CN113684195B - 一种甾醇酯酶和其编码基因及其突变体 - Google Patents
一种甾醇酯酶和其编码基因及其突变体 Download PDFInfo
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- CN113684195B CN113684195B CN202010422452.4A CN202010422452A CN113684195B CN 113684195 B CN113684195 B CN 113684195B CN 202010422452 A CN202010422452 A CN 202010422452A CN 113684195 B CN113684195 B CN 113684195B
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Abstract
本发明提供了一种甾醇酯酶和其编码基因及其突变体,所述的甾醇酯酶的氨基酸序列如SEQ ID NO.4所示的氨基酸序列;或将SEQ ID NO.4的氨基酸序列经一个或几个氨基酸的取代、缺失或添加由SEQ ID NO.4衍生的,且具有与所述甾醇酯酶相同功能的氨基酸序列;其编码基因的核苷酸序列如SEQ ID NO.3所示的核苷酸序列;或将SEQ ID NO.3的核苷酸序列经一个或几个核苷酸取代、缺失或添加由SEQ ID NO.3衍生的,且具有与所述甾醇酯酶基因相同功能的核苷酸序列。本发明的甾醇酯酶的最高酶活力可达11.66 U/mL,纯度高且酶学性质优良,为甾醇酯酶的规模化生产及推广应用提供了良好的前景。
Description
技术领域
本发明属于生物技术领域,具体涉及一种甾醇酯酶和其编码基因及其突变体。
背景技术
酯酶是指能够催化酯键水解或合成的一类酶的总称。酯酶可在水性介质中水解胆固醇脂肪酸酯和其他甾醇酯,又能在有机介质中催化酯化合成以及相应酯的交换反应。甾醇酯酶(EC 3.1.1.13)是酯酶的一种,可以定向降解体系中的甾醇酯键,生成甾醇与脂肪酸。甾醇酯酶作为一种重要的酶制剂,可以帮助有机体消化吸收油脂类营养成分,还可以解决机械制浆造纸工艺中的树脂障碍问题,同时还是检测血液中总胆固醇含量的主要反应物之一,因此甾醇酯酶在食品、医药及化工行业具有广泛的应用。
甾醇酯酶广泛分布于包括人体在内的高等动物与微生物中。由于在哺乳动物体内,甾醇酯酶主要存在于胰脏、肝脏、肾脏及肠粘膜等部位,而酶蛋白又多分离自动物内脏组织,所以通过内脏组织制备甾醇酯酶的方法不仅原料来源少,成本高,所产目标蛋白量也低,并且在分离过程中,因内脏组织营养丰富,往往会携带很多其他难以去除的蛋白酶,从而进一步降低甾醇酯酶的纯度。相比之下,因为微生物生长周期较短,生长条件简单,易于规模化生产,所以微生物来源的甾醇酯酶具有良好的制备优势,并且还可以通过分子生物学手段,从基因水平对微生物进行定向改造,提高单位产酶活力。
随着生物技术与化工产业的发展与融合,多种产酯酶微生物被筛选挖掘,不同菌种来源的酯酶性质不同。专利201611104306.7公开了一种通过基因分子改造而得到的造纸用甾醇脂酶的基因,但该酶的酶活力很低,仅为0.4U/mL;专利201710359673.X公开了一种通过宏基因组筛选等手段制备的酯酶,但该酶适用温度较低,因此这些酶都不利于工业化的应用。而如何以现有微生物为基础,辅以分子生物学手段,发现新型的甾醇酯酶基因,实现酶活力高、酶学性质良好的甾醇酯酶的制备,是拓宽其应用的主要技术瓶颈。
发明内容
为了克服现有技术中存在的缺陷,本发明的目的是提供了一种甾醇酯酶和其编码基因及其突变体。本发明通过基因的提取克隆、重组载体的构建、宿主细胞的构建以及分离纯化,获得酶活力高达11.66U/mL的甾醇酯酶,其具有优良的酶学性质,属于新型的甾醇酯酶。
为实现上述发明目的,本发明采用以下技术方案予以实现:
本发明提供了一种甾醇酯酶,所述甾醇酯酶的氨基酸序列为:
(a)如SEQ ID NO.4所示的氨基酸序列;或
(b)将SEQ ID NO.4的氨基酸序列经一个或几个氨基酸的取代、缺失或添加由SEQID NO.4衍生的,且具有与所述甾醇酯酶相同功能的氨基酸序列。
进一步的,所述甾醇酯酶的最适底物为丁酸对硝基苯酯。
进一步的,所述甾醇酯酶的适宜反应温度为30-55℃。
进一步的,所述甾醇酯酶的最适反应温度为50℃。
进一步的,所述甾醇酯酶在40-45℃具有良好的温度稳定性。
进一步的,所述甾醇酯酶的适宜反应pH为7.0-9.0。
进一步的,所述甾醇酯酶的最适反应pH为8.0。
进一步的,所述甾醇酯酶在pH 6.0-9.0时具有良好的pH稳定性。
进一步的,所述甾醇酯酶的酶活力为11.66U/mL。
进一步的,所述甾醇酯酶的制备方法包括以下步骤:
(1)甾醇酯酶基因的提取、克隆与分析:提取Cladosporium sp.菌株基因组,根据基因组序列与功能基因的分析并进行引物设计,以提取的基因组DNA为模板,经PCR,获得甾醇酯酶基因;
(2)甾醇酯酶重组载体的构建:将步骤(1)获得的甾醇酯酶基因的核苷酸序列经双酶酶切后与重组载体pPICZαA连接,得到重组载体pPICZαA-DZ16;
(3)甾醇酯酶重组宿主细胞的构建:将步骤(2)的重组载体pPICZαA-DZ16电转至毕赤酵母X33感受态细胞内,得到毕赤酵母甾醇酯酶重组菌株DZ16-X33;
(4)甾醇酯酶的表达与分离纯化步骤:将步骤(3)的重组菌株DZ16-X33接种至生物反应器中培养,培养过程中诱导表达,收集产物,通过层析技术分离纯化甾醇酯酶,并最终制备甾醇酯酶活性蛋白。
本发明还提供了一种编码所述甾醇酯酶的甾醇酯酶基因,所述甾醇酯酶基因的核苷酸序列为:
(a)如SEQ ID NO.3所示的核苷酸序列;或
(b)将SEQ ID NO.3的核苷酸序列经一个或几个核苷酸取代、缺失或添加由SEQ IDNO.3衍生的,且具有与所述甾醇酯酶基因相同功能的核苷酸序列。
本发明还提供了含有所述的甾醇酯酶基因的重组载体。
进一步的,所述的重组载体为pPICZαA。
本发明还提供了含有所述的甾醇酯酶基因的重组菌株。
进一步的,所述的重组菌株为毕赤酵母表达菌株X33。
本发明还提供了一种提高所述的甾醇酯酶的酶活的突变体G143A,所述突变体的氨基酸序列如SEQ ID NO:27所示;所述的突变体由氨基酸序列为SEQ ID NO:4的甾醇酯酶的第143位氨基酸由Gly变为Ala获得。
进一步的,编码所述突变体G143A的基因的核苷酸序列如SEQ ID NO:25所示。
进一步的,所述编码所述突变体G143A的基因通过如SEQ ID NO:5和SEQ ID NO:6所示的突变引物扩增获得。
进一步的,所述编码所述突变体G143A的酶活力为14.32U/mL。
本发明还提供了一种提高所述的甾醇酯酶的酶活的突变体A231D,所述突变体的氨基酸序列如SEQ ID NO:28所示;所述的突变体由氨基酸序列为SEQ ID NO:4的甾醇酯酶的第231位氨基酸由Ala变为Asp获得。
