CN114958793B - 一种制备高催化活性mNampt突变体的方法及其突变体、重组菌以及应用 - Google Patents
一种制备高催化活性mNampt突变体的方法及其突变体、重组菌以及应用 Download PDFInfo
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- CN114958793B CN114958793B CN202210606976.8A CN202210606976A CN114958793B CN 114958793 B CN114958793 B CN 114958793B CN 202210606976 A CN202210606976 A CN 202210606976A CN 114958793 B CN114958793 B CN 114958793B
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Abstract
本发明提供了一种制备高催化活性mNampt突变体的方法及其突变体、重组菌以及应用，属于基因突变技术领域。本发明提供的制备高催化活性mNampt突变体的方法，包括如下步骤：以mNampt作为模板，采用引物序列进行定点突变PCR扩增，筛选mNampt催化活性提高的突变体，所述引物序列的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。采用本发明方法制备所得的10株突变体除第二株突变体外其余9株突变体催化活性均有所提高。其中，第六株突变体(SEQ ID NO.7)在相同条件下催化底物NAM所得产物NMN的含量高达357.084μmol/L，同野生型mNampt相比提高79.05％。
Description
技术领域
本发明属于基因突变技术领域，尤其涉及一种制备高催化活性mNampt突变体的方法及其突变体、重组菌以及应用。
背景技术
烟酰胺单核苷酸(NMN)作为辅酶烟酰胺腺嘌呤二核苷酸(NAD+)的关键前体，对人类身体健康具有重要意义，如NMN具有改善缺血性心脑组织损伤、修复氧化相关的身体机能障碍、治疗代谢性疾病等重要作用。人体临床试验表明，NMN具有良好的安全性，且可有效促进身体机能。基于其人体安全性和生物活性，NMN被广泛地应用于食品原料、药品、保健品及护肤品等行业。因此有必要探寻经济且高效的NMN生产方法降低NMN原料成本。
目前技术较为成熟的化学法合成NMN易出现手性问题，且有机溶剂的残留也限制了其在市场中的应用。而酶法制备NMN具备有反应简便、产品活性高及污染程度低等优点，代表了制备NMN的发展趋势。根据底物不同，酶法合成NMN的路径主要分为烟酰胺(NAM)路径和烟酰胺核糖(NR)路径两种。其中烟酰胺路径的限速酶为烟酰胺磷酸核糖转移酶(Nampt)，Nampt催化底物烟酰胺和磷酸核糖焦磷酸(PRPP)合成NMN。该路径在工业化酶法制备NMN有一定的应用，但有其瓶颈所在：Nampt转化效率低，导致烟酰胺路径制备的NMN产率低下，故亟需获得一种酶活显著提高的Nampt，解决烟酰胺路径制备NMN在工业化生产上的瓶颈。
发明内容
有鉴于此，本发明的目的在于提供一种制备高催化活性mNampt突变体的方法，能够获得酶活提高的突变体。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种制备高催化活性mNampt突变体的方法，包括如下步骤：以mNampt作为模板，采用引物序列进行定点饱和突变PCR扩增，筛选mNampt催化活性提高的突变体，所述引物序列的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
优选的，所述PCR扩增程序为98℃3min，98℃10s，58℃10s，72℃80s，30个循环，72℃10min。
优选的，所述PCR扩增体系为Prime STAR Max Premix 12.5μL，正反引物序列各0.5μL，模板0.5ng，ddH2O补足至25μL。
本发明还提供了一种利用上述方法获得的小鼠来源的mNampt突变体，所述突变体的氨基酸序列如SEQ ID NO.3-11所示。
本发明还提供了一种表达上述突变体的重组菌。
优选的，所述菌包括E.coli BL21(DE3)。
本发明还提供了一种上述重组菌的制备方法，包括如下步骤：将上述方法所得的扩增产物进行DpnI酶切，获得突变后的重组质粒，将突变后的重组质粒转化至感受态细胞E.coli BL21(DE3)内，培养基培养，长出的单菌落即为突变体菌株。
本发明还提供了一种上述突变体或上述重组菌在制备烟酰胺单核苷酸中的应用。
本发明还提供了一种上述小鼠来源的mNampt突变体的固定化方法，包括如下步骤：对上述重组菌进行诱导表达得菌液，将菌液破碎离心得粗酶液；将浓度为1％-3％的壳聚糖凝胶颗粒与浓度为0.5％-2.5％的戊二醛交联制备获得壳聚糖微球，将所述壳聚糖微球与所述粗酶液混合，交联8h-16h，洗去多余酶液后得固定化酶。
优选的，所述诱导表达包括如下步骤：将所述重组菌接种于LB培养基中扩大培养，在菌液的OD600值达到0.5-0.7时加入终浓度为0.2mM的IPTG，30℃、200r/min条件下诱导表达12h。
优选的，所述壳聚糖凝胶颗粒的制备方法包括如下步骤：将1％-3％的壳聚糖粉末溶于2％醋酸溶液中，搅拌获得凝胶液体，将凝胶液体滴加到4mol/L的NaOH溶液中获得凝胶颗粒，洗涤凝胶颗粒至pH呈中性。
本发明的有益效果：
采用本发明方法制备所得的10株突变体除第二株突变体外其余9株突变体催化活性均有所提高。其中，第六株突变体(SEQ ID NO.7)在相同条件下催化底物NAM所得产物NMN的含量高达357.084μmol/L，同野生型mNampt相比提高79.05％。
采用本发明提供的mNampt突变体的固定化方法，固定的蛋白含量为635.74μg/mL，可催化生成23.58μmol/LNMN。对固定化条件进行优化后，壳聚糖微球催化得到的NMN产量为35.26μmol/L，约为游离酶的39.5％，相对于优化前提高了50％；酶蛋白固定量为1159.06μg/mL，相对于优化前提高了80％。
附图说明
图1为菌落PCR验证结果，其中M为250DLmarker；1-10分别对应第一株-第十株突变体；
图2为突变菌株测序结果比对图，其中1-10分别对应第一株-第十株突变体；
图3为HPLC各组分出峰图，其中(a)为NMN标准品出峰图，图(b)为ATP标准品出峰图，图(c)为NR标准品出峰，图(d)为NMN、ATP、NR混合样出峰图；
图4为HPLC法测定的NMN标准曲线；
图5为mNampt定点饱和突变的菌株对转化反应生成NMN的影响，其中1-10分别对应第一株至第十株突变体；
图6为壳聚糖微球形貌图；
图7为壳聚糖微球SEM电镜图，其中(a)为未固定酶的壳聚糖微球，(b)为已固定酶的壳聚糖微球；
图8为分光光度计法测定的NMN标准曲线；
图9为BSA标准曲线；
图10为交联剂浓度对固定化微球转化得到的NMN含量和固定蛋白量的影响，其中—■—表示NMN产量，—●—表示蛋白固定量；
图11为固定时间对固定化微球转化得到的NMN含量和固定蛋白量的影响，其中—■—表示NMN产量，—●—表示蛋白固定量；
图12为壳聚糖浓度对固定化微球转化得到的NMN含量和固定蛋白量的影响，其中—■—表示NMN产量，—●—表示蛋白固定量；
图13为模板质粒ppsumo-mNampt的图谱；
图14为固定化酶热稳定性图，其中—■—表示固定化酶—●—表示游离酶；
图15为固定化酶pH稳定性图，其中—■—表示固定化酶—●—表示游离酶；
图16为固定化酶操作稳定性图。
具体实施方式
本发明提供了一种制备高催化活性mNampt突变体的方法，包括如下步骤：以mNampt作为模板，采用引物序列进行定点饱和突变PCR扩增，筛选mNampt催化活性提高的突变体，所述引物序列的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
在本发明中，mNampt表示的是小鼠来源的Nampt基因，所述mNampt基因的核苷酸序列如SEQ ID NO.12所示。所述引物序列具体为
F：CAGGAAATTNNKGAAGGCATGAAACAG，
R：CACTGTATGAAGAAGATCATGGCCATATTC，其中N代表A/G/C/T，K代表G/T。
