JP7433246B2 - Thermostable isoamylase - Google Patents
Thermostable isoamylase Download PDFInfo
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- 108010028688 Isoamylase Proteins 0.000 title claims description 98
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 31
- 102000004190 Enzymes Human genes 0.000 claims description 24
- 229940088598 enzyme Drugs 0.000 claims description 24
- 230000035772 mutation Effects 0.000 claims description 24
- 108090000790 Enzymes Proteins 0.000 claims description 23
- 229920002472 Starch Polymers 0.000 claims description 21
- 235000019698 starch Nutrition 0.000 claims description 21
- 239000008107 starch Substances 0.000 claims description 21
- 150000001413 amino acids Chemical class 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102100022624 Glucoamylase Human genes 0.000 claims description 10
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 8
- 125000000539 amino acid group Chemical group 0.000 claims description 7
- 108090000637 alpha-Amylases Proteins 0.000 claims description 6
- 108010019077 beta-Amylase Proteins 0.000 claims description 6
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- 108010065511 Amylases Proteins 0.000 description 6
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000019418 amylase Nutrition 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 4
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- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
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- 238000003756 stirring Methods 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 229920000945 Amylopectin Polymers 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
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Description
本発明は、耐熱性が向上した変異イソアミラーゼ、及びその変異イソアミラーゼの製造法に関する。 The present invention relates to a mutant isoamylase with improved heat resistance and a method for producing the mutant isoamylase.
糖化工業において、デンプンやアミロペクチン中のα－１，６－グルコピラノシド結合を加水分解する酵素として、Klebsiella pneumoniaeなどが生産するプルラナーゼ及びイソアミラーゼが知られている。このうち、イソアミラーゼは、デンプン、アミロペクチン、グリコーゲン中のα－１，６－グルコピラノシド結合を加水分解する酵素であり、可逆的な反応が見られないため、他のアミラーゼやグルコアミラーゼなどを使用することで高純度のグルコースやマルトースが生産できることが知られている。イソアミラーゼ生産菌としては、Pseudomonas amyloderamosa（非特許文献１）などが報告されている。 In the saccharification industry, pullulanase and isoamylase produced by Klebsiella pneumoniae and the like are known as enzymes that hydrolyze α-1,6-glucopyranoside bonds in starch and amylopectin. Among these, isoamylase is an enzyme that hydrolyzes α-1,6-glucopyranoside bonds in starch, amylopectin, and glycogen, and since there is no reversible reaction, other amylases or glucoamylases are used. It is known that highly pure glucose and maltose can be produced by this process. Pseudomonas amyloderamosa (Non-Patent Document 1) and the like have been reported as isoamylase-producing bacteria.
しかし、Pseudomonas amyloderamosaなどが生産するイソアミラーゼの至適温度は他のアミラーゼの至適温度よりも低く、産業的に使用するレベルの反応温度帯では併用が困難であった。 However, the optimal temperature of isoamylase produced by Pseudomonas amyloderamosa and the like is lower than that of other amylases, and it has been difficult to use them together in the reaction temperature range of industrial use.
このような背景の下、本出願人は、イソアミラーゼのアミノ酸配列の一部を改変することによって耐熱性を向上できることを見出し、先に特許出願している（特許文献１）。耐熱性を向上するべく、新たな変異点を見い出すことが求められた。
従って、本発明の課題は、耐熱性の向上に寄与する別の変異点を見出し、新たなイソアミラーゼ、及びその製造法を提供することにある。
Against this background, the present applicant has discovered that heat resistance can be improved by modifying part of the amino acid sequence of isoamylase, and has previously filed a patent application (Patent Document 1). In order to improve heat resistance, it was necessary to find a new mutation point.
Therefore, an object of the present invention is to find another mutation point that contributes to improved heat resistance, and to provide a new isoamylase and a method for producing the same.
本発明者は、Pseudomonas amyloderamosaなどが生産するイソアミラーゼのアミノ酸配列のうち、後述する特定の位置のアミノ酸を他のアミノ酸に変異させることによって、耐熱性が一層向上した変異イソアミラーゼが得られることを見出し、本発明を完成した。 The present inventor has found that a mutant isoamylase with further improved heat resistance can be obtained by mutating the amino acids at specific positions described below to other amino acids in the amino acid sequence of isoamylase produced by Pseudomonas amyloderamosa and the like. The present invention has been completed.
すなわち、本発明は、以下の［１］～［１１］を提供するものである。 That is, the present invention provides the following [1] to [11].
［１］配列番号１で表されるアミノ酸配列からなるイソアミラーゼ、又は配列番号１で表されるアミノ酸配列において１～数個のアミノ酸残基が欠失、置換又は挿入されたアミノ酸配列であって、配列番号１で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるイソアミラーゼにおいて、Ｄ２６８Ｓ、Ａ２４１Ｔ及びＭ５７４Ｖから選ばれる１又は２以上のアミノ酸変異を有するイソアミラーゼ。
［２］前記配列同一性が９０％以上である［１］記載のイソアミラーゼ。
［３］さらに、Ａ５５４Ｐ、Ｍ２７７Ｉ、Ａ５４９Ｐ及びＡ５８０Ｔから選ばれる１又は２以上のアミノ酸変異を有する［１］又は［２］記載のイソアミラーゼ。
［４］さらに、Ａ５５４Ｐ、Ｍ２７７Ｉ、Ａ５４９Ｐ及びＡ５８０Ｔのアミノ酸変異を有する［１］又は［２］記載のイソアミラーゼ。
［５］アミノ酸変異が、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ、及びＡ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖから選ばれる変異を含む変異である［１］～［４］のいずれか１記載のイソアミラーゼ。
［６］［１］～［５］のいずれか１記載のイソアミラーゼをコードする遺伝子。
［７］［６］記載の遺伝子を有する組み換えベクター。
［８］［７］記載の組み換えベクターで形質転換した形質転換体。
［９］［８］記載の形質転換体を培養して、該培養物からイソアミラーゼを採取することを特徴とするイソアミラーゼの製造法。
［１０］［１］～［５］のいずれか１記載のイソアミラーゼを含有するデンプン糖化用酵素組成物。
［１１］さらに、β－アミラーゼ、α－アミラーゼ及びグルコアミラーゼから選ばれる酵素を含有する［１０］記載のデンプン糖化用酵素組成物。
[1] An isoamylase consisting of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one to several amino acid residues are deleted, substituted, or inserted in the amino acid sequence represented by SEQ ID NO: 1. , an isoamylase consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, which has one or more amino acid mutations selected from D268S, A241T, and M574V.
