CN102329745B - 高稳定性耐有机溶剂脂肪酶产生菌及脂肪酶和其基因与应用 - Google Patents
高稳定性耐有机溶剂脂肪酶产生菌及脂肪酶和其基因与应用 Download PDFInfo
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- CN102329745B CN102329745B CN201110211402.2A CN201110211402A CN102329745B CN 102329745 B CN102329745 B CN 102329745B CN 201110211402 A CN201110211402 A CN 201110211402A CN 102329745 B CN102329745 B CN 102329745B
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Abstract
本发明属于微生物工程与酶工程技术领域，具体涉及了一种高稳定性耐有机溶剂脂肪酶产生菌BurkholderiaambifariaYCJ01，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。该菌株为革兰氏阴性菌株，其保藏登记号为：CCTCCNO：M2011058，其耐有机溶剂脂肪酶产量高，具有较宽的pH适应范围，温度稳定性好，对多种有机溶剂有较好的耐受性，其在手性化合物拆分中具有良好的应用前景。
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产生菌，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中的应用。
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，在洗涤、制革、食品、生物化工、造纸、医药、环保等行业被广泛应用。
脂肪酶在有机相中可以完成酯化、交换及转酯等反应，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水解或拆分，如利用微生物脂肪酶可完成酮基布洛芬、反应停、心得安等手性药物的立体选择性水解或拆分。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。
虽然脂肪酶在有机溶剂中催化具有很多优势，但是酶一般在有机溶剂中不稳定。尤其是亲水性的有机溶剂往往会剥夺酶表面的水分子而使其失活。为了提高酶的稳定性，人们采用了化学修饰、固定化或者蛋白质工程对酶进行改造。然而如果酶天然在有机溶剂中稳定，这样就更利于其在有机相中的应用。因此寻找天然耐有机溶剂的脂肪酶，使其在有机溶剂或含有机溶剂的环境中具有较高的催化活性，已成为脂肪酶研究领域的一个重要方向。
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、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壤等样品中筛选获得一株耐有机溶剂脂肪酶产生菌，分类命名为Burkholderia ambifaria YCJ01，保藏日期为2011年3月6日，保藏单位全称为中国典型培养物保藏中心，简称CCTCC，保藏地址：中国武汉，武汉大学，保藏登记号为CCTCC NO：M 2011058。
本发明对洋葱伯克霍尔德氏菌Burkholderia ambifaria YCJ01的生物学特征进行鉴定，该菌株为革兰氏阴性菌株，菌落呈米白色，圆形，边缘整齐，光滑湿润；显微镜观察菌体为杆菌，0.8～1.0×1.6～3.2mm，专性好氧，生长的最适温度为30℃～35℃。其生理生化特性表现在：不出现反硝化反应、明胶反应结果为阳性，氧化酶阳性，可分解脂肪，可利用D-核糖、D-阿拉伯糖、海藻糖等，不可利用麦芽糖。
经BIOLOG全自动细菌鉴定仪鉴定，Sim值为0.576(24h培养)，结果显示该菌属于Burkholderia属；16S rDNA序列分析结果表明，该菌株与数据库中Burkholderia ambifariaMC40-6菌的同源性为99％。综合BIOLOG和16S rDNA分析结果，该菌株鉴定为Burkholderia ambifaria，命名为Burkholderia ambifariaYCJ01。
本发明对Burkholderia ambifaria YCJ01进行了产酶条件优化，优化后产酶量高达126.1U/mL，比优化前40.2U/mL提高了2.1倍。
本发明对该菌产生的胞外脂肪酶进行了纯化，经过两步分离纯化，得到电泳纯的耐有机溶剂脂肪酶，命名为脂肪酶YCJ01，纯化后脂肪酶的比活性达到4609.5U/mg，表观分子量为33kDa。
本发明对脂肪酶YCJ01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。脂肪酶YCJ01的最适反应pH为7.5，在pH 5.0～8.0的范围具有较高的稳定性。其最佳反应温度为60℃，此酶的最佳人工底物为对硝基苯酚棕榈酸酯(C16)。
