CN113444713A - 一种L-赖氨酸脱羧酶SpLDC及其在产1,5-戊二胺上的应用 - Google Patents
一种L-赖氨酸脱羧酶SpLDC及其在产1,5-戊二胺上的应用 Download PDFInfo
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- CN113444713A CN113444713A CN202110722212.0A CN202110722212A CN113444713A CN 113444713 A CN113444713 A CN 113444713A CN 202110722212 A CN202110722212 A CN 202110722212A CN 113444713 A CN113444713 A CN 113444713A
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- China
- Prior art keywords
- spldc
- lysine decarboxylase
- pentanediamine
- enzyme
- lysine
- Prior art date
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Abstract
本发明公开了一种L‑赖氨酸脱羧酶SpLDC及其在产1,5‑戊二胺上的应用。L‑赖氨酸脱羧酶SpLDC的氨基酸序列如SEQ ID NO.1所示,且该L‑赖氨酸脱羧酶能够在宿主中能够良好的表达,具有高催化L‑赖氨酸合成戊二胺的活性,且pH稳定性和温度稳定性良好,经测试可知,在pH5‑8时催化能力强,其中pH6.5时催化能力最优,且在pH8条件下放置12h,酶的合成戊二胺的能力依然能保持80%以上,具有良好的pH稳定性;在30‑52℃之间具有较高的催化活性,52℃下酶的最适催化能力最优;在52℃孵育12h酶催化活性依然保持在80%以上,具有良好的温度稳定性。
Description
技术领域
本发明涉及1,5-戊二胺的制备技术领域,具体涉及来一种L-赖氨酸脱羧酶SpLDC及其在产1,5-戊二胺上的应用。
背景技术
1,5-戊二胺(又名尸胺;1,5-二氨基戊烷;五亚甲基二胺;尸毒素),其广泛存在于原核生物及真核生物中,具有多种生物活性,尤其在植物和微生物的生理活动具有重要的调控作用。在生物体内戊二胺的产生主要是以赖氨酸为前体,在赖氨酸脱羧酶的脱羧作用下生成,在过程中会释放二氧化碳。
目前,戊二胺作为产品在农业,医学,化工,等领域得到广泛的应用,并且以其为原料生产的产品已经应用到我们生活的方方面面。在农业上,1,5-戊二胺可用于调控植物衰老过程、促进花蕊的发育、改善植物果实、提高果实产量。在医学上,戊二胺可以作为一种有效治疗痢疾的药物,并且其环化形成的化合物可以作为一种医药中间体合成多种药物;另外最新报道,戊二胺对肿瘤有一定抑制作用。在工业上,1,5-戊二胺是一种重要的化工原料。1,5-戊二胺与二元酸进行聚合反应合成特别的高分子材料聚酰胺,又称尼龙;其还可以聚合形成聚氨酯材料及合成异氰酸酯应用于涂料行业。目前尼龙材料多种多样,以己二酸和己二胺聚合而成的尼龙6,6,生产量为最大。而1,6-己二胺主要来源于不可再生资源石油。奇数碳的二元胺戊二胺和己二酸聚合而成尼龙56相对于尼龙66具有更好的物理性能。随着生物技术的发展,1,5-戊二胺可以以可再生资源生物质为原料进行生产。
1,5-戊二胺生产方法主要有微生物发酵法和全细胞催化法。微生物发酵法是以高产赖氨酸的大肠杆菌和谷氨酸棒状杆菌为菌种利用基因工程技术在细胞内表达赖氨酸脱羧酶基因直接合成戊二胺,并对戊二胺分解途径进行敲除;强化代谢上游基因来使得转化率最大化。原料多种多样,例如:葡萄糖,木糖,纤维素,半纤维素,甲醇等生物质资源。目前还在进行多种废弃生物质资源研究使得利益最大化,环境污染最小化。L-赖氨酸产能过剩促使全细胞催化法应用而生;这种方法是以L-赖氨酸盐酸盐为原料,以过表达赖氨酸脱羧酶的大肠杆菌作为催化剂催化脱羧生产戊二胺。这种方法相对发酵法工艺更为简单,调控步骤少。而且1,5-戊二胺的生产浓度更高,更有利于戊二胺下游的分离。