进一步的,编码所述突变体A231D的基因的核苷酸序列如SEQ IDNO:26所示。
进一步的,所述编码所述突变体A231D的基因通过如SEQ ID NO:13和SEQ ID NO:14所示的突变引物扩增获得。
进一步的,所述编码所述突变体A231D的酶活力为16.77U/mL。
本发明与现有技术相比,具有以下优点和有益效果:本发明通过设计特异性引物,获得野生菌Cladosporium sp.DZ16甾醇酯酶基因,该基因编码区长1620bp,编码539个氨基酸的甾醇酯酶,该酶基因序列与已有的甾醇酯酶基因序列相比存在显著差异,属于一种新型甾醇酯酶。本发明的技术方案可以实现所述的新型的甾醇酯酶重组载体的构建及毕赤酵母的高效表达,实现甾醇酯酶的规模化生产,表达后的甾醇酯酶酶活力高达11.66U/mL,相比于其他甾醇酯酶酶活力得到显著提高,且酶学性质优良,可有效拓宽其应用范围。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明,其中:
图1是甾醇酯酶基因的琼脂糖凝胶电泳图谱;其中,M为marker,泳道1为样品。
图2是甾醇酯酶的氨基酸序列与已知甾醇酯酶的氨基酸序列对比图。
图3是含有甾醇酯酶基因的重组载体pPICZαA-DZ16。
图4是甾醇酯酶的SDS-PAGE电泳图谱;其中,M为蛋白marker,泳道1为纯化后的甾醇酯酶蛋白。
图5是甾醇酯酶的底物专一性影响。
图6是温度对甾醇酯酶活性的影响。
图7是不同温度对甾醇酯酶稳定性的影响。
图8是pH对甾醇酯酶活性的影响。
图9是不同pH对甾醇酯酶稳定性的影响。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步详细的说明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为市售材料。
本发明所述的“甾醇酯酶活性”是以酶的活力单位来定义的,即利用紫外法测定酶活力。测定酶活力反应具体操作为:标准反应体系为1.03mL,在1 mL pH 8.0的Tris-HCl缓冲液中加入50mM乙酸对硝基苯酯溶液20μL,再加入10μL酶液,以不加酶的反应体系作为对照,50℃下反应10min,加入1ml1%SDS溶液终止反应,405nm处测定吸光值。酶活力定义为,在50℃和pH 8.0条件下,每分钟水解底物生成1μmoL对硝基苯酚所用的酶量为一个活力单位(U/mL)。
本发明包括甾醇酯酶的片段是指基本上保持与本发明的甾醇酯酶相同功能的蛋白或多肽。本发明的蛋白或多肽片段可以是以下三种情况之一:
(1)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白或多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的;
(2)在一个或多个氨基酸残基中具有取代基团的蛋白或多肽;
(3)附加的氨基酸序列融合到此蛋白或多肽序列而形成的蛋白或多肽(如前导序列、分泌序列、用来纯化此多肽的序列、蛋白原序列或融合蛋白)。
基于本发明目的,只要所得的蛋白质显示有甾醇酯酶活性,氨基酸替代可以在任何的氨基酸进行。而氨基酸替代包括改变蛋白质性质的替代、保守性替代和非保守性替代。
(1)改变蛋白质特性的替代,一般是结合位点或催化中心的替代,此类替代一般是保守性。甾醇酯酶的改变蛋白质性质的替代主要为,G143A:第143位G替代为A;G144R:第144位G替代为R;F217W:第217位F替代为W;S230T:第230位S替代为T;A231D:第231位A替代为D;G316S:第316位G替代为S;E362D:第362位E替代为D;F366Y:第366位F替代为Y;H475R:第475位H替代为R;N489G:第489位N替代为G等,其他的氨基酸替代也是允许的,并可以经验性的确定或根据已知的保守替代来确定。
(2)合适的氨基酸保守性替代,一般来说,在多肽的保守区域进行的单个氨基酸的替代可能会改变生物学活性,可以进行的保守的氨基酸替代也属于本发明保护范围。甾醇酯酶可以进行的保守氨基酸替代如表1所示:如G143A:氨基酸序列第143位G替代为A;G144R:氨基酸序列第144位G替代为R;A211V:氨基酸序列第211位A替代为V等,其他的保守氨基酸替代也是允许的,并可以经验性的确定或根据已知的保守替代来确定。
表1保守位点替代
(3)合适的氨基酸非保守性替代,一般来说,在多肽的非必需区域进行的单个氨基酸的替代基本不会改变生物学活性,可以进行的非保守的氨基酸替代也属于本发明保护范围。可以进行的保守氨基酸替代如表2所示:如V79A:氨基酸序列第79位V替代为A;F217W:氨基酸序列第217位F替代为W;F255Y:氨基酸序列第255位F替代为Y等,其他的非保守氨基酸替代也是允许的,并可以经验性的确定或根据已知的保守替代来确定。
表2非保守位点替代
实施例1:甾醇酯酶基因的克隆
1、Cladosporium sp.DZ16 RNA的提取
取培养到对数生长期的Cladosporium sp.DZ16菌液根据Fungal RNA Kit试剂盒的操作说明进行RNA提取,然后在NanoDrop 2000进行基因组RNA浓度和纯度测定,经测定提取的RNA浓度为430ng/mL;再经过1%琼脂糖凝胶电泳中进行RNA条带检测,检测条件为上样量5μL,电压120V,电泳30min,在凝胶成像***中观察条带,RNA条带均一性良好。
将提取的RNA样品进行转录组测序。
本发明所用的菌株Cladosporium sp.DZ16保藏于中国典型培养物保藏中心(CCTCC);地址:中国,武汉,武汉大学;保藏日期:2016年11月28日;Cladosporium sp.DZ16的保藏编号为CCTCC NO:M 2016685。
2、甾醇酯酶基因的克隆与分析
取培养到对数生长期的Cladosporium sp.DZ16的菌液,根据Fungal DNA Kit试剂盒的操作说明进行DNA提取。
以Cladosporium sp.DZ16总DNA为模板,根据转录组测序结果和功能基因分析,以含有限制性内切酶酶切位点的引物F和R扩增甾醇酯酶基因(不含信号肽),所述引物序列为:
采用PCR进行扩增,50μL反应体系为:DNA模板,1μL;F(10μM),1μL;R(10μM),1μL;dNTP(各2.5mM),4μL;Taq(2U/μL),1 μL;10×Taq buffer,5μL;最后补充dd H2O至50μL。
PCR扩增程序为:94℃预变性5min,94℃变性30s,60℃退火30s,72℃延伸2min,循环扩增35次,72℃终延伸5min。
