CN100408680C - 谷氨酰胺酶,谷氨酰胺酶基因,新的重组dna及生产谷氨酰胺酶的方法 - Google Patents
谷氨酰胺酶,谷氨酰胺酶基因,新的重组dna及生产谷氨酰胺酶的方法 Download PDFInfo
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- CN100408680C CN100408680C CNB021272557A CN02127255A CN100408680C CN 100408680 C CN100408680 C CN 100408680C CN B021272557 A CNB021272557 A CN B021272557A CN 02127255 A CN02127255 A CN 02127255A CN 100408680 C CN100408680 C CN 100408680C
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- glutamine deaminase
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- glutaminase
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Abstract
谷氨酰胺酶,该酶包括(a)一种含有如SEQ ID NO:2所示的氨基酸序列的蛋白,或(b)一种含有相对于氨基酸序列(a)进行缺失、取代或增加一个或多个氨基酸并具有谷氨酰胺酶活性的蛋白。一种谷氨酰胺酶基因,该基因编码(a)一种含有如SEQ ID NO:2所示的氨基酸序列的蛋白;或(b)一种含有相对于氨基酸序列(a)进行缺失、取代或增加一个或多个氨基酸并具有谷氨酰胺酶活性的蛋白。本发明使得能够高效地生产谷氨酰胺酶,从而有助于相关产业。
Description
技术领域
本发明涉及谷氨酰胺酶,谷氨酰胺酶基因,新的重组DNA,和生产谷氨酰胺酶的方法
背景技术
谷氨酰胺酶是一种酶,它能将L-谷氨酰胺水解成氨和L-谷氨酸,L-谷氨酸也是一种增甜剂(甜味剂)成分。谷氨酰胺酶在食品加工业起着重要的作用,例如,它可以应用于酱油或其它通过酶水解蛋白生产的调味品的制造业。已经从不同的物种中分离出谷氨酰胺酶,有关它的酶学特性和基因也已有报道(如日本专利公开(Kokoku)号6-38748)。
在使用日本酒曲麦芽的酱油制造业和调味品制造业中,为了通过遗传工程技术改进谷氨酰胺酶及提高酶的产量,从日本酒曲麦芽中获得酶是重要的。
采用这种方式能容易地改进水解蛋白产物(如酱油)的质量并可降低价格。
发明内容
因此本发明的目的是提供一种由日本酒曲麦芽中获得的谷氨酰胺酶,谷氨酰胺酶基因,新的重组DNA,和制备谷氨酰胺酶的方法。
经过多方面的研究和分析,本发明成功地分离出了源于酱油曲霉(Aspergillussojae)的一种谷氨酰胺酶,并确定了它的结构,完成了本发明。
即,第一项发明是根据如下(a)或(b)的一种谷氨酰胺酶:
(a)一种含有如SEQ ID NO:2所示的氨基酸序列的蛋白;
(b)一种含有相对于氨基酸序列(a)进行缺失、取代或增加一个或多个氨基酸的氨基酸序列,该蛋白具有谷氨酰胺酶活性。
第二项发明是编码根据如下(a)或(b)的蛋白的谷氨酰胺酶基因:
(a)一种含有如SEQ ID NO:2所示的氨基酸序列的蛋白;
(b)一种含有相对于氨基酸序列(a)进行缺失、取代或增加一个或多个氨基酸的蛋白,该蛋白具有谷氨酰胺酶活性。
第三项发明是含有根据如下列(a)或(b)DNA的谷氨酰胺酶基因:
(a)一种含有SEQ ID NO:1所示的碱基序列的DNA;
(b)一种在严格条件下能与含有(a)所示的碱基序列的DNA杂交的DNA,该DNA编码具有谷氨酰胺酶活性的蛋白。
第四项发明是一种新的重组DNA,其特征在于上述谷氨酰胺酶基因插入载体DNA。
第五项发明是一种含有上述重组DNA的转化体或转导体。
第六项发明是制备谷氨酰胺酶的方法,包括在培养基上培养上述转化体或转导体,从培养产物中收集谷氨酰胺酶。
发明内容
下面将对本发明进行详细描述。
1.一种谷氨酰胺酶和编码它的基因
本发明的谷氨酰胺酶是根据如下列(a)或(b)的一种谷氨酰胺酶:
(a)一种含有如SEQ ID NO:2所示的氨基酸序列的蛋白;
(b)一种含有相对于氨基酸序列(a)进行缺失、取代或增加一个或多个氨基酸的氨基酸序列的蛋白,该蛋白具有谷氨酰胺酶活性。
可通过将源于曲霉属的丝状真菌的染色体DNA或cDNA的天然存在的谷氨酰胺酶基因进行克隆,然后将克隆引入适宜的载体-宿主系统进行表达而获得蛋白(a)。