[2] The isoamylase according to [1], wherein the sequence identity is 90% or more.
[3] The isoamylase according to [1] or [2], further having one or more amino acid mutations selected from A554P, M277I, A549P, and A580T.
[4] The isoamylase according to [1] or [2], further having amino acid mutations of A554P, M277I, A549P, and A580T.
[5] A mutation in which the amino acid mutation includes a mutation selected from A554P/M277I/D268S/A549P/A580T, A554P/M277I/D268S/A549P/A580T/A241T, and A554P/M277I/D268S/A549P/A580T/A241T/M574V The isoamylase according to any one of [1] to [4].
[6] A gene encoding the isoamylase according to any one of [1] to [5].
[7] A recombinant vector having the gene described in [6].
[8] A transformant transformed with the recombinant vector described in [7].
[9] A method for producing isoamylase, which comprises culturing the transformant according to [8] and collecting isoamylase from the culture.
[10] An enzyme composition for starch saccharification containing the isoamylase according to any one of [1] to [5].
[11] The enzyme composition for starch saccharification according to [10], further comprising an enzyme selected from β-amylase, α-amylase, and glucoamylase.
本発明のイソアミラーゼは、ネイティブのイソアミラーゼと比べて耐熱性が２℃以上向上している（一重変異体）。本発明のイソアミラーゼは、この耐熱性を６℃以上向上させることもできる（五重変異体～七重変異体）。そして、本発明のイソアミラーゼの至適温度は他の各種アミラーゼの至適温度と重複する。従って、本発明のイソアミラーゼは、各種アミラーゼと併用してデンプン等に作用させることにより、高純度のグルコースやマルトース等を工業的に有利に生産することができる。本発明において「併用」とは、本発明のイソアミラーゼと、それ以外の１種以上の酵素を混合した状態において、少なくとも２種以上の酵素が活性を示す状態にあることをいう。 The isoamylase of the present invention has improved thermostability by 2°C or more compared to native isoamylase (single mutant). The isoamylase of the present invention can also improve this thermostability by 6°C or more (quintuple to sevenfold mutants). The optimum temperature of the isoamylase of the present invention overlaps with the optimum temperature of other various amylases. Therefore, the isoamylase of the present invention can advantageously industrially produce highly purified glucose, maltose, etc. by acting on starch etc. in combination with various amylases. In the present invention, "combination" refers to a state in which the isoamylase of the present invention and one or more other enzymes are mixed, and at least two or more enzymes exhibit activity.
本発明のイソアミラーゼは、配列番号１で表されるアミノ酸配列からなるイソアミラーゼ、又は配列番号１で表されるアミノ酸配列において１～数個のアミノ酸残基が欠失、置換又は挿入されたアミノ酸配列であって、配列番号１で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるイソアミラーゼにおいて、Ｄ２６８Ｓ、Ａ２４１Ｔ及びＭ５７４Ｖから選ばれる１又は２以上のアミノ酸変異を有するイソアミラーゼである。 The isoamylase of the present invention is an isoamylase consisting of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid in which one to several amino acid residues are deleted, substituted, or inserted in the amino acid sequence represented by SEQ ID NO: 1. An isoamylase consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and having one or more amino acid mutations selected from D268S, A241T and M574V. It is amylase.
ここで、配列番号１で表されるアミノ酸配列からなるイソアミラーゼは、非特許文献１に記載された、Pseudomonas amyloderamosaが産生するイソアミラーゼである。このイソアミラーゼには、同一のアミノ酸配列を有する限り、Pseudomonas amyloderamosa由来でないイソアミラーゼが含まれる。また、同一アミノ酸配列を有する限り、ポリペプチドだけでなく、糖ペプチドも含まれる。なお、配列番号１は、成熟タンパク質のアミノ酸配列を示す。 Here, the isoamylase consisting of the amino acid sequence represented by SEQ ID NO: 1 is an isoamylase produced by Pseudomonas amyloderamosa , which is described in Non-Patent Document 1. This isoamylase includes isoamylases that are not derived from Pseudomonas amyloderamosa as long as they have the same amino acid sequence. Furthermore, as long as they have the same amino acid sequence, not only polypeptides but also glycopeptides are included. Note that SEQ ID NO: 1 shows the amino acid sequence of the mature protein.
