CN114410669B - 重组腈水解酶的生产及固定化方法及其对乙腈的降解应用 - Google Patents
重组腈水解酶的生产及固定化方法及其对乙腈的降解应用 Download PDFInfo
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Abstract
本发明公开了一种重组腈水解酶的生产及固定化方法及其对乙腈的降解应用,涉及环境生物技术领域,该酶水解酶融合多肽为高温腈水解酶突变后,与纤维素结合域共整合后生成的新型工业酶,可以特异结合到木浆纤维素,实现酶的固定化及高效特异的污染物降解。本发明公布了表达该多肽的重组表达系统,包括重组载体、表达宿主、以及纤维素结合域以及连接肽段的序列,以及用于重组多肽表达的分泌肽序列。枯草芽孢杆菌重组表达的腈水解酶融合蛋白经一步法固定到木浆纤维素,提高酶催化的稳定性,实现对流动污水中乙腈的生物去除。
Description
技术领域
本发明属于环境生物技术领域,具体涉及一种重组腈水解酶的生产及固定及其对乙腈的降解应用。
背景技术
乙腈是丙烯腈生产的副产物,丙烯腈是一种重要的化工原料,它是合成人工羊毛、丁基橡胶及合成树脂的化学单体,还可以生产丙烯酰胺和丙烯酸等化学品,每年我国的丙烯腈的原料生产量巨大。乙腈自身也是一种的工业原料,可以用于生产有机腈类农药、维生素B1等化合物,也是丁二烯及芳烃等化工产品的抽提溶剂,近年来,乙腈也被大量用于甲硝羟乙唑等医药产品的合成原料或溶剂。随着乙腈在现代工业中的大量应用,一些工业园区的水体环境中可检测到超标的乙腈污染。
常温常压下乙腈是一种带甜味的有机液体,与水的互溶性非常好,也是很多有机物的溶剂,因此是有机物分析色谱中最常用的溶剂。但其具有一定的生物毒性,尤其是在生命体中,可由细胞内的酶降解产生无机氰离子,带来更大的毒性,因此污染水体中乙腈的排放控制十分严格,一般在5.0毫克每升以下。乙腈化学性能较为活泼,但是非特异性的降解或转化通常不能解除这类有机腈化合物带来的生物毒性。高浓度的乙腈污染可以通过吸附、臭氧氧化或加入高浓度碱液进行处理,但是在污染物浓度为克每升以下时,这类物理化学处理方法不仅使用成本高,而且还会带来新的污染物,例如,臭氧处理后会生成氰酸盐,其毒性下降,但并没有彻底降解为无害物。
许多微生物,如假单胞菌、玫瑰红球菌等均可在水体中对乙腈进行降解利用,这些微生物作为降解低浓度乙腈等有机腈化物的高效生物降解系统虽已被研究多年,但受活体微生物个体在污水系统中的存活率影响,该生物催化方法的持久性及其效率依然无法满足实际污染处理应用的需求。腈水解酶可见于自然界的各种生命体中,如真菌、细菌及植物细胞中,腈水解酶作为一种重要的工业酶,可以降解无机氰化物,也能将将有毒腈化物催化为有机酸和氨,因此,腈水解酶在氨基酸、维生素等化工产品或医药中间体的生物合成中具有重要的用途。但是为了更好的工业应用,腈水解的酶学性能往往需要进行工程改造或诱变训化。此外,由于游离酶对底物的敏感性,针对腈水解酶进行固定化,也是提高该酶催化稳定性,延长酶的使用周期,降解使用成本的关键技术之一。
酶的固定化技术按照固定方式分吸附、包埋、交联、耦合等方式,利用酶对天然材料的吸附是其中固定成本最低的方式之一。纤维素酶往往带有一段可以识别并结合纤维素的结构域,该多肽可以帮助其它蛋白对纤维素进行结合。在纤维素结合域与催化蛋白之间往往需要设计连接肽,使纤维素结合与酶的催化功能不受彼此干扰,由于酶的催化性能及特异性的不同,连接肽的序列及长度均需要进行优化及实验验证。
发明内容
为了能够利用腈水解酶,实现对污染水体中乙腈等有机腈污染物的生物处理,本发明公开了一种编码腈水解酶的融合蛋白的DNA及其多肽序列,以及用于重组生产该重组酶的实验方法;本发明还公开了一种基于纤维素特异性结合的生物固定方法,并提供了利用该纤维素固定系统开展乙腈降解的应用方法,该重组酶及其固定方法的应用可以极大地提升腈水解酶对水体中乙腈污染物的降解效率。
为了实现上述目的以及其它相关目的,本发明采用如下技术方案:
本发明的第一方面,公布一种编码腈水解酶重组蛋白Cbd-Nit20的核苷酸序列,所述编码腈水解酶重组蛋白Cbd-Nit20的核苷酸序列为SEQ ID NO.1所示,其编码的氨基酸序列为SEQ ID NO.2所示。所述的编码腈水解酶重组蛋白Cbd-Nit20的核苷酸的用途是将SEQID NO.1所示的核苷酸与其它DNA元件经基因合成后构成如SEQ ID NO.3所示的完整表达框,构建至重组表达质粒,并转入枯草芽孢杆菌宿主细胞,用于重组腈水解酶的分泌表达。所述其它DNA元件序列编码的多肽序列为三段不同功能肽的结合,第一段为蛋白分泌的信号肽,第二段为识别并结合纤维素的结合域,第三段为连接腈水解酶催化结构域与纤维素结合域的连接肽,所述的多肽序列如SEQ ID NO. 5所示。Cbd-Nit20可以结合多种纤维素材料,包括不溶于水的木浆纤维素多孔材料。
本发明的第二方面提供了所述腈水解酶重组蛋白Cbd-Nit20在枯草芽孢杆菌菌株168中开展重组表达及分泌生产的方法。
具体的,芽孢杆菌属枯草芽孢杆菌菌种Bacillu subtilis 168被用作微生物宿主细胞进行Cbd-Nit20的分泌表达和生产,包括步骤:将重组表达质粒pMK-CbN20转入枯草芽孢杆菌菌种Bacillu subtilis 168,将其作为表达分泌蛋白的宿主细胞,开展重组蛋白的诱导表达,收集发酵培养上清,获取上清中游离的腈水解酶重组蛋白。
进一步地,将重组表达质粒pMK-CbN20转化至枯草芽孢杆菌菌种Bacillu subtilis 168的感受态细胞中,筛选并获得重组枯草芽孢杆菌菌株,该重组枯草芽孢杆菌用于表达腈水解酶重组蛋白Cbd-Nit20。