如(b)中所表明,该蛋白可以包含相对于氨基酸序列(a)进行缺失、取代或增加一个或多个氨基酸的氨基酸序列,只要该氨基酸序列具有谷氨酰胺酶活性。在本发明中,“多个”通常意味着2至300,优选2-170,更优选2-50,最优选为2-10,这些取决于谷氨酰胺酶蛋白的三维结构中氨基酸残基的位置或氨基酸的种类。
这种突变的谷氨酰胺酶,即蛋白(b)的获得,可以通过在天然存在的谷氨酰胺酶基因的碱基序列中导入一种突变来产生出突变的谷氨酰胺酶基因,这种突变包括取代、缺失、插入、增加或倒置,然后将该突变基因导入一个适宜的载体-宿主系统进行表达。
将突变导入基因的方法包括,例如位点特异性诱变,PCR方法引入的随机突变,和其中基因被选择性剪切并在去掉或增加选择的核苷酸后被重新连接的方法。
依据本发明的谷氨酰胺酶基因是含有编码蛋白(a)或(b)DNA的基因。依据本发明的谷氨酰胺酶基因可以是一种在严格条件下能与编码蛋白(a)或(b)的DNA杂交的基因,并且该DNA编码的蛋白具有谷氨酰胺酶活性。在本发明中,“严格条件”指这样的条件,其中钠离子浓度在50-300mM,优选150mM,同时温度在42-68℃,优选65℃。
含有编码蛋白(a)的DNA的基因的例子是含有SEQ ID NO:1所示碱基序列的DNA。该DNA是一种天然存在的谷氨酰胺酶基因。
这种天然存在的谷氨酰胺酶基因能通过将源于曲霉属中的丝状真菌的染色体DNA或cDNA的天然存在的谷氨酰胺酶基因进行克隆获得。基因克隆的方法包括,例如,其中一种在纯化谷氨酰胺酶并确定部分的氨基酸序列后合成适合的探针DNA,然后用该探针DNA来筛选酱油曲霉染色体DNA。另一种方法包括基于部分的氨基酸序列设计适宜的引物DNA,接着通过适合的如5’-RACE或3’-RACE法等聚合酶链式反应(PCR)扩增出含有上述基因片段的DNA,这些片段连接起来从而产生含有全长基因的DNA。
具体地,天然存在的谷氨酰胺酶基因可以按照以下方法获得。首先,培养酱油曲霉FERM BP-6820,将所得到的细菌体在液氮中冷冻,接着用研钵之类将之机械研磨或挤碎获得细粉状的细菌体片段,用常规方法从中提取总RNA部分。在提取的过程中,可以使用商业上可获得的RNA抽提试剂盒。
可以选择地,可以从经乙醇沉淀的RNA的抽提液中收集RNA,然后用常规手段将带有多聚A链的RNA分馏出来,在分馏的过程中,可以使用商业上可使用的寡dT柱。
参照SEQ ID NO:2中的DNA序列合成PCR引物,利用引物DNA和由上述方法得到的RNA,通过一种适宜的RT-PCR反应如5’-RACE法和3’-RACE法扩增出含有基因片段的DNA,将这些片段连接就可以获得含有全长基因的DNA。在使用5’-RACE法和3’-RACE法合成部分cDNA的过程中,可以使用商业上可获得的试剂盒。
将上述cDNA作为模板,用合成的与5’-端和3’-端序列互补的引物进行PCR反应从而扩增出DNA,可以根据传统的方法克隆扩增的DNA。
将扩增出的DNA插入适宜的载体获得重组DNA。克隆可以使用商业上可获得的试剂盒如TA克隆试剂盒(Invitrogen Corporation),商业上可获得的质粒载体DNAs如pUC119(Takara Shuzo Co.,Ltd.),pBR322(Takara Shuzo Co.,Ltd.)和pBluescript SK+(Stratagene),和商业上可获得的噬菌体载体DNA如λEMBL3(Stratagene),或其它。
通过将所得到的重组DNA转化或转导入大肠杆菌K-12可获得转化体或转导体,优选如大肠杆菌JM109(Takara Shuzo Co.,Ltd.)或XL-Blue(Stratagene)。转化可以使用如D.MMorrison的方法(酶学方法,68,326-331,1979)。另一方面,转导可以使用如B.Hohn的方法(酶学方法,68,299-309,1979)。作为宿主细胞,除了大肠杆菌,可用其它微生物如细菌、酵母、丝状真菌和放线菌类和以及动物细胞。
可以对扩增DNA的全部碱基序列(见SEQ ID NO:1)进行分析,例如其中可以使用Li-COR MODEL 4200L测序仪(购自ALOKA CO.,LTD.)、370DNA测序仪(PerkinElmer Inc.)或CEQ2000XL DNA分析系统(Beck man Coulter)。通过比较碱基序列和部分氨基酸序列的信息,可以确定是否获得了天然存在的谷氨酰胺酶基因。
通过分析天然存在的谷氨酰胺酶基因,可以确定所翻译的多肽即蛋白(a)的氨基酸序列。
2.制备谷氨酰胺酶的方法
在制备本发明的谷氨酰胺酶时,首先生产含有谷氨酰胺酶基因的重组DNA,然后生产和培养含有该重组DNA的转化体或转导体,从培养产物中可以收集谷氨酰胺酶。
