Method for producing alkaline phosphatase, alkaline phosphatase obtained by using the same, and vector and transformant for producing the same

Abstract

The present invention provides a method for producing alkaline phosphatase (ALP), which comprises the step of culturing an Aspergillus transformant capable of producing ALP.

Description

Technical Field

[0001] The present invention relates to a method for producing alkaline phosphatase, alkaline phosphatase obtained by using the same, and a vector and a transformant for its production.

Background Art

[0002] Alkaline phosphatase (hereinafter abbreviated as ALP) is used in the field of molecular biology for dephosphorylation of DNA in gene recombination experiments and for labeling antibodies in Western blotting, etc. Further, in the industrial field, it is used as a labeling enzyme in immunological methods for clinical tests. In order to efficiently and homogeneously label an antibody, and further, in order to obtain accurate clinical test results using the antibody labeled in such a manner (labeled antibody), it is preferable that the antibody itself to be labeled is as homogeneous as possible. And for that purpose, it is desirable that the labeling enzyme itself is also as homogeneous as possible. For example, as a method for making the labeled antibody homogeneous, in a subsequent step of the step of labeling the antibody, the labeled antibody, the antibody that has not been completely labeled, and the excess labeling enzyme are separated using a physical separation method, and only the labeled antibody is obtained. It is necessary to work. In this operation, if the labeling enzyme itself is in a state such as a heterogeneous mixture in terms of, for example, molecular weight or performance as an enzyme, the labeled antibody may also have a heterogeneous molecular weight and performance as an enzyme.

[0003] Regarding the molecular weight, it can be said that there is a countermeasure method of obtaining only the labeled antibody having a specific molecular weight in the separation step of the subsequent step, but even in that case, there is a limit to the molecular weight separation. In addition, since the yield in obtaining the target labeled antibody fraction in the post-treatment deteriorates, there is still a need to improve the homogeneity of the initial labeling enzyme itself. In addition, it is also known that there are multiple isoforms of ALP, and they have variations in specific activity. Therefore, for example, when an ALP composition composed of a mixture of multiple isoforms having different specific activity characteristics or the like is used as a labeling enzyme to label an antibody, heterogeneity in terms of activity will occur in the resulting labeled antibody. From this perspective as well, there is a need to enhance the homogeneity of the labeling enzyme itself.

[0004] As ALP conventionally used in the production of labeled antibodies and the like, ALP produced by extraction from bovine intestinal tract can be mentioned. This ALP has been used for many years because of its high specific activity and usefulness as a labeling enzyme. However, since it is derived from mammals, there are problems such as restrictions on raw material import and export due to infectious diseases such as mad cow disease, and significant fluctuations in quality due to individual differences, and it is recognized that its stable supply is inferior. In addition, bovine intestinal tract contains multiple ALP isoforms, and in fact, even commercially available ALP products produced through extraction and purification are still mixtures containing multiple isoforms. Therefore, for the purpose of labeling an antibody with the above-mentioned labeling enzyme and using it in clinical tests, this ALP has the problem of inferior homogeneity as an enzyme product.

[0005] Based on the above recognition, various methods for obtaining a more homogeneous ALP product have been studied so far. As this method, for example, a method of recombinantly expressing bovine intestinal tract-derived ALP in mammalian cells has been disclosed (for example, see Patent Documents 1 and 2). In this method, for example, if a gene encoding a specific isoform is selected and recombinant production is carried out using only that gene, the expressed protein can be only that specific single isoform, so that the activity heterogeneity caused by the coexistence of multiple isoforms with greatly different specific activities can be eliminated.

[0006] In addition, as a method for mass-producing recombinant ALP by using yeast as a host instead of mammalian cells as an expression host, there is also known a method (see, for example, Patent Document 3). Further, as a method for recombinant production in yeast, there has also been proposed a method in which, after obtaining ALP obtained by using yeast as an expression host, deglycosylation treatment is performed as a subsequent step to obtain ALP with a reduced amount of sugar chains (see, for example, Patent Document 4).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, the ALP obtained by recombinant expression of bovine intestinal tract-derived ALP as disclosed in Patent Documents 1 and 2 has a problem that the production amount of ALP is small because mammalian cells such as CHO cells are used as an expression host.

[0009] In addition, when yeast is used as an expression host as disclosed in Patent Document 3, there is a problem of hyperglycosylation in that sugar chains are excessively added to the expressed protein. In fact, in the case of ALP recombinantly produced using yeast as a host, compared to ALP produced by extraction from bovine intestinal tract, the amount of sugar added is extremely large, and moreover, since it is added in a large amount in a non-uniform manner, the resulting ALP has the problem of being an enzyme with low sugar chain uniformity. The problem of enzyme non-uniformity is not only an issue in the step of labeling an antibody with an enzyme, but also when ALP is applied to conjugate preparation or sensor applications, it can be a problem from the perspective of handling in various steps, and the excessive addition of sugar chains to ALP in the method of expressing it in yeast is considered industrially unfavorable. In addition, in the ALP expression system using yeast cells as a host, although it can be said that the production amount is higher than when expressed using mammalian cells as a host, the produced ALP is hardly secreted extracellularly and accumulates in the cells. That is, in order to obtain ALP, it is necessary to disrupt the yeast cells and purify the target ALP from the mixed state with all other components in the cells, and there is still room for improvement in terms of work efficiency in the manufacturing process.

[0010] The post-treatment deglycosylation of yeast-expressed ALP disclosed in Patent Document 4 can reduce the amount of sugar chain addition by enzymatic treatment, and is considered preferable in that it partially solves the problems of yeast-expressed ALP caused by excessive sugar chain addition disclosed in Patent Document 3. However, in reality, the cleavage of sugar chains by enzymatic treatment is random, and it is difficult to perform deglycosylation while leaving the necessary amount of sugar chains, and it is also difficult to achieve homogeneous cleavage for each treatment batch. Therefore, in terms of the homogeneity of the sugar chain addition state, it can still be said that there are still problems in the supply of continuous and reproducible products. In addition, after purifying ALP, an enzymatic treatment as a subsequent process is added, and then, the deglycosylated ALP, the ALP that cannot be completely deglycosylated, and the enzyme for deglycosylation are further separated by a separation process such as gel filtration to obtain only the desired deglycosylated ALP. There is a problem that a complicated deglycosylation process is newly required.

[0011] The problem to be solved by the present invention is to provide a method for producing ALP that can produce a single isoform of ALP with high productivity and secretory ability, in which sugar chains are not added excessively and non-uniformly, in the production of recombinant ALP, and ALP II or IV obtained by using the same, as well as a vector and a transformant for its production.

Means for Solving the Problems

[0012] Therefore, as a result of intensive research to solve the above problems, the present inventors used a method for producing ALP including a step of culturing an Aspergillus transformant having the ability to produce glycosylated ALP, and in particular, for the N-terminal side of ALP derived from bovine tissue, a base sequence encoding an amino acid sequence having a secretion signal peptide derived from a specific Aspergillus bacterium was used. By using a method for producing ALP for culturing an Aspergillus transformant transformed with a vector for Aspergillus transformation, a single isoform of ALP with an excessive and non-uniform glycosylation can be expressed with high productivity, and moreover, the expressed ALP is very efficiently secreted extracellularly from the Aspergillus transformant, and the present invention has been completed.

[0013] That is, the present invention provides the following inventions. [1] A method for producing ALP, comprising a step of culturing an Aspergillus transformant having the ability to produce alkaline phosphatase (ALP). [2] ALP is an ALP containing a mutation in which a sugar chain is not added to a part of the N-type sugar chain addition motif. The method for producing ALP according to [1]. [3] The Aspergillus transformant has a gene encoding an amino acid sequence having a secretion signal peptide derived from an Aspergillus bacterium selected from the group consisting of CDHss, CDHcytbkex, CDHcytbKexmut1-4, CDHall, GlaB, GlaBss, CelBss, CelB, SKIK_CDHss, AsAP1ss54, and AsAP3ss72 on the N-terminal side of ALP. The method for producing ALP according to [1] or [2]. [4] The Aspergillus transformant has, on the 5'-terminal side with respect to the base sequence encoding ALP: A gene consisting of a base sequence to which any of the following base sequences (a) to (c) is bound: (a) A base sequence of any one of SEQ ID NOs: 2 to 15; (b) A base sequence consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of any one of SEQ ID NOs: 2 to 15, and encoding a secretory signal peptide having the secretory activity of ALP; (c) A base sequence having 90% or more homology with the base sequence of any one of SEQ ID NOs: 2 to 15, and encoding a secretory signal peptide having the secretory activity of ALP; Or, A gene consisting of a base sequence to which a base sequence encoding any one of the following secretory signal peptides (d) to (f) is bound: (d) A secretory signal peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 17 to 30; (e) A secretory signal peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of any one of SEQ ID NOs: 17 to 30, and having the secretory activity of ALP; (f) A secretory signal peptide having 90% or more homology with the amino acid sequence of any one of SEQ ID NOs: 17 to 30, and having the secretory activity of ALP; Having, The method for producing ALP according to [1] or [2]. [5] ALP is ALPII or IV, The method for producing ALP according to any one of [1] to [4]. [6] ALP is any one of the following proteins (i) to (iii): (i) A protein consisting of the amino acid sequence of SEQ ID NO: 31 or 32; (ii) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 31 or 32, and having ALP activity; (iii) A protein having 90% or more homology with the amino acid sequence of SEQ ID NO: 31 or 32, and having ALP activity; Or, A protein encoded by any one of the following base sequences (iv) to (vi): (iv) The base sequence of SEQ ID NO: 33 or 34; (v) A base sequence consisting of the base sequence of SEQ ID NO: 33 or 34 in which one or several bases are deleted, substituted or added, and which encodes a protein having ALP activity; (vi) A base sequence having 90% or more homology with the base sequence of SEQ ID NO: 33 or 34 and which encodes a protein having ALP activity; having, The method for producing ALP according to any one of [1] to [5]. [7] ALP is ALPII which has a mutation in one or two of the three N-glycosylation addition motifs, and no sugar chain is added to the mutated N-glycosylation addition motif, The method for producing ALP according to any one of [1] to [6]. [8] In ALPII, the mutation in the N-glycosylation addition motif is one or two of positions 122, 249, 251, and 410 of SEQ ID NO: 31, The method for producing ALP according to [7]. [9] In ALPII, the mutation in the N-glycosylation addition motif is one or two of the mutation of Asn to Gln or Lys at position 122 of SEQ ID NO: 31, the mutation of Asn to Gln, Asp or His at position 249, the mutation of Thr to Cys at position 251, and the mutation of Asn to Gln or Lys at position 410, The method for producing ALP according to [7] or [8].