在本发明中，所述PCR扩增程序优选为98℃3min，98℃10s，58℃10s，72℃80s，30个循环，72℃10min，所述PCR扩增体系优选为Prime STAR Max Premix 12.5μL，正反引物序列各0.5μL，模板0.5ng，ddH2O补足至25μL。
本发明还提供了一种利用上述方法获得的小鼠来源的mNampt突变体，所述突变体的氨基酸序列如SEQ ID NO.3-11所示，分别对应图5中的第一株和第三至第十株，氨基酸序列如SEQ ID NO.3-11所示的9株突变体所对应的核苷酸序列如SEQ ID NO.13-19所示(不包含图5中第一株和第四株的核苷酸序列)，其中图5中的第六株突变体(氨基酸序列如SEQ IDNO.7所示，核苷酸序列如SEQ ID NO.15所示)生成NMN的量最多。
本发明还提供了一种表达上述突变体的重组菌，优选的，所述菌为E.coli BL21(DE3)。
本发明还提供了一种上述重组菌的制备方法，包括如下步骤：将上述方法所得的扩增产物进行DpnI酶切，获得突变后的重组质粒，将突变后的重组质粒转化至感受态细胞E.coli BL21(DE3)内，培养基培养，长出的单菌落即为突变体菌株。
在本发明中，可以mNampt作为模板，采用引物序列进行定点饱和突变PCR扩增后，进行DpnI酶切，获得突变后mNampt，将突变后mNampt连接至质粒ppsumo中，获得突变后的重组质粒。本发明对于突变后mNampt连接至质粒ppsumo的具体方法没有特殊限定，采用本领域常规连接方法均可。在本发明中，也可以质粒ppsumo-mNampt(图谱如图13所示)作为模板，采用引物序列进行定点饱和突变PCR扩增后，进行DpnI酶切，便能直接获得突变后重组质粒。本发明对于将突变后的重组质粒转化至感受态细胞E.coli BL21(DE3)内的具体转化方法没有特殊限定，采用本领域常规转化方法均可。优选的将导入突变质粒的感受态细胞均匀涂布于含有kana抗性的LB固体培养基(50μg/mL)进行培养，所述培养的条件优选为37℃培养14h，长出的单菌落即为突变体。
本发明还提供了一种上述突变体或上述重组菌在制备烟酰胺单核苷酸中的应用。
本发明还提供了一种上述小鼠来源的mNampt突变体的固定化方法，包括如下步骤：对上述重组菌进行诱导表达得菌液，将菌液破碎离心得粗酶液；将浓度为1％-3％的壳聚糖凝胶颗粒与浓度为0.5％-2.5％的戊二醛交联制备获得壳聚糖微球，将所述壳聚糖微球与所述粗酶液混合，交联8h-16h，洗去多余酶液后得固定化酶。
在本发明中，所述诱导表达优选的包括如下步骤：将所述重组菌接种于LB培养基中扩大培养，在菌液的OD600值达到0.5-0.7时加入终浓度为0.2mM的IPTG，30℃、200r/min条件下诱导表达12h。
在本发明中，对重组菌进行扩大培养前，优选的需对重组菌进行活化，本发明对于活化的具体方法没有特殊限定。本发明对于LB培养基的具体组成没有特殊限定。在本发明中，优选的在菌液的OD600值达到0.6时加入终浓度为0.2mM的IPTG。本发明对于IPTG的具体制备方法没有特殊限定。
在本发明中，所述破碎优选的为超声破碎，所述超声破碎的条件优选为功率30％、工作3s、间歇5s，破碎8min。本发明所述壳聚糖凝胶颗粒的制备方法优选的包括如下步骤：将1％-3％的壳聚糖粉末溶于2％醋酸溶液中，搅拌获得凝胶液体，将凝胶液体滴加到4mol/L的NaOH溶液中获得凝胶颗粒，洗涤凝胶颗粒至pH呈中性。在本发明中，优选的将浓度为2％的壳聚糖凝胶颗粒与浓度为1.5％的戊二醛交联制备获得壳聚糖微球，所述壳聚糖微球与所述粗酶液混合交联的时间优选为10h-14h，更优选为12h。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
以质粒ppsumo-mNampt作为模板，采用引物序列(由广州艾基生物技术有限公司合成)V365-F：CAGGAAATTNNKGAAGGCATGAAACAG，V365-R：CACTGTATGAAGAAGATCATGGCCATATTC(其中N代表A/G/C/T，K代表G/T)进行定点饱和突变PCR扩增。PCR扩增体系如表1所示，扩增程序如表2所示。
表1定点饱和突变PCR扩增体系
表2定点饱和突变PCR扩增程序
实施例2
DpnI酶为核酸限制酶(识别序列为甲基化的GATC)，通常用于消除亲本DNA。使用Takara公司的Dpn I快切酶去除模板DNA(未突变)中的甲基化，DpnI酶切体系见表3，酶切程序见表4。
表3 DpnI酶切体系(50μL)
表4 DpnI酶切程序
将实施例1所得PCR扩增产物经DpnI酶酶切后，得突变后的重组质粒，将突变后的重组质粒转化至感受态细胞E.coli BL21(DE3)内，具体转化方法参照《分子克隆指南》。将导入突变质粒的感受态细胞均匀涂布于含有kana抗性的LB固体培养基(50μg/mL)，并将其倒置于37℃生化培养箱培养14h，长出的单菌落即为突变菌株。
突变菌株验证：
PCR验证：
将挑取的单克隆活化培养12h后进行PCR验证，使用煮沸裂解法提取质粒DNA：无菌枪头从平板中挑选单菌落并浸入10μL无菌ddH2O中，使用沸水浴加热10min裂解细胞。将裂解后的悬液在13000r/min转速下离心1min，取上清液作为PCR验证模板。克隆验证PCR体系如表5所示，程序如表6所示，克隆子PCR验证使用引物如下：
mNampt-F：TAATCCTTATTCAGTGGTGGTGGTGGTGGTGCTC(SEQ ID NO.20)
mNampt-R：AGGAAGCTTGCATATGAACGCTGCTGCTG(SEQ ID NO.21)
表5克隆子验证PCR体系(50μL)
表6 PCR验证程序
PCR产物琼脂糖电泳验证：
(1)在25mL1×的TAE溶液中加入0.25g琼脂糖，使用微波炉中高火档位加热约2min促进溶解。(2)待锥形瓶降温至室温，吸取2.5μL的10000×Gel Red核酸染料加入锥形瓶并摇匀。在制胶板A插上合适尺寸的梳子后缓慢倒入胶液。(3)将凝胶于室温静置20min，期间将1μL 6×Loading Buffer与5μL PCR产物混合准备上样。(4)将凝固态凝胶浸没于含电泳液的电泳槽内，使用点样吸头吸取5μL(3)中的混合样品加入胶孔，在120V电泳下电泳25min后结束电泳。
PCR扩增产物的琼脂糖凝胶电泳结果如图1所示，可见10个目的条带均在1500bp附近，与理论值1482bp基本相符。
测序验证：
挑取上述PCR验证出具有阳性条带的单克隆菌落接种至10mL含kana抗性的LB液体培养基，置于37℃、200r/min的恒温摇床培养12h。吸取3mL培养后的菌液于5mL无菌离心管，封口膜密封后委托艾基生物技术有限公司进行测序，测序引物如下所示。剩余菌液与50％甘油溶液1:1充分混匀，分装在冻存管冷冻保存于-20℃冰箱。
YZ-V365-F：AGAGGACCTTCGCCATCTTATTG(SEQ ID NO.22)
YZ-V365-R：AGGACTTCGTTACCTTGCCATTC(SEQ ID NO.23)
使用Snapgene将突变体序列与原始的mNampt序列进行比对，结果如图2所示。图中深色序列的白色缺失部位为突变位点所在位置，可见10株突变体均突变成功。
实施例3
突变后mNampt酶活测试
菌株诱导表达
吸取活化后的菌液400μL，按照2％(v/v)的比例接种于LB发酵培养基锥形瓶(20mL/50mL)，在37℃、220r/min条件下扩大培养。在菌液的OD600值达到0.6附近时加入终浓度为0.2mM的IPTG，于30℃、200r/min条件下诱导表达12h。
浓缩粗酶液的制备
将诱导表达后的菌液进行超声处理，制备15×浓缩酶液。具体操作如下：
(1)将菌液倒入50mL离心管内，离心机提前预冷至4℃，在13000r/min转速下离心1.5min，弃除上清液保留湿菌体沉淀。
(2)向含有湿菌体的离心管内加入5mLpH 7.4的PBS缓冲液，轻柔吹打侧壁内湿菌体使其充分悬浮。
(3)吸取100μL重悬液，加入900μLpH 7.4的PBS溶液将其稀释10倍。使用分光光度计调整细胞液OD600为1.0，若吸光度大于1.0则加入PBS缓冲液稀释；反之则重新离心并加入更少量的PBS吹打混匀。
(4)吸取5mL OD600为1.0细胞液于超声专用离心管，使用冰水浴进行超声。设定超声细胞破碎仪功率为30％，工作3s、间歇5s，对细胞液进行8min超声破碎。
