物工程學(xué)報(bào)2005年1月Chinese Journal of Biotechnology非解朊梄熱菌HG102耐熱β-糖苷酶的結(jié)構(gòu)與功能研究The Structure-function Relationship of Thermostable B-glycosidase from the Thermophilic Eubacterium Thermusnonproteolyticus HG102楊雪鹛2,楊壽鈞,韓北忠2,金城YANG Xue-Peng. 2, YANG Shou-Jun, HAN Bei-Zhong and JIN Cheng1.中國(guó)科學(xué)院微生物研究所微生物資源前期開發(fā)國(guó)家重點(diǎn)實(shí)驗(yàn)室,北京1000802.中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京1000831. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beying 100080, China2. College of Food Science and Nutritional Engineering, Beying 100083, China摘要非解朊棲熱莤HGl0耐熱β糖苷酶為(βα)桶狀結(jié)構(gòu),是具有水解功能和轉(zhuǎn)糖苷功能的單體酶。該酶可以作為一個(gè)很好的模型來研究糖苷酶的反應(yīng)機(jī)制、底物特異性和耐熱的分子基礎(chǔ)。根據(jù)對(duì)該酶的晶體結(jié)構(gòu)解析和同家族酶的結(jié)構(gòu)比較,推測(cè)Chul64和Glu38分別是質(zhì)子供體和親核基團(tuán)兩個(gè)活性位點(diǎn);在α-螺旋N端第一位的脯氡酸和蛋白質(zhì)外周的精氣酸是耐熱機(jī)制的關(guān)鍵位點(diǎn)和關(guān)鍵氯基酸殘基。為確定這些氨基酸殘基的功能,通過基因定點(diǎn)突變的方法分別把Gul64、Glu338Pmo316、Pm356、Pm344和Arg325置換成Cln、Ala、、Ala、Phe和Leu,同時(shí)還對(duì)Pm316和Pm356進(jìn)行了雙置換。突變酶經(jīng)過純化得到電泳純,用CD光譜進(jìn)行了野生酶和突變酶的結(jié)構(gòu)比較。通過突變酶的酶功能和酶學(xué)性質(zhì)分析,結(jié)果表明Gul64和Gu38分別是質(zhì)子供體和親核基團(tuán),親核基國(guó)的突變酶 TnglyE38A可以合威混合型糖苷健寡糖類似物;在α螺旋N端第一位的Pmo316和Pmo356以及在蛋白質(zhì)外周形成離子鍵的Arg325均是對(duì)耐熱性有貢獻(xiàn)的關(guān)鍵氨基酸殘基關(guān)鍵詞β-糖苷酶,轉(zhuǎn)糖苷活性,熱穩(wěn)定性,定點(diǎn)突變中圖分類號(hào)Q814文獻(xiàn)標(biāo)識(shí)碼A文章編號(hào)10003061(2005)01-008408Abstract B-Glycosidase(Tngly)from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostablemonomeric protein and adopts the(pla) barrel fold, is an excellent model system to be investigated for the thermostable mecha-nism,activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly( Wang et al2003)and comparison of other proteins structure of homofamily, Glul64 and Glu338 may act as proton donor and nucleophile inthe hydrolysis reaction respectively; proline located at NI of a-helix and arginine which can form ion link may