为了利用本发明的谷氨酰胺酶基因制备具有谷氨酰胺酶活性的蛋白,必须选择一个适宜的载体-宿主系统。这样的系统,这里可能提及的是,如pST14系统(Unkles等,1989,Mol.Gen.Genet.,218,99-104)和丝状真菌(酱油曲霉,米曲霉,构巢曲霉,黑色曲霉,产黄青霉等),酵母表达载体pYES2(Invitrogen)和啤酒糖酵母系统,和一种大肠杆菌表达载体pTE(Stratagene)与大肠杆菌系统。优选地使用一种丝状真菌或酵母系统,其中蛋白中出现了添加的糖链。
重组DNA可以通过将谷氨酰胺酶基因插入一个适宜载体获得,作为载体,可以使用商业上可得到的产品,如酵母表达载体pYES2、pYD1(Invitrogen),pUR123(Takara Shuzo Co.,Ltd.),pYEX-BX,pYEX-S1,pYEX-4T(CLONTECH),大肠杆菌表达载体如pSET(Invitrogen),和pTE(Stratgene)等。
然后将重组DNA转化或转导进宿主细胞。可以使用如Becker DM等的方法(酶学方法,194,182-187,1991)转导酵母。宿主细胞除了大肠杆菌和酵母外,还可以使用其它的微生物,如其它细菌、丝状真菌、放线菌或动物细胞。
这样就可以获得一种具有生产谷氨酰胺酶能力的转化体或转导体,虽然这种转化体或转导体可以通过常规固体培养方法进行培养,但优选尽可能采用液体培养方法。
当用酵母作宿主时,可以使用普通富养培养基如YPD培养基和YM培养基。当考虑宿主的遗传特性而使用选择性培养基时,可以使用作为极限培养基的SD培养基。当使用选择性培养基时,由于选择压力依赖于所用的宿主-载体系统而不同,除了选择压力还要根据宿主遗传需要向极限培养基中加入氨基酸或核酸。
另外,可以根据需要向培养基中加入无机盐、糖类物质、维生素等。培养基的初始PH值适当调至大约PH6-9。可以通过使用的载体控制蛋白的表达。当使用这样的载体时,可以加入适合于载体的诱导物如半乳糖或铜离子来诱导谷氨酰胺酶。
当培养酵母时,优选采用以下的方法,通气搅拌深层培养,振荡培养和静止培养等等,培养温度在25℃-30℃,优选30℃左右,培养时间为2448个小时。
可以使用部分改进的日本专利申请公开(Kokai)号11-332553中描述的方法对表达的谷氨酰胺酶进行纯化。
就酵母的情况而言,在采用上述适合的方法培养转化的酵母后,离心培养液以获得酵母菌体,在酵母菌体中加入一种细胞壁裂解酶使细胞壁充分裂解后离心取上清液。接着在上清液中加入硫酸铵进行盐析,并进一步离心该上清液,去掉不能溶解的蛋白后获得含有谷氨酰胺酶的粗酶溶液。
使用苯基琼脂糖柱、DEAE琼脂糖柱、凝胶过滤柱和HPLC从粗酶溶液中纯化出谷氨酰胺酶活性部分,从而获得纯化的谷氨酰胺酶。
本发明的遗传工程方法可以根据下列文献的描述实施,“分子克隆:实验手册第二版”(1989),冷泉港实验室出版社,ISBN 0-8769-309-6,“现代分子生物学方法”(1989),John Wiley & Sons,Inc.,ISBN 0-471-50338-X,等。
(实施例)
通过后面的实施例对本发明作进一步详细的描述。
实施例1:谷氨酰胺酶cDNA的获得
(1)从曲霉属细菌提取谷氨酰胺酶同源基因
对保藏于独立行政法人产业技术综合研究所特许生物寄托中心的日本酒曲霉菌EST文库进行筛选,以寻找与已知来源于隐球菌的谷氨酰胺酶基因(日本专利申请公开号2000-270371)高度同源的基因,结果得到一个含有711个碱基的EST克隆Contig Mix00101100037751。推测该克隆为谷氨酰胺酶基因的一个片段,并克隆了该基因的cDNA。
从酱油曲霉中提取总RNA
将酱油曲霉FERM BP-6820的孢子接种入50ml的大豆粉培养基(30%大豆粉,1%KH2PO4,pH 6.0),使其密度达到3×105/ml,孢子放入150ml Erlenmeyer烧瓶中在30C以150r.p.m振荡培养48小时。
用Miracloth(Calbiochem)过滤得到的混合培养液收集菌体。用灭菌水漂洗收集的菌体后,将菌体放入液氮冷冻,然后在研钵里将菌体机械研磨,从而获得细粉状菌体片段。用ISOGEN(Nippon Gene Co.,Ltd.)从菌体片段中提取总RNA。整个过程按照所附的说明进行。
(2)通过RACE法获得谷氨酰胺酶cDNA
使用Oligotex-dT30<Super>mRNA纯化试剂盒(Takara Shuzo Co.,Ltd.)从200μg这样获得的总RNA中得到4μg的mRNA。使用Marathon cDNA扩增试剂盒(Clontech)和高级cDNA PCR试剂盒(Clontech)对1μg的mRNA进行5’-RACE和3’-RACE法。RACE法中使用的引物是合成的,分别由SEQID NOS:3-6表示的寡DNAs,即用于对EST克隆Contig Mix00101100037751进行5’-RACE法的反义引物(SEQ ID NOS:34)和用于3’-RACE法的有义引物(SEQ ID NOS:5-6)。整个过程按照所附说明进行。其中PCR装置使用的是GeneAmp5700DNA检测系统(PerkinElmer)。结果扩增出了相应于谷氨酰胺酶cDNA 5’-端约1.7kb的DNA片段和相应于DNA 3’-端大约0.8kb的DNA片段。
