CN105400750B - 一种深海新型低温耐盐酯酶及应用 - Google Patents
一种深海新型低温耐盐酯酶及应用 Download PDFInfo
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Abstract
本发明公开了一种深海细菌来源低温耐盐酯酶E10及其编码基因与应用。本发明涉及酯酶基因e10来自深海细菌Croceicoccus marinus E4A9T,核苷酸序列如SEQ ID NO.1所示。本发明所述的酯酶基因经异源表达后,底物为对硝基苯酚己酸酯(C6)时催化活性最高,酶活为29.4U/mg。酯酶E10酯酶催化水解最适温度为15~20℃;在1mol/L NaCl中仍能保持80%以上活性;在添加有机溶剂二甲基亚砜、甘油和异丙醇条件下,酶学活性增大。该酯酶具有低温和耐盐的特征,可应用于手性药物合成、食品加工及食品风味改良、废水处理和洗涤工业等低温含盐条件下的工业生产。
Description
技术领域
本发明属于基因工程领域,具体涉及一种深海细菌来源低温耐盐酯酶、其编码基因及其应用。
背景技术
脂类水解酶是一类能够催化酯类化合物的水解和合成的酶。根据脂类水解酶的生化特征和序列特征,微生物来源的脂类水解酶主要分为8大家族。大多数脂类水解酶的活性位点Ser附近有一段相似的氨基酸序列Gly-x-Ser-x-Gly,但第II家族的活性位点附近序列为Gly-Asp-Ser-Leu(GDSL),因此该家族又称为GDSL家族。由于其结构的可变性,GDSL家族酯酶通常具有多种水解功能和较宽的底物谱,GDSL家族酯酶具有脂肪酶、蛋白酶、硫酯酶、磷脂酶、芳香酯酶和酰基转移等多种活性,在工业中具有广泛的应用潜力。
近年来,随着分子生物学技术特别是测序技术的快速发展,微生物全基因组测序已十分廉价和高效。基于全基因组测序数据并利用生物信息学分析手段,直接从基因组序列中获得酶基因成为了获得新型酶的一种重要方法,称为“in silicon分析”。
低温酯酶由于在低温下具有较高或最好的活性,在食品风味改良和废水处理等工业中比高温酯酶更具应用潜力,已受到广泛关注。海洋中大部分区域为低温环境,微生物在海洋环境中演化出了适应低温的独特代谢方式,因此海洋微生物是低温酯酶的巨大宝库。目前授权的低温细菌酯酶专利还较少。中国专利201110208211.0提供了一种深海沉积物来源的低温酯酶,其最适反应条件为20℃,最适催化底物为对硝基苯酚丁酸酯。此外,中国专利201510015698.7提供了一种最适反应条件为45℃的海洋来源适冷酯酶,该酯酶在20℃以下具有较高催化活性和稳定性。
发明内容
本发明的目的是提供一种新的深海细菌来源酯酶、其编码基因及其制备方法,该酯酶可用于酯类降解及其他酯类化合物的生物催化和转化。
本发明针对分离自深海沉积物的细菌Croceicoccus marinus E4A9T(公众可以从中国普通微生物菌种保藏管理中心购买获得),基于全基因组序列分析筛选获得酯酶基因e10,其核苷酸序列如SEQ ID No.1所示。酯酶基因e10大小为618bp,碱基组成为:106A(17.15%)、97T(15.70%)、203C(32.85%)和212G(34.30%),编码蛋白大小为205个氨基酸残基,其氨基酸序列如SEQ ID No.2所示。将该酯酶序列在GenBank中进行同源搜索,与之相似性最高的是同属细菌Croceicoccus naphthovorans中的酯酶,相似性为64%(其在GenBank数据库中的注册号为WP_047820200)。***发育分析结果表明,酯酶E10属于酯酶家族中的第II家族。氨基酸序列分析结果显示,酯酶E10活性位点丝氨酸附近序列为具有甘氨酸、天冬氨酸、丝氨酸和亮氨酸组成的保守区(氨基酸位置为27至30),29位丝氨酸与178位天冬氨酸和181位组氨酸共同构成酯酶催化中心,说明E10属于酯酶第II家族。综上所述,E10应为酯酶家族中的一名新成员。
