CN106434707B - 一种来源于枯草杆菌znxh1的细菌漆酶基因、细菌漆酶及其应用 - Google Patents
一种来源于枯草杆菌znxh1的细菌漆酶基因、细菌漆酶及其应用 Download PDFInfo
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- CN106434707B CN106434707B CN201610969808.XA CN201610969808A CN106434707B CN 106434707 B CN106434707 B CN 106434707B CN 201610969808 A CN201610969808 A CN 201610969808A CN 106434707 B CN106434707 B CN 106434707B
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Abstract
一种来源于枯草杆菌ZNXH1的细菌漆酶基因、细菌漆酶及其应用,属于生物技术领域,具体涉及一种来源于枯草杆菌ZNXH1的细菌漆酶基因cahh1、由该基因表达的细菌漆酶CAHH1以及该细菌漆酶CAHH1在纺织工业和环境保护等领域中的应用。与传统漆酶相比,细菌漆酶CAHH1具有明显的应用稳定性优势和作用广谱性优点,主要包括:(1)细菌漆酶CAHH1在pH=3‑9的范围内具有较好的酸碱稳定性,(2)细菌漆酶CAHH1在温度为30℃‑90℃的范围内均具有较好的温度稳定性,(3)细菌漆酶CAHH1对靛蓝类染料、偶氮类染料和三苯甲烷类染料的脱色作用均强,可用于工业染料的脱色,有广阔的应用前景,而且易实现工程化制备,有较高的实用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种来源于枯草杆菌ZNXH1的细菌漆酶基因cahh1、由该基因表达的细菌漆酶CAHH1以及该细菌漆酶CAHH1在纺织工业和环境保护等领域中的应用。
背景技术
漆酶(EC 1.10.3.2)是一种含铜的多酚氧化酶,它可利用活性中心的铜离子催化氧化多种结构的芳香类化合物,同时将分子氧还原成水。由于漆酶作用的底物范围十分广泛、催化效率高,所以在造纸工业(生物制浆和漂白)、纺织工业(人工合成染料脱色)、食品工业(食品风味改良)、饲料营养改善、新型药物开发、土壤的生物修复、生物传感器制造及新型能源开发等领域具有潜在重要应用价值。
漆酶广泛存在于高等真菌(特别是担子菌)中。近年来,部分真菌漆酶已经应用于多个工业领域,如来自黑曲霉的漆酶DeniliteⅡS等。但是真菌漆酶受环境影响比较大,在碱性条件下活性较低,这严重影响了它在工业领域中的应用。随着全基因组测序技术的迅速发展,人们发现,漆酶在细菌中同样广泛存在。细菌漆酶在碱性环境和高温条件下具有良好的催化活性和较高的稳定性,并且具有生长快、易于培养的特点。由于工业染料废水等一般温度较高,碱性较强,真菌漆酶在实际应用中表现出稳定性较差,而这种环境却不影响细菌漆酶功能的发挥,因此细菌漆酶在工业上具有更好的应用前景。虽然目前对细菌漆酶资源的发掘还很有限,但迄今已经在地衣芽孢杆菌(Bacillus licheniformis),枯草芽孢杆菌(Bacillus subtilis)等微生物中发现了漆酶。
随着印染工业的迅速发展,染料的品种和数量不断增加,全球每年至少有10%的染料通过废水排放到自然环境中,这些废水中的染料很多是具有毒性或致癌性的,严重危害了水生动物以及人类本身。纺织、造纸和塑胶等行业产生的染料废水往往不能有效的零污染处理,导致了大量工业染料废水的排放。而工业使用的合成染料绝大多数是芳香族化合物,使用传统的方法无法有效去除废水中的染料,而且非常耗能,但相比之下,应用漆酶来处理工业染料废水既经济、有效、又环境友好,是目前和未来较理想的治理方法。真菌漆酶虽然对纺织染料的脱色及降解效果明显,但产漆酶的真菌因较细菌生长缓慢、发酵周期长、生长温度和pH范围较窄而限制了真菌漆酶的应用,而产漆酶的细菌因具有生长迅速、发酵周期短、耐温、在碱性条件下稳定等特点而在工业上具有更好的应用前景和实用价值。目前关于细菌漆酶在染料降解与脱色方面的研究报道还相对较少,通过研究细菌漆酶对不同合成染料的脱色效果,可以进一 步推动细菌漆酶的在工业上的应用。
靛蓝类染料、偶氮类染料、蒽醌染料和三苯甲院类染料是目前工业是上用量最大的四类染料,也是较难降解的染料。目前世界各国都在致力于开发能有效降解这四类染料的细菌漆酶,以期实现有效保护和修复生态环境,但是前提是要开发出理想的细菌漆酶。
发明内容
本发明的目的之一是提供一种来源于枯草杆菌ZNXH1的细菌漆酶基因cahh1;
本发明的另一个目的是利用上述细菌漆酶基因cahh1制备高效、广谱的细菌漆酶CAHH1,即应用基因工程技术利用细菌表达系统进行漆酶的表达与制备;
本发明的再一个目的是通过对细菌漆酶CAHH1的理化性质的分析,提供细菌漆酶CAHH1在工业染料降解和脱色中的用途。