本发明分离克隆到该菌株所产耐有机溶剂脂肪酶YCJ01的编码基因，它具有SEQ ID NO：1所示的核苷酸序列：atggccagat cgatgcgttc cagggtggtg gcaggggcagtggcttgcgc gatgagcgtc gcgccgttcg cggggacgac cgcgctgatg acgctcgcga cgacgcgcgcggcgatggcg gcaaccgcgc cggcggacga ctacgcgacg acgcgttatc cgatcatcct cgtgcacgggctcaccggca ccgacaagta cgcgggcgtg ctcgagtact ggtacggcat ccaggaggac ctgcagcagcatggcgcgac cgtctacgtc gcgaacctgt cgggattcca gagcgacgac gggccgaatg ggcgcggtgaacaactgctc gcttacgtca agacggtgct cgccgcgacc ggtgcgacca aggtgaacct ggtcggtcacagccagggcg gcctcacgtc gcgctatgtc gcggccgtcg cacccgatct cgtcgcgtcg gtgacgacgatcggcacgcc gcatcgcggc tcagagttcg ccgacttcgt gcagagcgta ctcgcctacg atccgaccggattgtcgtcg tcggtgatcg cagcgttcgc caatgtgttc ggtatcctga cgagcagcag caacaacaccaaccaggacg cgctcgccgc actgaagacg ctgacgactt cccaggccgc cacgtacaac cagaattacccgagcgcggg cctcggcgcg ccgggcagct gccagacggg tgcgccgacg gaaaccgtcg gtggcaatacgcacctgctg tattcgtggg ccggcacggc gatccagccg accatctccg cgttcggcgt gacgggcgcgacggatacca gcacgattcc gctgatcgac ccggcaaacg cgctggacct gtcgacgctt gcgttgttcggcacgggcac ggtgatgatc aatcgcgcct cgggccagaa cgacgggctc gtgtcgaagt gcagtgcgctgtacgggaag gtgctgagca cgagctacaa gtggaaccat ctcgatgaga tcaaccagtt gctcggtgtgcgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaaa ctcgcgggcgtgtga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1095个核苷酸，编码364个氨基酸，与Burkholderia cepacia S31脂肪酶基因同源性为92.4％，氨基酸序列同源性为96.4％。其氨基酸序列示于SEQ ID NO：2：Met Ala Arg Ser Met Arg Ser Arg Val Val Ala GlyAla Val Ala Cys Ala Met Ser Val Ala Pro Phe Ala Gly Thr Thr Ala Leu Met Thr LeuAla Thr Thr Arg Ala Ala Met Ala Ala Thr Ala Pro Ala Asp Asp Tyr Ala Thr Thr ArgTyr Pro Ile Ile Leu Val His Gly Leu Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Glu TyrTrp Tyr Gly Ile Gln Glu Asp Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn LeuSer Gly Phe Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln Leu Leu Ala Tyr ValLys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys Val Asn Leu Val Gly His Ser Gln GlyGly Leu Thr Ser Arg Tyr Val Ala Ala Val Ala Pro Asp Leu Val Ala Ser Val Thr Thr IleGly Thr Pro His Arg Gly Ser Glu Phe Ala Asp Phe Val Gln Ser Val Leu Ala Tyr AspPro Thr Gly Leu Ser Ser Ser Val Ile Ala Ala Phe Ala Asn