如图1所示,得到的PCR产物经1%琼脂糖电泳检验为单一特异性条带,然后测序,经过PCR扩增获得长度为1620bp的甾醇酯酶基因,其核苷酸序列如SEQ IDNO.3所示,该甾醇酯酶基因编码一个由539个氨基酸组成的蛋白质,其氨基酸序列如SEQ ID NO.4所示,经过NCBI数据库BLAST序列分析后确定本发明得到的蛋白为甾醇酯酶。将本发明甾醇酯酶的氨基酸序列与其他已报道的甾醇酯酶氨基酸序列进行分析比较,结果如图2所示,本发明甾醇酯酶的氨基酸序列与已知甾醇酯酶氨基酸序列之间具有显著差异,而本发明甾醇酯酶的氨基酸序列与炭团菌甾醇酯酶(OTA52505.1)的相似性最高且仅达60.67%,由此说明本发明的甾醇酯酶是一个新型甾醇酯酶,编码该蛋白的甾醇酯酶基因也属于一个新型甾醇酯酶基因。
实施例2:甾醇酯酶重组载体的构建
将甾醇酯酶基因PCR产物按照Cycle-Pure Kit纯化试剂盒要求的操作方法进行纯化后,利用EcoR I、Not I对纯化后的甾醇酯酶基因与载体pPICZαA在37℃条件下进行双酶切,酶切时间为2h;将获得的酶切产物在80V下进行琼脂糖电泳,时间1h,再按照GelExtraction Kit胶提取试剂盒要求的操作方法进行切胶回收。用T4连接酶将酶切后的甾醇酯酶基因序列片段在16℃条件下与pPICZαA载体连接,时长16h,得到含有甾醇酯酶基因的重组载体pPICZαA-DZ16(见图3)。
实施例3:重组甾醇酯酶在毕赤酵母中的表达
将重组载体pPICZαA-DZ16经热转化(42℃,60s)转入大肠杆菌DH5α感受态细胞,然后在含有25μg/mL Zeocin的LLB固体平板上37℃培养12h,未被转化的菌株无法在平板上生长,利用PCR进行阳性菌株筛选。PCR扩增程序为:94℃预变性5min,94℃变性30s,60℃退火30s,72℃延伸2min,循环扩增35次,72℃终延伸5min。
将阳性菌株接种于含有25μg/mL Zeocin的LLB液体培养基上37℃培养12h,使用Plasmid Mini Kit进行质粒提取后,通过限制性核酸内切酶Pme I对重组载体pPICZαA-DZ16进行线性化,线性化反应体系:重组载体,59μL;Pme I,3μL;10×buffer,8μL;ddH2Q,加至80μL;线性化条件:37℃,6h。经1%琼脂糖电泳检验单酶切线性化完全后,按照Cycle-Pure Kit纯化试剂盒要求的操作方法进行线性重组载体pPICZαA-DZ16的纯化。
选择毕赤酵母表达菌株X33作为宿主细胞,将线性化的重组载体pPICZαA-DZ16加入到新鲜的毕赤酵母X33感受态细胞中,轻轻吸打混匀,冰浴15-20min,然后转移至冰浴的电转杯中,瞬时电击后立即加入1mL预冷的1M山梨醇,吸打混匀并转移至灭菌1.5mL EP管中,然后30℃孵育1h,最后菌体涂布至含100μg/mL Zeocin的YPDZ平板上,30℃培养2-3d。挑取YPDZ平板的单菌落接种于含1mL BMGY培养基的深孔板中,30℃,200r/min培养,每隔24h添加终浓度为1%的甲醇进行产酶诱导,诱导96h;从第48h开始每隔12h取样,按照酶活测定方法检测酶活力,并挑选甾醇酯酶表达量高的甾醇酯酶重组菌株DZ16-X33。
将甾醇酯酶重组菌株DZ16-X33接种于YPD液体培养基,30℃,200r/min培养24h,按1-2%接种量再接种至BMGY培养基,30℃,200r/min培养至OD600为2-6,加入甲醇至终浓度为1%,每隔24h添加甲醇,诱导72h。将发酵液在4℃下,8000r/min离心10min,收集上清。按照酶活测定方法测定酶活,测得甾醇酯酶的酶活力为11.66U/mL。
实施例4:甾醇酯酶突变实验
根据对甾醇酯酶氨基酸序列(不含信号肽)的保守位点和空间结构的分析,根据如SEQ ID NO.4所示的甾醇酯酶的氨基酸序列和如SEQ ID NO.3所示的甾醇酯酶基因的核苷酸序列,设计甾醇酯酶基因突变位置及方式为:G143A、G144R、F217W、S230T、A231D、G316S、E362D、F366Y、H475R、N489G,参照菌株密码子偏好性,利用CE Design V1.04设计的突变引物序列如下:
G143A-F:GATTTGGGCTGGCGTTTATCAGAAAGGCTCC(SEQ ID NO.5)
G143A-R:AAACGCCAGCCCAAATCCAACCTTTACCGGG(SEQ ID NO.6)
G144R-F:TTGGGGCAGAGTTTATCAGAAAGGCTCCGTCG(SEQ ID NO.7)
G144R-R:GATAAACTCTGCCCCAAATCCAACCTTTACC(SEQ ID NO.8)
F217W-F:ATAACATTGCGGCCTGGGGAGGTGACCCTGACAAGGTT(SEQ ID NO.9)
F217W-R:CCAGGCCGCAATGTTATCTGCA(SEQ ID NO.10)
S230T-F:AGAGACTGCTGGAGCAATTTCTGTACTTGATC(SEQ ID NO.11)
S230T-R:TTGCTCCAGCAGTCTCTCCCCAGATGGTAACCTTG(SEQ ID NO.12)
A231D-F:AGAGTCTGATGGAGCAATTTCTGTACTTGATCAGAT(SEQ ID NO.13)
A231D-R:TTGCTCCATCAGACTCTCCCCAGATGGTAACCT(SEQ ID NO.14)
G316S-F:GTCTCTCCTCTCGTACAACTCGCTGG(SEQ ID NO.15)
G316S-R:TGTACGAGAGGAGAGACGGCACGGAGTTGGCGGC(SEQ ID NO.16)
E362D-F:GAGGATGATGCTAGCGTGTTTGGTCTTTTCC(SEQ ID NO.17)
E362D-R:ACGCTAGCATCATCCTCCGGGTTGCGGTT(SEQ ID NO.18)
F366Y-F:TAGCGTGTACGGTCTTTTCCAGCCGAACCT(SEQ ID NO.19)
F366Y-R:AAAGACCGTACACGCTAGCCTCATCCTCCG(SEQ ID NO.20)
H475R-F:ACTTTGAGAGGCTCGGATCTTATCCAGGTC(SEQ ID NO.21)
H475R-R:TCCGAGCCTCTCAAAGTGCCCAAAACGGGG(SEQ ID NO.22)
N489G-F:CGGTAACGCCATGAGGAGCATCC(SEQ ID NO.23)
N489G-R:TCCTCATGGCGTTACCGGGCAGGATGCCAAAGAAG(SEQ ID NO.24)
以含有甾醇酯酶基因的重组载体pPICZαA-DZ16为模板,利用上述引物,采用PCR扩增质粒全长的方法进行突变。PCR反应体系为:质粒模板,0.5μL;2×Phanta Max Buffer,25μL;dNTP Mix(10mM),1μL;F(10μM),2μL;R(10μM),2μL;Phanta Max Super-Fidelity DNAPolymerase,1μL;dd H2O,加至50μL。PCR扩增程序为:95℃预变性3min,95℃变性30s,60℃退火15s,72℃延伸5min,循环扩增35次,72℃终延伸7min。
