University of Groningen Biochemical characterization of β-galactosidases and engineering of their product specificity Yin, Huifang

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University of Groningen

Biochemical characterization of β-galactosidases and engineering of their product specificity

Yin, Huifang

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Publication date: 2017

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Yin, H. (2017). Biochemical characterization of β-galactosidases and engineering of their product specificity. University of Groningen.

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512682-L-bw-Yin 512682-L-bw-Yin 512682-L-bw-Yin 512682-L-bw-Yin Processed on: 17-8-2017 Processed on: 17-8-2017 Processed on: 17-8-2017

Processed on: 17-8-2017 PDF page: 191PDF page: 191PDF page: 191PDF page: 191

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ѝ᮷᪈㾱 192 վ㚊ॺң㌆ᱟ⭡н਼Ⲵॺң㌆অݳ䘈৏ㄟ䘎᧕ањ㪑㨴㌆ᡆॺң㌆࠶ ᆀᶴᡀⲴˈ㚊ਸᓖа㡜ѪҼࡠॱњঅսǄഐѪᆳԜо⇽ңѝⲴሑ㚊㌆ᴹ㊫ լⲴ⳺⭏ݳ࣏㜭ˈվ㚊ॺң㌆ᐢ㓿㻛ᒯ⌋␫࣐ࡠႤᒬݯྦ㊹઼伏૱ѝǄ_β-ॺ ң㌆㤧䞦൘儈⎃ᓖⲴң㌆֌Ѫᓅ⢙ᰦਟԕۜॆ䖜㌆ส৽ᓄ⭏ᡀվ㚊ॺң㌆ˈ ਇࡠҶᒯ⌋Ⲵޣ⌘઼⹄ウǄн਼Ⲵ _{β-ॺң㌆㤧䞦ӗ⭏Ⲵվ㚊ॺң㌆൘ᡀ࠶ǃ} 㔃ᶴǃ㚊ਸᓖǃԕ৺䘎᧕䭞රкᆈ൘ᖸབྷᐞᔲǄⴞࡽሩ_{β-ॺң㌆㤧䞦ӗ⢙} ⢩ᔲᙗⲴ㔃ᶴส⹰䘈н␵ᾊǄᵜ䇪᮷࡙⭘สҾ㔃ᶴⲴॺ⨶ᙗケਈᒦ㔃ਸሩ ӗ⢙㔃ᶴⲴ䈖㓶䀓᷀⹄ウҶ_{β-ॺң㌆㤧䞦Ⲵӗ⢙㔃ᶴ৺ަӗ⢙⢩ᔲᙗⲴ㔃} ᶴส⹰Ǆ ᡁԜ俆ݸ⹄ウሩ∄Ҷй⿽൘ᐕъкᓄ⭘ᒯ⌋Ⲵβ-ॺң㌆㤧䞦ۜॆӗ⭏ Ⲵվ㚊ॺң㌆Ⲵӗ䟿઼ᡀ࠶ˈᆳԜ࠶࡛ᶕⓀҾBacillus circulans,

Kluyveromyces lactis ઼ Aspergillus oryzaeǄ ⹄ウ㔃᷌㺘᰾ˈᶕⓀҾ Bacillus circulans Ⲵ β-ॺң㌆㤧䞦⭏ӗվ㚊ॺң㌆Ⲵӗ䟿ᴰ儈ˈ㘼фվ㚊ॺң㌆Ⲵ