Val Phe Gly Ile Leu Thr SerSer Ser Asn Asn Thr Asn Gln Asp Ala Leu Ala Ala Leu Lys Thr Leu Thr Thr Ser GlnAla Ala Thr Tyr Asn Gln Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys GlnThr Gly Ala Pro Thr Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp Ala GlyThr Ala Ile Gln Pro Thr Ile Ser Ala Phe Gly Val Thr Gly Ala Thr Asp Thr Ser Thr IlePro Leu Ile Asp Pro Ala Asn Ala Leu Asp Leu Ser Thr Leu Ala Leu Phe Gly Thr GlyThr Val Met Ile Asn Arg Ala Ser Gly Gln Asn Asp Gly Leu Val Ser Lys Cys Ser AlaLeu Tyr Gly Lys Val Leu Ser Thr Ser Tyr Lys Trp Asn His Leu Asp Glu Ile Asn GlnLeu Leu Gly Val Arg Gly Ala Asn Ala Glu Asp Pro Val Ala Val Ile Arg Thr His AlaAsn Arg Leu Lys Leu Ala Gly Val
用SingnalP3.0软件分析了脂肪酶蛋白质序列的结构，在网站http://www.cbs.dtu.dk/services/SignalP/上提交了脂肪酶蛋白质序列，预测参数选为革兰氏阴性菌、神经网络和隐马科夫模型，预测结果表明该蛋白有100％的几率拥有信号肽，信号肽最可能的切割微点在第40和41个氨基酸间，这与大部分Burkholderia属菌所产相近分子量大小的脂肪酶吻合。
本发明还提供耐有机溶剂脂肪酶YCJ01在有机相催化手性拆分的应用。所述的耐有机溶剂脂肪酶在有机溶剂异丙醚体系中，催化底物扁桃酸和乙酸乙烯酯进行酯交换转化为乙酰氧基扁桃酸，转化率达50％，产物为S型乙酰氧基扁桃酸，eep值＞99.9％；拆分后底物为R型扁桃酸，ees值＞99.9％。
本发明的有益效果在于菌株Burkholderia ambifaria YCJ01的耐有机溶剂脂肪酶YCJ01产率较高，发酵60h脂肪酶活力达到126.1U/mL；耐有机溶剂脂肪酶YCJ01易纯化、比活高，经过离子交换层析和疏水层析后比活达到4609.5U/mg；该脂肪酶温度稳定性良好，在疏水性和亲水性有机溶剂中都有较好的耐受性，表明其在双相和单相有机相催化尤其是手性拆分方面有很好的应用前景。
附图说明
图1显示耐有机溶剂脂肪酶YCJ01的SDS-PAGE电泳图，其中泳道1：Marker；泳道2：粗酶液；泳道3：DEAE阴离子交换层析后的酶液；泳道4：纯酶液(经疏水层析后得到的纯酶液)；
图2显示用25％有机溶剂处理60天后脂肪酶YCJ01的剩余活力；
图3显示40％亲水有机溶剂对脂肪酶YCJ01的影响。所用有机溶剂为异丙醇(■)、丙酮(●)、乙醇(▲)、甲醇
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对照(◆)；
图4显示耐有机溶剂脂肪酶YCJ01的最适反应pH；
图5显示耐有机溶剂脂肪酶YCJ01的pH稳定性(在各pH条件下放置1h后的剩余酶活)。缓冲液为0.05M Citiric acid-citrate sodium(pH 3.0-6.1)(■)、0.05M Na2HPO4-NaH2PO4(pH 6.06-8.11)(●)、0.05MTris-HCl(pH 7.5-8.9)(▲)、0.05M Glycine-NaOH(pH 8.79-10.6)
0.05M Na2HPO4-NaOH(10.98-11.95)
图6显示耐有机溶剂脂肪酶YCJ01的最适反应温度；
图7显示耐有机溶剂脂肪酶YCJ01的温度稳定性(在各温度条件下放置1h后的剩余酶活)；
图8显示耐有机溶剂脂肪酶YCJ01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 myristate(C14)为对硝基苯酚豆蔻酸酯，p-Nitrophenyl palmitate(C 16)为对硝基苯酚棕榈酸酯，p-Nitrophenyl stearate(C18)为对硝基苯酚硬脂酸酯(C18)。
具体实施方式
实施例一
本实验说明产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壤等样品中筛选获得耐有机溶剂极端微生物。采用吐温-80平板培养基，具体配方为：酵母膏5g/L，蛋白胨10g/L，NaCl 10g/L，吐温-8010mL/L，CaCl21g/L，琼脂20g/L。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。此方法筛选到脂肪酶产量高的耐有机溶剂极端微生物5株。