将上述得到的PCR反应产物用0.5μL Dpn I酶在37℃下酶解2h以消化模板质粒,酶解产物按Cycle-Pure Kit纯化试剂盒要求的操作方法进行PCR产物纯化。纯化产物用II One Step Cloning Kit进行质粒连接,20μL体系:5×CE II Buffer,4μL;Exnase II,2μL;纯化产物,5μL;dd H2O,加至20μL。质粒重组条件:37℃,30min。
突变后的重组质粒经热转化(42℃,60s)转入大肠杆菌DH5α感受态细胞,然后在含有25μg/mL Zeocin的LLB固体平板上37℃培养12h,未被转化的菌株无法在平板上生长,利用PCR进行阳性菌株筛选。PCR扩增程序为:94℃预变性5min,94℃变性30s,60℃退火30s,72℃延伸2min,循环扩增35次,72℃终延伸5min;并测序鉴定突变序列。将阳性菌株接种于含有25μg/mL Zeocin的LLB液体培养基,37℃培养12h,用Plasmid Mini Kit进行质粒提取后,通过限制性核酸内切酶Pme I对突变重组质粒进行线性化,线性化反应体系:重组质粒,59μL;Pme I,3μL;10×buffer,8μL;dd H2O,加至80μL。线性化条件:37℃,6h。经1%琼脂糖电泳检验单酶切线性化完全后,按照Cycle-Pure Kit纯化试剂盒要求的操作方法进行线性化突变重组质粒的纯化。
选择毕赤酵母表达菌株X33作为宿主细胞,将线性化突变重组质粒加入到新鲜的毕赤酵母X33感受态细胞中,轻轻吸打混匀,冰浴15-20min,然后转移至冰浴的电转杯中,瞬时电击后立即加入1mL预冷的1M山梨醇,吸打混匀并转移至灭菌1.5mL EP管中,然后30℃孵育1h,最后菌体涂布至含100μg/mL Zeocin的YPDZ平板上,30℃培养2-3d。挑取YPDZ平板的单菌落接种于含1mL BMGY培养基的深孔板中,30℃,200r/min培养,每隔24h添加终浓度为1%的甲醇进行产酶诱导,诱导96h;从第48h开始每隔12h取样,按照酶活测定方法检测酶活力,并挑选甾醇酯酶表达量高的甾醇酯酶突变重组菌株。
将甾醇酯酶突变重组菌株接种于YPD液体培养基,30℃,200r/min培养24h,按1-2%接种量接种至BMGY培养基,30℃,200r/min培养至OD600为2-6,加入甲醇至终浓度为1%,每隔24h添加甲醇,诱导72h。将发酵液在4℃下,8000r/min离心10min,收集上清。按照酶活测定方法测定酶活,测得甾醇酯酶的酶活力结果见表3。
表3氨基酸突变及酶活力
氨基酸保守替代结果显示,突变体G143A、A231D的甾醇酯酶的酶活力较突变前菌株摇瓶发酵酶活力有显著升高,突变体G144R和G316S的酶活力较突变前菌株摇瓶发酵酶活力有一定降低,突变体S230T、E362D、H475R的酶活力较突变前菌株摇瓶发酵酶活力有明显下降,说明氨基酸位点143、231、144、316、230、362和475是酶促反应中的重要氨基酸位点,对甾醇酯酶的活性影响较大。
氨基酸非保守替代结果显示,突变体F217W、F366Y和N489G的甾醇酯酶的酶活力较突变前菌株的酶活力基本不变,说明Phe和Asn是维持酶的空间结构必须氨基酸,但对甾醇酯酶的酶活力基本没有影响。
其中,突变体G143A和A231D的核苷酸序列如SEQ ID NO.25和SEQ ID NO.26所示,其编码的氨基酸序列如SEQ ID NO.27和SEQ ID NO.28所示。
实施例5:重组甾醇酯酶的纯化
将实施例3制得的甾醇酯酶重组菌株DZ16-X33发酵液用镍柱进行亲和层析,按照镍柱纯化说明书进行初步分离纯化,收集分离纯化产物,纯化甾醇酯酶可以作为样品酶液;将纯化后的甾醇酯酶进行SDS-PAGE电泳,如图4所示,电泳得到单一蛋白条带,且位置与预测分子量相吻合,分子量约为62kDa,其酶活回收率23.68%,比活262.3U/mg,纯化倍数18.14倍。
实施例6:甾醇酯酶的酶学性质
1、甾醇酯酶的底物专一性
分别选取相同浓度的不同碳链长度的对硝基苯酯化合物(乙酸对硝基苯酯、丁酸对硝基苯酯、己酸对硝基苯酯、辛酸对硝基苯酯、癸酸对硝基苯酯、月桂酸对硝基苯酯、豆蔻酸对硝基苯酯和棕榈酸对硝基苯酯)作为反应底物,测定实施例5中制备的样品酶液的底物专一性。将最适碳链长度下的酶活力定义为100%,结果如图5所示,说明甾醇酯酶的最适底物为丁酸对硝基苯酯,其对短链长度的对硝基苯酚酯化合物的特异性较差,对碳链长度大于10的对硝基苯酯化合物水解活性较弱,且会随着碳链长度的增长而减弱。
2、温度对甾醇酯酶的影响
(1)分别在30、35、40、45、50、55℃下,按照酶活测定方法测定实施例5中制备的样品酶液的酶活。以相对酶活做比较(最高酶活为100%),实验结果如图6所示,说明样品酶液在30-55℃的温度范围内均具有良好的酶活,最适反应温度为50℃。
(2)将样品酶液分别在40、45、50、55℃下分别孵育5、15、30、60、120、180、240、300min,按照酶活力测定方法测定酶活来考察甾醇酯酶的温度稳定性,并将未经温度处理(0min)的酶液酶活力定义为100%。以相对酶活做比较,实验结果如图7所示,说明甾醇酯酶在低于45℃具有良好的稳定性,但是在55℃以上,甾醇酯酶温度稳定性较差,酶易失活。
3、pH对甾醇酯酶的影响
(1)分别配置pH 4.0、5.0、6.0、7.0、8.0、9.0、10.0的50mM Tris-HCl缓冲液,按照酶活力测定方法测定实施例5中制备的样品酶液在不同pH下的酶活。以相对酶活做比较(最高酶活为100%),实验结果如图8所示,说明甾醇酯酶的最适pH为8.0,且在pH 7.0-9.0范围内,酶活力较好。
(2)在4℃条件下,将样品酶液在pH 6.0、7.0、8.0、9.0孵育2h,按照酶活力方法测定酶活以考察酶的pH稳定性,将未经pH孵育处理的酶液酶活力定义为100%。以相对酶活做比较,实验结果如图9所示,结果表明在不同pH条件下,甾醇酯酶的酶活力较稳定(相对酶活≥80%),因此甾醇酯酶适合于工业化应用。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 中国海洋大学
<120> 一种甾醇酯酶和其编码基因及其突变体
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccggaattcc ggatggcttt ggagaaccag cc 32
<210> 2
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaggaaaaaa gcggccgcta taaacacggg ctgggaagcg 40
<210> 3
<211> 1620
<212> DNA
<213> Cladosporium sp.