配列番号１で表されるアミノ酸配列において１～数個のアミノ酸残基が欠失、置換又は挿入されたイソアミラーゼにおける、アミノ酸残基の欠失、置換又は挿入の数は、配列番号１で表されるアミノ酸配列からなるイソアミラーゼと同等の酵素活性を示すものであれば限定されないが、１～２０個が好ましく、１～１０個がさらに好ましく、１～８個がさらに好ましい。
また、当該欠失、置換又は挿入されたイソアミラーゼと配列番号１のアミノ酸配列との配列同一性は、８０％以上が好ましく、８５％以上がより好ましく、９０％以上がさらに好ましく、９５％以上がさらに好ましく、９９％以上がさらに好ましい。このような配列の同一性パーセンテージは、基準配列を照会配列として比較するアルゴリズムをもった公開又は市販されているソフトウエアを用いて計算することができる。例として、ＢＬＡＳＴ、ＦＡＳＴＡ又はＧＥＮＥＴＹＸ（ソフトウエア開発社製）などを用いることができる。
The number of deletions, substitutions, or insertions of amino acid residues in the isoamylase in which one to several amino acid residues have been deleted, substituted, or inserted in the amino acid sequence represented by SEQ ID NO: 1 is as shown in SEQ ID NO: 1. Although there are no limitations as long as the enzymatic activity is equivalent to that of the isoamylase consisting of the amino acid sequence shown in FIG.
Further, the sequence identity between the deleted, substituted or inserted isoamylase and the amino acid sequence of SEQ ID NO: 1 is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and 95% or more. is more preferable, and even more preferably 99% or more. The percentage identity of such sequences can be calculated using publicly available or commercially available software with algorithms that compare a reference sequence to a query sequence. For example, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) can be used.
本発明のイソアミラーゼは、Ｄ２６８Ｓ、Ａ２４１Ｔ及びＭ５７４Ｖから選ばれる１又は２以上のアミノ酸変異を有するが、耐熱性向上の観点から、さらにＡ５５４Ｐ、Ｍ２７７Ｉ、Ａ５４９Ｐ及びＡ５８０Ｔから選ばれる１又は２以上のアミノ酸変異を有するのが好ましい。本発明における好ましいアミノ酸変異は、Ｄ２６８Ｓ、Ａ２４１Ｔ及びＭ５７４Ｖからから選ばれる１又は２以上の変異と、Ａ５５４Ｐ、Ｍ２７７Ｉ、Ａ５４９Ｐ及びＡ５８０Ｔの４変異を含む５～７重変異である。 The isoamylase of the present invention has one or more amino acid mutations selected from D268S, A241T, and M574V, and from the viewpoint of improving heat resistance, it further has one or more amino acid mutations selected from A554P, M277I, A549P, and A580T. It is preferable to have a mutation. Preferred amino acid mutations in the present invention are one or more mutations selected from D268S, A241T, and M574V, and quintuple to sevenfold mutations including four mutations: A554P, M277I, A549P, and A580T.
具体的なアミノ酸変異としては、Ｄ２６８Ｓ、Ａ２４１Ｔ、Ｍ５７４Ｖ、Ｄ２６８Ｓ／Ａ２４１Ｔ、Ｄ２６８Ｓ／Ｍ５７４Ｖ、Ａ２４１Ｔ／Ｍ５７４Ｖ、Ｄ２６８Ｓ／Ａ２４１Ｔ／Ｍ５７４Ｖ、Ｄ２６８Ｓ／Ａ５５４Ｐ、Ｄ２６８Ｓ／Ｍ２７７Ｉ、Ｄ２６８Ｓ／Ａ５４９Ｐ、Ｄ２６８Ｓ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ａ５５４Ｐ、Ａ２４１Ｔ／Ｍ２７７Ｉ、Ａ２４１Ｔ／Ａ５４９Ｐ、Ａ２４１Ｔ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ａ５５４Ｐ、Ｍ５７４Ｖ／Ｍ２７７Ｉ、Ｍ５７４Ｖ／Ａ５４９Ｐ、Ｍ５７４Ｖ／Ａ５８０Ｔ、Ｄ２６８Ｓ／Ａ５５４Ｐ／Ｍ２７７Ｉ、Ｄ２６８Ｓ／Ａ５５４Ｐ／Ａ５４９Ｐ、Ｄ２６８Ｓ／Ａ５５４Ｐ／Ａ５８０Ｔ、Ｄ２６８Ｓ／Ｍ２７７Ｉ／Ａ５４９Ｐ、Ｄ２６８Ｓ／Ｍ２７７Ｉ／Ａ５８０Ｔ、Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ａ５５４Ｐ／Ｍ２７７Ｉ、Ａ２４１Ｔ／Ａ５５４Ｐ／Ａ５４９Ｐ、Ａ２４１Ｔ／Ａ５５４Ｐ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ｍ２７７Ｉ／Ａ５４９Ｐ、Ａ２４１Ｔ／Ｍ２７７Ｉ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ａ５５４Ｐ／Ｍ２７７Ｉ、Ｍ５７４Ｖ／Ａ５５４Ｐ／Ａ５４９Ｐ、Ｍ５７４Ｖ／Ａ５５４Ｐ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ｍ２７７Ｉ／Ａ５４９Ｐ、Ｍ５７４Ｖ／Ｍ２７７Ｉ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ｄ２６８Ｓ／Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５４９Ｐ、Ｄ２６８Ｓ／Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５８０Ｔ、Ｄ２６８Ｓ／Ａ５５４Ｐ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ｄ２６８Ｓ／Ｍ２７７Ｉ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５４９Ｐ、Ａ２４１Ｔ／Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ａ５５４Ｐ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ２４１Ｔ／Ｍ２７７Ｉ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５４９Ｐ、Ｍ５７４Ｖ／Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ａ５５４Ｐ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ｍ５７４Ｖ／Ｍ２７７Ｉ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ｍ５７４Ｖ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ｍ５７４Ｖ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖ等が挙げられる。
さらに好ましいアミノ酸変異としては、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ、Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖが挙げられる。