在不影响酯酶E10蛋白活性前提下,可对SEQ ID NO:2所示的远离催化中心氨基酸位置(优选远离27-30、178-181氨基酸位置)的氨基酸序列进行各种取代、添加和/或缺失一个或几个氨基酸获得具有酯酶E10活性的衍生蛋白质。根据本领域技术的公知常识,蛋白质的生物学活性是和其功能结构域密切相关的。一般来说,只有发生在功能结构域的位点突变可能对蛋白质的二维和三维结构产生影响,从而影响其生物学活性。而对于发生在远离功能结构域(优选27-30、178和181氨基酸位置)的氨基酸位点,由于这一区域不参与蛋白功能构象,因而氨基酸的个别点突变不会对蛋白质的生物学活性产生实质性影响,从而能够基本保留原蛋白质的生物学功能。优选的酯酶E10突变体具有至少与SEQ ID NO:2所示的氨基酸序列90%以上的同源性,更优选具有至少95%以上的同源性,最优选具有至少99%以上的同源性。
同理,本发明还提供了编码如SEQ ID NO.2所示氨基酸序列的基因序列,其与SEQID NO.1所示的核苷酸序列一致;本发明还提供对SEQ ID NO.1所示的核苷酸序列中除79-90、532-543位核苷酸外的其他核苷酸进行替换、添加和/或缺失一个或几个核苷酸从而获得编码能基本保留酯酶E10蛋白生物学活性的突变体基因。优选的酯酶E10突变体基因具有至少与SEQ ID NO:1所示的核苷酸序列90%以上的同源性,更优选具有至少95%以上的同源性,最优选具有至少99%以上的同源性。
利用基因克隆技术,可将克隆到的酯酶E10基因连接到合适的载体上,并转化或转染到原核生物或真核生物宿主表达制备重组酯酶E10。合适的原核生物宿主包括各种细菌如E.coli等,合适的真核生物宿主包括酵母(如甲醇酵母)及哺乳动物细胞(如中国仓鼠卵巢细胞)等,优选采用原核表达***E.coli。
合适的载体为本领域技术人员所熟知的各种可商业化购买的原核或真核表达载体,原核表达载体如pET系列载体,pQE系列载体;酵母表达载体pPICZ-α-A,pHIL-D2,pPIC9,pHIL-S1(Invitrogen Corp.San Diego.California.USA);动物细胞表达载体pSVK3、pMSG(Amersham Pharmacia Biotech Inc.USA)等。一个优选的例子是将本发明筛选到的酯酶基因e10连接到大肠杆菌表达载体pSMT3上,并转化到大肠杆菌Rosetta(DE3)中,经诱导表达出高活性的重组酯酶。
本发明还提供了酯酶E10或能表达酯酶E10的宿主菌在工业上的应用,例如可用于催化酯类水解。通过酯酶活力测定表明,酯酶E10或上述能表达E10酯酶的宿主菌可用于水解短链脂肪酸酯,例如C2-C8短碳链脂肪酸酯,同时对C10-C16的长碳链脂肪酸酯也具有一定降解作用。优选的短链脂肪酸脂为具有C2-C8短碳链的对硝基苯酚酯,例如对硝基苯酚乙酸酯、对硝基苯酚丁酸酯、对硝基苯酚己酸酯、对硝基苯酚辛酸酯和对硝基苯酚癸酸酯等,其中底物为对硝基苯酚己酸酯(C6)时催化活性最高,酶活为29.4U/mg。
E10酯酶催化水解温度范围为15~35℃,优选为15~20℃;所述水解的pH值为4.0~9.0,优选为7.0~7.5。在1mol/L NaCl中仍能保持80%以上活性;在添加EDTA、Ba2+、Ca2+、Co2+、Mg2+和Sr2+条件下,对酶活影响不大;在添加有机溶剂二甲基亚砜、甘油和异丙醇条件下,酶学活性增大。
