CN112921044A - 解淀粉芽孢杆菌脲酶在食品级枯草芽孢杆菌中的重组表达 - Google Patents
解淀粉芽孢杆菌脲酶在食品级枯草芽孢杆菌中的重组表达 Download PDFInfo
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- CN112921044A CN112921044A CN202110399002.2A CN202110399002A CN112921044A CN 112921044 A CN112921044 A CN 112921044A CN 202110399002 A CN202110399002 A CN 202110399002A CN 112921044 A CN112921044 A CN 112921044A
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Abstract
本发明公开了解淀粉芽孢杆菌脲酶在食品级枯草芽孢杆菌中的重组表达,属于酶工程技术领域。本发明通过枯草密码子优化、核糖体结合位点优化重构基因簇,使脲酶活力从6.85U/mL提升至9.01U/mL;通过调整基因ureC至ureA、ureB前,强化了UreC的表达,脲酶活力进一步提升至10.15U/mL,相对于原始脲酶活力提高了48%。重组脲酶最适反应温度为37℃,最适反应pH范围为6.5‑7.0。经过摇瓶发酵条件优化,确定TB培养基、5%接种体积分数、28℃诱导温度、4mmol/L Ni2+添加量为最佳发酵条件,酶活力达到了12.5U/mL,相对于原始脲酶活力提高了82%。本发明技术在生物酶法降低黄酒尿素含量具有应用潜力。
Description
技术领域
本发明涉及解淀粉芽孢杆菌脲酶在食品级枯草芽孢杆菌中的重组表达,属于酶工程技术领域。
背景技术
氨基甲酸乙酯(Ethyl carbamate或Urethane,简称EC),是一种存在于发酵食品和酒精饮料(奶酪、酱油;黄酒、清酒、威士忌)中的氨类危害物。2007年,国际癌症研究机构IARC(International Agency for Research on Cancer)正式将EC上升为2A类致癌物,与丙烯酰胺同等危险,引起人们对其食品安全的广泛关注。
尿素是黄酒中与乙醇反应生成EC的最主要前体物质。因而,去除黄酒中EC的主要策略是直接降低EC的含量和间接降低前体物质尿素的含量。脲酶(Urease,EC 3.5.1.5)因其不需更换酵母或生产工艺、直接高效的特性而在解决黄酒中EC的食品安全问题中占据优势。
目前,已报道的脲酶通常来自于发酵乳杆菌、运动节杆菌、肠杆菌属、罗伊氏乳杆菌、副地衣芽孢杆菌、葡萄球菌等,然而,这些微生物来源的脲酶基因簇的基因组成较为复杂,编码相应脲酶的活化过程需要所有辅助亚基UreE、UreF、UreG、UreD/UreH的参与,缺一不可,且相关辅助亚基的功能并不清晰。而来源于解淀粉芽孢杆菌的基因簇通常包含UreA、UreB、UreC三个结构亚基,基因异源表达及调控的可行性较高,然而,已报道来源于解淀粉芽孢杆菌JP-21的脲酶基因簇仅在大肠杆菌系统中重组表达,难以用于传统发酵食品加工体系。
为达到食品安全应用,脲酶基因的异源表达备受关注。枯草芽孢杆菌是当前研究得最好的食品安全级微生物(GRAS),已被广泛用于生产各种工业酶。但,尚无利用枯草系统表达重组脲酶。
发明内容
[技术问题]
本发明要解决的技术问题是现有利用大肠杆菌表达的脲酶不适用于传统发酵食品加工体系。
[技术方案]
本发明在枯草芽孢杆菌中实现了基因挖掘所得的解淀粉芽孢杆菌IT-45来源的脲酶基因簇(ureABC)的表达,并通过密码子优化、核糖体结合位点优化以及调整基因ureC的位置,进一步提升脲酶活力,本发明制备得到的脲酶在酶法降解黄酒中的尿素应用上具有潜力。所述解淀粉芽孢杆菌IT-45来源的脲酶基因簇(ureABC)的序列如SEQ ID NO.1所示,该脲酶有三个结构亚基:UreA、UreB、UreC,三个亚基的氨基酸序列分别如SEQ ID NO.2、SEQID NO.3、SEQ ID NO.4所示。
本发明提供经过重构的编码脲酶的基因,其核苷酸序列如SEQ ID NO.5或SEQ IDNO.6所示。
本发明提供携带编码脲酶基因的载体。
本发明提供表达所述经过重构的编码脲酶的基因的重组枯草芽孢杆菌,宿主可选自:B.subtilis WB600、B.subtilis WB700、B.subtilis WB800或B.subtilis WB168,表达载体可选自:pP43NMK、pHT43、pMA5或pTTB。
本发明提供一种构建表达经过重构的编码脲酶的基因的重组枯草芽孢杆菌的方法,包括以下步骤:
(1)将来源于Bacillus amyloliquefaciens IT-45的脲酶基因簇Ba-urease(ureABC)序列进行枯草芽孢杆菌密码子优化、核糖体结合位点优化,获得如SEQ ID NO.5所示的脲酶基因Ba-urease-1;
(2)在如SEQ ID NO.5所示的脲酶基因Ba-urease-1的基础上,将ureC移置至ureA和ureB的上游,从而获得序列如SEQ ID NO.6所示的脲酶基因Ba-urease-2,将其与表达载体pP43NMK连接,构建脲酶表达载体pP43NMK-Ure-2;
(3)将pP43NMK-Ure-2转化至B.subtilis WB600中,获得重组表达菌株B.subtilisWB600/pP43NMK-Ure-2。
本发明还提供应用所述重组枯草芽孢杆菌制备脲酶的方法,主要包括以下步骤:
挑取重组B.subtilis单菌落,接种于含有50μg/mL卡那霉素的5mL LB液体培养基中,在37℃、200r/min的摇床中振荡培养10h;按5%的接种体积分数,分别转接于含有50μg/mL卡那霉素、4mmol/L Ni2+的100mL TB液体培养基中,在37℃、200r/min的摇床中振荡培养8h,于28℃诱导培养42h。
本发明还提供利用食品级表达系统表达得到的脲酶在降解尿素中的应用。包括:利用脲酶来降低或消除发酵食品和酒精饮料中的尿素含量以降低氨基甲酸乙酯。
[有益效果]
(1)本发明在食品级系统枯草芽孢杆菌中实现了解淀粉芽孢杆菌来源脲酶(Ba-urease)的表达,采用枯草密码子优化、核糖体结合位点优化重构脲酶基因簇的策略,使脲酶活力从6.85U/mL提升至9.01U/mL;通过将基因ureC调整至ureA、ureB前,强化了UreC的表达,脲酶活力进一步提升至10.15U/mL,相对于原始脲酶活力提高了48%。
(2)本发明的重组脲酶最适反应温度为37℃,且在30-37℃范围内可以保持30%以上的酶活力;最适反应pH范围为6.5-7.0。