<400> 3
atggctttgg agaaccagcc cgtcgacact accgcctccg gcgcgatcaa ctctatcgaa 60
cgtgccgcca aggtcaccgt tgctgttcct tccggtaccg tcatcggctc cagcttgggc 120
aaggttgagt ctttcagggg catcccattt gcggaccccc ctactggctc tctccgtctc 180
aaacccccga agaggttgtc aaagcccctg ggcaattttg atgcctccgg tcttgttggc 240
ccatcatgcc ctcagatgtt tatctctact ggagctgagg atgtcatctc aaagttcctc 300
tccgactttc tgagcatccc cttcctccag gtcgttaccg gacaagagga ttgtctcacc 360
atgactgttc aacggcctgt gggcaccaag gccggggaca agctgcccgg taaaggttgg 420
atttggggcg gcgtttatca gaaaggctcc gtcgccatgt atgatggcac tagccttctg 480
ggtactggca ttgaccagaa gcaacccttt attttcgttg ctgtcaacta ccgtgttgcc 540
ggatttggct tcatgcccgg tgccgagctt gccaatgaag gcagcaccaa cctaggcttg 600
ctagatcagc gcatgggact tgaatgggtt gcagataaca ttgcggcctt tggaggtgac 660
cctgacaagg ttaccatctg gggagagtct gctggagcaa tttctgtact tgatcagatg 720
actctgttcg gtggtgatgc tgactacaag ggcaagcccc ttttccgtgg cgccatcatg 780
aactcgggca gtgtcgttcc ggcagagcca gtggacagcc ccaaggctca ggagattttc 840
gatactgttg tacagaatgc tggctgctcc agtgcctcca acagcctcga ctgtcttcga 900
ggtctcccct atgataagtt tttggacgcc gccaactccg tgccgggact cctctcgtac 960
aactcgctgg ccctctcctt cattcctcac agtaacgctg gtgtgcttcc cgaaagtccc 1020
gaagcactca tcgcggcggg gcgttaccat gcggttcctt gtttgaaccg caacccggag 1080
gatgaggcta gcgtgtttgg tcttttccag ccgaacctga ccacgacgga gaagcttgtc 1140
gattacctga aagagttcta cttttccgcc gccagcaaga cgcaactgac caacctcgtc 1200
aactcgtatt caagctccat cactgcaggc agccccttcc gtaccggcat cctcaacgag 1260
atcttcccag gctttaagcg acgtgctgcc atctttggcg atctcgtctt cacgctgact 1320
cgccgactct ttcttcagac cgccacggat accaacccag atgtccctgc gtggtcttac 1380
ttggcaagct acgactacgg aacccccgtt ttgggcactt tgcacggctc ggatcttatc 1440
caggtcttct ttggcatcct gcccaacaac gccatgagga gcatccggac gtactactac 1500
aactttttgt acaaccttga tcctaatgtt ggcgttacaa gcaagtggaa ttgggagcag 1560
ttcatcgact actttagaac caagaacgtc cgctacatgg gcgcttccca gcccgtgtga 1620
<210> 4
<211> 539
<212> PRT
<213> Cladosporium sp.
<400> 4
Met Ala Leu Glu Asn Gln Pro Val Asp Thr Thr Ala Ser Gly Ala Ile
1 5 10 15
Asn Ser Ile Glu Arg Ala Ala Lys Val Thr Val Ala Val Pro Ser Gly
20 25 30
Thr Val Ile Gly Ser Ser Leu Gly Lys Val Glu Ser Phe Arg Gly Ile
35 40 45
Pro Phe Ala Asp Pro Pro Thr Gly Ser Leu Arg Leu Lys Pro Pro Lys
50 55 60
Arg Leu Ser Lys Pro Leu Gly Asn Phe Asp Ala Ser Gly Leu Val Gly
65 70 75 80
Pro Ser Cys Pro Gln Met Phe Ile Ser Thr Gly Ala Glu Asp Val Ile
85 90 95
Ser Lys Phe Leu Ser Asp Phe Leu Ser Ile Pro Phe Leu Gln Val Val
100 105 110
Thr Gly Gln Glu Asp Cys Leu Thr Met Thr Val Gln Arg Pro Val Gly
115 120 125
Thr Lys Ala Gly Asp Lys Leu Pro Gly Lys Gly Trp Ile Trp Gly Gly
130 135 140
Val Tyr Gln Lys Gly Ser Val Ala Met Tyr Asp Gly Thr Ser Leu Leu
145 150 155 160
Gly Thr Gly Ile Asp Gln Lys Gln Pro Phe Ile Phe Val Ala Val Asn
165 170 175
Tyr Arg Val Ala Gly Phe Gly Phe Met Pro Gly Ala Glu Leu Ala Asn
180 185 190
Glu Gly Ser Thr Asn Leu Gly Leu Leu Asp Gln Arg Met Gly Leu Glu
195 200 205
Trp Val Ala Asp Asn Ile Ala Ala Phe Gly Gly Asp Pro Asp Lys Val
210 215 220
Thr Ile Trp Gly Glu Ser Ala Gly Ala Ile Ser Val Leu Asp Gln Met
225 230 235 240
Thr Leu Phe Gly Gly Asp Ala Asp Tyr Lys Gly Lys Pro Leu Phe Arg
245 250 255
Gly Ala Ile Met Asn Ser Gly Ser Val Val Pro Ala Glu Pro Val Asp
260 265 270
Ser Pro Lys Ala Gln Glu Ile Phe Asp Thr Val Val Gln Asn Ala Gly
275 280 285
Cys Ser Ser Ala Ser Asn Ser Leu Asp Cys Leu Arg Gly Leu Pro Tyr
290 295 300
Asp Lys Phe Leu Asp Ala Ala Asn Ser Val Pro Gly Leu Leu Ser Tyr
305 310 315 320
Asn Ser Leu Ala Leu Ser Phe Ile Pro His Ser Asn Ala Gly Val Leu
325 330 335
Pro Glu Ser Pro Glu Ala Leu Ile Ala Ala Gly Arg Tyr His Ala Val
340 345 350
Pro Cys Leu Asn Arg Asn Pro Glu Asp Glu Ala Ser Val Phe Gly Leu
355 360 365
Phe Gln Pro Asn Leu Thr Thr Thr Glu Lys Leu Val Asp Tyr Leu Lys
370 375 380
Glu Phe Tyr Phe Ser Ala Ala Ser Lys Thr Gln Leu Thr Asn Leu Val
385 390 395 400
Asn Ser Tyr Ser Ser Ser Ile Thr Ala Gly Ser Pro Phe Arg Thr Gly
405 410 415
Ile Leu Asn Glu Ile Phe Pro Gly Phe Lys Arg Arg Ala Ala Ile Phe
420 425 430
Gly Asp Leu Val Phe Thr Leu Thr Arg Arg Leu Phe Leu Gln Thr Ala
435 440 445
Thr Asp Thr Asn Pro Asp Val Pro Ala Trp Ser Tyr Leu Ala Ser Tyr
450 455 460
