CN112592909B - 一种甘油酯脂肪酶smg1突变体及其编码基因与应用 - Google Patents
一种甘油酯脂肪酶smg1突变体及其编码基因与应用 Download PDFInfo
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- CN112592909B CN112592909B CN202110004120.9A CN202110004120A CN112592909B CN 112592909 B CN112592909 B CN 112592909B CN 202110004120 A CN202110004120 A CN 202110004120A CN 112592909 B CN112592909 B CN 112592909B
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- lipase
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- 101000864057 Homo sapiens Serine/threonine-protein kinase SMG1 Proteins 0.000 title claims abstract description 30
- 102100029938 Serine/threonine-protein kinase SMG1 Human genes 0.000 title claims abstract description 30
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Abstract
本发明公开了一种甘油酯脂肪酶SMG1突变体及其编码基因和应用。该突变体为甘油酯脂肪酶突变体Y260C‑G268C，其氨基酸序列包括如SEQ ID NO.1所示的序列，核苷酸序列包括如SEQ ID NO.2或其互补序列。与野生型对比，本发明获得的甘油酯脂肪酶突变体Y260C‑G268C的Tm值为53.0℃,50℃半衰期为92.4min。与野生型对比，所述甘油酯脂肪酶突变体Y260C‑G268C的Tm值提高3.0℃,50℃半衰期提高22.7倍；且比活力也相对野生型提高约2倍，其更适用于工业领域上的应用。
Description
技术领域
本发明属于酶工程领域，特别涉及一种甘油酯脂肪酶SMG1突变体及其编码基因与应用。
背景技术
脂肪酶(EC 3.1.1.3)是一类能在油水界面上催化甘油酯分解或合成的酶的总称，可以催化天然底物油脂水解。此外，脂肪酶还可以催化酸解、转酯、酯合成和酯交换等反应。而甘油酯脂肪酶只能分解甘油二酯或甘油单酯，不能分解甘油三酯，其参与的酯化反应也只合成甘油二酯或甘油单酯。甘油酯脂肪酶在制备新型结构脂和去除油脂中甘油酯领域发挥重要作用，因此在油脂加工、医药、饲料添加剂、食品与营养和化学工业等领域中展现出巨大的市场前景。目前只有一种来源于卡门柏青霉的甘油酯脂肪酶被日本天野公司商品化。因此，开发新型甘油酯脂肪酶具有现实意义。
而在寻找热稳定性较好的脂肪酶的时候，由于可突变的位点多且蛋白结构的复杂性，需要既兼顾热稳定性又要具有较好的酶活性，找到能够适应工业用的酶并不容易。本发明的申请人也一直致力于寻找更适合于工业化应用的脂肪酶，为此，多年来进行了一序列的相关研究。
发明内容
本发明的目的之一在于提供一种热稳定性提高的甘油酯脂肪酶突变体，该甘油酯脂肪酶突变体还具有很好的比活力。
本发明的目的通过下述技术方案实现：
一种甘油酯脂肪酶SMG1突变体，其氨基酸构成包括如SEQ ID NO.1所示序列；或其编码基因包括如SEQ ID NO.2所示序列或其反向互补序列；或其氨基酸构成包括如SEQ IDNO.1所示序列，且该序列存在一个或多个点突变，但生物活性不变。
本发明的另一目的在于提供所述热稳定性提高的甘油酯脂肪酶SMG1突变体的编码基因。
一种编码上述甘油酯脂肪酶突变体的核苷酸序列。
在其中一些实施例中，编码上述甘油酯脂肪酶突变体的核苷酸序列包括如
SEQ ID NO.2所示序列，或该序列的反向互补序列。
本发明还提供了一种含有上述编码甘油酯脂肪酶SMG1突变体的编码基因的重组表达载体。
本发明还提供了一种含有上述编码甘油酯脂肪酶SMG1突变体的编码基因的重组工程菌株。
本发明的又一目的在于提供所述热稳定性提高的甘油酯脂肪酶SMG1突变体的应用。
