CN114525268B - 一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 - Google Patents

一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 Download PDF

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CN114525268B
CN114525268B CN202210086048.3A CN202210086048A CN114525268B CN 114525268 B CN114525268 B CN 114525268B CN 202210086048 A CN202210086048 A CN 202210086048A CN 114525268 B CN114525268 B CN 114525268B
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饶志明
韩瑾
杨套伟
徐美娟
张显
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Abstract

本发明属于生物工程技术领域，具体涉及一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ‑氨基丁酸合成中的应用，该突变体通过将氨基酸序列如SEQ ID NO.3所示的谷氨酸脱羧酶突变得到。该突变体在pH 6.5时的酶活提高至原始(SEQ ID NO.3所示)的178％，在5L罐中12h分批补料1000g底物最终产量高达688.13g/L，比原始谷氨酸脱羧酶生产性能提高约52％，最终摩尔转化率可达98.2％。本发明不仅拓宽了GAD在最适pH下的酶活，还拓宽其在偏中性pH下的酶活，增强了该酶合成γ‑氨基丁酸的能力，使其更适应于工业生产。

Description

一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁 酸合成中的应用
技术领域
本发明属于生物工程技术领域，具体涉及一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用。
背景技术
GABA(γ-氨基丁酸)是一种天然存在的非蛋白质氨基酸，作为哺乳动物中枢神经系统中主要的抑制性神经递质，具有抗焦虑、降血压、镇定安神、增强记忆、预防肥胖及糖尿病等多种重要的生理功能，因而被广泛应用于医药及食品领域。
目前，有关GABA的制备方法主要有化学合成法和生物合成法两大类。根据生物催化剂种类的不同，后者又可进一步细分为植物转化法、微生物转化法和酶法。常见的GABA化学合成法主要有两种。一种是以邻苯二甲酰亚胺钾和γ－氯丁氰为底物，在180℃条件下反应后，产物再与浓硫酸回流水解，经结晶提纯得到GABA。另一种方法是以吡咯烷酮作为原料，在121℃-135℃下反应，经Ca(OH)2和NH4HCO3水解开环而制得GABA。由于工业生产反应条件苛刻，且反应过程容易产生有毒物质，因此GABA的生产主要为微生物酶法合成，利用谷氨酸脱羧酶(GAD)能催化L-谷氨酸脱羧生成γ-氨基丁酸，反应过程中消耗一个质子。
GAD广泛存在于细菌、酵母和丝状真菌等各种微生物中，介导了胞内GABA的合成。利用含有GAD的微生物转化制备GABA，不受资源、环境和空间等限制，成为工业化生产GABA最为有效的手段之一，受到人们普遍关注。目前，自然界中存在的GAD最适pH普遍在4.0-5.0之间，在中性pH下酶活力急剧下降，而在微生物自然发酵或转化过程中，由于菌体催化活性往往会随着酵液中GABA积累所导致pH值的上升而降低，从而致使底物转化率偏低。因此拓宽谷氨酸脱羧酶的pH作用范围可有助于底物转化率的提高。
发明内容
本发明旨在提供一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用，在偏中性条件下的酶活提高，能够提高制备γ-氨基丁酸的转化效率。
按照本发明的技术方案，所述pH耐受性提高的谷氨酸脱羧酶突变体，通过将氨基酸序列如SEQ ID NO.3所示的谷氨酸脱羧酶经以下任一方式突变得到：
(1)第24位丝氨酸突变为精氨酸，氨基酸序列如SEQ ID NO.7所示；
(2)第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸，氨基酸序列如SEQ ID NO.9所示；
(3)第24位丝氨酸突变为精氨酸，第309位的酪氨酸突变为赖氨酸，氨基酸序列如SEQ ID NO.11所示；
(4)第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸以及第309位的酪氨酸突变为赖氨酸，氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.3所示谷氨酸脱羧酶与GB 01-21菌株谷氨酸脱羧酶(氨基酸序列如SEQID NO.5所示，核苷酸序列如SEQ ID NO.6所示)相比具有四个氨基酸残基突变，分别是第72位脯氨酸突变为苏氨酸、第154位丙氨酸突变为苏氨酸、第185位甲硫氨酸突变为缬氨酸以及第412位亮氨酸突变为苯丙氨酸。
编码SEQ ID NO.3所示谷氨酸脱羧酶的基因的核苷酸序列如SEQ ID NO.4所示。
本发明的第二方面提供了编码上述pH耐受性提高的谷氨酸脱羧酶突变体的基因。
进一步的，所述基因的核苷酸序列如SEQ ID NO.2、SEQ ID NO.8、SEQ ID NO.10或SEQ ID NO.12所示。
具体的，编码氨基酸序列如SEQ ID NO.1所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.2所示；编码氨基酸序列如SEQ ID NO.7所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.8所示；编码氨基酸序列如SEQ ID NO.9所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.10所示；编码氨基酸序列如SEQ ID NO.11所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.12所示。
本发明的第三方面提供了携带上述基因的重组表达载体。
进一步的，以pET-28a、PMA5或PXMJ-19作为原始表达载体。
本发明的第四方面提供了含有上述重组表达载体的重组菌。
进一步的，所述重组菌以大肠杆菌、枯草芽孢杆菌或谷氨酸棒杆菌作为宿主菌。
进一步的，所述枯草芽孢杆菌包括枯草芽孢杆菌168(Bacillus subtilis 168，BS168)。
本发明的第五方面提供了上述pH耐受性提高的谷氨酸脱羧酶突变体、基因、重组表达载体或重组菌在合成γ-氨基丁酸中的应用。
进一步的，以L-谷氨酸或L谷氨酸钠为底物，上述pH耐受性提高的谷氨酸脱羧酶突变体或重组菌为催化剂，生成γ-氨基丁酸。
进一步的，以0.9％NaCl溶液为转化体系，不需要调节pH，转化以后产物提取简单。
进一步的，温度为25-60℃。
本发明的技术方案相比现有技术具有以下优点：
本发明通过将谷氨酸脱羧酶进行突变，不仅拓宽了GAD在最适pH下的酶活，还拓宽其在偏中性pH下的酶活，增强了该酶合成γ-氨基丁酸的能力，使其更适应于工业生产；
试验结果表明，在不同pH条件下本发明的谷氨酸脱羧酶突变体比亲本谷氨酸脱羧酶的酶活均有所上升，特别是突变方式为第24位丝氨酸突变为精氨酸，第88位天冬氨酸突变为精氨酸以及第309位酪氨酸残基突变成赖氨酸残基时，在pH 6.5时的酶活提高至原始(SEQ ID NO.3所示)的178％，在5L罐中12h分批补料1000g底物最终产量高达688.13g/L，比原始谷氨酸脱羧酶提高约52％，最终摩尔转化率可达98.2％。
附图说明
图1是突变株的SDS-PAGE图。
图2为不同突变体的相对酶活。
图3是为三组合突变株在不同pH下的相对酶活。
图4是为三组合突变株在不同温度下的相对酶活。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1含编码谷氨酸脱羧酶突变体基因的重组表达载体的构建
以含有SEQ ID NO：4所示核苷酸序列的pET-28a重组质粒为模板，分别以含有突变位点的引物扩增出突变位点上下游的基因片段，采用融合PCR法构建突变基因，其中组合突变体通过以单突变质粒为模板以含有突变位点的引物扩增出突变位点上下游基因片段，再采用融合PCR法，最终可得到一系列基因。具体如下：
P1：ATGGCAATGTTATACGGTAAACACAAT
P2：CTTAGGAAGATCATGTTGTTCGCGAGG
P3：GGTGCGCCTCGCGAACAACATGATCTT
P4：TCAGATTTCCGGATGGCATTCTTTCGC
P5：TGCCATCCGGAAATCTGAGTACCCCCG
