CN103131659A - 一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用 - Google Patents
一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用 Download PDFInfo
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- CN103131659A CN103131659A CN201310089859XA CN201310089859A CN103131659A CN 103131659 A CN103131659 A CN 103131659A CN 201310089859X A CN201310089859X A CN 201310089859XA CN 201310089859 A CN201310089859 A CN 201310089859A CN 103131659 A CN103131659 A CN 103131659A
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
本发明公开了一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用，属于微生物工程与酶工程技术领域。该菌株分类命名为PseudomonasstutzeriZS04，其保藏登记号为：CCTCCNO：M2012542，为革兰氏阴性菌株，能耐受一定浓度的多种有机溶剂。本发明分离克隆到该菌所产耐有机溶剂的脂肪酶编码基因及相应的折叠酶基因。该耐有机溶剂脂肪酶具有产量高、溶剂耐受性强、作用pH范围广等性质。该脂肪酶具有在有机相中催化手性化合物拆分等工业应用价值。
Description
技术领域
本发明属于微生物工程与酶工程技术领域，具体涉及一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用。
背景技术
脂肪酶（lipase, EC 3. 1. 1. 3 ,甘油三酰酯水解酶)是类脂化合物分解、合成和酯交换的重要催化剂，其催化反应具有选择性高、反应条件温和、无污染等特点，因此广泛应用于精细化工及手性拆分药物中间体的研究。
脂肪酶在有机相中可以完成酯化、转酯和酰胺化等反应，并具有区域选择性、立体选择性、较高的稳定性，尤其是它的立体选择催化特性，可以用于化学法难以进行的消旋化合物的拆分、不对称合成等。近年来，国内外学者已经致力于应用脂肪酶在有机体系中实现手性化合物，尤其是药物中间体的立体选择性拆分，如利用微生物脂肪酶可完成萘普生、布洛芬、西司他汀重要中间体2,2-二甲基丙烷甲酸等手性药物的立体选择性的拆分。与传统的化学方法相比，酶催化拆分外消旋化合物具有反应条件温和、节省能源、专一性强、副反应少、产品纯度高、反应步骤简单、不需手性源、产品成本低等优点。
脂肪酶在水环境中能够催化酯键的水解，当处于有机溶剂环境中能催化酯交换，酯化，醇解，酸解等反应。而脂肪酶作用的底物往往是水不溶性的，在有机相中才有较好的溶解度，但是在有机溶剂中，大多数酶类活力受到抑制甚至失活，所以其在生物催化中的应用受到很大限制。在有机介质中维持较高的脂肪酶活性及脂肪酶催化底物的广泛性是实际生物催化过程中的难题。因此开发耐有机溶剂脂肪酶在理论和工业应用上具有重要的意义。
发明内容
本发明的目的是提供一种耐有机溶剂脂肪酶的产生菌，其产脂肪酶能力高，纯化过程简单。
本发明的另一目的是提供一种耐有机溶剂脂肪酶，该脂肪酶具有优良的有机溶剂耐受性。
本发明的另一目的是提供上述脂肪酶的折叠酶。
本发明的再一目的是提供耐有机溶剂脂肪酶在有机相中催化手性化合物拆分的应用。
为了实现本发明的目的，本发明首先从油污土样中筛选获得一株有机溶剂脂肪酶生产菌，经BIOLOG全自动细菌鉴定仪鉴定和16S rDNA序列分析，确定该菌株的分类命名为斯氏假单胞菌（Pseudomonas stutzeri），株名为ZS04，保藏日期为2012年12月21日，保藏单位全称为中国典型培养物保藏中心，简称CCTCC，保藏编号：CCTCC NO：M 2012542，保藏单位地址为：中国，武汉，武汉大学。
本发明对斯氏假单胞菌Pseudomonas stutzeri ZS04的生物学特征进行鉴定，该菌株为革兰氏阴性菌株，无芽孢。在肉汤培养基中生长24 h后，菌落直径大小为0.6 mm～1.2mm，生长范围为24℃～37℃，最适生长温度为35℃，生长pH为6.0～9.0，最适生长pH为8.0。其生理生化特性表现在：过氧化氢酶反应、氧化酶反应、硝酸还原反应结果为阳性，明胶反应结果为阴性，在有氧条件下生长。
本发明对该菌产生的胞外酶进行了纯化，SP-sepharose FF 阳离子交换层析一步纯化得到电泳纯的耐有机溶剂脂肪酶，命名为耐有机溶剂脂肪酶ZS04，其比活性达到398.2 U/mg。
本发明对耐有机溶剂脂肪酶ZS04进行了酶学性质的研究。实验证明该脂肪酶对多种有机溶剂都具有良好的耐受性，有机溶剂中脂肪酶的稳定性提高。其中，亲水性有机溶剂可激活该脂肪酶，使得酶活提高。该耐有机溶剂脂肪酶的最适反应pH为9.0，在pH 7.5～11.0的范围具有很高的稳定性。其最适反应温度为50℃，在30℃以下具有较好的热稳定性。金属离子Ca2+，Zn2+，Co3+等离子可提高该脂肪酶的热稳定性。此酶的最适人工底物为对硝基苯酚月桂酸酯。
本发明提供斯氏假单胞菌（Pseudomonas stutzeri）ZS04所产耐有机溶剂脂肪酶ZS04的氨基酸序列，如SEQ ID NO：2所示，前24个氨基酸为信号肽。耐有机溶剂脂肪酶ZS04与Pseudomonas stutzeri A1501脂肪酶基因同源性为90%，氨基酸同源性为93%。
本发明提供耐有机溶剂脂肪酶ZS04的编码基因，如SEQ ID NO：1。该耐有机溶剂脂肪酶编码基因全长为936bp，编码311个氨基酸。
本发明提供耐有机溶剂脂肪酶ZS04的折叠酶氨基酸序列，如于SEQ ID NO：4所示。