[10] The Aspergillus transformant is a transformant of any one of Aspergillus sojae, Aspergillus oryzae (Ahlburg) Cohn, Aspergillus luchuensis, Aspergillus niger, Aspergillus nidulans, Aspergillus tamarii, Aspergillus kawachii, Aspergillus awamori, Aspergillus usamii, or Aspergillus saitoi. The method for producing ALP according to any one of [1] to [9].

[11] A step of obtaining a secretory fraction from a culture obtained by culturing the Aspergillus transformant, and A step of extracting ALP from the secretory fraction, further comprising The method for producing ALP according to any one of [1] to

[10] .

[12] Glycosylated alkaline phosphatase (ALP) II having a molecular weight of 80 to 150 kDa as measured by gel filtration. [12-1] The isolated ALPII according to

[12] .

[13] A vector for Aspergillus transformation having a nucleotide sequence encoding an amino acid sequence having a secretory signal peptide derived from Aspergillus selected from the group consisting of CDHss, CDHcytbkex, CDHcytbKexmut1-4, CDHall, GlaBss, CelBss, CelB, SKIK_CDHss, AsAP1ss54, and AsAP3ss72 on the N-terminal side of ALP.

[14] For the nucleotide sequence encoding ALP, on the 5'-terminal side thereof: A gene consisting of a nucleotide sequence to which any of the following nucleotide sequences (a) to (c) is bound: (a) The nucleotide sequence of any one of SEQ ID NOs: 2 to 15; (b) A nucleotide sequence consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in the nucleotide sequence of any one of SEQ ID NOs: 2 to 15, and which encodes a secretory signal peptide having the secretory activity of ALP; (c) A nucleotide sequence having 90% or more homology with the nucleotide sequence of any one of SEQ ID NOs: 2 to 15, and which encodes a secretory signal peptide having the secretory activity of ALP; Or A gene consisting of a nucleotide sequence to which any of the following nucleotide sequences (d) to (f) encoding a secretory signal peptide is bound: (d) A secretory signal peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 17 to 30; (e) A secretory signal peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of any one of SEQ ID NOs: 17 to 30, and which has the secretory activity of ALP; (f) A secretory signal peptide having 90% or more homology with the amino acid sequence of any one of SEQ ID NOs: 17 to 30, and which has the secretory activity of ALP; A vector for Aspergillus transformation having the same.

[15] An Aspergillus transformant transformed using the vector for Aspergillus transformation described in

[13] or

[14] .

[16] A glycosylated alkaline phosphatase obtained by the method for producing ALP described in any one of [1] to

[11] . [Effects of the Invention]

[0014] According to the present invention, there is provided a method for producing ALP capable of producing a single isoform of ALP with excessive and non-uniform sugar chain addition with high productivity and secretory ability. Further, novel ALPII, a vector, and a transformant are provided, which are industrially useful.

Brief Description of Drawings

[0015]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in detail. The method for producing ALP of the present invention (hereinafter abbreviated as the present invention production method) is a production method including a step of culturing an Aspergillus transformant having the ability to produce ALP (hereinafter abbreviated as the present invention transformant). More specifically, it is a method for producing ALP derived from bovine intestinal tract by recombinant expression, and includes a step of culturing the present invention transformant having the ability to produce ALP derived from bovine intestinal tract.

[0017] The present invention production method is different from at least any known method for producing ALP in that it uses an Aspergillus transformant having the ability to produce ALP. Further, by using such a present invention transformant, it is possible to produce ALP with a high secretion rate and productivity.

[0018] The ALP may be a known ALP or a newly discovered ALP, or may be an ALP obtained by modifying an ALP having different physicochemical properties using methods such as genetic engineering techniques or mutagenesis treatment. For example, known ALPs include ALP derived from bovine (Bos taurus) intestinal tract [bIAPII; described in Manes et al., J. Biol. Chem., 273, No. 36, 23353-23360 (1998)] and the like.

[0019] ALP may have a secretion signal peptide on its N-terminal side and a GPI (glycosylphosphatidylinositol) addition signal peptide on its C-terminal side. To secrete and express ALP, the GPI addition signal peptide may be removed, or it may be used while leaving the original secretion signal peptide intact, or a host-derived secretion signal peptide may be used in place of the original secretion signal peptide of ALP. After ALP having a secretion signal peptide on its N-terminal side is expressed in the transformant of the present invention, the secretion signal peptide portion is deleted intracellularly, and then ALP without a secretion signal peptide is secreted from the cell. In certain embodiments, the above secretion signal peptide may be a carrier protein having a peptide portion (hereinafter also referred to as "secretion signal peptide portion") having a function as a secretion signal peptide on its N-terminal side. The carrier protein may also include a carrier protein linked with a linker (e.g., Kex2 cleavage linker) at its C-terminal. Here, the secretion signal peptide portion may be the same as the secretion signal peptide itself. The carrier protein is all or a part of the N-terminal of a protein efficiently secreted by the host. The Kex2 cleavage linker is a peptide containing the cleavage motif KR (lysine-arginine dipeptide) of a serine endopeptidase defined as enzyme number EC3.4.21.61 at its C-terminal. The peptide bond on the C-terminal side of the cleavage motif KR present in the protein sequence in the secretory pathway is decomposed by a serine endopeptidase. After ALP having a secretion signal peptide portion consisting of a carrier protein and a Kex2 cleavage linker on its N-terminal side is expressed in the transformant of the present invention, the secretion signal peptide is deleted intracellularly, and then ALP not containing a secretion signal peptide is secreted from the cell. ALP may be an isolated ALP, and examples of the isolation method include the extraction step described later.

[0020] Examples of the secretion signal peptide and carrier protein that function in the transformant of the present invention include host-derived cellobiose dehydrogenase and its cytochrome domain, secretory proteins such as glucoamylase, endoglucanase, alkaline phosphatase, etc., and a part of the N-terminal side thereof (secretion signal peptide part).

[0021] The number of N-glycosylation motifs (amino acid sequence of Asn-Xaa-Thr or Asn-Xaa-Ser; Xaa represents an amino acid other than Pro) contained in the amino acid sequence of ALP varies depending on the isoform. For example, bIAPII has three N-glycosylation motifs. In the present invention, ALP may be ALP with a sugar chain added to at least one N-glycosylation motif. When there are two or more N-glycosylation motifs originally present, it may also be an N-glycosylation motif-modified ALP in which one of the N-glycosylation motifs is modified so that an N-type sugar chain does not bind to it. Examples of the modification for preventing the binding of a sugar chain to the N-glycosylation motif include mutating the first Asn residue of the N-glycosylation motif to another amino acid, mutating the second Xaa residue to Pro, or mutating the third Thr / Ser residue to another amino acid, etc.

[0022] Next, the method for obtaining the gene encoding ALP for producing the transformant of the present invention and the DNA containing the same will be described below using ALP derived from bovine intestinal tract as an example. To obtain the gene encoding ALP used in the present invention, commonly used gene cloning methods can be used. For example, chromosomal DNA or mRNA is extracted from bovine intestinal tract cells by a conventional method, for example, the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. A library of chromosomal DNA or cDNA thus obtained is prepared. Next, based on the amino acid sequence of bovine intestinal alkaline phosphatase (ALP), an appropriate probe DNA is synthesized and used to screen a chromosomal DNA or cDNA library, or based on the above amino acid sequence, an appropriate primer DNA is prepared, and a DNA containing the target gene fragment is amplified by an appropriate polymerase chain reaction (PCR method) such as the 5' RACE method or 3' RACE method, and these are ligated to obtain a DNA containing the full-length target gene.

[0023] As a preferred example of the gene encoding bovine intestinal ALP thus obtained, the bovine intestinal ALP gene [bIAPI; Patent Document 1 or Weissig et al., Biochem. J. 260, 503 - 508 (1993)], more preferably the bovine intestinal ALP gene of high specific activity type [bIAPII, etc.; Patent Document 2 or Manes et al., J. Biol. Chem., 273, No. 36, 23353 - 23360 (1998)] can be mentioned. Further, since the amino acid sequence of bovine intestinal ALP has been published, a polynucleotide encoding the entire amino acid sequence can be synthesized by a DNA synthesizer, the complementary strands are associated and then ligated to obtain a full-length gene, or a polynucleotide synthesized from a part of the entire gene is used, the complementary strands are partially associated, and the complementary parts are amplified by a polymerase to obtain a full-length gene.

[0024] The preferred gene sequence is a DNA sequence in which the codons that match the ALP gene sequence at the amino acid level are optimized. Optimization of codons means, for example, optimizing each codon of the ALP gene sequence by silent mutation, that is, adding mutations at the DNA level in order to increase translation as required by the selected expression host without any influence at the amino acid level. These genes are preferably cloned into various vectors selected according to the host to be transformed in the usual manner.

[0025] A recombinant vector (recombinant DNA) containing a gene encoding ALP can be constructed by binding a PCR amplification product containing the gene encoding ALP and various vectors in a form enabling the expression of the gene encoding ALP. For example, it can be constructed by excising a DNA fragment containing the gene encoding ALP with an appropriate restriction enzyme and ligating the DNA fragment to a plasmid cleaved with an appropriate restriction enzyme. Alternatively, it can be obtained by ligating a DNA fragment containing the gene with sequences homologous to the plasmid added to both ends and a DNA fragment derived from the plasmid amplified by inverse PCR using a commercially available recombinant vector preparation kit such as In-Fusion HD Cloning Kit (Clontech). Various vectors contain a promoter that functions in a transformant, and further may contain a terminator, a 5'-untranslated region, a 3'-untranslated region, an antibiotic resistance gene, and a nutritional requirement marker gene.