(5)经超声破碎后的细胞液在4℃、4000r/min的条件下离心20min，所得上清液即为粗酶液。
酶转化反应
(1)酶转化反应母液：配置含有1mMATP、1mM NR、1mM MgSO4·7H2O的溶液，溶剂选用pH为7.4的PBS缓冲液。
(2)酶转化反应方法：吸取50μL粗酶液与50μL母液于1.5mL EP内轻柔吹打混匀，置于37℃生化培养箱孵育反应15min。孵育结束后，立即将样品置于95℃金属浴灭活1min以终止酶转化反应。
HPLC法检测NMN
选择100％磷酸二氢钠溶液分离反应体系内的ATP、NMN、NR；选择甲醇与磷酸二氢钠流动相进行梯度洗脱将反应体系内的杂物洗脱干净，具体洗脱程序见表7。
HPLC检测条件：使用ChromCore C18反相色谱柱(5μM，4.6×250mm)；流动相：A＝0.1mol/L NaH2PO4·2H2O(pH 5.5)，B＝100％甲醇；柱温为25℃；流速为1.0mL/min；进样量为20μL；紫外检测为254nm。
表7 HPLC洗脱程序
(1)进样处理：吸取酶转化反应液100μL与700μLpH 7.4的PBS缓冲液混合，使用孔径为0.22μm的微孔滤膜将其过滤至色谱进样瓶内(进样瓶内样液体积应大于500μL)后冷冻保存于冰箱-20℃。考虑检测时长与仪器损耗，每个样品做2组平行实验。
(2)HPLC法检测NMN标准曲线的测定：分别配置浓度为8、10、15、20、25、50、100μmol/L的NMN标准品各1mL，溶剂使用pH为7.4的PBS缓冲液。利用软件Origin 2019b处理数据并绘制以NMN浓度为横坐标、样品峰面积为纵坐标的标准曲线。
结果如图3和图4所示。由图3可以看出产物NMN和底物ATP、NR三种标准样品的保留时间分别为4.161min、6.161min、6.491min。将这三种标准样品混合后进样，NMN、ATP、NR的出峰时间分别为4.150min、6.212min、6.569min，混合后各标准样品的保留时间与单针进样保留时间基本吻合，说明此HPLC检测方法具备良好可重复性。从图3中可以看出NMN样品峰宽较窄、峰型较好，峰面积准确度较高；且产物NMN与底物ATP、NR的保留时间间隔在2min上，达到了良好的分离效果。由图4可以看出，NMN标准品在0-100μmol/L范围内时，使用高效液相色谱仪测定的NMN拟合出的标准曲线线性良好，其R2达到0.9997。后续根据标准曲线方程y＝1.0102x-2.1334计算反应体系内的NMN含量。
采用HPLC法测量NMN的产量，各突变体粗酶液转化结果如图5所示。可见相对于野生型mNampt，除第二株突变体的催化活性略有降低外，其余9株突变体催化活性均有所提高。其中，第六株突变体在相同条件下催化底物NAM所得产物NMN的含量高达357.084μmol/L，同野生型mNampt催化所得到的NMN含量199.432μmol/L相比酶活提高了79.05％。
实施例4
小鼠来源的mNampt突变体的固定化(以第六株突变体(氨基酸序列SEQ ID NO.7，核苷酸序列SEQ ID NO.15)为例)
(1)壳聚糖凝胶颗粒的制备：将1.5％的壳聚糖粉末溶于2％醋酸溶液，制成壳聚糖-醋酸溶液。于30℃恒温水浴锅内，使用磁力搅拌子搅拌约30min使其形成均匀透明的凝胶液体。用1mL注射器吸取壳聚糖凝胶体，将其缓慢滴加到4mol/L的NaOH溶液中形成大小均匀的凝胶颗粒，此时液面要与针尖保持垂直状态。用蒸馏水充分洗涤制得的凝胶颗粒，直到pH试纸擦拭颗粒呈中性。
(2)凝胶颗粒与戊二醛交联：将该凝胶颗粒加入到浓度为2％的戊二醛溶液中，在30℃恒温振荡摇床中混合3h，再用蒸馏水反复洗去戊二醛。将制备好的凝胶颗粒在40℃烘箱静置3min脱去多余水分后置于4℃冰箱内保存待用。
(3)微球与粗酶液交联：吸取10mL粗酶液(粗酶液制备同实施例3)与0.5g凝胶颗粒于50mL离心管内充分混合，于4℃恒温振荡摇床中交联16h，用蒸馏水洗去多余酶液后制得固定化酶。
以海藻酸钠和HPD700大孔吸附树脂对mNampt(第六株，核苷酸序列SEQ ID NO.17)进行固定化，作为对照组。
海藻酸钠-mNampt的固定化方法：
(1)海藻酸钠-mNampt凝胶的制备：准确称取0.25g海藻酸钠加入5mL无菌水内，完全溶解后与5mL粗酶液1:1混合。将混合体系置于30℃恒温水浴锅内，搅拌约30min使其形成均匀透明的海藻酸钠-mNampt凝胶液体。
(2)海藻酸钠-mNampt凝胶的硬化：以1ml注射器吸入上述凝胶液体，于5cm高度处缓慢滴入质量分数为1％的氯化钙溶液形成大小均匀的凝胶颗粒。缓慢搅拌后静置片刻，置于4℃冰箱内硬化2h。用去离子水将凝胶颗粒洗涤后置于40℃烘箱干燥3min即获得固定化酶小球，放置于4℃冰箱内保存待用。
HPD700大孔吸附树脂的固定化方法：
(1)大孔树脂的预处理：称取1g HPD700大孔树脂于无水乙醇，在200r/min、37℃恒温振荡摇床浸泡12h。用无菌水将大孔树脂颗粒充分洗涤掉无水乙醇，用滤纸擦干水分后放入4℃冰箱内储存待用。
(2)大孔树脂与酶液交联：将mNampt粗酶液与HPD700大孔树脂充分浸润，于220r/min、37℃恒温振荡摇床吸附4h(确保树脂在粗酶液中充分交联，防止树脂沉淀)，用蒸馏水洗涤2-3次后放于37℃恒温干燥箱中干燥，干燥后密封于1.5mLEP管内置于4℃冰箱中保存。
本实施例分别尝试壳聚糖、海藻酸钠以及HPD700大孔吸附树脂三种不同材料对mNampt粗酶液进行固定化，结果其中壳聚糖微球的固定化效果最高。壳聚糖本身为多孔网状的天然高分子粉粒材料，其分子中的羟基和氨基可形成活泼界面，对蛋白质有显著的亲合力，酶蛋白可通过离子键、氢键及范德华力与壳聚糖载体结合从而实现酶的固定化。
采用本发明壳聚糖固定化工艺所得的壳聚糖微球的外观形貌如图6所示。从图6中可以看出，球体光滑有弹性，呈淡黄色，具有一定的机械强度，微球大小均匀且无拖尾，直径在1.5mm左右。球体比表面积大，使得固定化酶与底物接触更为充分。
经冷冻干燥后壳聚糖微球切面的扫描电子显微镜结果如图7所示。从图7中可以看出，壳聚糖微球微观结构疏松多孔且排布密致、孔径大小均匀，其稳定的结构和适当的孔径得以充分装载酶液。对比微球与酶液固化前后差异：与酶液固定化后微球依旧具有良好的表观结构，其孔径由固定化之前的0.6(±0.4)μm增大至1.3(±0.6)μm，而孔径的膨胀则会导致酶液更易流出。推测孔径的膨胀是不恰当的固定化时间造成的，因此有必要对于固定化条件进行优化以达到更好的固定化效果。
实施例5
使用分光光度计法测定实施例4固定化酶催化底物转化生成的NMN含量，具体为：酶转化反应后，向黑色酶标板内加入50μL反应液、10μL20％的苯乙酮和20μL 2M KOH溶液后将置于冰水浴反应10min。随后继续加入90μL 88％的甲酸，并移至70℃金属浴反应10min，此时NMN已转化为荧光衍生物。设定分光光度计激发波长为382nm，发射波长445nm处测定荧光吸收值，gain值设为65，每个样品做3组平行实验。由于苯乙酮具有较强挥发性，该实验操作均在通风橱内进行。
分光光度计法检测NMN标准曲线的测定
分别配制浓度为0、5、10、20、70、80、100μmol/L的NMN标准样品，溶剂选用pH为7.4的PBS缓冲液。将不同浓度的NMN标准样品进行荧光转化反应检测不同NMN浓度荧光值。利用软件Origin 2019b处理数据并绘制以NMN浓度为横坐标、对应的荧光值为纵坐标的标准曲线。结果如图8所示。
对实验数据整理分析得：固定化酶可催化生成23.58μmol/LNMN，相同条件下游离酶催化生成的NMN为89.31μmol/L。
实施例6
使用Bradford法测定壳聚糖微球内固定的蛋白含量，做3组平行实验。在酸性条件下，考马斯亮蓝染料G-250(Bradford染色液)的三苯甲烷基团可与蛋白质的非极性结构特异性结合。此外，染色液中的磺酸盐基团也可与蛋白质的芳香类氨基酸和阳离子结合。结合后的蛋白样品吸收高峰产生红移，由465nm转换至595nm处，相应地溶液颜色由褐色转变为蓝色。可通过比色法检测溶液在595nm处的吸光值进而对蛋白定量。
Bradford蛋白定量法绘制BSA标准曲线结果如图9所示。BSA蛋白标准浓度在0-2000μg/mL范围内时，使用Bradford蛋白定量法拟合出的标准曲线线性良好，其R2达到0.9915。后续根据标准曲线方程y＝0.000607x+0.971计算体系内的蛋白浓度。其中，微球蛋白固定量＝固定前酶液中蛋白浓度-固定后酶液中蛋白浓度(μg/mL)。对实验数据整理分析得：壳聚糖微球内固定的蛋白含量为635.74μg/mL。