本发明从深海沉积物来源细菌Croceicoccus marinus E4A9T中筛选获得新的低温耐盐酯酶基因,发现了该基因编码蛋白具有优良的酶学特性,可应用于催化解酯和酶法合成酯产品生产过程中。获得的酯酶基因可克隆到合适的宿主中实现异源表达,实现工业化生产低温耐盐酯酶,为后续的工业应用提供成本低廉的低温耐盐酯酶起始材料。该酶的生产可在食品加工、洗涤剂和废水处理等低温或含盐生产工艺中显示出重要的经济和社会价值。
附图说明
图1为纯化酯酶E10的聚乙酰胺凝胶电泳分析图。
图2为酯酶E10的底物特异性图。C2:对硝基苯酚乙酸酯;C4:对硝基苯酚丁酸酯、C6:对硝基苯酚己酸酯;C8:对硝基苯酚辛酸酯;C10:对硝基苯酚癸酸酯;C12:对硝基苯酚十二酸酯;C14:对硝基苯酚十四酸酯;C16:对硝基苯酚十六酸酯;定义底物为C6时测定值为100%。
图3为酯酶E10最适反应温度图。
图4为酯酶E10最适反应pH图。
图5为二价阳离子对酯酶E10活性影响图。
图6为有机溶剂和去垢剂对酯酶E10活性影响图。
图7为NaCl对酯酶E10活性影响图。
具体实施方式
实施例1酯酶基因e10的获取
基于深海沉积物来源细菌Croceicoccus marinus E4A9T全基因组、开放阅读框预测及基因注释结果,筛选脂类水解酶相关基因。通过Blastx(http://blast.ncbi.nlm.nih.gov/)比对序列与数据库中已知酯酶基因序列的同源性。经数据库比对分析获得e10基因,大小为618bp,碱基组成为106A(17.15%)、97T(15.70%)、203C(32.85%)和212G(34.30%),其核苷酸序列如SEQ ID No:1所示。编码蛋白大小为205个氨基酸残基,其氨基酸序列如SEQ ID No.2所示。将该基因序列在GenBank中进行同源搜索,与之相似性最高的是同属细菌Croceicoccus naphthovorans中的酯酶,相似性为64%,其在GenBank数据库中的注册号为WP_047820200。
氨基酸序列分析结果显示,酯酶E10活性位点丝氨酸附近序列为具有甘氨酸、天冬氨酸、丝氨酸和亮氨酸组成的保守区(氨基酸位置为27至30),29位丝氨酸与178位天冬氨酸和181位组氨酸共同构成酯酶催化中心,说明E10属于酯酶第II家族。综上所述,E10应为酯酶家族中的一名新成员。
综上所述,E10应为酯酶家族中的一名新成员。
实施例2酯酶基因e10的重组表达质粒和重组菌株的构建
将本发明获得的酯酶基因e10克隆到表达载体上,构建重组表达菌株。基于NCBIORF Finder的ORF分析获得的酯酶基因的开放阅读框序列,设计扩增酯酶全基因的上游引物E10F(5’-TCGCGGATCCGTGGCGGACGGCGAGGC-3’,BamHI)和下游引物E10R(5’-TCCGCTCGAGCTAGAGGTCGTCGATCCTGTC-3’,XhoI),PCR扩增确认基因全长序列。采用酶切克隆的方法构建表达质粒,即用BamHI和XhoI双酶切PCR产物,纯化后的片段与经BamHI和XhoI双酶切的质粒pSMT3连接,采用CaCl2转化法转化至E.coli DH5α中,卡那霉素抗性筛选阳性克隆。采用质粒抽提试剂盒(Axygen,美国)提取阳性克隆的质粒,经BamHI和XhoI双酶切鉴定,获得700bp左右的DNA片段,经测序鉴定为酯酶基因e10。将重组表达质粒转化到E.coliRosetta(DE3)表达菌株中,构建表达重组菌株。
实施例3利用重组表达菌株表达重组酯酶基因e10