上述热稳定性提高的甘油酯脂肪酶SMG1突变体、所述热稳定性提高的甘油酯脂肪酶SMG1突变体的编码基因、重组表达载体、重组工程菌株在油脂制备中的应用。
在其中一些实施例中，所述应用是在制备甘油二酯或甘油单酯中的应用。
本发明的另一目的是提供一种制备甘油二酯或甘油单酯的方法。
一种制备甘油二酯或甘油单酯的方法，所用脂肪酶是上述的甘油酯脂肪酶SMG1突变体。
所述甘油酯脂肪酶SMG1突变体优选为甘油酯脂肪酶突变体Y260C-G268C。
本发明通过在野生型甘油酯脂肪酶SMG1中合适的位点引入二硫键获得了热稳定性高和比活力大幅度提高的脂肪酶突变体Y260C-G268C。与野生型对比，脂肪酶突变体Y260C-G268C包含突变位点Tyr260Cys和Gly268Cys。在相同条件下，野生型SMG1的Tm为50.0℃，50℃半衰期为3.9min；而脂肪酶突变体Y260C-G268C的Tm值为53.0℃,50℃半衰期为92.4min。与野生型对比，甘油酯脂肪酶突变体Y260C-G268C的Tm值提高3.0℃,50℃半衰期提高22.7倍。而且，所述甘油酯脂肪酶突变体Y260C-G268C的比活力比野生型约提高2倍。
综上所述，本发明的获得的甘油酯脂肪酶突变体Y260C-G268C的热稳定性和比活力均得以提高，为产业化应用奠定基础。
附图说明
图1野生型甘油酯脂肪酶SMG1在473k中Cα的RMSF计算结果。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明的一个实施例中，提供了一种甘油酯脂肪酶突变体，它在野生型的甘油酯脂肪酶SMG1中通过定点突变以引入二硫键，采用“原始氨基酸-位置-替换的氨基酸”来表示甘油酯脂肪酶突变体中突变的氨基酸(以下实施例相同)，所述甘油酯脂肪酶突变体为：甘油酯脂肪酶突变体Y260C-G268C。
甘油酯脂肪酶突变体Y260C-G268C，其氨基酸构成包括有如SEQ ID NO.1所示的序列；或其编码基因如SEQ ID NO.2或其反向互补序列所示；或其氨基酸构成如SEQ ID NO.1所示且存在一个或多个点突变，但生物活性不变，即生物活性与SEQ ID NO.1所示的突变体相同。
在实际应用中，本领域技术人员可以在SEQ ID NO.1所示的序列的一端或两端增加一个或多个氨基酸不影响该突变体的活性，也不改变本发明中所公开的该突变体的特性。
所述存在一个或多个点突变，指SEQ ID NO.1所示的序列存在一个或多个碱基的突变，或者缺失，但是不影响该突变体的活性，也不改变本发明中所公开的该突变体的特性。
所述生物活性是指用于水解或合成甘油二酯、甘油单酯。
相对野生型而言，该突变体具有更好的热稳定性和比活力。更具体指在相同条件下，该突变体热稳定性显著提升，在相同条件下，甘油酯脂肪酶突变体Y260C-G268C的Tm值为53.0℃,50℃半衰期为92.4min。与野生型对比，甘油酯脂肪酶突变体Y260C-G268C的Tm值提高3.0℃,50℃半衰期提高22.7倍；且比活力也相对野生型提高约2倍。
甘油酯脂肪酶突变体Y260C-G268C的氨基酸序列如SEQ ID NO.1所示：
MLFSRFVLLAFGSVAAVSASSIYARGRGGSSTDQPVANPYNTKEISLAAGLVQQTYCDSTENGLKIGDSELLYTMGEGYARQRVNIYHSPSLGIAVAIEGTNLFSLNSDLHDAKFWQEDPNERYIQYYPKGTKLMHGFQQAYNDLMDDIFTAVKKYKKEKNEKRVTVIGHSLGAAMGLLCAMDIELRMDGGLYKTYLFGLPRLGNPTFASFVDQKIGDKFHSIINGRDWVPTVPPRALGYQHPSDYVWIYPGNSTSAKLCPGQENVHCILTVAREFNFDDHQGIYFHTQIGAVMGECPAQVGAH
甘油酯脂肪酶突变体Y260C-G268C的编码核苷酸序列如SEQ ID