P6：CATTGCTTTACCGCCTAACATACAAGC
P7：AGGCGGTAAAGCAATGAAATTCGCCTG
P8：AAACTTACGCCAGCAAACTTGATAGCC
P9：TTGCTGGCGTAAGTTTTGTGTCTACTG
P10：TAAGACACGGTTAACGTCAAGGACCAT
P11：GTTAACCGTGTCTTAGACTACGTGGAC
P12：ATCGAGTTTGGCTAGGTCGTCATATTG
P13：CTAGCCAAACTCGATAAGGTCGTTACT
P14：CCCACCTAATTTACTAACTTTGAAGAC
P15：AAAGTTAGTAAATTAGGTGGGGAGTTG
P16：CAGAGCGCGTGCCAGGTAGCGGGCAAC
P17：CTGGCACGCGCTCTGGATAAAGTTGGT
P18：TTGTTGTTTCAGATTAGCAGGGAAAGG
P19：AATCTGAAACAACAAGTCATCCAACGA
P20：TCAGTGTGTGAATAGGTATTTCTTAGG
利用限制性内切酶EcoR I/Hind III对表达质粒pET-28a进行酶切处理以获得线性化载体，目的基因与线性化载体连接后获得重组质粒，将重组质粒用化学转化法转化至大肠杆菌JM109中，涂布于含卡那霉素抗性的LB平板，37℃下培养过夜，随机挑取克隆进行菌落PCR鉴定和测序验证，结果表明含有编码谷氨酸脱羧酶突变体的基因的重组表达载体成功转化至克隆载体大肠杆菌JM109中，经测序验证突变成功的菌液提取重组质粒保存于-20℃冰箱。测序工作由苏州金唯智完成。
实施例2在大肠BL21中进行GAD突变体的表达及纯化
将含有突变基因的pET-28a重组质粒采用化学转化法转化至大肠杆菌BL21中。重组大肠杆菌经过16℃诱导12h后离心收集菌体破碎细胞，采用镍柱进行蛋白纯化，所得纯化酶添加10％的甘油于4℃贮存备用。纯化后的酶用SDS-PAGE进行分析，其中E.coli BL21/pET-28a-lpgadS24R/D88R/Y309K结果参见图1，其中M，蛋白质Marker；泳道1，pET28a；泳道2，lpgadS24R/D88R/Y309K突变体上清；泳道3，纯化lpgadS24R/D88R/Y309K突变体。结果表明得到电泳纯的重组谷氨酸脱羧酶突变体。
实施例3谷氨酸脱羧酶的酶活测定及γ-氨基丁酸的HPLC检测
测定了实施例2中所得一系列纯化酶(谷氨酸脱羧酶突变体)在最适pH 4.5以及pH6.0时与L-谷氨酸反应时的相对酶活(以SEQ ID NO.3所示酶活为100％)，如图2所示，结果显示：lpgadS24R、lpgadS24R/D88R、lpgadS24R/Y309K和lpgadS24R/D88R/Y309K的相对酶活较高，且pH 6.0条件下相对酶活高于pH 4.5。
同时测定谷氨酸脱羧酶lpgadS24R/D88R/Y309K突变体在pH为4.0、4.5、5.0、5.5、6.0、6.5、7.0(如图3所示，可以看出其在pH6.5时相对酶活最高，pH5.0-7.0时相对酶活均处于较高水平)以及在25℃、30℃、35℃、40℃、50℃、55℃、60℃时的相对酶活(如图4所示)。反应30min后取样用HPLC法测产物γ-氨基丁酸的含量。
定义酶活单位为：1个酶活单位(U)等于在30℃下，在1min内催化L-谷氨酸生成1nmolγ-氨基丁酸所需的酶量。比酶活为1mg湿细胞的酶活，单位为U/mg。酶与底物发生脱羧反应的1mL反应体系采用的各物质浓度为：0.01mM PLP、100mM L-谷氨酸钠。在反应之前分别把缓冲液和酶液在30℃下预热5min，然后混匀，在30℃下反应30min，最后迅速煮沸终止反应，离心。用5％三氯乙酸(TCA)稀释5倍，在4℃冰箱沉淀蛋白3h左右，然后用HPLC检测。
HPLC：取反应液用5％三氯乙酸(TCA)稀释5倍，在4℃冰箱沉淀蛋白3h左右离心，取上清液用0.22μ膜进行过滤处理。然后进样。色谱柱：dimosoil C18(5μL,250mm×4.6mm)，流动相：A：0.1％甲酸水溶液，B：100％乙腈，检测器：UV Detector，检测波长：360nm，柱温：25℃，进样量：10μL，流速：1.0mL/min。过程：0～22min:15％B→50％B；22～22.1min:50％B→15％B；22.1～26min:15％B。
实施例4lpgadS24R/D88R/Y309K在枯草芽孢杆菌中的构建
以实施例1构建的pET-28a-lpgadS24R/D88R/Y309K质粒为模板，以引物P1/P2扩增获得目的基因，并利用限制性内切酶BamHI/Mlu I对表达质粒PMA5进行酶切处理以获得线性化载体，目的基因与线性化载体连接后获得重组质粒PMA5-lpgadS24R/D88R/Y309K，将重组质粒用化学转化法转化至大肠杆菌JM109中，涂布于含氨苄霉素抗性的LB平板，37℃下培养过夜，随机挑取克隆进行菌落PCR鉴定，结果表明含有编码谷氨酸脱羧酶突变体基因的重组质粒成功转化至克隆载体大肠杆菌JM109中，挑取经验证为阳性菌落接种于5mL LB液体培养基中于37℃下培养过夜，菌液提取重组质粒保存于-20℃冰箱。
P1：ATGGCAATGTTATACGGTAAACACAAT
P2：TCAGTGTGTGAATAGGTATTTCTTAGG
采用化学转化法将构建成功的PMA5-lpgadS24R/D88R/Y309K重组质粒转化至枯草芽孢杆菌BS168中构建BS168/PMA5-lpgadS24R/D88R/Y309K重组菌株，涂布于含卡那霉素抗性的LB平板，37℃下培养过夜，随机挑取克隆进行菌落PCR鉴定，验证转化成功的菌液加入甘油并于-80℃冰箱保藏。
实施例5BS168/PMA5-lpgadS24R/D88R/Y309K工程菌制备γ-氨基丁酸
将实施例4中的BS168/PMA5-lpgadS24R/D88R/Y309K突变型工程菌进行L-谷氨酸底物的转化。在5L罐中以1L 0.9％NaCl溶液为缓冲体系，菌量OD600nm控制为16，分批添加100g/l L-谷氨酸，在30℃下反应12h，lpgadS24R/D88R/Y309K工程菌分批补料1000g底物最终产量高达688.13g/L，比原始酶提高约52％，摩尔转化率最高可达98.2％。结果表明该突变酶的催化效率明显提升，具有广阔的工业化应用前景。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
SEQUENCE LISTING
<110> 江南大学
<120> 一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用
<130> 1.25
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 469
<212> PRT
<213> （人工合成）
<400> 1
Met Ala Met Leu Tyr Gly Lys His Asn His Glu Ala Glu Glu Tyr Leu
1 5 10 15
Glu Pro Val Phe Gly Ala Pro Arg Glu Gln His Asp Leu Pro Lys Tyr
20 25 30
Arg Leu Pro Lys His Ser Leu Ser Pro Arg Glu Ala Asp Arg Leu Val
35 40 45
Arg Asp Glu Leu Leu Asp Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr
50 55 60
Phe Cys Gln Thr Tyr Met Glu Thr Glu Ala Val Glu Leu Met Lys Asp
65 70 75 80
Thr Leu Arg Lys Asn Ala Ile Arg Lys Ser Glu Tyr Pro Arg Thr Ala
85 90 95
Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala
100 105 110
Pro Asp Asp Glu His Phe Thr Gly Thr Ser Thr Thr Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg
130 135 140
Ala Gln Ala Ala Gly Leu Asp Leu Asn Thr His Arg Pro Asn Leu Val
145 150 155 160
Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp
165 170 175
Asp Val Asp Met His Val Val Pro Val Asp Glu Gln His Met Val Leu
180 185 190
Asp Val Asn His Val Leu Asp Tyr Val Asp Glu Tyr Ile Ile Gly Ile
195 200 205
Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala
210 215 220
Ala Leu Asp Lys Val Val Thr His Tyr Asn His Gln His Pro Lys Leu
225 230 235 240
Pro Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro
245 250 255
Phe Ile Glu Pro Gln Leu Ile Trp Asp Phe Arg Leu Ala Asn Val Val
260 265 270
Ser Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Val Trp Arg Asp Arg Gln Phe Leu Pro Pro Glu Leu Val
290 295 300
Phe Lys Val Ser Lys Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn
305 310 315 320
Phe Ser His Ser Ala Ala Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile
325 330 335
Arg Phe Gly Met Asp Gly Tyr Arg Glu Ile Gln Thr Lys Thr His Asp
340 345 350
Val Ala Arg Tyr Leu Ala Ala Ala Leu Asp Lys Val Gly Glu Phe Lys
355 360 365
Met Ile Asn Asn Gly His Gln Leu Pro Leu Ile Cys Tyr Gln Leu Ala
370 375 380
Pro Arg Glu Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu
385 390 395 400
Leu Met Asn Gly Trp Gln Val Pro Thr Tyr Pro Phe Pro Ala Asn Leu
405 410 415
Glu Gln Gln Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met
420 425 430
Asn Met Ala His Asp Phe Met Asp Asp Leu Thr Lys Ala Val His Asp
435 440 445
Leu Asn His Ala His Ile Val Tyr His His Asp Ala Ala Pro Lys Lys
450 455 460
Tyr Leu Phe Thr His
465
<210> 2
<211> 1410
<212> DNA
<213> （人工合成）
<400> 2
atggcaatgt tatacggtaa acacaatcat gaagctgaag aatacttgga accagtcttt 60
ggtgcgcctc gggaacaaca tgatcttcct aagtatcggt taccaaagca ttcattatcc 120
cctcgagaag ccgatcgctt agttcgtgat gaattattag atgaaggcaa ttcacgactg 180
aacctggcaa ctttttgtca gacctatatg gaaaccgaag ccgttgaatt gatgaaggat 240
acgctgcgaa agaatgccat ccggaaatct gagtaccccc gcacggccga gattgaaaat 300
cggtgtgtga acattattgc caatctgtgg cacgcacctg atgacgaaca ctttacgggt 360
acctctacga caggctcctc tgaagcttgt atgttaggcg gtttagcaat gaaattcgcc 420
tggcgtaaac gcgctcaagc ggcaggttta gatctgaata cccatcgacc taacctcgtt 480
atttcggctg gctatcaagt ttgctgggaa aagttttgtg tctactggga cgttgacatg 540
cacgtggtcc cagtggatga gcaacacatg gtccttgacg ttaaccacgt cttagactac 600
gtggacgaat acattattgg tatcgtcggt atcatgggca tcacttatac cggtcaatat 660
gacgacctag ccgcactcga taaggtcgtt actcactaca atcatcagca tcccaaatta 720
ccagtctaca ttcacgttga cgcagcgtca ggtggcttct ataccccatt tattgagccg 780
caactcatct gggacttccg gttggctaac gtcgtttcga tcaacgcctc cgggcacaag 840
tacggtttag tttatcccgg ggtcggctgg gtcgtttggc gtgatcgtca gtttttaccg 900
ccagaattag tcttcaaagt tagtaaatta ggtggggagt tgccgacaat ggcgatcaac 960
ttctcacata gtgcagccca gctcattgga caatactata atttcattcg ctttggtatg 1020
gacggttacc gcgagattca aacaaagact cacgatgttg cccgctacct ggcagccgct 1080
ctggataaag ttggtgagtt taagatgatc aataacggac accaactccc cctgatttgt 1140
taccaactag ccccgcgcga agatcgtgaa tggacccttt atgatttatc ggatcgccta 1200
ttaatgaacg gttggcaagt accaacgtat cctttccctg ctaatctgga acaacaagtc 1260
atccaacgaa tcgtcgttcg ggctgacttt ggcatgaata tggcccacga tttcatggat 1320
gacctgacca aggctgtcca tgacttaaac cacgcccaca ttgtctatca tcatgacgcg 1380
gcacctaaga aatacctatt cacacactga 1410
<210> 3
<211> 469
<212> PRT
<213> （人工合成）
<400> 3
Met Ala Met Leu Tyr Gly Lys His Asn His Glu Ala Glu Glu Tyr Leu
1 5 10 15
Glu Pro Val Phe Gly Ala Pro Ser Glu Gln His Asp Leu Pro Lys Tyr
20 25 30
Arg Leu Pro Lys His Ser Leu Ser Pro Arg Glu Ala Asp Arg Leu Val
35 40 45
Arg Asp Glu Leu Leu Asp Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr
50 55 60
Phe Cys Gln Thr Tyr Met Glu Thr Glu Ala Val Glu Leu Met Lys Asp
65 70 75 80
Thr Leu Arg Lys Asn Ala Ile Asp Lys Ser Glu Tyr Pro Arg Thr Ala
85 90 95
Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala
100 105 110
Pro Asp Asp Glu His Phe Thr Gly Thr Ser Thr Thr Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg
130 135 140
Ala Gln Ala Ala Gly Leu Asp Leu Asn Thr His Arg Pro Asn Leu Val
145 150 155 160
Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp
165 170 175
Asp Val Asp Met His Val Val Pro Val Asp Glu Gln His Met Val Leu
180 185 190
Asp Val Asn His Val Leu Asp Tyr Val Asp Glu Tyr Ile Ile Gly Ile
