CN113046335B - 一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体及其应用 - Google Patents

一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体及其应用 Download PDF

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CN113046335B
CN113046335B CN201911376254.2A CN201911376254A CN113046335B CN 113046335 B CN113046335 B CN 113046335B CN 201911376254 A CN201911376254 A CN 201911376254A CN 113046335 B CN113046335 B CN 113046335B
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游淳
孟冬冬
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Abstract

本发明公开了一种仿生辅酶偏好性的葡萄糖6‑磷酸脱氢酶突变体，属于基因工程和酶工程领域。本发明采用半理性设计的方法，经过多轮定点饱和突变，提供了一系列来源于运动发酵单胞菌的葡萄糖6‑磷酸脱氢酶突变体。与野生型葡萄糖6‑磷酸脱氢酶相比，突变体利用仿生辅酶NMN+进行氧化还原反应的比酶活明显提高。本发明所示葡萄糖6‑磷酸脱氢酶将在糖产氢、糖产电等的工业生产中显示出巨大的潜在价值。

Description

一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体及其应用
技术领域
本发明涉及一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体，具体的说是一种能够以仿生辅酶作为辅因子进行氧化还原反应的葡萄糖6-磷酸脱氢酶突变体及其应用，属于基因工程和酶工程领域。
背景技术
天然辅酶（NAD(P)+）依赖型氧化还原酶被广泛的用于可替代传统有机合成的生物催化反应中。然而，天然辅酶昂贵的价格和不稳定性，限制了类似生物催化反应的规模化应用。
天然辅酶NAD(P)+是由负责电子传递的烟酰胺区域和负责在酶活性中心进行识别和定位的磷酸化或非磷酸化的腺苷二核苷酸组成。仿生辅酶（biomimetic cofactor）主要是以烟酰胺为母核，利用一些较稳定结构如糖、苯基、苯甲基等替换天然辅酶中腺苷二核苷酸而形成的。目前，报道过的仿生辅酶包括NFCD+（nicotinamide flucytosinedinucleotide）、NR+（nicotinamide mononucleoside）、NMN+（nicotinamidemononucleotide）和BNA+（1-benzyl nicotinamide）等。使用高稳定、低成本的仿生辅酶替代不稳定、昂贵的天然辅酶是降低辅酶使用成本的有效方法。然而，氧化还原酶利用仿生辅酶的催化能力及还原态仿生辅酶的再生，是目前亟待解决的问题。
体外多酶分子机器催化单糖、多糖等生产氢气、生物电的反应体系中，辅酶是介导电子和质子传递进而生成产物的关键（Zhu, Z., et al. (2014). A high-energy-density sugar biobattery based on a synthetic enzymatic pathway. Nat Commun.5: 3026; Kim, E.J., et al. (2018). Ultra-rapid rates of water splitting forbiohydrogen gas production through in vitro artificial enzymatic pathways.Energ Environ Sci. 11(8): 2064-2072）。对产氢和产电体外多酶体系中的葡萄糖6-磷酸脱氢酶（glucose 6-phosphate dehydrogenase, G6PDH, EC 1.1.1.49）和6-磷酸葡萄糖酸脱氢酶（6-phosphogluconate dehydrogenase，6PGDH，EC 1.1.1.44）进行仿生辅酶偏好性改造，具有重要的应用价值。
近期，有研究报道通过对6-磷酸葡萄糖酸脱氢酶进行多轮定向进化，极大提高了该酶利用仿生辅酶NMN+进行氧化还原反应的能力（Huang, R., et al. (2019). A high-throughput method for directed evolution of NAD(P)+-dependent dehydrogenasesfor the reduction of biomimetic nicotinamide analogues. ACS catalysis. 11(18): 3120-3130）。因此，在基于NMN+的体外多酶体系生产氢气和生物电的工业生产中，亟需对葡萄糖6-磷酸脱氢酶进行NMN+偏好性改造。
发明内容
本发明的目的在于提供一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体及其应用。