本发明所述的一种来源于枯草杆菌ZNXH1的细菌漆酶基因cahh1,其核苷酸序列如SEQ ID NO.1所示。
本发明所述的一种来源于枯草杆菌ZNXH1的细菌漆酶CAHH1,其氨基酸序列如SEQID NO.2所示。
本发明所述的细菌漆酶基因cahh1来自枯草杆菌ZNXH1,我们发现,该菌所分泌的蛋白具有漆酶的活力,经进一步研究确定该菌的基因组中含有漆酶基因。
将枯草杆菌ZNXH1通过菌体培养、目的基因钓取,得到本发明所述的细菌漆酶基因cahh1。我们委托上海生工生物技术有限公司对该基因进行测序,测序结果如图3所示,其核苷酸序列如SEQ ID NO.1所示。
将本发明的细菌漆酶基因cahh1与表达载体重组,形成重组表达载体,本发明不限于特定的表达载体,优选的表达载体是原核表达载体[pET-28a、pET-41b等系列的pET表达载体(通用的大肠杆菌表达载体,含有T7强启动子、N-或C-末端组氨酸标签以及卡那霉素抗性基因等,Novagen、Invitrogen等公司有售)或pPZW103等表达载体(枯草杆菌表达载体,含有P43强启动子以及卡那霉素抗性基因等,参见黄鹤等,“重组青霉素G酰化酶在枯草芽孢杆菌中的表达条件优化”,中国生物化学与分子生物学报,2001,17(2):173-177)]。
上述重组表达载体按生物学常规方法导入适宜的宿主细胞,适宜的宿主细胞包括真核表达细胞和原核表达细胞。本发明不局限于任何特定的宿主细胞,优选的宿主细胞是大肠杆菌或枯草杆菌,进一步优选的大肠杆菌宿主细胞为E.coli BL21(DE3)或E.coliBL21(DE3)pLysS、E.coli BL21(DE3)pLysE(通用的大肠杆菌表达宿主细胞,Novagen、Invitrogen等公司有售),进一步优选的枯草杆菌宿主细胞为WB600(参见 黄鹤等,“重组青霉素G酰化酶在枯草芽孢杆菌中的表达条件优化”,中国生物化学与分子生物学报,2001,17(2):173-177),表达载体导入宿主细胞后得到表达细菌漆酶CAHH1的工程菌。
工程菌表达的漆酶为可溶性漆酶蛋白。针对不同的宿主细胞,表达产物的纯化有不同的方法。1、针对大肠杆菌宿主细胞表达的胞内漆酶蛋白(胞内酶),通过表达细胞超声破碎和加热失活大肠杆菌杂蛋白即可分离得到细胞可溶性漆酶蛋白,再使用Ni2+-NTA亲和柱纯化得到纯化的漆酶蛋白;2、针对枯草杆菌宿主细胞表达的胞外漆酶蛋白(胞外酶),通过表达体系细胞的离心分离获得枯草杆菌表达细胞的培养液,再通过加热失活培养液中的杂蛋白即可分离纯化得到表达细胞分泌的漆酶蛋白。
表达的漆酶蛋白经活性实验鉴定,具有漆酶的活性,可催化多种染料等底物的脱色。该种漆酶蛋白即为本发明所述的细菌漆酶CAHH1,其氨基酸序列如SEQ ID NO.2所示。与传统漆酶相比,它具有明显的应用稳定性优势和作用广谱性优点,主要包括:(1)本发明的细菌漆酶CAHH1在pH=3-9的范围内具有较好的酸碱稳定性,(2)本发明的细菌漆酶CAHH1在温度为30℃-90℃的范围内均具有较好的温度稳定性,(3)本发明的细菌漆酶CAHH1对靛蓝类染料、偶氮类染料和三苯甲烷类染料的脱色作用均强,可用于工业染料的脱色。
以上技术方案中所有分子生物学操作均参照“分子克隆实验指南”(第三版,科学出版社,2002年)进行。
附图说明
图1:细菌漆酶基因cahh1产物琼脂糖凝胶电泳图谱,其中M:DNA分子Marker;S:细菌漆酶基因cahh1,在分子量1542bp处有明亮条带;
图2:重组表达载体pET28a-CAHH1构建示意图;
图3:细菌漆酶基因cahh1测序谱图,共测二个反应,进一步使用Gene-man软件组装得到全长的细菌漆酶基因(1542bp);
图4:纯化得到的细菌漆酶CAHH1的SDS-PAGE电泳图谱,其中M:蛋白质分子Marker;1:纯化的细菌漆酶CAHH1;
图5:细菌漆酶CAHH1的pH-活力曲线(A)和pH稳定性曲线(B);
图6:细菌漆酶CAHH1的温度-活力曲线(A)和温度稳定性水平(B);
图7:Co2+和Mn2+对细菌漆酶CAHH1酶活力的影响;
图8:细菌漆酶CAHH1对染料活性亮蓝19、刚果红、靛蓝胭脂红、甲基红、结晶紫的脱色效率曲线。
具体实施方式
实施例1:细菌漆酶CAHH1基因克隆及细菌漆酶CAHH1的表达与制备,包括以下步骤:
(1)枯草杆菌ZNXH1的培养及其染色体DNA的提取
将枯草芽孢杆菌BS168(上海北诺科技有限公司、北京天辰泽生物技术有限公司等有售)菌株接种于0.5mL的LB液体培养基(每100毫升培养基组分如下:0.5g酵母粉,1.0g蛋白胨,1.0g NaCl,其余为水,pH=7.6)中,37℃,180rpm,震荡培养2h后,将其倒入空培养皿中,置于紫外灯(20W)30cm处照射20s,间隔20s,共照射3次。然后取100μL菌液,涂布于LB琼脂培养平板(每100毫升培养基组分如下:0.5g酵母粉,1.0g蛋白胨,1.0g NaCl,0.5g琼脂粉,其余为水,pH=7.6)上,37℃培养10h后,挑取单克隆菌株,即为枯草杆菌ZNXH1。将枯草杆菌ZNXH1接种于1mL LB液体培养基中,37℃、180rpm震荡培养8h后,再转至100mL新鲜的LB液体培养基中扩大培养8h,8000rpm离心5min收集培养的菌体,-20℃保存菌体备用。