实施例7
不同交联剂浓度对壳聚糖固定化的影响
将浓度为1.5％的壳聚糖凝胶颗粒分别与浓度为0.5％、1％、1.5％、2％、2.5％的戊二醛交联制备壳聚糖微球，微球与粗酶液固定化时间为16h。以粗酶液作为对照，测定微球催化得到的NMN产量以及酶蛋白固定量。具体操作同实施例4。做3组平行样进行分析处理。
结果如图10所示。随着戊二醛浓度从0.5％增加至2.5％，微球的蛋白固定量以及催化转化得到的NMN含量均呈现先增长、后降低的趋势，且均在戊二醛浓度为1.5％时达到峰值。因此，使用1.5％浓度的戊二醛交联最佳。此时微球催化得到的NMN产量为32.41μmol/L，酶蛋白固定量为720.259μg/mL。
实施例8
不同固定时间对壳聚糖固定化的影响
将浓度为1.5％的壳聚糖凝胶颗粒与浓度为1.5％戊二醛交联制备壳聚糖微球。分别让微球与粗酶液固化8h、10h、12h、14h、16h。以粗酶液作为对照，测定微球催化得到的NMN产量以及酶蛋白固定量。具体操作同实施例4。
结果如图11所示。随着固定化时间从8h增加至16h，微球的蛋白固定量以及催化转化得到的NMN含量均呈现先增长、后降低的趋势，且均在交联12h时达到峰值。因此，壳聚糖微球与酶液固定时间为12h最佳。此时微球催化得到的NMN产量为28.46μmol/L，酶蛋白固定量为1022.7μg/mL。
实施例9
不同壳聚糖浓度与对壳聚糖固定化的影响
分别将浓度为1％、1.5％、2％、2.5％、3％的壳聚糖凝胶颗粒与浓度为1.5％戊二醛交联制备壳聚糖微球，微球与粗酶液固定化时间为12h。以粗酶液作为对照，测定微球催化得到的NMN产量以及酶蛋白固定量。具体操作同实施例4。
结果如图12所示。随着壳聚糖浓度从1％增加至3％，微球的蛋白固定量以及催化转化得到的NMN含量均呈现先增长、后降低的趋势，且均在壳聚糖浓度为2％时达到峰值。在制备微球的过程在也明显地发现当壳聚糖浓度低于1.5％时，形成的微球机械强度较差，易发生形变；当壳聚糖浓度高于2％时，从针头挤出的微球出现拖尾，呈水滴状。因此，使用2％浓度的壳聚糖进行固定化效果最佳。此时微球催化得到的NMN产量为35.26μmol/L，酶蛋白固定量为1159.06μg/mL。
由以上实施例可知，本发明针对mNampt设计简并引物进行定点饱和突变。将阳性克隆子进行16S rDNA测序验证，结果显示共获得10个突变体。对10株突变体进行酶活检测，除第二株突变体外其余9株突变体催化活性均有所提高。其中，氨基酸序列如SEQ ID NO.7所示、核苷酸序列如SEQ ID NO.15所示的第六株突变体在相同条件下催化底物NAM所得产物NMN的含量高达357.084μmol/L，同野生型mNampt相比提高79.05％。
分别使用壳聚糖、海藻酸钠以及HPD700大孔吸附树脂三种不同材料对mNampt进行固定化，最终壳聚糖微球对mNampt的固定化效果最好，其固定的蛋白含量为635.74μg/mL，可催化生成23.58μmol/LNMN。分别从交联剂浓度、固定化时间、壳聚糖浓度三个方面进行优化，得到最佳固定化条件为：2％的壳聚糖凝胶颗粒与1.5％的戊二醛交联3h制备壳聚糖微球，微球与mNampt粗酶液固定时间为12h。此条件下壳聚糖微球催化得到的NMN产量为35.26μmol/L，约为游离酶的39.5％，相对于优化前提高了50％；酶蛋白固定量为1159.06μg/mL，相对于优化前提高了80％。
实施例10
固定化酶的稳定性
在实际应用方面，酶的实际使用条件和反应环境大不相同。与游离酶对比，更好的稳定性和更高的回收率是固定化酶的关键意义。本实施例从固定化酶的热稳定性、pH稳定性、操作稳定性三个方面进行探究，目的是使壳聚糖固定化酶能更好地运用于社会生活中。
固定化酶热稳定性探究
分别取50uL的粗酶液(上述最优固定化条件下所得的粗酶液)于EP管中，再向每支EP管加入50uL的母液，充分摇匀后，分别在20℃、25℃、30℃、37℃、40℃、45℃水浴中反应15min计算相对酶活力。分别取0.01g固定化酶小球于EP管中，再向每支EP管加入50uL的母液，充分摇匀后，分别在20℃、25℃、30℃、37℃、40℃、45℃水浴中反应15min，以分光光度计法计算相对酶活力。其中固定化小球蛋白固定浓度＝游离酶蛋白浓度-转化反应后剩余酶液的蛋白浓度。
结果如图14所示，在温度20-45℃时固定化酶和游离酶的相对酶活力都呈现先增后减的趋势，游离酶只在30-37℃下较为稳定，继续提高温度后酶转化反应明显下降，在45℃时相对酶活力为37.37％。固定化酶在25-45℃中的变化较为平和，在37℃时相对酶活力为68.37％。在45℃下为相对酶活力为55.74％，酶活保持率为81.52％。实验结果表明壳聚糖固定化酶能使mNampt的热稳定性增强。
固定化酶pH稳定性探究
分别取50uL游离在不同pH(5、6、7、7.4、8、9)的缓冲溶液中的粗酶液于EP管中，再向每支EP管加入50uL相应pH的母液，充分摇匀后，分别在37℃水浴中反应15min计算相对酶活力。分别取0.01g的固定化酶于EP管中，再依次向每支EP管加入50uLpH分别为5、6、7、7.4、8、9的PBS缓冲溶液和50uL对应pH的母液，充分摇匀后，在37℃水浴中反应15min后以分光光度计法计算相对酶活力。
结果如图15所示，在pH值为5-9时都呈现先增后减的趋势。游离酶对pH较为敏感，在pH值＝7-8中酶活较高，在pH＝7.4时最佳。但pH增加到9时相对酶活力仅为31.36％，而固定化酶相对于游离酶下pH为5-9时影响较小，在pH＝7.4为最佳，相对酶活力为72.33％，pH＝9时相对酶活力为58.38％，酶活保持率为80.7％，实验结果表明壳聚糖固定化酶能降低较高或较低pH值对酶蛋白活性的影响，提高了酶的pH稳定性。
固定化酶的操作稳定性研究
取0.01g的固定化酶于EP管中，加入50uLpH值为7.4的PBS缓冲液和50uL母液，充分摇匀后，在37℃水浴中反应15min，计算相对酶活力。将此固定化酶按上述步骤重复利用10次，以分光光度计法分别计算相对酶活力。
结果如图16所示。前两次转化反应保持着较好的固定化效果，第三次转化反应之后相对酶活力开始下降明显，推测原因是在操作过程中使壳聚糖小球表面受到一定的损坏而导致酶蛋白流失，在第五次转化后相对于第一次酶转化实验来说，固定化酶的相对酶活力为42.5％。第十次为9.58％。而游离酶生产一次后便难以再回收利用，因此固定化酶较游离酶有可重复利用性、更好的稳定性，比游离酶更有利于运用于社会生活中。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 深圳大学
<120> 一种制备高催化活性mNampt突变体的方法及其突变体、重组菌以及应用
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> mutation
<222> (10)..(12)
<223> n=a/t/c/g,k=g/t
<220>
<221> misc_feature
<222> (10)..(10)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (11)..(11)
<223> n is a, c, g, t or u
<400> 1
caggaaattn nkgaaggcat gaaacag 27
<210> 2
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cactgtatga agaagatcat ggccatattc 30
<210> 3
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Cys Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 4
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Gly Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 5