为实现上述目的，本发明采取的技术方案为：以氨基酸序列为SEQ ID NO.2所示的野生型G6PDH为出发蛋白，该蛋白已经经过热稳定性改造（Huang, R., et al. (2018).Engineering a thermostable highly active glucose 6-phosphate dehydrogenaseand its application to hydrogen production in vitro. Appl MicrobiolBiotechnol. 102(7): 3203-3215）。在半理性设计基础上对其进行多轮定点饱和突变，采用文献报道的高通量筛选方法（Huang, R., et al. (2019). A high-throughput methodfor directed evolution of NAD(P)+-dependent dehydrogenases for the reductionof biomimetic nicotinamide analogues. ACS catalysis. 11(18): 3120-3130）进行阳性克隆挑选。本发明所述仿生辅酶NMN+偏好性的葡萄糖6-磷酸脱氢酶突变体为：
（a）第一轮改造：以野生型G6PDH为出发蛋白，通过定点饱和突变、实验验证后，获得编码NMN+偏好性提高的葡萄糖6-磷酸脱氢酶突变体的多肽的核酸，该核酸编码的蛋白质序列由SEQ ID NO.4中氨基酸序列所示（R1）;
（b）第二轮改造：以第一轮最优突变体R1作为出发蛋白，通过定点饱和突变、组合突变、实验验证后，获得编码NMN+偏好性提高的葡萄糖6-磷酸脱氢酶突变体的多肽的核酸，该核酸编码的蛋白质序列由SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10中氨基酸序列所示。SEQ ID NO.10所示氨基酸序列编码蛋白质为本轮最优突变体（R2）;
（c）第三轮改造：以第二轮最优突变体R2作为出发蛋白，通过定点饱和突变、实验验证后，获得编码NMN+偏好性提高的葡萄糖6-磷酸脱氢酶突变体的多肽的核酸，该核酸编码的蛋白质序列由SEQ ID NO.12中氨基酸序列所示（R3）;
（d）第四轮改造：以第三轮最优突变体R3作为出发蛋白，通过定点饱和突变、实验验证后，获得编码NMN+偏好性提高的葡萄糖6-磷酸脱氢酶突变体的多肽的核酸，该核酸编码的蛋白质序列由SEQ ID NO.14中氨基酸序列所示（R4）;
（e）NMN+偏好性的葡萄糖6-磷酸脱氢酶突变体，其与SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14中所示氨基酸序列具有至少50%的同源性，特别是至少70%同源性，更特别是至少80%同源性的氨基酸序列。
本发明所述获得NMN+偏好性的葡萄糖6-磷酸脱氢酶突变体的方法，以pET28a-g6pdh为模板，通过同源建模获得酶的3D结构，经半理性设计，利用定点饱和突变和组合突变将编码氨基酸序列如SEQ ID NO：2所示的G6PDH的基因进行突变改造，将携带编码突变体的基因的载体导入大肠杆菌BL21(DE3)，培养重组菌，经过热处理纯化或镍柱纯化获得葡萄糖6-磷酸脱氢酶突变体。
编码所述野生型葡萄糖6-磷酸脱氢酶的核苷酸序列如SEQ ID NO：1所示，来源于运动发酵单胞菌（Zymomonas mobilis），由GenBank登录号为AHJ70511.1的序列经热稳定性改造获得，参考文献为Huang, R., et al. (2018). Engineering a thermostablehighly active glucose 6-phosphate dehydrogenase and its application tohydrogen production in vitro. Appl Microbiol Biotechnol. 102(7): 3203-3215。
本发明还提供了利用葡萄糖6-磷酸脱氢酶突变体及仿生辅酶NMN+催化葡萄糖6-磷酸（G6P）生产生物电的方法。以大肠杆菌BL21(DE3)为宿主，分别重组表达运动发酵单胞菌（Zymomonas mobilis）葡萄糖6-磷酸脱氢酶突变体R4和嗜热脂肪芽孢杆菌（Geobacillusstearothermophilus）黄递酶（diaphorase, DI, GenBank accessionnumberJQ040550.1），通过热处理或镍柱进行重组蛋白纯化，构建基于NMN+的体外多酶体系催化葡萄糖6-磷酸产电。
本发明的有益效果：经过4轮定点饱和突变和组合突变，获得了6个NMN+偏好性提高的G6PDH突变体，均实现了以NMN+为辅酶的G6PDH比酶活的提高。以出发蛋白G6PDH构建多酶反应体系，在60℃条件下以NMN+为辅酶产电的功率密度为0.13 mW/cm2，以NADP+为辅酶产电的功率密度为0.49 mW/cm2；突变体R4构建的多酶体系以NMN+为辅酶产电的功率密度为0.61 mW/cm2，以NADP+为辅酶产电的功率密度为0.18 mW/cm2。本发明提供的仿生辅酶NMN+偏好性的葡萄糖6-磷酸脱氢酶突变体是工业生产用酶制剂的有力候选者。
附图说明
图1 60℃下，以NADP+或NMN+为辅酶的体外多酶体系催化葡萄糖6-磷酸生产生物电。
实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的，不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
本发明实施例中用到如下材料
葡萄糖6-磷酸（G6P），sigma公司产品，产品编号：G5509；
pET28a载体，Novagen，Madison，WI；
大肠杆菌表达菌BL21(DE3)，Invitrogen，Carlsbad，CA；
G6PDH突变体的载体构建方法：利用Phusion定点突变方法，设计引物，其中待突变氨基酸密码子设计为NNK。以含有野生型G6PDH的载体pET28a-g6pdh或含有每轮最优突变体的载体为模板，进行PCR扩增。PCR反应体系均为：2﹡PrimerStar mixture 25 μL，正向引物(10μM)1μL，反向引物(10μM)1μL，模板DNA 1μL，加入双蒸水22 μL。PCR反应扩增条件均为：98℃预变性45 s；随后98℃ 15 s，55℃15s，72℃ 2 min进行30个循环，最后72℃保温10min。将PCR产物经过Dpn I消化30 min后，利用PCR产物纯化试剂盒进行纯化。纯化后的PCR产物3 μL，与T4 PNK 1 μL、10﹡T4 PNK buffer 1 μL、100 mM ATP 1 μL、双蒸水4 μL混匀，37℃磷酸化反应2 h。取磷酸化的PCR产物溶液10 μL与快速连接酶1 μL、快速连接酶缓冲液10 μL混匀，25℃温浴30 min，进行平末端连接。连接产物分别转入大肠杆菌E.coliTOP10的感受态细胞中，涂布到含有抗生素的LB固体培养基平板，培养过夜。
