CN1133341A - 腈水解酶基因表达的调控因子及其基因 - Google Patents
腈水解酶基因表达的调控因子及其基因 Download PDFInfo
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- CN1133341A CN1133341A CN95119459A CN95119459A CN1133341A CN 1133341 A CN1133341 A CN 1133341A CN 95119459 A CN95119459 A CN 95119459A CN 95119459 A CN95119459 A CN 95119459A CN 1133341 A CN1133341 A CN 1133341A
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Abstract
本发明涉及由具有SEQ ID NO:1氨基酸序列的多肽和具有SEQ ID NO:2的氨基酸序列的多肽两种成分组成的能激活腈水解酶基因启动子的调控因子以及编码它的DNA。腈水解酶能经过将编码该调控因子的基因与含启动子区的腈水解酶基因一起导入Rhodococcus属微生物来生产。
Description
本发明涉及与腈水解酶基因表达有关的调控因子和编码这一因子的DNA,特别是涉及来自菌株Rhodococcus erythropolis SK92并激活腈水解酶基因启动子的调控因子,以及编码该因子的DNA,含有该DNA的重组质粒和用所述重组质粒转化的转化体。
对于已知的从其相应的腈进行转化生产有机酸的方法,可提及的有化学合成方法和生物学方法。后者涉及使用微生物或来自微生物的酶作为一种催化剂来水解腈,因此,这种方法的优势在于有机酸可在温和的条件下生产。属于Rhodococcus属的微生物已知可作为这样一种催化剂,用于经过其相应的腈的水合作用或水解生产酰胺或有机酸(见日本公开的专利公开号:251,192/1991,91,189/1987,470/1990,和84,198/1990)。
与上面提及的常规方法相比,使用经遗传重组克隆的腈水解酶基因进行腈水解预期能大幅度改进微生物对腈水合的催化能力,因为微生物可改造成含这种基因的多个拷贝。为了获得这种具有较高催化活性的催化剂生物,本发明者从菌株Rhodococcus erythropolis SK92中成功地克隆出了腈水解酶基因并经过将所说基因插入E.coli乳糖启动子的下游区域构建质粒。经过将该质粒导入E.coli,在IPTG(异丙基-β-D-硫代半乳糖苷)存在条件下的培养过程中,该生物体表现出较高腈水解酶活性。本发明者进一步试图获得Rhodococcus属转化体,使其成为具有较高效力的生物体催化剂。在这种尝试中,将腈水解酶基因插入Rhodococcus-E.coli杂交质粒载体中(见日本公开的专利公开号64,589/1993和68,566/1993),接着将构建的载体导入Rhodococcus属微生物中。然而,无腈水解酶活性表达出来,因此需要一种使腈水解酶活性在Rhodococcus属转化体中得以表达的方法。
本发明者推测:来自Rhodococcus属的基因不表达是因为腈水解酶基因的启动子不能发挥功能,并且在SK92染色体DNA某处可能存在一种编码使启动子发挥功能的调控因子的基因。通过筛选,本发明者发现它在腈水解酶结构基因的上游区并以此成功地在Rhodococcus属转化体中表达出腈水解酶活性。
这就是说,本发明涉及由2种成份(即:具有SEQ ID NO:1氨基酸序列的多肽和具有SEQ ID NO:2氨基酸序列的多肽)组成的能激活腈水解酶基因启动子的调控因子,以及编码它们的DNA。
编码本发明调控因子的基因和包含其启动子的腈水解酶基因的导入使Rhodococcus属微生物产生腈水解酶。
图1显示了缺失质粒的图解,其中SK92 DNA片段的箭头分别表示编码本发明调控因子的基因和编码腈水解酶基因的位置和方向。
图2显示了重组质粒pSK108的限制性酶图谱。
下文详细描述本发明。本发明按下列步骤实施。(1)菌株SK92染色体DNA的制备:
从Rhodococcus erythropolis SK92中分离染色体DNA。(2)DNA文库的构建:
用限制性酶裂解染色体DNA,使用SK92腈水解酶基因作为探针以Southern杂交检测含靶基因的DNA片段。将该片段插入能在E.coli和Rhodococcus细胞中复制的杂种质粒载体中以制备文库。(3)E.coli的转化和重组体DNA的选择:
在步骤(2)中构建的重组体文库用于制备转化体。使用步骤(2)中获得的探针对其进行菌落杂交以选择携带靶重组体DNA的菌落。(4)重组体质粒的制备:
从步骤(3)获得的重组体制备质粒。(5)Rhodococcus属微生物的转化和转化体的腈水解酶活性:
将所得的质粒导入Rhodococcus属微生物中,并测定其腈水解酶活性。(6)缺失质粒和腈水解酶活性:
经过对步骤(4)获得的质粒进行不同区域的缺失制备缺失质粒以鉴定为腈水解酶结构基因表达所必需的区域。制备的质粒不必能在E.coli中复制并且如果它们包含能在Rhodococcus属细胞中复制的DNA区域就足够。(7)核苷酸测序:测定在步骤(6)中鉴定区域的核苷酸序列。
对于上面的杂种质粒载体,可称为PK1,PK2,PK3和PK4。将这些质粒导入R.rhodochrous ATCC12674并分别以R.rhodochrous ATCC 12674/pK1(FERM BP-3728),R.rhodochrous ATCC 12674/pK2(FERM BP-3729),R.rhodochrous ATCC 12674/pK3(FERM BP-3730)和R.rhodochrous ATCC 12674/pK4(FERM BP-3731)保藏于日本工业科学和技术机构的国立生物科学和人类技术研究所(见日本公开的专利公开号:68,556/1993)。