L-赖氨酸脱羧酶是一种5-磷酸吡哆醛(plp)依赖性酶,大多数生物体内都含有该酶基因序列。在部分微生物体内,该酶还分为组成型和诱导性两种。目前,已经过表达于并且应用于戊二胺生产的赖氨酸脱羧酶有多种微生物来源,例如大肠杆菌(E.coli)、蜂房哈夫尼菌(Hafniaalvei)、反刍动物月形单胞菌(Selenomonasruminantium)、肺炎克雷伯氏菌、产酸克雷伯氏菌等。在目前研究的赖氨酸脱羧酶中,大肠杆菌赖氨酸脱羧酶Cada和LdcC最为广泛,但是,大肠杆菌赖氨酸脱羧酶Cada在催化合成戊二胺过程中pH升到7.5时,酶活急剧下降,在催化过程中不稳定。大肠杆菌LdcC虽然最适催化pH在7.5,但是酶活较差;不适合工业化生产催化合成戊二胺。当然,目前有学者在基因文库中挖掘出相对活性较高的耐碱性pH较好的赖氨酸脱羧酶,物种的多样性是基因的多样性所决定。丰富的微生物资源使我们能够获得更为不同的赖氨酸脱羧酶,来满足目前生产中所遇到的困难。
发明内容
针对现有技术的不足,本发明提供了一种L-赖氨酸脱羧酶SpLDC及其在产1,5-戊二胺上的应用,所述L-赖氨酸脱羧酶可在pH 5-8条件下具有高的催化活性,具备优异的pH稳定性和温度稳定性,且在戊二胺生产过程中可以获得高浓度的1,5-戊二胺,通过对其测试其优异的性能适合戊二胺的产业化生产。
为解决现有技术问题,本发明采取的技术方案:
一种L-赖氨酸脱羧酶SpLDC,所述L-赖氨酸脱羧酶SpLDC的氨基酸序列如SEQ IDNO.1所示。
SEQIDNo.1:
MNVIAIMNHMGVYFKEEPIRELHNALEKLNFRIVYPNDREDLLKLIENNARLCGVIFDWDKYNLELCQEISELNEYMPLYAFANTYSTLDVSLNDLRMQVRFFEYALGAANDIADKIKQNTDEYVDTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNAMKSDVSISVSELGSLLDHSGPHKEAEEYIARVFNAERSYMVTNGTSTANKIVGMYSAPAGNTVLIDRNCHKSLTHLMMMSDITPIYFRPTRNAYGILGGIPQSEFSRATIAKRVKDTPNATWPVHAVITNSTYDGLLYNTDFIKSTLDVKSIHFDSAWVPYTNFHPIYKGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDINEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLNVESEGWFFDVWQPEHIDEAECWPLRSDSAWHGFKGIDNEHMYLDPIKVTMLTPGMSKDGEMESFGIPASLVAKYLDEHGIIVEKTGPYNLLFLFSIGIDKTKALSLLRGLTDFKRSFDLNLRVKNMLPSLYKEAPEFYENMRIQELAQNIHNLVKHHNLPDLMYRAFEVLPAMVMNPFQAFQKELHGEVEEVYLEDMVGKVNANMILPYPPGVPLVMPGEMLTEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKNDK
作为改进的是,编码所述L-赖氨酸脱羧酶SpLDC的核苷酸序列如SEQ ID NO.2所示。
SEQIDNo.2核苷酸序列:
ATGAACGTTATTGCTATCATGAACCACATGGGCGTCTACTTCAAAGAAGAGCCCATCCGCGAACTGCACAACGCGTTGGAAAAACTGAACTTCCGCATTGTTTACCCGAACGACCGCGAAGACCTGCTCAAATTGATCGAGAATAATGCCCGTCTGTGCGGGGTGATCTTCGACTGGGATAAATACAATCTTGAGCTGTGCCAGGAAATCAGCGAATTGAATGAGTACATGCCGCTGTATGCCTTTGCCAACACCTATTCAACCCTGGACGTCAGTCTTAACGATCTGCGTATGCAGGTACGTTTCTTTGAATATGCGCTCGGTGCGGCCAACGACATTGCCGACAAGATCAAACAGAACACCGATGAATACGTGGACACCATCCTGCCACCGCTGACCAAAGCGCTGTTCAAATATGTACGTGAAGGTAAATACACCTTCTGTACTCCTGGCCACATGGGCGGTACCGCCTTCCAGAAAAGCCCGGTCGGTAGCCTGTTCTACGATTTCTTTGGCCCGAACGCGATGAAATCCGACGTGTCAATTTCGGTCTCTGAGCTGGGTTCACTGCTGGATCACTCCGGCCCGCACAAAGAAGCTGAAGAATATATTGCCCGCGTGTTCAACGCTGAACGCAGCTATATGGTGACTAACGGCACTTCTACCGCCAACAAAATTGTTGGTATGTACTCTGCGCCTGCCGGCAACACGGTGCTGATTGACCGTAACTGCCACAAATCGCTGACCCATCTGATGATGATGAGCGACATTACGCCGATCTACTTCCGCCCGACCCGTAACGCTTACGGCATTCTCGGTGGTATTCCACAGAGCGAATTTTCCCGTGCCACTATCGCCAAACGCGTGAAAGATACCCCTAACGCTACCTGGCCGGTACACGCAGTGATCACCAACTCGACCTACGACGGTCTGTTGTATAACACCGATTTCATCAAGAGCACGCTGGATGTGAAATCCATCCACTTTGACTCTGCCTGGGTGCCTTACACCAACTTCCACCCGATCTACAAAGGCAAGTGCGGCATGAGCGGCGGCCGAGTGGAAGGGAAAGTGATCTATGAAACCCAATCCACCCATAAACTGCTGGCGGCGTTCTCACAGGCTTCGATGATCCACGTGAAAGGGGACATCAACGAAGAAACCTTCAATGAAGCCTACATGATGCACACCACCACTTCACCGCACTACGGCATCGTGGCCTCTACCGAAACCGCAGCGGCGATGATGAAAGGCAATGCCGGTAAGCGTCTGATTAACGGTTCAATTGAACGAGCTATCAAGTTCCGTAAGGAAATCAAACGTCTGAATGTCGAGTCTGAAGGTTGGTTCTTTGACGTTTGGCAGCCGGAACATATTGATGAAGCAGAGTGTTGGCCGCTGCGTTCCGACAGTGCATGGCATGGTTTCAAAGGTATTGATAACGAACATATGTACCTTGACCCGATCAAGGTCACCATGTTGACACCGGGGATGAGCAAAGACGGCGAAATGGAATCGTTTGGTATCCCGGCCAGCCTGGTTGCCAAATACCTTGATGAGCACGGCATCATTGTTGAGAAAACCGGCCCGTATAACCTGCTGTTCCTGTTCAGCATCGGCATCGACAAAACCAAAGCTCTCAGCCTGCTGCGCGGCCTGACTGACTTTAAACGCTCGTTCGACCTGAACCTGCGGGTGAAAAACATGCTGCCTTCGCTGTACAAAGAGGCACCAGAGTTTTATGAAAACATGCGTATCCAGGAACTGGCCCAGAATATCCACAACCTGGTGAAACATCATAACCTGCCTGACCTAATGTACCGCGCCTTTGAAGTGCTGCCGGCGATGGTAATGAACCCGTTCCAGGCCTTCCAGAAAGAGCTGCACGGTGAAGTGGAAGAGGTTTATTTGGAAGACATGGTGGGCAAAGTGAATGCCAACATGATCCTGCCATATCCTCCGGGTGTTCCACTGGTGATGCCGGGTGAAATGCTCACCGAAGAAAGCCGTCCGGTGCTGGAGTTCCTGCAAATGCTGTGCGAAATCGGCGCGCATTATCCAGGTTTCGAAACTGATATCCACGGCGCTTATCGCCAGGCGGATGGACGTTACACCGTTAAGGTATTGAAAAACGATAAA
一种重组质粒,含有编码权利要求1所述L-赖氨酸脱羧酶SpLDC的核苷酸序列的质粒。
一种重组工程菌,表达含有上述氨基酸序列的L-赖氨酸脱羧酶SpLDC质粒的菌株。
上述L-赖氨酸脱羧酶SpLDC、重组质粒或重组工程菌在催化L-赖氨酸盐酸盐产1,5-戊二胺上的应用。
作为改进的是,所述L-赖氨酸脱羧酶SpLDC在催化产1,5-戊二胺上的应用,具体步骤如下:
步骤1,通过基因合成L-赖氨酸脱羧酶SpLDC的核苷酸序列,并将该序列插入到载体pETdute-1上,获得重组载体pETdute-SpLDC;
步骤2,将重组载体pETdute-SpLDC转移到大肠杆菌DH5α中,涂在带有氨苄抗性的平板上,待长出菌落,挑取正确的克隆宿主,在LB培养基中进行扩培,使用质粒提取试剂盒提取重组质粒pETdute-SpLDC;