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Ile Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 6
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Lys Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 7
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Leu Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 8
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Asn Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro
485
<210> 9
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Pro Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 10
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Arg Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 11
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Asn Ala Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp Ser
1 5 10 15
Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys Val
20 25 30
Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys Val
35 40 45
Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr Ile
50 55 60
Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile Gln
65 70 75 80
Glu Ala Lys Glu Val Tyr Arg Glu His Phe Gln Asp Asp Val Phe Asn
85 90 95
Glu Arg Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu Pro
100 105 110
Ile Glu Val Lys Ala Val Pro Glu Gly Ser Val Ile Pro Arg Gly Asn
115 120 125
Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu Thr
130 135 140
Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr Val
145 150 155 160
Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu Leu
165 170 175
Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp Phe
180 185 190
Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala Ser
195 200 205
Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Ile Ala
210 215 220
Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr Ser
225 230 235 240
Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp His
245 250 255
Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val Pro
260 265 270
Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu Lys
275 280 285
Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr Glu
290 295 300
Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr Val
305 310 315 320
Leu Lys Val Leu Asp Ile Leu Gly Lys Lys Phe Pro Val Thr Glu Asn
325 330 335
Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln Gly
340 345 350
Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Ser Glu Gly Met Lys
355 360 365
Gln Lys Lys Trp Ser Ile Glu Asn Val Ser Phe Gly Ser Gly Gly Ala
370 375 380
Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys Cys
385 390 395 400
Ser Tyr Val Val Thr Asn Gly Leu Gly Val Asn Val Phe Lys Asp Pro
405 410 415
Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu His
420 425 430
Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly Asp
435 440 445
Leu Glu Glu Tyr Gly His Asp Leu Leu His Thr Val Phe Lys Asn Gly
450 455 460
Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Val Arg Lys Asn Ala Gln
465 470 475 480
Leu Asn Ile Glu Gln Asp Val Ala Pro His
485 490
<210> 12
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
catatgaacg ctgctgctga ggccgagttc aatatattgt tagcgaccga ctcgtacaag 60
gtcacgcatt ataaacagta tcctcctaac acatcaaagg tctactcata tttcgagtgc 120
cgcgagaaga agacggagaa ctcgaaagtc cgaaaggtga agtatgaaga aacagtgttc 180
tacgggcttc agtatattct taacaaatat cttaaaggca aagttgttac aaaggagaag 240
atccaggaag ctaaagaagt ttatcgcgaa catttccaag acgatgtctt caatgagcgc 300
ggctggaact atattcttga gaagtacgac ggccatcttc ctattgaagt taaagctgtt 360
cctgaaggct cagttattcc tcgcggcaac gtcctgttta ccgtcgagaa tacggatcct 420
gaatgttatt ggcttacaaa ctggattgaa acaattcttg ttcagtcatg gtatcctatt 480