平板阳性克隆高通量筛选方法：根据文献（Huang, R., et al. (2019). A high-throughput method for directed evolution of NAD(P)+-dependent dehydrogenasesfor the reduction of biomimetic nicotinamide analogues. ACS catalysis. 11(18): 3120-3130）报道的高通量筛选方法进行阳性克隆挑选。将上述转化后的平板70℃热处理1h，破碎细胞并杀死大肠杆菌内源蛋白，降解内源还原型辅酶。通过滤纸复印菌落，经PBS洗涤4次，每次50 mL。将滤纸放置到干净培养皿中，倒上显色溶液，进行平板显色。平板上层显色溶液：0.5%琼脂糖，50 mM Tris-HCl，0.1%叠氮化钠，50 μg/mL氯霉素，150 μMWST-1，0.13 μM DI (2.8 ug/mL)，2 mM G6P，1 mM NMN+，室温放置显色。显现黄色的菌落为待挑取的潜在阳性克隆。
G6PDH及其突变体的表达：挑取上述显现黄色的潜在阳性单克隆，利用质粒提取试剂盒提质粒，转化TOP10，挑取单克隆扩大培养并提质粒送测序。将测序后含有突变体基因的载体转化BL21(DE3)菌株，接种LB液体培养基，在37℃，200rpm下培养到OD600nm为0.6-0.8，加入终浓度0.1 mM IPTG诱导，25℃继续培养12h。收集菌体，经超声破碎获得突变体蛋白粗酶液，经镍柱纯化、超滤浓缩和置换缓冲液，获得电泳纯突变体蛋白。
以NADP+或NMN+作为辅酶，G6PDH及其突变体的比酶活测定方法：酶活测定反应在37℃、100 mM HEPES缓冲液（pH 7.5）中进行。反应体系包括5 mM MgCl2、0.5 mM MnCl2、1mMNADP+或NMN+、2 mM G6P。通过测定340nm下的吸光度，根据NADPH、NMNH的摩尔吸光系数6.22 mM-1cm-1计算生成的NADPH或NMNH的量，进而计算比酶活。
实施例1 待突变位点的选择
利用I-TASSER（https://zhanglab.ccmb.med.umich.edu/I-TASSER/）以肠系膜明串珠菌来源的G6PDH（PDB: 1H9A）为模板，经同源建模获得运动单胞菌来源的G6PDH与NADP+分子对接信息。G6PDH与NADP+距离在5Å以内的残基包括F11、G12、S13、T14、G15、D16、L17、S18、R20、M21、S45、R46、S47、V88、D89、I90、S115、T116、S117、P118、L120、F121、E143、K144、P145、Y417、M421。通过设计引物，对上述位点进行定点饱和突变。
实施例2第一轮定点饱和突变
以核酸序列为SEQ ID NO.1、氨基酸序列为SEQ ID NO.2所示的野生型G6PDH为出发蛋白，对上述分析获得的与NADP+距离在5Å以内的残基分别进行定点饱和突变。利用平板高通量筛选方法进行阳性克隆筛选。经蛋白表达，获得突变体蛋白，以1 mM NADP+或NMN+作为辅酶，测定突变体的比酶活。出发蛋白G6PDH在37℃下对1 mM NADP+和NMN+的比酶活分别为152 U/mg和4.3 mU/mg。本轮突变获得将115位丝氨酸突变为丙氨酸的突变体S115A，在37℃下对1 mM NADP+和NMN+的比酶活分别为108 U/mg和52 mU/mg。本轮获得的突变体S115A命名为R1，其核酸序列为SEQ ID NO.3所示，氨基酸序列为SEQ ID NO.4所示。
实施例3第二轮定点饱和突变及组合突变
以R1为出发蛋白，对上述分析获得的与NADP+距离在5Å以内的残基（除了115位氨基酸）分别进行定点饱和突变。利用平板高通量筛选方法进行阳性克隆筛选。经蛋白表达，获得突变体蛋白，以1 mM NADP+和NMN+作为辅酶，测定突变体的比酶活。本轮突变获得突变体S115A/P118S及S115A/Y417H。在37℃下S115A/P118S对1 mM NADP+和NMN+的比酶活分别为73.8 U/mg和138 mU/mg；在37℃下S115A/Y417H对1 mM NADP+和NMN+的比酶活分别为43.6U/mg和122 mU/mg。对本轮获得的阳性突变体进行组合突变，获得突变体S115A/P118S/Y417H，在37℃下对1 mM NADP+和NMN+的比酶活分别为22.4 U/mg和180 mU/mg。本轮获得的突变体S115A/P118S，其核酸序列为SEQ ID NO.5所示，氨基酸序列为SEQ ID NO.6所示。本轮获得的突变体S115A/Y417H，其核酸序列为SEQ ID NO.7所示，氨基酸序列为SEQ ID NO.8所示。本轮获得的最优突变体S115A/P118S/Y417H命名为R2，其核酸序列为SEQ ID NO.9所示，氨基酸序列为SEQ ID NO.10所示。
实施例4 第三轮定点饱和突变
以R2为出发蛋白，对上述分析获得的与NADP+距离在5Å以内的残基（除了115、118、417位氨基酸）分别进行定点饱和突变。利用平板高通量筛选方法进行阳性克隆筛选。经蛋白表达，获得突变体蛋白，以1 mM NADP+或NMN+作为辅酶，测定突变体的比酶活。本轮突变获得突变体S115A/P118S/Y417H/M421L。在37℃下对1 mM NADP+和NMN+的比酶活分别为10.2U/mg和237 mU/mg。本轮获得的最优突变体S115A/P118S/Y417H/M421命名为R3，其核酸序列为SEQ ID NO.11所示，氨基酸序列为SEQ ID NO.12所示。
