專利名稱：：納豆芽孢桿菌谷氨酸脫氫酶基因的突變基因及編碼蛋白的制作方法
技術領域：
：本發(fā)明涉及一種生物
技術領域：
的基因及編碼蛋白，具體是一種納豆芽孢桿菌谷氨酸脫氫酶基因的突變基因及編碼蛋白。
背景技術：
：納豆芽孢桿菌(BacillusSubtilisNatto)是日本納豆的發(fā)酵菌，發(fā)酵后能產(chǎn)生多種獨特的保健功效成份，例如納豆激酶、多聚谷氨酸、維生素K2等，納豆激酶已被證實具有良好的溶解血栓功能，且無毒副作用。另外，納豆還具有預防骨質(zhì)疏松、抗氧化、抗菌、調(diào)節(jié)腸胃等生理預防和調(diào)節(jié)功能。除預防骨質(zhì)疏松與大豆本身含有的異黃酮有關外，其他功能主要取決于發(fā)酵后產(chǎn)生的新物質(zhì)。日本人將每年的7月10日定為納豆節(jié)。但納豆在中國市場卻難覓身影，究其原因，主要是發(fā)酵過程中產(chǎn)氨，氨在產(chǎn)品中累積使其風味讓國人難以接受。然而，當今世界，食療越來越受到人們的認可和追捧，治未病的思想也開始成為各國政府對于國民健康的主導政策。因此作為一種功能性食品，如果能改良其風味，納豆一定會有極大的市場需求。納豆芽孢桿菌氨基酸代謝產(chǎn)氨主要是聯(lián)合脫氨基作用，GDH為納豆菌代謝產(chǎn)氨途徑中的關鍵酶。ShigekiKada等在〈〈Bioscience，BiotechnologyandBiochemistry))(生物科學、生物技術和生物化學)2008年第72卷第7期18691876頁發(fā)表了題為《IdentificationofTwoMajorAmmonia-ReleasingReactionsInvolvedinSecondaryNattoFermentation》(納豆二次發(fā)酵過程中與氨還原釋放相關的兩個關鍵酶的鑒定)一文，認為GDH和尿素酶是產(chǎn)氨的關鍵酶，GDH編碼基因敲除后氨的含量可降低50%左右，若將編碼尿素酶的基因同時敲除，幾乎可完全抑制產(chǎn)氨。BORISR.BELITSKY等在《JournalofBacterology》(細菌學雜志)1998年第180卷第23期62986305頁發(fā)表了題為《RoleandRegulationofBacillussubtilisGlutamateDehydrogenaseGenes))(枯草芽抱桿菌谷氨酸脫氫酶基因的作用和調(diào)節(jié))一文，文中研究結果表明，如果敲除編碼GDH的基因，菌種的生長速度會下降，在普通的營養(yǎng)肉湯培養(yǎng)基中，代增時間由36min增加到52min。因此如果僅僅通過敲除GDH編碼基因來達到減少產(chǎn)氨量的目的，會付出發(fā)酵時間延長及底物要求更高的代價。所以通過研究GDH的活性位點，利用定點突變技術獲得不同GDH酶活的突變體，進而獲得具有不同GDH酶活的發(fā)酵菌株，有可能達到在不影響發(fā)酵周期的前提下，減少產(chǎn)氨量，改善納豆風味。納豆芽孢桿菌GDH完全失活雖然可以降低產(chǎn)氨量，但同時延長了發(fā)酵周期，增加了對底物的要求，因此有必要對此酶進行結構改造。經(jīng)對現(xiàn)有技術的文獻檢索發(fā)現(xiàn)，尚未見與本發(fā)明的主題有關的報道。
發(fā)明內(nèi)容本發(fā)明的目的在于克服現(xiàn)有技術中的不足，提供一種納豆芽孢桿菌谷氨酸脫氫酶基因的突變基因及編碼蛋白。通過同源重組將該本發(fā)明的突變引入野生菌株后，可獲得低產(chǎn)氨且發(fā)生長速度不變的基因工程菌，從而生產(chǎn)出無氨味納豆。本發(fā)明是通過以下的技術方案實現(xiàn)的，本發(fā)明涉及一種納豆芽孢桿菌谷氨酸脫氫酶編碼基因的突變基因，該突變基因的堿基序列如SEQIDNO:3、SEQIDNO:5、SEQIDNO:7、SEQIDNO:9、SEQIDNO:11、SEQIDNO13或SEQIDNO15所示。本發(fā)明還涉及一種納豆芽孢桿菌谷氨酸脫氫酶基因的突變基因的編碼蛋白，所述蛋白的氨基酸序列如SEQIDN0:4、SEQIDNO:6、SEQIDNO:8、SEQIDNO:10、SEQIDNO:12、SEQIDN0:14或SEQIDNO:16所示。本發(fā)明還涉及一種重組載體，該重組載體包含前述的突變基因。所述重組載體使用的載體為pET_28a。本發(fā)明確定了納豆芽孢桿菌的GDH的三維結構，利用SAMM分子模擬分析等軟件分析了GDH的活性位點和突變方案，利用QuikChange定點突變試劑盒對活性位點進行定點突變，得到7個突變體。通過同源重組將該突變引入野生菌株后，可獲得低產(chǎn)氨且發(fā)生長速度不變的基因工程菌，從而生產(chǎn)出無氨味納豆。本發(fā)明涉及的納豆芽孢桿菌具體為納豆芽孢桿菌(BacillusNatto)SJTU1-4CGMCCN02801，已于2008年12月9日提交中國微生物菌種保藏管理委員會普通微生物中心(CGMCC)保藏，該單位的地址為北京市朝陽區(qū)大屯路，中國科學院微生物研究所，郵編，10010圖1模型min的拉式圖檢驗結果；圖2谷氨酸脫氫酶的三維模型；圖3酶活性中心氫鍵結構的模擬；圖4氨基酸殘基化學修飾對酶活性影響的分析；圖5突變質(zhì)粒轉化子菌落PCR產(chǎn)物電泳分析；圖6GDH表達的SDS-PAGE分析；圖7純化蛋白的SDS-PAGE分析；圖8野生型和突變型谷氨酸脫氫酶活性的比較。具體實施例方式以下對本發(fā)明的實施例作詳細說明本實施例在以本發(fā)明技術方案為前提下進行實施，給出了詳細的實施方式和過程，但本發(fā)明的保護范圍不限于下述的實施例。下列實施例中未注明具體條件的實驗方法，通常按照常規(guī)條件，例如Sambrook等著的《分子克隆實驗室手冊》(NewYork=ColdSpringHarborLaboratoryPress,1989)中所述的條件，或按照制造廠商所建議的條件。實施例步驟一，納豆芽孢桿菌⑶H的同源建模；首先在PDB中搜尋與⑶H蛋白質(zhì)(SEQIDNO2)序列的高度相似的蛋白質(zhì)序列，在Swissmodel上嘗試性建模，經(jīng)PrOsa2003殘基能量圖分析后，最后選擇了來自強烈熾熱球菌的IGTM蛋白，嗜溫球菌的IEUZ蛋白作為同源建模的模版，建模，利用SAMM分子模擬分析軟件對得到的蛋白模型進行優(yōu)化；具體過程如下利用NCBI的BLASTn對得到的納豆芽孢桿菌⑶H編碼基因序列(SEQIDNO1)進行比對分析，使用ORFFinder模塊搜尋序列中可能的開放式閱讀框，并將所找到的一個含有422個氨基酸殘基的蛋白質(zhì)在BLASTp中進行比對分析；在PDB中得到六段與該蛋白質(zhì)序列的高度相似的蛋白質(zhì)序列(相似度在50%左右)，分別為1GTM、IEUZ,1PZ0、1HWY、1V9L和2RIR。