CN102174422A - 一种耐有机溶剂脂肪酶生产菌及该脂肪酶的基因和应用 - Google Patents
一种耐有机溶剂脂肪酶生产菌及该脂肪酶的基因和应用 Download PDFInfo
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- CN102174422A CN102174422A CN2010105532923A CN201010553292A CN102174422A CN 102174422 A CN102174422 A CN 102174422A CN 2010105532923 A CN2010105532923 A CN 2010105532923A CN 201010553292 A CN201010553292 A CN 201010553292A CN 102174422 A CN102174422 A CN 102174422A
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- lipase
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- 239000003960 organic solvent Substances 0.000 title claims abstract description 107
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 14
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Abstract
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生产菌，其耐有机溶剂脂肪酶的基因，以及该耐有机溶剂脂肪酶在有机相中催化拆分手性化合物的应用。该菌株分类命名为PseudomonasstutzeriLC2-8，其保藏登记号为：CCTCC M2010279，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。该耐有机溶剂脂肪酶具有产量高、比活高、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生产菌，其耐有机溶剂脂肪酶的基因，以及该耐有机溶剂脂肪酶在有机相中催化拆分手性化合物的应用。
背景技术
脂肪酶（lipase, EC 3. 1. 1. 3 ,甘油三酰酯水解酶) 是一类能催化长链脂肪酸甘油酯水解为甘油和长链脂肪酸的酶类。它是一类重要的水解酶，在医药、洗涤剂、食品和皮革等许多工业领域中均有广泛应用。
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。
由于脂肪酶催化的反应具有高立体选择性、底物专一性和位置选择性，近年来，在非水体系中应用脂肪酶实现手性化合物的拆分成为研究热点。如利用微生物脂肪酶可进行二芳基丙酸类药物：奈普森、布洛芬等的立体选择性水解或拆分。生物法手性拆分比化学及物理拆分法的反应条件温和，很少或几乎没有副反应，能耗低，极具应用潜力。然而，催化反应所用的脂肪酶大多是商品酶，价格昂贵，且现有的酶在有机溶剂中的稳定性较差。因此开发出在有机溶剂中具有高活性的脂肪酶是推进脂肪酶工业化应用的重要环节。
天然具有有机溶剂稳定性的脂肪酶为近年来发现的一类新型脂肪酶，这类脂肪酶通常由耐有机溶剂极端微生物产生。耐有机溶剂极端微生物由于能适应有机溶剂对细胞的毒性或结构破坏而具有独特的抵抗恶劣环境的能力。由此产生的酶，尤其是胞外酶多数具有极强的耐有机溶剂的特性。虽然已经有文章公开一些极端微生物所产的耐有机溶剂脂肪酶，但有机溶剂耐受能力较低，酶产量较低。
发明内容
本发明的目的是提供一种耐有机溶剂脂肪酶的生产菌、耐有机溶剂脂肪酶、耐有机溶剂脂肪酶基因以及该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，分类命名为斯氏假单胞菌Pseudomonas stutzeri LC2-8，保藏日期为2010年10月25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 2010279。
本发明对斯氏假单胞菌Pseudomonas stutzeri LC2-8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1.5 mm～2 mm，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。
本发明对经BIOLOG全自动细菌鉴定仪鉴定和16S rDNA序列分析，表明该菌株为Pseudomonas stutzeri，命名为Pseudomonas stutzeri LC2-8。