Asp Tyr Gly Thr Pro Val Leu Gly Thr Leu His Gly Ser Asp Leu Ile
465 470 475 480
Gln Val Phe Phe Gly Ile Leu Pro Asn Asn Ala Met Arg Ser Ile Arg
485 490 495
Thr Tyr Tyr Tyr Asn Phe Leu Tyr Asn Leu Asp Pro Asn Val Gly Val
500 505 510
Thr Ser Lys Trp Asn Trp Glu Gln Phe Ile Asp Tyr Phe Arg Thr Lys
515 520 525
Asn Val Arg Tyr Met Gly Ala Ser Gln Pro Val
530 535
<210> 5
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gatttgggct ggcgtttatc agaaaggctc c 31
<210> 6
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aaacgccagc ccaaatccaa cctttaccgg g 31
<210> 7
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttggggcaga gtttatcaga aaggctccgt cg 32
<210> 8
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gataaactct gccccaaatc caacctttac c 31
<210> 9
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ataacattgc ggcctgggga ggtgaccctg acaaggtt 38
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccaggccgca atgttatctg ca 22
<210> 11
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
agagactgct ggagcaattt ctgtacttga tc 32
<210> 12
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ttgctccagc agtctctccc cagatggtaa ccttg 35
<210> 13
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agagtctgat ggagcaattt ctgtacttga tcagat 36
<210> 14
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ttgctccatc agactctccc cagatggtaa cct 33
<210> 15
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gtctctcctc tcgtacaact cgctgg 26
<210> 16
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tgtacgagag gagagacggc acggagttgg cggc 34
<210> 17
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gaggatgatg ctagcgtgtt tggtcttttc c 31
<210> 18
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acgctagcat catcctccgg gttgcggtt 29
<210> 19
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tagcgtgtac ggtcttttcc agccgaacct 30
<210> 20
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aaagaccgta cacgctagcc tcatcctccg 30
<210> 21
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
actttgagag gctcggatct tatccaggtc 30
<210> 22
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tccgagcctc tcaaagtgcc caaaacggg 29
<210> 23
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
cggtaacgcc atgaggagca tcc 23
<210> 24
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcctcatggc gttaccgggc aggatgccaa agaag 35
<210> 25
<211> 1620
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atggctttgg agaaccagcc cgtcgacact accgcctccg gcgcgatcaa ctctatcgaa 60
cgtgccgcca aggtcaccgt tgctgttcct tccggtaccg tcatcggctc cagcttgggc 120
aaggttgagt ctttcagggg catcccattt gcggaccccc ctactggctc tctccgtctc 180
aaacccccga agaggttgtc aaagcccctg ggcaattttg atgcctccgg tcttgttggc 240
ccatcatgcc ctcagatgtt tatctctact ggagctgagg atgtcatctc aaagttcctc 300
tccgactttc tgagcatccc cttcctccag gtcgttaccg gacaagagga ttgtctcacc 360
atgactgttc aacggcctgt gggcaccaag gccggggaca agctgcccgg taaaggttgg 420
atttgggctg gcgtttatca gaaaggctcc gtcgccatgt atgatggcac tagccttctg 480
ggtactggca ttgaccagaa gcaacccttt attttcgttg ctgtcaacta ccgtgttgcc 540
ggatttggct tcatgcccgg tgccgagctt gccaatgaag gcagcaccaa cctaggcttg 600
ctagatcagc gcatgggact tgaatgggtt gcagataaca ttgcggcctt tggaggtgac 660
cctgacaagg ttaccatctg gggagagtct gctggagcaa tttctgtact tgatcagatg 720
actctgttcg gtggtgatgc tgactacaag ggcaagcccc ttttccgtgg cgccatcatg 780
aactcgggca gtgtcgttcc ggcagagcca gtggacagcc ccaaggctca ggagattttc 840
gatactgttg tacagaatgc tggctgctcc agtgcctcca acagcctcga ctgtcttcga 900
ggtctcccct atgataagtt tttggacgcc gccaactccg tgccgggact cctctcgtac 960
aactcgctgg ccctctcctt cattcctcac agtaacgctg gtgtgcttcc cgaaagtccc 1020
gaagcactca tcgcggcggg gcgttaccat gcggttcctt gtttgaaccg caacccggag 1080
gatgaggcta gcgtgtttgg tcttttccag ccgaacctga ccacgacgga gaagcttgtc 1140
gattacctga aagagttcta cttttccgcc gccagcaaga cgcaactgac caacctcgtc 1200
aactcgtatt caagctccat cactgcaggc agccccttcc gtaccggcat cctcaacgag 1260
atcttcccag gctttaagcg acgtgctgcc atctttggcg atctcgtctt cacgctgact 1320
cgccgactct ttcttcagac cgccacggat accaacccag atgtccctgc gtggtcttac 1380
ttggcaagct acgactacgg aacccccgtt ttgggcactt tgcacggctc ggatcttatc 1440
caggtcttct ttggcatcct gcccaacaac gccatgagga gcatccggac gtactactac 1500
aactttttgt acaaccttga tcctaatgtt ggcgttacaa gcaagtggaa ttgggagcag 1560
