CN112574975A - 甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用 - Google Patents
甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用 Download PDFInfo
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- CN112574975A CN112574975A CN202110004098.8A CN202110004098A CN112574975A CN 112574975 A CN112574975 A CN 112574975A CN 202110004098 A CN202110004098 A CN 202110004098A CN 112574975 A CN112574975 A CN 112574975A
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- mutant
- glyceride lipase
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- 239000004367 Lipase Substances 0.000 title claims abstract description 100
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- 125000005456 glyceride group Chemical group 0.000 title claims abstract description 93
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 28
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 150000001413 amino acids Chemical class 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 10
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Abstract
本发明公开了一种甘油酯脂肪酶突变体G28C‑P206C及其编码基因与应用。该甘油酯脂肪酶突变体G28C‑P206C是在甘油酯脂肪酶SMG1中引入二硫键，该突变体的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4。与野生型对比，本发明获得的甘油酯脂肪酶突变体G28C‑P206C的Tm提高9.0℃，50℃半衰期提高64.8倍，热稳定性显著提高，且相对于野生型，其酶活性更好，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突变体G28C-P206C及编码基因与应用。
背景技术
脂肪酶(EC 3.1.1.3)是一种丝氨酸水解酶，不仅能在油水界面水解油脂产生脂肪酸、甘油二酯、甘油单酯和甘油等产物，还可以催化酯化、酯交换、醇解和酸解等反应。甘油三酯脂肪酶能水解或酯化形成甘油三酯、甘油二酯或甘油单酯，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。目前文献报道的甘油酯脂肪酶数量相对较少，并且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业化。因此，开发甘油酯脂肪酶意义重大。本实验室前期筛选到一种来源于球形马拉色菌的甘油酯脂肪酶SMG1，能用于合成高纯度甘油二酯、油脂脱酸和环氧化反应等，但是在使用中发现，其热稳定性较差，在应用过程中容易失活，造成生产成本的增加，限制了应用范围，不利于产业化。因此，提高脂肪酶的热稳定性可以为其产业化应用奠定基础。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。
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G28C-P206C，该突变体还具有较好的比活力。
本发明的目的通过下述技术方案实现：
一种甘油酯脂肪酶突变体G28C-P206C，其氨基酸构成包括如SEQ ID NO.3所示序列；或其编码基因包括如SEQ ID NO.4所示序列或其反向互补序列；或其氨基酸构成包括如SEQ ID NO.3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突变体G28C-P206C的编码基因。
一种编码上述甘油酯脂肪酶突变体G28C-P206C的核苷酸序列。
所述的甘油酯脂肪酶突变体G28C-P206C，为在野生型甘油酯脂肪酶SMG1中通过定点突变以引入二硫键获得。
在其中一些实施例中，其如SEQ ID NO.4或其反向互补序列所示。
本发明还提供了一种含有上述编码甘油酯脂肪酶突变体G28C-P206C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突变体G28C-P206C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突变体G28C-P206C的应用。
上述热稳定性提高的甘油酯脂肪酶突变体、所述热稳定性提高的甘油酯脂肪酶突变体G28C-P206C的编码基因、重组表达载体、重组工程菌株在制备油脂中的应用。
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突变体G28C-P206C。
本发明通过在野生型甘油酯脂肪酶SMG1中引入合适的二硫键获得了热稳定性显著提高且比活力更高的甘油酯脂肪酶突变体G28C-P206C。与野生型对比，甘油酯脂肪酶突变体G28C-P206C包含突变位点Gly28Cys和Pro206Cys。同一条件下，野生型甘油酯脂肪酶SMG1的Tm为50.0℃，50℃半衰期为3.9min时，而本发明所述的甘油酯脂肪酶突变体G28C-P206C的Tm值为59.0℃,50℃半衰期为256.7min。与野生型对比，甘油酯脂肪酶突变体G28C-P206C的Tm值提高9.0℃,50℃半衰期提高64.8倍，具有显著的提高，而且，该甘油酯脂肪酶突变体G28C-P206C的比活力相对野生型的提高1倍多。因此，本发明获得的甘油酯脂肪酶突变体G28C-P206C更适合于工业化应用。
