CN101735967B - 一种耐有机溶剂脂肪酶、其应用及其产生菌株 - Google Patents
一种耐有机溶剂脂肪酶、其应用及其产生菌株 Download PDFInfo
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- CN101735967B CN101735967B CN2009102124474A CN200910212447A CN101735967B CN 101735967 B CN101735967 B CN 101735967B CN 2009102124474 A CN2009102124474 A CN 2009102124474A CN 200910212447 A CN200910212447 A CN 200910212447A CN 101735967 B CN101735967 B CN 101735967B
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- Enzymes And Modification Thereof (AREA)
Abstract
本发明的目的是提供一种耐有机溶剂脂肪酶的产生菌，以及用该产生菌制备而得的耐有机溶剂脂肪酶和该脂肪酶在有机相中催化合成生物柴油的应用。通过筛选获得菌株Pseudomonas aeruginosa LX1，其产生的耐有机溶剂LX1脂肪酶具有SEQ ID NO：2所示的氨基酸序列，编码基因具有SEQ ID NO：1所示的核苷酸序列。本发明还提供耐有机溶剂LX1脂肪酶在有机相酶催化反应中的应用，尤其是合成生物柴油的酯交换反应。所述的耐有机溶剂脂肪酶在有机溶剂叔丁醇体系或无溶剂中，催化底物大豆油和甲醇酯交换合成清洁能源生物柴油，转化率达80～90％。
Description
技术领域
本发明涉及一种耐有机溶剂脂肪酶，该耐有机溶剂脂肪酶在有机相中催化合成生物柴油的应用，属于微生物学与酶学领域。
技术背景
脂肪酶(Lipase，EC 3.1.1.3)是一类水解酶，能催化天然油脂底物水解，产生甘油二酯、甘油单酯，脂肪酸和甘油，在非水相中能催化合成、转酯、氨解等反应，因此被广泛运用于精细化工、洗涤、医药、食品、造纸、皮革加工、纺织和饲料工业等领域。
脂肪酶的很多底物不溶于，因此非水相中进行反应有利于增加底物溶解度，从而提高产率，降低成本。非水催化同时还具有以下优点：具有高度的立体选择性和区域选择性，有机介质可改变选择性；控制反应平衡向所需方向移动，如水解酶在有机介质中能催化脱水缩合反应；有效防止微生物污染，产物易于分离纯化等。近年来，采用动植物油脂与低碳醇进行酯交换反应制备生物柴油成为的研究热点，其中的反应物动植物油脂不溶于水，所以非水催化将大大提高产率。然而，催化反应所用的脂肪酶大多是商品酶，成本昂贵，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，具有能稳定存在于有机溶剂中的天然特性。这类脂肪酶通常由耐有机溶剂极端微生物产生。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，不易纯化，从而影响其应用于工业催化。
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，以及用该产生菌制备而得的耐有机溶剂脂肪酶和该脂肪酶在有机相中催化合成生物柴油的应用。
本发明提供一种耐有机溶剂脂肪酶产生菌，该菌为铜绿假单胞菌，命名为Pseudomonas aeruginosa LX1，其保藏登记号为CCTCC NO：M 209221。
为了实现本发明的目的，本发明首先从中国境内的油污土样中筛选获得一株耐有机溶剂脂肪酶产生菌株LX1。对其进行生物学特征鉴定，该菌株为革兰氏阴性菌株，无芽孢。在肉汤培养基中生长24h后，菌落直径大小为1.5mm～2mm，生长范围为24℃～37℃，最适生长温度为27℃，生长pH为6.0～11.0，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、明胶反应结果为阳性，在有氧条件下生长。
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 aeruginosa，命名为Pseudomonas aeruginosa LX1。
本发明对Pseudomonas aeruginosa LX1进行了产酶条件优化，优化后产酶量高达40.81U/mL，比优化前8.5U/mL提高了3.8倍。
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LX1脂肪酶，其比活性达到156.19U/mg。