(3)本发明确定了TB培养基、5%接种体积分数、28℃诱导温度、4mmol/L Ni2+添加量为最佳摇瓶发酵条件,酶活力达到了12.5U/mL,相对于原始脲酶活力提高了82%。
(4)本发明检测到反应体系中的尿素降解率约为36%,表明重组脲酶可有效降解尿素。
附图说明
图1:Ba-urease基因簇变换流程。
图2:重组菌株的核酸电泳。Lane 1:Ba-urease;Lane 2:Ba-urease-1;Lane 3:Ba-urease-2。
图3:重组菌株摇瓶发酵酶活力随时间变化曲线。
图4:重组酶的最适反应条件。(a)温度;(b)pH。
图5:重组菌株的发酵条件优化。(a)培养基;(b)接种体积分数;(c)诱导温度;(d)Ni2+添加量。
具体实施方式
脲酶酶活的测定方法:
脲酶酶活单位(U)定义:在常压、37℃、pH 7.0的条件下,每分钟分解1μmol尿素所需要的酶量。
采用Berthelot反应比色法测定脲酶活力:取粗酶液200μL,添加至200μL含有30g/L尿素的10mmol/L磷酸缓冲液(pH 7.0)中,混匀。在37℃水浴条件下反应20min后,立即加入200μL 10%三氯乙酸,混匀终止反应。随后,依次加入200μL显色剂Ⅰ(苯酚15g,亚硝基铁氰化钠0.625g,用超纯水定容至250mL)和200μL显色剂Ⅱ(NaOH 13.125g,NaClO7.5mL,用超纯水定容至250mL),混匀后在37℃水浴条件下反应20min。反应结束后,8000r/min离心5min,适当稀释。以灭活的酶液作为空白对照,测定其OD625nm的变化。使用0、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0mmol/L NH4Cl溶液作出氨离子标准曲线,用于计算反应体系生成氨的总量从而计算酶活。
实施例1:重组表达解淀粉芽孢杆菌来源脲酶的枯草芽孢杆菌的构建
步骤1质粒的构建
以基因合成所得的包含解淀粉芽孢杆菌IT-45中脲酶基因簇的质粒为模板,用引物U-F/U-R扩增脲酶基因簇Ba-urease(ureABC)序列SEQ ID NO.1。以质粒pP43NMK为模板,用引物P-F/P-R进行质粒线性化。利用II One Step Cloning Kit(购于诺唯赞公司)将经胶回收后的基因片段Ba-urease和线性化质粒pP43NMK进行连接,并转化至E.coli JM109中,挑取转化子验证正确,获得质粒pP43NMK-Ure。
如图1所示,通过密码子优化(http://www.kazusa.or.jp/codon/)以及核糖体结合位点(RBS)优化(https://salislab.net/software/)重新组装脲酶基因簇,获得基因Ba-urease-1序列SEQ ID NO.5。将ureC移置至ureA和ureB的上游,从而获得基因Ba-urease-2序列SEQ ID NO.6。
用引物U-F1/U-R1、U-F2/U-R2分别扩增基因Ba-urease-1、Ba-urease-2,用引物P-F1/P-R2、P-F2/P-R2分别进行质粒pP43NMK线性化,利用II One StepCloning Kit将经胶回收后的基因片段和线性化质粒进行连接,并转化至E.coli JM109中,挑取转化子验证正确,分别获得质粒pP43NMK-Ure-1、pP43NMK-Ure-2。
表1 质粒构建所用引物
注:下划线序列为用于组装的同源臂
步骤2重组菌株的构建
大肠感受态细胞的制备与转化:按照Competent Cell Preparation kit(购于TaKaRa公司)说明书,注意无菌操作。具体地,将长在无抗LB平板上的大肠杆菌E.coliJM109单菌落挑至LB试管,37℃、200r/min过夜培养8-12h;后以2%的接种量转接至装有50mL LB培养基的250mL三角瓶,培养条件如前所述;培养至OD600nm约为0.6-0.8后,将三角瓶于冰上放置30min;于超净台,将50mL菌液以25mL分装量分装至已灭菌的50mL离心管;4℃、6000r/min下离心5min后弃上清,加入10%A液轻轻将菌体重悬;再次离心后弃上清,加入10%B液轻轻将菌体重悬后,以100μL分装量分装至1.5mL无菌EP管中,所得E.coli JM109感受态于-80℃下保存。将E.coli JM109感受态至于冰上融化后,于超净台加入10μL的同源重组产物(重组质粒);轻轻混匀后,冰上放置30min左右;于金属浴中,42℃热击45-90s后冰上放置5min;加入800mL LB培养基,37℃、200r/min培养1h;4℃、6000r/min离心5min后于超净台弃800μL上清;剩余培养液重悬菌体后涂布至抗性平板,37℃静置过夜培养12h;挑菌落进行菌落PCR验证后送测序。获得测序正确的质粒pP43NMK-Ure、pP43NMK-Ure-1、pP43NMK-Ure-2。
B.subtilis WB600枯草感受态细胞的制备与转化:取长在无抗LB平板上的枯草芽孢杆菌WB600单菌落于5mL GM I培养基中,37℃、200r/min培养12-16h。取1mL培养液于9mLGM I培养基中,37℃、200r/min培养3.5h。取10mL培养液接种于装有90mL GM II培养基的500mL三角瓶中,37℃、200r/min培养1.5h。将GM II培养液分装于50mL无菌离心管中,4℃、3000r/min离心5min。取1mL GM II上清液,于1.5mL无菌EP管中轻轻重悬菌体,加入终浓度10%的甘油,以250μL分装量分装至1.5mL无菌EP管中,所得枯草芽孢杆菌WB600感受态于-80℃下保存。加入1-20μg DNA片段(质粒),45℃热击90s,冰浴1min。37℃、200r/min培养1.5-2h,4℃、6000r/min离心5min后于超净台弃部分上清;剩余培养液重悬菌体后涂布至抗性平板,37℃静置过夜培养12h;挑菌落进行菌落PCR验证后(扩增结果如图2所示)送测序。获得重组表达菌株B.subtilis WB600/pP43NMK-Ure、B.subtilis WB600/pP43NMK-Ure-1、B.subtilis WB600/pP43NMK-Ure-2。
实施例2:重组脲酶的表达
挑取实施例1所构建的B.subtilis WB600/pP43NMK-Ure、B.subtilis WB600/pP43NMK-Ure-1、B.subtilis WB600/pP43NMK-Ure-2的单菌落,分布接种于含有50μg/mL卡那霉素的5mL LB液体培养基中,在37℃、200r/min的摇床中振荡培养10h。按照3%的接种体积分数,转接于含有50μg/mL卡那霉素的100mL TB液体培养基中,在37℃、200r/min的摇床中振荡培养50h,每隔一定时间取样测定菌体浓度及脲酶活力(菌体浓度及酶活力随时间变化曲线如图3所示)。所得菌液在4℃、8000r/min条件下离心5min收集菌体,用20mmol/L PBS缓冲液(pH 7.4)洗涤2次后,等体积重悬于20mmol/L PBS缓冲液(pH7.4),获得粗酶液。