取0.2g上述收集的枯草杆菌ZNXH1菌体,用500μL PBS缓冲液(每1L PBS组分:8gNaCl、0.2g KCl、1.44g KH2PO4,其余为水,pH=7.4)洗涤菌体一次后,离心弃上清液;再用200μL上述缓冲液重悬菌体,加入50μL、50mg/mL溶菌酶,室温消化5min;加入50μL的SDS溶液{终浓度为2%(g/mL)}、25μg蛋白酶K,55℃恒温水浴消化3h;加入2μL的1mg/mL RNase A,37℃恒温水浴再消化30min;加入与上述溶液等体积(300μL)的酚:氯仿:异戊醇(体积比为25:24:1),颠倒混匀,10000rpm离心10min(4℃),将上清液转移至另一支离心管中,向上清液中加入2倍体积的无水乙醇,-20℃静置30min后,10000rpm离心10min(4℃),收集菌体染色体DNA沉淀;用200μL的70%乙醇(体积比)漂洗沉淀,10000rpm离心10min(4℃),收集沉淀,吸干,室温静置10min以除尽乙醇;最后用100μL的TE溶液(pH=8.0)重新溶解DNA沉淀,取5μL溶液用0.7%(g/mL)的琼脂糖凝胶电泳鉴定所得染色体DNA的浓度和大小,只在分子量20Kb附近有一条亮带,为所制得的染色体DNA,即本发明所述的细菌漆酶基因的PCR扩增模板,-20℃保存备用。
(2)细菌漆酶CAHH1基因的钓取及重组克隆载体的构建
本发明所述的细菌漆酶CAHH1基因通过PCR方法从步骤(1)所制得的染色体DNA中扩增而得到。所需引物依据枯草杆菌漆酶基因中的保守序列及表达载体的多克隆位点序列而设计,并委托上海生工生物技术有限公司合成,其序列如下:
上游引物:5’CGGGATCCATGACACTTGAAAAATTTGTG 3’,划线的GGATCC序列是内切酶BamH I识别位点;
下游引物:5’CCCTCGAGTTATTTATGGGGATCAGTTAT 3’,划线的CTCGAG 序列是内切酶XhoI识别位点。
上述两个引物所设置的酶切位点与本发明所用的表达载体pET-28a(通用的大肠杆菌表达载体,含有T7强启动子、N-末端组氨酸标签以及卡那霉素抗性基因等,Novagen、Invitrogen等公司有售)的BamH I和XhoI相匹配,适合于在大肠杆菌中高效表达。
PCR反应:100μL反应体系中含1μL Ex-Taq DNA聚合酶、10μL Ex-Taq DNA聚合酶缓冲液、1.5μL dNTP混合物(每种核苷酸浓度为25mmol/L)、4μL染色体DNA、1μL上游引物、1μL下游引物、81.5μL超纯水。每个循环中94℃变性0.5min,58℃退火1min,72℃延伸2min,最后一次循环延至10min,共30个循环。用1.0%(g/mL)的琼脂糖凝胶电泳检测PCR产物,分子量约为1542bp,如图1中S所示。
使用DNA纯化试剂盒(Bio Basic公司生产的EZ Spin Column DNA GelExtraction Kit)纯化PCR产物,步骤如下:向PCR溶液中加入4倍体积的结合缓冲液II(Binding Buffer II),加入EZ-10柱子中,静止2min,10000rpm离心2min;加入500μL的洗脱缓冲液(Wash Solution),10000rpm离心1min,重复1次,将EZ-10柱转移到干净的1.5mL微量离心管(microfuge tube)中,加入30μL无菌去离子水,室温放置2min,然后10000rpm离心2min,得到要纯化的目的DNA片段。取3μL上述纯化的目的DNA片段,加入1μL pUcm-T载体(通用的克隆载体,含有氨苄青霉素抗性基因等,上海生工生物技术有限公司等有售)、1μL 10×T4DNA连接酶缓冲液,加去离子水定容至10μL,最后加入1单位的的T4DNA连接酶,混匀并瞬间离心以使液滴聚集管底,置16℃水浴过夜或25℃水浴4小时后,得到连接好的重组克隆载体pUcm-T-CAHH1。
(3)细菌漆酶CAHH1基因的重组表达载体的构建
重组克隆载体pUcm-T-CAHH1转入E.coli DH5α:取10μl连接好的重组表达载体pUcm-T-CAHH1加入100μl E.coli DH5α感受态细胞【E.coli DH5α感受态细胞的制备方法参照“分子克隆实验指南”(第二板,科学出版社)49页】中,冰浴30分钟后置入42℃水浴中保温1分钟,立即取出并置冰浴中冷却2分钟。加入200μl 37℃预热的SOC液体培养基(1.0mg酵母粉,4.0mg蛋白胨,0.1mg NaCl,0.19mg MgCl2,0.037mg KCl,0.72mg葡萄糖,其余为水,培养基的pH=7.0),37℃、150rpm振摇培养60分钟后,取出100μl培养液涂布于含氨苄青霉素(Amp)的LB琼脂培养平板上,37℃培养16小时后,出现转化菌落。挑取单菌落加入2mL含有氨苄青霉素的LB培养液中,37℃,180rpm培养过夜。提取扩增的重组克隆载体pUcm-T-CAHH1,经BamH I和XhoI双酶切,琼脂糖凝胶电泳分析切割产物与理论值一致。