以前五個蛋白質(zhì)序列作為模版，在Swissmode1上得到蛋白模型mode11，經(jīng)PrOsa2003殘基能量圖分析后，來自強烈熾熱球菌的IGTM蛋白，嗜溫球菌的IEUZ蛋白與modell的殘基能量變化趨勢最為接近。選取IEUZUGTiHt為同源建模的模版，在Swissmodel上再一次建模得到蛋白模型swiss；SwissModel和PredictProtein在Swissmodel中使用IGTM禾ΠIEUZ作為模版，按默認設置進行同源建模，在PredictProtein中，按默認設置對納豆枯草芽孢桿菌⑶H的二級結構特點進行逐一分析；步驟二，納豆芽孢桿菌GDH活性位點和定點突變方案的確定，利用SAMM分子模擬分析軟件對蛋白模型進行能量最小化分析，并經(jīng)分子動力學模擬后得到可能與底物谷氨酸(Glu)存在氫鍵連接的氨基酸殘基，對其中出現(xiàn)概率較高的幾個氨基酸殘基作突變模擬；具體如下ClustalX利用ClustalX，以1GTM、1EUZ、1PZ0、1HWY、1V9L、和2RIR作為比對對象，對納豆枯草芽孢桿菌GDH進行多序列比對，得到四段高度一致性的氨基酸序列，分別為80-82號殘基，112-120號殘基，195-200號殘基，351-358號殘基，該四段序列均為與底物谷氨酸空間距離十分接近(<10)的區(qū)域，酶活中心很有可能包含于其中；SAMM利用SAMM分子模擬分析軟件對Swissmodel自動生成⑶H的蛋白模型利用PAMM94力場進行能量最小化，得到一個蛋白模型min。經(jīng)ProSa2003殘基能量分析后，模型min中幾段GDH的保守序列的氨基酸殘基的殘基能量略小于蛋白模型swiss。經(jīng)拉氏圖檢驗，模型min中只有3個氨基酸殘基的Ψ，Φ角在非核心區(qū)域內(nèi)，如圖1所示，圖中較亮的十字形點為在非核心區(qū)域內(nèi)的氨基酸殘基；在SAMM中固定蛋白骨架，利用ClustalX得到的信息，選擇CHAM22力場，進行⑶H與其輔酶NADP+以及底物谷氨酸的分子對接，得到該酶與底物谷氨酸和輔酶NADP+對接結果，如圖2所示；以2ns為步長，環(huán)境溫度為310Κ，經(jīng)分子動力學模擬后得到1000個分子模型，以時間t對模型能量E作圖。模型的能量隨著模擬時間增長而逐漸下降，并趨于穩(wěn)定，表明該蛋白與底物和輔酶的作用模型是穩(wěn)定而可信的。統(tǒng)計模型中與底物Glu存在氫鍵連接的氨基酸殘基，如圖3所示；模擬結果表明Lys80，Lys116，Argl96，Thr200,Ser351這五個氨基酸殘基與谷氨酸形成氫鍵連接的可能性較大，見表1，即意味著它們很有可能是酶活中心直接與底物發(fā)生連接或催化反應的位點，故選取它們進行突變模擬實驗。Lys，Ser,Arg這三個氨基酸殘基種類與谷氨酸形成氫鍵的概率最大，故對其進行特異性的蛋白質(zhì)化學修飾研究，由于Thr沒有特異性的修飾劑，故只進行定點突變模擬試驗。利用SAMM軟件中的定點突變模塊，以突變后的蛋白AG變化最小，突變模型的分子動力學曲線與原蛋白模型的變化趨勢一致，與底物的結合能變化盡可能大為原則，選出了五對突變體，分別為LysSOAla，Lysll6Gln,Argl96Phe,Thr200Ala,Ser351Ala，其AG變化值如表2。原酶與底物的結合能在7800J/mol左右，突變后基本在14000J/mol左右，證明這些位點的確對酶活有一定的作用。表1氨基酸殘基與底物形成氫鍵的概率模擬分析<table>tableseeoriginaldocumentpage6</column></row><table>步驟三，納豆芽孢桿菌GDH的純化和化學修飾；提取納豆芽孢桿菌發(fā)酵液中的粗酶液后，用硫酸銨分級沉淀、DE32離子交換層析、SephacrylS-300凝膠層析和羥基磷灰石層析對GDH進行純化。TNBS、PMSF和PG分別對純化酶中氫鍵出現(xiàn)概率較高的賴氨酸殘基、絲氨酸殘基和精氨酸殘基進行特異性化學修飾，并以底物保護后再進行修飾的效果作對照，對GDH的活性位點進行初步驗證；具體如下1、納豆枯草芽孢桿菌NADP+依賴型⑶H的純化(結果見表3)在37°C下培養(yǎng)9.5小時后，用差速離心法(1800rpm和4800rpm)收集對數(shù)生長后期的菌種，以下所有純化步驟均在常溫下進行粗酶液的提取，將收集的菌體置于細胞裂解液中25°C裂解1小時，料液比(W/V)為14；裂解液配方為0.05MTris-HCl,pH7.0,ImMEDTA,ImMDDT，IOOmMNaCl，lmg/L溶菌酶。裂解后超聲處理20min，每次5s，工作與停息比為14;8000rpm離心5min后收集上清液，即為粗酶液。硫酸銨分級沉淀在粗酶液中加入25.8g/mL的硫酸銨(40%飽和度)，在04°C靜止過夜，IOOOOrpm離心IOmin去沉淀。再在上清液中添加硫酸銨至38.lg/ml(65%飽和度)，04°C靜止過夜，12000rpm離心15min取沉淀，并溶于IOml緩沖液A(50mmol/LTris-HCl緩沖液、pH8.0，內(nèi)含1.Ommol/LEDTA、1.Ommol/LDDT)中，在4°C下保存。DE32離子交換層析將鹽析后得到的粗酶液4.5mL在緩沖液A中透析6小時后用PEG20000透析脫水至3mL；DE32柱(2.6cmX50cm)層析條件為上樣量為3mL，采用00.6MNaCl線性梯度洗脫，流速為20mL/h，緩沖體系為pH8.0的緩沖液A。SephacrylS-300凝膠層析將離子交換柱分離到的高酶活組分合并后經(jīng)PEG20000透析濃縮至3ml；SephacrylS-300凝膠柱(2.6cmX90cm)層析條件為上樣量為3mL，0.15MNaCl等梯度洗脫，流速為40mL/h，緩沖體系為pH8.0的緩沖液A。羥基磷灰石層析將凝膠柱分離到的高酶活組分合并后經(jīng)PEG20000透析濃縮至Iml；羥基磷灰石柱(1.6cmX10cm)層析條件為上樣量為lmL，00.3MKCl線性梯度洗脫，流速為4mL/h，緩沖體系為pH8.0的磷酸緩沖液。酶活檢測，25°C下，取酶液與酶活測定液各1.5毫升測定⑶H催化輔酶NADP+還原所引起的光吸收值在340nm波長處的變化。脫氨酶活力測定液配制lmmol/LNADP,30mmol/L谷氨酸，緩沖液pH8.050mmol/LTrisHCl；每分鐘催化1μmolNADP+還原所需的酶量定義為一個酶活力單位(μ)。所有測定都在BeckmanDU800分光光度計上完成。