NO.2所示ATGCTCTTCAGTCGCTTTGTTCTTCTTGCGTTCGGTTCGGTGGCCGCCGTCTCGGCCAGCAGTATTTACGCCCGTGGCCGTGGTGGTAGCTCTACCGACCAGCCAGTGGCAAACCCTTACAACACCAAAGAGATTTCTCTGGCTGCCGGTCTTGTCCAGCAAACTTACTGTGACAGCACGGAAAATGGTCTGAAGATTGGCGACAGCGAGCTCCTTTACACCATGGGAGAGGGTTACGCTCGCCAGCGTGTCAACATCTATCACTCGCCTAGCCTTGGTATTGCTGTGGCCATCGAGGGCACGAACCTTTTCTCGCTTAACTCGGACTTGCATGATGCGAAGTTCTGGCAAGAAGACCCGAACGAGCGTTACATCCAGTACTACCCGAAGGGTACAAAGCTTATGCACGGTTTCCAGCAAGCCTACAATGACTTGATGGATGATATCTTCACTGCAGTTAAGAAGTACAAGAAAGAGAAGAATGAAAAGCGCGTGACTGTCATTGGCCACTCGCTTGGTGCCGCTATGGGTTTGCTTTGCGCTATGGACATTGAGCTGCGTATGGATGGTGGTCTGTACAAGACGTACCTGTTTGGACTTCCCCGTCTTGGTAACCCAACATTTGCTTCGTTCGTTGACCAAAAGATTGGCGACAAGTTCCACTCAATTATCAATGGTCGCGACTGGGTTCCAACGGTGCCGCCGCGCGCTCTTGGTTACCAGCACCCATCTGACTATGTTTGGATCTACCCAGGCAACAGCACGAGCGCGAAGCTTTGCCCTGGCCAAGAGAATGTCCACTGTATCCTCACTGTTGCTCGCGAGTTCAACTTTGACGACCACCAAGGTATCTACTTCCACACCCAGATCGGTGCTGTTATGGGTGAGTGCCCAGCTCAGGTTGGTGCTCAT。
甘油酯脂肪酶SMG1的氨基酸序列如SEQ ID NO.3所示：
MLFSRFVLLAFGSVAAVSASSIYARGRGGSSTDQPVANPYNTKEISLAAGLVQQTYCDSTENGLKIGDSELLYTMGEGYARQRVNIYHSPSLGIAVAIEGTNLFSLNSDLHDAKFWQEDPNERYIQYYPKGTKLMHGFQQAYNDLMDDIFTAVKKYKKEKNEKRVTVIGHSLGAAMGLLCAMDIELRMDGGLYKTYLFGLPRLGNPTFASFVDQKIGDKFHSIINGRDWVPTVPPRALGYQHPSDYVWIYPGNSTSAKLYPGQENVHGILTVAREFNFDDHQGIYFHTQIGAVMGECPAQVGAH。
甘油酯脂肪酶SMG1的编码核苷酸序列如SEQ ID NO.4所示：
ATGCTCTTCAGTCGCTTTGTTCTTCTTGCGTTCGGTTCGGTGGCCGCCGTCTCGGCCAGCAGTATTTACGCCCGTGGCCGTGGTGGTAGCTCTACCGACCAGCCAGTGGCAAACCCTTACAACACCAAAGAGATTTCTCTGGCTGCCGGTCTTGTCCAGCAAACTTACTGTGACAGCACGGAAAATGGTCTGAAGATTGGCGACAGCGAGCTCCTTTACACCATGGGAGAGGGTTACGCTCGCCAGCGTGTCAACATCTATCACTCGCCTAGCCTTGGTATTGCTGTGGCCATCGAGGGCACGAACCTTTTCTCGCTTAACTCGGACTTGCATGATGCGAAGTTCTGGCAAGAAGACCCGAACGAGCGTTACATCCAGTACTACCCGAAGGGTACAAAGCTTATGCACGGTTTCCAGCAAGCCTACAATGACTTGATGGATGATATCTTCACTGCAGTTAAGAAGTACAAGAAAGAGAAGAATGAAAAGCGCGTGACTGTCATTGGCCACTCGCTTGGTGCCGCTATGGGTTTGCTTTGCGCTATGGACATTGAGCTGCGTATGGATGGTGGTCTGTACAAGACGTACCTGTTTGGACTTCCCCGTCTTGGTAACCCAACATTTGCTTCGTTCGTTGACCAAAAGATTGGCGACAAGTTCCACTCAATTATCAATGGTCGCGACTGGGTTCCAACGGTGCCGCCGCGCGCTCTTGGTTACCAGCACCCATCTGACTATGTTTGGATCTACCCAGGCAACAGCACGAGCGCGAAGCTTTACCCTGGCCAAGAGAATGTCCACGGTATCCTCACTGTTGCTCGCGAGTTCAACTTTGACGACCACCAAGGTATCTACTTCCACACCCAGATCGGTGCTGTTATGGGTGAGTGCCCAGCTCAGGTTGGTGCTCAT。