分别将1.0μg pUcm-T-CAHH1及pET-28a载体与适量去离子水混匀,使其总体积分别为18μL,各加入2单位限制性内切酶BamH I和XhoI及2μl相应的10×H缓冲 液,混匀,置37℃水浴保温2小时后,分别使用DNA纯化试剂盒回收约1540bp的目的DNA片段和线性化的pET28a载体,按上述pUcm-T-CAHH1相同的方法连接后转化E.coli DH5α,经BamH I和XhoI双酶切,琼脂糖凝胶电泳分析鉴定正确后获得阳性克隆pET28a-CAHH1。重组表达载体pET28a-CAHH1的构建过程如图2所示。
(4)细菌漆酶基因的序列分析
将酶切鉴定正确的重组表达载体pET28a-CAHH1进行核酸序列分析,结果如图3(pET28a-CAHH1载体测序谱图)所示,其核苷酸序列如SEQ ID NO.1所示(1542bp),起始密码子为ATG,终止密码子为TAA。其中目的基因cahh1的5’端融合了载体上的编码6×HIS标签序列(6×HIS标签的核苷酸序列见pET-28a图谱,网址为http://www.merckbiosciences.co.uk/docs/docs/PROT/TB074.pdf),它是表达产物的纯化标签。
(5)细菌漆酶CAHH1的表达与制备
重组表达载体pET28a-CAHH1可转化到E.coli BL21(DE3)系列细胞中,如E.coliBL21(DE3)、E.coli BL21(DE3)pLysS、E.coli BL21(DE3)pLysE细胞等,获得高表达的细菌漆酶CAHH1。
在本实施例中使用E.coli BL21(DE3)为宿主细胞,并使用卡那霉素筛选出能高效稳定表达的单细胞株。具体操作:将1.0μg表达载体pET28a-CAHH1加入到100μL E.coliBL21(DE3)感受态细胞中,轻轻混匀,冰浴30分钟,42℃水浴保温90秒,冰浴冷却2分钟,加入800μL 37℃预热的LB液体培养基,37℃150rpm振摇培养60分钟。取200μL培养液涂布于含卡那霉素的LB琼脂培养平板上,37℃培养16小时后,获得转化的单菌落。
挑取转化的单菌落,接种于2mL LB培养基中,37℃振摇培养过夜。以1:100(体积比)的比例接种10mL LB培养基(含30μg/mL Kan),37℃180rpm振荡培养至OD600=0.4-0.6(约2-3小时),加异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度0.2mM,加CuSO4·5H2O至终浓度0.25mM。25℃、160rpm振荡诱导12小时后,4℃、5000rpm离心5分钟收集菌体。
用1/10培养液体积的1×PBS(pH=7.8)重悬菌体,置冰浴中超声波处理破碎菌体。破碎菌体4℃,5000rpm离心15分钟,获得上清液。然后使用Ni2+-NTA亲和柱纯化得到本发明的细菌漆酶CAHH1:将上清液缓慢流过Ni2+-NTA柱,然后分别用5倍柱体积的BindingIIbuffer(20mM磷酸钠缓冲液,500mM氯化钠,20mM咪唑,pH调节至7.8)和WashingI buffer(20mM磷酸钠缓冲液,500mM氯化钠,20mM咪唑,pH调节至6.0)洗柱,最后用WashingIIbuffer(20mM磷酸钠缓冲液,500mM氯化钠,250mM咪唑,pH调节至6.0)洗脱本发明的细菌漆酶CAHH1。应用SDS-聚丙烯酰胺凝胶电泳检测 所得到的细菌漆酶CAHH1的纯度和分子量,结果如图4所示,可见只在约62kDa处有一蛋白带,说明纯化得到的细菌漆酶CAHH1的分子量约为62kDa,与理论值相符。
本发明从野生型产酶菌株枯草杆菌ZNXH1中钓取并克隆细菌漆酶CAHH1的基因,使用该基因表达出细菌漆酶CAHH1。所述的细菌漆酶CAHH1以可溶性蛋白形式表达,使用Ni2+-NTA亲和层析法纯化制备了本发明的细菌漆酶CAHH1。
实施例2:细菌漆酶CAHH1的特性
(1)最适反应pH和pH稳定性
pH值是影响酶催化活力的一个重要因素。通常酶在一定的pH值下表现出最大的催化活力,而偏离此pH值越远,酶的催化活力越低,对本发明所述细菌漆酶CAHH1的催化活力检测的最大pH范围为2.0-9.0。
以ABTS为底物,测定细菌漆酶CAHH1在不同pH值的缓冲液中(2.0-9.0)的活力,以最高酶活力为100%,计算其它pH条件下的相对酶活。以酶的相对活力对pH值作图(图5A),结果表明:细菌漆酶CAHH1催化ABTS的最适pH值为4.6。