ttcatcgact actttagaac caagaacgtc cgctacatgg gcgcttccca gcccgtgtga 1620
<210> 26
<211> 1620
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atggctttgg agaaccagcc cgtcgacact accgcctccg gcgcgatcaa ctctatcgaa 60
cgtgccgcca aggtcaccgt tgctgttcct tccggtaccg tcatcggctc cagcttgggc 120
aaggttgagt ctttcagggg catcccattt gcggaccccc ctactggctc tctccgtctc 180
aaacccccga agaggttgtc aaagcccctg ggcaattttg atgcctccgg tcttgttggc 240
ccatcatgcc ctcagatgtt tatctctact ggagctgagg atgtcatctc aaagttcctc 300
tccgactttc tgagcatccc cttcctccag gtcgttaccg gacaagagga ttgtctcacc 360
atgactgttc aacggcctgt gggcaccaag gccggggaca agctgcccgg taaaggttgg 420
atttggggcg gcgtttatca gaaaggctcc gtcgccatgt atgatggcac tagccttctg 480
ggtactggca ttgaccagaa gcaacccttt attttcgttg ctgtcaacta ccgtgttgcc 540
ggatttggct tcatgcccgg tgccgagctt gccaatgaag gcagcaccaa cctaggcttg 600
ctagatcagc gcatgggact tgaatgggtt gcagataaca ttgcggcctt tggaggtgac 660
cctgacaagg ttaccatctg gggagagtct gatggagcaa tttctgtact tgatcagatg 720
actctgttcg gtggtgatgc tgactacaag ggcaagcccc ttttccgtgg cgccatcatg 780
aactcgggca gtgtcgttcc ggcagagcca gtggacagcc ccaaggctca ggagattttc 840
gatactgttg tacagaatgc tggctgctcc agtgcctcca acagcctcga ctgtcttcga 900
ggtctcccct atgataagtt tttggacgcc gccaactccg tgccgggact cctctcgtac 960
aactcgctgg ccctctcctt cattcctcac agtaacgctg gtgtgcttcc cgaaagtccc 1020
gaagcactca tcgcggcggg gcgttaccat gcggttcctt gtttgaaccg caacccggag 1080
gatgaggcta gcgtgtttgg tcttttccag ccgaacctga ccacgacgga gaagcttgtc 1140
gattacctga aagagttcta cttttccgcc gccagcaaga cgcaactgac caacctcgtc 1200
aactcgtatt caagctccat cactgcaggc agccccttcc gtaccggcat cctcaacgag 1260
atcttcccag gctttaagcg acgtgctgcc atctttggcg atctcgtctt cacgctgact 1320
cgccgactct ttcttcagac cgccacggat accaacccag atgtccctgc gtggtcttac 1380
ttggcaagct acgactacgg aacccccgtt ttgggcactt tgcacggctc ggatcttatc 1440
caggtcttct ttggcatcct gcccaacaac gccatgagga gcatccggac gtactactac 1500
aactttttgt acaaccttga tcctaatgtt ggcgttacaa gcaagtggaa ttgggagcag 1560
ttcatcgact actttagaac caagaacgtc cgctacatgg gcgcttccca gcccgtgtga 1620
<210> 27
<211> 539
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Met Ala Leu Glu Asn Gln Pro Val Asp Thr Thr Ala Ser Gly Ala Ile
1 5 10 15
Asn Ser Ile Glu Arg Ala Ala Lys Val Thr Val Ala Val Pro Ser Gly
20 25 30
Thr Val Ile Gly Ser Ser Leu Gly Lys Val Glu Ser Phe Arg Gly Ile
35 40 45
Pro Phe Ala Asp Pro Pro Thr Gly Ser Leu Arg Leu Lys Pro Pro Lys
50 55 60
Arg Leu Ser Lys Pro Leu Gly Asn Phe Asp Ala Ser Gly Leu Val Gly
65 70 75 80
Pro Ser Cys Pro Gln Met Phe Ile Ser Thr Gly Ala Glu Asp Val Ile
85 90 95
Ser Lys Phe Leu Ser Asp Phe Leu Ser Ile Pro Phe Leu Gln Val Val
100 105 110
Thr Gly Gln Glu Asp Cys Leu Thr Met Thr Val Gln Arg Pro Val Gly
115 120 125
Thr Lys Ala Gly Asp Lys Leu Pro Gly Lys Gly Trp Ile Trp Ala Gly
130 135 140
Val Tyr Gln Lys Gly Ser Val Ala Met Tyr Asp Gly Thr Ser Leu Leu
145 150 155 160
Gly Thr Gly Ile Asp Gln Lys Gln Pro Phe Ile Phe Val Ala Val Asn
165 170 175
Tyr Arg Val Ala Gly Phe Gly Phe Met Pro Gly Ala Glu Leu Ala Asn
180 185 190
Glu Gly Ser Thr Asn Leu Gly Leu Leu Asp Gln Arg Met Gly Leu Glu
195 200 205
Trp Val Ala Asp Asn Ile Ala Ala Phe Gly Gly Asp Pro Asp Lys Val
210 215 220
Thr Ile Trp Gly Glu Ser Ala Gly Ala Ile Ser Val Leu Asp Gln Met
225 230 235 240
Thr Leu Phe Gly Gly Asp Ala Asp Tyr Lys Gly Lys Pro Leu Phe Arg
245 250 255
Gly Ala Ile Met Asn Ser Gly Ser Val Val Pro Ala Glu Pro Val Asp
260 265 270
Ser Pro Lys Ala Gln Glu Ile Phe Asp Thr Val Val Gln Asn Ala Gly
275 280 285
Cys Ser Ser Ala Ser Asn Ser Leu Asp Cys Leu Arg Gly Leu Pro Tyr
290 295 300
Asp Lys Phe Leu Asp Ala Ala Asn Ser Val Pro Gly Leu Leu Ser Tyr
305 310 315 320
Asn Ser Leu Ala Leu Ser Phe Ile Pro His Ser Asn Ala Gly Val Leu
325 330 335
Pro Glu Ser Pro Glu Ala Leu Ile Ala Ala Gly Arg Tyr His Ala Val
340 345 350
Pro Cys Leu Asn Arg Asn Pro Glu Asp Glu Ala Ser Val Phe Gly Leu
355 360 365
Phe Gln Pro Asn Leu Thr Thr Thr Glu Lys Leu Val Asp Tyr Leu Lys
370 375 380
Glu Phe Tyr Phe Ser Ala Ala Ser Lys Thr Gln Leu Thr Asn Leu Val
385 390 395 400
Asn Ser Tyr Ser Ser Ser Ile Thr Ala Gly Ser Pro Phe Arg Thr Gly
405 410 415
Ile Leu Asn Glu Ile Phe Pro Gly Phe Lys Arg Arg Ala Ala Ile Phe