[0026] Next, the transformant of the present invention will be described. The transformant of the present invention is not particularly limited, but as described above, using various recombinant DNAs obtained, an Aspergillus strain can be transformed or transduced to obtain an Aspergillus transformant containing recombinant DNA carrying various modified ALP gene fragments.

[0027] In the present invention, the Aspergillus strain is not particularly limited as long as it belongs to the genus Aspergillus. Examples of the Aspergillus strain include Aspergillus sojae, Aspergillus oryzae (Ahlburg) Cohn, Aspergillus luchuensis, Aspergillus niger, Aspergillus nidulans, Aspergillus tamarii, Aspergillus kawachii, Aspergillus awamori, Aspergillus usamii, Aspergillus saitoi and other strains. Among these, the strains of Aspergillus sojae and Aspergillus oryzae are preferable from the viewpoint of more surely obtaining ALP with a high secretion rate and productivity.

[0028] In one aspect of the present invention, the transformed organism of the present invention has a gene encoding an amino acid sequence having a secretion signal peptide derived from a fungus belonging to the genus Aspergillus on the N-terminal side of ALP. Examples of the secretion signal peptide derived from a fungus belonging to the genus Aspergillus include CDHss, CDHcytbkex, CDHcytbKexmut1-4, CHDHall, GlaBss, GlaB, CelBss, CelB, SKIK_CDHss, AsAP1ss54, and AsAP3ss72. From the viewpoint of obtaining more excellent secretory properties, those selected from the group consisting of CDHss, CDHcytbkex, CDHcytbKexmut1-4, CDHall, GlaB, GlaBss, CelBss, CelB, SKIK_CDHss, AsAP1ss54, and AsAP3ss72 are preferable, those selected from the group consisting of CDHss, CDHcytbkex, CDHcytbKexmut1, CDHcytbKexmut2, GlaB, GlaBss, CelBss, CelB, AsAP1ss54, and AsAP3ss72 are more preferable, those selected from the group consisting of CDHss, CDHcytbkex, CDHcytbKexmut1, CelBss, and CelB are even more preferable, and those selected from the group consisting of CDHss and CDHcytbkex are particularly preferable.

[0029] Here, the amino acid sequence having a secretion signal peptide on the N-terminal side of ALP may be an amino acid sequence in which the secretion signal peptide is directly bound to the N-terminal side of ALP, or may be an amino acid sequence in which the secretion signal peptide moiety is indirectly bound via a linker (for example, a carrier protein having a Kex2 cleavage linker bound to the C-terminal) on the N-terminal side of ALP.

[0030] In one aspect of the transformed organism of the present invention, with respect to the base sequence encoding ALP, at its 5'-terminal side: A gene consisting of a base sequence to which any of the following base sequences (a) to (c) is bound: (a) Any of the base sequences of SEQ ID NOs: 2 to 15; (b) A base sequence consisting of a base sequence in which one or several bases are deleted, substituted or added in any of the base sequences of SEQ ID NOs: 2 to 15, and encoding a secretory signal peptide having the secretory activity of ALP; (c) A base sequence having 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more homology with any of the base sequences of SEQ ID NOs: 2 to 15, and encoding a secretory signal peptide having the secretory activity of ALP; Or, A gene consisting of a base sequence to which a base sequence encoding any of the following secretory signal peptides (d) to (f) is bound: (d) A secretory signal peptide consisting of any of the amino acid sequences of SEQ ID NOs: 17 to 30; (e) A secretory signal peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in any of the amino acid sequences of SEQ ID NOs: 17 to 30, and having the secretory activity of ALP; (f) A secretory signal peptide having 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more homology with any of the amino acid sequences of SEQ ID NOs: 17 to 30, and having the secretory activity of ALP; Having.

[0031] In the case of "one or several bases are deleted, substituted or added", the number of bases to be deleted, substituted or added is not particularly limited as long as the resulting base sequence encodes a secretory signal peptide having the secretory activity of ALP. Here, "several" means an integer of 2 or more, preferably 2 to 20, more preferably 2 to 10, still more preferably 2 to 5, and even more preferably 2, 3 or 4. The position of deletion, substitution or addition in each base sequence may be at the 5' end, 3' end or in the middle as long as the resulting base sequence encodes a secretory signal peptide having the secretory activity of ALP.

[0032] "Having homology of Y% or more with the nucleotide sequence of SEQ ID NO: X" means that when the two nucleotide sequences are aligned so that the match is maximized, the ratio of the number of common nucleotides to the total number of nucleotides shown in SEQ ID NO: X is Y% or more.

[0033] In the case of "one or several amino acids being deleted, substituted or added", the number of amino acids to be deleted, substituted or added is not particularly limited as long as the resulting secretory signal peptide has the secretory activity of ALP. Here, "several" means an integer of 2 or more, preferably 2 to 20, more preferably 2 to 10, still more preferably 2 to 5, and even more preferably 2, 3 or 4. The position of deletion, substitution or addition in each secretory signal peptide may be at the N-terminus, C-terminus or in the middle as long as the resulting secretory signal peptide has the secretory activity of ALP.

[0034] "Having homology of Y% or more with the amino acid sequence of SEQ ID NO: X" means that when the amino acid sequences of the two secretory signal peptides are aligned so that the match is maximized, the ratio of the number of common amino acid residues to the total number of amino acid residues shown in SEQ ID NO: X is Y% or more.

[0035] The nucleotide sequence of SEQ ID NO: 1 is a nucleotide sequence encoding the secretory signal peptide (bIAPIVss) on the N-terminal side of bovine intestinal alkaline phosphatase IV (bIAPIV). The nucleotide sequence of SEQ ID NO: 2 is a nucleotide sequence encoding the secretory signal peptide (CDHss) derived from Aspergillus niger cellobiose dehydrogenase (CDH). The nucleotide sequence of SEQ ID NO: 3 is a nucleotide sequence encoding the secretory signal peptide (CDHcytbkex) consisting of the cytochrome b domain and the Kex2 cleavage linker derived from the carrier protein Aspergillus niger CDH. The nucleotide sequence of SEQ ID NO: 4 is a nucleotide sequence encoding a secretion signal peptide (CDHcytbkexmut1) consisting of a cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and Kex2 cleavage linker variant 1. The nucleotide sequence of SEQ ID NO: 5 is a nucleotide sequence encoding a secretion signal peptide (CDHcytbkexmut2) consisting of a cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and Kex2 cleavage linker variant 2. The nucleotide sequence of SEQ ID NO: 6 is a nucleotide sequence encoding a secretion signal peptide (CDHcytbkexmut3) consisting of a cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and Kex2 cleavage linker variant 3. The nucleotide sequence of SEQ ID NO: 7 is a nucleotide sequence encoding a secretion signal peptide (CDHcytbkexmut4) consisting of a cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and Kex2 cleavage linker variant 4. The nucleotide sequence of SEQ ID NO: 8 is a nucleotide sequence encoding a secretion signal peptide carrier protein (CDHall) consisting of Aspergillus oryzae CDH, which is a carrier protein, and a Kex2 cleavage linker. The nucleotide sequence of SEQ ID NO: 9 is a nucleotide sequence encoding a secretion signal peptide (GlaBss) derived from Aspergillus oryzae glucoamylase B. The nucleotide sequence of SEQ ID NO: 10 is a nucleotide sequence encoding a secretion signal peptide (GlaB) consisting of Aspergillus oryzae glucoamylase B, which is a carrier protein, and a Kex2 cleavage linker. The nucleotide sequence of SEQ ID NO: 11 is a nucleotide sequence encoding a secretion signal peptide (CelBss) derived from Aspergillus oryzae endoglucanase. The nucleotide sequence of SEQ ID NO: 12 is a nucleotide sequence encoding a secretion signal peptide (CelB) consisting of Aspergillus oryzae endoglucanase, which is a carrier protein, and a Kex2 cleavage linker. The nucleotide sequence of SEQ ID NO: 13 is a nucleotide sequence encoding a secretion signal peptide (SKIK_CDHss) in which the amino acid sequence of SKIK is inserted immediately after the start codon of the secretion signal peptide derived from Aspergillus oryzae CDH. The nucleotide sequence of SEQ ID NO: 14 is a nucleotide sequence encoding the secretion signal peptide 1 (AsAP1ss54) derived from Aspergillus oryzae ALP. The nucleotide sequence of SEQ ID NO: 15 is a nucleotide sequence encoding the secretion signal peptide 3 (AsAP1ss72) derived from Aspergillus oryzae ALP.