进一步地,将含有表达质粒pMK-CbN20的枯草芽孢杆菌菌种Bacillu subtilis 168质粒进行摇瓶或发酵罐培养,在诱导表达结束时,分离细胞,收集发酵上清,获得游离的腈水解酶重组蛋白。
进一步地,将含有重组腈水解酶的发酵上清流经装有木浆纤维素多孔材料的制备柱,将腈水解酶重组蛋白Cbd-Nit20固定到制备柱中。
进一步地,利用固定了腈水解酶重组蛋白Cbd-Nit20的制备柱,对污染水中乙腈进行污染物降解处理。
所述重组表达质粒pMK-CbN20的完整序列如SEQ ID NO.4所示。
本发明的第三方面提供了一种利用木浆纤维素多孔材料对腈水解酶重组蛋白Cbd-Nit20进行吸附固定的方法,腈水解酶重组蛋白的水溶液流经多孔木浆纤维素柱,利用纤维素结合域与纤维素进行自主亲合结合,特异性高,可以有效提高酶的稳定性和催化效率。
所述的腈水解酶重组蛋白为表达于枯草芽孢杆菌Bacillu subtilis 168的腈水解酶,所述的酶固定利用了重组蛋白与纤维素多孔材料间的特异亲合吸附力。
进一步地,所述方法包括:测定重组表达的腈水解酶的浓度后,以每1000毫升柱体积的木浆纤维素多孔材料吸附50毫克的腈水解酶的比例进行重组腈水解酶Cbd-Nit20的亲合柱吸附,在常温下完成酶-纤维素的固定化。
进一步地,所述方法包括:将结合了50毫克重组腈水解酶Cbd-Nit20的多孔木浆纤维素柱固定并连通含有乙腈的水体中,进行生物吸附后酶稳定性的测定以及乙腈降解的应用。
进一步地,所述的酶稳定性测定溶液的反应温度为25℃-60℃。
进一步地,所述的乙腈的浓度为50 mg/L-1000 mg/L。
与现有技术相比,本发明具有如下有益效果:
本发明利用枯草芽孢杆菌和表达质粒进行一种重组腈水解酶生产菌的构建,并通过发酵培养获得分泌至上清的重组酶,该酶融合了纤维素结合域,可以高效地与木浆纤维素多孔材料结合,从而将酶固定到天然生物聚合材料中,不仅提升了酶的催化稳定性,而且延长了酶的使用周期,降低了酶的使用成本,对污染水体中乙腈处理后可以将其转化为无害的乙酸根,实现绿色高效降解。
本发明中所用的枯草芽孢杆菌菌种Bacillu subtilis 168的来源信息如下:
菌株名称:Bacillu subtilis 168
原始来源: ATCC ® Catalog No. 23857™。
附图说明
图1为质粒pMK-CbN20的结构示意图。
图2为融合蛋白腈水解酶Cbd-Nit20分泌表达后的聚丙烯酰胺凝胶电泳结果。
图3重组腈水解酶的固定化流程示意图。
图4为结合了腈水解酶的木浆纤维素柱对乙腈的降解结果。
具体实施方式
本发明具体实施方式有以下理解,即,本发明的保护范围不局限于下列所述特定的具体实施方案;本发明实施例中所用术语是为了描述特定的具体实施方案,而非为了限制本发明的保护范围;下列所述实施例中未注明的其它试验方法或实验条件,按照各制造商所建议的实施方法。
实施例中给出相应的数值范围时,应理解为,除非另有所述的说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。进一步,除非另有定义,本发明中使用的相应的技术和科学术语与本技术领域技术人员通常理解的意义相同。此外,除了实施例中公开的具体方法、设备、材料及使用条件外,根据本技术领域的技术人员对相应技术的掌握及本发明公开的记载,还可以使用与以下发明实施例中所述的方法、设备、材料及使用条件类同或等同的涉及本技术的其它方法、设备和材料来实现本发明。
除非另有说明,本发明中所公开的实验方法、条件、检测方法、制备方法均采用本技术领域常规的分子生物学、微生物学、生物化学、分析化学、蛋白质研究、发酵培养、重组表达等技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等MOLECULAR CLONING :A LABORATORY MANUAL,Second edition,Cold SpringHarbor Laboratory Press,1989 and Third edition,2001 ;METHODS IN ENZYMO- LOGY,Vol.304,Chromatin (P.M.Wassarman and A.P.Wolffe,eds.),Academic Press,SanDiego,1999;Ausubel 等,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley &Sons,New York,1987 and periodic updates ;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego ;Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Thirdedition,Academic Press,San Diego,1998 ;METHODS IN MOLECULAR BIOLOGY,Vol.119,Chromatin Protocols (P.B.Becker,ed.) Humana Press,Totowa,1999等。
实施例1 重组腈水解酶的合成及克隆
