca tgg cta tat gac acc agc ttc aag ttg cgc tac cag 1440 Pro Asn Ala Ala Trp Leu Tyr Asp Thr Ser Phe Lys Leu Arg Tyr Gln 445 450 455 ctt aag aca ctc aaa gag gca ttc aac tat gac aag atc tac atc tca 1488 Leu Lys Thr Leu Lys Glu Ala Phe Asn Tyr Asp Lys Ile Tyr Ile Ser 460 465 470 475 gag ttt gga ttt gct cgg cct tac gaa tac ctc tac cct tac ggc ttc 1536 Glu Phe Gly Phe Ala Arg Pro Tyr Glu Tyr Leu Tyr Pro Tyr Gly Phe 480 485 490 gac gtc cctg tac gac aca gac cgt gcc att tac tac caa gac tac atg 1584 Asp Val Leu Tyr Asp Thr Asp Arg Ala Ile Tyr Gln Tyr Met 495,500,505 gct gag gcc ttg gat gcc att cat gac gac ggc att cct ctg gct ggt 1632 Ala Glu Ala Leu Asp Ala Ile His Asp Asp Gly Ile Pro Leu Ala Gly 510,515,520 gtc ttt gct tgg tcc ttc gtt gac aat ttc gaa tgg gct tcc gtt ctt 1680 Val Phe Ala Trp Ser Phe Val Asp Asn Phe Glu Trp Ala Ser Gly Leu 525 530 535 gaa cag cga ttc ggc atg cag ttc gtg aac tac acg aca ctg gaa aga 1728 Glu Gln Arg Phe Gly Met Gln Phe Val With Tyr Thr Thr Leu Glu Arg 540 545 550 555 gag tac aag ctc tcc ttc ctg ctt tat cgt gac ttc att gaa aac cac 1776 Glu Tyr Lys Leu Ser Phe Leu Leu Tyr Arg Asp Phe Ile Glu Asn His 560 565 570 agt tgc gaa gat taa 1791 Ser Cys Glu Asp 575 <210> 22 <211> 596 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 22 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ile Pro Ala Phe Pro Ile Thr Pro Asp Leu Ala -5 -1 1 5 10 Gly Gly Leu Glu Ser Val Thr Asn Thr Gln Thr Ser Leu Pro Ser Ala 15 20 25 Ser Ala Val Ser Ser Pro Tyr Asn Gln Asp Ala Leu Asp Lys Leu Trp 30 35 40 Ala Glu Val Glu Lys Asp Ile Pro Val Glu Thr Pro Ser Ile Ser Ser 45 50 55 Val Val Pro Val Asn Asn Ser Phe Ala Val Pro Lys Thr Pro Thr Leu 60 65 70 75 Pro Arg Ser Leu Gln Asp His Ala Thr Ser Gly Arg Lys Phe Pro Lys 80 85 90 Gly Phe Lys Phe Gly Val Ala Thr Ala Asp Gln Gln Tyr Glu Gly Ala 95 100 105 Val Lys Ala Asp Gly Arg Gly Pro Ser His Trp Asp Tyr Leu Cys His 110 115 120 Arg Leu Pro Gln Gln Cys Asn Asn Tyr Thr Ser Asp Ile Thr Asp Leu 125 130 135 Gly Arg Tyr Tyr Tyr Lys Gln Asp Ile Ala Arg Ile Lys Ala Met Gly 140 145 150 155 Val Asn Thr Val Ser Leu Thr Leu Ser Trp Ser Arg Ile Lys Pro Phe 160 165 170 Gly Thr Ala Asp Ser Pro Val Ser Lys Glu Gly Leu Gln Phe Tyr Asp 175 180 185 Asp Phe Ile Asn Glu Leu Ile Asp Asn Gly Ile Glu Pro Val Val Thr 190 195 200 Leu Phe His Trp Ser Thr Pro Leu Asn Leu Val Phe Glu Tyr Gly Ala 205 210 215 Phe Leu Asn Gly Ser Ser Val Glu Asp Phe Ala Ser Tyr Ala Lys Leu 220 225 230 235 Val Phe Glu His Phe Gly Asp Arg Val Thr Thr Phe Leu Thr Phe Asn 240 245 250 Glu Pro Arg Val Tyr Cys Ser Glu Tyr Thr Gly Glu Pro Phe Asn Asp 255 260 265 Tyr Trp His Phe Gly Gly Pro Asn Ile Asn Ala Thr Thr Ala Pro Tyr 270 275 280 Pro Cys Thr Tyr Asn Ile Leu Lys Ala His Gly Arg Ala Val Gln Glu 285 290 295 Tyr