[0036] The amino acid sequence of SEQ ID NO: 16 is the amino acid sequence of the secretion signal peptide (bIAPIVss) on the N-terminal side of bovine intestinal alkaline phosphatase IV (bIAPIV). The amino acid sequence of SEQ ID NO: 17 is the amino acid sequence of the secretion signal peptide (CDHss) derived from Aspergillus oryzae cellobiose dehydrogenase. The amino acid sequence of SEQ ID NO: 18 is the amino acid sequence of the secretion signal peptide (CDHcytbkex) consisting of the cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and the Kex2 cleavage linker. The amino acid sequence of SEQ ID NO: 19 is the amino acid sequence of the secretion signal peptide (CDHcytbkexmut1) consisting of the cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and the Kex2 cleavage linker mutant 1. The amino acid sequence of SEQ ID NO: 20 is the amino acid sequence of the secretion signal peptide (CDHcytbkexmut2) consisting of the cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and the Kex2 cleavage linker mutant 2. The amino acid sequence of SEQ ID NO: 21 is the amino acid sequence of the secretion signal peptide (CDHcytbkexmut3) consisting of the cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and the Kex2 cleavage linker mutant 3. The amino acid sequence of SEQ ID NO: 22 is the amino acid sequence of the secretion signal peptide (CDHcytbkexmut4) consisting of the cytochrome b domain derived from Aspergillus oryzae CDH, which is a carrier protein, and the Kex2 cleavage linker mutant 4. The amino acid sequence of SEQ ID NO: 23 is the amino acid sequence of the secretion signal peptide (CDHall) consisting of Aspergillus oryzae CDH, which is a carrier protein, and the Kex2 cleavage linker. The amino acid sequence of SEQ ID NO: 24 is the amino acid sequence of the secretion signal peptide (GlaBss) derived from Aspergillus oryzae glucoamylase B. The amino acid sequence of SEQ ID NO: 25 is the amino acid sequence of the secretion signal peptide (GlaB) consisting of the Aspergillus oryzae glucoamylase B, which is a carrier protein, and the Kex2 cleavage sequence. The amino acid sequence of SEQ ID NO: 26 is the amino acid sequence of the secretion signal peptide (CelBss) derived from Aspergillus oryzae endoglucanase. The amino acid sequence of SEQ ID NO: 27 is the amino acid sequence of the secretion signal peptide (CelB) consisting of the Aspergillus oryzae endoglucanase, which is a carrier protein, and the Kex2 cleavage sequence. The amino acid sequence of SEQ ID NO: 28 is the amino acid sequence of the secretion signal peptide (SKIK_CDHss) in which the amino acid sequence of SKIK is inserted immediately after the start codon of the secretion signal peptide derived from Aspergillus oryzae CDH. The amino acid sequence of SEQ ID NO: 29 is the amino acid sequence of the secretion signal peptide 1 (AsAP1ss54) derived from Aspergillus oryzae ALP. The amino acid sequence of SEQ ID NO: 30 is the amino acid sequence of the secretion signal peptide 3 (AsAP1ss72) derived from Aspergillus oryzae ALP.

[0037] In this specification, when recombinantly expressing ALP, the function of the secretion signal peptide to secrete ALP extracellularly is referred to as the "secretion activity of ALP". In the obtained transformant of the present invention, to examine the secretion activity of ALP of the secretion signal peptide, for example, it can be carried out as follows. Inoculate the transformant into a natural medium or synthetic medium containing a carbon source, nitrogen source, inorganic salts, etc. that can be utilized by Aspergillus strains and that can efficiently culture Aspergillus strains, and incubate with shaking at a temperature of 20 to 42 °C, preferably about 30 °C for 10 to 120 hours. Examples of the carbon source include sugars such as glucose, fructose, and sucrose, and carbohydrates such as dextran and starch. Among these, dextran is preferred. Examples of the nitrogen source include yeast extract, peptone, meat extract, etc. Among them, yeast extract and peptone are preferred. Examples of the inorganic salts include magnesium phosphate, magnesium sulfate, and sodium chloride. Among them, magnesium sulfate is preferred. Also, it is desirable to add magnesium ions and zinc ions as metal ions necessary for ALP activity to the culture solution. Next, collect the obtained culture and separate it into a culture supernatant and a residue containing cells by centrifugation or centrifugation, etc. Disrupt the cells in the residue of the cells with a cell wall-lysing enzyme, a surfactant, a drug such as EDTA, ultrasonic waves, a multi-bead shocker, etc., and obtain a cell disruption supernatant by centrifugation, etc. Measure the ALP activity using the obtained culture supernatant and cell disruption supernatant. In the present invention, when the ALP activity of the culture supernatant fraction is confirmed, it is said to have the secretion activity of ALP. Also, the intensity of the secretion activity of ALP can be shown as the secretion rate of the following formula. Secretion rate = (total activity in culture supernatant [U] ÷ (total activity in culture supernatant [U] + total activity in intracellular fraction [U])) × 100 The secretion activity of ALP of the transformant of the present invention is not particularly limited, but for example, it is preferably a secretion activity equal to or higher than the secretion rate when APIVss described later is used as the secretion signal peptide in the examples described later. More specifically, as the secretion rate measured in the examples described later, it is more preferably 10% or more, further preferably 20% or more, and even more preferably 30% or more.

[0038] In this way, the transformant of the present invention having the ability to produce ALP can be obtained. The transformant of the present invention as one aspect of the present invention has a gene designed such that no sugar chain is added to a part of the N-type sugar chain addition motif in ALP. More specifically, the bovine intestinal ALP isoform II (bIAPII or ALPII) has a mutation in one or two of the three N-type sugar chain addition motifs, and has a gene designed such that no sugar chain is added to the N-type sugar chain addition motif having the mutation. More specifically, in the bovine intestinal ALPII, the mutation in the N-type sugar chain addition motif is one or two of positions 122, 249, 251, and 410 of SEQ ID NO: 31. More specifically, in the bovine intestinal ALPII, the mutation in the N-type sugar chain addition motif is one or two of the mutations from Asn to Gln or Lys at position 122 of SEQ ID NO: 31, from Asn to Gln, Asp, or His at position 249, from Thr to Cys at position 251, and from Asn to Gln or Lys at position 410 of SEQ ID NO: 31.

[0039] As an example of the gene possessed by such a transformant of the present invention, a gene encoding a protein consisting of an amino acid sequence in which Asn at position 122 of SEQ ID NO: 31 is substituted with Gln and Asn at position 410 is substituted with Gln can be mentioned.

[0040] Further, the transformant of the present invention as one aspect of the present invention has a gene designed to be ALPII or IV. Specific examples of these ALPs are any of the following proteins (i) to (iii): (i) A protein consisting of the amino acid sequence of SEQ ID NO: 31 or 32; (ii) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 31 or 32 and having ALP activity; (iii) A protein having an amino acid sequence of SEQ ID NO: 31 or 32 and having a homology of 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more, and having ALP activity; Or, A protein encoded by any of the following nucleotide sequences (iv) to (vi): (iv) The nucleotide sequence of SEQ ID NO: 33 or 34; (v) A nucleotide sequence consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 33 or 34 and encoding a protein having ALP activity; (vi) A nucleotide sequence having a homology of 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more with the nucleotide sequence of SEQ ID NO: 33 or 34 and encoding a protein having ALP activity; It has.

[0041] When "one or several amino acids are deleted, substituted or added", the number of amino acids to be deleted, substituted or added is not particularly limited as long as the resulting protein has ALP activity. Also, "several" here means an integer of 2 or more, preferably 2 to 20, more preferably 2 to 10, still more preferably 2 to 5, and even more preferably 2, 3 or 4. The position of deletion, substitution or addition in each protein may be at the N-terminus, C-terminus or in the middle thereof as long as the resulting protein has ALP activity.

[0042] "Having a homology of Y% or more with the amino acid sequence of SEQ ID NO: X" means that when the amino acid sequences of two proteins are aligned to maximize the match, the ratio of the number of common amino acid residues to the total number of amino acid residues shown in SEQ ID NO: X is Y% or more.

[0043] When it is stated that "one or several bases are deleted, substituted or added", the number of bases to be deleted, substituted or added is not particularly limited as long as the resulting base sequence encodes a protein having ALP activity. Here, "several" means an integer of 2 or more, preferably 2 to 20, more preferably 2 to 10, still more preferably 2 to 5, and even more preferably 2, 3 or 4. The position of deletion, substitution or addition in each base sequence may be at the 5'-end, 3'-end or in the middle thereof as long as the resulting base sequence encodes a protein having ALP activity.

[0044] "Having Y% or more homology with the base sequence of SEQ ID NO: X" means that when the two base sequences are aligned so that the match is maximized, the ratio of the number of common bases to the total number of bases shown in SEQ ID NO: X is Y% or more.

[0045] To examine the ALP activity of the obtained transformant of the present invention, the ALP activity is measured as described above to examine the secretion activity of ALP. The ALP activity of the transformant of the present invention is not particularly limited, but is preferably equal to or higher than the activity of the secreted fraction obtained by culturing the wild strain before transformation. More specifically, it is more preferably that the enzyme activity value (U / ml) described later in the secreted fraction is 10 (U / ml) or more.

[0046] The amino acid sequence of SEQ ID NO: 31 is the amino acid sequence of bovine intestinal alkaline phosphatase II (bIAPII). The amino acid sequence of SEQ ID NO: 32 is the amino acid sequence of bovine intestinal alkaline phosphatase IV (bIAPIV).

[0047] The base sequence of SEQ ID NO: 33 is the base sequence encoding bovine intestinal alkaline phosphatase II (bIAPII). The base sequence of SEQ ID NO: 34 is the base sequence encoding bovine intestinal alkaline phosphatase IV (bIAPIV).

[0048] Next, each step that the production method of the present invention may include will be described. The step of culturing the transformant of the present invention is not particularly limited, but the mutant of the present invention may be cultured by either a solid culture method or a liquid culture method, and it is preferably cultured by the liquid culture method.

[0049] As the medium for culturing the transformant of the present invention, for example, one or more nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean, and leaching solution of wheat koji are added with one or more inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride, ferrous sulfate, magnesium chloride, zinc chloride, cobalt chloride, and manganese sulfate, and further, if necessary, a saccharide raw material, vitamins, etc. are appropriately added and used.

[0050] The culturing is preferably carried out at 20 to 37°C, specifically around 30°C for 10 to 120 hours by aeration agitation deep culture, shaking culture, etc.

[0051] The production method of the present invention can produce bovine intestinal ALP by collecting bovine intestinal ALP from the culture obtained by culturing the transformant of the present invention in a medium. The production method of the present invention can collect bovine intestinal ALP from the culture obtained by culturing the transformant of the present invention by ordinary enzyme collection means, and preferably further includes the step of obtaining a secretory fraction from the culture obtained by culturing the transformant of the present invention, and the step of extracting bovine intestinal ALP from the secretory fraction.

[0052] To obtain a secretory fraction from the culture obtained by culturing the transformant of the present invention, for example, the intracellular fraction may be separated from the culture, and the intracellular fraction is separated by operations such as filtration and centrifugation. Since the transformant of the present invention has a high secretion rate of bovine intestinal ALP, bovine intestinal ALP tends to be present more in the secretory fraction that can be more easily extracted than in the intracellular fraction. Thereby, the production method of the present invention can easily obtain bovine intestinal ALP from the culture.