将构建好的3ml重组表达菌株转接到100ml含有20μg/ml卡那霉素和34μg/ml氯霉素的LB液体培养基中,37℃振荡培养至OD600达到0.6,加入终浓度为0.5mM的IPTG进行诱导表达,转入25℃以150r/min振荡培养8h。低温离心收集菌体,重悬于NTA-10溶液(500mM氯化钠,10mM咪唑,20mM Tris盐酸,pH 8.0)中,在冰上进行超声波破碎处理。低温离心收集上清,采用NTA-Ni2+亲和柱层析纯化表达蛋白。所表达的重组蛋白含有N端的6×His tag,可亲和吸附到层吸柱上,经过不同浓度的咪唑溶液梯度洗脱,收集洗脱液。经SDS-PAGE检测,得到电泳纯的重组酯酶蛋白E10,分子量约37kDa。用Lowry法测定蛋白质浓度,得到约0.64mg/100ml发酵液的表达量。
实施例4重组酯酶基因e10的活性检测
利用对硝基苯酚已酸酯法测定纯化的重组酯酶E10活性。具体操作:1ml反应体系中包括1mM对硝基苯酚己酸酯,100mM磷酸盐缓冲液(pH 7.5)和18ng纯酶蛋白(为10μl经稀释的纯化酶液),采用紫外可见光分光光度计(Beckman DU800型,美国)于20℃条件下连续测定吸光值A4052min,使用失活的酶液作为对照用于调零。一个酶活力单位定义为每分钟从对硝基苯酚酯催化产生lμmol对硝基苯酚的所需要的酶量。测得的酯酶活性为29.4U/mg。
实施例5酯酶E10底物特异性分析
酯酶E10的底物特异性分析采用体系:100mM Tris-HCl缓冲液(pH 7.5),1mM底物,加入1000ng纯酶蛋白,在20℃下连续测定吸光值A4052min。测定采用的底物为:对硝基苯酚乙酸酯(C2),对硝基苯酚丁酸酯(C4),对硝基苯酚己酸酯(C6),对硝基苯酚辛酸酯(C8),对硝基苯酚癸酸酯(C10),对硝基苯酚十二酸酯(C12),对硝基苯酚十四酸酯(C14),对硝基苯酚十六酸酯(C16)。经测定表明,酯酶E10对酰基碳链较短的对硝基苯酚酯(C2、C4、C6、C8和C10)具有较好的催化活性,其中底物为对硝基苯酚乙酸酯(C2)时催化活性最高,对硝基苯酚十四酸酯(C12)、对硝基苯酚十四酸酯(C14)和对硝基苯酚十六酸酯(C16)也具有一定的催化活性(图2)。结果表明,酯酶E10对酰基碳链较短脂类物质具有较好催化活性,对于短链脂类的水解活力优于长链脂类。
实施例6酯酶E10最适反应条件分析
酯酶E10最适反应温度在15~35℃范围内测定。具体操作为:100mM Tris-HCl缓冲液(pH 7.5),1mM对硝基苯酚己酸酯,加入1000ng纯酶蛋白,分别在15、20、25、30、35和40℃条件下连续测定吸光值A4052min。测定结果表明E10的反应温度范围为15~35℃,最适反应温度为15~20℃,说明酯酶E10具有低温特性(图3)。
酯酶E10最适反应pH在3.0~9.5范围内测定。具体操作为:在不同pH缓冲液中加入1mM对硝基苯酚己酸酯和1000ng纯酶蛋白,在20℃下连续测定吸光值A3482min。测定使用的缓冲液为:100mM柠檬酸-柠檬酸钠缓冲液(pH 3.0~6.0),100mM磷酸二氢钾-氢氧化钠缓冲液(pH 6.0~7.5),100mM Tris盐酸缓冲液(pH 7.5~8.5)和100mM 2-环己胺基乙磺酸-氢氧化钠缓冲液(pH 8.5~9.5)。测定结果表明,酯酶E10最适反应pH为7.5,在pH 4.0~9.0范围内具有活性(图4)。
实施例7酯酶E10酶学稳定性分析
