DK2687597T3 - Modificeret alpha-glucosidase og anvendelser af samme - Google Patents
Modificeret alpha-glucosidase og anvendelser af samme Download PDFInfo
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- DK2687597T3 DK2687597T3 DK12758032.2T DK12758032T DK2687597T3 DK 2687597 T3 DK2687597 T3 DK 2687597T3 DK 12758032 T DK12758032 T DK 12758032T DK 2687597 T3 DK2687597 T3 DK 2687597T3
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Claims (13)
1. Modificeret α-glucosidase, i hvilken transglykosyleringsaktiviteten er forbedret og/eller balancen mellem hydrolyseaktiviteten og transglykosyleringsaktiviteten er ændret ved sænkning af hydrolyseaktiviteten sammenlignet med a-glucosidasen som skal modificeres, bestående afen aminosyresekvens i hvilken en eller to eller flere af aminosyrerne valgt fra gruppen bestående af de følgende (1) til (14) er blevet substitueret afen anden/andre aminosyrer i aminosyresekvensen af a-glucosidasen: (1) en aminosyre svarende til aminosyren i position 385 af aminosyresekvensen af SEQ ID NO. 1; (2) en aminosyre svarende til aminosyren i position 491 af aminosyresekvensen af SEQ ID NO. 1; (3) en aminosyre svarende til aminosyren i position 535 af aminosyresekvensen af SEQ ID NO. 1; (4) en aminosyre svarende til aminosyren i position 450 af aminosyresekvensen af SEQ ID NO. 1; (5) en aminosyre svarende til aminosyren i position 534 af aminosyresekvensen af SEQ ID NO. 1; (6) en aminosyre svarende til aminosyren i position 537 af aminosyresekvensen af SEQ ID NO. 1; (7) en aminosyre svarende til aminosyren i position 538 af aminosyresekvensen af SEQ ID NO. 1; (8) en aminosyre svarende til aminosyren i position 554 af aminosyresekvensen af SEQ ID NO. 1; (9) en aminosyre svarende til aminosyren i position 556 af aminosyresekvensen af SEQ ID NO. 1; (10) en aminosyre svarende til aminosyren i position 579 af aminosyresekvensen af SEQ ID NO. 2; (11) en aminosyre svarende til aminosyren i position 585 af aminosyresekvensen af SEQ ID NO. 2; (12) en aminosyre svarende til aminosyren i position 630 af aminosyresekvensen af SEQ ID NO. 1; (13) en aminosyre svarende til aminosyren i position 683 af aminosyresekvensen af SEQ ID NO. 1; og (14) en aminosyre svarende til aminosyren i position 689 af aminosyresekvensen af SEQ ID NO. 1, (a) hvor α-glucosidasen, som skal modificeres, er human maltase-glucoamylase, Aspergillus niger a/pfta-glucosidase, human neutral alpha-glucosidase C, muse-lysosomal alpha-glucosidase, gær GLU2A, Aspergillus nidulans a/pfta-glucosidase AgdA, Aspergillus nidulans A/pfta-glucosidase AgdB, Mucor javanicus alpha-glucosidase, Aspergillus oryzae alpha-glucosidase, Mortierella alliacea alpha-glucosidase, Schizosaccharomyces pombe alpha-glucosidase, Debaryomyces occidentalis alpha-giucosidase, Hordeum vulgare subsp. vulgare alpha-glucosidase, Arabidopsis thaliana alpha-glucosidase, Spinacia oleracea alpha-glucosidase, Beta vulgaris alpha-glucosidase eller Solanum tuberosum alpha-glucosidase, og/eller (b) hvor aminosyresekvensen af α-glucosidasen, som skal modificeres, er aminosyresekvensen af SEQ ID NO. 2 og aminosyren af (1) er aminosyren i position 343 af aminosyresekvensen, aminosyren af (2) er aminosyren i position 452 af aminosyresekvensen, aminosyren af (3) er aminosyren i position 496 af aminosyresekvensen, aminosyren af (4) er aminosyren i position 410 af aminosyresekvensen, aminosyren af (5) er aminosyren i position 495 af aminosyresekvensen, aminosyren af (6) er aminosyren i position 498 af aminosyresekvensen, aminosyren af (7) er aminosyren i position 499 af aminosyresekvensen, aminosyren af (8) er aminosyren i position 531 af aminosyresekvensen, aminosyren af (9) er aminosyren i position 533 af aminosyresekvensen, aminosyren af (12) er aminosyren i position 662 af aminosyresekvensen, aminosyren af (13) er aminosyren i position 715 af aminosyresekvensen, og aminosyren af (14) er aminosyren i position 721 af aminosyresekvensen.