为了进一步检测所分泌脂肪酶的溶剂耐受性，对5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：蛋白胨5g/L，酵母粉5g/L，尿素5g/L，K2HPO4·3H2O 1.8g/L，MgSO4·7H2O 0.7g/L，橄榄油5mL/L，TritonX-1000.5mL/L，pH 8.0。培养温度为30℃，培养时间为60h，摇床转速为180rmp。发酵结束后，10000rmp 4℃离心15min，取上清为粗酶液。取粗酶液1.5mL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℃、150rpm震荡处理24h，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产生菌株。
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：50mM的Na2HPO4·12H2O-NaH2PO4·2H2O缓冲液(pH 7.5)，其中含有0.6％(m/v)TritonX-100和0.1％(m/v)的阿拉伯树胶；B溶液：称取3mg的对硝基苯酚棕榈酸酯(p-Nitrophenylpalmitate，pNPP)，溶解在1mL的异丙醇中；A溶液与B溶液按体积比9∶1制成浓度为16.5mM的对硝基苯酚棕榈酸酯底物溶液。反应体系中先加入10μL稀释适当倍数的酶液，以灭活的酶液为空白对照，再加入240μL底物溶液，在酶标仪中进行反应，反应温度为40℃，反应时间为10min，在410nm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，每分钟催化产生1μmol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产生菌株所产脂肪酶活力达到40.2U/mL，该菌株编号YCJ01。
实施例二
本实验说明耐有机溶剂脂肪酶产生菌YCJ01的生物学性质、鉴定及其产酶条件研究。
菌株YCJ01的生物学性质：该菌株为革兰氏阴性菌株，菌落呈米白色，圆形，边缘整齐，光滑湿润；菌体为杆菌，0.8～1.0×1.6～3.2mm，专性好氧，生长的最适温度为30℃～35℃。其生理生化特性表现在：不出现反硝化反应、明胶反应结果为阳性，氧化酶阳性，可分解脂肪，可利用D-核糖、D-阿拉伯糖、海藻糖等，不可利用麦芽糖。
菌株YCJ01的菌种鉴定：经BIOLOG自动细菌鉴定仪鉴定，Sim值为0.576(24h培养)，鉴定结果显示该菌属于Burkholderia属；后经16S rDNA序列分析，表明该菌株与数据库中Burkholderia ambifaria MC40-6菌的同源性为99％。综合BIOLOG和16S rDNA分析结果，该菌株鉴定为Burkholderia ambifaria，命名为Burkholderia ambifaria YCJ01。
Burkholderia ambifaria YCJ01产酶条件研究：采用单因素替换法研究发酵培养基的碳源(葡萄糖、蔗糖、麦芽糖、果糖、淀粉、糊精)、氮源(蛋白胨、酵母膏、牛肉膏、尿素、大豆粉)、诱导剂(花生油、橄榄油、大豆油、玉米油、菜籽油、葵花籽油)、表面活性剂(Tween 80、SDS、TritonX-100、阿拉伯树胶)、初始pH，接种量，发酵温度，装液量等对Burkholderia ambifaria YCJ01产脂肪酶的影响，得出优化后的培养基组分为：糊精8g/L，酵母粉5.05g/L，牛肉膏7.71g/L，尿素5g/L，MgSO4·7H2O 0.7g/L，K2HPO4·3H2O 1.8g/L，菜籽油5mL/L，TritonX-1000.75mL/L，发酵初始pH 8.0；优化后的培养条件为：接种量5％(V/V)，发酵温度30℃，摇床转速180rpm，装液量40mL/250mL。在此优化条件下，发酵60h后，酶活力达到126.1U/mL，与初始培养基及培养条件下酶活40.2U/mL相比，提高了2.1倍。
实施例三
本实验说明耐有机溶剂脂肪酶YCJ01的纯化程序。
将Burkholderia cepacia YCJ01在产酶培养基中培养60h后，发酵液在10,000rmp在4℃离心15min，取上清为粗酶液；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0.05M的Tris-HCl(pH 7.25，NaCl 1mol/L)缓冲液阶段洗脱，收集脂肪酶活力峰，用0.05M Tris-HCl(pH 7.25，(NH4)2SO4含量为1mol/L)透析，再采用Phenyl-Sepharose FF疏水层析进行纯化，以含20％乙醇的0.05M Tris-HCl(pH 7.25)和含40％乙醇的0.05M Tris-HCl(pH7.25)溶液进行洗脱，收集脂肪酶活力峰。通过SDS-PAGE电泳图，发现两步纯化后的耐有机溶剂脂肪酶(命名为耐有机溶剂YCJ01脂肪酶)已达电泳纯(图1)，该脂肪酶表观分子量约为33kDa。纯化倍数为12.5倍，回收率为24.2％，纯化后脂肪酶比活达到4609.5U/mg，汇总见表1。