420 425 430
Gly Asp Leu Val Phe Thr Leu Thr Arg Arg Leu Phe Leu Gln Thr Ala
435 440 445
Thr Asp Thr Asn Pro Asp Val Pro Ala Trp Ser Tyr Leu Ala Ser Tyr
450 455 460
Asp Tyr Gly Thr Pro Val Leu Gly Thr Leu His Gly Ser Asp Leu Ile
465 470 475 480
Gln Val Phe Phe Gly Ile Leu Pro Asn Asn Ala Met Arg Ser Ile Arg
485 490 495
Thr Tyr Tyr Tyr Asn Phe Leu Tyr Asn Leu Asp Pro Asn Val Gly Val
500 505 510
Thr Ser Lys Trp Asn Trp Glu Gln Phe Ile Asp Tyr Phe Arg Thr Lys
515 520 525
Asn Val Arg Tyr Met Gly Ala Ser Gln Pro Val
530 535
<210> 28
<211> 539
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Met Ala Leu Glu Asn Gln Pro Val Asp Thr Thr Ala Ser Gly Ala Ile
1 5 10 15
Asn Ser Ile Glu Arg Ala Ala Lys Val Thr Val Ala Val Pro Ser Gly
20 25 30
Thr Val Ile Gly Ser Ser Leu Gly Lys Val Glu Ser Phe Arg Gly Ile
35 40 45
Pro Phe Ala Asp Pro Pro Thr Gly Ser Leu Arg Leu Lys Pro Pro Lys
50 55 60
Arg Leu Ser Lys Pro Leu Gly Asn Phe Asp Ala Ser Gly Leu Val Gly
65 70 75 80
Pro Ser Cys Pro Gln Met Phe Ile Ser Thr Gly Ala Glu Asp Val Ile
85 90 95
Ser Lys Phe Leu Ser Asp Phe Leu Ser Ile Pro Phe Leu Gln Val Val
100 105 110
Thr Gly Gln Glu Asp Cys Leu Thr Met Thr Val Gln Arg Pro Val Gly
115 120 125
Thr Lys Ala Gly Asp Lys Leu Pro Gly Lys Gly Trp Ile Trp Gly Gly
130 135 140
Val Tyr Gln Lys Gly Ser Val Ala Met Tyr Asp Gly Thr Ser Leu Leu
145 150 155 160
Gly Thr Gly Ile Asp Gln Lys Gln Pro Phe Ile Phe Val Ala Val Asn
165 170 175
Tyr Arg Val Ala Gly Phe Gly Phe Met Pro Gly Ala Glu Leu Ala Asn
180 185 190
Glu Gly Ser Thr Asn Leu Gly Leu Leu Asp Gln Arg Met Gly Leu Glu
195 200 205
Trp Val Ala Asp Asn Ile Ala Ala Phe Gly Gly Asp Pro Asp Lys Val
210 215 220
Thr Ile Trp Gly Glu Ser Asp Gly Ala Ile Ser Val Leu Asp Gln Met
225 230 235 240
Thr Leu Phe Gly Gly Asp Ala Asp Tyr Lys Gly Lys Pro Leu Phe Arg
245 250 255
Gly Ala Ile Met Asn Ser Gly Ser Val Val Pro Ala Glu Pro Val Asp
260 265 270
Ser Pro Lys Ala Gln Glu Ile Phe Asp Thr Val Val Gln Asn Ala Gly
275 280 285
Cys Ser Ser Ala Ser Asn Ser Leu Asp Cys Leu Arg Gly Leu Pro Tyr
290 295 300
Asp Lys Phe Leu Asp Ala Ala Asn Ser Val Pro Gly Leu Leu Ser Tyr
305 310 315 320
Asn Ser Leu Ala Leu Ser Phe Ile Pro His Ser Asn Ala Gly Val Leu
325 330 335
Pro Glu Ser Pro Glu Ala Leu Ile Ala Ala Gly Arg Tyr His Ala Val
340 345 350
Pro Cys Leu Asn Arg Asn Pro Glu Asp Glu Ala Ser Val Phe Gly Leu
355 360 365
Phe Gln Pro Asn Leu Thr Thr Thr Glu Lys Leu Val Asp Tyr Leu Lys
370 375 380
Glu Phe Tyr Phe Ser Ala Ala Ser Lys Thr Gln Leu Thr Asn Leu Val
385 390 395 400
Asn Ser Tyr Ser Ser Ser Ile Thr Ala Gly Ser Pro Phe Arg Thr Gly
405 410 415
Ile Leu Asn Glu Ile Phe Pro Gly Phe Lys Arg Arg Ala Ala Ile Phe
420 425 430
Gly Asp Leu Val Phe Thr Leu Thr Arg Arg Leu Phe Leu Gln Thr Ala
435 440 445
Thr Asp Thr Asn Pro Asp Val Pro Ala Trp Ser Tyr Leu Ala Ser Tyr
450 455 460
Asp Tyr Gly Thr Pro Val Leu Gly Thr Leu His Gly Ser Asp Leu Ile
465 470 475 480
Gln Val Phe Phe Gly Ile Leu Pro Asn Asn Ala Met Arg Ser Ile Arg
485 490 495
Thr Tyr Tyr Tyr Asn Phe Leu Tyr Asn Leu Asp Pro Asn Val Gly Val
500 505 510
Thr Ser Lys Trp Asn Trp Glu Gln Phe Ile Asp Tyr Phe Arg Thr Lys
515 520 525
Asn Val Arg Tyr Met Gly Ala Ser Gln Pro Val
530 535
Claims (6)
1.一种甾醇酯酶,其特征在于,所述甾醇酯酶的氨基酸序列为如SEQ ID NO.4所示的氨基酸序列。
2.一种编码权利要求1所述的甾醇酯酶的甾醇酯酶基因,其特征在于,所述甾醇酯酶基因的核苷酸序列为如SEQ ID NO.3所示的核苷酸序列。
3.含有权利要求2所述的甾醇酯酶基因的重组载体。
4.含有权利要求2所述的甾醇酯酶基因的重组菌株。
5.一种提高权利要求1所述的甾醇酯酶酶活的突变体G143A,其特征在于,所述突变体G143A的氨基酸序列如SEQ ID NO:27所示;所述的突变体由氨基酸序列为SEQ ID NO:4的甾醇酯酶的第143位氨基酸由Gly变为Ala获得。
6.一种提高权利要求1所述的甾醇酯酶酶活的突变体A231D,其特征在于,所述突变体A231D的氨基酸序列如SEQ ID NO:28所示;所述的突变体由氨基酸序列为SEQ ID NO:4的甾醇酯酶的第231位氨基酸由Ala变为Asp获得。
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CN108753637A (zh) * | 2018-04-25 | 2018-11-06 | 大连大学 | 一种生产低温甾醇酯酶的菌株及其发酵方法 |
CN109337821A (zh) * | 2018-09-29 | 2019-02-15 | 中国海洋大学 | 一种产甾醇酯酶的枝孢菌及产酶方法 |
CN110241097A (zh) * | 2019-05-24 | 2019-09-17 | 山东大学 | Pet降解酶及其突变体和应用 |
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CN108753637A (zh) * | 2018-04-25 | 2018-11-06 | 大连大学 | 一种生产低温甾醇酯酶的菌株及其发酵方法 |
CN109337821A (zh) * | 2018-09-29 | 2019-02-15 | 中国海洋大学 | 一种产甾醇酯酶的枝孢菌及产酶方法 |
CN110241097A (zh) * | 2019-05-24 | 2019-09-17 | 山东大学 | Pet降解酶及其突变体和应用 |
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