[0053] To extract bovine intestinal tract-derived ALP from the obtained secretory fraction, methods used in ordinary enzyme purification can be employed. For example, it is preferable to perform ammonium sulfate salting-out, organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, electrophoresis, etc. either alone or in appropriate combinations. In this way, bovine intestinal tract-derived ALP can be extracted until it shows almost a single band on SDS-PAGE. Also, by appropriately combining such methods, it is possible to adjust to preparations with different degrees of purification according to the intended use.

[0054] In addition, bovine intestinal tract-derived ALP may also be collected from the intracellular fraction in the culture obtained by the culturing step. In this case, it is preferable to wash the culture or the separated intracellular fraction to remove bacteria, and this can be used as it is. It is also possible to use various disruption means such as an ultrasonic disrupter, a French press, a Dyno-Mill, etc. to disrupt the cells, a method of lysing the cell wall of the cells using a cell wall-lysing enzyme, a method of extracting the enzyme from the cells using a surfactant such as Triton X-100, etc. to collect bovine intestinal tract-derived ALP from the cells.

[0055] The specific activity of the bovine intestinal tract-derived ALP of the present invention is preferably 6500 U / mg or more. As a method for measuring the specific activity of ALP, a method of measuring the increase or decrease in the color development amount of the substrate converted by the reaction of the enzyme, etc. are mainly mentioned as the measurement methods. Hereinafter, as an example, a method of measuring the increase in absorbance of p-nitrophenol generated by the conversion of p-nitrophenyl phosphate will be shown. Note that the enzyme titer is defined as 1 U when the amount of enzyme that generates 1 μmol of p-nitrophenol per minute is measured using p-nitrophenyl phosphate as the substrate. A. Preparation of reagents (1) Reagent 1: 1.0 M MgCl2 solution Dissolve 2.03 g of MgCl2·6H2O in ion-exchanged water and make up the volume to 10 ml. (2) Reagent 2: Diethanolamine (DEA) buffer (prepared before use) Dissolve 52.2 g DEA (Sigma - Aldrich) in 400 mL of ion - exchanged water, and add 0.25 mL of MgCl₂ solution (Reagent 1). Then, warm it to 37 °C, adjust the pH to 9.8 with 2N HCl, and make up the volume to 500 mL with ion - exchanged water. (3) Reagent 3: 0.65 M p - nitrophenyl phosphate solution Dissolve 247 mg of p - nitrophenyl phosphate (Sigma - Aldrich) in 1 mL of ion - exchanged water. (4) Enzyme dilution solution Dilute with DEA buffer (Reagent 2) so that the activity measurement value is 0.10 - 0.20 U / mL. B. Activity measurement method Mix 2.90 mL of DEA buffer (Reagent 2) and 0.05 mL of p - nitrophenyl phosphate solution (Reagent 3), and warm at 37 °C for 5 minutes. Then, add 0.05 mL of the enzyme dilution solution. After mixing, measure the absorbance at 405 nm using a spectrophotometer (U - 3010, Hitachi High - Technologies). The measured value (ΔODtest) is the change in absorbance per minute from 2 minutes to 4 minutes at 405 nm. The control solution (ΔODblank) is prepared in the same way as above except that 0.05 mL of DEA buffer is added instead of the enzyme solution. Calculate the value according to the following formula and use it as the enzyme activity value (U / mL).

Number

[0056] In the present invention, the molecular weight of ALPII measured by gel filtration is preferably 150 kDa or less, more preferably 80 to 150 kDa, and even more preferably 90 to 135 kDa. The molecular weight measured by gel filtration is measured according to the method described in the examples below or according to a method analogous thereto.

[0057] In one aspect of the Aspergillus transformation vector of the present invention, this has a nucleotide sequence encoding an amino acid sequence having the above-described Aspergillus - derived secretion signal peptide with respect to the N - terminal side of ALP derived from bovine intestine. Further, in one aspect of the Aspergillus transformation vector of the present invention, this has a gene consisting of a nucleotide sequence in which the above - described nucleotide sequence is linked to the 5'-terminal side with respect to the nucleotide sequence encoding ALP derived from bovine intestine. The transformant of the present invention can be produced by being transformed using these Aspergillus transformation vectors of the present invention.

[0058] Hereinafter, the present invention will be described in detail by way of examples.

Examples

[0059] [Production of DNA construct inserted with CDHss - bIAPII] (1 - 1) Production of plasmid for construct In the same manner as the method described in "[(3) Production of plasmid for construct] in [Example 1. Production of DNA construct inserted with gene AsEgtA, AsEgtB or AsEgtC]" of Pamphlet of International Publication No. 2016 / 121285, a plasmid for construct was prepared by ligating the promoter sequence Ptef of the translation elongation factor gene tef1, the terminator sequence Talp of the alkaline protease gene alp, and the transformation marker gene pyrG that complements uridine requirement to the In - Fusion Cloning Site in the multiple cloning site of pUC19 linearized Vector attached to the In - Fusion HD Cloning Kit (Clontech).

[0060] (1-2) Preparation of gene insertion DNA construct A DNA construct in which the target gene bIAPII was ligated between Ptef and Talp of the construct plasmid was prepared as follows. The template DNA was the plasmid for construct obtained above, the PCR enzyme was KOD-Plus-DNA Polymerase (Toyobo), the reaction reagents were those that came with this enzyme, and the equipment was T100. TM A vector fragment of the construct plasmid was obtained by performing inverse PCR using a thermal cycler (BIO-RAD) according to the protocol attached to the enzyme. The primers used are shown below. The amplified vector fragment was separated in a 1% (w / v) agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0061] [Table 1]

[0062] Instead of the N-terminal secretory signal peptide (SEQ ID NO: 36) of alkaline phosphatase II (bIAPII) derived from bovine intestine, the secretory signal peptide (CDHss) derived from Aspergillus cellobiose dehydrogenase was fused to the bIAPII gene, and the CDHss-bIAPII synthetic gene (SEQ ID NO: 37) was codon-optimized for Aspergillus oryzae (Genscript). The template DNA was the CDHss-bIAPII synthetic gene obtained above, the PCR enzyme was KOD-Plus-DNA Polymerase (Toyobo), the reaction reagents were those included with this enzyme, and the equipment was T100. TMPCR was performed using a thermal cycler (BIO-RAD) according to the protocol attached to this enzyme to obtain an inserted gene DNA fragment. The primers used for amplification are shown below. Among the sequences, the lowercase sequences indicate additional sequences for ligation into the construct plasmid (between Ptef and Talp). The amplified DNA fragment was separated in a 1% (w / v) agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0063]

Table 2

[0064] The amplified vector fragment and the CDHss-bIAPIIDNA fragment were ligated using an In-Fusion HD Cloning Kit according to the protocol attached to the kit to obtain a gene-inserted DNA construct (pbIAPII) into which CDHss-bIAPII was inserted. The DNA construct thus obtained is a ligation of a DNA fragment derived from pUC19, a DNA fragment of Ptef, a DNA fragment of CDHss, a DNA fragment of bIAPII, a DNA fragment of Talp, and a DNA fragment of pyrG from upstream. That is, a DNA construct in which the sequence of Ptef-CDHss-bIAPII-Talp-pyrG was ligated in order to the In-Fusion Cloning Site of pUC19 linearized Vector (Clontech) was obtained. Subsequently, 1 / 10 volume of the DNA construct solution was mixed with Escherichia coli competent cells ECOS Competent E. coli JM109 (Nippon Gene) in ice water, left in ice water for 5 minutes, and then transformed by treatment at 42°C for 45 seconds. Then, it was spread on an LB (Luria-Bertani) plate containing 50 μg / ml of ampicillin. This was statically cultured overnight at 37°C to form colonies. The obtained colonies were cultured overnight with shaking at 37°C in LB liquid medium containing 50 μg / ml of ampicillin. After culturing, the culture solution was centrifuged to recover the bacterial cells. For the obtained bacterial cells, plasmid DNA (DNA construct) was extracted using the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.) according to the protocol attached to the kit. By determining the nucleotide sequences of each DNA inserted into the extracted plasmid DNA, it was confirmed that a DNA construct (DNA construct name: pbIAPII) into which CDHss-bIAPII was inserted was obtained.

[0065] (1-3) Preparation of N-glycosylation motif-modified ALP gene insertion DNA construct by site-directed mutagenesis To generate N-glycosylation motif-modified ALP_Asn122Gln (Δ1), the gene insertion DNA construct (pbIAPII) into which CDHss-bIAPII was inserted was used as a template, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) was used as the enzyme for PCR, the reagents attached to this enzyme were used as reaction reagents, and T100 TM Thermal Cycler (BIO-RAD) was used to perform inverse PCR according to the protocol attached to this enzyme. The primers used are shown below. The amplified DNA fragment was separated in a 1% (w / v) agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen). The above DNA fragment was ligated using the In-Fusion HD Cloning Kit according to the protocol attached to the kit to obtain a gene insertion DNA construct (pΔ1) into which CDHss-bIAPII containing the mutation Asn122Gln to the N-glycosylation motif was inserted. Thereafter, various N-glycosylation motif-modified ALP gene insertion DNA constructs were obtained by sequentially introducing site-directed mutations based on the following table.

[0066]

Table 3

[0067] [Example 2. Preparation of Transformed Aspergillus sojae] (2-1) pyrG Disruptant Derived from Aspergillus sojae NBRC4239 Strain After ethanol precipitation of each gene insertion DNA construct, it was dissolved in TE buffer, and the DNA solution adjusted to the desired concentration was used for the transformation of a pyrG disruptant (a strain lacking 48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene) derived from Aspergillus sojae NBRC4239 strain according to the following procedure.