表1耐有机溶剂脂肪酶YCJ01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
本实验说明耐有机溶剂脂肪酶YCJ01编码基因的分离克隆程序。
将纯化的耐有机溶剂脂肪酶YCJ01(委托国家生物医学分析中心，NBCA)进行LC-MS/MS分析测定其氨基酸片段，结果表明该酶与Burkholderia属中多株菌的脂肪酶序列相近，其中相似度最高的为Burkholderia cenocepacia AU 1054脂肪酶基因(Score：535，emPAI：0.96)，参照Burkholderia cenocepaciaAU 1054的脂肪酶基因，在其脂肪酶基因CDS两端设计引物，以Burkholderiaambifaria YCJ01的总基因为模板，扩增耐有机溶剂脂肪酶YCJ01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)：CATGCATGGCCAAATCGATGC
LD1(SEQ ID：4)：TCACACGCCCGCGAGTTTCAA
将扩增到了11kb的PCR片段连接到pMD18-T载体，序列测定表明，此片段有一个全长为1095bp的阅读框。与Burkholderia cepaciaS31脂肪酶基因同源性为92.4％，所编码氨基酸序列同源性为96.4％。其中成熟肽氨基酸同源性为96.6％。
实施例五
本实验说明耐有机溶剂脂肪酶YCJ01的酶学性质。
脂肪酶YCJ01的有机溶剂耐受性：向脂肪酶YCJ01酶液中分别加入12种有机溶剂(按照实施三制备)，其混合比例分别为纯酶液∶有机溶剂＝3∶1(V/V)与纯酶液∶有机溶剂＝3∶2(V/V)，疏水有机溶剂组中对照不添加有机溶剂，亲水有机溶剂组中对照添加与溶剂等体积的缓冲液。30℃，150rpm振荡每隔24h取样，以对硝基苯酚棕榈酸酯为底物检测脂肪酶活力。脂肪酶YCJ01具有良好的有机溶剂耐受性，其在25％的异丙醚、正十六烷、壬烷、正辛烷、异辛烷、正庚烷、正己烷、异丙醇、丙酮、乙醇、甲醇、DMSO、DMF等有机溶剂中处理60天，显示了很好的耐受性(图2)，在40％的丙酮、甲醇、DMSO等亲水有机溶剂中仍有较好的耐受性。如图3所示。
耐有机溶剂脂肪酶YCJ01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，pH7.5条件下的脂肪酶活力为对照(100％)，不同pH体系中的酶活如图4所示。耐有机溶剂脂肪酶YCJ01的最适反应pH为7.5。以原始酶液的脂肪酶活力为对照，检测该酶的pH稳定性(图5)。将耐有机溶剂脂肪酶YCJ01在不同pH的缓冲溶液中30℃保温1h后测残余酶活，实验表明该脂肪酶在pH 5.5～8.5的范围内具有较高的稳定性，在pH 11.0的溶液中保温1h后，仍然保留最高活力的60％。
耐有机溶剂脂肪酶YCJ01最适反应温度和热稳定性的检测：最适反应温度的测定在0.05M 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60℃(图6)。热稳定性测定：耐有机溶剂脂肪酶YCJ01在30℃、40℃、50℃、60℃、70℃、80℃下处理1h以后，测定残留酶活。由图7可以看出该脂肪酶具有较好的热稳定性，60℃以下处理1h后活力基本不变，在70℃处理1h后仍然保留最高活力的75％。
耐有机溶剂脂肪酶YCJ01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，底物分别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8，耐有机溶剂脂肪酶YCJ01的最适人工底物为对硝基苯酚棕榈酸酯(C16)。
实施例六
本实验说明耐有机溶剂脂肪酶YCJ01在有机相中拆分手性化合物的应用。以30mM的扁桃酸和215mM的乙酸乙烯酯作为反应底物；以异丙醚作为溶剂，溶剂的加入量为2mL；加入1％(w/v)耐有机溶剂YCJ01脂肪酶酶粉，在45℃，转速180rpm条件下反应，定时取样。样品吹干后用流动相(正己烷∶异丙醇∶三氟乙酸＝94∶6∶0.2)稀释适当倍数，采用HPLC进行检测。结果表明，反应36h后，转化率达到50％，产物为S型乙酰氧基扁桃酸，eep＞99.9％；拆分后底物为R型扁桃酸，ees＞99.9％。达到了理论极限拆分效果，表明YCJ01脂肪酶具有很好的对映体选择性，在手性化合物拆分中具有良好的应用前景。
序列表
<110> 南京工业大学
<120> 一种高稳定性耐有机溶剂脂肪酶产生菌及脂肪酶和其基因与应用
<160> 4
<210> 1
<211> 1095
<212> DNA
<213> Burkholderia ambifaria YCJ01
<400> 1
atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcttgcgc gatgagcgtc 60