关于酶学性质
(1)最适反应温度:参照酶活测定方法,将粗酶液和尿素溶液混合,放置在不同温度(20、25、28、30、35、37、40℃)下反应后测定酶活,以最高酶活为100%,分别计算不同温度下酶活的相对值。
(2)最适反应pH:参照酶活测定方法,用10mmol/L的柠檬酸、柠檬酸三钠溶液以及10mmol/L的磷酸氢二钾、磷酸二氢钾溶液调节反应体系的pH(4.0、4.5、5.0、5.5、6.0、6.5、7.0)并测定酶活,以最高酶活为100%,分别计算不同pH下酶活的相对值。
如图4所示,重组脲酶最适反应温度为37℃,且在30-37℃范围内可以保持30%以上的酶活力;最适反应pH范围为6.5-7.0。
实施例3重组脲酶的表达条件优化
挑取所构建的重组B.subtilis单菌落,接种于含有50μg/mL卡那霉素的5mL LB液体培养基中,在37℃、200r/min的摇床中振荡培养10h。
然后,
(1)按3%的接种体积分数,分别转接于含有50μg/mL卡那霉素的100mL LB、TB、M9液体培养基中,在37℃、200r/min的摇床中振荡培养50h,每隔一定的时间测定脲酶活力。
或,
(2)分别按3、5、7、9%的接种体积分数,转接于含有50μg/mL卡那霉素的100mL TB液体培养基中,在37℃、200r/min的摇床中振荡培养50h,每隔一定的时间测定脲酶活力。
或,
按3%的接种体积分数,转接于含有50μg/mL卡那霉素的100mL TB液体培养基中,在37℃、200r/min的摇床中培养8h后,变温(17、25、28、30、37℃)诱导,每隔一定的时间测定脲酶活力。
或,
(4)按3%的接种体积分数,转接于含有50μg/mL卡那霉素、不同Ni2+浓度(0、1.0、2.0、3.0、4.0、5.0、6.0、7.0mmol/L)的100mL TB液体培养基中,在37℃、200r/min的摇床中振荡培养50h,每隔一定的时间测定脲酶活力。
如图5所示,对重组脲酶Ba-urease在100mL摇瓶发酵的基础上进行条件优化,最终确定TB培养基、5%接种体积分数、28℃诱导温度、4mmol/L Ni2+添加量的最佳发酵条件,酶活力达到了12.5U/mL,相对于原始脲酶活力提高了82%。
实施例4重组脲酶在尿素降解中的应用
反应体系中的尿素含量通过高效液相色谱法(HPLC)测定,反应体系:将500μL粗酶液加至500μL尿素溶液(300mg/L)中混匀,37℃反应1h。HPLC-FLD法测定尿素是通过衍生剂9-羟基吨可以与尿素发生衍生反应,所产生衍生物具有荧光特性。该衍生物在经过色谱柱分离后可以通过荧光检测器测定,进而通过外标法定量反应体系中的尿素。
(1)样品前处理:将500μL反应体系用色谱纯乙醇稀释至1000μL,加入100μL1.5mol/L HCl和400μL 0.02mol/L 9-羟基吨,漩涡震荡使其混匀,室温避光衍生30min,采用0.22μm有机滤膜过滤后准备进样。
(2)色谱条件:检测器:Waters 2695高效液相色谱仪和Waters 2475荧光检测器,色谱柱:C18色谱柱(Zorbax SB-Aq 250mm×4.6mm,粒径5μm),柱温:35℃,样品温度:25℃,荧光检测器:λex=234nm,λem=600nm,进样量:10μL。流动相A:0.02m ol/L乙酸钠(pH7.2),流动相B:乙腈,流速:0.8mL/min。进行梯度洗脱,计算尿素含量。
将重组菌株B.subtilis WB600/pP43NMK-Ure-2在实施例3所得最佳条件下发酵培养,取该条件发酵下得到的粗酶液500μL,加至500μL尿素溶液(300mg/L)中混匀,37℃反应1h。通过高效液相色谱法检测尿素含量,采用灭活后的粗酶液反应体系(对照)中尿素含量为180.20mg/L,粗酶液反应体系中残余的尿素含量为116.13mg/L,计算得出反应体系中的尿素降解率约为36%。这说明本发明制备的重组脲酶可以有效降解尿素。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 解淀粉芽孢杆菌脲酶在食品级枯草芽孢杆菌中的重组表达
<130> BAA210458A
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 2395
<212> DNA
<213> 解淀粉芽孢杆菌IT-45
<400> 1
atgaaactga caccggttga acaagaaaaa ttgcttattt ttacggcggg agagctcgct 60
aagcagcgga aggcgcgcgg cgttctgctg aattatcccg aagccgccgc atatttgacc 120
tgttatctga tggaaggcgc gagagacgga aaaagcgttg ctgagctgat ggaatccggc 180
cgcaatgtat tgactgaaaa agacgttatg gaaggcgttg cggaaatgct ggacagcatt 240
caggtggaag cgacgttccc ggacggggtt aagcttgtca ccgttcatca gccgatcaaa 300
gcggaggtga agtcatgaag ccgggagcga ttcaagtcgc aaaggggacc atcaccatta 360
acgaaggccg gaagacgctg gaggtgtcag tcaccaataa cggaacgcgg tcagtgcagg 420
tcggatcgca ttttcatttt gccgaagcca acggcgccct ttccttcaat cgggacaaag 480
ccatcggcat gcgccttgat atcccgtcag gcacatctgt ccgttttgaa ccgggagaag 540
agaaaaccgt cacgctcgtg gaaatcggag ggcgaaaaac ggtcagaggt ctcaacggca 600
tggccgatac gtacatggat gagcggggaa aagagaagac gctgtcaaat cttaaaaaag 660
ccggatggat ggaggaggcg atccgatgaa aatgtcgcgt gagcaatacg cagaactgtt 720
cggaccgaca acgggagaca aagtcagact cggagatacg gatttatgga ttgaagtcga 780