分别将细菌漆酶CAHH1置于不同pH值(2.0-9.0)的缓冲液中孵育1h后,以ABTS为底物在pH4.6条件下测定CAHH1活力,结果如图5B所示,表明细菌漆酶CAHH1在pH值3.0-9.0范围内相对稳定,放置1h后依然维持60%以上的活性,而当pH值超出此范围时,活力下降明显。这说明细菌漆酶CAHH1在pH值3.0-9.0范围内具有较好的pH稳定性。
(2)最适反应温度和温度稳定性
温度是影响酶催化活力的另一个重要因素。通常酶在高温下的反应活力远高于低温。对本发明所述细菌漆酶CAHH1的催化活力检测的温度范围为30-90℃。
以ABTS为底物,在30-90℃的温度范围内分别测定细菌漆酶CAHH1的活力,以最高酶活力为100%,计算其它温度条件下的相对酶活。以酶的相对活力对温度作图,如图6A所示,表明细菌漆酶CAHH1的活力在约55-70℃范围内活力较好,超过或低于这个范围,活力则下降,最适温度为60℃。
分别将细菌漆酶CAHH1置于不同温度(30℃-90℃)下孵育1h后,以ABTS为底物,在60℃条件下测定CAHH1活力,结果如图6B所示,表明细菌漆酶CAHH1在40℃-70℃范围内,放置1h后依然能维持70%以上的活性。这说明细菌漆酶CAHH1具有非常好的温度稳定性。
实施例3:Co2+与Mn2+对细菌漆酶CAHH1催化活性的影响
以ABTS为底物,阳离子Co2+和Mn2+(10mM)对细菌漆酶的催化活性影响如图 7所示,以对照组酶活力为100%,计算其它条件下的相对酶活。结果证明:Co2+和Mn2+均有增强细菌漆酶CAHH1的催化活性的效果。
实施例4:细菌漆酶CAHH1催化染料脱色的效力分析
以ABTS为介体底物,分析细菌漆酶CAHH1对五种染料(40mg/L):活性蓝19(RemazolBrilliant Blue R,RBBR)、刚果红(Congo red)、靛蓝胭脂红(Indigo carmine)、甲基红(MethyI red)和结晶紫(Crystal violet)的脱色效力,脱色率通过下述公式计算:
脱色率%=(初始吸光值-催化后吸光值)/初始吸光值×100%
结果如图8所示。在以ABTS为介体底物时,细菌漆酶CAHH1能使刚果红、靛蓝胭脂红和结晶紫有效脱色,脱色率达80%以上,这显示出该漆酶在染料的脱色和处理中具有广阔的应用前景。相比之下,该酶对甲基红和活性亮蓝19等染料的脱色效力略低。
本发明所公开的内容,相信本领域技术人员可最大限度地应用本发明。因此前面的具体实施方案应被理解为仅是举例说明,而非以任何方式限制本发明的范围。
<110> 吉林大学
<120> 一种来源于枯草杆菌ZNXH1的细菌漆酶基因、细菌漆酶及其应用
<160> 2
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<212> DNA
<213> 枯草杆菌ZNXH1
<220>
<221> CDS
<222> (1)...(1542)
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atg aca ctt gaa aaa ttt gtg gat gct ctc cca atc cca gat aca cta
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu
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aag cca gta cag caa tca aaa gaa aaa aca tac tac gaa gtc acc atg
Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met
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gag gaa tgc act cat cag ctc cat cgc gat ctc cct cca acc cgc ctg
Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu
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tgg ggc tac aac ggc tta ttt ccg gga ctg acc att gag gtt aaa aga
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ttc ctt ccg att gat cac acc att cat cac agt gac agc cag cat gaa
Phe Leu Pro Ile Asp His Thr Ile His His Ser Asp Ser Gln His Glu
85 90 95
gag ccc gag gta aag act gtt gtt cat tta cac ggc ggc gtc acg cca
Glu Pro Glu Val Lys Thr Val Val His Leu His Gly Gly Val Thr Pro