对于上述能在Rhodococcus属细胞中复制的DNA区域,可称为来自质粒pRC001,pRC002,pRC003和pRC004的DNA区域,它们可以是整个质粒或其部分片段。上述质粒分别得自菌株R.rhodochrous ATCC4276,ATCC 14349,ATCC 14348和IFO 3338(见日本公开的专利公开号:68,556/1993)。
Rhodococcus erythro polis SK92以FERM BP-3324进行了保藏,含腈水解酶基因和调控基因的质粒pSK108以携带所说质粒pSK108的转化体JM109/pSK108(FERM BP-5322)进行了保藏,两者都保藏于工业科学和技术机构的生物科学和人类技术国立研究所。菌株SK92首先根据其细菌特征鉴定为属于Rhodococcus属(见日本公开的专利公开号280,889/1991)。根据下列详细特征将该生物体进一步鉴定为Rhodococcus erythropolis:
检查项目 结果
腺嘌呤的分解 +
酪氨酸的分解 +
尿素的分解 +
利用:
肌醇 +
麦芽糖 -
甘露醇 +
鼠李糖 -
山梨醇 +
m-羟基苯甲酸钠 -
苯甲酸钠 +
柠檬酸钠 +
乳酸钠 +
睾丸素 +
乙酰胺 +
丙酮酸钠 +
在0.02%叠氮化钠存在的条件下生长 +
在10℃生长 +
在40℃生长 -
在0.001%结晶紫存在的条件下生长 -
在0.3%苯乙醇存在的条件下生长 -
在5%NaCl存在的条件下生长 +
在7%NaCl存在的条件下生长 +
实施例
下文将以下列实施例为参考(而不是用于限制本发明的范围)详细说明本发明。
SK92腈水解酶基因的克隆及其在E.cli和Rhodococcus中的表达将在参考实施例中进一步说明。(1)从SK92制备染色体DNA:
菌株SK92在100mlMY培养基(0.5%多肽胨,0.3%Bacto-酵母提取物,0.3%Bacto-蜕皮提取物)中30℃摇动培养72小时。收获细胞并将细胞沉淀悬浮于4ml盐水-EDTA溶液(0.1M EDTA,0.15M NaCl,pH8.0)中。向悬液中加入8mg溶菌酶。悬液在37℃摇动培养1至2小时,然后冷冻。在轻轻摇动的条件下加入10ml Tris-SDS溶液(1%SDS,0.1M NaCl,0.1MTris,pH9.0),接着加入蛋白酶K(MerK)达到终浓度为0.1mg。混合物在37℃下摇动培养1小时,然后置于60℃。向混合物中加入等量的以TE(TE:10mMTris,1mMEDTA,pH8.0)饱和的酚,搅拌并离心。向上层加入超出2倍量的乙醇,并用玻璃棒回收DNA。用90%,80%和70%的乙醇连续地去掉酚。然后,DNA溶于3ml TE缓冲液中,以10μg/ml的量向其中加入核糖核酸酶A(以100℃加热预先处理15分钟)。混合物37℃摇动培养30分钟,接着加入蛋白酶K。混合物37℃摇动培养30分钟。向混合物中加入等量的TE饱和酚,离心将它分成上下两层。对上层以相同的程序操作两次,接着用等量的含4%异戊醇的氯仿进行相同的提取程序(这些程序在下文称为酚处理)。然后向上层加入超出2倍量的乙醇并用玻璃棒回收DNA,以此获得染色体DNA。(2)DNA文库的构建:
将含有菌株SK92腈水解酶基因的DNA片段插入载体pUC118中制备的10μl质粒pSK002(见参考实施例)与2μl限制性酶SacI,10μl反应缓冲液(10倍浓缩)和78μl无菌水混合37℃裂解2小时。将消化物在0.7%琼脂糖凝胶上电泳以分离出1.1kb长的SacI片段。
另外,将步骤(1)获得的SK92染色体DNA用Eco RI消化,在琼脂糖凝胶上电泳并用于Southern杂交,其中上述1.1kb SacI片段用DIG DNA标记试剂盒(Boehringer Mannheim)标记并用作探针(SouthernE.M.,Mol.Bionl.98,503(1975))以检测一条大约14kb的DNA片段。将与探针杂交的含14kb片段的DAN部分从琼脂糖凝胶上切下来,然后将该DNA部分插入单独制备的Eco RI裂解的杂种质粒载体pK4(含Rhodococcus属质粒pRC004和E.coli载体pHSG299的FERM BP-3731(见日本公开的专利公开号64,589/1993和68,566/1993))。
用作载体的上述pK4片段制备如下:向10μl载体pK4中加入10μl反应缓冲液(10倍浓缩),77μl无菌水和2μl限制性酶Eco RI。混合物在37℃反应2小时,然后用酚处理,乙醇沉淀,干燥并溶于50μl无菌水中。向其中加入1μl碱性磷酸酶(Takara Shuzo Co.Ltd.),10μl反应缓中液(10倍浓缩)和39μl无菌水。混合物在65℃反应,用酚处理,乙醇沉淀,干燥并溶于无菌水中。
按上面所述,使用连接试剂盒(Takara ShuzoCo.Ltd.)4℃反应过夜将含14kb片段的1μl上述DNA部分插入上述Eco RI裂解的pK4中以制备DNA文库。(3)E.coli的转化和重组体DNA的选择:
将E.coli JM109(可从Takara Shuzo Co.Ltd.获得)接种到1ml LB培养基(1%Bacto胰蛋白胨提取物,0.5%Bacto-酵母提取物,0.5%NaCl)中并在37℃预先培养5小时。将100μl培养物接种到50ml SOB培养基(2%Bacto-胰蛋白胨,0.5%Bacto-酵母提取物,10mM NaCl,2.5mM KCl,1mM MgSO4,1mM MgCl2)中并在18℃培养20小时。离心回收细胞,将细胞沉淀悬浮于13ml冷的TF溶液(20mM PIPES-KOH,pH6.0,200mM KCl,10mM CaCl2,40mM MnCl2)中,0℃静置10分钟后再离心。去掉上清液后,将E.coli沉淀悬浮于3.2ml冷的TF溶液中,接着加入0.22ml二甲亚砜。将悬液在0℃静置10分钟。向由此制备的200μl感受态细胞中加入步骤(2)制备的10μl重组质粒(DNA文库)。混合物在0℃培养30分钟,然后在42℃热休克30秒并在0℃冷却2分钟,接着加入0.8ml SOC培养基(2% Bacto-胰蛋白胨,0.5%Bacto-酵母提取物,20mM葡萄糖,10mM NaCl,2.5mM KCl,1mMMgSO4,1mM MgCl2)。混合物在37℃摇动培养60分钟。将培养物以200μl/每板的量涂布到含100μg/ml氨苄青霉素的LB琼脂培养基上。平板在37℃培养。经过以下面方式进行菌落杂交从生长于平板上的菌落中选择出携带腈水解酶基因的转化体。将生长于平板上的菌落转移到尼龙膜(Biodyne A由Nippon Paul生产)上并裂解微生物。将DNA固定在膜上。然后用步骤(2)构建的探针(1.1kb片段)杂交,使用DIG发光检测试剂盒(Boehringer Mannheim)选择含靶重组DNA的菌落。(4)重组质粒的制备:
