一種葡聚糖蔗糖酶及其應用的製作方法

2023-12-01 11:59:21 5

本發明涉及基因工程和酶工程領域，具體涉及一種新型的葡聚糖蔗糖酶及其催化製備水不溶性葡聚糖的方法。
背景技術：
：葡聚糖（Glucan）是一種以葡萄糖為單體的聚合物，在醫藥、精細化工、石油開採、食品、化妝品等領域有著廣泛的應用，其中根據糖苷鍵的類型可分為α,葡聚糖和β-葡聚糖。α-葡聚糖中研究使用較多的為右旋糖酐（dextran），右旋糖酐具有較高的分子量，主要由D-葡糖吡喃糖以α,1-6鍵連接，支鏈點有α,1-2、1-3、1-4糖苷鍵連接形成的。隨著微生物種類和生長條件的不同，其結構也有差別。由於結構的不同，葡聚糖可分為水不溶性葡聚糖和水溶性葡聚糖。水不溶性葡聚糖在代血漿、藥物載體、凝膠分離、色譜分離柱等領域都有重要的應用。葡聚糖蔗糖酶（Glucansucrase，亦稱蔗糖-6-葡萄糖基轉移酶）（EC.2.4.15）是一種糖苷轉移酶，屬於糖苷水解酶第70家族。腸膜明串珠菌分泌的葡聚糖蔗糖酶屬於誘導型酶，誘導物和底物都為蔗糖，且在一定範圍內，酶的產量與蔗糖濃度成正比。葡聚糖的生產方法有兩種：微生物直接發酵法和酶合成法。直接發酵生產葡聚糖時，發酵液成分複雜，導致產物分離困難、葡聚糖分子大小難以控制、細胞核蛋白類雜質多等缺點，而利用純酶催化製備葡聚糖，可以克服這些不足，生產出高產品質量的葡聚糖。技術實現要素：本發明的第一目的是提供一種新型的葡聚糖蔗糖酶。為實現上述目的，本發明採用如下技術方案：一種葡聚糖蔗糖酶DsrU，其胺基酸序列如SEQIDNO.2所示。本發明還提供了編碼該葡聚糖蔗糖酶DsrU的基因，其核苷酸序列如SEQIDNO.1所示。本發明所述葡聚糖蔗糖酶DsrU獲取自腸膜明串珠菌LeuconostocmesenteroidesM8，所述腸膜明串珠菌LeuconostocmesenteroidesM8為本實驗室從泡菜發酵液中，通過自然篩選、鑑定得到的菌株。本發明對腸膜明串珠菌LeuconostocmesenteroidesM8進行了全基因組測序，通過對其基因組序列分析和已報導的葡聚糖蔗糖酶的基因序列比對，得到一種未見報導的新型葡聚糖蔗糖酶基因。本發明的另一目的是提供含有上述DsrU編碼基因的表達載體及基因工程菌。所述表達載體的出發載體選用pET-28a，構建含DsrU基因的表達質粒pET28a-DsrU作為表達載體。選用宿主菌E.coliBL21(DE3)構建含有DsrU基因的基因工程菌E.coliBL21(DE3)-pET28a-DsrU，高效表達該葡聚糖蔗糖酶基因。所述的基因工程菌E.coliBL21(DE3)-pET28a-DsrU的構建方法為：將葡聚糖蔗糖酶DsrU基因導入到載體pET28a中進行外源表達。構建得到pET28a-DsrU表達質粒，然後將表達質粒pET28a-DsrU轉化到大腸桿菌E.coliBL21(DE3)中，，通過選擇性培養基挑選得到重組菌E.coliBL21(DE3)-pET28a-DsrU。本發明的又一目的是提供上述葡聚糖蔗糖酶DsrU在製備水不溶性葡聚糖中的應用。本發明提供了一種葡聚糖蔗糖酶DsrU製備水不溶性葡聚糖的具體方法，其技術路線包括如下步驟：（1）將葡聚糖蔗糖酶DsrU基因導入到載體pET28a中進行外源表達。構建得到pET28a-DsrU表達質粒，將該質粒轉入大腸桿菌感受態細胞中，通過選擇性培養基挑選得到正確的基因工程菌。（2）將重組菌E.colipET28a-DsrU接種至種子培養基，進行種子培養。（3）將培養好的種子培養液接種至發酵培養基中，30℃培養0~4h，加0.1~1.0mMIPTG，繼續在25~30℃下培養24h後停止發酵，離心菌液後棄上清，用磷酸緩衝液重懸沉澱下來的細胞，超聲破碎細胞，獲得葡聚糖蔗糖酶粗酶液。（4）向葡聚糖蔗糖酶的粗酶液中加入不同濃度的蔗糖，並調節pH至5~7，反應一段時間後6~12h後，向反應液中加入冷乙醇，離心得葡聚糖沉澱，40-60℃下乾燥得葡聚糖。（5）將得到的葡聚糖加水復溶，溶解12h後，離心，取上清液，加入冷乙醇，離心，無沉澱，可判斷上清液中無葡聚糖，及所述的葡聚糖為水不溶性葡聚糖。所述步驟（1）中含葡聚糖蔗糖酶基因導入的載體為pET28a，胞內表達葡聚糖蔗糖酶DsrU有利於酶的回收和純化。所述步驟（1）中基因工程菌的構建方法：將葡聚糖蔗糖酶基因DsrU連接到質粒pET28a上，得到包含葡聚糖蔗糖酶基因的重組質粒pET28a-DsrU，然後將重組質粒pET28a-DsrU轉化到大腸桿菌E.coliBL21(DE3)中，得到重組菌E.coliBL21(DE3)-pET28a-DsrU。所述步驟（3）IPTG濃度為0.1mM~1.0mM。所述步驟（4）中pH的初始值為7.0。所述步驟（4）中需控制pH不低於5.0。