所述折叠酶的编码基因，如SEQ ID NO：3所示，长度为1014bp，编码337个氨基酸。与Pseudomonas stutzeri CCUG 29243折叠酶的基因同源性为91%，氨基酸同源性为83%。
采用耐有机溶剂脂肪酶ZS04上游引物LipAF（SEQ ID NO:6）和其相应折叠酶下游引物LifR（SEQ ID NO:11），PCR扩增得到耐有机溶剂脂肪酶ZS04及其折叠酶的基因，它具有SEQ ID NO：5所示的核苷酸序列。耐有机溶剂脂肪酶ZS04及其折叠酶基因全长为2013bp，折叠酶基因在耐有机溶剂脂肪酶ZS04基因下游63bp处。
本发明还提供耐有机溶剂脂肪酶在有机相中催化手性化合物拆分反应中的应用。所述手性化合物拆分反应可以为拆分1-苯乙醇反应、拆分1-（4-甲氧基苯基）-乙醇反应以及拆分3-甲氧基苯氧基-1,2-丙二醇。所述的耐有机溶剂脂肪酶在有机溶剂正己烷体系或异丙醚体系中，催化底物1-（4-甲氧基苯基）-乙醇和乙酸乙烯酯进行酯交换反应，转化率达42～52％，ees值99.9%。
有益效果：斯氏假单胞菌（Pseudomonas stutzeri）ZS04发酵制备耐有机溶剂脂肪酶ZS04的产率高，发酵60 h脂肪酶活力达到210 U/mL；耐有机溶剂脂肪酶ZS04易纯化，经过一步离子交换层析后即可达到电泳纯，比活达到398.2U/mg；该脂肪酶为碱性中温脂肪酶，对有机溶剂耐受性强，尤其是对疏水性有机溶剂的耐受性使其能够应用于1-（4-甲氧基苯基）-乙醇的拆分。耐有机溶剂脂肪酶ZS04也可在医药、洗涤剂、食品和皮革等许多工业领域中应用。
附图说明
图1为耐有机溶剂脂肪酶的SDS-PAGE电泳图，其中泳道1-粗酶液；泳道2-纯酶液（经离子交换层析后得到的纯酶液）；泳道3-Marker。
图2显示耐有机溶剂脂肪酶ZS04的最适反应pH。
图 3 显示耐有机溶剂脂肪酶ZS04的pH稳定性。
图4显示耐有机溶剂脂肪酶ZS04的最适反应温度。
图 5 显示耐有机溶剂脂肪酶ZS04的温度稳定性及金属离子对温度稳定性的影响。
图6显示耐有机溶剂脂肪酶ZS04的底物特异性，其中C2为对硝基苯酚乙酸酯,C4为对硝基苯酚丁酸酯，C8为对硝基苯酚辛酸酯，C10为对硝基苯酚癸酸酯，C12为对硝基苯酚月桂酸酯，C14为对硝基苯酚肉豆蔻酸酯，C16为对硝基苯酚棕榈酸酯，C18为对硝基苯酚硬脂酸酯。
图7显示耐有机溶剂脂肪酶ZS04及其相应折叠酶的基因示意图。
图8是BL21-pET-Lip菌体裂解液沉淀的SDS-PAGE电泳图，其中M是蛋白分子量Mark，泳道1是含有空载体pET22b的E.coli BL21的菌体裂解液沉淀，泳道2是BL21-pET-Lip菌体裂解液沉淀。
图 9是BL21-pET-LipB菌体裂解液上清SDS-PAGE电泳后的活性染色图，其中M是蛋白分子量Mark，泳道4、6、8、10、12和21分别是诱导4、6、8、10、12和21h的BL21-pET-LipB菌体裂解液上清。
图10是BL21-pET-LipB菌体裂解液沉淀的SDS-PAGE电泳图，其中M是蛋白分子量Mark，泳道4、6、8、10、12和21分别是诱导4、6、8、10、12和21h的BL21-pET-LipB菌体裂解液沉淀。
具体实施方式
本发明中吐温-80平板培养基配方为：酵母膏5 g/L，蛋白胨 10 g/L，NaCl 10 g/L，吐温-80 10 mL/L，CaCl2 1 g/L，琼脂20 g/L，溶剂为水。
产酶发酵培养基配方为：蛋白胨5 g/L，酵母粉5 g/L，尿素5 g/L， K2HPO43H2O 1.8 g/L，MgSO47H2O 0.7 g/L，橄榄油5 mL/L，TritonX-100 0.5 mL/L ，pH 8.0，溶剂为水。
脂肪酶活力检测方法（以对硝基苯酚棕榈酸酯为底物）为：A溶液：50mM的Na2HPO412H2O-NaH2PO42H2O缓冲液（pH 7.0），其中含有6g/L的riton X-100和1g/L的阿拉伯树胶；B溶液：称取3 mg的对硝基苯酚棕榈酸酯(p-Nitrophenylpalmitate, pNPP)，溶解在1 mL的异丙醇中；A溶液与B溶液按体积比9:1制成浓度为16.5 mM的对硝基苯酚棕榈酸酯底物溶液。反应体系中先加入10 μL稀释适当倍数的酶液，以灭活的酶液为空白对照，再加入240 μL底物溶液，在酶标仪中进行反应，反应温度为40℃，反应时间为10 min，在410 nm波长下检测反应结束时生成的对硝基苯酚(pNP)的量。每1个单位（U）脂肪酶酶活定义为，在上述条件下，每分钟催化产生1 μmol对硝基苯酚(pNP)所需的酶量。
实施例一 耐有机溶剂脂肪酶产生菌的筛选
初筛采用如下方法：以植物油为唯一碳源，15%环己烷、甲苯、DMSO等有机溶剂为筛选压力从油污土壤等样品中筛选获得耐有机溶剂极端微生物。将所筛选到的耐有机溶剂微生物接种于吐温-80平板，根据沉淀圈与菌落大小的比值，初步筛选脂肪酶产量高的菌株。通过初筛获得5株脂肪酶产量高的耐有机溶剂极端微生物。
复筛：为了进一步检测脂肪酶的溶剂耐受性，对初筛获得的5株菌脂肪酶产生能力和所产脂肪酶的耐有机溶剂性质进行全面的检测。将5株菌分别接种到产酶发酵培养基，培养温度为30℃，培养时间为60 h，摇床转速为180 rmp。发酵结束后，将发酵液在10000 rmp 、4℃条件下离心15 min，取上清为粗酶液。取1.5 mL粗酶液分别加入0.5mL的异丙醚、十六烷、壬烷、正辛烷、异辛烷、正庚烷、正己烷、异丙醇、丙酮、乙醇、甲醇、二甲基甲酰胺（DMF）和二甲基亚砜（DMSO），于30℃、150 rpm条件下振荡处理24 h，以pNPP为底物检测脂肪酶剩余酶活；选取具有最高剩余活力的脂肪酶的产生菌株ZS04。菌株ZS04发酵60h，粗酶液的脂肪酶活力达到210U/ml。
实施例二耐有机溶剂脂肪酶产生菌ZS04的生物学性质及鉴定
菌株ZS04的生物学性质：革兰氏染色表明此菌株为革兰氏阴性菌株，无芽孢。在肉汤培养基中生长24 h后，菌落直径大小为0.6 mm～1.2mm，生长范围为24℃～37℃，最适生长温度为35℃，生长pH为6.0～9.0，最适生长pH为8.0。其生理生化特性表现在：过氧化氢酶反应、氧化酶反应、硝酸还原反应结果为阳性，明胶反应结果为阴性，在有氧条件下生长。