扩增的DNA片段在0.7%的琼脂糖凝胶上进行分离,使用QIAquick凝胶提取试剂盒(QIAGEN)提取DNA片段。步骤按照所附说明进行。使用TOPOTA克隆试剂盒(Invitrogen)将提取的DNA片段插入pCR2.1-TOPO载体,将所得到的重组质粒进行序列反应,该反应使用了Thermo序列循环测序试剂盒(Amersham Pharmacia Biotech),碱基序列的测定是在LI-COR MODEL4200L测序仪(购自Aloka)上进行。结果测定出了由SEQ ID NO:1表示的约1.9-kb的开放式阅读框(ORF)的碱基序列,并且已经清楚EST克隆CONTIGMIX00101100037751是其部分片段。
该DNA编码含有由SEQ ID NO:2表示的643个氨基酸的蛋白,而且在一个已知的氨基酸序列数据库中进行了关于该氨基酸序列的同源性检索,使用NCBI BLAST进行同源性检索(http://www.ncbi.nlm.nih.gov/BLAST/)。结果表明没有已知蛋白与上述ORF相匹配。
然而,当对来源于浅白色隐球酵母和Cryptococcus nodaensis的谷氨酰胺酶进行同源性检索时,在认为是活性中心的一个区域发现同源性,意味着谷氨酰胺酶由ORF编码。
将5’-RACE法产生的cDNA作为模板进行PCR反应,由此克隆出全长的谷氨酰胺酶cDNA,其中使用了SEQ ID NOS:7和8代表的寡DNA作为引物。用上述方法提取出约2.1kb的扩增DNA片段,并使用TOPO TA克隆试剂盒(Invitrogen)将该DNA片段插入pCR2.1-TOPO载体,从而获得含有全长谷氨酰胺酶cDNA的重组质粒pASgln。
对重组质粒pASgln的碱基序列也进行了分析,从而确定谷氨酰胺酶cDNA的碱基序列(SEQ ID NO:1)。
全长的谷氨酰胺酶cDNA,如,含有由SEQ ID NO:1第1-1932个碱基所代表的碱基序列的质粒pASgln,已经在独立行政法人产业技术综合研究所特许生物寄托中心进行了保藏,保藏号为FERM BP-7634。
实施例2:谷氨酰胺酶cDNA的表达
上述质粒pASgln先用限制性内切酶Bam HI和Sph I(均来自Takara ShuzoCo.,Ltd.)进行酶处理,接着将酶切产物在0.7%的琼脂糖凝胶上进行电泳,然后切下所要大小(约2.0kb)的DNA片段并进行纯化。
将这些DNA片段导入已经由上述限制性内切酶进行酶处理的酵母表达载体pYES2(Invitrogen),从而产生重组质粒pYES-ASgln。该重组质粒能够诱导靶蛋白谷氨酰胺酶表达。宿主使用了所附的INVSc1(基因型:MATa,his3Δ1,leu2,trp1-289,ura3-52/MATα,his3Δ1,leu2,trp1-289,ura3-52),使用醋酸锂方法用上述质粒pYES-ASgln转化宿主酵母。选择性培养基使用了不含氨基酸的0.67%的酵母氮基(Difco)、2%的棉子糖(Wako Pure Chemicals Industries,Ltd.)和0.192%的不含尿嘧啶的酵母合成D析出培养基补料(SIGMA)。醋酸锂方法参见“Tanpakushitsu Jikken Purotokoru-Kino Kaiseki-hen-”(“蛋白试验手册——功能分析”)(Saibo Kogaku杂志的增补本(细胞技术;Shujun-sha))。
然后,根据pYES2载体(Invitrogen)附的方法,使用所得到的转化体来表达蛋白。用带有凸缘的200ml Erlenmeyer烧瓶将菌落中的转化体移入20ml选择性培养基中,在30℃以140rpm的速度振荡培养约14小时,从而获得种子培养物。
接着测定种子培养物的浑浊度(OD600),将该种子培养物接种到一种蛋白表达诱导培养基,其初始浑浊度为OD600=0.4。在蛋白表达诱导培养基的培养中使用了500ml的Sakaguchi烧瓶,在50ml培养基中于30℃在140rpm下振荡培养。蛋白表达诱导培养基使用了1%的酵母提取物(Difco),2%的聚蛋白胨(Nippon Seiyaku KK),1%的棉子糖和2%的半乳糖(均是SIGMA)。经离心收集的酵母体悬浮于蒸馏水中作为提供的酶溶液。