aaaagatttc aggaattacg gggaagaaat gattttcggc ggagggaaaa caatccggga 840
cggcatgggg cagaacgggc gtatcaccgg gaaagacgga gcgcttgatc tggtcattac 900
aaacgccgtc atcctcgatt atatcggaat cgtcaaggcg gatatcgggg tgaaggacgg 960
ccggattgtc ggcgtcggga aaagcggcaa ccctgatatg atggatgggg tggacccgca 1020
catgattatc ggtgccggaa cagaagtcat ttccggcgaa gggaaaatcg taacggccgg 1080
cggggtggat acgcatatcc actttatctg cccgcagcag atggaagtcg cgctttcttc 1140
aggcgtgact acgcttctcg gcggcggaac aggtcctgcg acgggaagta aagcgacgac 1200
ttgtacatcc ggtgcatggt acatgtcgag aatgctggaa gcggccgagg agtttccgat 1260
caatgtcggt ttcttaggaa aaggaaatgc atccgataaa gcgccgctga tcgagcaggt 1320
ggaagcaggc gcaatcggcc tgaagctgca tgaagattgg ggatcaacgc caagcgccat 1380
taaagcttgc atggaagcag cggatgaggc ggacattcag gtggcgatcc acacagacac 1440
gataaatgaa gcgggctttt tagaaaatac gcttgatgcg atcggcgacc gggttatcca 1500
tacatatcac atagagggag ccggcggagg ccatgcaccg gatattatga aactcgcatc 1560
ttacgccaat atcctgccgt cctctacgac gccgacgatt ccatataccg tcaacacgat 1620
ggacgagcat cttgatatga tgatggtctg ccatcattta gattcaaaag tgcctgaaga 1680
cgtggcgttc agtcattcac gcatcagagc ggccaccatt gcggcggagg atattctgca 1740
cgatatcggc gcgatcagca tgacgtcatc tgactcgcag gcgatgggca gggtgggaga 1800
agtgattatc cggacatggc aggtggccga taaaatgaaa aaacagcgcg gcgctctatc 1860
gggagaaaac ggcaatgaca atgtgcgcgc caaacgctat atcgccaaat acacgatcaa 1920
cccggctgtc actcacggtc tgagccatga agtcggttcc gttgaaaaag gaaagctcgc 1980
cgacctcgta ctatgggacc cggttttctt cggcgtcaaa cctgaacttg tgctcaaagg 2040
cggcatgatt gcccgcgccc agatgggaga tccgaatgct tccattccga cgcctgagcc 2100
cgtgtttatg cggcagatgt acgcatcata cggtaaagca aaccgcaaca cctctattac 2160
atttatgtcc caggccggta tcgcaaacgg tgtgccggaa aagctcggcc ttgaaaaaat 2220
gatttctccc gtacggaata tccgtaagct gagtaagctc gacatgaagc tgaatgacgc 2280
gatgccgaat atacatgtcg atccgaaaac ctatcaggta ttcgccgacg gagaagagct 2340
ggcatgccag cccgtcagct atgttccgct aggacagcgt tatttcttat tttaa 2395
<210> 2
<211> 105
<212> PRT
<213> 解淀粉芽孢杆菌IT-45
<400> 2
Met Lys Leu Thr Pro Val Glu Gln Glu Lys Leu Leu Ile Phe Thr Ala
1 5 10 15
Gly Glu Leu Ala Lys Gln Arg Lys Ala Arg Gly Val Leu Leu Asn Tyr
20 25 30
Pro Glu Ala Ala Ala Tyr Leu Thr Cys Tyr Leu Met Glu Gly Ala Arg
35 40 45
Asp Gly Lys Ser Val Ala Glu Leu Met Glu Ser Gly Arg Asn Val Leu
50 55 60
Thr Glu Lys Asp Val Met Glu Gly Val Ala Glu Met Leu Asp Ser Ile
65 70 75 80
Gln Val Glu Ala Thr Phe Pro Asp Gly Val Lys Leu Val Thr Val His
85 90 95
Gln Pro Ile Lys Ala Glu Val Lys Ser
100 105
<210> 3
<211> 124
<212> PRT
<213> 解淀粉芽孢杆菌IT-45
<400> 3
Met Lys Pro Gly Ala Ile Gln Val Ala Lys Gly Thr Ile Thr Ile Asn
1 5 10 15
Glu Gly Arg Lys Thr Leu Glu Val Ser Val Thr Asn Asn Gly Thr Arg
20 25 30
Ser Val Gln Val Gly Ser His Phe His Phe Ala Glu Ala Asn Gly Ala
35 40 45
Leu Ser Phe Asn Arg Asp Lys Ala Ile Gly Met Arg Leu Asp Ile Pro
50 55 60
Ser Gly Thr Ser Val Arg Phe Glu Pro Gly Glu Glu Lys Thr Val Thr
65 70 75 80