所述步驟（4）中加入冷乙醇的量是上清液的2-3倍，離心條件9,000rpm，10min。所述步驟（5）中加入冷乙醇的量是上清液的2-3倍，離心條件9,000rpm，10min。本發明的積極進步效果在於：本發明提供了一種來自於腸膜明串珠菌LeuconostocmesenteroidesM8的葡聚糖蔗糖酶DsrU，並提供了利用葡聚糖蔗糖酶DsrU合成水不溶性葡聚糖的方法。該酶在特定的催化條件下可高效催化製備水不溶性葡聚糖，同時葡聚糖得率高達90%以上。本發明提供的新型葡聚糖蔗糖酶應用前景廣闊，和以其製備的水不溶性葡聚糖的可被引用於藥物載體、凝膠分離系統、分離色譜柱製備等領域。附圖說明圖1是E.coliBL21(DE3)-pET28a-DsrU重組菌株鑑定圖；其中，Line1為DNAmarker，Line2為重組菌菌落PCR產物。具體實施方式下面通過實施例的方式進一步說明本發明，但並不因此將本發明限制在所述的實施例範圍之中。實施例1產葡聚糖蔗糖酶的菌株篩選與鑑定步驟1：取泡菜發酵液1mL於9mL的滅菌生理鹽水中，並梯度稀釋為10-4、10-5、10-6、10-7、10-8，取0.1mL的各梯度稀釋液接入選擇性平板中，於28℃培養2~3d，菌落長出後，挑取典型菌落，在含有萬古黴素（30μl/mL）的MRS平板上劃線，置於28℃培養箱中厭氧培養2~3d，觀察菌落生長狀況，若菌落生長，則進一步選取單菌落於含有萬古黴素（30μl/mL）的MRS固體培養基上劃線培養，重複上述步驟2~3次後，通過鏡檢確認為純培養物、並具有典型明串珠菌細胞形態的菌株轉入MRS固體培養基，低溫保存備用。步驟2：將步驟1中分離得到各菌株分別劃線接種於葡聚糖生成固體培養基表面，置於28℃培養1~3d，隨時觀察葡聚糖生成情況，選取能夠在培養基表面形成粘稠狀多糖的菌株。最終挑取一株在能夠在葡聚糖生成培養基上產生較多粘稠狀的菌株，接入MRS液體培養基，30℃培養8-10h後，轉入含有15%的甘油的MRS中，低溫保存，備用，命名為腸膜明串珠菌LeuconostocmesenteroidesM8。實施例2產葡聚糖蔗糖酶重組大腸桿菌的構建步驟1：將腸膜明串株菌LeuconostocmesenteroidesM8進行試管培養活化，試管裝液量為5mL，接種量為1%，培養溫度為30℃，培養時間為10-12h，得到菌株生長旺盛的菌液，10000rpm離心獲取菌泥，利用革蘭氏陽性菌的提基因組試劑盒提取腸膜明串株菌LeuconostocmesenteroidesM8的基因組作為PCR模板。步驟2：產葡聚糖蔗糖酶DsrU基因的獲取及線性化克隆載體的製備上遊引物：5』-CGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTC-3』下遊引物：5』-ACGGAGCTCGAATTCTTATATAGTATTTAAACGCTGTCTCTCTC-3』以LeuconostocmesenteroidesM8的基因組DNA為模板，以上述引入PCR擴增DsrU基因片段，反應條件為：94℃，5min；（94℃45s，50℃45s，72℃300s，35個循環），72℃，10min，擴增出DsrU基因，將PCR反應液進行純化回收，備用。將pET28a質粒用EcoRⅠ酶進行單酶切，酶切產物進行膠回收，作為線性化克隆載體，備用。步驟3：製備E.coliBL21(DE3)感受態細胞將取5μL的E.coliBL21菌體加入5mL的LB試管中，37℃，200rpm，培養12h，取1mL培養液加入裝有100mLLB培養基的搖瓶中，37℃，200rpm，待菌體OD600為0.5-0.6時，取出搖瓶，冰上放置10min，4℃，4100rpm離心10min，棄上清，用2mL含有15%甘油的0.05MCaCl2溶液混懸沉澱，分裝後於-80℃保存，備用。步驟4：利用一步克隆技術構建目標菌株將步驟2中回收的DsrU基因和線性化pET28a質粒載體，通過一部克隆轉入E.coliBL21(DE3)中，具體方法如下：將裝有200μL製作好的E.coliBL21(DE3)感受態細胞冰盒上解凍5-10min；加入20μL冷卻的一步克隆反應液：手指輕彈，混勻，冰上放置30min；42℃，水浴90s，冰浴2min；加入900μL新鮮LB培養基，復甦細胞，37℃，200rpm，培養1h。取100μL塗布在抗性平板上，37℃培養箱中培養10-12h，挑取菌落，使用步驟2中的引物進行PCR驗證，能擴增出目的基因片段的菌落即為pET28a-DsrU質粒成功表達的菌株。如圖1所示，DsrUPCR產物大小約為4500bp與預期一致，說明構建菌株正確。實施例3構建菌株發酵製備葡聚糖蔗糖酶DsrU種子培養基（g/L）：酵母粉5，蛋白腖10，NaCl10，pH7.0，121℃滅菌15min。