对日本专利申请公开(Kokai)号11-332553中描述的方法进行部分改进以用来测定谷氨酰胺酶的活性。具体地,将500μl 0.2M磷酸缓冲溶液(pH6.5)和500μl酶溶液加入到250μl 2%(W/V)的L-谷氨酸溶液中并于37℃反应30分钟。加入250μl 0.75N高氯酸溶液终止反应,并加入125μl1.5N氢氧化钠溶液来中和反应溶液。反应溶液经过离心(10000r.p.m.,10min),取100μl上清液,在该上清液中加入0.1M的盐酸羟胺缓冲液1.0ml(pH 8.0)、1.0ml 20mM NAD+溶液(Oriental Yeast Co.,Ltd)和50μl 500单位/ml的L-谷氨酸脱氢酶溶液(SHIGMA)。然后上清液在37℃反应30分钟后,用分光光度计测定其在340nm的吸收值。在这种条件下,每一个单位(U)的谷氨酰胺酶的活性是根据每分钟产生1μmol谷氨酸的酶的数量来定义。
转化体谷氨酰胺酶活性的测定结果列于表1。表中的数值表明在培养开始(OD600=15)后24小时每1ml培养液中谷氨酰胺酶的活性(mU/ml)。名称“pYES2”指质粒pYES2转化的转化体,“pYES-ASgln”指质粒pYES-ASgln转化的转化体,符号“-”和“+”分别指通过不含半乳糖的非蛋白表达诱导型培养基的诱导和含有半乳糖的蛋白表达诱导型培养基的诱导。
表1
质粒/半乳糖 | - | + |
pYES2 | 0.33 | 2.50 |
pYES-Asgln | 4.64 | 32.87 |
当在含有半乳糖的蛋白表达诱导型培养基上培养时,质粒pYES-ASgln的转化体的谷氨酰胺酶活性高于质粒pYES2的转化体。另一方面,当在不含半乳糖的非蛋白表达诱导型培养基上培养时,质粒pYES-ASgln的转化体没有表现出谷氨酰胺酶活性的提高。这表明质粒pYES-ASgln的转化体的谷氨酰胺酶活性源于导入的谷氨酰胺酶基因(表1)。当INVScl作为表达谷氨酰胺酶的宿主时,在酵母体表面表达的谷氨酰胺酶活性也如曲霉菌属细菌的情况一样。
这样可以根据本发明高效地制备谷氨酰胺酶,因此本发明具有巨大的工业实用性。
序列表
<110>龟甲万株式会社
<120>谷氨酰胺酶、谷氨酰胺酶基因、新的重组DNA及生产谷氨酰胺酶的方法
<130>PH-1498
<160>8
<210>1
<211>1932
<212>DNA
<213>酱油曲霉(Aspergillus sojae)
<400>1
atg ttt ctt agt aca ctc ctc tca ctg gcg gcg gtc gtt gcc ggc gct 48
gcc atc ccc aat ggc cag acg ctt tct ctc aat gac att cct tac tat 96
gtg agc ggc att cct gtg tca act ttg caa ggg tac aat gcc tct gca 144
tat gct gct ttg aca aag gga ata gat ttg gtg cca tta act gtc att 192
cct gta act cct acc acg aac ttg gag tcg ctg cta tcg gac tat gtt 240
gaa cgc gat gat gtc ttc cag ccg gct ttt ctg cgt gca gtc tat ctc 288
aca gct tcc act gct gat gac att gac tcc caa ctg agc aat tat gcg 336
tca att ctc aag tct tcc ggc acc gac gtg ctg ctg gtt gat tca gaa 384
gta cac acc gct tcg tca gat tcc aca atc aca gcg cag ttg acc aaa 432
gag ctg ccg agt ggg cct tat ttt gtc tcc ttg tat act gga gag gtg 480
ttt aga gcg tac cgg ttg tac cct gac gac aac cta gct ttc att caa 528
gca gga atc agt gac gag aag gga ggt gtc ctg ccc cta cca gcc gtg 576
aca gaa aac gcg atg acc aaa gac gtt gcc gtg cct tca cgt ctc tat 624
tat aca ccg acc gca gaa aag cca tta gcc ggt ctg agg tta ggt gtc 672
aag gat atc tac cac gtt aaa ggt ctc aag acg agt ggc ggc agt cgc 720
tcc tat tat tat tta tac gga act cag aat gtc act gcc cca tct att 768