對照為不含GDH的緩沖液Α。表3谷氨酸脫氫酶的純化結果<table>tableseeoriginaldocumentpage7</column></row><table>蛋白含量測定，280nm和260nm的吸收差法，用下面的經(jīng)驗公式算出蛋白質(zhì)的濃度蛋白質(zhì)濃度=1.45XA280-0.74XA260(mg/ml)。蛋白質(zhì)化學修飾實驗賴氨酸殘基的修飾將純化后的蛋白在pH9.0的重碳酸緩沖液中透析12小時，每6小時換一次緩沖溶液，取50μL純化后的酶溶液(0.lmg/mL,10U)，加入等體積的含有不同濃度TNBS溶液(0.051.5mM)。在黑暗中反應半小時后用5μL的4MHCl和10%SDS溶液中止反應，取10μL按酶活性測定方法測定修飾后的酶活，其余部分用來測定每個蛋白分子中被TNBS修飾的賴氨酸殘基數(shù)。方法為用分光光度計測定反應體系在346nm處吸收值，三硝基苯賴氨酸的吸收系數(shù)14.Gmr1CnT1，對照為不含TNBS的重碳酸緩沖液。精氨酸殘基的修飾將純化后的蛋白在pH9.0的重碳酸緩沖液中透析12小時，每6小時換一次緩沖溶液，取50μL純化后的酶溶液(0.lmg/mL,10U)，加入等體積的不同濃度PG溶液(0.055mM)。PG先溶于DMSO(DMS0對酶活沒有影響)，再用重碳酸緩沖液定容。反應半小時后用5μL4ΜHCl中止反應，用分光光度計測定反應體系在250nm處吸收值以確定每個蛋白分子中被修飾的精氨酸殘基數(shù)，PG修飾的精氨酸在250nm處的吸收系數(shù)為ILSmr1CnT1tj然后將反應體系16000rpm超速離心，取上清按酶活性測定方法測定酶的殘活，對照為重碳酸緩沖液。絲氨酸殘基的修飾將純化后的蛋白在pH9.0的重碳酸緩沖液中透析12小時，每6小時換一次緩沖溶液，取50μL純化后的酶溶液(0.lmg/mL,10U)，加入等體積的不同濃度PMSF溶液(0.055mM)。PMSF先溶于DMS0，再用重碳酸緩沖液進行定容，反應半小時后測定殘活，對照為重碳酸緩沖液。結果表明⑶H對于TNBS、PMSF存在濃度依賴性的失活。經(jīng)濃度為1.5mM的TNBS修飾后，⑶H便完全失活，ImM的TNBS可以抑制約90%的酶活，如圖4(A)所示。經(jīng)濃度為0.2mM的PMSF修飾后，谷氨酸脫氫酶便完全失活。0.ImM的PMSF可以抑制約90%的酶活，如圖4(C)所示。PG能造成小部分的酶活性喪失，ImM的PG可以抑制約17%的酶活力，更高的濃度也無法提升其對酶活性的抑制效果，如圖4(B)所示。說明該酶的酶促反應中心存在對酶活性有貢獻的賴氨酸和絲氨酸殘基。底物保護實驗所有底物保護實驗均先在酶溶液中加入谷氨酸至濃度為0.lmol/L，再與不同的修飾劑(濃度至可使酶基本失活)反應，殘活用酶活測定方法測定。純酶以及修飾后的酶(半失活)的動力學系數(shù)Km，Kcat均通過測定底物谷氨酸含量為0.0130mM時酶的活力計算得到，計算方法為LB法作圖，使用Excel線性回歸模塊作圖分析。進行底物保護后進行TNBS修飾，⑶H活性為原酶活性的98.7%。進行底物保護后進行PMSF修飾，⑶H活性幾乎不變。因此可以推測，酶活性中心必定含有Lys和Ser殘基，并且其與酶的催化作用有重要的關聯(lián)。而Arg殘基的特異性抑制劑PG對于該酶的抑制效果不明顯，經(jīng)底物保護后，進行PG修飾酶活可恢復到原水平的97%，所以Arg殘基并不在酶的活性位點上，不參與底物結合與催化反應。根據(jù)先前的計算模擬分析，Arg殘基處于酶活性中心的非核心區(qū)域，其可能通過與其他在酶活性中心氨基酸殘基形成氫鍵作用以穩(wěn)定酶活性中心結構，Argl96Phe,Argl96Glu兩個模擬突變體對于底物的結合能都大幅上升。然而Arg殘基并不直接參與到酶促反應中，對它進行化學修飾后，由于其側鏈與PG相連，很有可能對酶的底物結合產(chǎn)生一定的空間位阻效應，使酶的活性部分下降。步驟四，定點突變，針對三個有效活性位點設計突變擴增引物，利用QuikChange定點突變試劑盒對活性位點進行定點突變，得到單位點和多位點突變的突變體；具體步驟如下1、引物設計根據(jù)測序得到的序列，結合遺傳密碼子表，針對Lys80、Lysll6及Ser351三個活性位點，設計了3對突變引物Lys(aaa)80_Ala(gca)，引物如下5'-gtgaaaacgtatcccgcctgccgttaccgacagag-3'gcccacttttgcatagggcggaaagcaatggctgtctcagtcgggtgaaaacgtatccctcctttcgttaccgacagagtca,3，-cacttttgcatagggaggacggcaatggctgtctc-5，；Lys(aaa)116_Gln(caa)，引物如下5'-ggatcacaaacaattccgccttgaccaccgccatat-3'gaacctagtgtttgttaaggcggaaatggtggcggtatacctcttggatcacaaacaattccgcctttaccaccgccatatgga,3'-cctagtgtttgttaaggcggaactggtggcggtata-5'；Ser(TCA)351-Ala(GCT)5’-AACTGTTACGCCACCGGCAGCTGCCAGCAGC-3，TCTTTGACAATGCGGTGGCCGTCAACGGTCGTCGAGAAGAAACTGTTACGCCACCGGGAGTTGCCAGCAGCTCT，3，-TTGACAATGCGGTGGCCGTCGACGGTCGTCG-5，；2、定點突變以pMD19-T為克隆載體，經(jīng)TA克隆得到納豆芽孢桿菌⑶H編碼基因。利用Quikchange試劑盒對⑶H進行基因定點突變。Quikchange試劑盒定點突變體系5μLIOXreactionBuffer3μL(10IOOng)質(zhì)粒DNA(pMD19_T/GDH)2·5μL突變引物(lOOng/μL)1μLdNTP混合物1·5μLQuikSolution36μLdH20(使整個體系體積為50μ1)1μLQuikChangeLighting聚合Bl；3、PCR擴增將混合物在95°C加熱Imin后，95°C變性20s，60°C退火10s，68°C延伸4min，循環(huán)18輪，進行PCR。最后一輪循環(huán)結束后，于68°C保溫5min，使反應產(chǎn)物擴增充分。PCR擴增后加2μLDpnI限制性酶直接降解其中經(jīng)甲基化修飾與半甲基化修飾的原質(zhì)粒模板與質(zhì)粒雜環(huán)，輕輕混勻，離心后，放在37°C5min。