附图说明
图1是还原性SDS-PAGE检测蛋白纯度的结果图：泳道1-7分别为，野生型甘油酯脂肪酶SMG1、甘油酯脂肪酶突变体T74C-N85C、甘油酯脂肪酶突变体Y127C-L186C、甘油酯脂肪酶突变体N38C-A257C、甘油酯脂肪酶突变体G28C-P206C，甘油酯脂肪酶突变体T42C-F286C和甘油酯脂肪酶突变体S5蛋白条带。
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G28C-P206C，它在野生型甘油酯脂肪酶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)，即所述甘油酯脂肪酶突变体G28C-P206C包含突变位点Gly28Cys和Pro206Cys。
一种甘油酯脂肪酶突变体G28C-P206C，其氨基酸构成包括有如SEQ ID NO.3所示的序列；或其编码基因如SEQ ID NO.4或其反向互补序列所示；或其氨基酸构成如SEQ IDNO.3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.3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.3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.3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，如Tm值为59.0℃左右,50℃半衰期为256.7min左右，即热稳定高于野生型(氨基酸构成如SEQ ID NO.1所示,编码基因如SEQ ID NO.2或其反向互补序列所示)，且比活力也相对野生型提高1倍多。
甘油酯脂肪酶SMG1的氨基酸序列如SEQ ID NO.1所示：
MLFSRFVLLAFGSVAAVSASSIYARGRGGSSTDQPVANPYNTKEISLAAGLVQQTYCDSTENGLKIGDSELLYTMGEGYARQRVNIYHSPSLGIAVAIEGTNLFSLNSDLHDAKFWQEDPNERYIQYYPKGTKLMHGFQQAYNDLMDDIFTAVKKYKKEKNEKRVTVIGHSLGAAMGLLCAMDIELRMDGGLYKTYLFGLPRLGNPTFASFVDQKIGDKFHSIINGRDWVPTVPPRALGYQHPSDYVWIYPGNSTSAKLYPGQENVHGILTVAREFNFDDHQGIYFHTQIGAVMGECPAQVGAH
甘油酯脂肪酶SMG1的编码核苷酸序列如SEQ ID NO.2所示：
ATGCTCTTCAGTCGCTTTGTTCTTCTTGCGTTCGGTTCGGTGGCCGCCGTCTCGGCCAGCAGTATTTACGCCCGTGGCCGTGGTGGTAGCTCTACCGACCAGCCAGTGGCAAACCCTTACAACACCAAAGAGATTTCTCTGGCTGCCGGTCTTGTCCAGCAAACTTACTGTGACAGCACGGAAAATGGTCTGAAGATTGGCGACAGCGAGCTCCTTTACACCATGGGAGAGGGTTACGCTCGCCAGCGTGTCAACATCTATCACTCGCCTAGCCTTGGTATTGCTGTGGCCATCGAGGGCACGAACCTTTTCTCGCTTAACTCGGACTTGCATGATGCGAAGTTCTGGCAAGAAGACCCGAACGAGCGTTACATCCAGTACTACCCGAAGGGTACAAAGCTTATGCACGGTTTCCAGCAAGCCTACAATGACTTGATGGATGATATCTTCACTGCAGTTAAGAAGTACAAGAAAGAGAAGAATGAAAAGCGCGTGACTGTCATTGGCCACTCGCTTGGTGCCGCTATGGGTTTGCTTTGCGCTATGGACATTGAGCTGCGTATGGATGGTGGTCTGTACAAGACGTACCTGTTTGGACTTCCCCGTCTTGGTAACCCAACATTTGCTTCGTTCGTTGACCAAAAGATTGGCGACAAGTTCCACTCAATTATCAATGGTCGCGACTGGGTTCCAACGGTGCCGCCGCGCGCTCTTGGTTACCAGCACCCATCTGACTATGTTTGGATCTACCCAGGCAACAGCACGAGCGCGAAGCTTTACCCTGGCCAAGAGAATGTCCACGGTATCCTCACTGTTGCTCGCGAGTTCAACTTTGACGACCACCAAGGTATCTACTTCCACACCCAGATCGGTGCTGTTATGGGTGAGTGCCCAGCTCAGGTTGGTGCTCAT
甘油酯脂肪酶突变体G28C-P206C的氨基酸序列如SEQ ID NO.3所示：
MLFSRFVLLAFGSVAAVSASSIYARGRCGSSTDQPVANPYNTKEISLAAGLVQQTYCDSTENGLKIGDSELLYTMGEGYARQRVNIYHSPSLGIAVAIEGTNLFSLNSDLHDAKFWQEDPNERYIQYYPKGTKLMHGFQQAYNDLMDDIFTAVKKYKKEKNEKRVTVIGHSLGAAMGLLCAMDIELRMDGGLYKTYLFGLPRLGNCTFASFVDQKIGDKFHSIINGRDWVPTVPPRALGYQHPSDYVWIYPGNSTSAKLYPGQENVHGILTVAREFNFDDHQGIYFHTQIGAVMGECPAQVGAH
甘油酯脂肪酶突变体G28C-P206C的编码核苷酸序列如SEQ ID NO.4所示