cag aga ctg ttg gac tta ggc gcg gtc ttt gtc ggt aaa act ggg acc 816
gtt cag ttt gct aac ggt gat cga cct act gcc gac tgg gtg gat ttc 864
cac tgt cca ttc aac caa cgc gga gaa gga tat cag gca cct agc ggt 912
tcc tcc tcc ggc tca ggt gtg gct att gca gcc tac gac tgg ttg gac 960
ctt gct gtc ggt agt gac act ggc ggt tca atg cgt tcc cca gct gca 1008
gtt caa ggg ata tat ggc aac agg cca tct act ggc gct atc tct cta 1056
gat cat gtc tta cct ctc tcg ccg gct ctg gat aca gcg ggc gtc ttt 1104
gcc cga agt gcc tca cta tgg tcc cat act gtg caa gct tgg tat cct 1152
cat ctc cag cac aat ttt acg tcc ttc cct cgg cag ctg ctc cta gcc 1200
ggt ggt gga tgg gat ggt aaa ggc atc agt ccc gag gcc cat cag agt 1248
ctt acc aca ttc aca cgt ggg ctt gag gca ttc ctc gga aca aac cat 1296
acc aat gtc gac gtg tcg cag cga tgg ctt gac aca cac tct ccc acc 1344
aca cca agc ctg gaa gag atg ctc aac ctg acc tat gcc aca ctt act 1392
tct gtg gat cag ttc aac cac cta gcc gtc cct ctc ttt gct gac tat 1440
aaa gcc gtc cac cgc ggt cgt cag cct ttc att aac ccc ggc cca tta 1488
gcg cgt tgg cag tgg ggc cag gcg aat ggc gga aac acc tcg tac gat 1536
gca gct ctg cgc aac atg act act ttc cga aac tgg tgg gag aag tcc 1584
ggg tat ggt cag tcc gat aat gcc tct tgc tcc agg tcg ctt ttc gtc 1632
agt gtg tat tcg gtc ggc acc act gac tac cgt aac caa tat tat gag 1680
gcg ccc act aca ccc cca ctg gga ttc tcg atc gga cgc atc gcg gta 1728
tta ggt gga gca cct gag gtt gtt gtt cct gtg gga gag tcc cca tac 1776
aat agt act atc tct ttg cag acc gag tat ttg ccg gtc agt gtt gcg 1824
ctg cag atg gcg cga gga tgt gac cat gtt ctg gct tcc ttg gtc gct 1872
ggc ctt gag aag aag ggc gtc ctc cga cct gtc agt acc ggc tct cgc 1920
cta tac tcc taa 1932
<210>2
<211>643
<212>PRT
<213>酱油曲霉(Aspergillus sojae)
<400>2
Met Phe Leu Ser Thr Leu Leu Ser Leu Ala Ala Val Val Ala Gly
1 5 10 15
Ala Ala Ile Pro Asn Gly Gln Thr Leu Ser Leu Asn Asp Ile Pro
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Tyr Tyr Val Ser Gly Ile Pro Val Ser Thr Leu Gln Gly Tyr Asn
35 40 45
Ala Ser Ala Tyr Ala Ala Leu Thr Lys Gly Ile Asp Leu Val Pro