gcgccgttcg cggggacgac cgcgctgatg acgctcgcga cgacgcgcgc ggcgatggcg 120
gcaaccgcgc cggcggacga ctacgcgacg acgcgttatc cgatcatcct cgtgcacggg 180
ctcaccggca ccgacaagta cgcgggcgtg ctcgagtact ggtacggcat ccaggaggac 240
ctgcagcagc atggcgcgac cgtctacgtc gcgaacctgt cgggattcca gagcgacgac 300
gggccgaatg ggcgcggtga acaactgctc gcttacgtca agacggtgct cgccgcgacc 360
ggtgcgacca aggtgaacct ggtcggtcac agccagggcg gcctcacgtc gcgctatgtc 420
gcggccgtcg cacccgatct cgtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480
tcagagttcg ccgacttcgt gcagagcgta ctcgcctacg atccgaccgg attgtcgtcg 540
tcggtgatcg cagcgttcgc caatgtgttc ggtatcctga cgagcagcag caacaacacc 600
aaccaggacg cgctcgccgc actgaagacg ctgacgactt cccaggccgc cacgtacaac 660
cagaattacc cgagcgcggg cctcggcgcg ccgggcagct gccagacggg tgcgccgacg 720
gaaaccgtcg gtggcaatac gcacctgctg tattcgtggg ccggcacggc gatccagccg 780
accatctccg cgttcggcgt gacgggcgcg acggatacca gcacgattcc gctgatcgac 840
ccggcaaacg cgctggacct gtcgacgctt gcgttgttcg gcacgggcac ggtgatgatc 900
aatcgcgcct cgggccagaa cgacgggctc gtgtcgaagt gcagtgcgct gtacgggaag 960
gtgctgagca cgagctacaa gtggaaccat ctcgatgaga tcaaccagtt gctcggtgtg 1020
cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaaa 1080
ctcgcgggcg tgtga 1095
<210> 2
<211> 364
<212> PRT
<213> Burkholderia ambifaria YCJ01
<400> 2
Met Ala Arg Ser Met Arg Ser Arg Val Val Ala Gly Ala Val Ala Cys
1 6 11 16
Ala Met Ser Val Ala Pro Phe Ala Gly Thr Thr Ala Leu Met Thr Leu
21 26 31
Ala Thr Thr Arg Ala Ala Met Ala Ala Thr Ala Pro Ala Asp Asp Tyr
36 41 46
Ala Thr Thr Arg Tyr Pro Ile Ile Leu Val His Gly Leu Thr Gly Thr
51 56 61
Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly Ile Gln Glu Asp
66 71 76
Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn Leu Ser Gly Phe
81 86 91 96
Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln Leu Leu Ala Tyr
101 106 111
Val Lys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys Val Asn Leu Val
116 121 126
Gly His Ser Gln Gly Gly Leu Thr Ser Arg Tyr Val Ala Ala Val Ala
131 136 141
Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Thr Pro His Arg Gly
146 151 156
Ser Glu Phe Ala Asp Phe Val Gln Ser Val Leu Ala Tyr Asp Pro Thr
161 166 171 176