本发明对Pseudomonas stutzeri LC2-8进行了产酶条件优化，优化后产酶量高达352.9 U/mL。
本发明对该菌产生的胞外酶进行了纯化，经过两步分离纯化得到电泳纯的耐有机溶剂脂肪酶，命名为耐有机溶剂LC2-8脂肪酶，其比活性达到6204.6 U/mg。
本发明对该耐有机溶剂LC2-8脂肪酶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，半衰期延长。同时，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8.0，在pH 5.5～9.5的范围具有很高的稳定性。其最适反应温度为30℃，属低温脂肪酶，有机溶剂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辛酸酯。
本发明分离克隆到该菌株所产耐有机溶剂脂肪酶的编码基因，序列为： atgaacaaga acaaaacctt gctcgctctc tgtctcggta gcgccatggc gcttgccggt caggctcatg ctgctactgg cagcggctac a ccgctacga agtacccgat cgtgctcgcc cacggcatgc tcggcttcga cagcctgctg ggcatcgatt actggtacgg catccccagc gc gctgcgcc gcgacggtgc gcaggtctac gtcaccgaag tcagtcagct caatacgtcc gaattgcgcg gtgaggaact gcttgcgcag gtg gaggaaa tcgtcgccat cagcggcaag ccaaaggtca atctggttgg ccacagccac ggcggcccaa ccgtgcgcta tgtagccggt gtcc ggccgg acctgatcgc ctcggtaacc agcgtgggcg cacctcacaa gggttcggac gtcgccgatc tgatccgcaa gatccccgag ggctc ctctg gcgaggcgat catcgccgga ctggtgaacg ccatgggcag cttcatcaat ttcgtttccg gcagctcaag cacggccccg caggac tctc tcggctcgct ggagtcactc aacagcgaag gcgccgcccg cttcaacgcc aaattcccgc aaggcattcc caccaccgct tgcggcg agg gcgcctacag ggtcaatggc gtgcgctact actcctggag tggcaccagc ccgctgacca acccactgga tatcagcgac gccatgat gg gtgccggagc cctggcattc agcgggccca acgatggact ggtcgggcgc tgcagctcgc acctgggcat ggtgatccgc gataactac c ggatgaacca cctggacgag gtcaatcagt tcatggggct gaccagcctg ttcgagaccg atccggtcag tgtctatcgc
为此基因的改造并在各种外源基因表达系统中高效表达提供了优良的基因材料。通过PCR法分离克隆了这一耐有机溶剂脂肪酶基因，DNA 全序列分析结果表明，该耐有机溶剂脂肪酶基因全长936个核苷酸，编码311个氨基酸，与斯氏假单胞菌Pseudomonas stutzeri A1501脂肪酶基因同源性为87.1%，蛋白质一级结构同源性为96.2%。其氨基酸序列为: Met Asn Lys Asn Lys Thr Leu Leu Ala Leu Cys Leu Gly Ser Ala Met Ala Leu Ala Gly Gln Ala His Ala Ala Thr Gly Ser Gly Tyr Thr Ala Thr Lys Tyr Pro Ile Val Leu Ala His Gly Met Leu Gly Phe Asp Ser Leu Leu Gly Ile Asp Tyr Trp Tyr Gly Ile Pro Ser Ala Leu Arg Arg Asp Gly Ala Gln Val Tyr Val Thr Glu Val Ser Gln Leu Asn Thr Ser Glu Leu Arg Gly Glu Glu Leu Leu Ala Gln Val Glu Glu Ile Val Ala Ile Ser Gly Lys Pro Lys Val Asn Leu Val Gly His Ser His Gly Gly Pro Thr Val Arg Tyr Val Ala Gly Val Arg Pro Asp Leu Ile Ala Ser Val Thr Ser