脂肪酶突变体S5的氨基酸序列如SEQ ID NO.25所示：
MLFSRFVLLAFGSVAAVSASSIYARGRGGSSTDPPVPNPYNTKEISLAAGLVQQTYCDSTENGLKIGDSELLYTMGEGYARQRVNIYHSPSLGIAVAIEGTNLFSLNSDLHDAKFWQEDPNERYIQYYPKGTKLMHGFQQAYNDLMDDIFTAVKKYKKEKNEKRVTVIGHSLGAAVALLCAMDIELRMDGGLYKTYLFGLPRLGNPTFASFVDQKIGDKFHSIINGRDWVPTVPPRALGYQHPSDYVWIYPGNSTSAKLYPGQENVHGILTVAREFNFDDHQGIYFHTQIGAVRGECPAQVGAH。
脂肪酶突变体S5的编码核苷酸序列如SEQ ID NO.26所示
ATGCTCTTCAGTCGCTTTGTTCTTCTTGCGTTCGGTTCGGTGGCCGCCGTCTCGGCCAGCAGTATTTACGCCCGTGGCCGTGGTGGTAGCTCTACCGACCCGCCAGTGCCGAACCCTTACAACACCAAAGAGATTTCTCTGGCTGCCGGTCTTGTCCAGCAAACTTACTGTGACAGCACGGAAAATGGTCTGAAGATTGGCGACAGCGAGCTCCTTTACACCATGGGAGAGGGTTACGCTCGCCAGCGTGTCAACATCTATCACTCGCCTAGCCTTGGTATTGCTGTGGCCATCGAGGGCACGAACCTTTTCTCGCTTAACTCGGACTTGCATGATGCGAAGTTCTGGCAAGAAGACCCGAACGAGCGTTACATCCAGTACTACCCGAAGGGTACAAAGCTTATGCACGGTTTCCAGCAAGCCTACAATGACTTGATGGATGATATCTTCACTGCAGTTAAGAAGTACAAGAAAGAGAAGAATGAAAAGCGCGTGACTGTCATTGGCCACTCGCTTGGTGCCGCTGTGGCTTTGCTTTGCGCTATGGACATTGAGCTGCGTATGGATGGTGGTCTGTACAAGACGTACCTGTTTGGACTTCCCCGTCTTGGTAACCCAACATTTGCTTCGTTCGTTGACCAAAAGATTGGCGACAAGTTCCACTCAATTATCAATGGTCGCGACTGGGTTCCAACGGTGCCGCCGCGCGCTCTTGGTTACCAGCACCCATCTGACTATGTTTGGATCTACCCAGGCAACAGCACGAGCGCGAAGCTTTACCCTGGCCAAGAGAATGTCCACGGTATCCTCACTGTTGCTCGCGAGTTCAACTTTGACGACCACCAAGGTATCTACTTCCACACCCAGATCGGTGCTGTTAGGGGTGAGTGCCCAGCTCAGGTTGGTGCTCAT。
以下通过具体实施例，对本发明做进一步的阐述，但是不用于限制本发明的保护范围。
材料与试剂：质粒提取试剂盒购自Omega贸易有限公司，Primer STAR试剂盒购自TAKARA公司，Sypro Orange dye购自Sigama公司；TOP10大肠杆菌感受态细胞购自天根生物公司，突变引物由上海生工生物工程公司合成；PmeⅠ限制性内切酶购自New EnglandBiolabs公司；PCR产物纯化回收试剂盒购自大连宝生物公司；电转仪购自Bio-Rad公司；LLB、LLB+Zeocin、YPD、BMGY、BMMY培养基均按照Invitrogen毕赤酵母表达试剂盒操作手册配制，其余试剂均为国内外购买的分析纯级别。
实施例1脂肪酶SMG1的分子动力学模拟和二硫键设计
1)利用GROMACS 2018.06对脂肪酶SMG1的晶体结构(PDB ID:3UUE)进行分子动力学模拟，使用Amberff99SB-NMR-ILDN力场参数，将蛋白(甘油酯脂肪酶SMG1)放置于正方体盒子中央并填充TIP3P水分子与3个Na+中和的体系电荷，以Steepest算法对整个体系进行能量优化。随后在等温等压体系(NPT)体系下，限制蛋白骨架重原子的运动，在500ps内缓慢地从0K升温至303K，充分平衡酶周围的水分子，体系的温度和压力偶联使用Berendsen算法，得到预平衡模型；将预平衡模型在2ns内从303K缓慢地升温至473K，然后在等温等压体系(NPT)下进行100ns模拟。范德华力截断半径、静电相互作用力截断半径、邻近搜索截断半径均根据AMBER力场推荐，设置为1.0nm。所有的键长采用LINCS算法约束，长程静电相互作用力采用PME算法，计算步长设置为2fs；通过Gromacs自带的分析工具gmx_rmsf计算Cα原子的RMSF，以寻找脂肪酶SMG1的柔性区域。RMSF分析结果如图1所示，RMSF越大，柔性越大。