Leu Val Glu Ile Gly Gly Arg Lys Thr Val Arg Gly Leu Asn Gly Met
85 90 95
Ala Asp Thr Tyr Met Asp Glu Arg Gly Lys Glu Lys Thr Leu Ser Asn
100 105 110
Leu Lys Lys Ala Gly Trp Met Glu Glu Ala Ile Arg
115 120
<210> 4
<211> 569
<212> PRT
<213> 解淀粉芽孢杆菌IT-45
<400> 4
Met Lys Met Ser Arg Glu Gln Tyr Ala Glu Leu Phe Gly Pro Thr Thr
1 5 10 15
Gly Asp Lys Val Arg Leu Gly Asp Thr Asp Leu Trp Ile Glu Val Glu
20 25 30
Lys Asp Phe Arg Asn Tyr Gly Glu Glu Met Ile Phe Gly Gly Gly Lys
35 40 45
Thr Ile Arg Asp Gly Met Gly Gln Asn Gly Arg Ile Thr Gly Lys Asp
50 55 60
Gly Ala Leu Asp Leu Val Ile Thr Asn Ala Val Ile Leu Asp Tyr Ile
65 70 75 80
Gly Ile Val Lys Ala Asp Ile Gly Val Lys Asp Gly Arg Ile Val Gly
85 90 95
Val Gly Lys Ser Gly Asn Pro Asp Met Met Asp Gly Val Asp Pro His
100 105 110
Met Ile Ile Gly Ala Gly Thr Glu Val Ile Ser Gly Glu Gly Lys Ile
115 120 125
Val Thr Ala Gly Gly Val Asp Thr His Ile His Phe Ile Cys Pro Gln
130 135 140
Gln Met Glu Val Ala Leu Ser Ser Gly Val Thr Thr Leu Leu Gly Gly
145 150 155 160
Gly Thr Gly Pro Ala Thr Gly Ser Lys Ala Thr Thr Cys Thr Ser Gly
165 170 175
Ala Trp Tyr Met Ser Arg Met Leu Glu Ala Ala Glu Glu Phe Pro Ile
180 185 190
Asn Val Gly Phe Leu Gly Lys Gly Asn Ala Ser Asp Lys Ala Pro Leu
195 200 205
Ile Glu Gln Val Glu Ala Gly Ala Ile Gly Leu Lys Leu His Glu Asp
210 215 220
Trp Gly Ser Thr Pro Ser Ala Ile Lys Ala Cys Met Glu Ala Ala Asp
225 230 235 240
Glu Ala Asp Ile Gln Val Ala Ile His Thr Asp Thr Ile Asn Glu Ala
245 250 255
Gly Phe Leu Glu Asn Thr Leu Asp Ala Ile Gly Asp Arg Val Ile His
260 265 270
Thr Tyr His Ile Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Met
275 280 285
Lys Leu Ala Ser Tyr Ala Asn Ile Leu Pro Ser Ser Thr Thr Pro Thr
290 295 300
Ile Pro Tyr Thr Val Asn Thr Met Asp Glu His Leu Asp Met Met Met
305 310 315 320
Val Cys His His Leu Asp Ser Lys Val Pro Glu Asp Val Ala Phe Ser
325 330 335
His Ser Arg Ile Arg Ala Ala Thr Ile Ala Ala Glu Asp Ile Leu His
340 345 350
Asp Ile Gly Ala Ile Ser Met Thr Ser Ser Asp Ser Gln Ala Met Gly
355 360 365
Arg Val Gly Glu Val Ile Ile Arg Thr Trp Gln Val Ala Asp Lys Met
370 375 380
Lys Lys Gln Arg Gly Ala Leu Ser Gly Glu Asn Gly Asn Asp Asn Val
385 390 395 400
Arg Ala Lys Arg Tyr Ile Ala Lys Tyr Thr Ile Asn Pro Ala Val Thr
405 410 415
His Gly Leu Ser His Glu Val Gly Ser Val Glu Lys Gly Lys Leu Ala
420 425 430
Asp Leu Val Leu Trp Asp Pro Val Phe Phe Gly Val Lys Pro Glu Leu
435 440 445
Val Leu Lys Gly Gly Met Ile Ala Arg Ala Gln Met Gly Asp Pro Asn
450 455 460
Ala Ser Ile Pro Thr Pro Glu Pro Val Phe Met Arg Gln Met Tyr Ala
465 470 475 480
Ser Tyr Gly Lys Ala Asn Arg Asn Thr Ser Ile Thr Phe Met Ser Gln
485 490 495
Ala Gly Ile Ala Asn Gly Val Pro Glu Lys Leu Gly Leu Glu Lys Met
500 505 510
Ile Ser Pro Val Arg Asn Ile Arg Lys Leu Ser Lys Leu Asp Met Lys
515 520 525
Leu Asn Asp Ala Met Pro Asn Ile His Val Asp Pro Lys Thr Tyr Gln
530 535 540
Val Phe Ala Asp Gly Glu Glu Leu Ala Cys Gln Pro Val Ser Tyr Val
545 550 555 560