SEQ ID NO.1所示的核苷酸序列编码完成的腈水解酶融合蛋白,其氨基端含有来自木霉纤维素酶的纤维素结合域(CBD),其编码序列与随后的连接肽(linker)由SEQ IDNO.5所示,腈水解酶的羧基端为来自古细菌的同源蛋白,其编码序列由SEQ ID NO.2所示。腈水解酶融合蛋白还包括来自枯草芽孢杆菌分泌肽。编码SEQ ID NO.5氨基酸的核苷酸序列与腈水解酶的多肽序列经密码子优化后,通过化学合成法制备。利用分子生物学技术扩增获取PCR产物并连接到Thermo Fisher pET101 Topo质粒系统中,然后转化到大肠杆菌DH5α中,得到具有氨苄抗性的阳性克隆;挑取6个大肠杆菌单克隆,利用3毫升过夜培养的大肠杆菌细胞进行质粒提取。提取的质粒再以EcoRI 和BamHI进行DNA酶切,37℃酶切约3小时,然后65℃灭活 20 min,再加入同样处理的模板质粒,以T4 DNA 连接酶,常温处理1小时,将连接液转化到枯草芽孢杆菌168中,涂板,过夜生长,得到具有氯霉素抗性的阳性克隆。
将枯草芽孢杆菌阳性克隆接种到LB液体培养基(每升含有10 克NaCl,10 克胰蛋白胨,5克酵母提取物)中,细胞密度在3.0左右时,收集并破碎细胞,提取表达质粒pMK-CbN20,进行DNA测序验证,其序列如SEQ ID NO.4所示,质粒图如图1所示。
实施例2 融合腈水解酶的表达及其酶活测定
挑取3个得到验证的枯草芽孢杆菌转化株,分别接种到LB液体培养基(每升含有10克NaCl,10 克胰蛋白胨,5克酵母提取物)中,细胞密度在1.5左右时,加入10 g/L的木糖作为诱导物,继续培养12小时,37℃,取20 μL培养上清进行腈水解酶性能的鉴定。另外取20 μL培养上清用于聚丙烯酰胺凝胶电泳。蛋白电泳结果如图2所示,表达上清中蛋白成分单一,基本只有一条分子量对应为腈水解酶融合蛋白的表达产物。
酶活测定过程具体如下:10 μL上清置于 30-70℃水浴摇床 2 min,活化重组酶,加入40 μL 500 mM的3-氰基吡啶,恒温反应5-10分钟;迅速取10 μL 反应液上清至装有1.0mL蒸馏水的10 mL反应管,再依次加入1.5 mL 亚硝基铁氰化钠溶液、1.0 mL苯酚钠溶液,以及1.5 mL 次氯酸钠溶液,再继续于原先设定的反应温度反应20分钟,然后吸取 200 μL反应液到96孔微孔板中,再利用酶标仪测定OD630。酶活定义为每分钟生成1 μmol 烟酸或氨所需要的酶量。由图3可看出,融合腈水解酶具有高温酶的特征,其最佳催化温度为60℃,发酵上清的最高酶活达到137 U/mL。
实施例3 融合腈水解酶的固定化及对乙腈降解的应用
挑取单克隆的枯草芽孢杆菌转化子168/ pMK-CbN20,进行摇瓶培养,将其接种到装有50 ml LB,含有5 μg/mL氯霉素的培养管,37 ℃,200 r/min 摇床培养16小时左右,将1-3%的过夜培养菌液转接到装有2.5 L液体LB培养基的5L发酵罐中,在培养基中另加10 g/L的木糖、5 μg/mL氯霉素,保持好氧培养,通2-10 L/min空气,转速自动控制在100-700rpm,溶氧值保持在40%或以下,持续培养 72 h,pH维持在6.8附近,培养后期补加10 g/L(NH4)2PO4以及 20 g/L葡萄糖。发酵后离心去除细胞,收集上清中重组酶,测定其蛋白浓度,并进行酶的生物固定,如图3所示。酶的固定流程如下:将装有200 mL的多根木浆纤维素多孔材料层析柱中,以50 mM,1% NaCl的Tris-HCl缓冲液,2倍柱体积平衡流洗;再将25 mL,含有约10 mg的重组融合蛋白的上清液以1:10的比例与50 mM,1% NaCl的Tris-HCl缓冲液混合后流经多孔木浆纤维素柱,再以4倍柱体积平衡流洗50 mM,1% NaCl的Tris-HCl缓冲液,封闭层析柱两端,4℃保存待用。
将所有层析柱放入37℃恒温房,固定后,分别以蠕动泵接入600 mL含有乙腈的污染水,浓度分别为50、100、200、400、600、800及1000 mg/L,流速设定为20 ml/min,持续运转3小时,30分钟测定一次乙腈浓度,结果如图4所示。固定后的重组腈水解酶能够高效地降解水体中的乙腈,浓度为600 mg/L以下时,在2小时内可得到完全降解,浓度为1000 mg/L时,3小时后其降解率也达到90%以上。因此,利用纤维素腈水解酶固定系统可实现乙腈污染物的高效快速降解。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员,在不脱离本发明的精神和范围的情况下,当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化,均为本发明的等效实施例;同时,凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变,均仍属于本发明的技术方案的范围内。
序列表
<110> 佛山市玉凰生态环境科技有限公司
<120> 重组腈水解酶的生产及固定化方法及其对乙腈的降解应用
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gtgtccgggc atacatgtgt ttcaattaat cagtggtata gccagtgtca gccggctgga 180
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aaggtccgcg ctgctaaaga aggggcaaag ttggtggtgc ttccagaact gtttgacacc 360
ggatataact ttgaatctgg cgatgaagtg tatgctatcg cccaaccgat cccaaatgga 420
aaaactaccg attttctgat ggaattagca gaagagttgg acgtctttat cgtagcaggt 480
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gaaattatcg cgcacccgag caatctcgtt atgccgtacg ctccgcgagc aatgccgatt 780