2)利用Disulfide by Design软件预测潜在的二硫键突变位点，然后在RMSF较大的区域设计二硫键。经过发明人根据以往积累的经验，反复比较，最终留下了五个脂肪酶突变体：脂肪酶突变体S69C-S89C，脂肪酶突变体K133C-N205C，脂肪酶突变体D213C-S244C，脂肪酶突变体Y260C-G268C或脂肪酶突变体H287C-H304C。
实施例2甘油酯脂肪酶SMG1突变体载体的构建
脂肪酶SMG1的表达载体pPICZaA-SMG1由本实验前期所构建，即将SMG1的基因(Genbank ID:XM_001732152.1)插入至载体pPICZaA中，限制性酶切位点为EcoRⅠ与SalⅠ。以pPICZaA-SMG1为模板，表1中的引物进行PCR反应，构建甘油酯脂肪酶突变体S69C-S89C，甘油酯脂肪酶突变体K133C-N205C，甘油酯脂肪酶突变体D213C-S244C，甘油酯脂肪酶突变体Y260C-G268C或甘油酯脂肪酶突变体H287C-H304C。
表1突变引物汇总
PCR扩增条件为：98℃3min；98℃10s、55℃30s、72℃5min，30个循环；72℃延伸5min。反应体系：引物各为1μL，Primer Star(2X)为12.5μL，模板为1μL，ddH2O为9.5μL。扩增产物以DnpⅠ酶消化模板，在琼脂糖凝胶电泳检测突变条带大小后，随后使用热激法将突变质粒转入E.coli TOP10感受态细胞，并涂布于LLB(Zeocin浓度为25μg/ml)固体平板37℃过夜培养，挑选阳性转化子进行质粒的测序。
此外，为进行对比筛选，将本实验室前期筛选的SMG1最优突变体S5(《基于生物计算对SMG1和PCL脂肪酶热稳定性改造的应用基础研究》)的基因插入至载体pPICZaA中，限制性酶切位点为EcoRⅠ与SalⅠ。该脂肪酶突变体含有突变位点Q34P、A37P、M176V、G177A和M294R。
实施例3：甘油酯脂肪酶突变体工程菌株的构建、表达和纯化
将测序正确的阳性转化子于LLB液体培养基过夜扩增培养后，提取质粒，以PmeⅠ线性化处理并纯化回收，以总量为5μg的质粒线性化产物与X33毕赤酵母感受态混合电击转化。毕赤酵母感受态制备参照Invitrogen公司操作手册。电转程序按照Bio-Rad公司推荐参数设置。
电转完毕立刻加入1mL 1mol/L山梨醇溶液，将菌液在30℃孵育复苏1h后，均匀涂布于YPDS+Zeocin(Zeocin浓度为100μg/ml)抗性平板上筛选；培养72h后，挑选阳性转化子。
工程菌株摇床发酵：
把工程菌株单菌落接种至50mL BMGY培养基，30℃250rpm培养至OD0.8-1.0，按10％接种量接种至400mL BMMY培养基30℃250rpm发酵96h,每24h以1％甲醇进行诱导。
酶的分离和纯化:
将发酵液于4℃7000r/min离心40min后，收集上清液。上清液用0.45μm的滤膜抽滤后，以镍柱亲和层析柱进行纯化。层析柱先用含20mM咪唑的磷酸缓冲液(pH 7.4)进行平衡，平衡结束后再将上清液与层析柱结合，接着用含20mM咪唑的磷酸缓冲液(pH 7.4)进行洗脱杂蛋白，然后用含300mM咪唑的磷酸缓冲液(pH 7.4)洗脱目标蛋白。将洗脱下来的目的蛋白进一步以G-25脱盐柱脱除咪唑，最后以还原性SDS-PAGE垂直电泳检测酶纯度。在得到纯度在90％以上的蛋白后进行后续的实验。
实施例4：甘油酯脂肪酶突变体的DSF筛选
将蛋白的浓度稀释至0.3mg/ml，染料Sypro Orange dye稀释100倍。取蛋白20μL与5μL染料混合，于Bio-Rad实时荧光定量PCR仪中进行蛋白热失活曲线测定，得出蛋白质的Tm值。
结果如表2所示，与野生型SMG1对比，甘油酯脂肪酶突变体Y260C-G268C和S5的Tm值分别提高了3.0和6.0℃。甘油酯脂肪酶SMG1的的核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示；甘油酯脂肪酶突变体Y260C-G268C的核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO.4所示；甘油酯脂肪酶突变体S5的核苷酸序列如SEQ IDNO.25所示，氨基酸序列如SEQ ID NO.26所示。