2. Den modificerede α-glucosidase ifølge krav 1(b), hvor aminosyren, som skal substitueres, er (1) aminosyren af (1), og aminosyren er blevet substitueret af cystein, asparaginsyre, methionin, histidin, alanin, phenylalanin, glycin, threonin, glutaminsyre, valin, glutamin, asparagin eller isoleucin; (2) aminosyren af (2), og aminosyren er blevet substitueret af glycin, asparaginsyre eller glutaminsyre; (3) aminosyren af (3), og aminosyren er blevet substitueret af valin, asparagin eller glutamin; (4) aminosyren af (1) og aminosyren af (2), og aminosyrerne er blevet substitueret af asparaginsyre for aminosyren af (1) og alanin eller glycin for aminosyren af (2); (5) aminosyren af (1) og aminosyren af (3), og aminosyrerne er blevet substitueret af asparaginsyre eller methionin for aminosyren af (1) og isoleucin, arginin, cystein eller threonin for aminosyren af (3); (6) aminosyren af (5), og aminosyren er blevet substitueret af glycin, proline eller valin; eller (7) aminosyren af (6), og aminosyren er blevet substitueret af leucin eller serin.
3. Den modificerede α-glucosidase ifølge krav 1, (1) hvilken består af en hvilken som helst af aminosyresekvenserne af SEQ ID Nos. 18 til 42 og 77 til 78; eller (2) hvilken består af en hvilken som helst af aminosyresekvenserne af SEQ ID Nos. 74 til 76.
4. Gen, som koder for den modificerede α-glucosidase ifølge et hvilket som helst af kravene 1 til 3.
5. Genet ifølge krav 4, hvilket omfatter en hvilken som helst af basesekvenserne af SEQ ID Nos. 43 til 67 og 79 til 83.
6. Rekombinant DNA omfattende genet ifølge krav 4 eller 5.
7. Mikroorganisme med det rekombinante DNA ifølge krav 6.
8. Enzymmiddel omfattende den modificerede α-glucosidase ifølge et hvilket som helst af kravene 1 til 3.
9. Fremgangsmåde til fremstilling af et oligosaccharid, omfattende at reagere (i) den modificerede α-glucosidase ifølge et hvilket som helst af kravene 1, 2 (1) til 2 (5), 2 (7) eller 3 (1) med et oligosaccharid eller polysaccharid af di- eller flere saccharider med a-1,4 glucosidbinding, eventuelt omfattende anvendelse af et vild type-enzym i kombination; (ii) den modificerede α-glucosidase ifølge krav 2 (6) eller 3 (2) med et oligosaccharid eller polysaccharid af di- eller flere saccharider med a-1,6 glucosidbinding, eventuelt omfattende anvendelse af et vild type-enzym i kombination; eller (iii) den modificerede α-glucosidase ifølge krav 2 (6) eller 3 (2) og et vild type-enzym med et oligosaccharid eller polysaccharid af di- eller flere saccharider med a-1,4 glucosidbinding(er).
10. Farmaceutisk sammensætning, en kvasi-lægemiddelssammensætning, en kosmetisk sammensætning, en fødevaresammensætning eller en fodersammensætning, omfattende den modificerede α-glucosidase ifølge et hvilket som helst af kravene 1 til 3 eller enzymmidlet ifølge krav 8.