将在步骤(3)中选择的转化体在100mlLB培养基中37℃培养过夜,收获细胞并用无菌水洗涤。向细胞中加入5ml溶液I(2mM葡萄糖,10mM ED TA,25mMTris-HCl缓冲液,pH8.0)和25mg溶菌酶。0℃静置30分钟。然后加入10ml溶液II(1N NaOH,5%SDS),将混合物在0℃静置5分钟。接着加入7.5ml溶液III(3M乙酸钠,pH4.8),将混合物在0℃静置30分钟并离心。向上清液中加入50ml乙醇。再离心去掉上清液。接着加入5ml溶液IV(10mM乙酸钠。50mM Tris-HCl缓冲液,pH8.0)和2.5μl 10mg/ml的核糖核酸酶A。将混合物在室温下静置20分钟,接着加入12ml乙醇。离心,干燥并溶于无菌水中。(5)Rhodococcus属微生物的转化及转化体的腈水解酶活性:
离心收获对数生长期的Rhodococcus rhodochrousATCC 12674,用冰冷无菌水洗涤3次并悬浮于无菌水中。将1μg步骤(4)获得的质粒pSK104与10μl细胞悬液混合,混合物在冰上冷却。将DNA与微生物的混合物放入电穿孔装置CET-200(Japan SpectroscopicCo.Ltd.)的槽中,用3.8kV/cm的电场密度和1ms的脉冲宽度将样品脉冲20次。然后将处理的细胞悬液在冰上放置10分钟并在37℃热休克10分钟。接着加入500μlMYK培养基(0.5%多肽胨,0.3%Bacto蜕皮提取物,0.3%Bacto-酵母提取物,0.2%KH2PO4,0.2%K2HPO4(pH7.0))。然后将细胞悬液在26℃下摇动培养3小时。将悬液涂布到含75μg/ml卡那霉素的MYK琼脂板上并在26℃培养3天。
将所得的Rhodococcus属转化体接种到10ml含50μg/ml卡那霉素的MYK培养基中并在30℃预先培养24小时。将1ml培养物加到100ml含1.5%乙烯氰醇(ECH)(作诱导剂)和75μg/ml卡那霉素的GGP培养基(1.5%葡萄糖,0.1%Bacto-酵母提取物,1.0%谷氨酸钠,0.05%KH2PO4,0.05%K2HPO4,0.05%MgSO4·7H2O(pH7.2))中。将微生物在30℃培养48小时后收获,细胞沉淀悬浮于50mM磷酸盐缓冲液(pH7.7)中,取部分悬液在含100mM丙烯腈的50mM磷酸缓冲液(pH7.7)中30℃反应20分钟。以加入1N HCl终止反应,用高效液相色谱(HPLC)测定反应溶液中形成的丙烯酸量。结果表明:在转化体ATCC12674/pSK104中有8mM的丙烯酸形成。它揭示了腈水解酶表达所必需的调控因子编码基因存在于腈水解酶结构基因的上游或下游。(6)缺失质粒和腈水解酶活性:
由于估计pSK104仍含有许多对表达腈水解酶不需要的区域,因而由它制备了各种各样的缺失质粒。检查用缺失质粒转化的微生物的腈水解酶活性(表1,图1)。
表1.缺失质粒和丙烯酸的形成
形成的丙烯酸量(mM) | ||
诱导剂(ECH) | ||
不存在 | 存在 | |
1)pSK102 | 0 | 0 |
2)pSK104 | 0.77 | 8.00 |
3)pSK105 | 0 | 1.71 |
4)pSK123 | 0 | 0 |
5)pSK124 | 0 | 0 |
6)pSK106 | 1.14 | 6.38 |
7)pSK107 | 0 | 3.40 |
8)pSK125 | 0 | 0 |
9)pSK126 | 0 | 0 |
10)pSK127 | 0 | 0 |
11)pSK109 | 0 | 0 |
12)pSK108 | 0 | 8.05 |
从表中可明显看出,ATCC 12674/pSK108(6.2kbHind III-EcoRV片段)(图2)具有高腈水解酶活性。
构建了其它的缺失质粒并检查编码调控因子的基因。结果表明:该基因位于距腈水解酶结构基因较远的上游区域内(大约3kb Bam HI-EcoRV片段)。(7)核苷酸测序:
使用荧光序列仪ALFII(Pharmacia)测定了在步骤(6)揭示的,对腈水解酶表达所必需的调控因子编码基因的序列。序列分析揭示出SEQ ID NO:5的核苷酸序列,发现存在2个分别编码氨基酸序列SEQ ID NO:1和2的开架阅读框。与氨基酸序列资料库NBRF(国立生物医学研究基金会)进行比较表明:该调控因子属于2元调控因子家族。这些开架阅读框的核苷酸序列如SEQ ID NOs:3和4所示。
参考实施例
(1)菌株SK92染色体DNA的制备:
按与实施例步骤(1)相似的方法制备SK92染色体DNA。(2)探针的制备和DNA文库的构建:
使用100μl含10μl DNA作底物(稀释20倍),10μl反应缓冲液(10倍浓缩),4μl 5mM dNTP,作为引物#1的5’-AACTGCTGGGA(AG)CACTTCCA-3’(与氨基酸序列NCWEHFQ相对应的20个核苷酸)和作为引物#2的5’-GA(AG)TA(AG)TG(AG)CC(CG)AC(ACTG)GG(AG)TC-3′(与氨基酸序列DPVGHYS相对应的20个核苷酸)各5μl(500pmol)以及1μl Tth DNA聚合酶(TOYO Boseki)的溶液进行聚合酶链反应。上述2个引物根据与已知的各种腈水解酶具有高度同源性的氨基酸序列制备。反应包含50次循环,每次组成为:样品在93℃温育30秒(变性步骤),45℃30秒(退火步骤)和72℃2分钟(延长步骤)。从反应溶液中获得编码SK92腈水解酶的410bp的DNA片段。使用DIG DNA标记试剂盒(Boehringer Mannheim)标记该DNA片段作为探针。