菌株ZS04的菌种鉴定：经BIOLOG自动细菌鉴定仪鉴定，菌株ZS04与Pseudomonas stutzeri属的Sim值为0.507。菌株ZS04的16S rDNA序列与斯氏假单胞菌同源性为99%。因此，将菌株ZS04命名为斯氏假单胞菌（Pseudomonas stutzeri）ZS04。
实施例三 耐有机溶剂脂肪酶ZS04的纯化方法。
将斯氏假单胞菌（Pseudomonas stutzeri）ZS04在产酶培养基中培养60 h，取发酵液在8,000rmp、4℃条件下离心20 min，取上清作为粗酶液。向粗酶液中加入0.05 M、pH 6.0的Na2HPO4-NaH2PO4缓冲液, 透析平衡。将以上处理得到的酶液，采用SP-Sepharose FF离子交换层析柱进行纯化，以0.05 M的Na2HPO4-NaH2PO4（pH 6.0，NaC1含量为0 mol/L -2 mol/L）缓冲液进行梯度洗脱，收集脂肪酶活力峰。通过SDS-PAGE电泳图（图1），发现一步纯化后的耐有机溶剂脂肪酶（命名为耐有机溶剂脂肪酶ZS04）已达电泳纯，该脂肪酶亚基分子量约为32 kDa。纯化倍数为7.1倍，回收率为15.1%，最终脂肪酶比活达到398.2U/mg，汇总见表1。
表1 耐有机溶剂脂肪酶ZS04的纯化步骤及结果
注：蛋白质浓度采用考马斯亮兰法测定
实施例四耐有机溶剂脂肪酶ZS04及其折叠酶基因的分离克隆及异源共表达
采用酚－氯仿法抽提斯氏假单胞菌（Pseudomonas stutzeri）ZS04菌体总DNA。
委托国家生物医学分析中心(NBCA)，采用LC-MS/MS测定耐有机溶剂脂肪酶ZS04氨基酸片段。结果表明该酶与Pseudomonas mendocina PK-12CS的脂肪酶序列最相近，因此根据Pseudomonas mendocina PK-12CS脂肪酶CDS两端外各约100 bp处设计引物LipAF和LipAR，扩增耐有机溶剂脂肪酶ZS04的CDS编码序列。
LipAF（SEQ ID NO:6）：ATCGTTGCGATCCGCCAGTTAG
LipAR（SEQ ID NO:7）：ATTCGGGCCGGGCGATAAAGAA
PCR扩增体系：菌体总DNA 1μL，LipAF 1μL，LipAR 1μL，10*buffer 2.5μL，2.5mM dNTPs 2μL，LA taq 0.3μL，ddH2O 补足体积至 25μL。
PCR反应参数为：94℃预变性2 min；94℃变性30 sec，60℃退火30 sec，72℃延伸1 min 30 sec，循环30轮；72℃保温10 min。
将含有CDS编码序列的PCR片段电泳回收后克隆到pMD18-T载体，进行序列分析。耐有机溶剂脂肪酶ZS04的基因序列如SEQ ID NO: 1所示，大小为936 bp。编码311个氨基酸（SEQ ID NO: 2），与Pseudomonas stutzeri A1501脂肪酶基因同源性为90%。
由于Pseudomonas stutzeri A1501脂肪酶有其对应的折叠酶，推测耐有机溶剂脂肪酶ZS04也有其相对应的折叠酶ZS04，因此根据Pseudomonas stutzeri A1501折叠酶CDS两端外各约100 bp处设计引物LipBF和LipBR，扩增耐有机溶剂脂肪酶ZS04的折叠酶的CDS编码序列。
LipBF（SEQ ID NO:8）：CAGCACGCGAACCGATTGAA；
LipBR（SEQ ID NO:9）：CGTTCATCCTCGCTGCCTACAA。
PCR扩增体系：菌体总DNA 1μL，LipBF 1μL，LipBR 1μL，10*buffer 2.5μL，2.5mM dNTPs 2μL，LA taq 0.3μL，ddH2O 补足体积至 25μL。
PCR反应参数为：94℃预变性2 min；94℃变性30 sec，60℃退火30 sec，72℃延伸1 min 30 sec，循环30轮；72℃保温10 min。
扩增得到的片段为1.4 kb，回收后克隆到pMD18-T载体。通过序列分析发现，折叠酶的基因序列如SEQ ID NO: 3所示，全长为1011 bp，编码337个氨基酸，具体的氨基酸序列如SEQ ID NO: 4所示。该折叠酶的基因与Pseudomonas stutzeri CCUG 29243脂肪酶折叠酶基因同源性为91%。
采用耐有机溶剂脂肪酶ZS04上游引物LipAF（SEQ ID NO:6）和其相应折叠酶下游引物LifR（SEQ ID NO:11），PCR扩增得到耐有机溶剂脂肪酶ZS04其相应的折叠酶的基因，它具有SEQ ID NO：5所示的核苷酸序列。耐有机溶剂脂肪酶及其折叠酶基因全长长度为2013bp，该折叠酶基因在耐有机溶剂脂肪酶ZS04基因下游63bp处，如图7所示。
对上述克隆到的耐有机溶剂脂肪酶ZS04及其折叠酶进行异源共表达。采用引物LipdF和LifR从斯氏假单胞菌（Pseudomonas stutzeri）ZS04总DNA中扩增耐有机溶剂脂肪酶ZS04及其折叠酶。
LipdF（SEQ ID NO:10）: gaccatatggccaccggcagcggctatac；
LifR（SEQ ID NO:11）: aaaggatcctcagcgctccgccacttcctg。
PCR反应体系：菌体总DNA 1μL，LipdF 1μL，LifR 1μL，10*buffer 2.5μL，2.5mM dNTPs 2μL，LA taq 0.3μL，ddH2O 补足体积至 25μL。
PCR反应参数为：94℃预变性2 min；94℃变性30 sec，60℃退火30 sec，72℃延伸2 min，循环30轮；72℃保温10 min。
将扩增得到的片段及载体pET_22b(+)分别进行双酶切。双酶切体系：目的片段/载体pET_22b(+)25μL，NdeⅠ1.5μL，BamHⅠ1.5μL，10*K Buffer 5μL，ddH2O 17μL。双酶切5小时后电泳纯化连接。连接体系：双酶切后的目的片段6.6μL，双酶切后的载体pET_22b(+) 2μL，T4 ligase 0.4μL，T4 Buffer 1μL。将连接产物经电泳验证，发现载体中插入了耐有机溶剂脂肪酶ZS04及其折叠酶，记为pET-LipB。另外，构建仅插入耐有机溶剂脂肪酶ZS04的重组载体,记为pET-Lip。