实施例5 第四轮定点饱和突变
以R3为出发蛋白，对上述分析获得的与NADP+距离在5Å以内的残基（除了115、118、417、421位氨基酸）分别进行定点饱和突变。利用平板高通量筛选方法进行阳性克隆筛选。经蛋白表达，获得突变体蛋白，以1 mM NADP+或NMN+作为辅酶，测定突变体的比酶活。本轮突变获得突变体S25A/S115A/P118S/Y417H/M421。在37℃下对1 mM NADP+和NMN+的比酶活分别为6.6 U/mg和336 mU/mg。本轮获得的最优突变体S25A/S115A/P118S/Y417H/M421命名为R4，其核酸序列为SEQ ID NO.13所示，氨基酸序列为SEQ ID NO.14所示。
经过4轮定点饱和突变和组合突变，相比G6PDH，最优突变体R4在37℃下对1 mMNADP+的比酶活下降23倍，对1 mM NMN+的比酶活提高78倍。各轮突变后最优突变体的辅酶偏好性及动力学参数如表1所示。
表1 G6PDH及其突变体的动力学参数
Figure SMS_1
实施例6 60℃下体外多酶催化葡萄糖6-磷酸生产生物电
构建体外多酶体系产电系统。阳极溶液包含100 mM HEPES缓冲液(pH 7.5)、5 mMMgCl2, 0.5 mM MnCl2、20 mMNADP+或NMN+、40 mM G6P、100 mM NaCl、2 g/L G6PDH或突变体R4、2 g/L DI。含有固定化电子传递体VK3的1 cm2炭毡作为阳极浸入阳极溶液中，浸润在50mM铁氰化钾（100 mM PBS, pH 7.5）溶液中的炭毡作为阴极。阳极与阴极之间通过质子交换膜Nafion 212进行分隔。在60℃下，利用线性伏安扫描法，以1 mV/s扫速进行扫描，测定整个体系的产电能力。包含G6PDH的多酶体系以NMN+为辅酶产电的功率密度为0.13 mW/cm2，以NADP+为辅酶产电的功率密度为0.49 mW/cm2；突变体R4构建的多酶体系以NMN+为辅酶产电的功率密度为0.61 mW/cm2，以NADP+为辅酶产电的功率密度为0.18 mW/cm2（图1）。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体及其应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1458
<212> DNA
<213> 人工序列()
<400> 1
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc tttcgacttc gccttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctta tgaacgcctg 1260
atgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 2
<211> 485
<212> PRT
<213> 人工序列()
<400> 2
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ser Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ser Thr Ser Pro Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
Tyr Glu Arg Leu Met Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485
<210> 3
<211> 1458
<212> DNA
<213> 人工序列()
<400> 3
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc ttgcgacttc gccttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctta tgaacgcctg 1260
atgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 4
<211> 485
<212> PRT
<213> 人工序列()
<400> 4
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ser Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ala Thr Ser Pro Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
Tyr Glu Arg Leu Met Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485
<210> 5
<211> 1458
<212> DNA
<213> 人工序列()
<400> 5
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc ttgcgacttc gagttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctta tgaacgcctg 1260
atgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 6
<211> 485
<212> PRT
<213> 人工序列()
<400> 6
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ser Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ala Thr Ser Ser Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