Val Gly Ala Pro His Lys Gly Ser Asp Val Ala Asp Leu Ile Arg Lys Ile Pro Glu Gly Ser Ser Gly Glu Ala Ile Ile Ala Gly Leu Val Asn Ala Met Gly Ser Phe Ile Asn Phe Val Ser Gly Ser Ser Ser Thr Ala Pro Gln Asp Ser Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala Ala Arg Phe Asn Ala Lys Peh Pro Gln Gly Ile Pro Thr Thr Ala Cys Gly Glu Gly Ala Tyr Arg Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser Gly Thr Ser Pro Leu Thr Asn Pro Leu Asp Ile Ser Asp Ala Met Met Gly Ala Gly Ala Leu Ala Phe Ser Gly Pro Asn Asp Gly Leu Val Gly Arg Cys Ser Ser His Leu Gly Met Val Ile Arg Asp Asn Tyr Arg Met Asn His Leu Asp Glu Val Asn Gln Phe Met Gly Leu Thr Ser Leu Phe Glu Thr Asp Pro Val Ser Val Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Ala Gly Leu
本发明还提供耐有机溶剂LC2-8脂肪酶在有机相酶催化手性化合物拆分反应中的应用。所述的有机相酶催化手性化合物拆分反应可以为拆分1-苯乙醇反应、拆分安息香反应以及拆分扁桃酸的反应。所述的耐有机溶剂脂肪酶在有机溶剂正己烷体系或无溶剂中，催化底物1-苯乙醇和乙酸乙烯酯进行酯交换反应，转化率达52～65％，ees值99.9%。
本发明的有益效果在于菌株Pseudomonas stutzeri LC2-8的耐有机溶剂LC2-8脂肪酶产率高，发酵54 h脂肪酶活力达到352.9 U/mL；耐有机溶剂LC2-8脂肪酶易纯化、比活高，经过丙酮沉淀和离子交换层析后比活达到6204.6U/mg；该脂肪酶为碱性低温脂肪酶，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-苯乙醇的拆分。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LC2-8脂肪酶的SDS-PAGE电泳图，其中泳道1，Marker；泳道2，粗酶液；泳道3，丙酮沉淀后酶液；泳道4，纯酶液（经丙酮沉淀、离子交换层析后得到的纯酶液）；
图2显示耐有机溶剂脂肪酶LC2-8的最适反应pH；
图 3 显示耐有机溶剂脂肪酶LC2-8的pH稳定性；
图4显示耐有机溶剂脂肪酶LC2-8的最适反应温度；
图 5 显示耐有机溶剂脂肪酶LC2-8的温度稳定性及有机溶剂对温度稳定性的影响；
图6显示耐有机溶剂脂肪酶LC2-8的底物特异性，其中p-Nitrophenyl acetate (C2)为对硝基苯酚乙酸酯,p-Nitrophenyl butyrate (C4)为对硝基苯酚丁酸酯，p-Nitrophenyl caprate (C8)为对硝基苯酚辛酸酯，p-Nitrophenyl decanoate (C10)为对硝基苯酚癸酸酯，p-Nitrophenyl palmitate (C16)为对硝基苯酚棕榈酸酯，p-Nitrophenyl stearate (C18)为对硝基苯酚硬脂酸酯。
具体实施方式
1．耐有机溶剂脂肪酶天然菌株的筛选程序。
初筛采用如下方法：以植物油为唯一碳源，不同浓度环己烷、甲苯、DMSO等有机溶剂为筛选压力从油污土样中筛选获得耐有机溶剂极端微生物。采用橄榄油罗丹明B平板培养基，具体配方为：酵母膏1 g/L，玉米浆5 mL/L，K2HPO4 1 g/L，MgSO4·7H2O 0.5 g/L，橄榄油60 mL/L，Rhodamin B 0.024 g/L。将所筛选到的耐有机溶剂微生物接种于橄榄油罗丹明B平板，根据菌落与橙黄色荧光圈大小的比值，初步筛选脂肪酶产量高的菌株。此方法筛选到具有脂肪酶产量高的耐有机溶剂极端微生物6株。