将重组载体pET-LipB和pET-Lip分别采用热休克法转入E.coli BL21，采用氨苄抗性的LB平板筛选阳性克隆子BL21-pET-LipB和BL21-pET-Lip。将阳性克隆子分别转入加有50mM 氨苄的LB液体培养基，20℃，1mM IPTG低温诱导21小时。BL21-pET-LipB菌体裂解液上清脂肪酶活达到0.73U/mL。BL21-pET-Lip菌体裂解液上清和沉淀均无脂肪酶活力。将BL21-pET-Lip菌体裂解液上清和沉淀进行SDS-PAGE电泳，发现菌体裂解液沉淀在目的分子量处有明显条带，说明形成了大量的包涵体（图8），将胶条进行乙酸萘酯-快蓝活性染色，没有出现染色带。从这里可以看出，耐有机溶剂脂肪酶ZS04单独表达（不考虑脂肪酶折叠酶ZA04）不会有活性表达，也侧面说明了耐有机溶剂脂肪酶ZS04合成以后，必须在其折叠酶的帮助下正确折叠，才能够有脂肪酶活性。
将BL21-pET-LipB发酵液离心获得菌体，采用超声破碎后，获得菌体裂解液。将菌体裂解液离心，分别得到菌体裂解液上清和沉淀。将菌体裂解液上清进行SDS-PAGE电泳后，将胶条放入pH7.0的Na2HPO4-NaH2PO4（含有0.6%Triton X-100）进行复性，采用乙酸萘酯-快蓝活性染色，在分子量约为29 kDa处有明显的条带，说明重组脂肪酶ZS04表达成功（图9）。同时，将菌体裂解液沉淀进行SDS-PAGE电泳后，发现有有大量的包涵体（图10），说明了虽然发明人成功进行了耐有机溶剂脂肪酶ZS04的异源活性表达，但只是达到低量的活性表达，还有大量的蛋白没有进行正确的折叠，后续工作将研究脂肪酶及其折叠酶互相作用机制，从而实现耐有机溶剂脂肪酶ZS04大量的活性表达。
实施例五 耐有机溶剂脂肪酶ZS04的酶学性质
耐有机溶剂脂肪酶ZS04的有机溶剂耐受性：向12种有机溶剂中分别加入耐有机溶剂脂肪酶ZS04（按照实施三制备），其混合比例为1：3（V/V），对照中不添加有机溶剂。30℃，180 rpm振荡24 h后取样，以对硝基苯酚棕榈酸酯为底物检测脂肪酶活力。结果如表2所示。耐有机溶剂脂肪酶ZS04具有良好的有机溶剂耐受性。其中,亲水性有机溶剂对该脂肪酶具有一定的激活作用，例如DMSO、乙醇等显著激活脂肪酶活力。
表2 有机溶剂对脂肪酶ZS04的影响
| 有机溶剂 | LogP | 残余活力（%） |
| 对照 | - | 100 |
| 十六烷 | 8.8 | 97.9 |
| 正辛烷 | 4.5 | 108.1 |
| 正庚醇 | 2.4 | 75.0 |
| 正己烷 | 3.5 | 90.1 |
| 正辛醇 | 2.9 | 73.1 |
| 正丁醇 | 0.88 | 26.7 |
| 异丙醇 | 0.05 | 97.1 |
| 乙醇 | -0.24 | 111.6 |
| 甲醇 | -0.69 | 88.7 |
| DMF | -1 | 102.0 |
| DMSO | -1.35 | 141.2 |
| 丙酮 | -0.23 | 67.0 |
耐有机溶剂脂肪酶ZS04最适反应pH和pH稳定性的检测：为了研究耐有机溶剂脂肪酶ZS04的最适反应pH，以不同pH缓冲液溶解的对硝基苯酚棕榈酸酯为底物， pH 7.0条件下的脂肪酶活力为对照（100％）。不同pH体系中的相对酶活如图2所示。从图2可以看出，耐有机溶剂脂肪酶ZS04的最适反应pH为9.0，是碱性脂肪酶。以原始酶液的脂肪酶活力为对照，检测该酶的pH稳定性（图3）。将耐有机溶剂脂肪酶ZS04加入不同pH的缓冲溶液中30℃保温1 h后测残余酶活，实验表明该脂肪酶在pH 7.5～11.0的范围内具有较高的稳定性。
耐有机溶剂脂肪酶ZS04最适反应温度和热稳定性的检测：最适反应温度的测定在0.05 M Na2HPO412H2O-NaH2PO42H2O缓冲体系（pH 7.0）中进行，在不同的温度下以对硝基苯酚棕榈酸酯为底物进行酶促反应。结果显示该酶的最适反应温度为50℃(图4)。热稳定性测定：耐有机溶剂脂肪酶ZS04在30℃、40℃、50℃、60℃、70℃下处理1 h以后，测定残留酶活（图5）。在耐有机溶剂脂肪酶ZS04中分别添加终浓度为10mM Ca2+，Zn2+，Co3+等离子对该脂肪酶活力有明显的激活作用，10mmol/L的Ca2+可以提高其热稳定性，60℃保温一小时酶活几乎不下降（对照下降为原来的40%）。
耐有机溶剂脂肪酶ZS04底物特异性的检测：参照以对硝基苯酚棕榈酸酯为底物的脂肪酶活力测定方法来检测底物特异性，将对硝基苯酚棕榈酸酯（C16）分别替换为对硝基苯酚乙酸酯（C2）,对硝基苯酚丁酸酯（C4），对硝基苯酚辛酸酯（C8），对硝基苯酚癸酸酯（C10），对硝基苯酚月桂酸酯（C12），对硝基苯酚肉豆蔻酸酯（C14），对硝基苯酚棕榈酸酯（C16），对硝基苯酚硬脂酸酯（C18）。结果如图6，耐有机溶剂脂肪酶ZS04的最适人工底物为对硝基苯酚月桂酸酯。
实施例六 耐有机溶剂脂肪酶ZS04在有机相中拆分手性化合物的应用。
采用实施例三制备的耐有机溶剂脂肪酶ZS04进行下面的拆分试验。
将0.12 mmol的1-苯乙醇和0.48mmol的乙酸乙烯酯作为反应底物溶于4mL正己烷，加入20mg耐有机溶剂脂肪酶ZS04，在30℃，转速180rpm条件下反应，定时取样。样品用正己烷稀释适当倍数，采用HPLC进行检测。结果表明，反应24 h后，1-苯乙醇的转化率达到48.9%，产物对映体过剩值（eep）为99.9%。
以0.12mmol的1-（4-甲氧基苯基）-乙醇的和0.48 mmol的乙酸乙烯酯作为反应底物溶于4mL正己烷，加入20mg耐有机溶剂脂肪酶ZS04，在30℃，转速180rpm条件下反应，定时取样，采用HPLC进行检测。结果表明，反应48 h后，1-（4-甲氧基苯基）-乙醇的转化率达到49.1%,底物对映体过剩值（ees）为99.9%。