步骤3,将重组质粒pETdute-SpLDC导入大肠杆菌BL21(DE3),得到含有重组质粒pETdute-SpLDC的大肠杆菌BL21(DE3),命名为重组菌;
步骤4,挑选重组菌的单克隆,摇床过夜培养后,加入诱导剂诱导培养,收集菌液,离心后,收集沉淀;
步骤5,向沉淀中加入缓冲液重悬菌株,再超声破碎后,离心,收集上清后冷冻备用;
步骤6,采用AKTA蛋白纯化系统和该公司的HIS-TAG柱重组蛋白进行蛋白纯化获得纯酶,使用纯酶对L-赖氨酸盐酸盐进行催化反应,即得1,5-戊二胺溶液。
作为改进的是,步骤6中在催化反应的pH为4.0-9.5都具有催化合成戊二胺的能力;在温度为37-55℃具有较高的催化合成戊二胺的能力。
进一步改进的是,步骤6中催化反应研究中,纯酶在pH为5.0-8.0的缓冲中孵育12h,酶任然保持原有酶活的80%以上的催化合成戊二胺的活性,说明改酶具有良好的pH稳定性;纯酶SpLDC在pH 7,在温度为37℃-52℃的水浴锅放置12 h,检测纯酶催化合成戊二胺的能力任然保持在80% 以上,说明该酶具有良好的温度稳定性。
进一步改进的是,步骤6中在催化反应的pH为6.5时,催化效果佳;催化反应的温度为52℃,催化效果佳。
有益效果:
与现有技术相比,本发明一种L-赖氨酸脱羧酶SpLDC及其在产1,5-戊二胺上的应用,所用L-赖氨酸脱羧酶SpLDC的催化pH区间宽泛,满足L-赖氨酸合成戊二胺的pH急剧体系,且该酶的pH稳定性和温度稳定性,用于1,5-戊二胺的生产,催化过程稳定,且效率高,适合产业化应用。纯酶在pH为5.0-8.0的缓冲中孵育12 h,酶任然保持原有酶活的80%以上的催化合成戊二胺的活性,纯酶SpLDC在pH 7,在温度为37℃-52℃的水浴锅放置12 h,检测纯酶催化合成戊二胺的能力任然保持在80%,因此,L-赖氨酸脱羧酶SpLDC具有良好的pH稳定性和温度稳定性,适合应用催化生产1,5-戊二胺。
附图说明
图1为本发明L-赖氨酸脱羧酶的pH稳定性的情况;
图2为本发明L-赖氨酸脱羧酶的温度稳定性的情况;
图3为本发明实施例2中制备的戊二胺的气相色谱图。
具体实施方式
下面结合实施例对本发明的技术方案进行详细说明。
需要特殊说明的是,本发明各实施例中所用的材料为常规的,且可购买的,检测方法以及计算方式均为常规的,在此不需要做特殊说明。
实施例1
异源表达L-赖氨酸脱羧酶SpLDC的细胞的制备,步骤如下:
步骤1,通过通用生物公司(滁州)合成L-赖氨酸脱羧酶的核苷酸序列基因,并命名“SpLDC”,序列如SEQID-No.2所示;并将基因连接在pETdute-1质粒上,目的基因插入的酶切位点为分别为HindIII和XhoI, 在N-端具有6×His,并将重组质粒命名为“pETdute-SpLDC”;
步骤2,将重组质粒pETdute-SpLDC;转移到大肠杆菌DH5α中,涂在带有氨苄抗性的LB培养基平板(蛋白胨10 g/L;酵母粉5 g/L;NaCl 5 g/L;琼脂粉20 g/L)上;待长出菌落,挑取正确的克隆宿主,在LB培养基中进行扩培,使用质粒提取试剂盒提取重组质粒pETdute-SpLDC;
步骤3,将重组质粒pETdute-SpLDC导入大肠杆菌BL21(DE3),得到含有重组质pETdute-SpLDC的大肠杆菌BL21(DE3),命名为重组菌BL21(DE3)-pETdute-SpLDC;
步骤4,挑取重组菌BL21(DE3)- pETdute-SpLDC单菌落,接种于5mLLB种子液(蛋白胨10 g/L;酵母粉5 g/L;NaCl 5 g/L)中进行培养5-6h得种子液,将种子液按照1%的接种量转接到放有100ml含有100mg的氨苄青霉素的LB培养基的500 mL摇瓶中,37℃下200rpm培养,直至OD600生长到0.7左右,再加入0.5mM的IPTG进行诱导培养,诱导培养的温度为23℃,待培养15h后,6000rpm离心10min收集菌体获得过表达L-赖氨酸脱羧酶SpLDC的细胞。
实施例2
将实施例1中的细胞进行破碎,离心收集上清液;收集的上清液用AKTA蛋白纯化系统和该公司的HIS-TAG柱重组蛋白进行蛋白纯化。获得纯L-赖氨酸脱羧酶SpLDC,对其最适催化温度进行研究。在催化体系中有50 g/L的L-赖氨酸,200 mM磷酸盐缓冲液(pH7.0),反应体积200μL,加入酶量10μg/mL,0.1mM 辅因子5-磷酸吡哆醛(PLP),分别在27℃,32℃,37℃,42℃,47℃,52℃,57℃,62℃条件下进行反应,催化反应10 min后结束反应。