Specific amino acid mutations include D268S, A241T, M574V, D268S/A241T, D268S/M574V, A241T/M574V, D268S/A241T/M574V, D268S/A554P, D268S/M277I, D268S/A549P, D268S/A5 80T, A241T/ A554P, A241T/M277I, A241T/A549P, A241T/A580T, M574V/A554P, M574V/M277I, M574V/A549P, M574V/A580T, D268S/A554P/M277I, D268S/A554P/A54 9P, D268S/A554P/A580T, D268S/ M277I/A549P, D268S/M277I/A580T, D268S/A549P/A580T, A241T/A554P/M277I, A241T/A554P/A549P, A241T/A554P/A580T, A241T/M277I/A549P, A24 1T/M277I/A580T, A241T/A549P/ A580T, M574V/A554P/M277I, M574V/A554P/A549P, M574V/A554P/A580T, M574V/M277I/A549P, M574V/M277I/A580T, M574V/A549P/A580T, D268S/A55 4P/M277I/A549P, D268S/A554P/ M277I/A580T, D268S/A554P/A549P/A580T, D268S/M277I/A549P/A580T, A241T/A554P/M277I/A549P, A241T/A554P/M277I/A580T, A241T/A554P/A54 9P/A580T, A241T/M277I/A549P/ A580T, M574V/A554P/M277I/A549P, M574V/A554P/M277I/A580T, M574V/A554P/A549P/A580T, M574V/M277I/A549P/A580T, A554P/M277I/D268S/A54 9P/A580T, A554P/M277I/A549P/ A580T/A241T, A554P/M277I/A549P/A580T/M574V, A554P/M277I/D268S/A549P/A580T/A241T, A554P/M277I/D268S/A549P/A580T/M574V, A554P/M27 7I/A549P/A580T/A241T/M574V, Examples include A554P/M277I/D268S/A549P/A580T/A241T/M574V.
More preferred amino acid mutations include A554P/M277I/D268S/A549P/A580T, A554P/M277I/D268S/A549P/A580T/A241T, and A554P/M277I/D268S/A549P/A580T/A241T/M574V.
本発明の変異イソアミラーゼは、配列番号１で表されるアミノ酸配列からなるイソアミラーゼ、又は配列番号１で表されるアミノ酸配列において１～数個のアミノ酸残基が欠失、置換又は挿入されたアミノ酸配列であって、配列番号１で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるイソアミラーゼにおいて、Ｄ２６８Ｓ、Ａ２４１Ｔ及びＭ５７４Ｖから選ばれる１又は２以上をアミノ酸変異することにより構築した遺伝子を用いて製造することができる。 The mutant isoamylase of the present invention is an isoamylase consisting of the amino acid sequence represented by SEQ ID NO: 1, or one to several amino acid residues are deleted, substituted, or inserted in the amino acid sequence represented by SEQ ID NO: 1. In an isoamylase consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids selected from D268S, A241T and M574V are mutated. It can be produced using a gene constructed by.
本発明の変異イソアミラーゼを製造するための遺伝子は、前記の変異イソアミラーゼをコードする塩基配列を有する遺伝子であり、例えば前記の配列番号１で示されるアミノ酸配列をコードする遺伝子において、置換すべきアミノ酸配列をコードする塩基配列を、所望のアミノ酸残基をコードする塩基に置換することにより構築することができる。このような部位特異的塩基配列置換のための種々の方法は、当該技術分野においてよく知られており、例えば適切に設計されたプライマーを用いるＰＣＲによって行うことができる。あるいは、改変型のアミノ酸配列をコードする遺伝子を全合成してもよい。 The gene for producing the mutant isoamylase of the present invention is a gene having a nucleotide sequence encoding the mutant isoamylase, for example, in the gene encoding the amino acid sequence shown in SEQ ID NO: 1, which should be replaced. It can be constructed by substituting a base sequence encoding an amino acid sequence with a base encoding a desired amino acid residue. Various methods for such site-specific base sequence substitution are well known in the art, and can be performed, for example, by PCR using appropriately designed primers. Alternatively, a gene encoding a modified amino acid sequence may be totally synthesized.
このようにして得た遺伝子を適当な発現ベクターに挿入し、これを適当な宿主（例えば大腸菌）に形質転換する。外来性蛋白質を発現させるための多くのベクター・宿主系が当該技術分野において知られている。変異イソアミラーゼ遺伝子を組み込むための発現ベクターとしては、プラスミドベクターが挙げられ、例えば大腸菌用としてはｐＥＴ－１４ｂ、ｐＢＲ３２２等が挙げられる。枯草菌用としては、ｐＵＢ１１０等が挙げられる。糸状菌用としては、ｐＰＴＲＩ等が挙げられる。また、酵母用としては、ｐＲＳ４０３等が挙げられる。 The gene thus obtained is inserted into an appropriate expression vector, and this is transformed into an appropriate host (eg, E. coli). Many vector-host systems for expressing foreign proteins are known in the art. Examples of expression vectors for incorporating the mutant isoamylase gene include plasmid vectors, such as pET-14b and pBR322 for E. coli. Examples for Bacillus subtilis include pUB110. Examples for filamentous fungi include pPTRI. Further, examples for yeast include pRS403 and the like.