为了进一步检测所分泌脂肪酶的溶剂耐受性，对6株菌的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。将所筛选到的高活力菌株接种到产酶发酵培养基，具体配方为：玉米浆15 mL/L，尿素5 g/L，葡萄糖8 g/L，玉米油5 mL/L，K2HPO4 2 g/L，MgSO4·7H2O 0.5 g/L，pH 8.0。培养温度为30℃，培养时间为54 h，摇床转速为180 rmp。发酵结束后，10,000 rmp 4℃离心15 min，取上清为粗酶液。取粗酶液1.5 mL分别加入0.5mL的十六烷、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震荡处理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菌株作为耐有机溶剂脂肪酶生产菌株。
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：0.05 M的Na2HPO4·12H2O-NaH2PO4·2H2O缓冲液（pH 7.0），其中含有0.6%（m/v）Triton X-100和0.1%（m/v）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，在相应条件下，每毫升酶液每分钟催化产生1 μmol对硝基苯酚(pNP)所需的酶量。通过酶活性检测以及有机溶剂稳定性检测，其中一株具有优良耐受性的脂肪酶生产菌株经初步优化后脂肪酶活力高达352.9 U/mL，经过BIOLOG自动细菌鉴定仪鉴定和16SrDNA序列分析，表明该菌株属于斯氏假单胞菌属，并命名为Pseudomonas stutzeri LC2-8，该菌即为进一步研究的材料。
2．耐有机溶剂脂肪酶产生菌LC2-8的生物学性质及鉴定。
菌株LC2-8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，菌落大小直径为1.5 mm～2 mm，生长温度范围为24℃～37℃，最适生长温度为35℃，生长pH范围为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LC2-8的菌种鉴定：经BIOLOG自动细菌鉴定仪鉴定，Sim值为0.375，鉴定结果显示该菌属于Pseudomonas属。后经16S rDNA序列分析，表明该菌株与斯氏假单胞菌同源性为99%，将其命名为Pseudomonas stutzeri LC2-8。
3．耐有机溶剂LC2-8脂肪酶的纯化方法。
首先将菌株在产酶培养基中培养54 h后，10,000rmp在4℃离心15 min，取上清作为粗酶液，将粗酶液置于冰浴中，一边搅拌一边缓慢加入预先冰冻的丙酮，到终浓度为V发酵液：V丙酮=1：1 .5，搅拌4h后离心取沉淀。沉淀用0.05 M的pH 8.0的Tris-HCl缓冲液溶解, 透析除盐。将以上处理得到的酶液，采用DEAE-Sepharose FF离子交换层析柱进行纯化，以0.05 M的Tris-HCl（pH 8.0，NaC1含量为0 mol/L -1 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），发现两步纯化后的耐有机溶剂脂肪酶（命名为耐有机溶剂LC2-8脂肪酶）已达电泳纯，该脂肪酶亚基分子量约为32 kDa。纯化倍数为22.77倍，回收率为32.8%，最终脂肪酶比活达到6204.6U/mg，汇总见表1。
表1 耐有机溶剂LC2-8脂肪酶的纯化步骤及结果
| 总活力(U) | 总蛋白(mg) | 比活力(U/mg) | 回收率(%) | 纯化倍数 | |
| 粗酶液 | 2900 | 12.9 | 223.42 | 100 | 1 |
| 丙酮沉淀 | 1992 | 3.12 | 637.9 | 68.7 | 2.85 |
| DEAE-Sepharose FF | 951 | 0.153 | 6204.6 | 32.8 | 22.77 |
注：蛋白质浓度采用考马斯亮兰法测定
4．耐有机溶剂LC2-8脂肪酶编码基因的分离克隆程序。
采用酚－氯仿法抽提菌体总DNA。将纯化的耐有机溶剂LC-8脂肪酶做LC-MS/MS（委托国家生物医学分析中心(NBCA)进行LC/MS/MS序列分析）测定其氨基酸片段，结果表明该酶与斯氏假单胞菌Pseudomonas stutzeri A1501中脂肪酶序列最相近，因此在此酶CDS两端外各约100 bp处设计引物，扩增耐有机溶剂LC2-8脂肪酶的CDS编码序列。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。设计的引物为：
LU1（SEQ ID:3）: TGCCCCATAGCCCGATTCACAT
LD1（SEQ ID:4）: ATGCTCTGTTTGAGCGGCTCCT
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轮后，72℃保温10 min。根据该反应条件，扩增到了1.0 kb的PCR片段。将此片段连接到pMD18-T载体，进行序列测定。结果表明，此片段有一个全长为936 bp的阅读框，编码311个氨基酸。与斯氏假单胞菌Pseudomonas stutzeri A1501脂肪酶基因同源性为87.1%。