11. Fremgangsmåde til fremstilling afen modificeret α-glucosidase i hvilken transglykosyleringsaktiviteten er forbedret og/eller balancen mellem hydrolyseaktiviteten og transglykosyleringsaktiviteten ændres ved sænkning af hydrolyseaktiviteten sammenlignet med α-glucosidase som skal modificeres, omfattende de følgende trin (i) og (ii): (i) et trin til at identificere en eller to eller flere aminosyrer valgt fra gruppen bestående af de følgende (1) til (14) i aminosyresekvensen af a-glucosidase som et enzym der skal muteres: (1) en aminosyre svarende til aminosyren i position 385 af aminosyresekvensen af SEQ ID NO. 1; (2) en aminosyre svarende til aminosyren i position 491 af aminosyresekvensen af SEQ ID NO. 1; (3) en aminosyre svarende til aminosyren i position 535 af aminosyresekvensen af SEQ ID NO. 1; (4) en aminosyre svarende til aminosyren i position 450 af aminosyresekvensen af SEQ ID NO. 1; (5) en aminosyre svarende til aminosyren i position 534 af aminosyresekvensen af SEQ ID NO. 1; (6) en aminosyre svarende til aminosyren i position 537 af aminosyresekvensen af SEQ ID NO. 1; (7) en aminosyre svarende til aminosyren i position 538 af aminosyresekvensen af SEQ ID NO. 1; (8) en aminosyre svarende til aminosyren i position 554 af aminosyresekvensen af SEQ ID NO. 1; (9) en aminosyre svarende til aminosyren i position 556 af aminosyresekvensen af SEQ ID NO. 1; (10) en aminosyre svarende til aminosyren i position 579 af aminosyresekvensen af SEQ ID NO. 2; (11) en aminosyre svarende til aminosyren i position 585 af aminosyresekvensen af SEQ ID NO. 2; (12) en aminosyre svarende til aminosyren i position 630 af aminosyresekvensen af SEQ ID NO. 1; (13) en aminosyre svarende til aminosyren i position 683 af aminosyresekvensen af SEQ ID NO. 1; og (14) en aminosyre svarende til aminosyren i position 689 af aminosyresekvensen af SEQ ID NO. 1; (ii) et trin til at danne en aminosyresekvens i hvilken aminosyrerne identificeret i trin (i) er blevet substitueret med en anden/andre aminosyrer, baseret på aminosyresekvensen af enzymet der skal muteres.
12. Fremgangsmåden ifølge krav 11, hvor a-glucosidasen (1) er human ma/tase-glucoamylase, Aspergillus niger a/påa-glucosidase, human neutral a/påa-glucosidase C, muse-lysosomal alpha-glucosidase, gær GLU2A, Aspergillus nidulans A/påa-glucosidase AgdA, Aspergillus nidulans A/påa-glucosidase AgdB, Mucor javanicus a/påa-glucosidase, Aspergillus oryzae a/påa-glucosidase, Mortierella alliacea a/påa-glucosidase, Schizosaccharomyces pombe a/påa-glucosidase, Debaryomyces occidentalis a/påa-glucosidase, Hordeum vulgare subsp. vulgare a/påa-glucosidase, Arabidopsis thaliana a/påa-glucosidase, Spinacia oleracea alpha-glucosidase, Beta vulgaris alpha-glucosidase eller Solanum tuberosum a/påa-glucosidase; (2) omfatter en hvilken som helst af aminosyresekvenserne af SEQ ID Nos. 1 til 17; eller (3) består af aminosyresekvensen af SEQ ID NO. 2, og aminosyrer der er substitueret i trin (i) er én aminosyre valgt fra (1) til (14), to aminosyrer valgt fra (1) til (14) eller tre aminosyrer valgt fra (1) til (14).
13. Fremgangsmåde til fremstilling afen modificeret α-glucosidase, omfattende de følgende trin (I) til (III): (I) et trin til at fremstille en nukleinsyre der koder for en hvilken som helst af aminosyresekvenserne af SEQ ID Nos. 18 til 42 og 74 til 78 eller en aminosyresekvens sammensat ved fremgangsmåden ifølge et hvilket som helst af kravene 11 eller 12; (II) et trin til at udtrykke nukleinsyren, og (III) et trin til at opsamle et ekspressionsprodukt.