得られた組み換えプラスミドは、大腸菌、枯草菌、カビ、酵母、放線菌、酢酸菌、シュードモナス属菌等の微生物に形質転換し、当該形質転換体を培養すれば、本発明変異イソアミラーゼが得られる。当該形質転換体は、変異イソアミラーゼ遺伝子をプラスミドのまま保持しても良いし、ゲノム内に取り込んでも良い。 The obtained recombinant plasmid is transformed into microorganisms such as Escherichia coli, Bacillus subtilis, mold, yeast, actinomycetes, acetic acid bacteria, Pseudomonas bacteria, etc., and the transformant is cultured to obtain the mutant isoamylase of the present invention. . The transformant may retain the mutant isoamylase gene as a plasmid or may incorporate it into the genome.
本発明のイソアミラーゼは、Pseudomonas amyloderamosaなどが生産するイソアミラーゼに比べて耐熱性が２℃以上向上しており、かつ至適ｐＨ、イソアミラーゼ活性等はPseudomonas amyloderamosaなどが生産するイソアミラーゼと同等である。特に、本発明のイソアミラーゼは、この耐熱性を６℃以上向上させることも可能である（五重変異体～七重変異体）。従って、デンプンに、β－アミラーゼ、α－アミラーゼ、グルコアミラーゼなどから選ばれる酵素と、本発明イソアミラーゼとを作用させれば、高純度のグルコースやマルトースが容易に得られる。ここで、β－アミラーゼとしては、ＧＯＤＯ－ＧＢＡ２（合同酒精株式会社）、オプチマルトＢＢＡ（ダニスコジャパン株式会社）、β－アミラーゼＬ／Ｒ（ナガセケムテックス株式会社）、ハイマルトシンＧＬ（エイチビィアイ株式会社）等を用いることができる。また、α－アミラーゼとしては、例えば、クライスターゼＴ１０（大和化成株式会社）を用いることができる。グルコアミラーゼとしては、例えば、グルクザイム（天野エンザイム株式会社）、ＧＯＤＯ－ＡＮＧＨ（合同酒精株式会社）を用いることができる。 The isoamylase of the present invention has improved heat resistance by 2°C or more compared to isoamylase produced by Pseudomonas amyloderamosa , etc., and has the same optimum pH, isoamylase activity, etc. as the isoamylase produced by Pseudomonas amyloderamosa , etc. be. In particular, the isoamylase of the present invention can improve this thermostability by 6°C or more (quintuple to sevenfold mutants). Therefore, highly pure glucose or maltose can be easily obtained by allowing an enzyme selected from β-amylase, α-amylase, glucoamylase, etc., and the isoamylase of the present invention to act on starch. Here, examples of β-amylase include GODO-GBA2 (Godo Shusei Co., Ltd.), Optimalt BBA (Danisco Japan Co., Ltd.), β-amylase L/R (Nagase ChemteX Co., Ltd.), Hymaltocin GL (HBI Co., Ltd.), etc. can be used. Furthermore, as the α-amylase, for example, clystase T10 (Daiwa Kasei Co., Ltd.) can be used. As glucoamylase, for example, gluczyme (Amano Enzyme Co., Ltd.) and GODO-ANGH (Godo Shusei Co., Ltd.) can be used.
本発明のイソアミラーゼは、糖化用のイソアミラーゼとして使用することが好ましく、デンプン糖化用のイソアミラーゼとして使用することがさらに好ましい。
本発明のイソアミラーゼは、必要により他の１種以上の酵素と混合したデンプン糖化用酵素組成物とすることも好ましい。他の１種以上の酵素は、上述したβ－アミラーゼ、α－アミラーゼ、グルコアミラーゼから選択することができる。
The isoamylase of the present invention is preferably used as an isoamylase for saccharification, and more preferably used as an isoamylase for starch saccharification.
It is also preferable that the isoamylase of the present invention is mixed with one or more other enzymes as necessary to form an enzyme composition for starch saccharification. The other one or more enzymes can be selected from the above-mentioned β-amylases, α-amylases, and glucoamylases.
反応は、例えば、アミラーゼ等のデンプン糖化酵素に本発明のイソアミラーゼを添加し、当該デンプン糖化酵素と当該イソアミラーゼが作用するｐＨ、温度条件にて混合撹拌することにより行なわれる。本発明方法によれば、高純度のグルコースやマルトースを工業的に有利に製造することができる。 The reaction is carried out, for example, by adding the isoamylase of the present invention to a starch saccharifying enzyme such as amylase, and mixing and stirring under pH and temperature conditions where the starch saccharifying enzyme and the isoamylase act. According to the method of the present invention, highly purified glucose and maltose can be industrially advantageously produced.
次に実施例を挙げて本発明をより詳細に説明するが、本発明は何らこれに限定されるものではない。 EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.