Arg Ala Leu Val Asn Ser Gly Lys Ile Lys Lys Gly Glu Val Ala 300 305 310 315 Ile Lys Asn Asp Asp Ser Tyr Pro Val Pro Val Asn Pro Asp Ser Glu 320 325 330 Ala Asp Val Glu Ala Ala Lys Arg His Phe Asp Phe Tyr Ile Gly Ile 335 340 345 Phe Ser Gln Pro Ile Tyr Gly Asp Gly Lys Phe Pro Asp Thr Val Arg 350 355 360 Asn Thr Ile Ser Thr Glu Phe Leu Pro Tyr Leu Thr Asp Asp Glu Lys 365 370 375 Ala Met Ile Lys Gly Ser Gly Asp Phe Phe Ala Ile Asp Ala Tyr Arg 380 385 390 395 Thr Asn Leu Ala Arg Ala Ala Pro Asn Gly Ile Gln Ala Cys Val Ala 400 405 410 Asn Ile Ser Asp Pro Asn Trp Pro Val Cys Gln Asp Asn Ser Pro Glu 415 420 425 Gly Gln Tyr Gln Thr Met Asp Gly Phe Ala Phe Gly Pro Pro Ala Asp 430 435 440 Pro Asn Ala Ala Trp Leu Tyr Asp Thr Ser Phe Lys Leu Arg Tyr Gln 445 450 455 Leu Lys Thr Leu Lys Glu Ala Phe Asn Tyr Asp Lys Ile Tyr Ile Ser 460 465 470 475 Glu Phe Gly Phe Ala Arg Pro Tyr Glu Tyr Leu Tyr Pro Tyr Gly Phe 480 485 490 Asp Val Leu Tyr Asp Thr Asp Arg Ala Ile Tyr Tyr Gln Asp Tyr Met 495 500 505 Ala Glu Ala Leu Asp Ala Ile His Asp Asp Gly Ile Pro Leu Ala Gly 510 515 520 Val Phe Ala Trp Ser Phe Val Asp Asn Phe Glu Trp Ala Ser Gly Leu 525 530 535 Glu Gln Arg Phe Gly Met Gln Phe Val Asn Tyr Thr Thr Leu Glu Arg 540 545 550 555 Glu Tyr Lys Leu Ser Phe Leu Leu Tyr Arg Asp Phe Ile Glu Asn His 560 565 570 Ser Cys Glu Asp 575 <210> twenty three <211> 1791 <212> DNA <213> Artificial sequence <220> <223> β-Galactosidase <220> <221> CDS <222> (1)..(1791) <220> <221> Signal peptide <222> (1)..(63) <220> <221> Mature peptide <222> (64)..(1788) <400> twenty three atg atg gtc gcg tgg tgg tct cta ttt ctg tac ggc ctt cag gtc gcg 48 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 gca cct gct ttg gct att ccc gct ttc cca atc ​​act ccc gat ttg gcc 96 Ala Pro Ala Leu Ala Ile Pro Ala Phe Pro Ile Thr Pro Asp Leu Ala -5 -1 1 5 10 gga ggc ctg gaa agc gtc aca aac acc caa acg tcc ctc cca tct gct 144 Gly Gly Leu Glu Ser Val Thr Asn Thr Gln Thr Ser Leu Pro Ser Ala 15 20 25 tcc gct gtt tcc agc ccg tac aac caa gac gct ctt gat aag ctg tgg 192 Ser Ala Val Ser Ser Pro Tyr Asn Gln Asp Ala Leu Asp Lys Leu Trp 30 35 40 gca gag gtt gag aag gat atc ccc gtt gaa aca cca tcc atc agc tct 240 Ala Glu Val Glu Lys Asp Ile Pro Val Glu Thr Pro Ser Ile Ser Ser 45 50 55 gtg gtg ccc gtg aac aac tcc ttc gcc gtg cct aag acg ccg acc ctc 288 Val Val Pro Val Asn Asn Ser Phe Ala Val Pro Lys Thr Pro Thr Leu 60 65 70 75 ccg cgc tcc ctg cag gat cat gct act tcg ggg cgg aag ttc cca aaa 336 Pro Arg Ser Leu Gln Asp His Ala Thr Ser Gly Arg Lys Phe Pro Lys 80 85 90 ggg ttc aag ttc gga gtt gcc act gcg gac cag cag tac gaa ggc gcc 384 Gly Phe Lys Phe Gly Val Ala Thr Ala Asp Gln Gln Tyr Glu Gly Ala 95 100 105 gtc aag gcc gac ggc cgc ggg ccg agc cac tgg gat tac ctg tgc cac 432 