表2DSF测定结果
实施例5：二硫键数目测定
1)将纯化蛋白样品以含8M尿素的Tris-Gly缓冲液(50mM，pH＝8.00)稀释至0.5mg/mL。混合均匀后，分装2mL至两支4mL离心管中，A管用于测定游离巯基的浓度，B管用于测定总半胱氨酸含量，并在B管中加入50μLβ-巯基乙醇还原分子内的二硫键，于37℃保温1h。
2)在B管中加入2mL 30％(w/v)三氯乙酸溶液，37℃水浴1h，充分沉淀蛋白，随后以12000r/min离心30min，弃上清，沉淀使用2mL 30％三氯乙酸溶液重悬润洗并再次离心，晾干5min，挥发残余的β-巯基乙醇。最后用2mL含8M尿素的Tris-Gly缓冲液(50mM，pH＝8.00)重新溶解沉淀。每管吸取1mL做为空白组，并在剩余反应液中加入10μL DTNB(4mg/mL)显色。孵育30min后以12000r/min离心10min，除去沉淀，吸取上清，并稀释一定的倍数，于412nm测定吸光值，保证其吸光值在0.2-0.8之间。二硫键数量由以下公式计算：
SH＝73.53×A412×D/V----------------(公式1)
SH3＝SH1－SH2----------------(公式2)
SS＝N/2×(SH3/SH1)----------------(公式3)
其中SH为巯基浓度(μmol/mL)，SH3为成键半胱氨酸巯基浓度(μmol/mL)，SH2为游离半胱氨酸巯基浓度(μmol/mL)，SH1为总半胱氨酸巯基浓度(μmol/mL)，A412为除去空白样品后的吸光光度值，D为稀释倍数，V为溶液体积(mL)，SS为蛋白质内部二硫键的数目。
二硫键测定结果如表3所示，脂肪酶突变体G28C-P206C成功引入了二硫键。
表3二硫键数目测定
实施例6：甘油酯脂肪酶的热稳定性质测定
半衰期测定方法：将纯化酶液稀释到0.2mg/mL，于50℃中孵育，在一定的时间间隔取出样品，并以pNP比色法测定残留酶活。获得残留酶活后，进一步计算半衰期：以热处理时间(分钟)为横坐标，相对酶活力的ln对数值为纵坐标，然后求其拟合直线，所得拟合直线斜率的相反数kd为失活常数，根据半衰期公式T1/2＝ln2/kd求出其半衰期。
pNP比色法测定酶活：量取24.23μL的p-Nitrophenylcaprylate(C8)溶于10mL无水乙醇中，涡旋振荡充分溶解。反应体系为10μL10 mM pNP-酯、10μL酶液、80μL反应缓冲液，于室温孵育5min后添加100μL异丙醇终止反应的进行，并且在405nm波长测定其吸光值。
表4半衰期测定结果
半衰期测定结果如表4所示，与野生型脂肪酶SMG1对比，甘油酯脂肪酶突变体Y260C-G268C和S5的50℃半衰期分别提高22.7和40.1倍。
实施例7：酶的比活力测定
以p-Nitrophenylcaprylate(C8)和甘油二酯为底物分别测定酶的活性。以p-Nitrophenylcaprylate(C8)为底物时，一个脂肪酶活力单位(U)定义为在一定条件下单位时间内脂肪酶生成1μmol对硝基苯酚所需的酶量。
以富含甘油二酯乳的油脂的乳化液为底物测酶活性：称取4％PVA溶液150g，甘油二酯50g，用高速匀浆机处理6min制成乳化液。取100mL锥形瓶，加入4g乳化液和5mL 20mM磷酸缓冲液(pH为6.0)于25℃孵育5min。将纯化的酶稀释，向锥形瓶中加入1mL酶液反应10min，以15mL 95％乙醇终止反应。加入酚酞，用0.05M氢氧化钠溶液进行滴定，以测定反应过程中释放的游离脂肪酸的量。对照组：将上述加入的酶溶液用相同体积的缓冲液代替即可。酶活性U定义为每分钟产生1μmol脂肪酸所消耗酶量。
表5比活力测定结果(U/mg)
比活测定结果如表4所示，与野生型对比，突变体S5的比活无明显变化。而以p-Nitrophenylcaprylate和甘油二酯为底物时，突变体Y260C-G268C的比活均提高两倍左右，比突变体S5的比活力更高。这比通常的突变体的比活力都更好。
实施例8甘油二酯的合成
称取20g油酸和甘油的混合物(油酸与甘油摩尔比为4:1)于锥形瓶中，并置于转速为200rpm的恒温磁力搅拌器上35℃预热10min，预热结束后，分别加入120U/g的野生型甘油酯脂肪酶、甘油酯脂肪酶突变体S5或甘油酯脂肪酶突变体Y260C-G268C，然后将其置于35℃的恒温振荡器中配合磁力搅拌子进行抽真空反应，转速为200rpm，于不同的反应时间提取油样，使用HPLC分析反应产物的甘油酯组成。反应24小时后，野生型甘油酯脂肪酶的酯化产物DAG(甘油二酯)含量为42％，甘油酯脂肪酶突变体S5的DAG含量为51％，甘油酯脂肪酶突变体Y260C-G268C的DAG含量为63％。结果说明甘油酯突变体Y260C-G268C合成甘油二酯的效果更好，具有更好的应用前景。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 广州永华特医营养科技有限公司
华南理工大学
<120> 一种甘油酯脂肪酶SMG1突变体及其编码基因与应用
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Ala Lys Phe Trp Gln Glu Asp Pro Asn Glu Arg Tyr Ile Gln Tyr Tyr
115 120 125
Pro Lys Gly Thr Lys Leu Met His Gly Phe Gln Gln Ala Tyr Asn Asp
130 135 140
Leu Met Asp Asp Ile Phe Thr Ala Val Lys Lys Tyr Lys Lys Glu Lys
145 150 155 160
Asn Glu Lys Arg Val Thr Val Ile Gly His Ser Leu Gly Ala Ala Val
165 170 175
Ala Leu Leu Cys Ala Met Asp Ile Glu Leu Arg Met Asp Gly Gly Leu
180 185 190
Tyr Lys Thr Tyr Leu Phe Gly Leu Pro Arg Leu Gly Asn Pro Thr Phe
195 200 205
Ala Ser Phe Val Asp Gln Lys Ile Gly Asp Lys Phe His Ser Ile Ile
210 215 220
Asn Gly Arg Asp Trp Val Pro Thr Val Pro Pro Arg Ala Leu Gly Tyr
225 230 235 240
Gln His Pro Ser Asp Tyr Val Trp Ile Tyr Pro Gly Asn Ser Thr Ser
245 250 255
Ala Lys Leu Tyr Pro Gly Gln Glu Asn Val His Gly Ile Leu Thr Val
260 265 270
Ala Arg Glu Phe Asn Phe Asp Asp His Gln Gly Ile Tyr Phe His Thr
275 280 285
Gln Ile Gly Ala Val Arg Gly Glu Cys Pro Ala Gln Val Gly Ala His
290 295 300
<210> 26
<211> 912
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atgctcttca gtcgctttgt tcttcttgcg ttcggttcgg tggccgccgt ctcggccagc 60
agtatttacg cccgtggccg tggtggtagc tctaccgacc cgccagtgcc gaacccttac 120
aacaccaaag agatttctct ggctgccggt cttgtccagc aaacttactg tgacagcacg 180
gaaaatggtc tgaagattgg cgacagcgag ctcctttaca ccatgggaga gggttacgct 240
cgccagcgtg tcaacatcta tcactcgcct agccttggta ttgctgtggc catcgagggc 300
acgaaccttt tctcgcttaa ctcggacttg catgatgcga agttctggca agaagacccg 360
aacgagcgtt acatccagta ctacccgaag ggtacaaagc ttatgcacgg tttccagcaa 420
gcctacaatg acttgatgga tgatatcttc actgcagtta agaagtacaa gaaagagaag 480