cgggccctcg aaaaccgggt ttacacggtc acagcaaatc gtgtcggcga agagcgccaa 840
cgtaaacagg gtgaagcact tacctttatc ggccagagtc aaataacaag ccctaaagca 900
gagatcttat tacgggcagg aaatgacgag gagattctta tgacagcaga cattgacctt 960
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Ile Asn Gln Trp Tyr Ser Gln Cys Gln Pro Ala Gly Ala Pro Ser Asn
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Asp Asn Leu Met Lys Val Arg Ala Ala Lys Glu Gly Ala Lys Leu Val
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His Val Phe Asn Ile Gly Ile Ala Lys Val Gly Val Met Ile Cys Phe
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Asp Trp Phe Phe Pro Glu Ser Ala Arg Thr Leu Ala Leu Lys Gly Ala
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Glu Ile Ile Ala His Pro Ser Asn Leu Val Met Pro Tyr Ala Pro Arg
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Asp Arg Arg Pro Lys Tyr Tyr Phe Val
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tcacgccgaa aagtgcagtg tgtccgggca tacatgtgtt tcaattaatc agtggtatag 480
ccagtgtcag ccggctggag ccccgtctaa taatgcatca aacaacaaca acggcggcgg 540
aggctctgga ggcggagggt caaaggcaag cttctttcaa tttaatccgg tatttgggcg 600
caagtcagac aacttgatga aggtccgcgc tgctaaagaa ggggcaaagt tggtggtgct 660
tccagaactg tttgacaccg gatataactt tgaatctggc gatgaagtgt atgctatcgc 720
ccaaccgatc ccaaatggaa aaactaccga ttttctgatg gaattagcag aagagttgga 780
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ggttatcgtg ggacctattg gttggggata tattggcaaa tacagaaagg tccacttgtt 900
ttatcgagaa aaacttttct ttgaaccggg gaatctgggc ttccatgtat ttaacattgg 960
catcgcaaaa gtaggtgtaa tgatatgttt tgattggttc tttccggaaa gtgctcgcac 1020
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gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
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ctgcaggtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac 300
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cggcggaggc tctggaggcg gagggtcaaa ggcaagcttc tttcaattta atccggtatt 840
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ggtgcttcca gaactgtttg acaccggata taactttgaa tctggcgatg aagtgtatgc 960
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gttggacgtc tttatcgtag caggtacggc cgaaaaagat gaaaaaggga gattgtacaa 1080
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cttgttttat cgagaaaaac ttttctttga accggggaat ctgggcttcc atgtatttaa 1200