acagttgcta caaactcacg cgaacagaag aagatcctag ctaaatatct tcttgaaaca 540
tcaggcaacc ttgatggcct tgaatataaa cttcatgatt tcgggtaccg cggcgtttca 600
tcacaggaaa cagctggcat tggcgcttca gctcatcttg ttaactttaa aggcacagat 660
acagttgctg gcattgctct tattaagaag tactacggca caaaggaccc agttcctggt 720
tattcagttc ctgctgctga acattcaaca attacagctt ggggaaagga tcatgagaag 780
gacgcgttcg agcacattgt tacacagttc agtagtgttc ctgtttcagt tgtttcagat 840
tcttatgata tttataacgc ttgtgagaag atctggggag aggaccttcg ccatcttatt 900
gtttcacgct caacagaagc tcctcttatt attcgccctg attcaggcaa ccctcttgat 960
acagttctta aagttcttga tattcttggc aagaagttcc cggttaccga gaattccaag 1020
ggttataaac ttcttcctcc ttatcttcgc gttattcagg gcgatggcgt tgatattaac 1080
acacttcagg aaattgttga aggcatgaaa cagaagaagt ggtccattga gaatgtctca 1140
tttggctcag gcggcgctct tcttcagaaa cttacacgcg atcttcttaa ctgttcattt 1200
aaatgttctt atgttgttac aaacggcctt ggcgttaacg tgttcaaaga tcccgtagca 1260
gaccctaaca aacgctcaaa gaagggtcga ctttcacttc atcgcacacc tgctggcaac 1320
tttgttacac ttgaagaagg caaaggcgat cttgaagaat atggccatga tcttcttcat 1380
acagtgttca agaatggcaa ggtaacgaag tcctactcat ttgatgaagt tcgcaagaat 1440
gcgcagctta acattgaaca ggatgttgct cctcataagc tt 1482
<210> 13
<211> 979
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
accatcgctt ctttcgggct ttgttagcag ccggatctca gtggtggtgg tggtggtgct 60
cgagtgcggc cgcaagctta tgaggagcaa catcctgttc aatgttaagc tgcgcattct 120
tgcgaacttc atcaaatgag taggacttcg ttaccttgcc attcttgaac actgtatgaa 180
gaagatcatg gccatattct tcaagatcgc ctttgccttc ttcaagtgta acaaagttgc 240
cagcaggtgt gcgatgaagt gaaagtcgac ccttctttga gcgtttgtta gggtctgcta 300
cgggatcttt gaacacgtta acgccaaggc cgtttgtaac aacataagaa catttaaatg 360
aacagttaag aagatcgcgt gtaagtttct gaagaagagc gccgcctgag ccaaatgaga 420
cattctcaat ggaccacttc ttctgtttca tgccttcacc aatttcctga agtgtgttaa 480
tatcaacgcc atcgccctga ataacgcgaa gataaggagg aagaagttta taacccttgg 540
aattctcggt aaccgggaac ttcttgccaa gaatatcaag aactttaaga actgtatcaa 600
gagggttgcc tgaatcaggg cgaataataa gaggagcttc tgttgagcgt gaaacaataa 660
gatggcgaag gtcctctccc cagatcttct cacaagcgtt ataaatatca taagaatctg 720
aacaactgaa acaggaacac tactgaactg tgtaacaatg tgctcgaacg cgtccttctc 780
atgatccttt ccccaagctg taattgttga atgttcagca gcagaactga ataaccagaa 840
ctgggtcctt tgtgccgtag tacttcttaa taagagcaat gccagcaact gtatctgtgc 900
ctttaaagtt aacaagatga gctgaagcgc caatgccagc tgtttcctgt gatgaaacgc 960
cgcggtaccc gaaatcatg 979
<210> 14
<211> 977
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
agaacatcag cttctttcgg gctttgttag cagccggatc tcagtggtgg tggtggtggt 60
gctcgagtgc ggccgcaagc ttatgaggag caacatcctg ttcaatgtta agctgcgcat 120
tcttgcgaac ttcatcaaat gagtaggact tcgttacctt gccattcttg aacactgtat 180
gaagaagatc atggccatat tcttcaagat cgcctttgcc ttcttcaagt gtaacaaagt 240
tgccagcagg tgtgcgatga agtgaaagtc gacccttctt tgagcgtttg ttagggtctg 300
ctacgggatc tttgaacacg ttaacgccaa ggccgtttgt aacaacataa gaacatttaa 360
atgaacagtt aagaagatcg cgtgtaagtt tctgaagaag agcgccgcct gagccaaatg 420
agacattctc aatggaccac ttcttctgtt tcatgccttc cttaatttcc tgaagtgtgt 480
taatatcaac gccatcgccc tgaataacgc gaagataagg aggaagaagt ttataaccct 540
tggaattctc ggtaaccggg aacttcttgc caagaatatc aagaacttta agaactgtat 600
caagagggtt gcctgaatca gggcgaataa taagaggagc ttctgttgag cgtgaaacaa 660
taagatggcg aaggtcctct ccccagatct tctcacaagc gttataaata tcataagaat 720
ctgaaacaac tgaaacagga acactactga actgtgtaac aatgtgctcg aacgcgtcct 780
tctcatgatc ctttccccaa gctgtaattg ttgaatgttc agcagcagaa ctgaataacc 840
atgaactggg tcctttgtgc cgtagtactt cttaataaga gcaatgccag caactgtatc 900
tgtgccttta aagttaacaa gatgagctga agcgccaatg ccagctgttt cctgtgatga 960
aacgccgcgg tacccga 977
<210> 15