以0.08 mmol的3-甲氧基苯氧基-1,2-丙二醇和0.32mmol的乙酸乙烯酯作为反应底物溶于4mL异丙醚，加入25mg耐有机溶剂脂肪酶ZS04，在30℃，转速180rpm条件下反应，定时取样，采用HPLC进行检测。结果表明，反应48 h后，3-甲氧基苯氧基-1,2-丙二醇的转化率达到48.5%,eep为99.9%。
结果表明脂肪酶ZS04具有良好的对映体选择性，在手性化合物拆分中具有良好的应用，并在医药、洗涤剂、食品和皮革等许多工业领域中应用。
SEQUENCE LISTING
<110> 南京工业大学
<120> 一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用
<130> 20130320
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 936
<212> DNA
<213> 斯氏假单胞菌（Pseudomonas stutzeri）ZS04
<400> 1
atgaacaaga acaaaacctt gctcgccctc tgtctcggaa gcgccctcgc gctttccggt 60
caggcattcg ctgccaccgg cagcggctat accgccacga agtacccaat cgtcctcacc 120
cacggcatgc ttggcttcga cagcctattg ggcatcgact actggtacgg catccccagc 180
gctttgcgcc gcgacggcgc gcaggtctac atcaccgaag tcagccaact caacacctcc 240
gaactgcgtg gcgaggaact gctggcccag gtcgaggaaa tcgttgctat cagcggcaag 300
ccgaaggtca acctgatcgg ccacagccaa ggcggaccgg acatccgtta cgtggcaggc 360
gtacggccgg acctgatcgc ctcggtcacc agtgtcggcg cgccccacaa gggctcggac 420
gtggcggatc tgatccgcaa ggttccagag ggttcatccg gtgaggccat aatcgccggc 480
ttggtgaacg ccatgggcgc actgatcaac tttctttccg gcagcaacag cagcgctccg 540
cagaatgcac tcggcgcgct cgaatcgctc aacagcgagg gcgctgcgcg cttcaacgcg 600
aagtatcccc agggcatccc gaccagcgcc tgcggcgaag gggcctacgt ggtcaagggt 660
gtgcgctact actcctggag cgggacgagc ccgctgacca acccgctgga tgtcagcgac 720
gcagtgatgg tcgcgggctc gctcgcgttc gacgaagcca atgacggcct ggtcggacgc 780
tgcagctcgc atctgggcat ggtgatccgc gacaactacc ggatgaacca cctggacgag 840
gtcaaccagg tgctcggcct gaccagcctc ttcgaaacag acccagtgag cgtctatcgc 900
cagcacgcga accgattgaa gaacgcgggg ctctga 936
<210> 2
<211> 311
<212> PRT
<213> 斯氏假单胞菌（Pseudomonas stutzeri）ZS04
<400> 2
Met Asn Lys Asn Lys Thr Leu Leu Ala Leu Cys Leu Gly Ser Ala Leu
1 5 10 15
Ala Leu Ser Gly Gln Ala Phe Ala Ala Thr Gly Ser Gly Tyr Thr Ala
20 25 30
Thr Lys Tyr Pro Ile Val Leu Thr His Gly Met Leu Gly Phe Asp Ser
35 40 45
Leu Leu Gly Ile Asp Tyr Trp Tyr Gly Ile Pro Ser Ala Leu Arg Arg
50 55 60
Asp Gly Ala Gln Val Tyr Ile Thr Glu Val Ser Gln Leu Asn Thr Ser
65 70 75 80
Glu Leu Arg Gly Glu Glu Leu Leu Ala Gln Val Glu Glu Ile Val Ala
85 90 95
Ile Ser Gly Lys Pro Lys Val Asn Leu Ile Gly His Ser Gln Gly Gly
100 105 110
Pro Asp Ile Arg Tyr Val Ala Gly Val Arg Pro Asp Leu Ile Ala Ser
115 120 125
Val Thr Ser Val Gly Ala Pro His Lys Gly Ser Asp Val Ala Asp Leu
130 135 140
Ile Arg Lys Val Pro Glu Gly Ser Ser Gly Glu Ala Ile Ile Ala Gly
145 150 155 160
Leu Val Asn Ala Met Gly Ala Leu Ile Asn Phe Leu Ser Gly Ser Asn
165 170 175
Ser Ser Ala Pro Gln Asn Ala Leu Gly Ala Leu Glu Ser Leu Asn Ser
180 185 190
Glu Gly Ala Ala Arg Phe Asn Ala Lys Tyr Pro Gln Gly Ile Pro Thr
195 200 205