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| JP2011057386 | 2011-03-16 | ||
| PCT/JP2012/055558 WO2012124520A1 (ja) | 2011-03-16 | 2012-03-05 | 改変型α-グルコシダーゼ及びその用途 |
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| EP3211076A4 (en) * | 2014-10-20 | 2018-03-21 | Showa Sangyo Co., Ltd. | Novel alpha-glucosidase |
| CN104480127B (zh) * | 2014-12-14 | 2017-04-05 | 长春中医药大学 | 超嗜热糖苷酶突变体及其在人参皂苷ck制备中的应用 |
| CN105925550B (zh) * | 2016-06-23 | 2019-06-04 | 福州大学 | α-葡萄糖苷酶及同步糖化转苷制备低聚异麦芽糖的方法 |
| DK3635009T3 (da) | 2017-06-07 | 2026-03-30 | Regeneron Pharma | Sammensætninger og fremgangsmåder til internalisering af enzymer |
| US12258597B2 (en) | 2018-02-07 | 2025-03-25 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for therapeutic protein delivery |
| BR112020021962A2 (pt) | 2018-04-30 | 2021-01-26 | Amicus Therapeutics, Inc. | construtos para terapia gênica e métodos de uso |
| MY207355A (en) | 2018-05-16 | 2025-02-21 | Spark Therapeutics Inc | Codon-optimized acid lpha-glucosidase expression cassettes and methods of using same |
| BR112020023145A2 (pt) | 2018-05-17 | 2021-02-02 | Regeneron Pharmaceuticals, Inc. | anticorpo anti-cd63 ou fragmento de ligação ao antígeno do mesmo, molécula de ligação ao antígeno biespecífica, proteína terapêutica de múltiplos domínios, polinucleotídeo composição farmacêutica, e, composto |
| CN113631182B (zh) | 2018-10-10 | 2025-03-21 | 阿米库斯治疗学公司 | 二硫键稳定的多肽组合物和使用方法 |
| CN109576246B (zh) * | 2019-01-11 | 2021-01-29 | 江南大学 | 一种α-葡萄糖苷酶突变体及其应用 |
| CN111500558B (zh) * | 2019-01-31 | 2022-01-25 | 东莞泛亚太生物科技有限公司 | 具提升活性的葡萄糖苷酶 |
| CN110004128A (zh) * | 2019-03-18 | 2019-07-12 | 中粮集团有限公司 | 复合糖化酶制剂和淀粉糖化的方法 |
| CN112695021B (zh) * | 2020-12-02 | 2023-02-28 | 南京工业大学 | 一种α-糖苷酶基因突变体及在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用 |
| CN113980932B (zh) * | 2021-07-15 | 2023-08-01 | 暨南大学 | 一种定点突变的α-葡萄糖苷酶 |
| WO2025164587A1 (ja) * | 2024-01-29 | 2025-08-07 | 天野エンザイム株式会社 | 改変型トランスグルコシダーゼ |
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| JP2001046096A (ja) | 1999-08-09 | 2001-02-20 | Lotte Co Ltd | α−グルコシダーゼによる配糖体の製造方法及び新規なα−グルコシダーゼ並びにその製造方法 |
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| JP2009022204A (ja) | 2007-07-19 | 2009-02-05 | Hokkaido Univ | 改変デキストラングルコシダーゼ及びその製造方法 |
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| US9493753B2 (en) | 2016-11-15 |
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| WO2012124520A1 (ja) | 2012-09-20 |
| JP5992902B2 (ja) | 2016-09-14 |
| US20170009218A1 (en) | 2017-01-12 |
| EP2687597A4 (en) | 2014-12-10 |
| EP2687597B1 (en) | 2018-12-26 |
| CN103443273A (zh) | 2013-12-11 |
| EP2687597A1 (en) | 2014-01-22 |
| US20140087405A1 (en) | 2014-03-27 |
| CN103443273B (zh) | 2016-10-26 |
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