<211> 1008
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
acccatcggc ttctttcggg ctttgttagc agccggatct cagtggtggt ggtggtggtg 60
ctcgagtgcg gccgcaagct tatgaggagc aacatcctgt tcaatgttaa gctgcgcatt 120
cttgcgaact tcatcaaatg agtaggactt cgttaccttg ccattcttga acactgtatg 180
aagaagatca tggccatatt cttcaagatc gcctttgcct tcttcaagtg taacaaagtt 240
gccagcaggt gtgcgatgaa gtgaaagtcg acccttcttt gagcgtttgt tagggtctgc 300
tacgggatct ttgaacacgt taacgccaag gccgtttgta acaacataag aacatttaaa 360
tgaacagtta agaagatcgc gtgtaagttt ctgaagaaga gcgccgcctg agccaaatga 420
gacattctca atggaccact tcttctgttt catgccttcc agaatttcct gaagtgtgtt 480
aatatcaacg ccatcgccct gaataacgcg aagataagga ggaagaagtt tataaccctt 540
ggaattctcg gtaaccggga acttcttgcc aagaatatca agaactttaa gaactgtatc 600
aagagggttg cctgaatcag ggcgaataat aagaggagct tctgttgagc gtgaaacaat 660
aagatggcga aggtcctctc cccagatctt ctcacaagcg ttataaatat cataagaatc 720
tgaaacaact gaaacaggaa cactactgaa ctgtgtaaca atgtgctcga acgcgtcctt 780
ctcatgatcc tttccccaag ctgtaattgt tgaatgttca gcagcatgaa ctgaataacc 840
aggaactggg tcctttgtgc cgtagtactt cttaataaga gcaatgccag caactgtatc 900
tgtgccttta aagttaacaa gatgagctga agcgccaatg ccagctgttt cctgtgatga 960
aacgccgcgg tacccgaaat catgaagttt atattcaagc catcaagt 1008
<210> 16
<211> 967
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
actcgcttct ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg 60
agtgcggccg caagcttatg aggagcaaca tcctgttcaa tgttaagctg cgcattcttg 120
cgaacttcat caaatgagta ggacttcgtt accttgccat tcttgaacac tgtatgaaga 180
agatcatggc catattcttc aagatcgcct ttgccttctt caagtgtaac aaagttgcca 240
gcaggtgtgc gatgaagtga aagtcgaccc ttctttgagc gtttgttagg gtctgctacg 300
ggatctttga acacgttaac gccaaggccg tttgtaacaa cataagaaca tttaaatgaa 360
cagttaagaa gatcgcgtgt aagtttctga agaagagcgc cgcctgagcc aaatgagaca 420
ttctcaatgg accacttctt ctgtttcatg ccttcattaa tttcctgaag tgtgttaata 480
tcaacgccat cgccctgaat aacgcgaaga taaggaggaa gaagtttata acccttggaa 540
ttctcggtaa ccgggaactt cttgccaaga atatcaagaa ctttaagaac tgtatcaaga 600
gggttgcctg aatcagggcg aataataaga ggagcttctg ttgagcgtga aacaataaga 660
tggcgaaggt cctctcccca gatcttctca caagcgttat aaatatcata agaatctgaa 720
acaactgaaa caggaacact actgaactgt gtaacaatgt gctcgaacgc gtccttctca 780
tgatcctttc cccaagctgt aattgttgaa tgttcagcag cagaactgaa taaccaggaa 840
ctgggtcctt tgtgccgtag tacttcttaa taagagcaat gccagcaact gtatctgtgc 900
ctttaaagtt aacaagatga gctgaagcgc caatgccagc tgtttcctgt gatgaaacgc 960
cgcggta 967
<210> 17
<211> 992
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
agtggcttct ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg 60
agtgcggccg caagcttatg aggagcaaca tcctgttcaa tgttaagctg cgcattcttg 120
cgaacttcat caaatgagta ggacttcgtt accttgccat tcttgaacac tgtatgaaga 180
agatcatggc catattcttc aagatcgcct ttgccttctt caagtgtaac aaagttgcca 240
gcaggtgtgc gatgaagtga aagtcgaccc ttctttgagc gtttgttagg gtctgctacg 300
ggatctttga acacgttaac gccaaggccg tttgtaacaa cataagaaca tttaaatgaa 360
cagttaagaa gatcgcgtgt aagtttctga agaagagcgc cgcctgagcc aaatgagaca 420
ttctcaatgg accacttctt ctgtttcatg ccttccggaa tttcctgaag tgtgttaata 480
tcaacgccat cgccctgaat aacgcgaaga taaggaggaa gaagtttata acccttggaa 540
ttctcggtaa ccgggaactt cttgccaaga atatcaagaa ctttaagaac tgtatcaaga 600
gggttgcctg aatcagggcg aataataaga ggagcttctg ttgagcgtga aacaataaga 660
tggcgaaggt cctctcccca gatcttctca caagcgttat aaatatcata agaatctgaa 720
acaactgaaa caggaacact actgaactgt gtaacaatgt gctcgaacgc gtccttctca 780
tgatcctttc cccaagctgt aattgttgaa tgttcagcag cagaactgaa taaccagaac 840
tgggtccttt gtgccgtagt acttcttaat aagagcaatg ccagcaactg tatctgtgcc 900