Tyr Glu Arg Leu Met Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485
<210> 7
<211> 1458
<212> DNA
<213> 人工序列()
<400> 7
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc ttgcgacttc gccttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctca tgaacgcctg 1260
atgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 8
<211> 485
<212> PRT
<213> 人工序列()
<400> 8
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ser Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ala Thr Ser Pro Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
His Glu Arg Leu Met Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485
<210> 9
<211> 1458
<212> DNA
<213> 人工序列()
<400> 9
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc ttgcgacttc gagttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctca tgaacgcctg 1260
atgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 10
<211> 485
<212> PRT
<213> 人工序列()
<400> 10
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ser Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ala Thr Ser Ser Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
His Glu Arg Leu Met Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485
<210> 11
<211> 1458
<212> DNA
<213> 人工序列()
<400> 11
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc ttgcgacttc gagttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctca tgaacgcctg 1260
ttgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 12
<211> 485
<212> PRT
<213> 人工序列()
<400> 12
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ser Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ala Thr Ser Ser Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
His Glu Arg Leu Leu Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485
<210> 13
<211> 1458
<212> DNA
<213> 人工序列()
<400> 13
atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct ttcacagcgt 60
atgctgttgc cggcgcttta tggtcttgat gccgatggtt tgcttgcgga tgatctgcgt 120
atcgtctgca cctctcgtag cgaatacgac acagatggtt tccgtgattt tgcagaaaaa 180
gctttagatc gctttgtcgc ttctgaccgg ttaaatgatg acgctaaagc taaattcctt 240
aacaagcttt tctacgcgac ggtcgatatt acggatccga cccaattcgg aaaaatagct 300
gacctttgtg gcccggtcga aaaaggtatc gccatttatc ttgcgacttc gagttctttg 360
tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac ttctcgcctg 420
gcgcttgaaa aacctttagg tcaagatctt gcttcttccg atcatattaa tgatgcggtt 480
ttgaaagttt tctctgaaaa gcaagtttat cgtattgacc attatctggg taaagaaacg 540
gttcagaatc ttctgaccct gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600