向50μl SK92染色体DNA中加入10μl反应缓冲液(10倍浓缩),37μl无菌水和3μl限制性酶SalI。混合物在37℃反应2小时,然后用乙醇沉淀并进行琼脂糖凝胶电泳。使用DNA PREP(DIA-IATRON)回收大约1.1kb的DNA片段。使用连接试剂盒(Takara ShuzoCo.Ltd.)将DNA片段插入E.coli载体pUC118的SacI位点,从而制备重组DNA文库。
以下述方式制备上面的pUC118片段。向10μlpUC118中加入10μl反应缓冲液(10倍浓缩),77μl无菌水和2μl限制性酶SacI。混合物在37℃反应2小时,然后用酚处理,乙醇沉淀,干燥并溶于50μl无菌水中。向其中加入1μl碱性磷酸酶(Takara Shuzo Co.Ltd.),10μl反应缓冲液(10倍浓缩)和39μl无菌水。样品溶液在65℃反应,用酚处理,乙醇沉淀,干燥并溶于无菌水中。(3)E.coli的转化和重组DNA的选择:
按与实施例步骤(3)相同的方式制备E.coli JM109的感受态细胞。含在步骤(2)中制备的重组质粒的10μl溶液(DNA文库)加入至200μl感受态细胞中。细胞在0℃静置30分钟,然后在42℃热休克30秒并在0℃冷却2分钟。向其中加入0.8ml SOC培养基,细胞在37℃摇动培养60分钟。以每板200μl的量将培养物涂布到含100μg/ml氨苄青霉素的LB琼脂培养基上,接着37℃培养。按下面方法进行菌落杂交以便从生长于琼脂培养基的菌落中挑选出携带腈水解酶基因的转化体。将生长于琼脂培养基上的转化体转移到尼龙膜(Biodaine A由Paul Co.Ltd.生产)上并裂解它们以固定DNA。用步骤(2)制备的探针(410bp的片段)处理DNA,使用DIG发光检测试剂盒(Boehringer Mannheim)选择含靶重组DNA的菌落。(4)重组质粒的构建和限制性酶切图谱的制备:
按实施例步骤(4)相同的方式处理步骤(3)挑选的转化体。将由此获得的重组质粒pSKO02用一些限制性酶裂解以制备限制性酶切图谱。(5)用转化的E.coli生产腈水解酶和将腈转变成酸:
将JM109/pSK002菌株接种到1ml含50μg/ml氨苄青霉素的2XYT培养基(1.6%Bacto-胰蛋白胨,1.0%Bacto-酵母提取物,0.5%NaCl)中,37℃培养8小时。将1ml培养物接种到100ml含50μg/ml氨苄青霉素和1mM IPTG的2XYT培养基中,接着37℃培养14小时。收获后,将微生物悬浮于50mM磷酸缓冲液(pH7.7)中,取部分悬液加入含100mM丙烯腈的50mM磷酸缓中液(pH7.7)中,30℃反应20分钟。经加入1NHCl终止反应,以HPLC测定反应溶液中形成的丙烯酸量。在对照试验中,使用转化前的菌株JM109。结果表明:在宿主JM109中检测不到丙烯酸,而在转化体JM109/pSK002中发现有18mM丙烯酸形成。(6)将含腈水解酶基因的DNA片段导入杂种质粒载体中:
将含腈水解酶结构基因的DNA片段(5.8kb BgIII-Hind III片段)及推测为包含其启动子的区域克隆到杂种质粒载体pK4中,以此构建质粒pSK120。(7)Rhodococcus属微生物的转化和转化体的腈水解酶活性:
离心收获对数生长期的Rhodococcus rhodochrousATCC 12674,用冰冷无菌水洗涤3次,悬浮于无菌水中,将10μg细胞悬液与1μg步骤(6)获得的质粒pSK120混合,然后将混合物在冰上冷却。将该DNA与微生物的混合物导入基因导入装置CET-200(NipponBunko)的槽中,以3.8kV/cm的电场密度和1ms的脉冲宽度将样品脉冲20次。
处理的细胞悬液在冰上放置10分钟并37℃热休克10分钟,向悬液中加入500μl MYK培养基,然后混合物在26℃摇动培养3小时。将培养物涂布到含75μg/ml卡那霉素的MYK琼脂培养基上,26℃培养3天。
将由此获得的Rhodococcus属转化体接种到含50μg/ml卡那霉素的10ml MYK培养基中,30℃预培养24小时。将1ml培养物加入100ml含75μg/ml卡那霉素的GGP培养基中。向其中加入1.5%ECH作为诱导剂。转化体在30℃培养48小时。回收后,将细胞悬浮于50mM磷酸缓冲液(pH7.7)中,以与步骤(5)相同的方式检查其腈水解酶活性。在其中未发现活性。
本文引用了许多参考文献,将其全文公开的内容引用作为本文的参考。序列表SEQ ID NO:1长度:244类型:氨基酸拓扑学:线型分子类型:蛋白质来源生物体:Rhodococcus erythropolis菌株:SK92序列:
15
MetAlaGlyAlaAspValHisAlaGlnGlyGlyThrAsnArgArg
30
AlaArgIleLeuValValAspAspGluLysHisValArgThrMet
45
ValThrTrpGlnLeuGluSerGluAsnPheAspValValAlaAla
60
AlaAspGlyAspAlaAlaLeuArgGlnValThrGluSerAlaPro
75
AspLeuMetValLeuAspLeuSerLeuProGlyLysGlyGlyLeu
90
GluValLeuAlaThrValArgArgThrAspAlaLeuProIleVal
105
ValLeuThrAlaArgArgAspGluThrGluArgIleValAlaLeu
120
AspLeuGlyAlaAspAspTyrVallleLysProPheSerProArg
135
GluLeuAlaAlaArgIleArgAlaValLeuArgArgThrThrAla
150
GluProProHisGluAlaAlaValGlnArgPheGlyAspLeuGlu
165
IleAspThrAlaAlaArgGluValArgLeuHisGlyIleProLeu
180
GluPheThrThrLysGluPheAspLeuLeuAlaTyrMetAlaAla
195
SerProMetGlnValPheSerArgArgArgLeuLeuLeuGluVal
210