[0068] (2-2) Transformation of pyrG Disruptant Derived from Aspergillus sojae NBRC4239 Strain 100 ml of a polypeptone dextrose liquid medium containing 20 mM uridine in a 500 ml Erlenmeyer flask was inoculated with conidia of a pyrG disruptant derived from Aspergillus sojae NBRC4239 strain, and after shaking culture at 30 °C for about 20 hours, the cells were collected. Protoplasts were prepared from the collected cells. Using the obtained protoplasts and 20 μg of the gene insertion DNA construct, transformation was carried out by the protoplast PEG method, and then using a Czapek-Dox minimal medium (Difco; pH 6) containing 0.5% (w / v) agar and 1.2 M sorbitol, incubation was carried out at 30 °C for 5 days or more, and transformed Aspergillus sojae was obtained as those having the ability to form colonies. The obtained transformed Aspergillus sojae could grow on a uridine-free medium due to the introduction of pyrG, a gene that complements uridine auxotrophy, and thus could be selected as a strain into which the target gene had been introduced.

[0069] [Example 3. Production of Various ALPs by Transformed Aspergillus sojae] In 1800 ml of the liquid medium (2% (w / v) Hyppo polypeptone (Nippon Pharmaceutical Co., Ltd.), 2% (w / v) Pindex #2 (Matsutani Chemical Industry Co., Ltd.), 1% (w / v) yeast extract (Oriental Yeast Co., Ltd.), 0.25% (w / v) KH2PO4 (Wako Pure Chemical Industries, Ltd.), 0.25% (w / v) K2HPO4 (Wako Pure Chemical Industries, Ltd.), 0.05% (w / v) MgSO4·7H2O (Wako Pure Chemical Industries, Ltd.), 3 mM MgCl2 (Wako Pure Chemical Industries, Ltd.), 0.1 mM ZnCl2 (Wako Pure Chemical Industries, Ltd.); pH not adjusted) in a 3 L jar fermenter, conidia of various transformant strains were inoculated and cultivation was started at 30 °C, stirring speed 250 rpm, aeration 25 L per minute, and under a pressure of 0.05 MPa. After 48 hours, the stirring speed was changed to 500 rpm and cultivation was carried out for a total of 5 days. Then, the cultured product after cultivation was filtered and separated with Miracloth (Calbiochem), and the culture supernatant and cells were recovered. The recovered cells were washed with distilled water, and then the cells were sandwiched between paper towels to squeeze out the moisture to obtain wet cells. The obtained wet cells were weighed, and Tris buffer (20 mM Tris-HCl pH 8.0, 3 mM MgCl2, 0.1 mM ZnCl2) was added to make it 50 mg wet cells / ml. After homogenization treatment, a part was put into a 2 ml screw cap vial together with zirconia beads, and disruption at 2500 rpm for 20 seconds was repeated twice using a bead shocker (MS-100R, Tomy Seiko Co., Ltd.). Then, centrifugation was carried out at 15000×g for 5 minutes, and the obtained centrifugal supernatant was used as the intracellular fraction. The ALP activity of the obtained culture supernatant fraction and intracellular fraction was measured according to the description below of the above-mentioned "B. Activity measurement method", and the secretion rate (total activity in culture supernatant [U] ÷ (total activity in culture supernatant [U] + total activity in intracellular fraction [U]) × 100) was calculated (Figure 1).

[0070] [Comparative example. Production of various bIAPII in transformed S. pombe] The culture broth of the ALP-producing yeast S. pombe obtained in Example 5 of JP-A-2008-5734 was prepared in the same manner. The culture broth was centrifuged to separate it into a cell pellet and a culture supernatant fraction. Subsequently, the cell pellet was suspended in Tris buffer (10 mM Tris-HCl pH 7.0, 5 mM MgCl2, 0.1 mM ZnCl2), and the cells were disrupted with a bead beater to obtain an intracellular fraction. The ALP activity of the obtained culture supernatant fraction and intracellular fraction was measured by the same method as in Example 3, and the secretion rate was calculated in the same manner as in Example 3 (Figure 1).

[0071] When expressed in Aspergillus sojae, the secretion rates of various bIAPIIs (ALP names: bIAPII, Δ1, Δ1k, Δ13, and Δ1k3) corresponding to each DNA construct (DNA construct names: pbIAPII, pΔ1, pΔ1k, pΔ13, and pΔ1k3) were very high at 75% or more. However, when expressed in yeast S. pombe, the secretion rate of bIAPII was about 7%. In addition, in yeast expression, the secretion rate decreased as the number of N-glycosylation motifs decreased, but in Aspergillus expression, the secretion rate was always high regardless of the number of N-glycosylation motifs.

[0072] [Example 4. Purification of Various ALPs] The culture supernatant was centrifuged at 8000 rpm for 90 minutes to recover the supernatant, which was concentrated using an ultrafiltration membrane AIP-1010 (Asahi Kasei Corporation) and dialyzed with Tris buffer. The inner solution was recovered, and the filtrate that passed through a 0.45 μm filter (material: PES, Millipore Corporation) was collected. After adsorbing the sample onto a Q-Sepharose Fast Flow (GE Healthcare) anion exchange column chromatography equilibrated with Tris buffer, gradient elution was performed with Tris buffer containing 1 M NaCl. Ammonium sulfate was added to a final concentration of 30%, and the filtrate that passed through a 0.2 μm filter (material: PES, Millipore Corporation) was collected. Subsequently, after adsorbing the sample onto a TOYOPearl Butyl-650C (Tosoh Corporation) hydrophobic column chromatography equilibrated with Tris buffer containing 30% ammonium sulfate, gradient elution was performed with Tris buffer, and it was concentrated using an ultrafiltration membrane Amicon Ultra-15 (molecular weight cut-off 30 kDa, Millipore Corporation). Then, fractionation and purification were performed by gel filtration column chromatography on a Hiload Superdex 200 pg (GE Healthcare) equilibrated with Tris buffer.

[0073] [Example 5. Measurement of Specific Activity] The ALP activities of various purified ALPs were measured, the protein amount was determined by the absorbance at 280 nm, and the specific activity was calculated (Figure 2). bIAPII, Δ1, Δ1k, Δ13, and Δ1k3 expressed by Aspergillus showed high specific activities of 6000 U / mg or more.

[0074] [Example 6. Production of bIAPII by Transformed Aspergillus oryzae] Transformed Aspergillus oryzae-expressed bIAPII was prepared in the same procedure as in Examples 2 to 5, except that Aspergillus oryzae RIB40 strain was used instead of Aspergillus sojae NBRC4239 strain.

[0075] [Example 7. Measurement of Molecular Weight] The molecular weights of various purified ALPs were measured by gel filtration chromatography using HPLC (Agilent 1220 Infinity LC, Agilent). 50 μL of the purified ALP solution was applied to a TSK-gel G3000SW (7.5 mm I.D. × 30 cm, Tosoh) equilibrated with 100 mM phosphate buffer pH 7.0 containing 0.1 M NaCl, and eluted with the same buffer at a column temperature of 25°C and a flow rate of 1 mL / min. The absorbance at 280 nm was measured. A calibration curve was created using a molecular weight marker (MW-Marker proteins, Oriental Yeast Co., Ltd.), and the molecular weight of the purified ALP was calculated (Table 4). As a control, commercially available yeast recombinant bovine intestinal ALP (Alkaline Phosphatase recombinant highly active: Roche_ALP, Roche), deglycosylated yeast recombinant bovine intestinal ALP (Alkaline Phosphatase recombinant highly active, Carbohydrate Reduced: Roche_ALP_CR, Roche), and highly specific activity bovine intestinal extract-derived ALP (ALP13G, BBI solutions) were used.

[0076]

Table 4

[0077] The number of N - type glycosylation motifs in the amino acid sequence is three in bIAPII, two in Δ1 and Δ1k, and one in Δ13 and Δ1k3. The amount of sugar chains added to various ALPs is considered to be proportional to the number of N - type glycosylation motifs, and it is considered that the molecular weight increases according to the amount of sugar chains. In fact, the molecular weight of bIAPII expressed in yeast S. pombe described in JP - A - 2008 - 5734 is 1100 kDa, the molecular weight of Δ1k is 440 kDa, and the molecular weights of Δ13 and Δ1k3 are 120 kDa (Table 5 of JP - A - 2008 - 5734), and the molecular weight of ALP decreased according to the decrease in the number of N - type glycosylation motifs. On the other hand, the same tendency was also observed in various ALPs obtained by expression in Aspergillus sojae, and furthermore, the molecular weights of various ALPs expressed in Aspergillus sojae or A. oryzae were significantly lower than those of ALPs having the same number of N - type glycosylation motifs expressed in yeast S. pombe. The molecular weights of bovine intestinal alkaline phosphatase (Roche_ALP) expressed in commercially available Pichia Patoris, deglycosylated Roche_ALP_CR, and highly specific activity bovine intestinal alkaline phosphatase (ALP13G; BBI solutions) were 220 kDa, 145 kDa, and 131 kDa, respectively. The molecular weights of various ALPs expressed in Aspergillus were smaller than those of commercially available bovine intestinal alkaline phosphatase.

[0078] [Example 8. Preparation of DNA constructs inserted with bIAPIV fused with various secretion signal peptides] (8 - 1) Preparation of plasmids for constructs In the same procedure as the plasmid for the DNA construct inserted with CDHss - bIAPII in Example 1 - 1, a plasmid for the DNA construct inserted with bIAPIV fused with various secretion signal peptides was prepared.