Val Lys Ala Asp Gly Arg Gly Pro Ser His Trp Asp Tyr Leu Cys His 110 115 120 cgg ctg cct cag caa tgc aac aac tac acc tct gac atc act gac ttg 480 Arg Leu Pro Gln Gln Cys Asn Asn Tyr Thr Ser Asp Ile Thr Asp Leu 125 130 135 gga cgt tac tac tac aag cag gat atc gcc cgg atc aag gcc atg ggc 528 Gly Arg Tyr Tyr Tyr Lys Gln Asp Ile Ala Arg Ile Lys Ala Met Gly 140 145 150 155 gtg aat act gtc tcg ctt acc ctg agc tgg tcg cgc atc aag ccc ttt 576 Val Asn Thr Val Ser Leu Thr Leu Ser Trp Ser Arg Ile Lys Pro Phe 160 165 170 ggc act gct gac tcg cct gtt tcg aag gag ggt ttg cag ttc tac gat 624 Gly Thr Ala Asp Ser Pro Val Ser Lys Glu Gly Leu Gln Phe Tyr Asp 175 180 185 gac ttc atc aac gaa ctc att gac aat gga atc gag cct gtt gtg aca 672 Asp Phe Ile Asn Glu Leu Ile Asp Asn Gly Ile Glu Pro Val Val Thr 190 195 200 ctc ttc cat tgg tct acc cct ctg aat ctg gtg ttt gag tac ggt gcc 720 Leu Phe His Trp Ser Thr Pro Leu Asn Leu Val Phe Glu Tyr Gly Ala 205 210 215 ttt ctc aac ggc agc agc gtg gaa gac ttt gcg tcc tat gca aag ctg 768 Phe Leu Asn Gly Ser Ser Val Glu Asp Phe Ala Ser Tyr Ala Lys Leu 220 225 230 235 gtc ttc gaa cac ttt ggg gac cgc gtg aca acc ttc ctt acc ttc aac 816 Val Phe Glu His Phe Gly Asp Arg Val Thr Thr Phe Leu Thr Phe Asn 240 245 250 gag ccg cgt gtg tac tgc agc gag tat acg ggc gaa ccc ttc aac gac 864 Glu Pro Arg Val Tyr Cys Ser Glu Tyr Thr Gly Glu Pro Phe Asn Asp 255 260 265 tac tgg cat ttc ggt gga ccg aac atc aac gca acg acc gca ccc tat 912 Tyr Trp His Phe Gly Gly Pro Asn Ile Asn Ala Thr Thr Ala Pro Tyr 270 275 280 cca tgc acg tac aac atc ctt aag gcg cat ggg cga gct gtt cag gag 960 Pro Cys Thr Tyr Asn Ile Lys Ala His Gly Arg Ala Val Gln Glu 285,290,295 tat cgg gct ctg gtc aac tcg ggg aag atc aag aag ggt gaa gtt gca 1008 Tyr Arg Ala Leu Val Asn Ser Gly Lys Ile Lys Gly Glu Val Ala 300 305 310 315 atc aag aac gat gac tcc tac cct gtg ccc gtt aac cca gac tct gaa 1056 Ile Lys Asn Asp Asp Ser Tyr Pro Val Pro Val Asn Pro Asp Ser Glu 320 325 330 gca gat gtt gaa gct gcg aaa cga cat ttc gac ttc tac atc ggc atc 1104 Ala Asp Val Glu Ala Ala Lys Arg His Phe Asp Phe Tyr Ile Gly Ile 335 340 345 ttc tcg cag ccc atc tat ggc gat ggc aag ttc cct gat acc gtc cgc 1152 Phe Ser Gln Pro Ile Tyr Gly Asp Gly Lys Phe Pro Asp Thr Val Arg 350 355 360 aac aca atc agc aca gaa ttc ctg cct tac ctc act gat gac gag aag 1200 Asn Thr Ile Ser Thr Glu Phe Leu Pro Tyr Leu Thr Asp Asp Glu Lys 365 370 375 gcc atg atc aag ggt tcc ggc gac ttc ttc gca atc gat gct tac cgc 1248 Ala Met Ile Lys Gly Ser Gly Asp Phe Phe Ala Ile Asp Ala Tyr Arg 380 385 390 395 acc aac ctt gct cga gct gct cca aac gga atc cag gcc tgt gtt gcc 1296 Thr Asn Leu Ala Arg Ala Ala Pro Asn Gly Ile Gln Ala Cys Val Ala 400 405 410 aac atc tcg gat ccg aat tgg ccg gtc tgt caa gac aac agc cct gaa 