Pro Leu Gly Gln Arg Tyr Phe Leu Phe
565
<210> 5
<211> 2565
<212> DNA
<213> 人工序列
<400> 5
gacacgataa actttacacc cgtcacgcgg tttggaggag gtaatttaat gcaccaccac 60
caccaccaca aacttacacc tgttgaacaa gaaaaacttc ttatcttcac agctggcgaa 120
cttgctaaac aacgtaaagc tcgtggcgtt cttcttaact accctgaagc tgctgcttac 180
cttacatgct accttatgga aggcgctcgt gatggcaaat ctgttgctga acttatggaa 240
tctggccgta acgttcttac agaaaaagat gttatggaag gcgttgctga aatgcttgat 300
tctatccaag ttgaagctac attccctgat ggcgttaaac ttgttacagt tcatcaacct 360
atcaaagctg aagttaaatc ttaatgaact cgagtaatcg aaaaggaggt attttttatg 420
aaacctggcg ctatccaagt tgctaaaggc acaatcacaa tcaacgaagg ccgtaaaaca 480
cttgaagttt ctgttacaaa caacggcaca cgttctgttc aagttggctc tcatttccat 540
ttcgctgaag ctaacggcgc tctttctttc aaccgtgata aagctatcgg catgcgtctt 600
gatatccctt ctggcacatc tgttcgtttc gaacctggcg aagaaaaaac agttacactt 660
gttgaaatcg gcggccgtaa aacagttcgt ggccttaacg gcatggctga tacatacatg 720
gatgaacgtg gcaaagaaaa aacactttct aaccttaaaa aagctggctg gatggaagaa 780
gctatccgtt aatgcacttt ccggggaaat tcgccggaca aagactgagg tttatatatg 840
caccaccacc accaccacaa aatgtctcgt gaacaatacg ctgaactttt cggccctaca 900
acaggcgata aagttcgtct tggcgataca gatctttgga tcgaagttga aaaagatttc 960
cgtaactacg gcgaagaaat gatcttcggc ggcggcaaaa caatccgtga tggcatgggc 1020
caaaacggcc gtatcacagg caaagatggc gctcttgatc ttgttatcac aaacgctgtt 1080
atccttgatt acatcggcat cgttaaagct gatatcggcg ttaaagatgg ccgtatcgtt 1140
ggcgttggca aatctggcaa ccctgatatg atggatggcg ttgatcctca tatgatcatc 1200
ggcgctggca cagaagttat ctctggcgaa ggcaaaatcg ttacagctgg cggcgttgat 1260
acacatatcc atttcatctg ccctcaacaa atggaagttg ctctttcttc tggcgttaca 1320
acacttcttg gcggcggcac aggccctgct acaggctcta aagctacaac atgcacatct 1380
ggcgcttggt acatgtctcg tatgcttgaa gctgctgaag aattccctat caacgttggc 1440
ttccttggca aaggcaacgc ttctgataaa gctcctctta tcgaacaagt tgaagctggc 1500
gctatcggcc ttaaacttca tgaagattgg ggctctacac cttctgctat caaagcttgc 1560
atggaagctg ctgatgaagc tgatatccaa gttgctatcc atacagatac aatcaacgaa 1620
gctggcttcc ttgaaaacac acttgatgct atcggcgatc gtgttatcca tacataccat 1680
atcgaaggcg ctggcggcgg ccatgctcct gatatcatga aacttgcttc ttacgctaac 1740
atccttcctt cttctacaac acctacaatc ccttacacag ttaacacaat ggatgaacat 1800
cttgatatga tgatggtttg ccatcatctt gattctaaag ttcctgaaga tgttgctttc 1860
tctcattctc gtatccgtgc tgctacaatc gctgctgaag atatccttca tgatatcggc 1920
gctatctcta tgacatcttc tgattctcaa gctatgggcc gtgttggcga agttatcatc 1980
cgtacatggc aagttgctga taaaatgaaa aaacaacgtg gcgctctttc tggcgaaaac 2040
ggcaacgata acgttcgtgc taaacgttac atcgctaaat acacaatcaa ccctgctgtt 2100
acacatggcc tttctcatga agttggctct gttgaaaaag gcaaacttgc tgatcttgtt 2160
ctttgggacc ctgttttctt cggcgttaaa cctgaacttg ttcttaaagg cggcatgatc 2220
gctcgtgctc aaatgggcga tcctaacgct tctatcccta cacctgaacc tgttttcatg 2280
cgtcaaatgt acgcttctta cggcaaagct aaccgtaaca catctatcac attcatgtct 2340
caagctggca tcgctaacgg cgttcctgaa aaacttggcc ttgaaaaaat gatctctcct 2400