contribute to thethermostability. We aim to further identify the critical sites and the amino acid residue(s)responsible for the activity, the thermal stability and the substrate specificity. Mutations had been constructed by site-directed mutagenesis. They are Glu164GInGlu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified toSDS-PAGE purity. Changes in the conformations were examined by means of CD. The Glu338 Ala mutant showed no detectablehydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. TheReceived: June 17, 2004; Accepted: July 26, 2004中國(guó)煤化工CNMHGw Corresponding author. Tel: 86-10-62587206: E-mail: jinc@ sun. imaccn中國(guó)科學(xué)院知識(shí)創(chuàng)新工程項(xiàng)目基金資助(No.0103)。楊雪鵬等:非解朊棲熱菌HG102耐熱β-糖苷酶的結(jié)構(gòu)與功能研究Glul64 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-typewere determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is located at NI positions of a-helix, and Arg325 that form salt bridge between a-helices 5 and a-helices 6, are the critical sites toprotein thermostabilizaticKey words B-glycosidase, transglycosylation, thermostability, site-directed mutagenesisβ-糖苷酶(E.C.3.2.1.21)生物來源廣泛,可以Cu38來鑒定P糖苷酶Tngy水解反應(yīng)時(shí)的質(zhì)子供水解多種B構(gòu)型的糖苷鍵具有廣闊的應(yīng)用前景。體和親核基團(tuán);置換Pm316、Arg325、Po344和Pm356嗜熱細(xì)菌 Thermus nonproteolyticus HGI02的β糖苷酶來探討酶的耐熱分子基礎(chǔ)。Tngy基因已克隆、表達(dá)并進(jìn)行了酶學(xué)性質(zhì)的研究2。Tngy屬于糖苷酶家族1,具有葡萄糖苷酶、半1材料和方法乳糖苷酶、巖藻糖苷酶和甘露糖苷酶活性,在高溫下1.1材料還具有轉(zhuǎn)糖苷活性,它的最適水解反應(yīng)溫度和pH1.1.1菌株與質(zhì)粒:大腸桿菌(E.col)AS1.1739值分別為90℃和56,在90℃時(shí),酶的半衰期為25K12r△( lacIPOZY)×74]在中國(guó)科學(xué)院微生物研h。Tngy晶體結(jié)構(gòu)解析表明,該酶為(β/a)桶狀結(jié)究所購(gòu)買,重組質(zhì)粒pHY(在pUCl8載體HindⅢ克構(gòu),分別位于第四β-shet和第七β-shet上的Glu164隆位點(diǎn)含有β糖苷酶目的基因)為本實(shí)驗(yàn)室構(gòu)建。和Gu338,可能為水解反應(yīng)時(shí)的質(zhì)子供體和親核基BMH1-18購(gòu)自 Promega公司。11.2培養(yǎng)基及培養(yǎng)條件:LB培養(yǎng)基為大腸桿菌B糖苷酶可用來合成寡糖。,但反應(yīng)要在有機(jī)完全培養(yǎng)基,固體培養(yǎng)基加入1.5%的瓊脂粉。重相中并需要高濃度的糖基供體。 