TrpArgSerSerProAspTrpGlnGlnAspAlaThrValThrGlu
225
HisValHisArgIleArgArgLysIleGluGluAspProThrLys
240
ProThrIleLeuGlnThrValArgGlyAlaGlyTyrArgPheAsp
244
GlyGluArgAlaSEQ ID No:2长度:534类型:氨基酸拓扑学:线型分子类型:蛋白质来源生物体:Rhodococcus erythropolis菌株:SK92序列
15MetMetThrAspThrLeuProSerSerSerArgTrpThrLeuGlu
30GlyProHisLeuGlnProLeuGlnGlyGluAlaLeuAlaAspLeu
45HisAlaArgThrLeuGluMetlleThrSerGlyArgGluLeuHis
60GluThrLeuGluValValAlaArgGlyIleGluGluLeuMetPro
75GlyLysArgCysAlalleLeuLeuLeuAspAsnThrGlyProVal
90LeuArgCysGlyAlaAlaProThrMetSerAlaProTrpArgArg
105TrplleAspSerLeuValProGlyProMetSerGlyGlyCysGly
120ThrAlaValHisLeuGlyGluProValIleSerTyrAspValAla
135AspAspProLysPheArgGlyProPheArgAlaAlaAlaLeuHis
150GluGlylleArgAlaCysTrpSerThrProValThrSerGlyAsp
165GlyThrlleLeuGlyThrPheAlaIleTyrGlySerValProAla
180PheProAlaGlnGlnAspValAlaLeuValThrGlnCysThrAsp
195LeuThrAlaAlaValIleThrThrHisLysLeuHisGlnAspLeu
210SerMetSerGluGluArgPheArgArgAlaPheAspSerAsnVal
225ValGlyMetAlaLeuLeuAspGluSerGlySerSerIleArgVal
240AsnAspThrLeuCysAlaLeuThrAlaAlaProProArgArgLeu
255LeuGlyHisProMetGlnGluIleLeuThrAlaAspSerArgGlu
270ProPheAlaAsnGlnLeuSerSerIlcArgGluGlyLeuThrAsp
285GlyGlyGlnLeuAspGlyArgIleGlnThrThrGlyGlyArgTrp
300IleProValHisLeuSerIleSerGlyMetTrpThrThrGluArg
315GluPheMetGlyPheSerValHisValLeuAsplleSerGluArg
330LeuAlaAlaGluArgAlaArgGluGluGlnLeuGluAlaGluVal
345AlaArgHisThrAlaGluGluAlaSerArgAlaLysSerThrPhe
360LeuSerGlyMetThrHisGluValGlnThrProMetAlaValIle
375ValGlyPheSerGluLeuLeuGluThrLeuAspLeuAspGluGlu
390ArgArgGlnCysAlaTyrArgLysIleGlyGluAlaAlaLysHis
405ValIleSerLeuValAspAspValLeuAspIleAlaLysIleGlu
420AlaGlyAlaIleThrLeuGlnAspGluAspIleAspLeuSerGlu
435GluValAlaThrIleValGluMetLeuGluProIleAlaArgAsp
450ArgAspArgAspValCysLeuArgTyrValProProGlnThrPro
465ValHisValCysSerAspArgArgArgValArgGluValLeuLeu
480AsnlleValSerAsnGlyIleLysTyrAsnArgLeuGlyGlyVal
495ValAspProProThrGlySerGlyAlaAlaArgProArgGlnThr
510ArgAlaProAspTyrProAlaThrProThrThrAsnSerSerSer
525ProSerThrGlyTrpGluSerArgProArgGlyCysLysGlyArg
534GlySerValLeuArgSerProAlaArg SEQ ID No:3长度:735类型:核酸链型:双链拓扑学:线型来源生物体:Rhodococcus erythropolis菌株:SK92序列:ATG GCC GGA GCG GAC GTC CAC GCC CAG GGT GGC ACG AAT CGA CGT 45GCA CGC ATC CTC GTC GTC GAC GAC GAA AAA CAC GTG CGC ACG ATG 90GTG ACG TGG CAA CTC GAA TCG GAG AAT TTC GAT GTT GTC GCT GCG 135GCA GAC GGA GAT GCG GCA CTG CGT CAG GTC ACT GAG AGC GCA CCC 180GAT TTG ATG GTG CTC GAT CTG TCG CTC CCG GGG AAA GGT GGG TTG 225GAA GTG CTC GCT ACG GTC CGC AGA ACC GAT GCA CTG CCT ATC GTC 270GTG CTC ACA GCA CGC CGC GAT GAA ACC GAA CGG ATC GTC GCG CTG 315GAT CTC GGC GCC GAT GAC TAC GTC ATC AAA CCG TTC TCC CCG CGG 360GAA TTG GCC GCC CGT ATC CGG GCA GTG CTT CGT CGA ACC ACA GCT 405GAA CCC CCA CAC GAG GCG