Ser Ala Cys Gly Glu Gly Ala Tyr Val Val Lys Gly Val Arg Tyr Tyr
210 215 220
Ser Trp Ser Gly Thr Ser Pro Leu Thr Asn Pro Leu Asp Val Ser Asp
225 230 235 240
Ala Val Met Val Ala Gly Ser Leu Ala Phe Asp Glu Ala Asn Asp Gly
245 250 255
Leu Val Gly Arg Cys Ser Ser His Leu Gly Met Val Ile Arg Asp Asn
260 265 270
Tyr Arg Met Asn His Leu Asp Glu Val Asn Gln Val Leu Gly Leu Thr
275 280 285
Ser Leu Phe Glu Thr Asp Pro Val Ser Val Tyr Arg Gln His Ala Asn
290 295 300
Arg Leu Lys Asn Ala Gly Leu
305 310
<210> 3
<211> 1014
<212> DNA
<213> 斯氏假单胞菌（Pseudomonas stutzeri）ZS04
<400> 3
atgagcagat ccatccttct gttgccgctg gccatcgcgc tgggcctggg cttctttatc 60
gcccggcccg aatccaccgt gacgccggtg gcagaagcac ccgcatcgtc tcccgccgcc 120
aacctcactg ccgctcgtcc tgcccagcgc acaacgaccg gcgcggcacc gcaggtcatg 180
gcgaagctgc ccgcctcctt caagggcacc gaggtcgacg gccagttcca gctggacgcc 240
gccggcaacc tgatcatcgg tccagagctg cgtcagctgt tcgactactt cctcagcgcc 300
attggcgagg agccgctcaa acagagcatc gagcgcctgc ggcgccatat cgctgcgcaa 360
ttgccggagc ctgcccaggc gcaggcactt gccgtgctca accaatatct caactacaag 420
cgccaactgc ttgatctcga agcgacttat tcgcgaacca cggacatttc agcgctacgc 480
cagcgcctga gtgccgtgca ggcgttgcgc gcacgcgtgc tggagccggc cgttcaccag 540
gcgttcttcg ccccggacga ggcctatgat cgcttcagcc tggagcgcct agccattcaa 600
gccgattcag cactggacag cgacgccaag ggccgcgcca tcgaccagct gcgcgccgga 660
ttgcccggcg acctgcaaga gctgctcgta ccgcagctgc aaagcgagct gcgagagcaa 720
actgtcgcct tgcaggccca gggcgccaat gcgcagcaga tccgccagct gcgccagcag 780
ttggtgggca gcgaggctgc cacccgactc gaagcgctgg atcgacagcg cgagcagtgg 840
cagcaacggg tcgctgtcta tcggcaggaa cgtcagcgta tcgaagccac ccgcggcctg 900
gacgatgtcg agcgccgcag cgctatcgag caactagagg ccgagcagtt tgacgagggc 960
gaacgcttgc ggcttgtcgc tgcctttcag cagcaggaag tggcggagcg ctga 1014
<210> 4
<211> 337
<212> PRT
<213> 斯氏假单胞菌（Pseudomonas stutzeri）ZS04
<400> 4
Met Ser Arg Ser Ile Leu Leu Leu Pro Leu Ala Ile Ala Leu Gly Leu
1 5 10 15
Gly Phe Phe Ile Ala Arg Pro Glu Ser Thr Val Thr Pro Val Ala Glu
20 25 30
Ala Pro Ala Ser Ser Pro Ala Ala Asn Leu Thr Ala Ala Arg Pro Ala
35 40 45
Gln Arg Thr Thr Thr Gly Ala Ala Pro Gln Val Met Ala Lys Leu Pro
50 55 60
Ala Ser Phe Lys Gly Thr Glu Val Asp Gly Gln Phe Gln Leu Asp Ala
65 70 75 80
Ala Gly Asn Leu Ile Ile Gly Pro Glu Leu Arg Gln Leu Phe Asp Tyr
85 90 95
Phe Leu Ser Ala Ile Gly Glu Glu Pro Leu Lys Gln Ser Ile Glu Arg
100 105 110
Leu Arg Arg His Ile Ala Ala Gln Leu Pro Glu Pro Ala Gln Ala Gln
115 120 125
Ala Leu Ala Val Leu Asn Gln Tyr Leu Asn Tyr Lys Arg Gln Leu Leu
130 135 140
Asp Leu Glu Ala Thr Tyr Ser Arg Thr Thr Asp Ile Ser Ala Leu Arg
145 150 155 160
Gln Arg Leu Ser Ala Val Gln Ala Leu Arg Ala Arg Val Leu Glu Pro