Mackenzie等人將組菌培養(yǎng)時(shí)加入氨芐青霉素(100pg/mL),培養(yǎng)溫度Agrobacterium sp.β葡萄糖苷酶的親核基團(tuán)谷氨酸為37℃;誘導(dǎo)時(shí)液體培養(yǎng)基加入1%(W/V)乳糖固殘基突變成甘氨酸,使得突變的酶只能合成糖苷鍵,體培養(yǎng)基涂布4μLITG(200mg/mL)40 L X-gal(20不再具有水解功能,從而能使寡糖的產(chǎn)率達(dá)到mg/mL)。90%。B糖苷酶Tngy可以在65℃下水解乳糖或11.3酶及生化試劑:定點(diǎn)突變?cè)噭┖?Geneen纖維二糖生成三糖2),說明酶的活性中心適合轉(zhuǎn)糖 tor in vitro Site-Directed Mutagenesis System Kit購(gòu)自苷反應(yīng),可用來合成寡糖,置換耐熱β-糖苷酶的親核 Promega公司;T4 Polynucleotide Kinase購(gòu)自 Promega基團(tuán)來合成寡糖更具有優(yōu)勢(shì)。已知結(jié)構(gòu)的蛋白質(zhì)大公司;溶菌酶購(gòu)自華美公司;CMP3 Fluoro-neuram約10%為(pa)桶狀結(jié)構(gòu),或叫TM結(jié)構(gòu),因此以Acid購(gòu)自 Calbiochem-Novabiochem公司;胰化蛋白胨Tngy作為模型來研究(a桶狀結(jié)構(gòu)的穩(wěn)定機(jī)制非( TRYPTONE)和酵母提取物( YEAST EXTRACT)購(gòu)自常有意義。文獻(xiàn)報(bào)道,在 a-helix N端第一位脯氨酸 OXOID公司;PTG、Xgl,ONPG和乳糖購(gòu)自Sgma公的剛性結(jié)構(gòu)和蛋白質(zhì)外周精氨酸形成的離子鍵可能司;蛋白質(zhì)分子量標(biāo)準(zhǔn)購(gòu)自華美公司;其它試劑均為對(duì)蛋白質(zhì)熱穩(wěn)定性有一定的貢獻(xiàn)3分析純?cè)噭?。本?shí)驗(yàn)在Tngy晶體結(jié)構(gòu)的基礎(chǔ)上,用基因定所用誘變寡核苷酸為上海生工公司合成,見點(diǎn)突變的方法置換Tngy的氨基酸殘基Gn164和表1。表1置換氨基酸位點(diǎn)及其在蛋白質(zhì)二級(jí)結(jié)構(gòu)中的位置和相應(yīng)的誘變寡核苷酸設(shè)計(jì)Table 1 Oligonucleotides and mutagenic position in proteinGlu164GlnNo, 4, B-sheet5’- ACCCTGAACCAGCCCTGGTGO3′Glu338AlaNo. 7, B-sheet5.TACATCACGGCAAACGGGGCC-35'-GGGAGGTCTACGGCGAGGGGCTT-3lon linkNo.7,β-shee中國(guó)煤化工3No. 7. g-helix NICNMHG5'-GTGGAGGACGCCGACCGGGTG-3old and underlined nucleotides are the mutations sites86Chinese Journal of Biotechnology生物工程學(xué)報(bào)2005,vol.21,No.11.2方法SWISS- MODEL. htm.網(wǎng)站上完成801.2.1基因定點(diǎn)突變:以重組質(zhì)粒pHY單鏈DNA12.6轉(zhuǎn)糖苷反應(yīng):突變酶 TnglyE338A和各種底物為模板在誘變寡核苷酸介導(dǎo)下,用定點(diǎn)突變?cè)噭┖性?5℃,pH6.8下反應(yīng)2h,薄層層析檢測(cè)。相應(yīng)的試劑進(jìn)行突變和篩選。篩選出單菌株提取質(zhì)1.27薄層層析(mLC):展開劑為正丁醇:乙酸:水粒送交 TaKaRa Biotechnology( Dalian)測(cè)序鑒定突變=1:2:1;顯色劑為苯胺:二苯胺:磷酸=5:5:1(顯色結(jié)果。范圍為10g)。