1344 Asn Ile Ser Asp Pro Asn Trp Pro Val Cys Gln Asp Asn Ser Pro Glu 415 420 425 ggg cag tat cag acg atg gat ggc ttt gcc ttt ggc cca cca gcg gac 1392 Gly Gln Tyr Gln Thr Met Asp Gly Phe Ala Phe Gly Pro Pro Ala Asp 430 435 440 cct aac gcg gcg tgg ctc tac gac acg agc ttc aag ctg cgg tat cag 1440 Pro Asn Ala Ala Trp Leu Tyr Asp Thr Ser Phe Lys Leu Arg Tyr Gln 445 450 455 ctt aag act ctc aaa gag gcc ttc aac tac gac aag atc tac atc agc 1488 Leu Lys Thr Leu Lys Glu Ala Phe Asn Tyr Asp Lys Ile Tyr Ile Ser 460 465 470 475 gaa ttc ggt ttc gcc cga ccc tat gag tat ctg tat ccc tat ggt ttt 1536 Glu Phe Gly Phe Ala Arg Pro Tyr Glu Tyr Leu Tyr Pro Tyr Gly Phe 480 485 490 gac gtt ctc tat gac aca gac cga gct atc tac cag gac tac atg 1584 Asp Val Leu Tyr Asp Thr Asp Arg Ala Ile Tyr Gln Tyr Met 495,500,505 gcc gag gca ctg gat gct atc cac gac gat ggt att cca ttg gcg ggt 1632 Ala Glu Ala Leu Asp Ala Ile His Asp Asp Gly Ile Pro Leu Ala Gly 510,515,520 gtg ttc gct tgg tcc ttc gtc gac aac ttc gaa tgg gcg tcc ggc ctt 1680 Val Phe Ala Trp Ser Phe Val Asp Asn Phe Glu Trp Ala Ser Gly Leu 525 530 535 gag caa cgc ttt ggc atg cag ttc gtc aac tac aca acc ctc gag cgc 1728 Glu Gln Arg Phe Gly Met Gln Phe Val Asn Tyr Thr Thr Leu Glu Arg 540 545 550 555 gag tac aag ctc tct ttc ctg ctg tat cgt gat ttc atc gag aat cat 1776 Glu Tyr Lys Leu Ser Phe Leu Leu Tyr Arg Asp Phe Ile Glu Asn His 560 565 570 agc tgc gag gat taa 1791 Ser Cys Glu Asp 575 <210> 24 <211> 596 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala -20 -15 -10 Ala Pro Ala Leu Ala Ile Pro Ala Phe Pro Ile Thr Pro Asp Leu Ala -5 -1 1 5 10 Gly Gly Leu Glu Ser Val Thr Asn Thr Gln Thr Ser Leu Pro Ser Ala 15 20 25 Ser Ala Val Ser Ser Pro Tyr Asn Gln Asp Ala Leu Asp Lys Leu Trp 30 35 40 Ala Glu Val Glu Lys Asp Ile Pro Val Glu Thr Pro Ser Ile Ser Ser 45 50 55 Val Val Pro Val Asn Asn Ser Phe Ala Val Pro Lys Thr Pro Thr Leu 60 65 70 75 Pro Arg Ser Leu Gln Asp His Ala Thr Ser Gly Arg Lys Phe Pro Lys 80 85 90 Gly Phe Lys Phe Gly Val Ala Thr Ala Asp Gln Gln Tyr Glu Gly Ala 95 100 105 Val Lys Ala Asp Gly Arg Gly Pro Ser His Trp Asp Tyr Leu Cys His 110 115 120 Arg Leu Pro Gln Gln Cys Asn Asn Tyr Thr Ser Asp Ile Thr Asp Leu 125 130 135 Gly Arg Tyr Tyr Tyr Lys Gln Asp Ile Ala Arg Ile Lys Ala Met Gly 140 145 150 155 Val Asn Thr Val Ser Leu Thr Leu Ser Trp Ser Arg Ile Lys Pro Phe 160 165 170 Gly Thr Ala Asp Ser Pro Val Ser Lys Glu Gly Leu Gln Phe Tyr Asp 175 180 185 Asp Phe Ile Asn Glu Leu Ile Asp Asn Gly Ile Glu Pro Val Val Thr 190 195 200 Leu Phe His Trp Ser Thr Pro Leu Asn Leu Val Phe Glu Tyr Gly Ala 205 210 215 Phe Leu Asn Gly Ser Ser Val Glu Asp Phe Ala Ser Tyr Ala Lys Leu 220 225 230 235 Val Phe Glu His Phe Gly Asp Arg Val Thr Thr Phe Leu Thr Phe Asn 240 245 250 Glu Pro Arg Val Tyr