取10μl不同温度条件下的反应液,沸水浴5min后终止反应,稀释检测L-赖氨酸脱羧酶的消耗量。即赖氨酸脱羧酶的最优催化温度为52℃,比酶酶活可以达到为400U/mg。酶活定义为:1 min消耗1umolL-赖氨酸所使用的酶量为1U。
实施例3
将实施例1中的细胞进行破碎,离心收集上清液;收集的上清液用AKTA蛋白纯化系统和该公司的HIS-TAG柱重组蛋白进行蛋白纯化;获得纯L-赖氨酸脱羧酶,对其最适催化pH进行研究。催化体系 50 g/L的L-赖氨酸,反应体积200 μL,加入酶量10 μg/mL,0.1 mM 辅因子磷酸吡哆醛,分别在pH 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0条件下进行反应,反应时间为10 min后,分别取10μl反应液,沸水浴煮沸5 min后终止反应,检测L-赖氨酸剩余量,计算L-赖氨酸消耗量,计算酶活。可知L-赖氨酸脱羧酶的最适pH为6.5,该pH条件下比酶酶活为510U/mg。
实施例4
将实施例1中的细胞进行破碎,收集的上清液用AKTA蛋白纯化系统和该公司的HIS-TAG柱重组蛋白进行蛋白纯化,获得纯L-赖氨酸脱羧酶SpLDC,研究纯酶SpLDC的pH稳定性。
将纯酶液分别在pH为5.0-9.0条件下、37℃下孵育12h后,取酶液并测定酶活。测酶活反应体系为:10μg/mL纯L-赖氨酸脱羧酶,0.1mM辅因子5-磷酸吡哆醛,50g/LL-赖氨酸,反应体系200μL,在酶的最适的催化条件下进行催化反应,检测L-赖氨酸脱羧酶的消耗量。
经计算可知(图1),在pH为5.0-8.0范围内孵育后的酶,酶活依然能保持在70%以上,说明该酶具有良好的pH稳定性。该酶较好的pH稳定性,为其催化产1,5-戊二胺的工业化应用上提供了有力证据。
实施例5
将实施例1收集的细胞破碎处理,收集的上清液用AKTA蛋白纯化系统和该公司的HIS-TAG柱重组蛋白进行蛋白纯化,获得纯L-赖氨酸脱羧酶,然后对其温度稳定性进行研究。
纯酶液在温度梯度37℃、42℃、47℃、52℃、57℃和62℃孵育12 h,然后取酶液测定酶残留活性。其中,测酶活反应体系为:10μg/ml纯L-赖氨酸脱羧酶浓度,0.1mM辅因子5-磷酸吡哆醛,50g/L赖氨酸盐酸盐,反应体系200μL,在最适催化条件下,反应10min,检测L-赖氨酸脱羧酶的消耗量。
经计算可知,在52℃下,仍能保持初始酶活的80%以上,赖氨酸脱羧酶的温度稳定性为其催化产1,5-戊二胺的工业化应用上提供了有力证据。
实施例6
收集实施例1中的细胞,以细胞浓度为5 OD细胞破碎液催化含有310g/LL-赖氨酸的食品级380g/L L-赖氨酸盐酸盐溶液;
催化条件为:搅拌速度为200rpm,温度37℃,催化反应时间3h;
对催化后的反应液进行检测,得1,5-戊二胺转化液为220g/L。
调节所得反应液的pH至14,再加入等体积的正丁醇作为萃取液,对溶液中的戊二胺进行萃取,萃取后静置分层,取正丁醇相,进行气相色谱分析,鉴定产物为戊二胺,色谱图如图3所示。
实施例7
收集实施例1中的细胞,以细胞浓度为5 OD细胞破碎液加入到含有300g/L的L-赖氨酸的420g/L饲料级赖氨酸硫酸盐溶液;
催化条件为:搅拌速度为200rpm,温度37℃,催化反应5h;
对催化后的反应液进行检测,得1,5-戊二胺转化液浓度为205g/L。
实施例8
收集实施例1中的细胞,以细胞浓度为5OD的细胞破碎液催化含有320 g/L的450g/L饲料级赖氨酸硫酸盐溶液;
催化条件为:搅拌速度为350rpm,温度52℃,催化反应3h,催化反应进行时,需要将反应体系封闭,保证体系的pH通过自产的二氧化碳实现调节。
对催化后的反应液进行检测,得1,5-戊二胺转化液为230g/L。
从实验结果看出,封闭体系下催化反应比未封闭体系催化速率快,催化时间缩短2h,1,5-戊二胺的产量提高了12%。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 南京工业大学
<120> 一种L-赖氨酸脱羧酶SpLDC及其在产1,5-戊二胺上的应用
<141> 2021-06-28
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2139