5．本实验说明耐有机溶剂LC2-8脂肪酶的酶学性质。
耐有机溶剂LC2-8脂肪酶的有机溶剂耐受性：向12种有机溶剂中分别加入耐有机溶剂LC2-8脂肪酶（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℃，150 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LC2-8脂肪酶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，例如丙酮、乙醇等显著激活脂肪酶活力。同时，耐有机溶剂LC2-8脂肪酶在25%（v/v）有机溶剂中与对照（不加有机溶剂）相比半衰期均显著延长。
| 有机溶剂 | LogP | 残余活力(%) | 半衰期 |
| 对照 | - | 49.90% | 1d |
| 十六烷 | 8.8 | 42.0% | 1d |
| 壬烷 | 5.6 | 76.5% | 2d |
| 异辛烷 | 4.7 | 70.5% | 2d |
| 正辛烷 | 4.5 | 73.4% | 2d |
| 正庚烷 | 4 | 94.2% | ＞10d |
| 正己烷 | 3.5 | 89.8% | 7d |
| 异丙醇 | 0.28 | 132.6% | ＞10d |
| 丙酮 | -0.23 | 215.3% | ＞10d |
| 乙醇 | -0.24 | 168.9% | ＞10d |
| 甲醇 | -0.76 | 121.0% | ＞10d |
| DMF | -1 | 159.3% | 7d |
| DMSO | -1.35 | 106.0% | 7d |
耐有机溶剂LC2-8脂肪酶最适反应pH和pH稳定性的检测：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体系中的酶活如图2所示。耐有机溶剂LC2-8脂肪酶的最适反应pH为8.0，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LC2-8脂肪酶加入不同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 5.5～9.5的范围内具有较高的稳定性。
耐有机溶剂LC2-8脂肪酶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 Na2HPO4·12H2O-NaH2PO4·2H2O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30℃(图4)，在15℃条件下反应仍然具有最佳情况下45%的酶活力。热稳定性测定：耐有机溶剂LC2-8脂肪酶在25℃、30℃、35℃、40℃、45℃、50℃下处理1 h以后，测定残留酶活。在耐有机溶剂脂肪酶LC2-8中分别添加终浓度为25%的DMF和正己烷，在25℃、30℃、35℃、40℃、45℃、50℃下处理3 h以后，测定残留酶活。由图5可以看出在50℃处理1 h后未添加有机溶剂的LC2-8脂肪酶酶活迅速下降，而添加25%的DMF和正己烷使得耐有机溶剂LC2-8脂肪酶热稳定性大大提高，在50℃处理3 h后，仍可保持80%左右的酶活力。
耐有机溶剂LC2-8脂肪酶底物特异性的检测：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），对硝基苯酚硬脂酸酯（C18）。结果如图6，耐有机溶剂LC2-8脂肪酶的最适人工底物为对硝基苯酚辛酸酯。
6．耐有机溶剂LC2-8脂肪酶在有机相中拆分手性化合物的应用。
以0.3 mmol的1-苯乙醇和1.8 mmol的乙酸乙烯酯作为反应底物；以壬烷、正辛烷、异辛烷、正庚烷、正己烷作为溶剂，溶剂的加入量为1mL；加入10mg耐有机溶剂LC2-8脂肪酶，在30℃，转速200rpm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，正己烷为最佳溶剂，反应24 h后，转化率达到52%~65%，ees为99.9%。
以30mg的安息香和1.5mmol的乙酸乙烯酯作为反应底物；以THF作为溶剂，溶剂的加入量为1.5mL；加入15mg耐有机溶剂LC2-8脂肪酶，在30℃，转速200rpm条件下反应，定时取样，采用HPLC进行检测。结果表明，反应24 h后，转化率达到35%~48%,eep为99.9%。
以9.2mg的扁桃酸和2mmol的乙酸乙烯酯作为反应底物；以THF、异丙醚作为溶剂，溶剂的加入量为2mL；加入15mg耐有机溶剂LC2-8脂肪酶，在30℃，转速200rpm条件下反应，定时取样，采用HPLC进行检测。结果表明，反应24 h后，转化率达到38%~48%,eep为99.9%。
结果表明LC2-8脂肪酶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。