aaaggcattg accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc 660
ggttatttcg acagttctgg cagcttgcgc gatatggttc aaagccatat ccttcagttg 720
gtcgctttgg ttgcaatgga accaccggcg catatggaag ccaacgctgt tcgtgacgaa 780
aaggtaaaag ttttccgcgc tctgcgtccg atcaataacg acaccgtcat tacgcatacc 840
gttaccggtc aatatggtgc cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900
gaactgggtc agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac 960
tggcgttggc acggtgttcc gttctatatc cgcactggta agcgtttacc tgcacgtcgt 1020
tctgaaatcg tggttcagtt taaacctgtt ccgcattcga ttttctcttc ttcaggtggt 1080
atcttgcagc cgaacaagct gcgtattgtc ttacagcctg atgaaaccat ccagatttct 1140
ataatggtga aagaaccggg tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200
gatctttccc tcacggatgt gtttaaagac cgtaaacgtc gtatcgctca tgaacgcctg 1260
ttgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga agttgaggcg 1320
cagtggattt ggattgacgg aattcgtgaa ggctggaaag ccaacagtat gaagccaaaa 1380
acctatgtct ctggtacatg ggggcctatt actgctatag ctctggtcga acgtgatgga 1440
gtaacttggt atgactga 1458
<210> 14
<211> 485
<212> PRT
<213> 人工序列()
<400> 14
Met Thr Asn Thr Val Ser Thr Met Ile Leu Phe Gly Ser Thr Gly Asp
1 5 10 15
Leu Ser Gln Arg Met Leu Leu Pro Ala Leu Tyr Gly Leu Asp Ala Asp
20 25 30
Gly Leu Leu Ala Asp Asp Leu Arg Ile Val Cys Thr Ser Arg Ser Glu
35 40 45
Tyr Asp Thr Asp Gly Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg
50 55 60
Phe Val Ala Ser Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu
65 70 75 80
Asn Lys Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe
85 90 95
Gly Lys Ile Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile
100 105 110
Tyr Leu Ala Thr Ser Ser Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125
Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu Glu Lys
130 135 140
Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile Asn Asp Ala Val
145 150 155 160
Leu Lys Val Phe Ser Glu Lys Gln Val Tyr Arg Ile Asp His Tyr Leu
165 170 175
Gly Lys Glu Thr Val Gln Asn Leu Leu Thr Leu Arg Phe Gly Asn Ala
180 185 190
Leu Phe Glu Pro Leu Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile
195 200 205
Ser Val Ala Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp
210 215 220
Ser Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu
225 230 235 240
Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala Asn Ala
245 250 255
Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu Arg Pro Ile Asn
260 265 270
Asn Asp Thr Val Ile Thr His Thr Val Thr Gly Gln Tyr Gly Ala Gly
275 280 285
Val Ser Gly Gly Lys Glu Val Ala Gly Tyr Ile Asp Glu Leu Gly Gln