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgaacgtta ttgctatcat gaaccacatg ggcgtctact tcaaagaaga gcccatccgc 60
gaactgcaca acgcgttgga aaaactgaac ttccgcattg tttacccgaa cgaccgcgaa 120
gacctgctca aattgatcga gaataatgcc cgtctgtgcg gggtgatctt cgactgggat 180
aaatacaatc ttgagctgtg ccaggaaatc agcgaattga atgagtacat gccgctgtat 240
gcctttgcca acacctattc aaccctggac gtcagtctta acgatctgcg tatgcaggta 300
cgtttctttg aatatgcgct cggtgcggcc aacgacattg ccgacaagat caaacagaac 360
accgatgaat acgtggacac catcctgcca ccgctgacca aagcgctgtt caaatatgta 420
cgtgaaggta aatacacctt ctgtactcct ggccacatgg gcggtaccgc cttccagaaa 480
agcccggtcg gtagcctgtt ctacgatttc tttggcccga acgcgatgaa atccgacgtg 540
tcaatttcgg tctctgagct gggttcactg ctggatcact ccggcccgca caaagaagct 600
gaagaatata ttgcccgcgt gttcaacgct gaacgcagct atatggtgac taacggcact 660
tctaccgcca acaaaattgt tggtatgtac tctgcgcctg ccggcaacac ggtgctgatt 720
gaccgtaact gccacaaatc gctgacccat ctgatgatga tgagcgacat tacgccgatc 780
tacttccgcc cgacccgtaa cgcttacggc attctcggtg gtattccaca gagcgaattt 840
tcccgtgcca ctatcgccaa acgcgtgaaa gataccccta acgctacctg gccggtacac 900
gcagtgatca ccaactcgac ctacgacggt ctgttgtata acaccgattt catcaagagc 960
acgctggatg tgaaatccat ccactttgac tctgcctggg tgccttacac caacttccac 1020
ccgatctaca aaggcaagtg cggcatgagc ggcggccgag tggaagggaa agtgatctat 1080
gaaacccaat ccacccataa actgctggcg gcgttctcac aggcttcgat gatccacgtg 1140
aaaggggaca tcaacgaaga aaccttcaat gaagcctaca tgatgcacac caccacttca 1200
ccgcactacg gcatcgtggc ctctaccgaa accgcagcgg cgatgatgaa aggcaatgcc 1260
ggtaagcgtc tgattaacgg ttcaattgaa cgagctatca agttccgtaa ggaaatcaaa 1320
cgtctgaatg tcgagtctga aggttggttc tttgacgttt ggcagccgga acatattgat 1380
gaagcagagt gttggccgct gcgttccgac agtgcatggc atggtttcaa aggtattgat 1440
aacgaacata tgtaccttga cccgatcaag gtcaccatgt tgacaccggg gatgagcaaa 1500
gacggcgaaa tggaatcgtt tggtatcccg gccagcctgg ttgccaaata ccttgatgag 1560
cacggcatca ttgttgagaa aaccggcccg tataacctgc tgttcctgtt cagcatcggc 1620