195 200 205
Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala
210 215 220
Ala Leu Asp Lys Val Val Thr His Tyr Asn His Gln His Pro Lys Leu
225 230 235 240
Pro Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro
245 250 255
Phe Ile Glu Pro Gln Leu Ile Trp Asp Phe Arg Leu Ala Asn Val Val
260 265 270
Ser Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Val Trp Arg Asp Arg Gln Phe Leu Pro Pro Glu Leu Val
290 295 300
Phe Lys Val Ser Tyr Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn
305 310 315 320
Phe Ser His Ser Ala Ala Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile
325 330 335
Arg Phe Gly Met Asp Gly Tyr Arg Glu Ile Gln Thr Lys Thr His Asp
340 345 350
Val Ala Arg Tyr Leu Ala Ala Ala Leu Asp Lys Val Gly Glu Phe Lys
355 360 365
Met Ile Asn Asn Gly His Gln Leu Pro Leu Ile Cys Tyr Gln Leu Ala
370 375 380
Pro Arg Glu Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu
385 390 395 400
Leu Met Asn Gly Trp Gln Val Pro Thr Tyr Pro Phe Pro Ala Asn Leu
405 410 415
Glu Gln Gln Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met
420 425 430
Asn Met Ala His Asp Phe Met Asp Asp Leu Thr Lys Ala Val His Asp
435 440 445
Leu Asn His Ala His Ile Val Tyr His His Asp Ala Ala Pro Lys Lys
450 455 460
Tyr Leu Phe Thr His
465
<210> 4
<211> 1410
<212> DNA
<213> （人工合成）
<400> 4
atggcaatgt tatacggtaa acacaatcat gaagctgaag aatacttgga accagtcttt 60
ggtgcgcctt ctgaacaaca tgatcttcct aagtatcggt taccaaagca ttcattatcc 120
cctcgagaag ccgatcgctt agttcgtgat gaattattag atgaaggcaa ttcacgactg 180
aacctggcaa ctttttgtca gacctatatg gaaaccgaag ccgttgaatt gatgaaggat 240
acgctgcgaa agaatgccat cgacaaatct gagtaccccc gcacggccga gattgaaaat 300
cggtgtgtga acattattgc caatctgtgg cacgcacctg atgacgaaca ctttacgggt 360
acctctacga caggctcctc tgaagcttgt atgttaggcg gtttagcaat gaaattcgcc 420
tggcgtaaac gcgctcaagc ggcaggttta gatctgaata cccatcgacc taacctcgtt 480
atttcggctg gctatcaagt ttgctgggaa aagttttgtg tctactggga cgttgacatg 540
cacgtggtcc cagtggatga gcaacacatg gtccttgacg ttaaccacgt cttagactac 600
gtggacgaat acattattgg tatcgtcggt atcatgggca tcacttatac cggtcaatat 660
gacgacctag ccgcactcga taaggtcgtt actcactaca atcatcagca tcccaaatta 720
ccagtctaca ttcacgttga cgcagcgtca ggtggcttct ataccccatt tattgagccg 780
caactcatct gggacttccg gttggctaac gtcgtttcga tcaacgcctc cgggcacaag 840
tacggtttag tttatcccgg ggtcggctgg gtcgtttggc gtgatcgtca gtttttaccg 900
ccagaattag tcttcaaagt tagttattta ggtggggagt tgccgacaat ggcgatcaac 960
ttctcacata gtgcagccca gctcattgga caatactata atttcattcg ctttggtatg 1020
gacggttacc gcgagattca aacaaagact cacgatgttg cccgctacct ggcagccgct 1080
ctggataaag ttggtgagtt taagatgatc aataacggac accaactccc cctgatttgt 1140
taccaactag ccccgcgcga agatcgtgaa tggacccttt atgatttatc ggatcgccta 1200
ttaatgaacg gttggcaagt accaacgtat cctttccctg ctaatctgga acaacaagtc 1260
atccaacgaa tcgtcgttcg ggctgacttt ggcatgaata tggcccacga tttcatggat 1320
gacctgacca aggctgtcca tgacttaaac cacgcccaca ttgtctatca tcatgacgcg 1380
gcacctaaga aatacctatt cacacactga 1410
<210> 5
<211> 469
<212> PRT
<213> （人工合成）
<400> 5
Met Ala Met Leu Tyr Gly Lys His Asn His Glu Ala Glu Glu Tyr Leu
1 5 10 15
Glu Pro Val Phe Gly Ala Pro Ser Glu Gln His Asp Leu Pro Lys Tyr
20 25 30
Arg Leu Pro Lys His Ser Leu Ser Pro Arg Glu Ala Asp Arg Leu Val
35 40 45
Arg Asp Glu Leu Leu Asp Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr
50 55 60
Phe Cys Gln Thr Tyr Met Glu Pro Glu Ala Val Glu Leu Met Lys Asp
65 70 75 80
Thr Leu Arg Lys Asn Ala Ile Asp Lys Ser Glu Tyr Pro Arg Thr Ala
85 90 95
Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala
100 105 110
Pro Asp Asp Glu His Phe Thr Gly Thr Ser Thr Thr Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg
130 135 140
Ala Gln Ala Ala Gly Leu Asp Leu Asn Ala His Arg Pro Asn Leu Val
145 150 155 160
Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp
165 170 175
Asp Val Asp Met His Val Val Pro Met Asp Glu Gln His Met Val Leu
180 185 190