50 55 60
Leu Thr Val Ile Pro Val Thr Pro Thr Thr Asn Leu Glu Ser Leu
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Leu Ser Asp Tyr Val Glu Arg Asp Asp Val Phe Gln Pro Ala Phe
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Leu Arg Ala Val Tyr Leu Thr Ala Ser Thr Ala Asp Asp Ile Asp
95 100 105
Ser Gln Leu Ser Asn Tyr Ala Ser Ile Leu Lys Ser Ser Gly Thr
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Asp Val Leu Leu Val Asp Ser Glu Val His Thr Ala Ser Ser Asp
125 130 135
Ser Thr Ile Thr Ala Gln Leu Thr Lys Glu Leu Pro Ser Gly Pro
140 145 150
Tyr Phe Val Ser Leu Tyr Thr Gly Glu Val Phe Arg Ala Tyr Arg
155 160 165
Leu Tyr Pro Asp Asp Asn Leu Ala Phe Ile Gln Ala Gly Ile Ser
170 175 180
Asp Glu Lys Gly Gly Val Leu Pro Leu Pro Ala Val Thr Glu Asn
185 190 195
Ala Met Thr Lys Asp Val Ala Val Pro Ser Arg Leu Tyr Tyr Thr
200 205 210
Pro Thr Ala Glu Lys Pro Leu Ala Gly Leu Arg Leu Gly Val Lys
215 220 225
Asp Ile Tyr His Val Lys Gly Leu Lys Thr Ser Gly Gly Ser Arg
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Ser Tyr Tyr Tyr Leu Tyr Gly Thr Gln Asn Val Thr Ala Pro Ser
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Ile Gln Arg Leu Leu Asp Leu Gly Ala Val Phe Val Gly Lys Thr
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Gly Thr Val Gln Phe Ala Asn Gly Asp Arg Pro Thr Ala Asp Trp
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Val Asp Phe His Cys Pro Phe Asn Gln Arg Gly Glu Gly Tyr Gln
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Ala Pro Ser Gly Ser Ser Ser Gly Ser Gly Val Ala Ile Ala Ala
305 310 315
Tyr Asp Trp Leu Asp Leu Ala Val Gly Ser Asp Thr Gly Gly Ser
320 325 330
Met Arg Ser Pro Ala Ala Val Gln Gly Ile Tyr Gly Asn Arg Pro
335 340 345
Ser Thr Gly Ala Ile Ser Leu Asp His Val Leu Pro Leu Ser Pro
350 355 360
Ala Leu Asp Thr Ala Gly Val Phe Ala Arg Ser Ala Ser Leu Trp
365 370 375
Ser His Thr Val Gln Ala Trp Tyr Pro His Leu Gln His Asn Phe
380 385 390
Thr Ser Phe Pro Arg Gln Leu Leu Leu Ala Gly Gly Gly Trp Asp