[0079] (8 - 2) Preparation of DNA constructs inserted with bovine intestinal alkaline phosphatase IV (bIAPIV) gene A DNA construct in which the bIAPIV gene, which is the target gene, was ligated between Ptef and Talp of the plasmid for the construct was prepared as follows. The template DNA was the plasmid for construct obtained above, the PCR enzyme was KOD-Plus-DNA Polymerase (Toyobo), the reaction reagents were those included with this enzyme, and the equipment was T100. TM A vector fragment of the construct plasmid was obtained by performing inverse PCR using a thermal cycler (BIO-RAD) according to the protocol attached to the enzyme. The primers used are shown below. The amplified vector fragment was separated in a 1% (w / v) agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0080] [Table 5]

[0081] The bovine intestinal alkaline phosphatase IV (bIAPIV) gene (GenBank accession number sequence: AAC33854) was codon-optimized for Aspergillus oryzae, and a synthetic bIAPIV gene (SEQ ID NO: 60) was synthesized by contract (Genscript, Inc.) by removing the region encoding the C-terminal membrane-binding signal peptide. The synthetic bIAPIV gene obtained above was used as the template DNA, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) was used as the PCR enzyme, the reaction reagents were those attached to this enzyme, and the equipment was T100 TM PCR was performed using a thermal cycler (BIO-RAD) according to the protocol attached to the enzyme to obtain a bIAPIV synthetic gene DNA fragment. The primers used for amplification are shown below. Note that the sequences in lowercase indicate additional sequences for linking to the construct plasmid (between Ptef and Talp). The amplified DNA fragment was separated in a 1% (w / v) agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0082] [Table 6]

[0083] The amplified vector fragment and the bIAPIV synthetic gene DNA fragment as described above were ligated using the In-Fusion HD Cloning Kit according to the protocol attached to the kit to obtain a gene-inserted DNA construct (pbIAPIV) into which the bIAPIV synthetic gene was inserted. The DNA construct thus obtained is a ligation of a DNA fragment derived from pUC19, a DNA fragment of Ptef, a bIAPIV synthetic gene DNA fragment, a DNA fragment of Talp, a DNA fragment of pyrG, and a DNA fragment derived from pUC19 from upstream. That is, a DNA construct in which the sequences of Ptef-bIAPIV synthetic gene-Talp-pyrG were ligated in order was obtained at the In-Fusion Cloning Site of the pUC19 linearized Vector (Clontech). Thereafter, 1 / 10 volume of the DNA construct solution was mixed with Escherichia coli competent cells ECOS Competent E. coli JM109 (Nippon Gene) in ice water, left in ice water for 5 minutes, and then transformed by treatment at 42°C for 45 seconds. Thereafter, it was spread on an LB (Luria-Bertani) plate containing 50 μg / ml of ampicillin. This was left standing and cultured overnight at 37°C to form colonies. The obtained colonies were cultured with shaking overnight at 37°C in an LB liquid medium containing 50 μg / ml of ampicillin. After culturing, the culture solution was centrifuged to collect the cells. For the obtained cells, plasmid DNA (DNA construct) was extracted using the FastGene Plasmid Mini Kit (Nippon Genetics) according to the protocol attached to the kit. By determining the base sequences of each DNA inserted into the extracted plasmid DNA, it was confirmed that a DNA construct (pbIAPIV) into which the bIAPIV synthetic gene was inserted was obtained.

[0084] (8-3) Preparation of a DNA construct for inserting the bIAPIV synthetic gene having various secretion signal peptides The N-terminal secretory signal peptide encoded by the bIAPIV synthetic gene was replaced with the secretory signal peptides shown in Table 3 (including the secretory signal peptide as a carrier protein fused with the Kex2 cleavage linker), and various DNA constructs inserted with the bIAPIV synthetic gene were prepared as follows.

[0085]

Table 7

[0086] Using pbIAPIV as the template DNA, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, the reagents attached to this enzyme as the reaction reagents, and the T100 TM thermal cycler (BIO-RAD) to perform inverse PCR according to the protocol attached to this enzyme, a vector fragment of the plasmid for the construct was obtained in which the nucleotide sequence encoding the secretory signal peptide of pbIAPIV was removed. The primers used are shown below. The amplified vector fragment was separated in a 1% (w / v) agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen).

[0087]

Table 8

[0088] Using the chromosomal DNA obtained by the same operation as "[Example 1. Preparation of DNA Constructs Inserted with Gene AsEgtA, AsEgtB, or AsEgtC] (2) Extraction of Chromosomal DNA of Aspergillus sojae NBRC4239 Strain" in the pamphlet of International Publication No. 2016 / 121285 as the template, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, the reagents attached to this enzyme as the reaction reagents, and the T100 TMPCR was performed using a thermal cycler (BIO-RAD) according to the protocol attached to this enzyme. The primers used to amplify the genes of cellobiose dehydrogenase, glucoamylase B, and endoglucanase, which serve as carrier proteins (cdh, glab, and celb, respectively), are shown in the following table. By using each primer described in the following table, a DNA fragment encoding an amino acid sequence (SEQ ID NO: 23, 25, 27) in which a linker sequence (SEQ ID NO: 61) containing a Kex2 cleavage sequence is linked to the C-terminus of each carrier protein was obtained. The DNA fragments of the amplified CDH gene, GlaB gene, and CelB gene were separated in a 1% (w / v) agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0089]

Table 9

[0090] Using the In-Fusion HD Cloning Kit, DNA fragments of the CDH gene, GlaB gene, and CelB gene were ligated to vector fragments of construct plasmids, respectively, to obtain gene insertion DNA constructs (pCDHall, pGlaB, and pCelB) in which the CDH gene, GlaB gene, and CelB gene were inserted. The DNA constructs thus obtained are those in which, from the upstream, a pUC19-derived DNA fragment, a Ptef DNA fragment, DNA fragments of various carrier protein genes, a bIAPIV synthetic gene DNA fragment not containing a base encoding a secretory signal peptide, a Talp DNA fragment, a pyrG DNA fragment, and a pUC19-derived DNA fragment are ligated. That is, DNA constructs (pCDHall, pGlaB, and pCelB) were obtained in which the following sequences were linked in order to the In-Fusion Cloning Site of pUC19 linearized Vector (Clontech): Ptef, genes for various carrier proteins, a base sequence encoding a linker containing a Kex2 cleavage sequence, a bIAPIV synthetic gene not containing a base sequence encoding a secretory signal peptide, and the Talp-pyrG sequence.

[0091] The resulting DNA construct was used to transform E. coli JM109. It was then spread onto a Luria-Bertani (LB) plate containing 50 μg / ml ampicillin, and cultured overnight at 37° C. to form colonies. The colonies obtained were cultured overnight at 37°C with shaking in LB liquid medium containing 50 μg / ml ampicillin. The culture medium was centrifuged to collect the cells. Plasmid DNA (DNA construct) was extracted from the cells obtained using the FastGene Plasmid Mini Kit (Nihon Genetics) according to the protocol attached to the kit. By determining the base sequence of the DNA inserted into the extracted plasmid DNA, it was confirmed that the desired DNA constructs (pCDHall, pGlaB, and pCelB) were obtained.

[0092] Next, a DNA construct containing a bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with a secretion signal peptide (SEQ ID NO: 18) consisting of a linker sequence (SEQ ID NO: 61) containing a cellobiose dehydrogenase cytochrome b domain (CDHcytb) derived from Aspergillus sojae and a Kex2 cleavage sequence was prepared by the following procedure. Using pCDHall obtained above as the template DNA, primers of SEQ ID NOs: 71 and 72, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, reagents attached to this enzyme as the reaction reagents, and a T100 TM thermal cycler (BIO-RAD) and performing inverse PCR according to the protocol attached to this enzyme, a construct DNA fragment of CDHcytbkex-bIAPIV was obtained. The construct DNA fragment of CDHcytbkex_bIAPIV was transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was performed by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the cells. By determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA, it was confirmed that a DNA construct (pCDHcytbkex) was obtained.

[0093] Next, a DNA construct containing a bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with a secretion signal peptide derived from Aspergillus sojae cellobiose dehydrogenase (CDHss; SEQ ID NO: 17) was prepared by the following procedure. Using pCDHall obtained above as the template DNA, primers of SEQ ID NOs: 73 and 74, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, reagents attached to this enzyme as the reaction reagents, and a T100 TMInverse PCR was performed using a thermal cycler (BIO-RAD) according to the protocol attached to this enzyme to obtain a construct DNA fragment of CDHss-bIAPIV. The construct DNA fragment of CDHss_bIAPIV was transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was performed by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the cells. It was confirmed that a DNA construct (pCDHss) was obtained by determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA.

[0094] Next, a DNA construct containing a bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with a secretion signal (SKIK_CDHss; SEQ ID NO: 28) in which SKIK was inserted immediately after the initiation methionine of the secretion signal peptide of Aspergillus sojae cellobiose dehydrogenase (CDHss) was prepared by the following procedure. Using pCDHss obtained above as template DNA, KOD-Plus-DNA Polymerase (Toyobo) as the enzyme for PCR of primers of SEQ ID NOs: 75 and 76, reagents attached to this enzyme as reaction reagents, and T100 as the apparatus TM Inverse PCR was performed using a thermal cycler (BIO-RAD) according to the protocol attached to this enzyme to obtain a construct DNA fragment of SKIK_CDHss-bIAPIV. The construct DNA fragment of SKIK_CDHss-bIAPIV was transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was performed by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the cells. It was confirmed that a DNA construct (pSKIK_CDHss) was obtained by determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA.

[0095] Next, a DNA construct containing the bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with a secretion signal peptide (SEQ ID NOs: 19-22) consisting of a cellobiose dehydrogenase cytochrome b domain (CDHcytb) derived from Aspergillus sojae and a linker sequence (SEQ ID NOs: 62-65) containing Kex2 cleavage linker mutations 1-4 was prepared by the following procedure. Using pCDHcytbkex obtained above as the template DNA, primers of SEQ ID NOs: 77-81, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, reagents attached to this enzyme as the reaction reagents, and T100 TM as the apparatus, inverse PCR was carried out according to the protocol attached to this enzyme using a thermal cycler (BIO-RAD) to obtain construct DNA fragments of CDHcytbkexmut1_bIAPIV, CDHcytbkexmut2_bIAPIV, CDHcytbkexmut3_bIAPIV, and CDHcytbkexmut4_bIAPIV. The obtained construct DNA fragments were transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was carried out by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the bacterial cells. It was confirmed that DNA constructs (pCDHcytbkexmut1, pCDHcytbkexmut2, pCDHcytbkexmut3, pCDHcytbkexmut4) were obtained by determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA.