atcgacaaaa ccaaagctct cagcctgctg cgcggcctga ctgactttaa acgctcgttc 1680
gacctgaacc tgcgggtgaa aaacatgctg ccttcgctgt acaaagaggc accagagttt 1740
tatgaaaaca tgcgtatcca ggaactggcc cagaatatcc acaacctggt gaaacatcat 1800
aacctgcctg acctaatgta ccgcgccttt gaagtgctgc cggcgatggt aatgaacccg 1860
ttccaggcct tccagaaaga gctgcacggt gaagtggaag aggtttattt ggaagacatg 1920
gtgggcaaag tgaatgccaa catgatcctg ccatatcctc cgggtgttcc actggtgatg 1980
ccgggtgaaa tgctcaccga agaaagccgt ccggtgctgg agttcctgca aatgctgtgc 2040
gaaatcggcg cgcattatcc aggtttcgaa actgatatcc acggcgctta tcgccaggcg 2100
gatggacgtt acaccgttaa ggtattgaaa aacgataaa 2139
<210> 2
<211> 713
<212> PRT
<213> 氨基酸序列(Amino acid Sequence)
<400> 2
Met Asn Val Ile Ala Ile Met Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Asn Ala Leu Glu Lys Leu Asn Phe Arg
20 25 30
Ile Val Tyr Pro Asn Asp Arg Glu Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Gln Glu Ile Ser Glu Leu Asn Glu Tyr Met Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Met Gln Val Arg Phe Phe Glu Tyr Ala Leu Gly Ala Ala Asn Asp
100 105 110
Ile Ala Asp Lys Ile Lys Gln Asn Thr Asp Glu Tyr Val Asp Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Ala Met
165 170 175
Lys Ser Asp Val Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Glu Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Asn Thr Val Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Ile Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Ser Arg Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Asp Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Ser
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe His Pro Ile Tyr Lys Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Ile
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Asn Val Glu Ser Glu Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Glu Ala Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Ala Trp His Gly Phe Lys Gly Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Met Leu Thr Pro