發酵培養基（g/L）：酵母粉24，蛋白腖12，KH2PO42.31，K2HPO416.43，葡萄糖10，121℃滅菌15min。生產方法：取平板上單菌落接種於裝液量為5mL的試管中，30℃、200rpm培養12h。製備一級種子液，將一級種子液接種於裝有100mL發酵培養液的500mL三角瓶中，200rpm，30℃培養0~4h後加入0.1~1.0mMIPTG誘導DsrU目的蛋白的表達，繼續在25~30℃下培養至24h，停止發酵後，將培養液於10000r/min離心10min，棄上清，用pH為6.2的磷酸緩衝液重懸沉澱，超聲破碎細胞獲得粗酶液。如表1所示，不同IPTG誘導條件下，DsrU最高酶活可達4.9U。表1不同IPTG濃度誘導重組菌株對DsrU酶活的影響酶活測定方法：葡聚糖蔗糖酶的活力表示為在pH5.4，30℃的條件下，1min中內反應生成1µmol果糖所需的酶量為一個酶活單位U。酶活反應體系：30℃，20mM乙酸鈉緩衝液（pH5.4），0.05g/LCaCl2，1g/LNaNO3，100g/L蔗糖。具體方法為：（1）取7支比色管編號0-7，分別加入濃度為1mg/mL的果糖標準液0mL，0.2mL，0.4mL，0.6mL，0.8mL，1.0mL，1.2mL，補足蒸餾水至2.0mL，加入1.5mLDNS試劑，配成不同果糖濃度的反應液，將比色管搖勻後在沸水浴中加熱5min，取出，冷卻至室溫，蒸餾水定容至20mL，顛倒混勻，用0號管調零，540nm下測定吸光值，以吸光值為橫坐標，果糖含量（μmol）為縱坐標制定標準曲線。（2）取500μL待測酶液與2.5mL反應體系混合，混勻後放入30℃水浴中反應20min，採用DNS測定反應體系中0min和20min的還原糖（即為葡聚糖蔗糖酶催化反應生成的果糖）含量。（3）取葡聚糖蔗糖酶反應體系的樣品500μL，加蒸餾水至2.0mL，加入1.5mLDNS，沸水浴5min，取出冷卻至室溫，蒸餾水定容至20mL，顛倒混勻，測定540nm下測定吸光值，對照標曲計算得到相應還原糖的含量（μmol）。（4）葡聚糖蔗糖酶活力（U,μmol/min）=(20min反應體系中還原糖總量-0min反應體系中還原糖總量)/(20min*反應體系中加入的酶液體積)。實施例4葡聚糖蔗糖酶催化蔗糖生產不溶性葡聚糖步驟一：向葡聚糖蔗糖酶的粗酶液中加入不同濃度的蔗糖，使反應體系中蔗糖終濃度分別為50g/L，100g/L，150g/L，200g/L，30℃，反應時間為6~12h，優選8h；反應體系pH為5.0~7.0，優選6.0；反應溫度為30℃~40℃，優選30℃；酶添加量為1~10U，優選10U，利用葡聚糖蔗糖酶催化蔗糖製備葡聚糖的產量如表2所示。表2利用葡聚糖蔗糖酶催化蔗糖在優選條件下製備葡聚糖蔗糖濃度葡聚糖產量葡聚糖得率*50g/L23.6g/L94%100g/L48.2g/L96%150g/L72.7g/L97%200g/L93.5g/L95%*得率按照生產每g葡聚糖消耗的蔗糖中的葡萄糖量計算得出。該新型酶具有高葡聚糖催化得率的特性，在50~200g/L蔗糖條件下，葡聚糖得率均大於90%。步驟二：向步驟一的反應液中加入冷乙醇，加入冷乙醇的體積為反應液的2-3倍，冰上靜置30~60min，9000rpm離心10~20min得沉澱物，棄上清，50-55℃下乾燥至恆重（W1）得葡聚糖。步驟三：將步驟二中乾燥所得的葡聚糖，加水於40℃溶解12h，將溶解液9000rpm離心10min得沉澱物，50-55℃下乾燥至恆重（W2），並收集上清，加入2-3倍體積的冷乙醇，冰上靜置30~60min，9000rpm離心10~20min，經比較，W2和步驟三中的W1值基本一致，且離心後並無沉澱產生，由此可得，步驟二中所產生的葡聚糖為水不溶性葡聚糖。序列表南京工業大學一種葡聚糖蔗糖酶及其應用xb161104012PatentInversion3.514518DNAArtificialSequence1CDS(1)..(4518)1atgttagtaacagctggtattttttctgctgtgatatttggcgtttcc48MetLeuValThrAlaGlyIlePheSerAlaValIlePheGlyValSer151015atagctaacgtaagcgctgatagtattaacaatactagtattgcggtg96IleAlaAsnValSerAlaAspSerIleAsnAsnThrSerIleAlaVal202530gcgcaatcaaaaaatatagcagtggccacaacgacagctacaatggac144AlaGlnSerLysAsnIleAlaValAlaThrThrThrAlaThrMetAsp354045aaagtaactgatacgacagctacaacggacaaagtaactgatacgaca192LysValThrAspThrThrAlaThrThrAspLysValThrAspThrThr505560gctacgacagataaagtaactgatacgacagctacaacggacaaagta240AlaThrThrAspLysValThrAspThrThrAlaThrThrAspLysVal65707580actgatacgacagctacgacagataaagtaactgacacggcagctacg288ThrAspThrThrAlaThrThrAspLysValThrAspThrAlaAlaThr859095acggacaaagtggctgacacgacagctacaacggacaaagtaactgat336ThrAspLysValAlaAspThrThrAlaThrThrAspLysValThrAsp100105110acgacagctacaacgggcaaagtaactgacacgacagctacgacggac384ThrThrAlaThrThrGlyLysValThrAspThrThrAlaThrThrAsp115120125aaagtggctgacacgacagctacgacagataaagtggctgacacgaca432LysValAlaAspThrThrAlaThrThrAspLysValAlaAspThrThr130135140gctacaacggacaaagtaactgacacggcagctacaacggataaagca480AlaThrThrAspLysValThrAspThrAlaAlaThrThrAspLysAla145150155160actgacacggcagctacaacggacaaagtagctgatacaacagctacg528ThrAspThrAlaAlaThrThrAspLysValAlaAspThrThrAlaThr165170175acagataaagcagcgaacacaacagttacaccttcagaaaaatcaaaa576ThrAspLysAlaAlaAsnThrThrValThrProSerGluLysSerLys180185190agtattaagcaaatcgatggtaaaacatatttcattggtgatgatggt624SerIleLysGlnIleAspGlyLysThrTyrPheIleGlyAspAspGly195200205cagcccaagaaaaattttacagccattgtggacggtcaagtattatat672GlnProLysLysAsnPheThrAlaIleValAspGlyGlnValLeuTyr210215220ttcgacaaagataccggtactttgacatcaaatagtagtcaatatacc720PheAspLysAspThrGlyThrLeuThrSerAsnSerSerGlnTyrThr225230235240gatggtttggtcaatataggaaatgagcataatgcggcttattcattg768AspGlyLeuValAsnIleGlyAsnGluHisAsnAlaAlaTyrSerLeu245250255tcttcggacagttttacacaagttgatggctatctaacggctaacagt816SerSerAspSerPheThrGlnValAspGlyTyrLeuThrAlaAsnSer260265270tggtatcgacctaaagatatattgaaaaatggtacaacatggacagct864TrpTyrArgProLysAspIleLeuLysAsnGlyThrThrTrpThrAla275280285tcaacagcaaatgattttcgaccattgttgatgtcttggtggccggat912SerThrAlaAsnAspPheArgProLeuLeuMetSerTrpTrpProAsp290295300aaagatacccaagtttcatacttaaaatatatgcaatctgttggatta960LysAspThrGlnValSerTyrLeuLysTyrMetGlnSerValGlyLeu305310315320ttatcagatgacgttgtactatcaaacaaggatagtatgaatagtttg1008LeuSerAspAspValValLeuSerAsnLysAspSerMetAsnSerLeu325330335acagctatggcactgactgttcaaaaaaatattgaagagaaaataggc1056ThrAlaMetAlaLeuThrValGlnLysAsnIleGluGluLysIleGly340345350ctattaggtaccactgactggcttaagactgatatggaccaaatggtt1104LeuLeuGlyThrThrAspTrpLeuLysThrAspMetAspGlnMetVal355360365gactcacaatcaaattggaacattagtagtgagtctaaaggaacagat1152AspSerGlnSerAsnTrpAsnIleSerSerGluSerLysGlyThrAsp370375380catttgcaaggtggtgcgctcctatatgtgaatagtgatttgacacca1200HisLeuGlnGlyGlyAlaLeuLeuTyrValAsnSerAspLeuThrPro385390395400aatgctaattctgattatcgtttattaaatcgaacgccaacgaaccaa1248AsnAlaAsnSerAspTyrArgLeuLeuAsnArgThrProThrAsnGln405410415aaaggtcaaattacaacagacggtaatcaaggtggctatgagatgctg1296LysGlyGlnIleThrThrAspGlyAsnGlnGlyGlyTyrGluMetLeu420425430ttggccaacgatgttgataattctaaccctattgttcaagctgaacaa1344LeuAlaAsnAspValAspAsnSerAsnProIleValGlnAlaGluGln435440445ttgaattggttgtactacatgatgaatatcggtagtatcgtccaaaat1392LeuAsnTrpLeuTyrTyrMetMetAsnIleGlySerIleValGlnAsn450455460gatccaacggcaaattttgatggttacagagttgatgctgttgacaac1440AspProThrAlaAsnPheAspGlyTyrArgValAspAlaValAspAsn465470475480gtgaatgctgatttgttgcaaattgctggcgattactttaaggcagcg1488ValAsnAlaAspLeuLeuGlnIleAlaGlyAspTyrPheLysAlaAla485490495tatggaacggataaaagtgatgctaatgcaaacaatcacatttctatc1536TyrGlyThrAspLysSerAspAlaAsnAlaAsnAsnHisIleSerIle500505510ttagaagactgggataataatgatccggcgtatgtaaaatcacaggga1584LeuGluAspTrpAspAsnAsnAspProAlaTyrValLysSerGlnGly515520525aataaccaatcaaccatggattttccaatgcatttggcgttaaagtat1632AsnAsnGlnSerThrMetAspPheProMetHisLeuAlaLeuLysTyr530535540tcattaaatatgccaagtagtgctcgtagcgggttggaaccagatatt1680SerLeuAsnMetProSerSerAlaArgSerGlyLeuGluProAspIle545550555560gtaacaagtctagtaaatcgctcagaagattcaacagaaaatgaagca1728ValThrSerLeuValAsnArgSerGluAspSerThrGluAsnGluAla565570575caaccgaactattcatttattcgggcacatgatagtgaagtacaaacc1776GlnProAsnTyrSerPheIleArgAlaHisAspSerGluValGlnThr580585590gttattgcgcaaattattaaagataaaattaatcctagttctgatgat1824ValIleAlaGlnIleIleLysAspLysIleAsnProSerSerAspAsp595600605ggattgactgtttcaacggatgaaattgccaaagcattcgaaatatat1872GlyLeuThrValSerThrAspGluIleAlaLysAlaPheGluIleTyr610615620aatgccgacgaattaaaagctgataaagagtacactgcatataatata1920AsnAlaAspGluLeuLysAlaAspLysGluTyrThrAlaTyrAsnIle625630635640ccctcatcatatgcattg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