二价阳离子对酯酶E10活性影响的测定具体操作为:在反应体系中分别加入10mMCo2+、Cu2+、Ca2+、Mg2+、Zn2+、Sr2+、Mn2+、Ni2+、Ba2+和乙二胺四乙酸(EDTA),测定酶活性。测酶活体系为:100mM Tris-HCl缓冲液(pH 7.5),1mM对硝基苯酚己酸酯,1000ng纯酶蛋白,于20℃下连续测定吸光值A4052min。测定结果表明,酯酶E10活性会被Mn2+和Cu2+完全抑制,在EDTA、Ba2+、Ca2+、Co2+、Mg2+和Sr2+存在下对酶活影响不大(图5)。
有机溶剂和去垢剂对酯酶E10活性影响的测定具体操作为:在反应体系中分别加入15%(v/v)有机溶剂(异丙醇、乙腈、乙醇、甲醇、丙酮、二甲基亚砜和二甲基甲酰胺)和1%去垢剂(w/v或v/v)(SDS、吐温20、吐温80和Triton X-100)然后测定酶的活性。测活体系为:100mM Tris-HCl缓冲液(pH 7.5),1mM对硝基苯酚己酸酯,1000ng纯酶蛋白,于20℃下连续测定吸光值A4052min。测定结果表明,酯酶E10活性会被SDS和乙腈完全抑制,而二甲基亚砜、甘油和异丙醇可增强其活性(图6)。
NaCl对酯酶E10活性影响的测定具体操作为:在反应体系中分别加入0、1、2、3、4和5mol/L NaCl,然后测定酶的活性。测活体系为:100mM Tris-HCl缓冲液(pH 7.5),1mM对硝基苯酚己酸酯,1000ng纯酶蛋白,于20℃下连续测定吸光值A4052min。结果表明,在1mol/L和2mol/L NaCl条件下,E10仍能保持80%和40%以上活性,说明E10具有较好的耐盐特性。
Claims (19)
1.一种酯酶,其氨基酸序列与Seq ID NO.2所示序列一致。
2.编码权利要求1所述酯酶的基因,其核苷酸序列如SEQ ID NO.1所示。
3.携带有权利要求2所述基因的载体。
4.根据权利要求3所述的载体,其特征在于:所述的载体选自pET系列载体,pQE系列载体,酵母表达载体pPICZ-α-A,pHIL-D2,pPIC9,pHIL-S1,动物细胞表达载体pSVK3或pMSG。
5.根据权利要求3所述的载体,其特征在于:所述的载体为大肠杆菌表达载体pSMT3。
6.一种宿主细胞,其由权利要求3-5任一项所述的载体经转化或转染原核生物或真核生物宿主细胞得到。
7.根据权利要求6所述的宿主细胞,其为细菌、酵母或哺乳动物细胞。
8.根据权利要求7所述的宿主细胞,其为E.coli细菌、甲醇酵母或中国仓鼠卵巢细胞。
9.根据权利要求8所述的宿主细胞,其为E.coli细菌。
10.权利要求1所述的酯酶或权利要求6所述的能表达酯酶的宿主细胞在催化酯类水解中的应用。
11.根据权利要求10所述的应用,其特征在于,所述的酯类为短链脂肪酸酯。
12.根据权利要求11所述的应用,其特征在于,所述短链脂肪酸酯为C2-C8短碳链。
13.根据权利要求12所述的应用,其特征在于,所述的C2-C8短链脂肪酸酯为具有C2-C8短碳链的对硝基苯酚酯。
14.根据权利要求13所述的应用,其特征在于,所述的具有C2-C8短碳链的对硝基苯酚乙酸酯、对硝基苯酚丁酸酯、对硝基苯酚己酸酯、对硝基苯酚辛酸酯和对硝基苯酚癸酸酯。
15.根据权利要求10所述的应用,其特征在于,所述的酯类为C10-C16的长碳链脂肪酸酯。
16.根据权利要求10-15任一项所述的应用,其特征在于,所述的酯酶催化水解温度范围为15~35℃。
17.根据权利要求16所述的应用,其特征在于,所述的酯酶催化水解温度范围为15~20℃。
18.根据权利要求10-15任一项所述的应用,其特征在于,所述的酯酶催化水解的pH值为4.0~9.0。
19.根据权利要求18所述的应用,其特征在于,所述的酯酶催化水解pH值为7.0~7.5。
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