[0096] Next, a DNA construct containing the bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with a secretion signal peptide derived from Aspergillus sojae glucoamylase (SEQ ID NO: 24) was prepared by the following procedure. Using pbIAPIV obtained above as the template DNA, primers of SEQ ID NOs: 82 and 83, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, reagents attached to this enzyme as the reaction reagents, and T100 TMUsing a thermal cycler (BIO-RAD), inverse PCR was performed according to the protocol attached to this enzyme to obtain a construct DNA fragment of GlaBss_bIAPIV. The obtained construct DNA fragment was transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was performed by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the bacterial cells. By determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA, it was confirmed that a DNA construct (pGlaBss) was obtained.

[0097] Next, a DNA construct containing the bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with the secretion signal peptide derived from Aspergillus sojae endoglucanase (CelBss; SEQ ID NO: 26) was prepared by the following procedure. Using pCelB obtained above as the template DNA, primers of SEQ ID NOs: 84 and 85, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, reagents attached to this enzyme as the reaction reagents, and T100 as the apparatus TM Using a thermal cycler (BIO-RAD), inverse PCR was performed according to the protocol attached to this enzyme to obtain a construct DNA fragment of CelBss_bIAPIV. The construct DNA fragment of CelBss_bIAPIV was transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was performed by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the bacterial cells. By determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA, it was confirmed that a DNA construct (pCelBss) in which the gene encoding CelBss was inserted upstream of the bIAPIV gene was obtained.

[0098] Next, a DNA construct containing the bIAPIV synthetic gene in which the secretion signal peptide of bIAPIV was replaced with the secretion signal peptide derived from Aspergillus sojae alkaline phosphatase (SEQ ID NOs: 29 and 30) was prepared by the following procedure. Using pbIAPIV obtained above as the template DNA, KOD-Plus-DNA Polymerase (Toyobo Co., Ltd.) as the PCR enzyme, primers of SEQ ID NOs: 86 to 89, reagents attached to this enzyme as the reaction reagents, and T100 TM as the apparatus, inverse PCR was carried out according to the protocol attached to this enzyme using a thermal cycler (BIO-RAD) to obtain the construct DNA fragments of AsAP1ss_bIAPIV and AsAP3ss_bIAPIV. The obtained construct DNA fragments were transformed into Escherichia coli JM109 using the In-Fusion HD Cloning Kit. Plate culture was performed by the above method. After culturing the obtained colonies, plasmid DNA was recovered from the cells. It was confirmed that DNA constructs (pAsAP1ss54 and pAsAP3ss72) were obtained by determining the nucleotide sequence of the DNA inserted into the extracted plasmid DNA.

[0099] [Example 9. Preparation of Transformed Aspergillus sojae] Using the DNA constructs inserted with the bIAPIV synthetic genes having various secretion signal peptides obtained in Example 8, transformants of the pyrG disrupted strain derived from Aspergillus sojae NBRC4239 were obtained according to the method described in Example 2.

[0100] [Example 10. Flask Culture Test of Transformed Aspergillus sojae] In 20 ml of a liquid medium (2% (w / v) Hy-Polypeptone (Nippon Pharmaceutical Co., Ltd.), 2% (w / v) Pindex #2 (Matsutani Chemical Industry Co., Ltd.), 1% (w / v) yeast extract (Oriental Yeast Co., Ltd.), 0.25% (w / v) KH2PO4 (Wako Pure Chemical Industries, Ltd.), 0.25% (w / v) K2HPO4 (Wako Pure Chemical Industries, Ltd.), 0.05% (w / v) MgSO4·7H2O (Wako Pure Chemical Industries, Ltd.), 3 mM MgCl2 (Wako Pure Chemical Industries, Ltd.), 0.1 mM ZnCl2 (Wako Pure Chemical Industries, Ltd.); pH not adjusted) in a 50 mL Erlenmeyer flask, conidia of various transformants obtained in Example 9 were inoculated, and the culture was started at 30 °C with a stirring speed of 180 rpm and cultured for a total of 3 days. Then, the cultured product after culturing was filtered and separated with Miracloth (Calbiochem), and the culture supernatant and cells were recovered. The recovered cells were washed with distilled water, and then the cells were sandwiched between paper towels to squeeze out the moisture to obtain wet cells. The obtained wet cells were weighed, and Tris buffer (20 mM Tris-HCl pH 8.0, 3 mM MgCl2, 0.1 mM ZnCl2) was added to make it 50 mg wet cells / ml. After homogenization, a part was put into a 2 ml screw-cap vial together with zirconia beads, and disruption at 2500 rpm for 20 seconds was repeated twice using a bead shocker (MS-100R, Tomy Seiko Co., Ltd.). Then, centrifugation was performed at 15000×g for 5 minutes, and the obtained centrifugal supernatant was used as the intracellular fraction. The ALP activity of the obtained culture supernatant fraction and intracellular fraction was measured by the method described in JP-A-2008-5734, and the secretion rate (total activity in culture supernatant [U] ÷ (total activity in culture supernatant [U] + total activity in intracellular fraction [U]) × 100) was calculated (Figure 3).

[0101]

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

Table 36

[0102] This application claims priority based on Japanese Patent Application No. 2017-193624 filed on October 3, 2017, the content of which is incorporated herein by reference.

Industrial Applicability

[0103] According to the present invention, there is provided a method for producing ALP of a single isoform in which sugar chains are not excessively and non-uniformly added too much, with high productivity and secretory ability, and also provided are a novel ALPII, a vector, and a transformant, which are industrially useful.

Claims

1. A method for producing alkaline phosphatase (ALP), comprising a step of culturing a transformant of the genus Aspergillus having the ability to produce ALP, wherein the transformant of the genus Aspergillus has, on the 5'-terminal side with respect to the nucleotide sequence encoding ALP: A gene consisting of a nucleotide sequence to which any one of the following nucleotide sequences (a) and (c) is linked: (a) Any one of the nucleotide sequences of SEQ ID NOs: 2 to 8 and 11 to 15; (c) A nucleotide sequence having 90% or more homology with any one of the nucleotide sequences of SEQ ID NOs: 2 to 8 and 11 to 15 and encoding a secretory signal peptide having ALP secretion activity; Or A gene consisting of a nucleotide sequence to which any one of the following nucleotide sequences (d) and (f) encoding a secretory signal peptide is linked: (d) A secretory signal peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 17 to 23 and 26 to 30; (f) A secretory signal peptide having 90% or more homology with any one of the amino acid sequences of SEQ ID NOs: 17 to 23 and 26 to 30 and having ALP secretion activity; A method for producing ALP, which has.

2. ALP is an ALP containing a mutation in which no sugar chain is added to a part of the N-type sugar chain addition motif, The method for producing ALP according to claim 1.

3. ALP is ALP II or IV, The method for producing ALP according to claim 1 or 2.

4. ALP is any one of the following proteins (i) to (iii): (i) A protein consisting of the amino acid sequence of SEQ ID NO: 31 or 32; (ii) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 31 or 32 and having ALP activity; (iii) A protein having an amino acid sequence of SEQ ID NO: 31 or 32 and having a homology of 90% or more and having ALP activity; Or, A protein encoded by any of the following base sequences (iv) to (vi): (iv) The base sequence of SEQ ID NO: 33 or 34; (v) A base sequence consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of SEQ ID NO: 33 or 34 and encoding a protein having ALP activity; (vi) A base sequence having a homology of 90% or more with the base sequence of SEQ ID NO: 33 or 34 and encoding a protein having ALP activity; having, The method for producing ALP according to any one of claims 1 to 3.

5. ALP is ALP II which has a mutation in one or two of the three N-glycosylation addition motifs and no sugar chain is added to the N-glycosylation addition motif having the mutation, The method for producing ALP according to any one of claims 1 to 4.

6. In ALP II, the mutation in the N-glycosylation addition motif is one or two of positions 122, 249, 251, and 410 of SEQ ID NO: 31, The method for producing ALP according to claim 5.

7. In ALP II, the mutation in the N-glycosylation addition motif is one or two of the mutation from Asn to Gln or Lys at position 122, the mutation from Asn to Gln, Asp or His at position 249, the mutation from Thr to Cys at position 251, and the mutation from Asn to Gln or Lys at position 410 of SEQ ID NO: 31, The method for producing ALP according to claim 5 or 6.

8. The Aspergillus transformant is a transformant of any one of Aspergillus sojae, Aspergillus oryzae (Ahlburg) Cohn, Aspergillus luchuensis, Aspergillus niger, Aspergillus nidulans, Aspergillus tamarii, Aspergillus kawachii, Aspergillus awamori, Aspergillus usamii, or Aspergillus saitoi, The method for producing ALP according to any one of claims 1 to 7.

9. A step of obtaining a secreted fraction from a culture obtained by culturing an Aspergillus transformant, and A step of extracting ALP from the secreted fraction, further comprising The method for producing ALP according to any one of claims 1 to 8.

10. With respect to the nucleotide sequence encoding alkaline phosphatase (ALP), at its 5'-end side: A gene consisting of a nucleotide sequence to which any one of the following nucleotide sequences (a) and (c) is bound: (a) Any one of the nucleotide sequences of SEQ ID NOs: 2 to 8 and 11 to 15; (c) A nucleotide sequence having 90% or more homology with any one of the nucleotide sequences of SEQ ID NOs: 2 to 8 and 11 to 15 and encoding a secretion signal peptide having ALP secretion activity; or A gene consisting of a nucleotide sequence to which any one of the following secretion signal peptide-encoding nucleotide sequences (d) and (f) is bound: (d) A secretion signal peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 17 to 23 and 26 to 30; (f) A secretion signal peptide having 90% or more homology with any one of the amino acid sequences of SEQ ID NOs: 17 to 23 and 26 to 30 and having the secretion activity of ALP; A vector for Aspergillus transformation having the above.

11. An Aspergillus transformant transformed using the vector for Aspergillus transformation according to Claim 10.

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