Gly Leu Ser Ser Ser Val Ile Ala Ala Phe Ala Asn Val Phe Gly Ile
181 186 191
Leu Thr Ser Ser Ser Asn Asn Thr Asn Gln Asp Ala Leu Ala Ala Leu
196 201 206
Lys Thr Leu Thr Thr Ser Gln Ala Ala Thr Tyr Asn Gln Asn Tyr Pro
211 216 221
Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr Gly Ala Pro Thr
226 231 236
Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp Ala Gly Thr
241 246 251 256
Ala Ile Gln Pro Thr Ile Ser Ala Phe Gly Val Thr Gly Ala Thr Asp
261 266 271
Thr Ser Thr Ile Pro Leu Ile Asp Pro Ala Asn Ala Leu Asp Leu Ser
276 281 286
Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Ile Asn Arg Ala Ser
291 296 301
Gly Gln Asn Asp Gly Leu Val Ser Lys Cys Ser Ala Leu Tyr Gly Lys
306 311 316
Val Leu Ser Thr Ser Tyr Lys Trp Asn His Leu Asp Glu Ile Asn Gln
321 326 331 336
Leu Leu Gly Val Arg Gly Ala Asn Ala Glu Asp Pro Val Ala Val Ile
341 346 351
Arg Thr His Ala Asn Arg Leu Lys Leu Ala Gly Val
356 361
<210> 3
<211> 21
<212> DNA
<213> Artificial
<220>
<223> LU1
<400> 3
catgcatggccaaatcgatgc
<210> 4
<211> 21
<212> DNA
<213> Artificial
<220>
<223> LD1
<400> 4
tcacacgcccgcgagtttcaa
Claims (4)
1.一种Burkholderia属菌所产生的耐有机溶剂脂肪酶YCJ01，其特征在于，所述Burkholderia属菌是保藏登记号为CCTCC NO：M2011058的伯克霍尔德氏菌，命名为Burkholderia ambifaria YCJ01，所述耐有机溶剂脂肪酶YCJ01编码基因的核苷酸序列为SEQ ID NO:1。
2.根据权利要求1所述的Burkholderia属菌所产生的耐有机溶剂脂肪酶YCJ01，其特征在于所产耐有机溶剂脂肪酶YCJ01氨基酸序列为：SEQ ID NO:2，其由SEQ ID NO:1中所述的核苷酸序列编码。
3.根据权利要求1所述的Burkholderia属菌所产生的耐有机溶剂脂肪酶YCJ01在手性化合物拆分中的应用。
4.根据权利要求3所述的Burkholderia属菌所产生的耐有机溶剂脂肪酶YCJ01在手性化合物拆分中的应用，其特征在于所述耐有机溶剂脂肪酶在有机溶剂体系中，可完成药物中间体扁桃酸的手性拆分。
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| CN105400752A (zh) * | 2015-12-17 | 2016-03-16 | 中国科学院微生物研究所 | 一种具有转酯活性的脂肪酶Lip-1及其编码基因与应用 |
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| CN103275898B (zh) * | 2013-05-28 | 2015-04-08 | 安徽师范大学 | 一种脂肪酶高产菌株、其筛选方法及用该菌株发酵生产脂肪酶的方法 |
| CN105349507B (zh) * | 2015-12-15 | 2018-09-28 | 中国科学院南海海洋研究所 | 一种脂肪酶LIPDa6及其编码基因和应用 |
| CN105969836B (zh) * | 2016-05-11 | 2020-04-03 | 南京工业大学 | 一种酶法拆分阿巴卡韦手性中间体文斯内酯的方法 |
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| CN105400752B (zh) * | 2015-12-17 | 2019-02-01 | 中国科学院微生物研究所 | 一种具有转酯活性的脂肪酶Lip-1及其编码基因与应用 |
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