Asp Val Asn His Val Leu Asp Tyr Val Asp Glu Tyr Ile Ile Gly Ile
195 200 205
Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala
210 215 220
Ala Leu Asp Lys Val Val Thr His Tyr Asn His Gln His Pro Lys Leu
225 230 235 240
Pro Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro
245 250 255
Phe Ile Glu Pro Gln Leu Ile Trp Asp Phe Arg Leu Ala Asn Val Val
260 265 270
Ser Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Val Trp Arg Asp Arg Gln Phe Leu Pro Pro Glu Leu Val
290 295 300
Phe Lys Val Ser Tyr Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn
305 310 315 320
Phe Ser His Ser Ala Ala Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile
325 330 335
Arg Phe Gly Met Asp Gly Tyr Arg Glu Ile Gln Thr Lys Thr His Asp
340 345 350
Val Ala Arg Tyr Leu Ala Ala Ala Leu Asp Lys Val Gly Glu Phe Lys
355 360 365
Met Ile Asn Asn Gly His Gln Leu Pro Leu Ile Cys Tyr Gln Leu Ala
370 375 380
Pro Arg Glu Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu
385 390 395 400
Leu Met Asn Gly Trp Gln Val Pro Thr Tyr Pro Leu Pro Ala Asn Leu
405 410 415
Glu Gln Gln Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met
420 425 430
Asn Met Ala His Asp Phe Met Asp Asp Leu Thr Lys Ala Val His Asp
435 440 445
Leu Asn His Ala His Ile Val Tyr His His Asp Ala Ala Pro Lys Lys
450 455 460
Tyr Leu Phe Thr His
465
<210> 6
<211> 1410
<212> DNA
<213> （人工合成）
<400> 6
atggcaatgt tatacggtaa acacaatcat gaagctgaag aatacttgga accagtcttt 60
ggtgcgcctt ctgaacaaca tgatcttcct aagtatcggt taccaaagca ttcattatcc 120
cctcgagaag ccgatcgctt agttcgtgat gaattattag atgaaggcaa ttcacgactg 180
aacctggcaa ctttttgtca gacctatatg gaacccgaag ccgttgaatt gatgaaggat 240
acgctgcgaa agaatgccat cgacaaatct gagtaccccc gcacggccga gattgaaaat 300
cggtgtgtga acattattgc caatctgtgg cacgcacctg atgacgaaca ctttacgggt 360
acctctacga caggctcctc tgaagcttgt atgttaggcg gtttagcaat gaaattcgcc 420
tggcgtaaac gcgctcaagc ggcaggttta gatctgaatg cccatcgacc taacctcgtt 480
atttcggctg gctatcaagt ttgctgggaa aagttttgtg tctactggga cgttgacatg 540
cacgtggtcc caatggatga gcaacacatg gtccttgacg ttaaccacgt cttagactac 600
gtggacgaat acattattgg tatcgtcggt atcatgggca tcacttatac cggtcaatat 660
gacgacctag ccgcactcga taaggtcgtt actcactaca atcatcagca tcccaaatta 720
ccagtctaca ttcacgttga cgcagcgtca ggtggcttct ataccccatt tattgagccg 780
caactcatct gggacttccg gttggctaac gtcgtttcga tcaacgcctc cgggcacaag 840
tacggtttag tttatcccgg ggtcggctgg gtcgtttggc gtgatcgtca gtttttaccg 900
ccagaattag tcttcaaagt tagttattta ggtggggagt tgccgacaat ggcgatcaac 960
ttctcacata gtgcagccca gctcattgga caatactata atttcattcg ctttggtatg 1020
gacggttacc gcgagattca aacaaagact cacgatgttg cccgctacct ggcagccgct 1080
ctggataaag ttggtgagtt taagatgatc aataacggac accaactccc cctgatttgt 1140
taccaactag ccccgcgcga agatcgtgaa tggacccttt atgatttatc ggatcgccta 1200
ttaatgaacg gttggcaagt accaacgtat cctttacctg ctaatctgga acaacaagtc 1260
atccaacgaa tcgtcgttcg ggctgacttt ggcatgaata tggcccacga tttcatggat 1320
gacctgacca aggctgtcca tgacttaaac cacgcccaca ttgtctatca tcatgacgcg 1380
gcacctaaga aatacctatt cacacactga 1410
<210> 7
<211> 469
<212> PRT
<213> （人工合成）
<400> 7
Met Ala Met Leu Tyr Gly Lys His Asn His Glu Ala Glu Glu Tyr Leu
1 5 10 15
Glu Pro Val Phe Gly Ala Pro Arg Glu Gln His Asp Leu Pro Lys Tyr
20 25 30
Arg Leu Pro Lys His Ser Leu Ser Pro Arg Glu Ala Asp Arg Leu Val
35 40 45
Arg Asp Glu Leu Leu Asp Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr
50 55 60
Phe Cys Gln Thr Tyr Met Glu Thr Glu Ala Val Glu Leu Met Lys Asp
65 70 75 80
Thr Leu Arg Lys Asn Ala Ile Asp Lys Ser Glu Tyr Pro Arg Thr Ala
85 90 95
Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala
100 105 110
Pro Asp Asp Glu His Phe Thr Gly Thr Ser Thr Thr Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg
130 135 140
Ala Gln Ala Ala Gly Leu Asp Leu Asn Thr His Arg Pro Asn Leu Val
145 150 155 160
Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp
165 170 175
Asp Val Asp Met His Val Val Pro Val Asp Glu Gln His Met Val Leu