122突變基因的表達(dá)和蛋白質(zhì)純化:用突變的在數(shù)據(jù)的測(cè)定當(dāng)中都進(jìn)行3次或3次以上試重組質(zhì)粒轉(zhuǎn)化到大腸桿菌(E.coli)AS1.1739,從過驗(yàn),誤差范圍在5%以內(nèi)夜培養(yǎng)的 Amp-LB平皿上挑取單菌落,接種到5mL液體LB(Amp100g/mL)培養(yǎng)12h,1%接種量接種結(jié)果到100 mL Amp-LB液體培養(yǎng)基中培養(yǎng)12h;再以1%2.1基因定點(diǎn)突變構(gòu)建突變酶基因的接種量接種到4 L Amp-LB液體培養(yǎng)基(5L發(fā)酵以單鏈重組質(zhì)粒為模板,由誘變寡核苷酸和選罐)中培養(yǎng),加入1%的乳糖誘導(dǎo),37℃,300rmin的擇寡核苷酸介導(dǎo)突變和篩選突變基因)。突變質(zhì)攪拌速度,通無(wú)菌空氣,培養(yǎng)28h粒與野生質(zhì)粒大小相同。測(cè)序結(jié)果顯示突變質(zhì)粒收集發(fā)酵罐中的4L發(fā)酵液(4℃,6000g,15 pHYL64編碼164位上的clu堿基密碼子GAG突min)離心,菌體用磷酸緩沖液(50mml/L,pH66)懸變?yōu)閏n的密碼子CAG;突變質(zhì)粒pHYE338A編碼浮;冰浴超聲破碎,離心去除細(xì)胞碎片;在80℃水浴338位上的Gu堿基密碼子GAA突變?yōu)锳la的密碼恒溫加熱15mn,離心(4℃,1000g,15min)取上清。子GCA;突變質(zhì)粒pHYP36G編碼316位上的Pro堿向上清液中緩慢加入固體硫酸銨,收集30%~60%基密碼子CCC突變?yōu)镚y的密碼子GGC;突變質(zhì)粒飽和度的沉淀,溶于磷酸緩沖液中(50mmoL,pHYP356A編碼356位上的Pro堿基密碼子CCC突pH66),用同種緩沖液透析過夜。除鹽的粗酶凍干變?yōu)锳la的密碼子GCC;突變質(zhì)粒pHYP344F編碼濃縮后,在 AKTA FPLC蛋白質(zhì)純化系統(tǒng)上用DEAE344位上的Pmo堿基密碼子CCC突變?yōu)镻he的密碼離子交換柱進(jìn)行純化,緩沖液A為磷酸緩沖液(50子CTr;雙突變質(zhì)粒pHYP316G/P356A編碼316位和mmol/L,pH6.6),緩沖液B為1mol/LNaC溶于磷酸356位上的Pmo堿基密碼子CCC分別突變?yōu)镃y的緩沖液(50mmol/L,pH6.6),洗脫條件為在5個(gè)柱體密碼子GCC和Ala的密碼子GCC;突變質(zhì)粒積內(nèi)B溶液比例上升到30%,測(cè)酶活和 SDS-PAGE pHYR325L編碼325位上的Arg堿基密碼子CCC突檢測(cè)目的蛋白純度,收集合并酶活峰,用凍干機(jī)凍于變?yōu)長(zhǎng)eu的密碼子CTC。濃縮,用蒸餾水溶解凍干的酶蛋白,在磷酸緩沖液2.2突變基因的表達(dá)和突變酶的純化(50mmol/L,pH6.6)中透析,再用 Superdex G75分子含有突變基因的質(zhì)粒分別轉(zhuǎn)化到大腸桿菌篩柱層析純化,洗脫液為磷酸緩沖液(50mm/L,AS1.1739中,經(jīng)乳糖誘導(dǎo),發(fā)酵罐大量培養(yǎng),目的基pH6.6)。以上步驟均在常溫下進(jìn)行。因得到大量表達(dá),將表達(dá)產(chǎn)物分別經(jīng)加熱分離、硫酸123酶活測(cè)定:0.1mL4mmo/ L ONPG,0.1mL銨分級(jí)沉淀、DEAE和 Superdex G75分子篩層析純pH58磷酸緩沖液,0.7mLH2O,混勻后于85℃水浴化,得到電泳純,純度均達(dá)90%以上(圖1);除保溫5min,加入0.1mL酶液,反應(yīng)10min,加人4 Angle164Q和 TnglyE338A外,其他突變酶在在85℃mLNa2CO3溶液終止反應(yīng),420m測(cè)定吸光值。此時(shí)的比活均與野生酶相當(dāng)(表2)條件下每分鐘產(chǎn)生1pmo對(duì)硝基苯酚的酶量為一2.3突變酶與野生酶結(jié)構(gòu)的比較個(gè)酶活單位。野生和突變純酶由CD光譜儀直接掃描得到觀1.24蛋白含量測(cè)定:以牛血清白蛋白為標(biāo)準(zhǔn),察值(mdeg),把觀察值換算成平均殘基橢圓度(meanLowy法測(cè)定residue ellipticity)。結(jié)果顯示突變酶與野生酶結(jié)構(gòu)1.2.5CD光譜分析和同源建模:一定濃度的各種相同加圖?