序列表
<110> 南京工业大学
<120> 一种耐有机溶剂脂肪酶生产菌及该脂肪酶的基因和应用
<160> 2
<210> 1
<211> 936
<212> DNA
<213> Pseudomonas stutzeri LC2-8
<400> 1
atgaacaaga acaaaacctt gctcgctctc tgtctcggta gcgccatggc gcttgccggt 60
caggctcatg ctgctactgg cagcggctac accgctacga agtacccgat cgtgctcgcc 120
cacggcatgc tcggcttcga cagcctgctg ggcatcgatt actggtacgg catccccagc 180
gcgctgcgcc gcgacggtgc gcaggtctac gtcaccgaag tcagtcagct caatacgtcc 240
gaattgcgcg gtgaggaact gcttgcgcag gtggaggaaa tcgtcgccat cagcggcaag 300
ccaaaggtca atctggttgg ccacagccac ggcggcccaa ccgtgcgcta tgtagccggt 360
gtccggccgg acctgatcgc ctcggtaacc agcgtgggcg cacctcacaa gggttcggac 420
gtcgccgatc tgatccgcaa gatccccgag ggctcctctg gcgaggcgat catcgccgga 480
ctggtgaacg ccatgggcag cttcatcaat ttcgtttccg gcagctcaag cacggccccg 540
caggactctc tcggctcgct ggagtcactc aacagcgaag gcgccgcccg cttcaacgcc 600
aaattcccgc aaggcattcc caccaccgct tgcggcgagg gcgcctacag ggtcaatggc 660
gtgcgctact actcctggag tggcaccagc ccgctgacca acccactgga tatcagcgac 720
gccatgatgg gtgccggagc cctggcattc agcgggccca acgatggact ggtcgggcgc 780
tgcagctcgc acctgggcat ggtgatccgc gataactacc ggatgaacca cctggacgag 840
gtcaatcagt tcatggggct gaccagcctg ttcgagaccg atccggtcag tgtctatcgc 900
caacacgcga accgcctgaa gaacgcgggc ctttga 936
<210> 2
<211> 311
<212> PRT
<213> Pseudomonas stutzeri LC2-8
<400> 2
Met Asn Lys Asn Lys Thr Leu Leu Ala Leu Cys Leu Gly Ser Ala Met
1 6 11 16
Ala Leu Ala Gly Gln Ala His Ala Ala Thr Gly Ser Gly Tyr Thr Ala
21 26 31
Thr Lys Tyr Pro Ile Val Leu Ala His Gly Met Leu Gly Phe Asp Ser
36 41 46
Leu Leu Gly Ile Asp Tyr Trp Tyr Gly Ile Pro Ser Ala Leu Arg Arg
51 56 61
Asp Gly Ala Gln Val Tyr Val Thr Glu Val Ser Gln Leu Asn Thr Ser
66 71 76
Glu Leu Arg Gly Glu Glu Leu Leu Ala Gln Val Glu Glu Ile Val Ala
81 86 91 96
Ile Ser Gly Lys Pro Lys Val Asn Leu Val Gly His Ser His Gly Gly
101 106 111
Pro Thr Val Arg Tyr Val Ala Gly Val Arg Pro Asp Leu Ile Ala Ser
116 121 126
Val Thr Ser Val Gly Ala Pro His Lys Gly Ser Asp Val Ala Asp Leu
131 136 141
Ile Arg Lys Ile Pro Glu Gly Ser Ser Gly Glu Ala Ile Ile Ala Gly
146 151 156
Leu Val Asn Ala Met Gly Ser Phe Ile Asn Phe Val Ser Gly Ser Ser