本发明对该菌所产耐有机溶剂LX1脂肪酶进行固定化，通过载体硅藻土吸附后用戊二醛交联，制备的固定化酶活力为16.5U/g。实验表明该游离耐有机溶剂LX1脂肪酶和固定化耐有机溶剂LX1脂肪酶在多种有机溶剂中均具有较好的耐受性。
本发明对该耐有机溶剂LX1脂肪酶进行了酶学性质的研究。该耐有机溶剂脂肪酶的最适反应pH为7.0，为中性脂肪酶，该脂肪酶在pH 6.5～10.5的范围具有很高的稳定性，在pH 12.0的溶液中保温1h以后仍然保留65％以上酶活力。其最佳反应温度为40℃，60℃处理1h，其残余酶活为初始酶活的60％，表明其具有良好的热稳定性。此酶的最佳人工底物为对硝基苯酚棕榈酸酯。
本发明Pseudomonas aeruginosa LX1产生的耐有机溶剂LX1脂肪酶具有SEQ IDNO：2所示的氨基酸序列，含有536个氨基酸。该耐有机溶剂脂肪酶的编码基因具有SEQ ID NO：1所示的核苷酸序列，具有1611个核苷酸，与铜绿假单胞菌Pseudomonasaeruginosa PAO1氨肽酶基因同源性为99％，为首次报道的脂肪酶基因。
本发明还提供耐有机溶剂LX1脂肪酶在有机相酶催化反应中的应用。所述的有机相酶催化反应可以为合成生物柴油的酯交换反应。所述的耐有机溶剂脂肪酶在有机溶剂叔丁醇体系或无溶剂中，催化底物大豆油和甲醇酯交换合成清洁能源生物柴油，转化率达80～90％。
本发明的有益效果在于菌株Pseudomonas aeruginosa LX1的耐有机溶剂LX1脂肪酶产率高，发酵30h脂肪酶活力达到40.81U/mL；耐有机溶剂LX1脂肪酶易纯化、比活高，经过硫酸铵沉淀和离子交换层析后比活达到156.19U/mg；该脂肪酶作用pH范围广、耐高温，对有机溶剂耐受性强尤其是对甲醇、乙醇和叔丁醇的耐受性使其能够应用于生物柴油的合成。
附图说明
图1为耐有机溶剂LX1脂肪酶的SDS-PAGE电泳图，其中泳道1，Marker；泳道2，粗酶液；泳道3，硫铵沉淀后酶液；泳道4，纯酶液(经硫铵沉淀、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LX1的最适反应pH；
图3显示耐有机脂肪酶LX1的pH稳定性；
图4显示耐有机溶剂脂肪酶LX1的最适反应温度；
图5显示耐有机溶剂脂肪酶LX1的温度稳定性；
图6显示耐有机溶剂脂肪酶LX1的底物特异性，其中p-Nitrophenyl palmitate(C16)为对硝基苯酚棕榈酸酯，p-Nitrophenyl acetate(C2)为对硝基苯酚乙酸酯，p-Nitrophenylbutyrate(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 stearate(C18)为对硝基苯酚硬脂酸酯(C18)。
本发明的微生物分类命名为铜绿假单胞菌Pseudomonas aeruginosa LX1，保藏日期为2009年10月13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 209221。
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，然后采用橄榄油罗丹明B平板从其中筛选出6株脂肪酶高产菌株。橄榄油罗丹明B平板的具体配方为：酵母膏1g/L，玉米浆5mL/L，K2HPO4 1g/L，MgSO4·7H2O 0.5g/L，橄榄油60mL/L，Rhodamin B 0.024g/L。
为了获得优良的耐有机溶剂脂肪酶产生菌，通过检测摇瓶产酶能力及脂肪酶有机溶剂稳定性对上述6株菌进行复筛。将6株菌分别接种到产酶发酵培养基，具体配方为：玉米浆15mL/L，尿素5g/L，葡萄糖5g/L，葵花籽油5mL/L，K2HPO4 2g/L，MgSO4·7H2O 0.5g/L，pH 7.5。培养温度为30℃，培养时间为48h，摇床转速为180rpm。将发酵液在10,000rpm，4℃下离心10min，取上清为粗酶液。检测各菌所产粗酶液脂肪酶活力和有机溶剂稳定性，其中菌株LX1的粗酶液的脂肪酶活力达到8.5U/mL，具有优良的有机溶剂稳定性。
有机溶剂稳定性的检测方法：1.5mL粗酶液中分别加入0.5mL的十六烷、十四烷、十二烷、癸烷、壬烷、辛烷、庚烷、己烷、辛醇、庚醇、己醇、戊醇、异丙醇、丙酮、甲醇、甘油、二甲基甲酰胺(DMF)和二甲基亚砜(DMSO)中的一种有机溶剂，于30℃、150rpm震荡处理1h，检测脂肪酶残余酶活。
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：0.05M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，每毫升酶液每分钟催化产生1μmol对硝基苯酚(pNP)所需的酶量。
实施例二