180 185 190
Asp Val Asn His Val Leu Asp Tyr Val Asp Glu Tyr Ile Ile Gly Ile
195 200 205
Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala
210 215 220
Ala Leu Asp Lys Val Val Thr His Tyr Asn His Gln His Pro Lys Leu
225 230 235 240
Pro Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro
245 250 255
Phe Ile Glu Pro Gln Leu Ile Trp Asp Phe Arg Leu Ala Asn Val Val
260 265 270
Ser Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Val Trp Arg Asp Arg Gln Phe Leu Pro Pro Glu Leu Val
290 295 300
Phe Lys Val Ser Tyr Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn
305 310 315 320
Phe Ser His Ser Ala Ala Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile
325 330 335
Arg Phe Gly Met Asp Gly Tyr Arg Glu Ile Gln Thr Lys Thr His Asp
340 345 350
Val Ala Arg Tyr Leu Ala Ala Ala Leu Asp Lys Val Gly Glu Phe Lys
355 360 365
Met Ile Asn Asn Gly His Gln Leu Pro Leu Ile Cys Tyr Gln Leu Ala
370 375 380
Pro Arg Glu Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu
385 390 395 400
Leu Met Asn Gly Trp Gln Val Pro Thr Tyr Pro Phe Pro Ala Asn Leu
405 410 415
Glu Gln Gln Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met
420 425 430
Asn Met Ala His Asp Phe Met Asp Asp Leu Thr Lys Ala Val His Asp
435 440 445
Leu Asn His Ala His Ile Val Tyr His His Asp Ala Ala Pro Lys Lys
450 455 460
Tyr Leu Phe Thr His
465
<210> 8
<211> 1410
<212> DNA
<213> （人工合成）
<400> 8
atggcaatgt tatacggtaa acacaatcat gaagctgaag aatacttgga accagtcttt 60
ggtgcgcctc gcgaacaaca tgatcttcct aagtatcggt taccaaagca ttcattatcc 120
cctcgagaag ccgatcgctt agttcgtgat gaattattag atgaaggcaa ttcacgactg 180
aacctggcaa ctttttgtca gacctatatg gaaaccgaag ccgttgaatt gatgaaggat 240
acgctgcgaa agaatgccat cgacaaatct gagtaccccc gcacggccga gattgaaaat 300
cggtgtgtga acattattgc caatctgtgg cacgcacctg atgacgaaca ctttacgggt 360
acctctacga caggctcctc tgaagcttgt atgttaggcg gtttagcaat gaaattcgcc 420
tggcgtaaac gcgctcaagc ggcaggttta gatctgaata cccatcgacc taacctcgtt 480
atttcggctg gctatcaagt ttgctgggaa aagttttgtg tctactggga cgttgacatg 540
cacgtggtcc cagtggatga gcaacacatg gtccttgacg ttaaccacgt cttagactac 600
gtggacgaat acattattgg tatcgtcggt atcatgggca tcacttatac cggtcaatat 660
gacgacctag ccgcactcga taaggtcgtt actcactaca atcatcagca tcccaaatta 720
ccagtctaca ttcacgttga cgcagcgtca ggtggcttct ataccccatt tattgagccg 780
caactcatct gggacttccg gttggctaac gtcgtttcga tcaacgcctc cgggcacaag 840
tacggtttag tttatcccgg ggtcggctgg gtcgtttggc gtgatcgtca gtttttaccg 900
ccagaattag tcttcaaagt tagttattta ggtggggagt tgccgacaat ggcgatcaac 960
ttctcacata gtgcagccca gctcattgga caatactata atttcattcg ctttggtatg 1020
gacggttacc gcgagattca aacaaagact cacgatgttg cccgctacct ggcagccgct 1080
ctggataaag ttggtgagtt taagatgatc aataacggac accaactccc cctgatttgt 1140
taccaactag ccccgcgcga agatcgtgaa tggacccttt atgatttatc ggatcgccta 1200
ttaatgaacg gttggcaagt accaacgtat cctttccctg ctaatctgga acaacaagtc 1260
atccaacgaa tcgtcgttcg ggctgacttt ggcatgaata tggcccacga tttcatggat 1320
gacctgacca aggctgtcca tgacttaaac cacgcccaca ttgtctatca tcatgacgcg 1380
gcacctaaga aatacctatt cacacactga 1410
<210> 9
<211> 469
<212> PRT
<213> （人工合成）
<400> 9
Met Ala Met Leu Tyr Gly Lys His Asn His Glu Ala Glu Glu Tyr Leu
1 5 10 15
Glu Pro Val Phe Gly Ala Pro Arg Glu Gln His Asp Leu Pro Lys Tyr
20 25 30
Arg Leu Pro Lys His Ser Leu Ser Pro Arg Glu Ala Asp Arg Leu Val
35 40 45
Arg Asp Glu Leu Leu Asp Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr
50 55 60
Phe Cys Gln Thr Tyr Met Glu Thr Glu Ala Val Glu Leu Met Lys Asp
65 70 75 80
Thr Leu Arg Lys Asn Ala Ile Arg Lys Ser Glu Tyr Pro Arg Thr Ala
85 90 95
Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala
100 105 110
Pro Asp Asp Glu His Phe Thr Gly Thr Ser Thr Thr Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg
130 135 140
Ala Gln Ala Ala Gly Leu Asp Leu Asn Thr His Arg Pro Asn Leu Val
145 150 155 160
Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp
165 170 175
Asp Val Asp Met His Val Val Pro Val Asp Glu Gln His Met Val Leu