gttcgtaaca tccgtaaact ttctaaactt gatatgaaac ttaacgatgc tatgcctaac 2460
atccatgttg atcctaaaac ataccaagtt ttcgctgatg gcgaagaact tgcttgccaa 2520
cctgtttctt acgttcctct tggccaacgt tacttccttt tctaa 2565
<210> 6
<211> 2565
<212> DNA
<213> 人工序列
<400> 6
tgcactttcc ggggaaattc gccggacaaa gactgaggtt tatatatgca ccaccaccac 60
caccacaaaa tgtctcgtga acaatacgct gaacttttcg gccctacaac aggcgataaa 120
gttcgtcttg gcgatacaga tctttggatc gaagttgaaa aagatttccg taactacggc 180
gaagaaatga tcttcggcgg cggcaaaaca atccgtgatg gcatgggcca aaacggccgt 240
atcacaggca aagatggcgc tcttgatctt gttatcacaa acgctgttat ccttgattac 300
atcggcatcg ttaaagctga tatcggcgtt aaagatggcc gtatcgttgg cgttggcaaa 360
tctggcaacc ctgatatgat ggatggcgtt gatcctcata tgatcatcgg cgctggcaca 420
gaagttatct ctggcgaagg caaaatcgtt acagctggcg gcgttgatac acatatccat 480
ttcatctgcc ctcaacaaat ggaagttgct ctttcttctg gcgttacaac acttcttggc 540
ggcggcacag gccctgctac aggctctaaa gctacaacat gcacatctgg cgcttggtac 600
atgtctcgta tgcttgaagc tgctgaagaa ttccctatca acgttggctt ccttggcaaa 660
ggcaacgctt ctgataaagc tcctcttatc gaacaagttg aagctggcgc tatcggcctt 720
aaacttcatg aagattgggg ctctacacct tctgctatca aagcttgcat ggaagctgct 780
gatgaagctg atatccaagt tgctatccat acagatacaa tcaacgaagc tggcttcctt 840
gaaaacacac ttgatgctat cggcgatcgt gttatccata cataccatat cgaaggcgct 900
ggcggcggcc atgctcctga tatcatgaaa cttgcttctt acgctaacat ccttccttct 960
tctacaacac ctacaatccc ttacacagtt aacacaatgg atgaacatct tgatatgatg 1020
atggtttgcc atcatcttga ttctaaagtt cctgaagatg ttgctttctc tcattctcgt 1080
atccgtgctg ctacaatcgc tgctgaagat atccttcatg atatcggcgc tatctctatg 1140
acatcttctg attctcaagc tatgggccgt gttggcgaag ttatcatccg tacatggcaa 1200
gttgctgata aaatgaaaaa acaacgtggc gctctttctg gcgaaaacgg caacgataac 1260
gttcgtgcta aacgttacat cgctaaatac acaatcaacc ctgctgttac acatggcctt 1320
tctcatgaag ttggctctgt tgaaaaaggc aaacttgctg atcttgttct ttgggaccct 1380
gttttcttcg gcgttaaacc tgaacttgtt cttaaaggcg gcatgatcgc tcgtgctcaa 1440
atgggcgatc ctaacgcttc tatccctaca cctgaacctg ttttcatgcg tcaaatgtac 1500
gcttcttacg gcaaagctaa ccgtaacaca tctatcacat tcatgtctca agctggcatc 1560
gctaacggcg ttcctgaaaa acttggcctt gaaaaaatga tctctcctgt tcgtaacatc 1620
cgtaaacttt ctaaacttga tatgaaactt aacgatgcta tgcctaacat ccatgttgat 1680
cctaaaacat accaagtttt cgctgatggc gaagaacttg cttgccaacc tgtttcttac 1740
gttcctcttg gccaacgtta cttccttttc taagacacga taaactttac acccgtcacg 1800
cggtttggag gaggtaattt aatgcaccac caccaccacc acaaacttac acctgttgaa 1860
caagaaaaac ttcttatctt cacagctggc gaacttgcta aacaacgtaa agctcgtggc 1920
gttcttctta actaccctga agctgctgct taccttacat gctaccttat ggaaggcgct 1980
cgtgatggca aatctgttgc tgaacttatg gaatctggcc gtaacgttct tacagaaaaa 2040
gatgttatgg aaggcgttgc tgaaatgctt gattctatcc aagttgaagc tacattccct 2100
gatggcgtta aacttgttac agttcatcaa cctatcaaag ctgaagttaa atcttaatga 2160
actcgagtaa tcgaaaagga ggtatttttt atgaaacctg gcgctatcca agttgctaaa 2220
ggcacaatca caatcaacga aggccgtaaa acacttgaag tttctgttac aaacaacggc 2280