100 105 110
gat gat agt gac ggg tat ccg gag gct tgg ttt tcc aaa gac ttt gaa
Asp Asp Ser Asp Gly Tyr Pro Glu Ala Trp Phe Ser Lys Asp Phe Glu
115 120 125
caa aca gga cct tat ttc aaa aga gag gtt ttt cat tat cca aac cag
Gln Thr Gly Pro Tyr Phe Lys Arg Glu Val Phe His Tyr Pro Asn Gln
130 135 140
cag cgc ggg gct ata ttg tgg tat cac gat cat gcc cat acg ctc acc
Gln Arg Gly Ala Ile Leu Trp Tyr His Asp His Ala His Thr Leu Thr
145 150 155 160
agg cta aat gtc tat gcc gga ctt gtc ggt gca tat atc att cat gac
Arg Leu Asn Val Tyr Ala Gly Leu Val Gly Ala Tyr Ile Ile His Asp
165 170 175
cca aag gaa aaa cgc tta aaa ctg cct tca gac gaa tac gat gtg ccg
Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp Glu Tyr Asp Val Pro
180 185 190
ctt ctt att aca gac cgc acg acc aat gag gat ggt tct ttg ttt tat
Leu Leu Ile Thr Asp Arg Thr Thr Asn Glu Asp Gly Ser Leu Phe Tyr
195 200 205
ccg agc gca ccg gaa aac cct tct ccg tca ctg cct aat cct tca atc
Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile
210 215 220
gtt ccg gct ttt tgc gga gaa acc ata ctc gtc aac ggg aag gta tgg
Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp
225 230 235 240
cca tac ttg gaa gtc gag cca agg aaa tac cga ttc cgt gtc atc aac
Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile Asn
245 250 255
gcc tcc aat aca aga acc tat aac ctg tca ctc gat aat ggc gga gat
Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly Asp
260 265 270
ttt att cag att ggt tca gat gga ggg ctc ctg ccg cga tct gtt aaa
Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
ctg aat tct ttc agc ctt gcg cct gct gaa cgt tac gat atc atc att
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile
290 295 300
gac ttc aca gca tat gaa gga gaa tcg atc att ttg gca aac agc gcg
Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Ala
305 310 315 320
ggc tgc ggc ggt gac gtc aat cct gaa aca gat gcg aat atc atg caa
Gly Cys Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln
325 330 335
ttc aga gtc aca aaa cca ttg gca caa aaa gac gaa agc aga aag ccg
Phe Arg Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro
340 345 350
aag tac ctc gcc tca tac cct tcg gta cag cat gaa aga ata caa aac