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Gly Lys Gly Ile Ser Pro Glu Ala His Gln Ser Leu Thr Thr Phe
410 415 420
Thr Arg Gly Leu Glu Ala Phe Leu Gly Thr Asn His Thr Asn Val
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Asp Val Ser Gln Arg Trp Leu Asp Thr His Ser Pro Thr Thr Pro
440 445 450
Ser Leu Glu Glu Met Leu Asn Leu Thr Tyr Ala Thr Leu Thr Ser
455 460 465
Val Asp Gln Phe Asn His Leu Ala Val Pro Leu Phe Ala Asp Tyr
470 475 480
Lys Ala Val His Arg Gly Arg Gln Pro Phe Ile Asn Pro Gly Pro
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Leu Ala Arg Trp Gln Trp Gly Gln Ala Asn Gly Gly Asn Thr Ser
500 505 510
Tyr Asp Ala Ala Leu Arg Asn Met Thr Thr Phe Arg Asn Trp Trp
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Glu Lys Ser Gly Tyr Gly Gln Ser Asp Asn Ala Ser Cys Ser Arg
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Ser Leu Phe Val Ser Val Tyr Ser Val Gly Thr Thr Asp Tyr Arg
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Asn Gln Tyr Tyr Glu Ala Pro Thr Thr Pro Pro Leu Gly Phe 5er
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<210>3
<211>30
<212>DNA
<213>人工序列
<400>3
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<210>4
<211>30
<212>DNA
<213>人工序列
<400>4
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<210>5
<211>30
<212>DNA
<213>人工序列
<400>5
gca gcg caa cac tga ccg gca aat act cgg 30
<210>6
<211>30
<212>DNA
<213>人工序列
<400>6
aag agc gac ttg gag cag gag gca cat cgg 30
<210>7
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<213>人工序列
<400>7
ggt gac aga ctg gat cca tca tgt ttc tta 30
<210>8
<211>30
<212>DNA
<213>人工序列
<400>8
ttg ttt gaa ccg gca tgc tct act ttg tac 30
Claims (6)
1. 一种谷氨酰胺酶,其是含有SEQ ID NO:2所示氨基酸序列的蛋白。
2. 编码含有SEQ ID NO:2所示氨基酸序列的蛋白的谷氨酰胺酶基因。
3. 一种含有根据如下(a)或(b)的DNA的谷氨酰胺酶基因:
(a)一种含有SEQ ID NO:1所示碱基序列的DNA;
(b)一种在严格条件下能与含有SEQ ID NO:1所示碱基序列的DNA杂交,并且编码具有谷氨酰胺酶活性的蛋白的DNA,其中严格条件是指这样的条件,其中钠离子浓度为50-300mM,同时温度为42-68℃。
4. 一种新的重组DNA,其特征在于根据权利要求2或3的谷氨酰胺酶基因被插入到载体DNA中。
5. 一种含有根据权利要求4的重组DNA的转化体或转导体。
6. 一种制备谷氨酰胺酶的方法,包括:
在培养基上培养如权利要求5所述的转化体或转导体,以及
从培养产物中收集谷氨酰胺酶。
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