Cys Ser Glu Tyr Thr Gly Glu Pro Phe Asn Asp 255 260 265 Tyr Trp His Phe Gly Gly Pro Asn Ile Asn Ala Thr Thr Ala Pro Tyr 270 275 280 Pro Cys Thr Tyr Asn Ile Leu Lys Ala His Gly Arg Ala Val Gln Glu 285 290 295 Tyr Arg Ala Leu Val Asn Ser Gly Lys Ile Lys Lys Gly Glu Val Ala 300 305 310 315 Ile Lys Asn Asp Asp Ser Tyr Pro Val Pro Val Asn Pro Asp Ser Glu 320 325 330 Ala Asp Val Glu Ala Ala Lys Arg His Phe Asp Phe Tyr Ile Gly Ile 335 340 345 Phe Ser Gln Pro Ile Tyr Gly Asp Gly Lys Phe Pro Asp Thr Val Arg 350 355 360 Asn Thr Ile Ser Thr Glu Phe Leu Pro Tyr Leu Thr Asp Asp Glu Lys 365 370 375 Ala Met Ile Lys Gly Ser Gly Asp Phe Phe Ala Ile Asp Ala Tyr Arg 380 385 390 395 Thr Asn Leu Ala Arg Ala Ala Pro Asn Gly Ile Gln Ala Cys Val Ala 400 405 410 Asn Ile Ser Asp Pro Asn Trp Pro Val Cys Gln Asp Asn Ser Pro Glu 415 420 425 Gly Gln Tyr Gln Thr Met Asp Gly Phe Ala Phe Gly Pro Pro Ala Asp 430 435 440 Pro Asn Ala Ala Trp Leu Tyr Asp Thr Ser Phe Lys Leu Arg Tyr Gln 445 450 455 Leu Lys Thr Leu Lys Glu Ala Phe Asn Tyr Asp Lys Ile Tyr Ile Ser 460 465 470 475 Glu Phe Gly Phe Ala Arg Pro Tyr Glu Tyr Leu Tyr Pro Tyr Gly Phe 480 485 490 Asp Val Leu Tyr Asp Thr Asp Arg Ala Ile Tyr Tyr Gln Asp Tyr Met 495 500 505 Ala Glu Ala Leu Asp Ala Ile His Asp Asp Gly Ile Pro Leu Ala Gly 510 515 520 Val Phe Ala Trp Ser Phe Val Asp Asn Phe Glu Trp Ala Ser Gly Leu 525 530 535 Glu Gln Arg Phe Gly Met Gln Phe Val Asn Tyr Thr Thr Leu Glu Arg 540 545 550 555 Glu Tyr Lys Leu Ser Phe Leu Leu Tyr Arg Asp Phe Ile Glu Asn His 560 565 570 Ser Cys Glu Asp 575 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1 Primer <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, or t <220> <221> misc_feature <222> (27)..(27) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30)..(30) <223> n is a, c, g, or t <400> 25 gccggcgcgg ctathcargt ngarggngcn 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F2 Primer <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g, or t <220> <221> misc_feature <222> (18) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30)..(30) <223> n is a, c, g, or t <400> 26 gtcaagacnt ggttyacntt yaaygarccn 30 <210> 27 <211> 30 <212> DNA <213> Artificial sequence <220> <223> R1 Primer <220> <221> misc_feature <222> (19)..(19) <223> n is a, c, g, or t <400> 27 ctcggcccac ccraaytcns wraartadat 30 <210> 28 <211> 27 <212> DNA <213> Artificial sequence <220> <223> R2 Primer <220> <221> misc_feature <222> (16) <223> n is a, c, g, or t <220> <221> misc_feature <222> (22)..(22) <223> n is a, c, g, or t <400> 28 ccattcccar ttrtcnacra answcca 27 <210> 29 <211> 30 <212> DNA <213> Artificial sequence <220> <223> C-R70 primer <220> <221> misc_feature <222> (10)..(10) <223> n is a, c, g, or t <400> 29 gacgaggccn swrttccayt craarttrtc 30