因?yàn)椤竛半遞閔曲線擁擠,這里只顯示酶蛋白分別加入測(cè)量杯中,把測(cè)量杯放入光譜儀兩個(gè)中國(guó)煤化工圖)。說明單個(gè)氨( JASCO J-715)中,用200~250m的光進(jìn)行掃描。用基酸CNMHG的折疊。同源建模蛋白質(zhì)分子量和氨基酸殘基數(shù)目計(jì)算殘基橢圓度。結(jié)果也顯示置換位點(diǎn)的結(jié)構(gòu)域與野生酶相同(見圖同源建模在htp:/w, expasy,org/ swissmod/3),這與CD光譜結(jié)果一致。楊雪鵬等:非解朊棲熱菌HGI02耐熱β-糖苷酶的結(jié)構(gòu)與功能研究表2突變純酶的比活Table 2 The specific activity of purified TnglysPurified proteinsE338AP316GP356AP344FP316G/P56Aecific activity/(u/mg) 17.60.9820.0M IkD5000WT T66一Tngly P316G/p356A圖1 SDS-PAGE分析野生酶Tngy和突變酶的純化20000Fig. I SDS-PAGE pattern of purifed Tnglys211: Tngly E164Q: 2: Tngly P344F: 3: Tngly P316G4: Tngly P356A: 5: Tngly R325L: 6: Tngly P316G/P356A圖2突變酶和野生酶CD光譜圖7: wileM: standard marker proFig. 2 Circular dichroic spectra of wild-type Tngly2.4質(zhì)子供體與親核基團(tuán)的確定and two mutant TnglyR325L, Tngly P316G/P356A in the突變酶 TnglyE164Q的比活是野生酶比活的The spectra were measured on a JASCO J-715 spectrolarimeter at 25C55%,且?guī)缀醪皇芊磻?yīng)液pH值的影響(見圖4);突 The protein concentration of wild-type Tngly was 0.16 mg/mL, the Tng變酶 TnglyE338A檢測(cè)不到水解活性,在CMP3-F-hyB32Lwas0.053 mg/ml, the Tingly P316cP356Awas009mgmlNeu5Ac和甘露糖為底物的情況下表現(xiàn)出轉(zhuǎn)糖苷功p6.6,50mml能(見圖5),用相應(yīng)的糖苷酶Tngy和a2-(3,6)-和底物形成共價(jià)復(fù)合物,是雙置換反應(yīng)的關(guān)鍵步驟NANaseⅡ都沒能把反應(yīng)產(chǎn)物水解。根據(jù)以上結(jié)果突變酶 Ingle384失去水解功能而只有轉(zhuǎn)糖苷功確定Gu164為水解反應(yīng)時(shí)的質(zhì)子供體,介導(dǎo)水的攻能,可作為糖苷合成酶用于寡糖的合成。擊,Gu338為水解反應(yīng)時(shí)的親核基團(tuán),其側(cè)鏈羧基23314圖3突變點(diǎn)的結(jié)構(gòu)和野生酶的中國(guó)煤化工CNMHGwild-type and mutants structures are superimposed. A: Arg325L; B: P344F; C: P316G: D: P356AChinese Journal of Biotechnology生物工程學(xué)報(bào)2005,vol,2l,No.1· TnglyP3l6GiP356A圖4突變酶 TnglyEI64Q和野生酶的活性受反應(yīng)液pH值的影響圖6突變酶的最適反應(yīng)溫度Fig. 4 Comparison of the ph dependence on the ONPG substrateFig. 6 EHects of temperature on site-directedwild-type Tngly and TnglyE164Q at the same condition of reactionmutagenesis Tngly hydrolysis activities1254567The hydrolysis activity was determined as describedials and Methods at differentfrom50℃to95℃Tngly316G/P356A-Highlight圖5突變酶 TnglyE33A的轉(zhuǎn)糖苷反應(yīng)TLC圖譜Fig. 