Lys Tyr Leu Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn
355 360 365
atc aga acg tta aaa ctg gca ggc acc cag gac gaa tac ggc aga ccc
Ile Arg Thr Leu Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro
370 375 380
gtc ctt ctg ctt aat aac aaa cgc tgg cac gat ccc gtc aca gaa aca
Val Leu Leu Leu Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr
385 390 395 400
cca aaa gtc ggc aca act gaa ata tgg tcc att atc aac ccg aca cgc
Pro Lys Val Gly Thr Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg
405 410 415
gga aca cat ccg atc cac ctg cat cta gtc tcc ttc cgt gta tta gac
Gly Thr His Pro Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp
420 425 430
cgg cgg ccg ttt gat atc gcc cgt tat caa gaa agc ggg gaa ttg tcc
Arg Arg Pro Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser
435 440 445
tat acc ggt ccg gcc gtc ccg ccg ccg cca agt gaa aag ggc tgg aaa
Tyr Thr Gly Pro Ala Val Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys
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Asp Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr
465 470 475 480
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Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu Glu
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aaa taa
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<213> 枯草杆菌ZNXH1
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Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu
1 5 10 15
Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met
20 25 30
Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu
35 40 45
Trp Gly Tyr Asn Gly Leu Phe Pro Gly Leu Thr Ile Glu Val Lys Arg
50 55 60
Asn Glu Asn Val Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His
65 70 75 80
Phe Leu Pro Ile Asp His Thr Ile His His Ser Asp Ser Gln His Glu
85 90 95
Glu Pro Glu Val Lys Thr Val Val His Leu His Gly Gly Val Thr Pro
100 105 110
Asp Asp Ser Asp Gly Tyr Pro Glu Ala Trp Phe Ser Lys Asp Phe Glu
115 120 125
Gln Thr Gly Pro Tyr Phe Lys Arg Glu Val Phe His Tyr Pro Asn Gln
130 135 140
Gln Arg Gly Ala Ile Leu Trp Tyr His Asp His Ala His Thr Leu Thr
145 150 155 160