実施例１（イソアミラーゼの部位特異的変異導入）
Pseudomonas amyloderamosaゲノムを鋳型としてプライマーＰＳＴＰＩＡ－Ｆ（AAACTGCAGATGAAGTGCCCAAAGATTCTC（配列番号２））及びＨＩＮＤＰＩＡ－Ｒ（CCCAAGCTTCTACTTGGAGATCAACAGCAG（配列番号３））を用いて耐酸性イソアミラーゼ遺伝子配列を含む約２．３ｋｂの断片を取得した。この断片を制限酵素Ｐｓｔ Ｉ及びＨｉｎｄ IIIで消化し、ｐＨＳＧ３９８（タカラバイオ株式会社）を制限酵素Ｐｓｔ Ｉ及びＨｉｎｄ IIIで消化した約２．２ｋｂの断片と連結することにより、ｐ－ＰＩを取得した。ネイティブの耐酸性イソアミラーゼの発現プラスミドであるプラスミドｐ－ＰＩに部位特異的変異導入を行い、一重変異体（Ｄ２６８Ｓ）発現プラスミドｐ－ＰＩＤ２６８Ｓを取得した。同様に一重変異体（Ａ２４１Ｔ）発現プラスミドｐ－ＰＩＡ２４１Ｔ、一重変異体（Ｍ５７４Ｖ）発現プラスミドｐ－ＰＩＭ５７４Ｖ、五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ａ／Ａ５４９Ｐ／Ａ５８０Ｔ）発現プラスミドｐ－ＰＩ５Ｍを取得した。また、ｐ－ＰＩ５Ｍに部位特異的変異導入を行い、最適化 五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ）発現プラスミドｐ－ＰＩ５Ｍｓ、六重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ）発現プラスミドｐ－ＰＩ６Ｍ、七重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖ）発現プラスミドｐ－ＰＩ７Ｍを取得した。
Example 1 (site-directed mutagenesis of isoamylase)
An approximately 2.3 kb fragment containing the acid-resistant isoamylase gene sequence was obtained using the Pseudomonas amyloderamosa genome as a template and primers PSTPIA-F (AAACTGCAGATGAAGTGCCCAAAGATTCTC (SEQ ID NO: 2)) and HINDPIA-R (CCCAAGCTTCTACTTGGAGATCAACAGCAG (SEQ ID NO: 3)). . This fragment was digested with restriction enzymes Pst I and Hind III, and p-PI was obtained by ligating it with an approximately 2.2 kb fragment obtained by digesting pHSG398 (Takara Bio Inc.) with restriction enzymes Pst I and Hind III. . Site-directed mutagenesis was performed on plasmid p-PI, which is an expression plasmid for native acid-resistant isoamylase, to obtain a single mutant (D268S) expression plasmid p-PID268S. Similarly, a single mutant (A241T) expression plasmid p-PIA241T, a single mutant (M574V) expression plasmid p-PIM574V, and a quintuple mutant (A554P/M277I/D268A/A549P/A580T) expression plasmid p-PI5M were obtained. In addition, site-directed mutagenesis was introduced into p-PI5M, and optimization was performed. A580T/A241T) expression plasmid p-PI6M and heptad mutant (A554P/M277I/D268S/A549P/A580T/A241T/M574V) expression plasmid p-PI7M were obtained.
実施例２（酵素の生成）
ネイティブ耐酸性イソアミラーゼ発現プラスミドｐ－ＰＩ、一重変異体（Ｄ２６８Ｓ）発現プラスミドｐ－ＰＩＤ２６８Ｓ、一重変異体（Ａ２４１Ｔ）発現プラスミドｐ－ＰＩＡ２４１Ｔ、一重変異体（Ｍ５７４Ｖ）発現プラスミドｐ－ＰＩＭ５７４Ｖ、五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ａ／Ａ５４９Ｐ／Ａ５８０Ｔ）発現プラスミドｐ－ＰＩ５Ｍ、最適化 五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ）発現プラスミドｐ－ＰＩ５Ｍｓ、六重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ）発現プラスミドｐ－ＰＩ６Ｍ、七重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖ）発現プラスミドｐ－ＰＩ７Ｍにより大腸菌ＤＨ５α株を形質転換し、それぞれのイソアミラーゼを生産する大腸菌株を取得した。これらの大腸菌を３０μｇ／ｍＬのクロラムフェニコールを含むＬＢ培地（酵母エキス ０．５％、トリプトン１．０％、塩化ナトリウム ０．５％、ＩＰＴＧ ０．１ｍＭ ｐＨ７．２）で、３０℃３日培養し、培養液１Ｌを得た。超音波により菌体を破砕後、遠心分離（１０，０００ｇ、１０分間）を行い、上清をＵＦ濃縮（旭化成社製 ＡＩＰモジュール）により、１，０００Ｕ／ｍＬとなるように濃縮した。これらを０．２μｍのポアサイズの膜で除菌することにより、ネイティブ耐酸性イソアミラーゼ、一重変異体（Ｄ２６８Ｓ）イソアミラーゼ、一重変異体（Ａ２４１Ｔ）イソアミラーゼ、一重変異体（Ｍ５７４Ｖ）イソアミラーゼ、五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ａ／Ａ５４９Ｐ／Ａ５８０Ｔ）イソアミラーゼ、最適化 五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ）イソアミラーゼ、六重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ）イソアミラーゼ、七重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖ）イソアミラーゼの酵素溶液とした。
Example 2 (Enzyme production)
Native acid-resistant isoamylase expression plasmid p-PI, single mutant (D268S) expression plasmid p-PID268S, single mutant (A241T) expression plasmid p-PIA241T, single mutant (M574V) expression plasmid p-PIM574V, quintuple mutant (A554P/M277I/D268A/A549P/A580T) expression plasmid p-PI5M, optimized Quintuplet mutant (A554P/M277I/D268S/A549P/A580T) expression plasmid p-PI5Ms, sextuple mutant (A554P/M277I/ D268S/A549P/A580T/A241T) expression plasmid p-PI6M, heptad mutant (A554P/M277I/D268S/A549P/A580T/A241T/M574V) expression plasmid p-PI7M was used to transform E. coli DH5α strain, and each isoamylase We obtained an E. coli strain that produces . These E. coli were incubated at 30°C in LB medium (yeast extract 0.5%, tryptone 1.0%, sodium chloride 0.5%, IPTG 0.1mM pH 7.2) containing 30 μg/mL chloramphenicol. The cells were cultured for 1 day to obtain 1 L of culture solution. After crushing the bacterial cells with ultrasound, centrifugation (10,000 g, 10 minutes) was performed, and the supernatant was concentrated to 1,000 U/mL using UF concentration (AIP module manufactured by Asahi Kasei Corporation). By sterilizing these with a membrane with a pore size of 0.2 μm, native acid-resistant isoamylase, single mutant (D268S) isoamylase, single mutant (A241T) isoamylase, single mutant (M574V) isoamylase, and five Double mutant (A554P/M277I/D268A/A549P/A580T) isoamylase, optimized Quintuplet mutant (A554P/M277I/D268S/A549P/A580T) isoamylase, sextuple mutant (A554P/M277I/D268S/A549P/ An enzyme solution of A580T/A241T) isoamylase and a 7-fold mutant (A554P/M277I/D268S/A549P/A580T/A241T/M574V) isoamylase were prepared.