GCG GTT CAG CGA TTC GGT GAC CTA GAG 450ATC GAC ACC GCT GCG CGC GAG GTT CGG CTC CAC GGG ATA CCG CTC 495GAG TTC ACC ACC AAG GAG TTC GAT CTG CTG GCC TAT ATG GCC GCA 540TCA CCG ATG CAG GTC TTC AGC CGA CGC AGA TTG TTG CTC GAG GTG 585TGG CGA TCG TCG CCC GAC TGG CAG CAG GAC GCC ACC GTG ACC GAG 630CAC GTG CAC CGC ATT CGC CGC AAG ATC GAA GAA GAT CCC ACC AAA 675CCG ACG ATC CTG CAG ACA GTG CGG GGA GCC GGT TAC CGT TTC GAC 720GGA GAG CGT GCA TGA 735 SEQ ID N0:4长度:1605类型:核酸链型:双链拓扑学:线型来源生物体:Rhodococcus ervthropolis菌株:SK92序列:ATG ATG ACC GAC ACA CTG CCC TCC TCG TCC CGT TGG ACC CTT GAA 45GGC CCG CAT CTC CAG CCG CTG CAG GGT GAG GCC CTG GCG GAT CTC 90CAC GCC CGT ACG CTC GAG ATG ATC ACT TCC GGG AGA GAA TTG CAC 135GAG ACA CTC GAG GTG GTC GCC CGC GGC ATC GAG GAA CTG ATG CCG 180GGC AAA CGT TGC GCA ATT CTG TTG CTC GAC AAC ACC GGA CCG GTA 225TTG CGC TGC GGC GCG GCC CCA ACA ATG AGC GCG CCG TGG CGC CGG 270TGG ATC GAC AGC CTC GTC CCT GGT CCG ATG TCG GGT GGC TGC GGC 315ACA GCG GTT CAC CTC GGC GAG CCG GTT ATT TCC TAT GAC GTG GCC 360GAT GAC CCG AAA TTC CGC GGC CCC TTC CGC GCC GCA GCC CTC CAC 405GAG GGC ATA CGT GCC TGC TGG TCC ACC CCC GTC ACA AGC GGA GAC 450GGC ACG ATC CTC GGC ACT TTC GCG ATC TAC GGA TCC GTG CCG GCG 495TTC CCC GCA CAA CAG GAC GTT GCC CTG GTC ACC CAA TGC ACC GAC 540CTG ACC GCT GCC GTC ATC ACC ACC CAC AAA CTT CAT CAA GAT CTG 585AGC ATG AGC GAG GAG CGG TTC CGA CGC GCC TTC GAT TCC AAT GTC 630GTC GGC ATG GCA CTT CTC GAC GAA TCC GGC TCC AGC ATC CGC GTC 675AAC GAC ACC CTG TGC GCG TTG ACC GCA GCT CCG CCA CGG CGC CTC 720CTC GGC CAC CCC ATG CAG GAG ATA CTC ACC GCC GAC TCC CGG GAA 765CCG TTC GCC AAT CAG TTG TCC TCC ATC CGT GAG GGA TTG ACC GAC 810GGC GGA CAG CTC GAC GGA CGA ATC CAA ACC ACC GGA GGT CGG TGG 855ATT CCG GTG CAC CTG TCC ATC AGC GGT ATG TGG ACC ACG GAG CGG 900GAG TTC ATG GGA TTC AGC GTC CAT GTC CTG GAC ATC TCC GAG CGC 945CTG GCC GCC GAA CGC GCC CGC GAG GAA CAA CTC GAG GCC GAG GTT 990GCC CGC CAT ACC GCG GAG GAA GCC AGT CGC GCC AAG TCC ACG TTC 1035CTG TCC GGC ATG ACG CAC GAG GTC CAA ACG CCC ATG GCC GTT ATC 1080GTC GGA TTC AGT GAG CTA CTC GAG ACC CTG GAC CTG GAT GAA GAA 1125CGT CGT CAG TGC GCC TAC CGC AAG ATC GGC GAA GCC GCG AAA CAC 1170GTG ATC TCC CTG GTC GAC GAC GTT CTC GAT ATA GCC AAG ATC GAA 1215GCC GGC GCT ATC ACT CTG CAG GAC GAA GAC ATC CAC CTG TCC GAA 1260GAA GTT GCC ACC ATC GTG GAG ATG CTC GAG CCC ATC GCC CGT GAC 1305CGT GAC CGT GAC GTC TGC CTG CGG TAC GTC CCG CCG CAG ACA CCG 1350GTG CAC GTG TGC TCG GAC CGG CGG CGG GTG CGG GAA GTG CTG CTC 1395AAC ATC GTC TCC AAC GGG ATC AAG TAC AAT CGG CTC GGT GGT GTC 1440GTC GAC CCC CCA ACA GGA TCA GGG GCT GCT CGT CCG CGT CAG ACG 1485AGG GCC CCG GAC