Arg Leu Asn Val Tyr Ala Gly Leu Val Gly Ala Tyr Ile Ile His Asp
165 170 175
Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp Glu Tyr Asp Val Pro
180 185 190
Leu Leu Ile Thr Asp Arg Thr Thr Asn Glu Asp Gly Ser Leu Phe Tyr
195 200 205
Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile
210 215 220
Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp
225 230 235 240
Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile Asn
245 250 255
Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly Asp
260 265 270
Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile
290 295 300
Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Ala
305 310 315 320
Gly Cys Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln
325 330 335
Phe Arg Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro
340 345 350
Lys Tyr Leu Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn
355 360 365
Ile Arg Thr Leu Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro
370 375 380
Val Leu Leu Leu Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr
385 390 395 400
Pro Lys Val Gly Thr Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg
405 410 415
Gly Thr His Pro Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp
420 425 430
Arg Arg Pro Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser
435 440 445
Tyr Thr Gly Pro Ala Val Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys
450 455 460
Asp Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr
465 470 475 480
Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu Glu
485 490 495
His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile Thr Asp Pro His
500 505 510
Lys ***
Claims (6)
1.一种来源于枯草杆菌ZNXH1的细菌漆酶基因cahh1,其特征在于:其核苷酸序列如SEQID NO.1所示。
2.一种来源于枯草杆菌ZNXH1的细菌漆酶CAHH1,其特征在于:其氨基酸序列如SEQ IDNO.2所示。
3.如权利要求2所述的一种来源于枯草杆菌ZNXH1的细菌漆酶CAHH1,其特征在于:是由权利要求1所述的来源于枯草杆菌ZNXH1的细菌漆酶基因cahh1与表达载体重组,将重组后的表达载体导入原核表达细胞内得到。
4.如权利要求3所述的一种来源于枯草杆菌ZNXH1的细菌漆酶CAHH1,其特征在于:表达载体为pET-28a、pET-41b或pPZW103。
5.如权利要求3所述的一种来源于枯草杆菌ZNXH1的细菌漆酶CAHH1,其特征在于:原核表达细胞为大肠杆菌或枯草杆菌。
6.权利要求2~5任何一项所述的一种来源于枯草杆菌ZNXH1的细菌漆酶CAHH1在靛蓝类染料、偶氮类染料或三苯甲烷类染料的降解或脱色中的应用。
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