本实验说明耐有机溶剂脂肪酶产生菌LX1的生物学性质、鉴定及其产酶条件研究。
菌株LX1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24h后，菌落大小直径为1.5mm～2mm，生长温度范围为24℃～37℃，最适生长温度为27℃，生长pH范围为6.0～11.0，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、明胶反应结果为阳性，在有氧条件下生长。
菌株LX1的菌种鉴定：经BIOLOG自动细菌鉴定仪鉴定和16S rDNA序列分析，表明该菌株为铜绿假单胞菌，并将其命名为Pseudomonas aeruginosa LX1。
Pseudomonas aeruginosa LX1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、果糖、蔗糖、麦芽糖、乳糖、淀粉、糊精)、氮源(玉米浆、酵母膏、牛肉膏、酵母粉、胰蛋白胨、玉米粉)、诱导剂(葵花籽油、菜籽油、花生油、橄榄油、大豆油、玉米油、油酸、棕榈酸、三丁酸甘油酯)、初始pH，菌种的接种龄、接种量，发酵温度及摇床转速等对Pseudomonas aeruginosa LX1产脂肪酶的影响，并用响应曲面法优化其产酶水平，得出优化后的培养基组分为：葡萄糖5g/L，玉米浆20mL/L，牛肉膏10g/L，MgSO4·7H2O 0.5g/L，K2HPO4·3H2O 2g/L，菜籽油5mL/L，发酵初始pH 8.0；优化后的培养条件为：接种量5％(V/V)，接种龄10h，发酵温度27℃，摇床转速220rpm，装液量40mL/250mL。在此优化条件下，发酵30h后，酶活力达到40.81U/mL，与初始培养基及培养条件下酶活8.5U/mL相比，提高了3.8倍，高于已报道来源于Pseudomonas aeruginosa的耐有机溶剂脂肪酶活力。
实施例三
本实验说明耐有机溶剂LX1脂肪酶的纯化程序。
将Pseudomonas aeruginosa LX1在产酶培养基中培养30h后，发酵液在10,000rpm，4℃离心10min，取上清为粗酶液；将粗酶液置于冰浴中，先用20％饱和度的硫酸铵沉淀，离心后取上清，再用50％饱和度的硫酸铵沉淀，沉淀用0.01M的pH 7.10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0.01M的Tris-HCl(pH 7.10，NaCl含量为1mol/L)缓冲液进行洗脱，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LX1脂肪酶)已达电泳纯，该脂肪酶亚基分子量约为56kDa。纯化倍数为4.29倍，回收率为41.09％，最终脂肪酶比活达到156.19U/mg，汇总见表1。
表1耐有机溶剂LX1脂肪酶的纯化步骤及结果
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LX1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LX1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，结果表明该酶与铜绿假单胞菌Pseudomonas aeruginosa PAO1中氨肽酶序列最相近，因此在此酶CDS两端外各约150bp处设计引物，扩增耐有机溶剂LX1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)：GCTTATCGATCATCGCCTCAC
LD1(SEQ ID：4)：CGAACTGGGGCTGGACAT
PCR反应参数为：94℃预变性2min；94℃变性30sec；65℃退火30sec，72℃延伸1min30sec；循环30轮后，72℃保温10min。根据该反应条件，扩增到了1.9kb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1611bp的阅读框，含有信号肽序列24个氨基酸，编码成熟蛋白氨基酸498个。与铜绿假单胞菌Pseudomonas aeruginosa PAO1氨肽酶基因同源性为99％。
实施例五
本实验说明耐有机溶剂LX1脂肪酶的固定化方法。
在250mL摇瓶中加入1g硅藻土和5mL耐有机溶剂LX1脂肪酶(按照实施例3获得)，在水浴摇床中(20℃，120rpm)固定1h，倒出上清，并且用pH 7.0磷酸缓冲液洗涤3遍，再加入0.5％(v/v)的戊二醛溶液5mL，在水浴摇床中(20℃，120rpm)固定1h后倒出上清，冷冻干燥即制备得到交联的固定化脂肪酶。所制备的固定化脂肪酶活力为16.5U/g。
实施例六
本实验说明耐有机溶剂LX1脂肪酶的酶学性质。