180 185 190
Asp Val Asn His Val Leu Asp Tyr Val Asp Glu Tyr Ile Ile Gly Ile
195 200 205
Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala
210 215 220
Ala Leu Asp Lys Val Val Thr His Tyr Asn His Gln His Pro Lys Leu
225 230 235 240
Pro Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro
245 250 255
Phe Ile Glu Pro Gln Leu Ile Trp Asp Phe Arg Leu Ala Asn Val Val
260 265 270
Ser Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Val Trp Arg Asp Arg Gln Phe Leu Pro Pro Glu Leu Val
290 295 300
Phe Lys Val Ser Tyr Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn
305 310 315 320
Phe Ser His Ser Ala Ala Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile
325 330 335
Arg Phe Gly Met Asp Gly Tyr Arg Glu Ile Gln Thr Lys Thr His Asp
340 345 350
Val Ala Arg Tyr Leu Ala Ala Ala Leu Asp Lys Val Gly Glu Phe Lys
355 360 365
Met Ile Asn Asn Gly His Gln Leu Pro Leu Ile Cys Tyr Gln Leu Ala
370 375 380
Pro Arg Glu Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu
385 390 395 400
Leu Met Asn Gly Trp Gln Val Pro Thr Tyr Pro Phe Pro Ala Asn Leu
405 410 415
Glu Gln Gln Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met
420 425 430
Asn Met Ala His Asp Phe Met Asp Asp Leu Thr Lys Ala Val His Asp
435 440 445
Leu Asn His Ala His Ile Val Tyr His His Asp Ala Ala Pro Lys Lys
450 455 460
Tyr Leu Phe Thr His
465
<210> 10
<211> 1410
<212> DNA
<213> （人工合成）
<400> 10
atggcaatgt tatacggtaa acacaatcat gaagctgaag aatacttgga accagtcttt 60
ggtgcgcctc gcgaacaaca tgatcttcct aagtatcggt taccaaagca ttcattatcc 120
cctcgagaag ccgatcgctt agttcgtgat gaattattag atgaaggcaa ttcacgactg 180
aacctggcaa ctttttgtca gacctatatg gaaaccgaag ccgttgaatt gatgaaggat 240
acgctgcgaa agaatgccat ccggaaatct gagtaccccc gcacggccga gattgaaaat 300
cggtgtgtga acattattgc caatctgtgg cacgcacctg atgacgaaca ctttacgggt 360
acctctacga caggctcctc tgaagcttgt atgttaggcg gtttagcaat gaaattcgcc 420
tggcgtaaac gcgctcaagc ggcaggttta gatctgaata cccatcgacc taacctcgtt 480
atttcggctg gctatcaagt ttgctgggaa aagttttgtg tctactggga cgttgacatg 540
cacgtggtcc cagtggatga gcaacacatg gtccttgacg ttaaccacgt cttagactac 600
gtggacgaat acattattgg tatcgtcggt atcatgggca tcacttatac cggtcaatat 660
gacgacctag ccgcactcga taaggtcgtt actcactaca atcatcagca tcccaaatta 720
ccagtctaca ttcacgttga cgcagcgtca ggtggcttct ataccccatt tattgagccg 780
caactcatct gggacttccg gttggctaac gtcgtttcga tcaacgcctc cgggcacaag 840
tacggtttag tttatcccgg ggtcggctgg gtcgtttggc gtgatcgtca gtttttaccg 900
ccagaattag tcttcaaagt tagttattta ggtggggagt tgccgacaat ggcgatcaac 960
ttctcacata gtgcagccca gctcattgga caatactata atttcattcg ctttggtatg 1020
gacggttacc gcgagattca aacaaagact cacgatgttg cccgctacct ggcagccgct 1080
ctggataaag ttggtgagtt taagatgatc aataacggac accaactccc cctgatttgt 1140
taccaactag ccccgcgcga agatcgtgaa tggacccttt atgatttatc ggatcgccta 1200
ttaatgaacg gttggcaagt accaacgtat cctttccctg ctaatctgga acaacaagtc 1260
atccaacgaa tcgtcgttcg ggctgacttt ggcatgaata tggcccacga tttcatggat 1320
gacctgacca aggctgtcca tgacttaaac cacgcccaca ttgtctatca tcatgacgcg 1380
gcacctaaga aatacctatt cacacactga 1410
<210> 11
<211> 469
<212> PRT
<213> （人工合成）
<400> 11
Met Ala Met Leu Tyr Gly Lys His Asn His Glu Ala Glu Glu Tyr Leu
1 5 10 15
Glu Pro Val Phe Gly Ala Pro Arg Glu Gln His Asp Leu Pro Lys Tyr
20 25 30
Arg Leu Pro Lys His Ser Leu Ser Pro Arg Glu Ala Asp Arg Leu Val
35 40 45
Arg Asp Glu Leu Leu Asp Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr
50 55 60
Phe Cys Gln Thr Tyr Met Glu Thr Glu Ala Val Glu Leu Met Lys Asp
65 70 75 80
Thr Leu Arg Lys Asn Ala Ile Asp Lys Ser Glu Tyr Pro Arg Thr Ala
85 90 95
Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala
100 105 110
Pro Asp Asp Glu His Phe Thr Gly Thr Ser Thr Thr Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg
130 135 140
Ala Gln Ala Ala Gly Leu Asp Leu Asn Thr His Arg Pro Asn Leu Val
145 150 155 160
Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp
165 170 175