実施例３（各変異体の熱安定性向上）
実施例２で調製した各酵素溶液を４０℃、５０℃、５５℃、５７．５℃、６０℃、６２．５℃、６５℃に１０分間保ったのちに氷冷し、残存活性を測定した。各温度における残存活性のプロットより作成された近似式を用いて残存活性が５０％になる温度を算出し、この温度をネイティブ耐酸性イソアミラーゼと比較した場合に、増加する度合いを耐熱化度とした。七重変異体のアミノ酸配列を配列番号４に示す。
Example 3 (Improvement of thermal stability of each mutant)
Each enzyme solution prepared in Example 2 was kept at 40°C, 50°C, 55°C, 57.5°C, 60°C, 62.5°C, and 65°C for 10 minutes, cooled on ice, and residual activity was measured. . The temperature at which the residual activity becomes 50% is calculated using an approximate formula created from a plot of the residual activity at each temperature, and when this temperature is compared with that of the native acid-resistant isoamylase, the degree of increase is defined as the degree of thermostability. did. The amino acid sequence of the heptad mutant is shown in SEQ ID NO: 4.
＜活性測定方法＞
イソアミラーゼの活性測定法は、以下のとおりである。
０．５％ワキシーコーンスターチ溶液０．３５ｍＬに、０．５Ｍの酢酸緩衝液（ｐＨ４．５）０．１ｍＬを混合し、適時希釈した酵素液を０．１ｍＬ加え、４５℃で１５分間反応させる。その後、０．１Ｎ ＨＣｌにて５倍希釈したヨード溶液（０．０５Ｍヨウ素を含む０．５Ｍヨウ化カリウム溶液）０．５ｍＬを加えて酵素反応を止め、１０ｍＬの水を加えて十分に撹拌した後、分光光度計を用いて６１０ｎｍで測定する。酵素活性の単位は、上記条件下で１分間に０．０１吸光度を増加する酵素量を１単位とした。
その結果、図１及び表１に示すように、一重変異体（Ｄ２６８Ｓ）で約２．９℃、一重変異体（Ａ２４１Ｔ）で約２．１℃、一重変異体（Ｍ５７４Ｖ）で約３．４℃、五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ａ／Ａ５４９Ｐ／Ａ５８０Ｔ）で約６．０℃、最適化五重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ）で約６．８℃、六重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ）で約７．１℃、七重変異体（Ａ５５４Ｐ／Ｍ２７７Ｉ／Ｄ２６８Ｓ／Ａ５４９Ｐ／Ａ５８０Ｔ／Ａ２４１Ｔ／Ｍ５７４Ｖ）で約８．２℃の耐熱化度の向上が確認された。
<Activity measurement method>
The method for measuring the activity of isoamylase is as follows.
Mix 0.1 mL of 0.5 M acetate buffer (pH 4.5) with 0.35 mL of 0.5% waxy corn starch solution, add 0.1 mL of appropriately diluted enzyme solution, and react at 45° C. for 15 minutes. Thereafter, 0.5 mL of an iodine solution (0.5 M potassium iodide solution containing 0.05 M iodine) diluted 5 times with 0.1 N HCl was added to stop the enzyme reaction, and 10 mL of water was added and thoroughly stirred. Afterwards, it is measured at 610 nm using a spectrophotometer. The unit of enzyme activity was defined as the amount of enzyme that increases the absorbance by 0.01 per minute under the above conditions.
As a result, as shown in Figure 1 and Table 1, the single mutant (D268S) was approximately 2.9°C, the single mutant (A241T) was approximately 2.1°C, and the single mutant (M574V) was approximately 3.4°C. °C, about 6.0°C for the quintuple mutant (A554P/M277I/D268A/A549P/A580T), and about 6.8°C for the optimized quintuple mutant (A554P/M277I/D268S/A549P/A580T), sextuplet. The degree of heat resistance is approximately 7.1°C for the mutant (A554P/M277I/D268S/A549P/A580T/A241T) and approximately 8.2°C for the heptad mutant (A554P/M277I/D268S/A549P/A580T/A241T/M574V). improvement was confirmed.