cattggcatc gcaaaagtag gtgtaatgat atgttttgat tggttctttc cggaaagtgc 1260
tcgcactttg gcccttaaag gcgccgaaat tatcgcgcac ccgagcaatc tcgttatgcc 1320
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gagtcaaata acaagcccta aagcagagat cttattacgg gcaggaaatg acgaggagat 1500
tcttatgaca gcagacattg acctttccct ttcacgcgac aaaaagatta atgattataa 1560
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gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 1680
ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatca 1740
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cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 1860
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gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 1980
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tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 2100
ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 2160
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ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 2400
gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 2460
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acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 2640
caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 2700
taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 2760
ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 2820
aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 2880
agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 2940
atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 3000
tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 3060
tgaagatcca tatccttctt tttctgaacc gacttctcct ttttcgcttc tttattccaa 3120
ttgctttatt gacgttgagc ctcggaaccc ttaacaatcc caaaacttgt cgaatggtcg 3180
gcttaatagc tcacgctatg ccgacattcg tctgcaagtt tagttaaggg ttcttctcaa 3240
cgcacaataa attttctcgg cataaatgcg tggtctaatt tttattttta ataaccttga 3300
tagcaaaaaa tgccattcca atacaaaacc acatacctat aatcgataac cacataacag 3360
tcataaaacc actccttttt aacaaacttt atcacaagaa atatttaaat tttaaatgcc 3420
tttattttga attttaaggg gcattttaaa gatttagggg taaatcatat agttttatgc 3480
ctaaaaacct acagaagctt ttaaaaagca aatatgagcc aaataaatat attctaattc 3540
tacaaacaaa aatttgagca aattcagtgt cgatttttta agacactgcc cagttacatg 3600
caaattaaaa ttttcatgat tttttatagt tcctaacagg gttaaaattt gtataacgaa 3660
agtataatgt ttatataacg ttagtataat aaagcatttt aacattatac ttttgataat 3720
cgtttatcgt cgtcatcaca ataactttta aaatactcgt gcataattca acagctgacc 3780