5 Transglycosylation activity of mutantTngly E338A at 65C, pH6. 82: sialic acid: 3: CMP-3-F-Neu5Ac: 4: mannose and CMP-3.-Neu5Ac reacted with mutant Tngly E338A: 5: mannose and CMP-3-F.圖7突變酶的最適反應(yīng)pHNeusAc with no enzyme: 6: production of reaction of lane reacted withEfects of pH on site-directed mutagenesiswild-type Tngly: 7: production of reaction of laned reacted with a-2-(3Tngly hydrolysis activities6)-NANasc l (reaction of condition rely on the enzyme of optimum con-The hydrolysis activity was determined as describedin Materials and Methods at different pH from 4. 5 to 9.025熱穩(wěn)定性相關(guān)的突變酶酶學(xué)性質(zhì)分析252最適pH值:如圖7所示,6個(gè)突變酶的最適21最適溫度:在50-95℃的溫度范圍內(nèi)測(cè)定pH值都是大約pH5.8,與野生酶相似。說明置換的野生酶和突變酶的最適反應(yīng)溫度,發(fā)現(xiàn)突變酶氨基酸不影響酶活性中心的環(huán)境,也表明置換的點(diǎn)TnglyP34!F的最適反應(yīng)溫度與野生酶都為90℃,其在蛋白的外周不影響酶蛋白質(zhì)的活性。它突變酶最適反應(yīng)溫度都有所下降(如圖6),2.5.3熱穩(wěn)定性:突變酶和野生酶在85℃溫浴不Ingly316G和 TnglyP56A最適反應(yīng)溫度大約為同的時(shí)間,然后測(cè)剩余酶活,當(dāng)剩余酶活是原來的87℃,Tngy-P316G/P356A和 Ingly325L最適反應(yīng)溫50%時(shí),各種酶經(jīng)歷的時(shí)間(tm)分別是野生酶Tngy度大約為85℃。結(jié)果表明位于第六 a-helix的N端63min突變酶TnyP344F57min, Ingly316G43min,第一位的P316、位于第七 a-helix的N端第一位的TngyP356和位于第六a- helix的R325置換都降低了酶蛋P35中國(guó)煤化工min和 Ingly316c/在不同的溫度溫白的剛性結(jié)構(gòu),但在Loop區(qū)的P34對(duì)酶的結(jié)構(gòu)剛浴CNMH出相似的結(jié)果,Tm性影響較小。值是酶剩余50%活力時(shí)所對(duì)應(yīng)的溫度如圖9,結(jié)果楊雪鵬等:非解朊棲熱菌HGl02耐熱β糖苷酶的結(jié)構(gòu)與功能研究89力學(xué)常數(shù),底物濃度由2mmo/L到40mmol/L。結(jié)果iyP0356Aa顯示動(dòng)力學(xué)常數(shù)基本相同(見表3),表明突變酶置Pro316Gl換的氨基酸不影響酶的親和性。一Arg325Leu- Pro344Phe表3野生酶及其突變酶熱穩(wěn)定性和反應(yīng)動(dòng)力學(xué)常數(shù)Table 3 Thermodynamic and kinetic Parameters for(ken/K)▲0.010020030040050060.070.0wild-type63±0.594.0±0.5t/min圖8野生酶和突變酶的熱穩(wěn)定性分析Pro356Ala45±0.591.5±0.5-2.5and its mutants at 85C Values whose erreArg325Leu33±0.589.0±0.5-5.0±0.578,6within 