tttaaagtta acaagatgag ctgaagcgcc aatgccagct gtttcctgtg atgaaacgcc 960
gcggtacccg aaatcatgaa gtttatattc aa 992
<210> 18
<211> 959
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acatcgcttc tttcgggctt tgttagcagc cggatctcag tggtggtggt ggtggtgctc 60
gagtgcggcc gcaagcttat gaggagcaac atcctgttca atgttaagct gcgcattctt 120
gcgaacttca tcaaatgagt aggacttcgt taccttgcca ttcttgaaca ctgtatgaag 180
aagatcatgg ccatattctt caagatcgcc tttgccttct tcaagtgtaa caaagttgcc 240
agcaggtgtg cgatgaagtg aaagtcgacc cttctttgag cgtttgttag ggtctgctac 300
gggatctttg aacacgttaa cgccaaggcc gtttgtaaca acataagaac atttaaatga 360
acagttaaga agatcgcgtg taagtttctg aagaagagcg ccgcctgagc caaatgagac 420
attctcaatg gaccacttct tctgtttcat gccttcacga atttcctgaa gtgtgttaat 480
atcaacgcca tcgccctgaa taacgcgaag ataaggagga agaagtttat aacccttgga 540
attctcggta accgggaact tcttgccaag aatatcaaga actttaagaa ctgtatcaag 600
agggttgcct gaatcagggc gaataataag aggagcttct gttgagcgtg aaacaataag 660
atggcgaagg tcctctcccc agatcttctc acaagcgtta taaatatcat aagaatctga 720
aacaactgaa acaggaacac tactgaactg tgtaacaatg tgctcgaacg cgtccttctc 780
atgatccttt ccccaagctg taattgttga atgttcagca gcagaactga ataaccagga 840
actgggtcct ttgtgccgta gtacttctta ataagagcaa tgccagcaac tgtatctgtg 900
cctttaaagt taacaagatg agctgaagcg ccaatgccag ctgtttcctg tgatgaaac 959
<210> 19
<211> 942
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aactagcctt cctttcgggc tttgttagca gccggatctc agtggtggtg gtggtggtgc 60
tcgagtgcgg ccgcaagctt atgaggagca acatcctgtt caatgttaag ctgcgcattc 120
ttgcgaactt catcaaatga gtaggacttc gttaccttgc cattcttgaa cactgtatga 180
agaagatcat ggccatattc ttcaagatcg cctttgcctt cttcaagtgt aacaaagttg 240
ccagcaggtg tgcgatgaag tgaaagtcga cccttctttg agcgtttgtt agggtctgct 300
acgggatctt tgaacacgtt aacgccaagg ccgtttgtaa caacataaga acatttaaat 360
gaacagttaa gaagatcgcg tgtaagtttc tgaagaagag cgccgcctga gccaaatgag 420
acattctcaa tggaccactt cttctgtttc atgccttccg aaatttcctg aagtgtgtta 480
atatcaacgc catcgccctg aataacgcga agataaggag gaagaagttt ataacccttg 540
gaattctcgg taaccgggaa cttcttgcca agaatatcaa gaactttaag aactgtatca 600
agagggttgc ctgaatcagg gcgaataata agaggagctt ctgttgagcg tgaaacaata 660
agatggcgaa ggtcctctcc ccagatcttc tcacaagcgt tataaatatc ataagaatct 720
gaaacaactg aaacaggaac actactgaac tgtgtaacaa tgtgctcgaa cgcgtccttc 780
tcatgatcct ttccccaagc tgtaattgtt gaatgttcag cagcaggaac tgaataacca 840
cgagctgggt cctttgtgcc gtagtacttc ttaataaaga gcaatgccag caactgtatc 900
tgtgccttta aagttaacag atgagctgaa gcgccaatgc ca 942
<210> 20
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
taatccttat tcagtggtgg tggtggtggt gctc 34
<210> 21
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aggaagcttg catatgaacg ctgctgctg 29
<210> 22
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agaggacctt cgccatctta ttg 23
<210> 23
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
aggacttcgt taccttgcca ttc 23
Claims (2)
1.一种小鼠来源的mNampt突变体的固定化方法，其特征在于，包括如下步骤：对重组菌进行诱导表达得菌液，将菌液破碎离心得粗酶液；将浓度为2％的壳聚糖凝胶颗粒与浓度为1.5％的戊二醛交联3h制备获得壳聚糖微球，将所述壳聚糖微球与所述粗酶液混合，交联12h，洗去多余酶液后得固定化酶；
所述重组菌的制备方法，包括如下步骤：以mNampt作为模板，采用引物序列进行定点饱和突变PCR扩增，筛选mNampt催化活性提高的突变体，所述引物序列的核苷酸序列如SEQ IDNO.1和SEQ ID NO.2所示；所述PCR扩增程序为98℃3min，98℃10s，58℃10s，72℃80s，30个循环，72℃10min；所述PCR扩增体系为Prime STAR Max Premix 12.5μL，正反引物序列各0.5μL，模板0.5ng，ddH2O补足至25μL；将所得的突变体进行DpnI酶切，获得突变后的重组质粒，将突变后的重组质粒转化至感受态细胞E.coli BL21(DE3)内，培养基培养，长出的单菌落即为重组菌；
所述突变体的氨基酸序列如SEQ ID NO.3-11所示；
所述壳聚糖凝胶颗粒的制备方法包括如下步骤：将2％的壳聚糖粉末溶于2％醋酸溶液中，搅拌获得凝胶液体，将凝胶液体滴加到碱性溶液中获得凝胶颗粒，洗涤凝胶颗粒至pH呈中性。
2.根据权利要求1所述的固定化方法，其特征在于，所述诱导表达包括如下步骤：将所述重组菌接种于LB培养基中扩大培养，在菌液的OD600值达到0.5-0.7时加入终浓度为0.2mM的IPTG，30℃、200r/min条件下诱导表达12h。
Priority Applications (1)
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