165 170 175
Ala Val His Gln Ala Phe Phe Ala Pro Asp Glu Ala Tyr Asp Arg Phe
180 185 190
Ser Leu Glu Arg Leu Ala Ile Gln Ala Asp Ser Ala Leu Asp Ser Asp
195 200 205
Ala Lys Gly Arg Ala Ile Asp Gln Leu Arg Ala Gly Leu Pro Gly Asp
210 215 220
Leu Gln Glu Leu Leu Val Pro Gln Leu Gln Ser Glu Leu Arg Glu Gln
225 230 235 240
Thr Val Ala Leu Gln Ala Gln Gly Ala Asn Ala Gln Gln Ile Arg Gln
245 250 255
Leu Arg Gln Gln Leu Val Gly Ser Glu Ala Ala Thr Arg Leu Glu Ala
260 265 270
Leu Asp Arg Gln Arg Glu Gln Trp Gln Gln Arg Val Ala Val Tyr Arg
275 280 285
Gln Glu Arg Gln Arg Ile Glu Ala Thr Arg Gly Leu Asp Asp Val Glu
290 295 300
Arg Arg Ser Ala Ile Glu Gln Leu Glu Ala Glu Gln Phe Asp Glu Gly
305 310 315 320
Glu Arg Leu Arg Leu Val Ala Ala Phe Gln Gln Gln Glu Val Ala Glu
325 330 335
Arg
<210> 5
<211> 2013
<212> DNA
<213> 斯氏假单胞菌（Pseudomonas stutzeri）ZS04
<400> 5
atgaacaaga acaaaacctt gctcgccctc tgtctcggaa gcgccctcgc gctttccggt 60
caggcattcg ctgccaccgg cagcggctat accgccacga agtacccaat cgtcctcacc 120
cacggcatgc ttggcttcga cagcctattg ggcatcgact actggtacgg catccccagc 180
gctttgcgcc gcgacggcgc gcaggtctac atcaccgaag tcagccaact caacacctcc 240
gaactgcgtg gcgaggaact gctggcccag gtcgaggaaa tcgttgctat cagcggcaag 300
ccgaaggtca acctgatcgg ccacagccaa ggcggaccgg acatccgtta cgtggcaggc 360
gtacggccgg acctgatcgc ctcggtcacc agtgtcggcg cgccccacaa gggctcggac 420
gtggcggatc tgatccgcaa ggttccagag ggttcatccg gtgaggccat aatcgccggc 480
ttggtgaacg ccatgggcgc actgatcaac tttctttccg gcagcaacag cagcgctccg 540
cagaatgcac tcggcgcgct cgaatcgctc aacagcgagg gcgctgcgcg cttcaacgcg 600
aagtatcccc agggcatccc gaccagcgcc tgcggcgaag gggcctacgt ggtcaagggt 660
gtgcgctact actcctggag cgggacgagc ccgctgacca acccgctgga tgtcagcgac 720
gcagtgatgg tcgcgggctc gctcgcgttc gacgaagcca atgacggcct ggtcggacgc 780
tgcagctcgc atctgggcat ggtgatccgc gacaactacc ggatgaacca cctggacgag 840
gtcaaccagg tgctcggcct gaccagcctc ttcgaaacag acccagtgag cgtctatcgc 900
cagcacgcga accgattgaa gaacgcgggg ctctgacgca gccgtttcct tgaaccaggc 960
cgggatcatc gatcccggcc gctaccgaaa ggcgagtcca tgagcagatc catccttctg 1020
ttgccgctgg ccatcgcgct gggcctgggc ttctttatcg cccggcccga atccaccgtg 1080
acgccggtgg cagaagcacc cgcatcgtct cccgccgcca acctcactgc cgctcgtcct 1140
gcccagcgca caacgaccgg cgcggcaccg caggtcatgg cgaagctgcc cgcctccttc 1200
aagggcaccg aggtcgacgg ccagttccag ctggacgccg ccggcaacct gatcatcggt 1260
ccagagctgc gtcagctgtt cgactacttc ctcagcgcca ttggcgagga gccgctcaaa 1320
cagagcatcg agcgcctgcg gcgccatatc gctgcgcaat tgccggagcc tgcccaggcg 1380
caggcacttg ccgtgctcaa ccaatatctc aactacaagc gccaactgct tgatctcgaa 1440
gcgacttatt cgcgaaccac ggacatttca gcgctacgcc agcgcctgag tgccgtgcag 1500