tcccaataac tacatggtgt tatcgggagg tcagctgtta gcacttatat tttgttattg 3840
ttcttcctcg atttcgtcta tcattttgtg attaatttct cttttttctt gttctgttaa 3900
gtcataaagt tcactagcta aatactcttt ttgtttccaa atataaaaaa tttgatagat 3960
atattcggtt ggatcaattt cttttaagta atctaaatcc ccatttttta atttcttttt 4020
agcctcttta aataatcctg aataaactaa tacctgttta cctttaagtg atttataaaa 4080
tgcatcaaag actttttgat ttattaaata atcactatct ttaccagaat acttagccat 4140
ttcatataat tctttattat tattttgtct tattttttga acttgaactt gtgttatttc 4200
tgaaatgccc gttacatcac gccataaatc taaccattct tgttggctaa tataatatct 4260
tttatctgtg aaatacgatt tatttactgc aattaacaca tgaaaatgag gattataatc 4320
atctcttttt ttattatatg taatctctaa cttacgaaca tatcccttta taacactacc 4380
tacttttttt ctctttataa gttttctaaa agaattatta taacgtttta tttcattttc 4440
taattcatca ctcattacat taggtgtagt caaagttaaa aagataaact cctttttctc 4500
ttgctgctta atatattgca tcatcaaaga taaacccaat gcatcttttc tagcttttct 4560
ccaagcacag acaggacaaa atcgattttt acaagaatta gctttatata atttctgttt 4620
ttctaaagtt ttatcagcta caaaagacag aaatgtattg caatcttcaa ctaaatccat 4680
ttgattctct ccaatatgac gtttaataaa tttctgaaat acttgatttc tttgtttttt 4740
ctcagtatac ttttccatgt tataacacat aaaaacaact tagttttcac aaactatgac 4800
aataaaaaaa gttgcttttt cccctttcta tgtatgtttt ttactagtca tttaaaacga 4860
tacattaata ggtacgaaaa agcaactttt tttgcgctta aaaccagtca taccaataac 4920
ttaagggtaa ctagcctcgc cggcaatagt tacccttatt atcaagataa gaaagaaaag 4980
gatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa gaccacattt tttaatgtgg 5040
tcttttattc ttcaactaaa gcacccatta gttcaacaaa cgaaaattgg ataaagtggg 5100
atatttttaa aatatatatt tatgttacag taatattgac ttttaaaaaa ggattgattc 5160
taatgaagaa agcagacaag taagcctcct aaattcactt tagataaaaa tttaggaggc 5220
atatcaaatg aactttaata aaattgattt agacaattgg aagagaaaag agatatttaa 5280
tcattatttg aaccaacaaa cgacttttag tataaccaca gaaattgata ttagtgtttt 5340
ataccgaaac ataaaacaag aaggatataa attttaccct gcatttattt tcttagtgac 5400
aagggtgata aactcaaata cagcttttag aactggttac aatagcgacg gagagttagg 5460
ttattgggat aagttagagc cactttatac aatttttgat ggtgtatcta aaacattctc 5520
tggtatttgg actcctgtaa agaatgactt caaagagttt tatgatttat acctttctga 5580
tgtagagaaa tataatggtt cggggaaatt gtttcccaaa acacctatac ctgaaaatgc 5640
tttttctctt tctattattc catggacttc atttactggg tttaacttaa atatcaataa 5700
taatagtaat taccttctac ccattattac agcaggaaaa ttcattaata aaggtaattc 5760
aatatattta ccgctatctt tacaggtaca tcattctgtt tgtgatggtt atcatgcagg 5820