4、大腸桿菌(DH5α)感受態(tài)細胞的制備(CaCl2法)將_70°C冰箱內(nèi)凍存的DH5α宿主菌在冰上融化后，用接種環(huán)挑取菌液在LB平板上(無抗生素)上劃線，37°C倒置培養(yǎng)過夜；從平板上挑取DH5α單菌落，接入5mL滅菌液體LB培養(yǎng)基中，37°C，250rpm，振搖培養(yǎng)過夜；將宿主菌以的轉接量轉接至IOOmL液體LB培養(yǎng)基中，37°C，250rpm，培養(yǎng)菌液至0D600=0.40.6；在無菌條件下將菌液轉移至預冷的滅菌離心管中，4°C，5000rpm離心4min收集菌體；用30mL的預冷滅菌CaCl2(0.ImoL/L)重懸菌體，4°C過夜；4°C下5000rpm離心4min，倒掉上清，收集菌體；用9mL滅菌CaCl2(0.ImoL/L)重懸菌體，加甘油至終濃度為20%；取1.5mL離心管，按100μL/管分裝，超低溫保存。5、DH5a感受態(tài)細胞轉化取分裝的感受態(tài)細胞，在冰上融解；加入2μLβ-Me，輕輕混勻，在冰上放2min；力卩102μL質(zhì)粒，輕輕旋轉離心管混勻內(nèi)容物，在冰上培養(yǎng)30min；將離心管在42°C水浴中熱擊90s，然后快速轉移到冰上，冷卻靜置2min，該過程不要搖動離心管；加入滅菌LB培養(yǎng)液(無抗生素)500μL，混勻后在37°C下150rpm振蕩培養(yǎng)60min，使菌體復蘇；吸取100300μL離心管內(nèi)容物，涂布在含有l(wèi)OOng/mL氨芐青霉素的LB固體培養(yǎng)基平板上，37°C過夜培養(yǎng)(>16h)。6、菌落PCR檢測篩選突變質(zhì)粒陽性轉化子挑選突變質(zhì)粒轉化子為模板，進行菌落PCR，篩選陽性轉化子；PCR參數(shù)設置94°C下加熱5min后，94°C變性lmin，57°C退火lmin，72°C延伸lmin，循環(huán)35輪，進行PCR。最后一輪循環(huán)結束后，于72°C下保溫lOmin。用1%瓊脂糖凝膠電泳檢查擴增結果。引物如下Pl5'GCCTGCAAGAGTATGGTAAG3'和P2:5'GATAGTCCACAAGGTCCTCC3‘；電泳圖如圖5所示。圖中各泳道分別為M標準分子量(200bp)；N:陰性對照；泳道14:pMD19-T/⑶H/K80A突變質(zhì)粒轉化子的擴增結果；泳道58:pMD19-T/ffl)H/K116Q突變質(zhì)粒轉化子的擴增結果；泳道913:pMD19_T/⑶H/S351A突變質(zhì)粒轉化子的擴增結果。7、DNA測序將PCR擴增結果為陽性的轉化子送上海桑尼生物科技有限公司測序，測序結果用VectorNTI軟件進行比對分析，分析結果顯示突變實驗成功。單位點突變后，再以突變子為模板，利用相同的條件進行多位點突變。結果如下野生型⑶H的堿基序列如SEQIDNO1所示，⑶H的氨基酸序列如SEQIDNO2所示；突變型GDH的堿基序列和氨基酸序列具體為(1)K80A，在DNA序列為238240從AAA變?yōu)镚CA(DNA的堿基序列為SEQIDNO:3)，在氨基酸序列為第80位的賴氨酸變?yōu)楸彼?氨基酸序列為SEQIDNO4)；(2)Kl16Q，在DNA序列為346348從AAA變?yōu)镃AA(DNA的堿基序列為SEQIDNO:5)，在氨基酸序列為第116位的賴氨酸變?yōu)楣劝滨０?氨基酸序列為SEQIDNO6)；(3)S351A，在DNA序列為10511053從AGT變?yōu)镚CT(DNA的堿基序列為SEQIDNO:7)，在氨基酸序列為351位的絲氨酸變?yōu)楸彼?氨基酸序列為SEQIDNO8)；(4)K80A-K116Q是指第80位的賴氨酸變?yōu)楸彼幔瑫r第116位的賴氨酸變?yōu)楣劝滨０罚?DNA的堿基序列為SEQIDNO:9，氨基酸序列為SEQIDNO10)；(5)K80AS351A是指第80位的賴氨酸變?yōu)楸彼?，同時第351位的絲氨酸變?yōu)楸彼幔?DNA的堿基序列為SEQIDNO:11，氨基酸序列為SEQIDNO12)；(6)K116_S351A是指第116位的賴氨酸變?yōu)楣劝滨０?，同時第351位的絲氨酸變?yōu)楸彼幔?DNA的堿基序列為SEQIDNO13，氨基酸序列為SEQIDNO14)；(7)K80A-K116Q-S351A是指第80位的賴氨酸變?yōu)楸彼?，同時第116位的賴氨酸變?yōu)楣劝滨０?，?51位的絲氨酸變?yōu)楸彼?；DNA的堿基序列為SEQIDNO:15，氨基酸序列為SEQIDNO16)。步驟五，基因表達和酶活檢測；構建表達載體，將野生型基因和突變基因在大腸桿菌中表達，將得到的包涵體復性后測定⑶H酶活，檢測突變的有效性；具體步驟如下1、構建表達載體設計帶有酶切位點的引物F(含NheI酶切位點)CGTACGGCTAGCATGGCGGCCGATCGAAAC，R(含EcoRI酶切位點)CGCTAGGAATTCCGATTGGCATTTCACTTT。分別以野生型和突變型的pMD19-T/⑶H為模板，用上述引物和pyrobestDNA聚合酶進行PCR擴增。用NheI和EcoRI對純化后的PCR產(chǎn)物和原核表達載體pET28a分別進行雙酶切。酶切9小時后對酶切產(chǎn)物進行純化。在T4DNA連接酶的作用下將PCR產(chǎn)物和載體連接，轉化DH5α，構建含目的基因GDH的原核表達載體。重組質(zhì)粒經(jīng)PCR和酶切鑒定后送上海桑尼生物科技有限公司測序。2、誘導表達將重組質(zhì)粒pET28a_GDH轉化表達宿主菌E.coliBL21(DE3)。挑取單菌落接種于含50μg/mlKan的LB液體培養(yǎng)基，在37°C、ISOrpm振蕩過夜培養(yǎng)后，按1%比例轉接入新鮮的Kan-LB，37°C搖床培養(yǎng)3小時后，加IPTG至終濃度為0.4mM，28°C，180rpm振蕩培養(yǎng)7小時3、表達產(chǎn)物SDS-PAGE分析取誘導表達菌液lmL，12000rpm離心lmin，將細胞用50μL水重懸后，加入40μL的2XSDS上樣緩沖液，混勻，再加入10μL的DTT，99°C干浴5min，取15μL進行SDS-PAGE電泳。電泳條件如下IOmL12%的分離膠Η203.2mL，30%丙烯酰胺混合液4.OmL,1.5mol/ITris(pH8.8)2.6mL,10%SDS0.ImL,10%過硫酸銨0.ImL,TEMED4μL。2ml5%的積層膠H201.4mL，30%丙烯酰胺混合液0.33mL，1.Omol/ITris(pH6.8)0.25mL,10%SDS0.02mL,10%過硫酸銨0.02mL,TEMED2μL。