TAC CCA GCG ACG CCG ACG ACG AAC TCT TCG AGC 1530CCT TCA ACC GGC TGG GAG TCG AGG CCA CGG GGG TGC AAG GGT CGG 1575GGC TCG GTC TTG CGC TCT CCC GCG CGC TGA 1605SEQ ID No:5长度:2336类型:核酸链型:双链拓扑学:线型来源生物体:Rhodococcus erythropolis菌株:SK92序列:ATGGCCGGAG CGGACGTCCA CGCCCAGGGT GGCACGAATC GACGTGCACG 50CATCCTCGTC GTCGACGACG AAAAACACGT GCGGACGATG GTGACGTGGC 100AACTCGAATC GGAGAATTTC GATGTTGTCG CTGCGGCAGA CGGAGATGCG 150GCACTGCGTC AGGTCACTGA GAGCGCACCC GATTTGATGG TGCTCGATCT 200GTCGCTCCCG GGGAAAGGTG GGTTGGAAGT GCTCGCTACG GTCCGCAGAA 250CCGATGCACT GCCTATCGTC GTGCTCACAG CACGCCGCGA TGAAACCGAA 300CGGATCGTCG CGGTGGATCT CGGCGCCGAT GACTACGTCA TGAAACCGTT 350CTCCCCGCGG GAATTGGCCG CCCGTATCCG GGCAGTGCTT CGTCGAACCA 400CAGCTGAACC CCCACACGAG GCGGCGGTTC AGCGATTCGG TGACCTAGAG 450ATCGACACCG CTGCGCGCGA GGTTCGGCTC CACGGGATAC CGCTCGAGTT 500CACCACCAAG GAGTTCGATC TGCTGGCCTA TATGGCCGCA TCACCGATGC 550AGGTCTTCAG CCGACGCAGA TTGTTGCTCG AGGTGTGGCG ATCGTCGCCC 600GACTGGCAGC AGGACGCCAC CGTGACCGAG CACGTGCACC GCATTCGCCG 650CAAGATCGAA GAAGATCCCA CCAAACCGAC GATCCTGCAG ACAGTGCGGG 700GAGCCGGTTA CCGTTTCGAC GGAGAGCGTG CATGATGACC GACACACTGC 750CCTCCTCGTC CCGTTGGACC CTTGAAGGCC CGCATCTCCA GCCGCTGCAG 800GGTGAGGCCC TGGCGGATCT CCACGCCCGT ACGCTCGAGA TGATCACTTC 850CGGGAGAGAA TTGCACGAGA CACTCGAGGT GGTCGCCCGC GGCATCGAGG 900AACTGATGCC GGGCAAACGT TGCGCAATTC TGTTGCTCGA CAACACCGGA 950CCGGTATTGC GCTGCGGCGC GGCCCCAACA ATGAGCGCGC CGTGGCGCCG 1000GTGGATCGAC AGCCTCGTCC CTGGTCCGAT GTCGGGTGGC TGCGGCACAG 1050CGGTTCACCT CGGCGAGCCG GTTATTTCCT ATGACGTGGC CGATGACCCG 1100AAATTCCGCG GCCCCTTCCG CGCCGCAGCC CTCCACGAGG GCATACGTGC 1150CTGCTGGTCC ACCCCCGTCA CAAGCGGAGA CGGCACGATC CTCGGCACTT 1200TCGCGATCTA CGGATCCGTG CCGGCGTTCC CCGCACAACA GGACGTTGCC 1250CTGGTCACCC AATGCACCGA CCTGACCGCT GCCGTCATCA CCACCCACAA 1300ACTTCATCAA GATCTGAGCA TGAGCGAGGA GCGGTTCCGA CGCGCCTTCG 1350ATTCCAATGT CGTCGGCATG GCACTTCTCG ACGAATCCGG CTCCAGCATC 1400CGCGTCAACG ACACCCTGTG CGCGTTGACC GCAGCTCCGC CACGGCGCCT 1450CCTCGGCCAC CCCATGCAGG AGATACTCAC CGCCGACTCC CGGGAACCCT 1500TCGCCAATCA GTTGTCCTCC ATCCGTGAGG GATTGACCGA CGGCGGACAG 1550CTCGACGGAC GAATCCAAAC CACCGGAGGT CGGTGGATTC CGGTGCACCT 1600GTCCATCAGC GGTATGTGGA CCACGGAGCG GGAGTTCATG GGATTCAGCG 1650TCCATGTCCT GGACATCTCC GAGCGCCTGG CCGCCGAACG CGCCCGCGAG 1700GAACAACTCG AGGCCGAGGT TGCCCGCCAT ACCGCGGAGG AAGCCAGTCG 1750CGCCAAGTCC ACGTTCCTGT CCGGCATGAC GCACGAGGTC CAAACGCCCA 1800TGGCCGTTAT CGTCGGATTC AGTGAGCTAC TCGAGACGCT GGACCTGGAT 1850GAAGAACGTC GTCAGTGCGC CTACCGCAAG ATCGGCGAAG CCGCGAAACA 1900CGTGATCTCC CTGGTCGACG ACGTTCTCGA TATAGCCAAG ATCGAAGCCG 1950GCGCTATCAC TCTGCAGGAC GAAGACATCG ACCTGTCCGA AGAAGTTGCC 2000ACCATCGTGG AGATGCTCGA GCCCATCGCC CGTGACCGTG ACCGTGACGT 2050CTGCCTGCGG TACGTCCCGC CGCAGACACC GGTGCACGTG TGCTCGGACC 2100GGCGGCGGGT GCGGGAAGTG CTGCTCAACA TCGTCTCCAA CGGGATCAAG 2150TACAATCGGC TCGGTGGTGT CGTCGACCCC CCAACAGGAT CAGGGGCTGC 2200TCGTCCGCGT CAGACGAGGG CCCCGGACTA CCCAGCGACG CCGACGACGA 2250ACTCTTCGAG CCCTTCAACC GGCTGGGAGT CGAGGCCACG GGGGTGCAAG 2300GGTCGGGGCT CGGTCTTGCG CTCTCCCGCG CGCTGA 2336