5% were averaged15±0.586.5±0.5-7.5±0.51,478.2Pro356Alaa: Determined by kinetics of irreversible heat inactivity at 85Tc: Obtained by catalytic reaction of enzyme at optimum temperature3討論s0→ wild-type糖苷酶家族1β糖苷酶采用保留型雙置換催化/ Pro356A機(jī)制,在酶的活性部位存在兩個(gè)重要的羧酸部分,I Pro316Gly個(gè)質(zhì)子化,稱為質(zhì)子供體,另一個(gè)發(fā)生離子化,稱為10 -. Arg325Leu親核基團(tuán),所切糖苷鍵的氧原子被質(zhì)子化的羧基攻F Pro344Phe75080.0850擊,糖苷鍵斷裂,形成的碳正離子與離子化的另一個(gè)10950Incubation temperature/C羧基以離子鍵或共價(jià)鍵的形式形成中間產(chǎn)物,此中圖9野生酶和突變酶的熱穩(wěn)定性分析間產(chǎn)物不穩(wěn)定,當(dāng)親核試劑進(jìn)行親核攻擊時(shí),此中間Fig.9 Effect of temperature on the stability of產(chǎn)物不存在,取而代之的是一個(gè)新的糖苷鍵,當(dāng)親核試劑是水時(shí),發(fā)生水解反應(yīng),當(dāng)親核試劑是醇或某個(gè)Each purified enzyme was treated at different temperatures for 15 minutes糖的羥基時(shí),則發(fā)生轉(zhuǎn)糖苷反應(yīng)62。質(zhì)子供體在The remaining activities were expressed as percentages of the original activities. Values whosewithin 5% were averaged雙置換當(dāng)中起到酸/堿催化的雙功能,在第一步提供質(zhì)子,在第二步介導(dǎo)水的攻擊,親核基團(tuán)是形成顯示野生酶和突變酶 TnglyP344F、 Tngly-F316、酶與底物中間物的關(guān)鍵氨基酸殘基TnglyP356A、 TnglyR325L、 T'nglyP3l6G/P356A的T。值A(chǔ)grobacterium faecalis e-葡萄糖苷酶的質(zhì)子供體分別是949391、91、89和86℃。以上結(jié)果表明位和 Sulfolobus solfataricus糖苷酶兩個(gè)活性位點(diǎn)的谷于第六α-helⅸx的N端第一位的P316和位于第七氨酸殘基都已確定3·。目前,在糖苷酶家族1中α- helix的N端第一位的P356剛性結(jié)構(gòu)及位于第六除了葡萄糖硫苷酶外,質(zhì)子供體和親核基團(tuán)兩個(gè)活a-helix的R325所形成的離子鍵對(duì)酶蛋白的熱穩(wěn)定性位點(diǎn)都是谷氨酸,分別在第四 B-sheet和第七p性有一定的貢獻(xiàn),而在Lop區(qū)的P344對(duì)蛋白質(zhì)的het上。這兩個(gè)活性位點(diǎn)氨基酸殘基在家族1中很穩(wěn)定性影響較小。保守,靠近蛋白質(zhì)N端是T(F/LM)NE(P/L/I),靠近24突變酶米氏常數(shù)(K)和轉(zhuǎn)化數(shù)(k)的測(cè)C端是(n中國(guó)煤化工當(dāng)中,El64所在定:野生酶及突變酶可以水解 ONPGIe、 ONPGalTLNEPCNMHG域,分別位于第ONP-GFuc、 PNPGIc、 PNPGal、 PNPFuc和 PNPMan等多四shet相第七 B-sheet上,叮捱凋El64和E38可種底物,這里只選擇ONP作為底物來測(cè)定酶的動(dòng)能為糖苷酶Tngy的兩個(gè)活性位點(diǎn),在水解反應(yīng)當(dāng)Chinese Journal of Biotechnology生物工程學(xué)報(bào)2005,vl21,No.1中前者作為質(zhì)子供體,后者作為親核基團(tuán)。Glu164P344的B值分別為2527、28.51和29.97。