実施例４（デンプンの糖化）
デンプンの糖化として、還元糖遊離試験を行った。１０ｍＭ酢酸緩衝液（ｐＨ４．５）に溶性デンプンを３０％（重量/重量）となるように加温溶解し、（１）０．０５ｍｇ/ｍＬに溶解したグルコアミラーゼ（和光純薬株式会社製）を３０％溶性デンプン１ｇあたり２０ｍｇ及び五重変異体イソアミラーゼを３０％溶性デンプン１ｇあたり１２５Ｕ添加したもの、（２）グルコアミラーゼを３０％溶性デンプン１ｇあたり２０ｍｇ及び六重変異体イソアミラーゼを３０％溶性デンプン１ｇあたり１２５Ｕ添加したもの、（３）グルコアミラーゼを３０％溶性デンプン１ｇあたり２０ｍｇ及び七重変異体イソアミラーゼを３０％溶性デンプン１ｇあたり１２５Ｕ添加したものそれぞれを５５℃、６０℃、６２．５℃で１６時間反応させた。
１００℃、５分間加熱し反応を停止させ、生成した還元糖量について、以下に示すＤＮＳ法にて測定した。
Example 4 (Saccharification of starch)
As for starch saccharification, a reducing sugar release test was conducted. Dissolve soluble starch in 10 mM acetate buffer (pH 4.5) by heating to a concentration of 30% (weight/weight), and (1) glucoamylase (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved at 0.05 mg/mL. (2) glucoamylase at 20 mg/g of 30% soluble starch and quintuple mutant isoamylase at 125 U/g of 30% soluble starch; (2) glucoamylase at 20 mg/g of 30% soluble starch and 30% sextuple mutant isoamylase; (3) 20 mg of glucoamylase per 1 g of 30% soluble starch and 125 U of 7-fold mutant isoamylase per 1 g of 30% soluble starch were added at 55°C, 60°C, and 62.5°C, respectively. The reaction was carried out at ℃ for 16 hours.
The reaction was stopped by heating at 100° C. for 5 minutes, and the amount of reducing sugar produced was measured by the DNS method shown below.
＜測定方法（ＤＮＳ法）＞
還元糖量の測定法は、以下のとおりである。
ＤＮＳ（３，５－ジニトロサリチル酸）溶液１．５ｍＬに、適時希釈したサンプル液を０．５ｍＬ加えた後に撹拌し、沸騰水中で５分間反応させる。水冷後、４ｍＬの水を加えて十分に撹拌した後、分光光度計を用いて５４０ｎｍで測定する。還元糖量はグルコース溶液を用いて作製した検量線より算出した。ＤＮＳ溶液は、１％ＤＮＳ溶液４４００ｍＬに４．５％水酸化ナトリウム溶液を１５００ｍＬとロッセル塩１２７５ｇ溶解した後、別に調製したフェノール溶液（１％フェノール、２．４４％水酸化ナトリウム）を３４５ｍＬと、炭酸水素ナトリウムを３４．５ｇ加えて撹拌溶解し、２日間暗所保存後、ろ紙にて濾過したものを使用した。
その結果を表２に示した。五重変異体イソアミラーゼを用いた５５℃反応において得られた還元糖量を１００％としたとき、六重変異体イソアミラーゼを用いた際は１０２％、七重変異体イソアミラーゼを用いた際は１０２％に向上した。また、６０℃反応においては、五重変異体イソアミラーゼでは１００％と変化がないのに対して、六重変異体イソアミラーゼを用いた際は１０２％、七重変異体イソアミラーゼでは１０５％に向上した。さらに、６２．５℃反応においては、五重変異体イソアミラーゼでは９８％に低下したのに対して、六重変異体イソアミラーゼを用いた際は９９％、七重変異体イソアミラーゼでは９９％であった。したがって、五重変異体イソアミラーゼに比べて、六重変異体又は七重変異体イソアミラーゼを使用した場合の方が明らかに還元糖生成量の向上が認められた。さらに、５５℃から６０℃の温度上昇に伴って五重変異体イソアミラーゼは枝切り効果に変化が無いのに対し、六重変異体又は七重変異体イソアミラーゼは枝切り効果が向上し、歩留りの向上が認められたことから、変異による熱安定性効果が確認された。
<Measurement method (DNS method)>
The method for measuring the amount of reducing sugar is as follows.
Add 0.5 mL of the appropriately diluted sample solution to 1.5 mL of DNS (3,5-dinitrosalicylic acid) solution, stir, and react in boiling water for 5 minutes. After cooling with water, 4 mL of water is added and stirred thoroughly, followed by measurement at 540 nm using a spectrophotometer. The amount of reducing sugar was calculated from a calibration curve prepared using a glucose solution. The DNS solution was prepared by dissolving 1500 mL of 4.5% sodium hydroxide solution and 1275 g of Rosselle's salt in 4400 mL of 1% DNS solution, then adding 345 mL of a separately prepared phenol solution (1% phenol, 2.44% sodium hydroxide), 34.5 g of sodium hydrogen carbonate was added and dissolved with stirring, stored in the dark for 2 days, filtered through filter paper, and used.
The results are shown in Table 2. When the amount of reducing sugar obtained in the 55°C reaction using the 5-fold mutant isoamylase is taken as 100%, when the 6-fold mutant isoamylase is used, it is 102%, and when the 7-fold mutant isoamylase is used, it is 102%. This has improved to 102%. In addition, in the 60°C reaction, the 5-fold mutant isoamylase showed no change at 100%, while the 6-fold mutant isoamylase improved to 102%, and the 7-fold mutant isoamylase improved to 105%. did. Furthermore, in the reaction at 62.5°C, the rate decreased to 98% with the 5-fold mutant isoamylase, 99% with the 6-fold mutant isoamylase, and 99% with the 7-fold mutant isoamylase. there were. Therefore, compared to the five-fold mutant isoamylase, it was clearly observed that the amount of reducing sugar produced was improved when the six-fold or seven-fold mutant isoamylase was used. Furthermore, as the temperature increases from 55°C to 60°C, the debranching effect of the 5-fold mutant isoamylase does not change, whereas the debranching effect of the 6-fold or 7-fold mutant isoamylase improves and the yield increases. Since an improvement in the temperature was observed, the thermostability effect of the mutation was confirmed.
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| WO2017204317A1 (en) | 2016-05-27 | 2017-11-30 | 合同酒精株式会社 | Heat resistant isoamylase |
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| WO2017204317A1 (en) | 2016-05-27 | 2017-11-30 | 合同酒精株式会社 | Heat resistant isoamylase |
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