attgtttatg aactctattc aggaattgtc agataggcct aatgactggc ttttataata 5880
tgagataatg ccgactgtac tttttacagt cggttttcta atgtcactaa cctgccccgt 5940
tagttgaaga aggtttttat attacagctc cagatctagg tgaagatcct ttttgataat 6000
ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 6060
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 6120
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 6180
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 6240
tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 6300
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 6360
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 6420
agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 6480
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 6540
ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 6600
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 6660
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 6720
cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 6780
tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 6840
gcggaaga 6848
<210> 5
<211> 82
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Lys Asn Leu Ser Leu Ala Cys Val Leu Ser Leu Gly Leu Ala Gly Leu
1 5 10 15
Ala Asn Gly Ala Cys Gly Gly Ala Trp Ala Gln Cys Ala Gly Glu Asn
20 25 30
Phe His Ala Glu Lys Cys Ser Val Ser Gly His Thr Cys Val Ser Ile
35 40 45
Asn Gln Trp Tyr Ser Gln Cys Gln Pro Ala Gly Ala Pro Ser Asn Asn
50 55 60
Ala Ser Asn Asn Asn Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
Lys Ala
Claims (8)
1.一种编码腈水解酶重组蛋白Cbd-Nit20的核苷酸,其特征在于,所述编码腈水解酶重组蛋白Cbd-Nit20的核苷酸序列如SEQ ID NO.1所示。
2.腈水解酶重组蛋白Cbd-Nit20,其特征在于,所述腈水解酶重组蛋白Cbd-Nit20的氨基酸序列如SEQ ID NO.2所示。
3.权利要求1所述的编码腈水解酶重组蛋白Cbd-Nit20的核苷酸的用途,其特征在于,所述用途是将SEQ ID NO.1所示的核苷酸与其它DNA元件经基因合成后构成如SEQ ID NO.3所示的完整表达框,构建至重组表达质粒,并转入枯草芽孢杆菌宿主细胞,用于重组腈水解酶的分泌表达。
4.如权利要求3所述的用途,其特征在于,所述重组表达质粒为pMK-CbN20,pMK-CbN20的核苷酸序列如SEQ ID NO.4所示。
5.如权利要求3所述的用途,其特征在于,所述其它DNA元件编码的多肽序列为三段不同功能肽的结合,第一段为蛋白分泌的信号肽,第二段为识别并结合纤维素的结合域,第三段为连接腈水解酶催化结构域与纤维素结合域的连接肽,所述的多肽序列如SEQ ID NO. 5所示。
6.如权利要求4所述的用途,其特征在于,所述枯草芽孢杆菌宿主细胞为Bacillus subtilis 168。
7.如权利要求2所述的腈水解酶重组蛋白Cbd-Nit20的固定方法,其特征在于,所述方法是利用木浆纤维素多孔材料对腈水解酶重组蛋白Cbd-Nit20进行吸附固定,通过多孔木浆纤维素柱,利用腈水解酶重组蛋白Cbd-Nit20与纤维素的亲合能力进行酶的固定化。
8.如权利要求7所述的腈水解酶重组蛋白Cbd-Nit20的固定方法制备固定化后的腈水解酶重组蛋白Cbd-Nit20的用途,其特征在于,所述的固定化后的腈水解酶重组蛋白Cbd-Nit20用于对有机腈类物质的生物降解,将结合了重组腈水解酶Cbd-Nit20的多孔木浆纤维素柱固定并连通含有乙腈的水体中,对含乙腈污染水体进行降解处理。
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