Claims (6)
1.一种由具有SEQ ID NO:1氨基酸序列的多肽和具有SEQ ID NO:2的氨基酸序列的多肽两种成份组成的、能激活腈水解酶基因启动子的调控因子以及编码它的DNA。
2.根据权利要求1的调控因子和DNA,其中编码所说多肽的基因具有SEQ ID NO:3和4的核苷酸序列。
3.根据权利要求1或2的调控因子和DNA,其中在腈存在的条件下腈水解酶基因启动子的激活被增强。
4.一种重组质粒,含有编码权利要求1,2或3的调控因子的DNA,包含启动子区的腈水解酶基因和能在属于Rhodococcus属的微生物细胞中复制的DNA区域。
5.根据权利要求4的重组质粒,其中能在属于Rhodococcus属的微生物细胞中复制的DNA区选自质粒pRC001、pRC002、pRC003和pRC004。
6.属于被权利要求4或5的重组质粒转化的Rhodococcus属的微生物。
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US4920054A (en) * | 1986-07-25 | 1990-04-24 | Allelix, Inc. | Shuttle vectors from rhodococcus equi |
AU627648B2 (en) * | 1990-02-28 | 1992-08-27 | Teruhiko Beppu | Dna fragment encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant |
EP0502476B1 (en) * | 1991-03-04 | 2001-07-18 | Mitsubishi Rayon Co., Ltd. | Hybrid plasmid vectors containing genes encoding nitrile degrading enzymes and methods of producing amides and acids |
-
1994
- 1994-12-28 JP JP33765294A patent/JP3154633B2/ja not_active Expired - Lifetime
-
1995
- 1995-12-22 US US08/577,184 patent/US5602014A/en not_active Expired - Lifetime
- 1995-12-27 EP EP95309454A patent/EP0719862B1/en not_active Expired - Lifetime
- 1995-12-27 DE DE69522192T patent/DE69522192T2/de not_active Expired - Lifetime
- 1995-12-28 CN CN95119459A patent/CN1080306C/zh not_active Expired - Lifetime
- 1995-12-28 KR KR1019950061485A patent/KR100358532B1/ko not_active IP Right Cessation
-
1996
- 1996-01-30 TW TW085101136A patent/TW387895B/zh not_active IP Right Cessation
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105296512A (zh) * | 2002-05-15 | 2016-02-03 | 巴斯夫酶有限责任公司 | 腈水解酶、编码腈水解酶的核酸,以及制备和使用它们的方法 |
CN102260695B (zh) * | 2004-08-16 | 2012-09-05 | 纳幕尔杜邦公司 | 使用腈水解酶突变体生产3-羟基羧酸 |
CN113025601A (zh) * | 2019-12-25 | 2021-06-25 | 上海奥博生物医药技术有限公司 | 腈水解酶启动子优化表达及应用 |
Also Published As
Publication number | Publication date |
---|---|
DE69522192T2 (de) | 2002-04-18 |
KR100358532B1 (ko) | 2003-03-28 |
CN1080306C (zh) | 2002-03-06 |
JPH08173169A (ja) | 1996-07-09 |
KR960023057A (ko) | 1996-07-18 |
DE69522192D1 (de) | 2001-09-20 |
TW387895B (en) | 2000-04-21 |
JP3154633B2 (ja) | 2001-04-09 |
EP0719862B1 (en) | 2001-08-16 |
EP0719862A3 (en) | 1998-02-25 |
US5602014A (en) | 1997-02-11 |
EP0719862A2 (en) | 1996-07-03 |
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