Claims

1. A method for producing a secretory β-galactosidase, characterized in that: The non-secretory β-galactosidase gene from basidiomycete yeast was introduced into Aspergillus oryzae to produce secretory β-galactosidase. The non-secretory β-galactosidase gene from basidiomycete yeast consists of a signal sequence from Aspergillus oryzae and a sequence encoding β-galactosidase. The signal sequence and the sequence encoding β-galactosidase are any one of SEQ ID NOs: 3, 9, 15, 21, 5, 11, 17, and 23.

2. The method for producing secretory β-galactosidase according to claim 1, wherein Basidiomycete yeasts belong to the genera Sporobolomyces, Streptoceras, Rhodotorula, or Pseudomonas.

3. The method for producing secretory β-galactosidase according to claim 1, wherein The sequence encoding β-galactosidase is a sequence obtained by changing the codons of the sequence encoding natural β-galactosidase within the range that the amino acid sequence of β-galactosidase is not changed.

4. A transformant of Aspergillus oryzae, characterized in that A non-secretory β-galactosidase gene from basidiomycete yeast was introduced into Aspergillus oryzae to produce secretory β-galactosidase. The non-secretory β-galactosidase gene from basidiomycete yeast consists of a signal sequence from Aspergillus oryzae and a sequence encoding β-galactosidase. The signal sequence and the sequence encoding β-galactosidase are any one of SEQ ID NOs: 3, 9, 15, 21, 5, 11, 17, and 23.

5. A method for producing galacto-oligosaccharide, characterized in that: A β-galactosidase is produced by the method for producing a secretory β-galactosidase according to any one of claims 1 to 3, and is allowed to act on a substrate containing at least lactose.

6. A method for producing a secretory β-galactosidase, characterized in that: The signal sequence and the sequence encoding β-galactosidase from Sporobolomyces singularis were introduced into Aspergillus oryzae, and the Aspergillus oryzae was cultured in CDD medium to produce secretory β-galactosidase. The signal sequence and the sequence encoding β-galactosidase from Sporobolomyces singularis are the sequences set forth in SEQ ID NO: 3 or 5, The composition of the CDD medium is as follows: 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.

7. The method for producing secretory β-galactosidase according to claim 6, wherein The signal sequence is the secretion signal sequence of Aspergillus oryzae.

8. The method for producing secretory β-galactosidase according to claim 6, wherein Cultivation in CDD medium was performed at 30°C for 144 hours.

9. A method for producing galacto-oligosaccharide, characterized in that: A β-galactosidase is produced by the method for producing a secretory β-galactosidase according to any one of claims 6 to 8, and is allowed to act on a substrate containing at least lactose.

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