Asp Val Asp Met His Val Val Pro Val Asp Glu Gln His Met Val Leu
180 185 190
Asp Val Asn His Val Leu Asp Tyr Val Asp Glu Tyr Ile Ile Gly Ile
195 200 205
Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala
210 215 220
Ala Leu Asp Lys Val Val Thr His Tyr Asn His Gln His Pro Lys Leu
225 230 235 240
Pro Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro
245 250 255
Phe Ile Glu Pro Gln Leu Ile Trp Asp Phe Arg Leu Ala Asn Val Val
260 265 270
Ser Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Val Trp Arg Asp Arg Gln Phe Leu Pro Pro Glu Leu Val
290 295 300
Phe Lys Val Ser Lys Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn
305 310 315 320
Phe Ser His Ser Ala Ala Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile
325 330 335
Arg Phe Gly Met Asp Gly Tyr Arg Glu Ile Gln Thr Lys Thr His Asp
340 345 350
Val Ala Arg Tyr Leu Ala Ala Ala Leu Asp Lys Val Gly Glu Phe Lys
355 360 365
Met Ile Asn Asn Gly His Gln Leu Pro Leu Ile Cys Tyr Gln Leu Ala
370 375 380
Pro Arg Glu Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu
385 390 395 400
Leu Met Asn Gly Trp Gln Val Pro Thr Tyr Pro Phe Pro Ala Asn Leu
405 410 415
Glu Gln Gln Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met
420 425 430
Asn Met Ala His Asp Phe Met Asp Asp Leu Thr Lys Ala Val His Asp
435 440 445
Leu Asn His Ala His Ile Val Tyr His His Asp Ala Ala Pro Lys Lys
450 455 460
Tyr Leu Phe Thr His
465
<210> 12
<211> 1410
<212> DNA
<213> （人工合成）
<400> 12
atggcaatgt tatacggtaa acacaatcat gaagctgaag aatacttgga accagtcttt 60
ggtgcgcctc gcgaacaaca tgatcttcct aagtatcggt taccaaagca ttcattatcc 120
cctcgagaag ccgatcgctt agttcgtgat gaattattag atgaaggcaa ttcacgactg 180
aacctggcaa ctttttgtca gacctatatg gaaaccgaag ccgttgaatt gatgaaggat 240
acgctgcgaa agaatgccat cgacaaatct gagtaccccc gcacggccga gattgaaaat 300
cggtgtgtga acattattgc caatctgtgg cacgcacctg atgacgaaca ctttacgggt 360
acctctacga caggctcctc tgaagcttgt atgttaggcg gtttagcaat gaaattcgcc 420
tggcgtaaac gcgctcaagc ggcaggttta gatctgaata cccatcgacc taacctcgtt 480
atttcggctg gctatcaagt ttgctgggaa aagttttgtg tctactggga cgttgacatg 540
cacgtggtcc cagtggatga gcaacacatg gtccttgacg ttaaccacgt cttagactac 600
gtggacgaat acattattgg tatcgtcggt atcatgggca tcacttatac cggtcaatat 660
gacgacctag ccgcactcga taaggtcgtt actcactaca atcatcagca tcccaaatta 720
ccagtctaca ttcacgttga cgcagcgtca ggtggcttct ataccccatt tattgagccg 780
caactcatct gggacttccg gttggctaac gtcgtttcga tcaacgcctc cgggcacaag 840
tacggtttag tttatcccgg ggtcggctgg gtcgtttggc gtgatcgtca gtttttaccg 900
ccagaattag tcttcaaagt tagtaaatta ggtggggagt tgccgacaat ggcgatcaac 960
ttctcacata gtgcagccca gctcattgga caatactata atttcattcg ctttggtatg 1020
gacggttacc gcgagattca aacaaagact cacgatgttg cccgctacct ggcagccgct 1080
ctggataaag ttggtgagtt taagatgatc aataacggac accaactccc cctgatttgt 1140
taccaactag ccccgcgcga agatcgtgaa tggacccttt atgatttatc ggatcgccta 1200
ttaatgaacg gttggcaagt accaacgtat cctttccctg ctaatctgga acaacaagtc 1260
atccaacgaa tcgtcgttcg ggctgacttt ggcatgaata tggcccacga tttcatggat 1320
gacctgacca aggctgtcca tgacttaaac cacgcccaca ttgtctatca tcatgacgcg 1380
gcacctaaga aatacctatt cacacactga 1410

Claims (10)

1.一种pH耐受性提高的谷氨酸脱羧酶突变体，其特征在于，通过将氨基酸序列如SEQID NO.3所示的谷氨酸脱羧酶经以下方式突变得到：
第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸以及第309位的酪氨酸突变为赖氨酸。
2.编码权利要求1所述的pH耐受性提高的谷氨酸脱羧酶突变体的基因。
3.如权利要求2所述的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.2所示。
4.携带权利要求2或3所述基因的重组表达载体。
5.如权利要求4所述的重组表达载体，其特征在于，以pET-28a、PMA5或PXMJ-19作为原始表达载体。
6.含有权利要求4或5所述重组表达载体的重组菌。
7.如权利要求6所述的重组菌，其特征在于，所述重组菌以大肠杆菌、枯草芽孢杆菌或谷氨酸棒杆菌作为宿主菌。
8.如权利要求7所述的重组菌，其特征在于，所述枯草芽孢杆菌包括枯草芽孢杆菌168。
9.如权利要求1所述的pH耐受性提高的谷氨酸脱羧酶突变体、权利要求2或3所述的基因、权利要求4或5所述的重组表达载体或权利要求6、7或8所述的重组菌在合成γ-氨基丁酸中的应用。
10.如权利要求9所述的应用，其特征在于，以L-谷氨酸或L谷氨酸钠为底物，权利要求1所述的pH耐受性提高的谷氨酸脱羧酶突变体或权利要求8所述的重组菌为催化剂，生成γ-氨基丁酸。
CN202210086048.3A 2022-01-25 2022-01-25 一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 Active CN114525268B (zh)

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