161 166 171 176
Ser Thr Ala Pro Gln Asp Ser Leu Gly Ser Leu Glu Ser Leu Asn Ser
Claims (6)
1.一种耐有机溶剂脂肪酶生产菌，其特征是从油污土样中筛选获得一株有机溶剂脂肪酶生产菌，其分类命名为斯氏假单胞菌属的一个新菌株，命名为Pseudomonas stutzeri LC2-8，保藏日期为2010年10月25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 2010279。
2.根据权利要求1所述耐有机溶剂脂肪酶生产菌，其特征在于其所产耐有机溶剂脂肪酶的纯化方法为：
（1）获得粗酶液：Pseudomonas stutzeri LC2-8菌株在30℃，180rpm条件下发酵培养54h，发酵液离心去除菌体，上清液即为粗酶液；
（2）丙酮沉淀：取上清作为粗酶液，将粗酶液置于冰浴中，一边搅拌一边缓慢加入预先冰冻的丙酮，到终浓度为V发酵液：V丙酮=1：1 .5，搅拌4h后离心取沉淀；沉淀用0.05 M的pH 8.0的Tris-HCl缓冲液溶解, 透析除盐；
（3）DEAE-Sepharose FF离子交换层析：将以上处理得到的酶液，采用DEAE-Sepharose FF离子交换层析柱进行纯化，以0.05 M的Tris-HCl缓冲液进行梯度洗脱，收集脂肪酶活力峰，透析后冻干即为LC2-8纯酶。
3.根据权利要求1所述的耐有机溶剂脂肪酶生产菌，其特征在于其所产耐有机溶剂脂肪酶氨基酸序列为：
Met Asn Lys Asn Lys Thr Leu Leu Ala Leu Cys Leu Gly Ser Ala Met Ala Leu Ala Gly Gln Ala His Ala Ala Thr Gly Ser Gly Tyr Thr Ala Thr Lys Tyr Pro Ile Val Leu Ala His Gly Met Leu Gly Phe Asp Ser Leu Leu Gly Ile Asp Tyr Trp Tyr Gly Ile Pro Ser Ala Leu Arg Arg Asp Gly Ala Gln Val Tyr Val Thr Glu Val Ser Gln Leu Asn Thr Ser Glu Leu Arg Gly Glu Glu Leu Leu Ala Gln Val Glu Glu Ile Val Ala Ile Ser Gly Lys Pro Lys Val Asn Leu Val Gly His Ser His Gly Gly Pro Thr Val Arg Tyr Val Ala Gly Val Arg Pro Asp Leu Ile Ala Ser Val Thr Ser Val Gly Ala Pro His Lys Gly Ser Asp Val Ala Asp Leu Ile Arg Lys Ile Pro Glu Gly Ser Ser Gly Glu Ala Ile Ile Ala Gly Leu Val Asn Ala Met Gly Ser Phe Ile Asn Phe Val Ser Gly Ser Ser Ser Thr Ala Pro Gln Asp Ser Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala Ala Arg Phe Asn Ala Lys Peh Pro Gln Gly Ile Pro Thr Thr Ala Cys Gly Glu Gly Ala Tyr Arg Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser Gly Thr Ser Pro Leu Thr Asn Pro Leu Asp Ile Ser Asp Ala Met Met Gly Ala Gly Ala Leu Ala Phe Ser Gly Pro Asn Asp Gly Leu Val Gly Arg Cys Ser Ser His Leu Gly Met Val Ile Arg Asp Asn Tyr Arg Met Asn His Leu Asp Glu Val Asn Gln Phe Met Gly Leu Thr Ser Leu Phe Glu Thr Asp Pro Val Ser Val Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Ala Gly Leu。
4.根据权利要求1所述的耐有机溶剂生产菌，其特征在于所产耐有机溶剂脂肪酶LC2-8的编码基因的核苷酸序列为：