㔃ᶴҏᴰѠᇼǄᡁԜӾᶕⓀҾB. circulans, K. lactis ઼ A. oryzae Ⲵ β-ॺң㌆ 㤧䞦ӗ⭏Ⲵӗ⢙ѝ࠶࡛࠶⿫䢤ᇊҶ 21ǃ12 ઼ 11 ⿽н਼Ⲵվ㚊ॺң㌆㔃ᶴǄ ᶕⓀҾB. circulans Ⲵ β-ॺң㌆㤧䞦ӗ⭏Ⲵվ㚊ॺң㌆ѫ㾱䭞රवᤜ(β1→4)ǃ (β1→2)ǃ(β1→3)઼(β1→6)Ǆަѝˈ(β1→4)ᱟӗ⢙ѝᴰѠᇼⲴ䭞රǄᶕⓀҾ K. lactis ઼ A. oryzae Ⲵ β-ॺң㌆㤧䞦ӗ⢙ѝ(β1→6)ᱟᴰѫ㾱Ⲵ䭞රǄሩҾ й⿽_{β-ॺң㌆㤧䞦ѫ㾱ӗ⢙Ⲵࣘᘱ䐏䑚㺘᰾ˈվ㚊ॺң㌆Ⲵᡀ࠶ѫ㾱ਆߣ} Ҿ䞦ⲴᶕⓀ઼৽ᓄᰦ䰤Ǆ਼ᰦˈᡁԜ⭘13C ḷ䇠Ⲵॺң㌆઼㪑㨴㌆᧒㍒Ҷᆳ Ԝሩ䞦৽ᓄ䗷〻Ⲵᖡ૽Ǆ㔃᷌㺘᰾ˈሩҾᶕⓀҾ_{B. circulans ઼ K. lactis} β-ॺң㌆㤧䞦ˈ≤䀓৽ᓄӗ⭏Ⲵॺң㌆઼㪑㨴㌆䜭֌Ѫਇփᓅ⢙৲оҶ䖜㌆ ส䞦৽ᓄˈ⭏ᡀҶ䘈৏ㄟнѪң㌆Ⲵሑ㌆ӗ⢙ǄሩᶕⓀҾ_{A. oryzae Ⲵ β-ॺ} ң㌆㤧䞦ᶕ䈤ˈ㪑㨴㌆৲оҶ৽ᓄˈ㘼ॺң㌆ᱟ䖜㌆ส৽ᓄⲴᣁࡦࡲǄ ަ⅑ᡁԜԕᶕⓀҾ Bacillus circulans Ⲵ β-ॺң㌆㤧䞦Ѫ⹄ウሩ䊑䘹ਆҶ սҾ⍫ᙗѝᗳⲴ≘ส䞨↻สᒦ䘋㹼Ҷᇊ⛩ケਈˈᒦф⹄ウ઼䀓᷀Ҷケਈփ ᡰӗ⭏Ⲵվ㚊ॺңሑ㌆ˈԕ䱀᰾ަӗ⢙⢩ᔲᙗⲴ㔃ᶴส⹰Ǆ⹄ウ㔃᷌㺘᰾ սҾ⍫ᙗѝᗳⲴArg484 ሩҾվ㚊ॺң㌆Ⲵ䭞රᴹ⵰䶎ᑨޣ䭞Ⲵ֌⭘Ǆ価઼ ケਈ㺘᰾ᡰᴹ൘䈕ս⛩Ⲵケਈ䜭᭩ਈҶӗ⢙Ⲵ䭞රˈӾ㘼ӗ⭏Ҷਜ਼ᴹн਼ 㓴࠶Ⲵվ㚊ॺң㌆ӗ⢙ǄNMR ࠶᷀㺘᰾ˈ䘉⿽ӗ⢙Ⲵѫ㾱䭞රᱟ(β1→3)઼ (β1→4)ˈ㘼䟾⭏ර β-ॺң㌆㤧䞦ѫ㾱ӗ⢙ᱟ(β1→4)䭞රǄᡁԜ䙊䗷Ṩ⻱ޡ ᥟ˄_{NMR˅ǃ儈᭸⿫ᆀ㢢䉡˄HPAEC-PAD˅઼ส䍘䖵ࣙ◰ݹ䀓᷀⭥⿫伎}

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㹼ᰦ䰤䍘䉡˄MALDI-TOF-MS˅䢤ᇊҶ 10 ⿽ޘᯠⲴվ㚊ॺң㌆㔃ᶴˈӾ㘼 ֯վ㚊ॺң㌆Ⲵ㔃ᶴᮠᦞᓃӾ_{60 ⿽ᢙབྷࡠҶ 70 ⿽Ǆ䘉ҏᱟ俆⅑ᣕ䚃࡙⭘} 䞦࠶ᆀ᭩䙐Ⲵᯩ⌅᭩ਈ_{β-ॺң㌆㤧䞦Ⲵӗ⢙⢩ᔲᙗǄ਼ᰦˈᡁԜ⹄ウҶ༴} Ҿ⍫ᙗս⛩ѝᗳⲴަԆ8 њ≘ส䞨↻ส(Arg185, Asp481, Lys487, Tyr511, Trp570, Trp593, Glu601, and Phe616)Ⲵ֌⭘Ǆ≘ส䞨↻ส Arg185 ઼ Glu601 սҾ-1 ս⛩ˈ൘ GH2 ᇦ᯿Ⲵ β-ॺң㌆㤧䞦ѝ儈ᓖ؍ᆸǄሩҾ䞦⍫䎧ޣ䭞֌ ⭘ˈ᧘⍻৲оҶᓅ⢙Ⲵ㔃ਸ઼ᇊսǄ≘ส䞨↻ส_{Tyr511 оѤ䘁ⲴӢṨ䈅ࡲ} Glu532 ᴹ≒䭞֌⭘ˈ᧘⍻ᴹॿࣙۜॆⲴ֌⭘Ǆ≘ส䞨↻ส Trp570,Trp593 ઼ Phe616 ൘⍫ᙗս⛩ᖒᡀҶ⮿≤ਓ㺻ˈᖡ૽ᓅ⢙㔃ਸⲴս⛩Ǆ≘ส䞨↻ส Lys487 ઼ Asp481 սҾ+1 ս⛩ˈ㜭ཏᖡ૽ᓅ⢙઼䞦Ⲵ㔃ਸᯩᔿˈӾ㘼᭩ਈ ӗ⢙Ⲵ㓴ᡀ઼䭞රǄ ᴰਾᡁԜ∄䖳Ҷ β-ॺң㌆㤧䞦⭘ң㌆઼ң᷌㌆є⿽н਼ᓅ⢙ᡰӗ⭏Ⲵ ӗ⢙൘㔃ᶴкⲴᐞᔲᙗǄԕң᷌㌆֌Ѫᓅ⢙ˈB. circulans β-ॺң㌆㤧䞦䟾 ⭏ර઼R484H ケਈփⲴਟԕۜॆਸᡀվ㚊ॺң᷌㌆ǄᡁԜᙫޡ䢤ᇊҶ 8 ⿽ վ㚊ॺң᷌㌆㔃ᶴˈަѝ_{5 ⿽ᱟ俆⅑ᣕ䚃Ǆৼ↗ᵶ㧼˄Bifidobacterium}

dentium ઼ Bifidobacterium breve˅㜭࡙⭘վ㚊ॺң᷌㌆ᒦф⭏䮯⣦ߥ㢟ྭǄ

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