5XTris甘氨酸電泳緩沖液Tris15.lg，甘氨酸94.0g，10%SDS50mL，定容至IOOOmL0脫色液125mL乙醇，40mL冰醋酸，加水至500mL。電壓設定為80V，待染料前沿到達分離膠位置后，電壓加至120V。染料前沿遷移至凝膠的底部時，停止電泳。將膠放入考馬斯亮藍快速染色液中，輕輕震蕩30min；棄去染色液，將凝膠在水中漂洗數(shù)次，放入脫色液中，置于搖床輕輕震蕩脫色；將脫色完全的凝膠在成像系統(tǒng)內(nèi)拍照。4、⑶H純化超聲破菌誘導培養(yǎng)后的細菌在4°C以IOOOOrpm離心5min，收集菌體。用0.OlM磷酸鹽緩沖液(PBS)洗菌體一遍，再用PBS重懸一次，置于冰浴中，超聲破菌(350W，持續(xù)5S，間隔10S,共20次)。IOOOOrpm離心IOmin后，分別將上清和沉淀進行12%SDS-PAGE分析。分析結果顯示，超聲破菌后，重組蛋白主要以包涵體的形式存在于沉淀中，如圖6所示，圖中各泳道分別為泳道1誘導表達E.coliBL21(DE3)/pET28a；泳道2、3分別為誘導表達E.coliBL21(DE3)/pET28a/WT的上清和沉淀；泳道4、5分別為誘導表達E.coliBL21(DE3)/pET28a/K80A的上清和沉淀；泳道6、7分別為誘導表達E.coliBL21(DE3)/pET28a/K116Q的上清和沉淀；泳道8、9分別為誘導表達E.coliBL21(DE3)/pET28a/S351A的上清和沉淀；包涵體的變性、純化和復性變性條件純化所需緩沖液變性裂解/結合緩沖液B:8M尿素；0.IMNaH2PO4；0.OlMTris-CI,pH8.0；變性漂洗緩沖液C:8Μ尿素；0.IMNaH2PO4；0.OlMTris-CIρΗ6·3；變性洗脫緩沖液緩沖液D:8Μ尿素；0.IMNaH2PO4；0.OlMTris-CIρΗ5·9；緩沖液E:8Μ尿素；0.IMNaH2PO4；0.OlMTris-CIρΗ4·5；注意緩沖液B、C、D、E應在使用前調(diào)至適宜ρΗ值，以避免尿素解離。(1)取IOOmL經(jīng)適宜濃度IPTG誘導6h后的細菌培養(yǎng)物，8000Xg離心5min，收集菌體，以PBS(ρΗ=7.2)洗滌菌體1次；(2)將菌體重懸于1/10體積的PBS中，在冰上超聲破碎至溶液不再粘稠；(3)5，000\8離心15111土11;(4)移去上清，將IOOmL培養(yǎng)物的沉淀重懸于IOmLIX結合緩沖液(含6M尿素)；(5)冰浴孵育1小時，徹底溶解包涵體，離心去除不溶成分；(6)將ImL50%Ni-NTAHis·Bind樹脂懸液加到4mL細胞裂解液中，輕柔混勻(如旋轉混合器200rpm)，室溫結合1560min。(7)將裂解液與Ni-NTAHis·Bind樹脂的混合物小心加入下端封閉的空色譜柱中；(8)除去柱下端封閉蓋子，收集流出液，用于SDS-PAGE分析；(9)以4ml緩沖液C漂洗雜蛋白2次。保存漂洗組分用于SDS-PAGE分析；(10)以0.5mL緩沖液D洗脫目的蛋白4次，再以0.5mL緩沖液E洗4次，收集組分，并用SDS-PAGE分析。蛋白單體通常用緩沖液D即可洗脫，而多聚體、聚合物和含兩個His標簽的蛋白通常需由緩沖液E洗脫；(11)在收集的蛋白洗脫液中，加105μL20%的PEG4000、210μL50mmol/L的氧化型谷胱甘肽和210μL100mmol/L的還原型谷胱甘肽，室溫靜置約2h。轉至處理好的透析袋中，用TE(pH=7.2)透析23天，每天換液57次。純化后蛋白的電泳圖如圖7所示，圖中泳道M為標準蛋白，泳道1為野生型⑶H，泳道2為⑶H/K80A，泳道3為⑶H/Kl16Q，泳道4為⑶H/S351A，泳道5為未純化的野生型⑶H。5、蛋白濃度及⑶H酶活性測定測定⑶H氧化脫氨基作用的酶活性測定液為0.5mmol/LNADP+,15mmol/L谷氨酸，25mmol/LTris-Hcl，pH8.0。取酶液與酶活性測定液各1.5mL測定⑶H催化輔酶NADP+還原所引起的光吸收值在340nm波長處的變化。每分鐘催化1μmolNADP+還原所需的酶量定義為一個酶活力單位(U)。酶活性=ΔOD34tlX0.2X2/0.0486，蛋白濃度=1.45XOD280-O.74XOD26tl，單位酶活性=酶活性/蛋白濃度。酶活性測定結果如圖8所示。從圖8可以看出，野生型⑶HNADP+依賴酶活性為9.42U/mgprotein，單點突變的K80A、K116Q和S351A酶活性分別為4.35U/mgprotein,4.24U/mgprotein和3.60U/mgprotein,酶活性分別降至野生型的46.2%,45.0%和38.2%；K80A/K116Q、K80A/S351A、K116Q/S351A和K80A/K116Q/S351A的酶活性分別為1.77U/mgprotein,2.03U/mgprotein、1.62U/mgprotein和1.76U/mgprotein,即雙點突變和三點突變可使酶活性降低至18.8%,21.5%U7.2%和18.7%由此，可以證明K80、K116、S351位于谷氨酸脫氫酶的活性中心。也證明本發(fā)明采用的突變方案是有效的，突變后的蛋白有穩(wěn)定的結構，且表現(xiàn)出部分酶活性。序列表<110>上海交通大學<120>納豆芽孢桿菌谷氨酸脫氫酶基因的突變基因及編碼蛋白<130>01001<160>16<170>PatentInversion3.3<210>1<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>1atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacgaaa240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtaaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnIleAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcaagtgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>2<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>2MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>3<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>3atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacggca240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtaaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcaagtgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>4<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>4MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnIleAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>5<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>5atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacgaaa240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtcaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcaagtgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>6<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>6MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPheIleThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>7<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>7atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacgaaa240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtaaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcagetgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>8<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>8MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>9<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>9atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacggca240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtcaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcaagtgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>10<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>10MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaSerAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>11<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>11atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrValIleHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacggca240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtaaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPheIleThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnIleAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAspIleLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcagetgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>12<211>424[1007]<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>12MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyLysGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285[1046]GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320[1050]ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>13<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>13atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacgaaa240[1085]PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110ggcggtggtcaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270[1123]gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864[1124]AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcagetgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>14<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>14MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu[1163]354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrLys65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla[1201]340345350[1202]GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415AlaSerArgPheArgGlyTrplie420<210>15<211>1275<212>DNA<213>納豆芽孢桿菌(BacillusSubtillisNatto)<220><221>CDS<222>(1)..(1275)<400>15atggcggccgatcgaaacaccggtcatacagaaggggacaaacttgat48MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015gtattaaaatcgacccaaaccgtaatacataaggetctggaaaaattg96ValLeuLysSerThrGlnThrValIleHisLysAlaLeuGluLysLeu202530ggatatcccgaagaggtatacgaattgttaaaagagccgatgagatta144GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045ttaacggtaaaaatacctgttcgtatggacgacggtteagttaagatt192LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560ttcacaggatatcgtgcgeggcacaatgactctgtcggtccaacggca240PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580ggcgggatacgttttcacccgaatgtaacagaaaaagaggtgaaggcg288GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095cttteaatttggatgagtttaaaatgcggcataattgatcttccatat336LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr100105110[1241]ggcggtggtcaaggcggaattgtttgtgatccaagggatatgtcgttt384GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125agagagctggagcgtctgageagagggtatgtcagagcgateagecaa432ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln[1246]130135140attgtcggcccgacaaaagacgtgccggcaccggatgtatttacaaac480lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160teacaaateatggettggatgatggatgagtatteaagaattgatgaa528SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175tttaattcgcctggatttattacaggcaaaccgcttgtgcttggcgga576PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190tctcacgggagagaatctgcgacagcaaatggtgttaccatetgtatt624SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205aaagaagcggetaagaagagaggcategatattaaaggtgcgcgtgtc672LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220gttgtccaaggcttcggaaacgcgggaagetatttggcaaaatttatg720ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240catgatgcgggggcaaaagttgtcggcateteagatgcgtatggcgga768HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255ctttatgatccggaaggccttaatategattgtttactcgaccgacgc816LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270gacagettcggtaccgtaacaaagcttttcaacgataccattaccaac864AspSerPheGlyThrValThrLysLeuPheAsnAspThrlieThrAsn275280285caagagctgctggagctggattgtgatattctcgttcctgetgcgatt912GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300gaaaatcaaattacagaagaaaatgcccataatattegggetaaaatt960GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320[1280]gtcgttgaagcagcgaacggaccaacaacgcttgaaggaacaaagatt1008ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335cttteagacegggacattctgcttgtaccagacgtgctggcagetgcc1056LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350[1286]ggtggcgtaacagtttcttattttgaatgggttcagaataaccaaggc1104GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365ttctactggagtgaagaagaggtagaagaaaaattagaaaaaatgatg1152PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380gtcaaateatttaacaatatttatgaaatggetaacaaccgaagaatt1200ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400gacatgaggctcgetgcatatatggtcggcgttcgcaaaatggetgaa1248AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415gettcgcgttttagaggctggatataa1275AlaSerArgPheArgGlyTrplie420<210>16<211>424<212>PRT<213>納豆芽孢桿菌(BacillusSubtillisNatto)<400>16MetAlaAlaAspArgAsnThrGlyHisThrGluGlyAspLysLeuAsp151015ValLeuLysSerThrGlnThrVallieHisLysAlaLeuGluLysLeu202530GlyTyrProGluGluValTyrGluLeuLeuLysGluProMetArgLeu354045LeuThrValLyslieProValArgMetAspAspGlySerValLyslie505560PheThrGlyTyrArgAlaArgHisAsnAspSerValGlyProThrAla65707580GlyGlylieArgPheHisProAsnValThrGluLysGluValLysAla859095LeuSerlieTrpMetSerLeuLysCysGlylielieAspLeuProTyr[1319]100105110GlyGlyGlyGlnGlyGlylieValCysAspProArgAspMetSerPhe115120125ArgGluLeuGluArgLeuSerArgGlyTyrValArgAlalieSerGln130135140lieValGlyProThrLysAspValProAlaProAspValPheThrAsn145150155160SerGlnlieMetAlaTrpMetMetAspGluTyrSerArglieAspGlu165170175PheAsnSerProGlyPhelieThrGlyLysProLeuValLeuGlyGly180185190SerHisGlyArgGluSerAlaThrAlaAsnGlyValThrlieCyslie195200205LysGluAlaAlaLysLysArgGlylieAsplieLysGlyAlaArgVal210215220ValValGlnGlyPheGlyAsnAlaGlySerTyrLeuAlaLysPheMet225230235240HisAspAlaGlyAlaLysValValGlylieSerAspAlaTyrGlyGly245250255LeuTyrAspProGluGlyLeuAsnlieAspCysLeuLeuAspArgArg260265270AspSerPheGlyThrValThrLysLeuPheAsnAspThrIleThrAsn275280285GlnGluLeuLeuGluLeuAspCysAsplieLeuValProAlaAlalie290295300GluAsnGlnlieThrGluGluAsnAlaHisAsnlieArgAlaLyslie305310315320ValValGluAlaAlaAsnGlyProThrThrLeuGluGlyThrLyslie325330335LeuSerAspArgAsplieLeuLeuValProAspValLeuAlaAlaAla340345350GlyGlyValThrValSerTyrPheGluTrpValGlnAsnAsnGlnGly355360365PheTyrTrpSerGluGluGluValGluGluLysLeuGluLysMetMet370375380ValLysSerPheAsnAsnlieTyrGluMetAlaAsnAsnArgArglie385390395400AspMetArgLeuAlaAlaTyrMetValGlyValArgLysMetAlaGlu405410415[1358]AlaSerArgPheArgGlyTrplie[1359]420權利要求一種納豆芽孢桿菌谷氨酸脫氫酶編碼基因的突變基因，其特征在于，該突變基因的堿基序列如SEQIDNO3、SEQIDNO5、SEQIDNO7、SEQIDNO9、SEQIDNO11、SEQIDNO13或SEQIDNO15所示。2.—種權利要求1所述的納豆芽孢桿菌谷氨酸脫氫酶編碼基因的突變基因編碼的蛋白質(zhì)，其特征在于，所述蛋白質(zhì)的氨基酸序列如SEQIDN0:4、SEQIDNO:6、SEQIDNO:8、SEQIDNO:10、SEQIDNO:12、SEQIDNO:14或SEQIDNO:16所示。3.—種重組載體，其特征在于，所述重組載體包含權利要求1所述的突變基因，使用的載體為pET-28a。全文摘要一種生物
技術領域：
的納豆芽孢桿菌谷氨酸脫氫酶基因的突變基因及編碼蛋白；所述納豆芽孢桿菌谷氨酸脫氫酶編碼基因的突變基因的堿基序列如SEQIDNO3、SEQIDNO5、SEQIDNO7、SEQIDNO9、SEQIDNO11、SEQIDNO13或SEQIDNO15所示；所述納豆芽孢桿菌谷氨酸脫氫酶編碼基因的突變基因編碼的蛋白的氨基酸序列如SEQIDNO4、SEQIDNO6、SEQIDNO8、SEQIDNO10、SEQIDNO12、SEQIDNO14或SEQIDNO16所示；本發(fā)明還涉及一種包含前述突變基因的重組載體。通過同源重組將該本發(fā)明的突變引入野生菌株后，可獲得低產(chǎn)氨且發(fā)生長速度不變的基因工程菌，從而生產(chǎn)出無氨味納豆。文檔編號C12N15/63GK101824420SQ20101030028公開日2010年9月8日申請日期2010年1月14日優(yōu)先權日2010年1月14日發(fā)明者張建華,王家樂,陳麗麗申請人:上海交通大學