290 295 300
Pro Ser Asp Thr Glu Thr Phe Val Ala Ile Lys Ala His Val Asp Asn
305 310 315 320
Trp Arg Trp His Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu
325 330 335
Pro Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His
340 345 350
Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365
Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Ile Met Val Lys
370 375 380
Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg Glu Val Trp Leu
385 390 395 400
Asp Leu Ser Leu Thr Asp Val Phe Lys Asp Arg Lys Arg Arg Ile Ala
405 410 415
His Glu Arg Leu Leu Leu Asp Leu Ile Glu Gly Asp Ala Thr Leu Phe
420 425 430
Val Arg Arg Asp Glu Val Glu Ala Gln Trp Ile Trp Ile Asp Gly Ile
435 440 445
Arg Glu Gly Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser
450 455 460
Gly Thr Trp Gly Pro Ile Thr Ala Ile Ala Leu Val Glu Arg Asp Gly
465 470 475 480
Val Thr Trp Tyr Asp
485

Claims (15)

1.一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体，其特征在于，是将氨基酸序列如SEQ ID NO .2所示的G6PDH进行定点饱和突变和/或组合突变，获得以NMN+作为辅酶，比酶活提高的葡萄糖6-磷酸脱氢酶突变体，所述突变体的氨基酸序列为突变位点S115A或突变位点S115A与S25A、P118S、Y417H、M421其中的一个或多个位点的组合，其中，突变体为S115A，氨基酸序列如SEQ ID NO.4所示；S115A/P118S，氨基酸序列如SEQ ID NO.6所示；S115A/Y417H，氨基酸序列如SEQ ID NO.8所示；S115A/P118S/Y417H，氨基酸序列如SEQ IDNO.10所示；S115A/P118S/Y417H/M421L，氨基酸序列如SEQ ID NO.12所示；S25A/S115A/P118S/Y417H/M421L，氨基酸序列如SEQ ID NO.14所示。
2.编码权利要求1所述葡萄糖6-磷酸脱氢酶突变体的基因。
3.携带权利要求2所述基因的载体或细胞。
4.一种获得权利要求1所述葡萄糖6-磷酸脱氢酶突变体的方法，其特征在于，以含有野生型或突变体G6PDH的编码基因的载体为模版，将如序列SEQ ID NO .1、SEQ ID NO .3、SEQID NO .9和SEQ ID NO .11所示的基因进行定点饱和突变，将携带编码突变体的基因的载体转入大肠杆菌BL21(DE3)，培养重组菌得到葡萄糖6-磷酸脱氢酶突变体。
5.一种催化G6P生产生物电的方法，其特征在于，以大肠杆菌BL21(DE3)为宿主，分别重组表达运动发酵单胞菌权利要求1所述葡萄糖6-磷酸脱氢酶突变体和嗜热脂肪芽孢杆菌黄递酶，纯化获得重组酶，构建体外多酶体系，催化G6P生产生物电。
6.权利要求5所述产电体外多酶体系，其特征在于，还包括G6P、镁盐、锰盐、辅酶、电子传递体，所述镁盐选自氯化镁或硫酸镁；所述锰盐选自氯化锰或硫酸锰；所述辅酶为天然辅酶NAD+、NADP+或人工辅酶NMN+；所述电子传递体为维生素K3或9 ,10-蒽醌-2 ,7-二磺酸。
7.根据权利要求6所述产电体外多酶体系，其特征在于，所述镁盐的浓度为1-20mM，锰盐的浓度为0.01-5mM。
8.根据权利要求7所述产电体外多酶体系，其特征在于，所述镁盐的浓度为2-15mM，锰盐的浓度为0.1-2mM。
9.根据权利要求8所述产电体外多酶体系，其特征在于，所述镁盐的浓度为3-10mM，锰盐的浓度为0.2-1mM。
10.根据权利要求9所述产电体外多酶体系，其特征在于，所述镁盐的浓度为5mM；锰盐的浓度为0.5mM。
11.权利要求5-10之一所述体外多酶体系，其特征在于，酶组分可以是任何来源的具有相同功能的酶资源，所述多酶催化反应的温度为10-95℃；所述多酶催化反应中G6P底物的浓度为1-200mM；所述多酶催化反应中酶的用量为0.1-50U/mL。
12.根据权利要求11所述体外多酶体系，其特征在于，所述多酶催化反应的温度为20-80℃；所述多酶催化反应中G6P底物的浓度为5-100mM；所述多酶催化反应中酶的用量为0.5-10U/mL。
13.根据权利要求12所述体外多酶体系，其特征在于，所述多酶催化反应的温度为50-70℃；所述多酶催化反应中G6P底物的浓度为20-60mM；所述多酶催化反应中酶的用量为1-5U/mL。
14.根据权利要求13所述体外多酶体系，其特征在于，所述多酶催化反应的温度为60℃；所述多酶催化反应中G6P底物的浓度为40mM。
15.应用权利要求1-4任一所述葡萄糖6-磷酸脱氢酶突变体生产生物电的方法。其特征在于，所述体外多酶体系以葡萄糖6-磷酸脱氢酶突变体为催化剂、以NMN+作为辅酶，催化G6P生产生物电的应用。
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