485 490 495
Gly Met Ser Lys Asp Gly Glu Met Glu Ser Phe Gly Ile Pro Ala Ser
500 505 510
Leu Val Ala Lys Tyr Leu Asp Glu His Gly Ile Ile Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Gly Leu Thr Asp Phe Lys Arg Ser Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Lys Glu
565 570 575
Ala Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Asn Leu Val Lys His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Ala Met Val Met Asn Pro Phe Gln Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Glu Val Glu Glu Val Tyr Leu Glu Asp Met
625 630 635 640
Val Gly Lys Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Leu Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Asn Asp Lys
705 710
Claims (9)
1.一种L-赖氨酸脱羧酶SpLDC,其特征在于,所述L-赖氨酸脱羧酶SpLDC的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的L-赖氨酸脱羧酶SpLDC,其特征在于,编码所述L-赖氨酸脱羧酶SpLDC的核苷酸序列如SEQ ID NO.2所示。
3.一种重组质粒,含有编码权利要求1所述L-赖氨酸脱羧酶SpLDC的核苷酸序列的质粒。
4.一种重组工程菌,表达权利要求3所述重组质粒的菌株。
5.基于权利要求1所述L-赖氨酸脱羧酶SpLDC、权利要求3所述重组质粒或权利要求4所述重组工程菌在催化L-赖氨酸盐酸盐产1,5-戊二胺上的应用。
6.根据权利要求5所述的应用,其特征在于,所述L-赖氨酸脱羧酶SpLDC在催化产1,5-戊二胺上的应用,具体步骤如下:
步骤1,通过基因合成L-赖氨酸脱羧酶SpLDC的核苷酸序列,并将该序列插入到载体pETdute-1上,获得重组载体pETdute-SpLDC;
步骤2,将重组载体pETdute-SpLDC转移到大肠杆菌DH5α中,涂在带有氨苄抗性的平板上,待长出菌落,挑取正确的克隆宿主,在LB培养基中进行扩培,使用质粒提取试剂盒提取重组质粒pETdute-SpLDC;
步骤3,将重组质粒pETdute-SpLDC导入大肠杆菌BL21(DE3),得到含有重组质粒pETdute-SpLDC的大肠杆菌BL21(DE3),命名为重组菌;
步骤4,挑选重组菌的单克隆,摇床过夜培养后,加入诱导剂诱导培养,收集菌液,离心后,收集沉淀;
步骤5,向沉淀中加入缓冲液重悬菌株,再超声破碎后,离心,收集上清后冷冻备用;
步骤6,采用镍柱纯化方式获得纯酶,使用纯酶对L-赖氨酸盐酸盐进行催化反应,即得1,5-戊二胺溶液。
7.根据权利要求6所述的应用,其特征在于,步骤6中在催化反应的pH为4.0-9.5都具有催化合成戊二胺的能力;在温度为37-55℃具有较高的催化合成戊二胺的能力。
8.根据权利要求7所述的应用,其特征在于,步骤6纯酶在pH为5.0-8.0的缓冲中孵育12h,酶任然具有原有的80%以上的催化合成戊二胺的活性,说明改酶具有良好的pH稳定性;纯酶在pH 7,在温度为37℃-52℃的水浴锅放置12 h,检测纯酶催化合成戊二胺的能力任然保持在80% 以上,说明该酶具有良好的温度稳定性。
9.根据权利要求7所述的应用,其特征在于,步骤6中在催化反应的pH为6.5时,催化效果佳;催化反应的温度为52℃,催化效果佳。
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