gcgttgcgcg cacgcgtgct ggagccggcc gttcaccagg cgttcttcgc cccggacgag 1560
gcctatgatc gcttcagcct ggagcgccta gccattcaag ccgattcagc actggacagc 1620
gacgccaagg gccgcgccat cgaccagctg cgcgccggat tgcccggcga cctgcaagag 1680
ctgctcgtac cgcagctgca aagcgagctg cgagagcaaa ctgtcgcctt gcaggcccag 1740
ggcgccaatg cgcagcagat ccgccagctg cgccagcagt tggtgggcag cgaggctgcc 1800
acccgactcg aagcgctgga tcgacagcgc gagcagtggc agcaacgggt cgctgtctat 1860
cggcaggaac gtcagcgtat cgaagccacc cgcggcctgg acgatgtcga gcgccgcagc 1920
gctatcgagc aactagaggc cgagcagttt gacgagggcg aacgcttgcg gcttgtcgct 1980
gcctttcagc agcaggaagt ggcggagcgc tga 2013
<210> 6
<211> 22
<212> DNA
<213> artificial
<220>
<223> LipAF
<400> 6
atcgttgcga tccgccagtt ag 22
<210> 7
<211> 22
<212> DNA
<213> artificial
<220>
<223> LipAR
<400> 7
attcgggccg ggcgataaag aa 22
<210> 8
<211> 20
<212> DNA
<213> artificial
<220>
<223> LipBF
<400> 8
cagcacgcga accgattgaa 20
<210> 9
<211> 22
<212> DNA
<213> artificial
<220>
<223> LipBR
<400> 9
cgttcatcct cgctgcctac aa 22
<210> 10
<211> 29
<212> DNA
<213> artificial
<220>
<223> LipdF
<400> 10
gaccatatgg ccaccggcag cggctatac 29
<210> 11
<211> 30
<212> DNA
<213> artificial
<220>
<223> LifR
<400> 11
aaaggatcct cagcgctccg ccacttcctg 30
Claims (10)
1.一种耐有机溶剂脂肪酶产生菌株，其分类命名为：斯氏假单胞菌（Pseudomonas stutzeri）ZS04，保藏编号：CCTCC NO：M 2012542。
2.一种权利要求1所述菌株产生的耐有机溶剂脂肪酶，其氨基酸序列如SEQ ID NO: 2所示。
3.编码权利要求2所述耐有机溶剂脂肪酶的基因。
4.根据权利要求3所述基因，其特征在于：所述基因的序列如SEQ ID NO: 1所示。
5.含有权利要求3所述基因的重组表达载体。
6.含有权利要求3所述基因的重组菌。
7.一种权利要求2所述耐有机溶剂脂肪酶的折叠酶，其氨基酸序列如SEQ ID NO: 4。
8.编码权利要求7所述折叠酶的基因。
9.根据权利要求8所述基因，其特征在于：所述基因的序列如SEQ ID NO: 3所示。
10.一种权利要求2所述耐有机溶剂脂肪酶在手性化合物拆分中的应用。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486895A (zh) * | 2018-12-04 | 2019-03-19 | 南京工业大学 | 一种催化拆分制备异甘草酸的方法 |
| CN109486896A (zh) * | 2018-12-04 | 2019-03-19 | 南京工业大学 | 一种催化拆分制备异甘草酸的方法 |
| CN111662894A (zh) * | 2020-05-14 | 2020-09-15 | 中国农业科学院饲料研究所 | 一种嗜冷碱性脂肪酶PgLip1及其编码基因和应用 |
| CN114317491A (zh) * | 2022-01-04 | 2022-04-12 | 西南科技大学 | 一种新型脂肪酶AjLip970及其应用 |
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| CN102174422A (zh) * | 2010-11-22 | 2011-09-07 | 南京工业大学 | 一种耐有机溶剂脂肪酶生产菌及该脂肪酶的基因和应用 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486895A (zh) * | 2018-12-04 | 2019-03-19 | 南京工业大学 | 一种催化拆分制备异甘草酸的方法 |
| CN109486896A (zh) * | 2018-12-04 | 2019-03-19 | 南京工业大学 | 一种催化拆分制备异甘草酸的方法 |
| CN111662894A (zh) * | 2020-05-14 | 2020-09-15 | 中国农业科学院饲料研究所 | 一种嗜冷碱性脂肪酶PgLip1及其编码基因和应用 |
| CN114317491A (zh) * | 2022-01-04 | 2022-04-12 | 西南科技大学 | 一种新型脂肪酶AjLip970及其应用 |
| CN114317491B (zh) * | 2022-01-04 | 2023-05-23 | 西南科技大学 | 一种新型脂肪酶AjLip970及其应用 |
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