耐有机溶剂LX1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LX1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℃，150rpm振荡48h后取样，以对硝基苯酚棕榈酸酯为底物检测脂肪酶活力。固定化耐有机溶剂LX1脂肪酶(按照实施例四制备)在有机溶剂中处理1h，结果如表2所示。耐有机溶剂LX1脂肪酶具有良好的有机溶剂耐受性，游离耐有机溶剂LX1脂肪酶在25％(v/v)的正十六烷、异辛烷、正己烷、丙酮、DMF、DMSO和甘油中与对照(不加有机溶剂)相比半衰期变大，但固定化耐有机溶剂LX1脂肪酶有机溶剂耐受性发生了改变，在游离酶耐受性较差的有机溶剂叔丁醇和乙腈中固定化酶的半衰期大于10天。该脂肪酶对于甲醇、乙醇和叔丁醇的耐受性可以指导生物柴油的合成。
表2有机溶剂对LX1脂肪酶的影响
注：括号中的数据为耐有机溶剂LX1脂肪酶在不同有机溶剂中的半衰期
耐有机溶剂LX1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LX1脂肪酶的最适反应pH为7.0，是中性脂肪酶。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LX1脂肪酶加入不同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 6.5～10.5的范围内具有较高的稳定性，在pH 12.0的溶液中保温1h后，仍然保留65％的活力。
耐有机溶剂LX1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0.05M 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40℃(图4)，在60℃条件下反应仍然具有最佳情况下20％的酶活力。热稳定性测定：耐有机溶剂LX1脂肪酶在30℃、40℃、50℃、60℃、70℃下处理1h以后，测定残留酶活。由图5可以看出该脂肪酶具有较好的热稳定性，在60℃处理1h后仍然保留60％的活力。
耐有机溶剂LX1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LX1脂肪酶的最适人工底物为对硝基苯酚棕榈酸酯。
耐有机溶剂LX1脂肪酶催化性能检测：由于该脂肪酶与铜绿假单胞菌Pseudomonasaeruginosa PAO1氨肽酶基因同源性为99％，因此采用检测其催化性能的方式来进一步确定其为脂肪酶。以对硝基苯酚棕榈酸酯为底物(脂肪酶底物)时，该酶转化数为6.1*105S-1；以丙氨酸对硝基苯胺为底物(氨肽酶底物)时，该酶转化数6.0*103S-1。可以看出，该酶对对硝基苯酚棕榈酸酯的催化效率远远大于丙氨酸对硝基苯胺，因此该酶的天然底物为酯类(脂类)物质而不是酰胺类物质，我们所研究的耐有机溶剂LX1脂肪酶确实是新型的脂肪酶。
实施例七
本实验说明耐有机溶剂LX1脂肪酶在有机相中制备生物柴油合成的应用
以5mmol(4.5g)的大豆油和15mmol(600μL)的甲醇作为反应底物，其中甲醇一步加入；以叔丁醇、正己烷、异辛烷作为溶剂或无溶剂，溶剂的加入量为5mL；加入1g固定化耐有机溶剂LX1脂肪酶，在30℃，转速150rpm条件下反应，定时取样。样品用正己烷稀释适当倍数，采用GC进行检测(以不同的脂肪酸甲酯为标准品)。结果表明，叔丁醇为最佳溶剂，生物柴油的转化率24h后达到80％～90％。
SEQUENCE LISTING
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Claims (3)
1.一种耐有机溶剂脂肪酶产生菌，其特征在于该菌命名为铜绿假单胞菌LX1(Pseudomonas aeruginosa LX1)，其保藏登记号为CCTCC M 209221。
2.一种权利要求1所述菌株产生的耐有机溶剂脂肪酶在有机相酶催化合成生物柴油的酯交换反应中的应用，该耐有机溶剂脂肪酶的氨基酸序列为SEQ ID NO：2。
3.根据权利要求2所述的耐有机溶剂脂肪酶的应用，其特征在于所述耐有机溶剂脂肪酶在有机溶剂叔丁醇体系或无溶剂中，催化底物大豆油和甲醇进行酯交换合成清洁能源生物柴油，转化率达80～90％。
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| JP特开平10-234365A 1998.09.08 |
| Winsor, G. L.等.NC_002516.《GeneBank》.2009, * |
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