aatgaaaagc gcgtgactgt cattggccac tcgcttggtg ccgctgtggc tttgctttgc 540
gctatggaca ttgagctgcg tatggatggt ggtctgtaca agacgtacct gtttggactt 600
ccccgtcttg gtaacccaac atttgcttcg ttcgttgacc aaaagattgg cgacaagttc 660
cactcaatta tcaatggtcg cgactgggtt ccaacggtgc cgccgcgcgc tcttggttac 720
cagcacccat ctgactatgt ttggatctac ccaggcaaca gcacgagcgc gaagctttac 780
cctggccaag agaatgtcca cggtatcctc actgttgctc gcgagttcaa ctttgacgac 840
caccaaggta tctacttcca cacccagatc ggtgctgtta ggggtgagtg cccagctcag 900
gttggtgctc at 912
Claims (10)
1.一种甘油酯脂肪酶SMG1突变体，其特征是，其氨基酸构成如SEQ ID NO.1所示序列；或其编码基因如SEQ ID NO.2所示序列。
2.一种编码权利要求1所述甘油酯脂肪酶SMG1突变体的基因。
3.根据权利要求2所述的基因，其特征是，其包括如SEQ ID NO.2所示序列。
4.一种插入有包括权利要求2或3所述的编码甘油酯脂肪酶SMG1突变体的基因的重组表达载体。
5.一种转入有权利要求4所述重组表达载体的重组工程菌株。
6.权利要求1所述的甘油酯脂肪酶SMG1突变体、和/或权利要求2或3所述的基因在油脂制备中的应用。
7.权利要求4所述的重组表达载体、和/或权利要求5所述的重组工程菌株在油脂制备中的应用。
8.根据权利要求6或7所述的应用，其特征是，所述油脂是甘油二酯或甘油单酯。
9.一种制备油脂的方法，其特征是，所用脂肪酶是权利要求1所述的甘油酯脂肪酶SMG1突变体。
10.根据权利要求9所述的制备油脂的方法，其特征是，所述油脂是甘油二酯或甘油单酯。
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| CN102676592A (zh) * | 2012-02-29 | 2012-09-19 | 华南理工大学 | 脂肪酶smg1在单脂肪酸甘油酯制备中的应用 |
| CN103525784A (zh) * | 2013-09-30 | 2014-01-22 | 华南理工大学 | 一种偏甘油酯脂肪酶突变体、质粒、重组菌株及制备方法和应用 |
| CN103627685A (zh) * | 2013-11-20 | 2014-03-12 | 华南理工大学 | 一种活性提高的偏甘油酯脂肪酶突变体及其应用 |
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| CN102676592A (zh) * | 2012-02-29 | 2012-09-19 | 华南理工大学 | 脂肪酶smg1在单脂肪酸甘油酯制备中的应用 |
| CN103525784A (zh) * | 2013-09-30 | 2014-01-22 | 华南理工大学 | 一种偏甘油酯脂肪酶突变体、质粒、重组菌株及制备方法和应用 |
| CN103627685A (zh) * | 2013-11-20 | 2014-03-12 | 华南理工大学 | 一种活性提高的偏甘油酯脂肪酶突变体及其应用 |
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Denomination of invention: A Glycerol Ester Lipase SMG1 Mutant and Its Encoding Genes and Applications Granted publication date: 20230228 Pledgee: Bank of China Limited Foshan Nanhai Xiqiao Sub-branch Pledgor: Guangzhou Yonghua special medicine nutrition Technology Co.,Ltd. Registration number: Y2024980005359 |