B值和和Gu38分別置換為Ghn和Aa的突變?cè)囼?yàn)結(jié)論與原子的穩(wěn)定性有關(guān),B值越小原子群的穩(wěn)定性越大。上述推測(cè)一致。β-糖苷酶 Tingly熱穩(wěn)定性可能不只是含有脯氨糖苷酶水解反應(yīng)時(shí)糖苷鍵的水解有立體選擇酸的結(jié)果,在蛋白質(zhì)一級(jí)序列當(dāng)中含有96%的精性,但合成糖苷鍵時(shí),合成的鍵型是多樣的,可能是氨酸,在3D結(jié)構(gòu)當(dāng)中發(fā)現(xiàn)精氨酸大部分位于蛋白B鍵或a鍵,也可能是1-3、14或16等"。耐熱突質(zhì)外周,在a- helix之間形成離子鍵網(wǎng)絡(luò)。這樣的離變酶 TnglyE338A在65℃溫度下可催化底物CMP3-子鍵在其他耐熱蛋白質(zhì)中都被證明為重要的穩(wěn)定因F-Neu5AC和甘露糖的合成,合成產(chǎn)物不能用相應(yīng)的子0,別。較早Pent等人比較了同一種酶不同糖苷酶Tngy和a-2-(3,6)- NANase水解,表明產(chǎn)物來源的耐熱酶和常溫酶的結(jié)構(gòu)發(fā)現(xiàn),耐熱酶的分子的糖苷鍵鍵型可能發(fā)生了改變。這樣產(chǎn)生的混合鍵表面離子鍵比常溫酶的多。在以后的耐熱酶研型連接的寡糖( mixed-linkage)有可能作為寡糖類似究中也發(fā)現(xiàn)類似的情況,比如,蘋果酸脫氫酶、甘物用于糖苷水解酶的抑制劑1油醛-3-磷酸-脫氫酶和DNA聚合酶琍等。我們蛋白質(zhì)的耐熱因素包括離子鍵作用、氫鍵作把6a-heix的Amg325和5 a-helix的A甲p235之間形用、疏水作用、金屬鍵、二硫鍵、包裝效應(yīng)、Poie理成的離子鍵打斷,突變酶R2SL的熱穩(wěn)定性參數(shù)T論、a螺旋的穩(wěn)定作用和氨基酸組成等都經(jīng)過深入值下降大約5℃,表明蛋白質(zhì)外周的離子鍵對(duì)野生廣泛的分析3,從大量的研究中發(fā)現(xiàn),一個(gè)蛋白酶Tngy熱穩(wěn)定性也有貢獻(xiàn)。質(zhì)的耐熱機(jī)制可能是多種因素的結(jié)果。各個(gè)突變酶與野生酶純化方法相同,從SDSpone與其他的a氨基酸不同,由于其N原子PAGE電泳結(jié)果分析各個(gè)突變酶與野生酶純度相位于吡咯環(huán)上,使得前一個(gè)氨基酸與它形成肽鍵時(shí)當(dāng),然而突變酶 TnglyP316G、 TnglyP356A、 Ingly316G(C-N)不能自由旋轉(zhuǎn),另外吡咯環(huán)還具有疏水作P56A和 TnglyR32L的比活與野生酶相比有所上用。Poue分子的這些特點(diǎn),在形成肽鏈時(shí)導(dǎo)致它升,可能的原因是在85℃時(shí)突變酶結(jié)構(gòu)與野生酶相比其它氨基酸的構(gòu)型熵小22.2,從而降低蛋白質(zhì)比“剛性”下降“柔韌性”增加,有利于突變酶催化反的折疊熵。蛋白質(zhì)的折疊熵降低可以提高其穩(wěn)定應(yīng)。突變酶 TnglyP316G、 Ingly356A、 Ingly3l6G性33。較早,從統(tǒng)計(jì)的結(jié)果表明蛋白質(zhì)中脯氨酸含量增多,可以明顯提高蛋白質(zhì)的熱穩(wěn)定性),但P356A、 Ingly344F和 Ingly325L動(dòng)力學(xué)常數(shù)與野生酶基本相同,說明被置換的氨基酸殘基與酶的活性脯氨酸在蛋白質(zhì)二級(jí)結(jié)構(gòu)中的位置不同對(duì)穩(wěn)定性的貢獻(xiàn)不一樣,動(dòng)力學(xué)模擬試驗(yàn)證明脯氨酸位于a無(wú)關(guān),置換上來的氨基酸殘基也不影響酶活性中心heix的N端第一位最有利于蛋白質(zhì)的穩(wěn)定性結(jié)構(gòu)的正確折疊。統(tǒng)計(jì)結(jié)果也表明脯氨酸偏向位于α- helix的N端第 REFERENCES(參考文獻(xiàn))位和Bum的第二位,這可能有利于蛋白質(zhì)的[1] Ichikawa Y, Look GC, Wong CH. 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