atgaacaaga acaaaacctt gctcgctctc tgtctcggta gcgccatggc gcttgccggt caggctcatg ctgctactgg cagcggctac accgctacga agtacccgat cgtgctcgcc cacggcatgc tcggcttcga cagcctgctg ggcatcgatt actggtacgg catccccagc gcgctgcgcc gcgacggtgc gcaggtctac gtcaccgaag tcagtcagct caatacgtcc gaattgcgcg gtgaggaact gcttgcgcag gtggaggaaa tcgtcgccat cagcggcaag ccaaaggtca atctggttgg ccacagccac ggcggcccaa ccgtgcgcta tgtagccggt gtccggccgg acctgatcgc ctcggtaacc agcgtgggcg cacctcacaa gggttcggac gtcgccgatc tgatccgcaa gatccccgag ggctcctctg gcgaggcgat catcgccgga ctggtgaacg ccatgggcag cttcatcaat ttcgtttccg gcagctcaag cacggccccg caggactctc tcggctcgct ggagtcactc aacagcgaag gcgccgcccg cttcaacgcc aaattcccgc aaggcattcc caccaccgct tgcggcgagg gcgcctacag ggtcaatggc gtgcgctact actcctggag tggcaccagc ccgctgacca acccactgga tatcagcgac gccatgatgg gtgccggagc cctggcattc agcgggccca acgatggact ggtcgggcgc tgcagctcgc acctgggcat ggtgatccgc gataactacc ggatgaacca cctggacgag gtcaatcagt tcatggggct gaccagcctg ttcgagaccg atccggtcag tgtctatcgc caacacgcga accgcctgaa gaacgcgggc ctttga。
5.根据权利要求2所述方法生产的耐有机溶剂脂肪酶生产菌产生的耐有机溶剂脂肪酶LC2-8在手性化合物拆分体系中的应用。
6.根据权利要求2所述方法生产的耐有机溶剂脂肪酶生产菌产生的耐有机溶剂脂肪酶LC2-8在医药、洗涤剂、食品、皮革工业领域中的应用。
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| CN102659495A (zh) * | 2012-04-25 | 2012-09-12 | 天津大学 | 利用寡核苷酸拆分手性药物的方法 |
| CN103131659A (zh) * | 2013-03-20 | 2013-06-05 | 南京工业大学 | 一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用 |
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| CN101586086A (zh) * | 2009-05-15 | 2009-11-25 | 南京工业大学 | 一种耐有机溶剂蛋白酶及其产生菌株 |
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| CN101586086A (zh) * | 2009-05-15 | 2009-11-25 | 南京工业大学 | 一种耐有机溶剂蛋白酶及其产生菌株 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102659495A (zh) * | 2012-04-25 | 2012-09-12 | 天津大学 | 利用寡核苷酸拆分手性药物的方法 |
| CN102659495B (zh) * | 2012-04-25 | 2014-02-26 | 天津大学 | 利用寡核苷酸拆分手性药物的方法 |
| CN102643746A (zh) * | 2012-04-27 | 2012-08-22 | 淮海工学院 | 快速筛选分泌耐有机溶剂脂肪酶的海洋微生物的方法 |
| CN103131659A (zh) * | 2013-03-20 | 2013-06-05 | 南京工业大学 | 一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用 |
| CN103131659B (zh) * | 2013-03-20 | 2014-09-17 | 南京工业大学 | 一种耐有机溶剂脂肪酶、其编码基因、产生菌株及应用 |
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