acacgttctg ttcaagttgg ctctcatttc catttcgctg aagctaacgg cgctctttct 2340
ttcaaccgtg ataaagctat cggcatgcgt cttgatatcc cttctggcac atctgttcgt 2400
ttcgaacctg gcgaagaaaa aacagttaca cttgttgaaa tcggcggccg taaaacagtt 2460
cgtggcctta acggcatggc tgatacatac atggatgaac gtggcaaaga aaaaacactt 2520
tctaacctta aaaaagctgg ctggatggaa gaagctatcc gttaa 2565
<210> 7
<211> 42
<212> DNA
<213> 人工序列
<400> 7
ggtaagagag gaatgtacac atgaaactga caccggttga ac 42
<210> 8
<211> 42
<212> DNA
<213> 人工序列
<400> 8
gaccatgatt acgccaagct tttaaaataa gaaataacgc tg 42
<210> 9
<211> 39
<212> DNA
<213> 人工序列
<400> 9
aagcttggcg taatcatggt catagctgtt tcctgtgtg 39
<210> 10
<211> 40
<212> DNA
<213> 人工序列
<400> 10
gtgtacattc ctctcttacc tataatggta ccgctatcac 40
<210> 11
<211> 46
<212> DNA
<213> 人工序列
<400> 11
cgcgcgatta tgtaaaatat aagacacgat aaactttaca cccgtc 46
<210> 12
<211> 42
<212> DNA
<213> 人工序列
<400> 12
gaccatgatt acgccaagct tttagaaaag gaagtaacgt tg 42
<210> 13
<211> 36
<212> DNA
<213> 人工序列
<400> 13
aagcttggcg taatcatggt catagctgtt tcctgt 36
<210> 14
<211> 42
<212> DNA
<213> 人工序列
<400> 14
ttatatttta cataatcgcg cgcttttttt cacgcccatt tc 42
<210> 15
<211> 44
<212> DNA
<213> 人工序列
<400> 15
cgcgcgatta tgtaaaatat aatgcacttt ccggggaaat tcgc 44
<210> 16
<211> 44
<212> DNA
<213> 人工序列
<400> 16
gaccatgatt acgccaagct tttaacggat agcttcttcc atcc 44
Claims (10)
1.编码脲酶的基因,其特征在于,其核苷酸序列如SEQ ID NO.1、SEQ ID NO.5或SEQ IDNO.6所示。
2.表达权利要求1所述基因的重组枯草芽孢杆菌,其特征在于,宿主选自:B.subtilisWB600、B.subtilis WB700、B.subtilis WB800或B.subtilis WB168,表达载体选自:pP43NMK、pHT43、pMA5或pTTB。
3.一种构建表达脲酶基因的重组枯草芽孢杆菌的方法,其特征在于,包括以下步骤:
(1)将来源于Bacillus amyloliquefaciens IT-45的脲酶基因簇Ba-urease序列进行枯草芽孢杆菌密码子优化、核糖体结合位点优化,获得如SEQ ID NO.5所示的脲酶基因Ba-urease-1;
(2)在如SEQ ID NO.5所示的脲酶基因Ba-urease-1的基础上,将ureC移置至ureA和ureB的上游,从而获得序列如SEQ ID NO.6所示的脲酶基因Ba-urease-2,将其与表达载体pP43NMK连接,构建脲酶表达载体pP43NMK-Ure-2;
(3)将pP43NMK-Ure-2转化至B.subtilis WB600中,获得重组表达菌株B.subtilisWB600/pP43NMK-Ure-2。
4.一种表达脲酶基因的重组枯草芽孢杆菌,其特征在于,以B.subtilis WB600为宿主,以pP43NMK载体表达SEQ ID NO.6所示的脲酶基因。
5.应用权利要求4所述重组枯草芽孢杆菌制备脲酶的方法,其特征在于,包括以下步骤:
挑取重组枯草芽孢杆菌单菌落,接种于含有50μg/mL卡那霉素的5mL LB液体培养基中,在37℃、200r/min的摇床中振荡培养10h;
按5%的接种体积分数,转接于含有50μg/mL卡那霉素、4mmol/L Ni2+的100mL TB液体培养基中,在37℃、200r/min的摇床中振荡培养8h,于28℃诱导培养42h。
6.携带权利要求1所述基因的载体。
7.解淀粉芽孢杆菌脲酶基因的食品级表达方法,其特征在于,对解淀粉芽孢杆菌IT-45来源的脲酶基因簇进行密码子优化、核糖体结合位点优化重构以及将结构基因ureC调整至ureA、ureB上游,然后与表达载体连接,并转化至食品级表达宿主,培养重组宿主使之表达脲酶。
8.权利要求1所述基因编码的脲酶。
9.权利要求8所述脲酶在降解尿素中的应用。
10.根据权利要求9所述的应用,其特征在于,利用脲酶来降低或消除发酵食品和酒精饮料中的尿素含量以降低氨基甲酸乙酯。
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CN106011118A (zh) * | 2016-06-07 | 2016-10-12 | 江南大学 | 一种Fe3+依赖型食品级酸性脲酶及其在黄酒中的应用 |
CN111254135A (zh) * | 2020-03-03 | 2020-06-09 | 江南大学 | 一种应用性能提高的脲酶突变体 |
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CN106011118A (zh) * | 2016-06-07 | 2016-10-12 | 江南大学 | 一种Fe3+依赖型食品级酸性脲酶及其在黄酒中的应用 |
CN111254135A (zh) * | 2020-03-03 | 2020-06-09 | 江南大学 | 一种应用性能提高的脲酶突变体 |
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