ATGCTCTTCAGTCGCTTTGTTCTTCTTGCGTTCGGTTCGGTGGCCGCCGTCTCGGCCAGCAGTATTTACGCCCGTGGCCGTTGTGGTAGCTCTACCGACCAGCCAGTGGCAAACCCTTACAACACCAAAGAGATTTCTCTGGCTGCCGGTCTTGTCCAGCAAACTTACTGTGACAGCACGGAAAATGGTCTGAAGATTGGCGACAGCGAGCTCCTTTACACCATGGGAGAGGGTTACGCTCGCCAGCGTGTCAACATCTATCACTCGCCTAGCCTTGGTATTGCTGTGGCCATCGAGGGCACGAACCTTTTCTCGCTTAACTCGGACTTGCATGATGCGAAGTTCTGGCAAGAAGACCCGAACGAGCGTTACATCCAGTACTACCCGAAGGGTACAAAGCTTATGCACGGTTTCCAGCAAGCCTACAATGACTTGATGGATGATATCTTCACTGCAGTTAAGAAGTACAAGAAAGAGAAGAATGAAAAGCGCGTGACTGTCATTGGCCACTCGCTTGGTGCCGCTATGGGTTTGCTTTGCGCTATGGACATTGAGCTGCGTATGGATGGTGGTCTGTACAAGACGTACCTGTTTGGACTTCCCCGTCTTGGTAACTGCACATTTGCTTCGTTCGTTGACCAAAAGATTGGCGACAAGTTCCACTCAATTATCAATGGTCGCGACTGGGTTCCAACGGTGCCGCCGCGCGCTCTTGGTTACCAGCACCCATCTGACTATGTTTGGATCTACCCAGGCAACAGCACGAGCGCGAAGCTTTACCCTGGCCAAGAGAATGTCCACGGTATCCTCACTGTTGCTCGCGAGTTCAACTTTGACGACCACCAAGGTATCTACTTCCACACCCAGATCGGTGCTGTTATGGGTGAGTGCCCAGCTCAGGTTGGTGCTCAT。
本发明的一个实施例中，一种甘油二酯或甘油单酯的生产方法中，所用的甘油酯脂肪酶是上述的甘油酯脂肪酶突变体G28C-P206C。
所述甘油酯脂肪酶突变体G28C-P206C的热稳定性显著提升，适用于工业领域上的应用。
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、甘油酯突变脂肪酶表达载体的构建
甘油酯脂肪酶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。分析脂肪酶SMG1的晶体结构(PDB ID:3UUE)，根据发明人积累的经验，并利用Disulfide byDesign软件预测潜在的二硫键突变位点，经过发明人反复比较，最后留下了5个二硫键突变体，即甘油酯脂肪酶突变体T74C-N85C、甘油酯脂肪酶突变体Y127C-L186C、甘油酯脂肪酶突变体N38C-A257C、甘油酯肪酶突变体G28C-P206C或甘油酯脂肪酶突变体T42C-F286C。每个二硫键突变体涉及两个突变位点的组合，即将两个氨基酸残基突变成Cys，两个Cys将可能形会成二硫键。
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T74C-N85C、甘油酯脂肪酶突变体Y127C-L186C、甘油酯脂肪酶突变体N38C-A257C、甘油酯脂肪酶突变体G28C-P206C或甘油酯脂肪酶突变体T42C-F286C的表达载体。
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(柳艳华.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[D].华南理工大学,2018.)的基因插入至载体pPICZaA中，限制性酶切位点为EcoRⅠ与SalⅠ。该甘油酯脂肪酶突变体含有突变位点Q34P、A37P、M176V、G177A和M294R。
甘油酯脂肪酶突变体S5的氨基酸序列如SEQ ID NO.25所示：
MLFSRFVLLAFGSVAAVSASSIYARGRGGSSTDPPVPNPYNTKEISLAAGLVQQTYCDSTENGLKIGDSELLYTMGEGYARQRVNIYHSPSLGIAVAIEGTNLFSLNSDLHDAKFWQEDPNERYIQYYPKGTKLMHGFQQAYNDLMDDIFTAVKKYKKEKNEKRVTVIGHSLGAAVALLCAMDIELRMDGGLYKTYLFGLPRLGNPTFASFVDQKIGDKFHSIINGRDWVPTVPPRALGYQHPSDYVWIYPGNSTSAKLYPGQENVHGILTVAREFNFDDHQGIYFHTQIGAVRGECPAQVGAH
甘油酯脂肪酶突变体S5的编码核苷酸序列如SEQ ID NO.26所示
ATGCTCTTCAGTCGCTTTGTTCTTCTTGCGTTCGGTTCGGTGGCCGCCGTCTCGGCCAGCAGTATTTACGCCCGTGGCCGTGGTGGTAGCTCTACCGACCCGCCAGTGCCGAACCCTTACAACACCAAAGAGATTTCTCTGGCTGCCGGTCTTGTCCAGCAAACTTACTGTGACAGCACGGAAAATGGTCTGAAGATTGGCGACAGCGAGCTCCTTTACACCATGGGAGAGGGTTACGCTCGCCAGCGTGTCAACATCTATCACTCGCCTAGCCTTGGTATTGCTGTGGCCATCGAGGGCACGAACCTTTTCTCGCTTAACTCGGACTTGCATGATGCGAAGTTCTGGCAAGAAGACCCGAACGAGCGTTACATCCAGTACTACCCGAAGGGTACAAAGCTTATGCACGGTTTCCAGCAAGCCTACAATGACTTGATGGATGATATCTTCACTGCAGTTAAGAAGTACAAGAAAGAGAAGAATGAAAAGCGCGTGACTGTCATTGGCCACTCGCTTGGTGCCGCTGTGGCTTTGCTTTGCGCTATGGACATTGAGCTGCGTATGGATGGTGGTCTGTACAAGACGTACCTGTTTGGACTTCCCCGTCTTGGTAACCCAACATTTGCTTCGTTCGTTGACCAAAAGATTGGCGACAAGTTCCACTCAATTATCAATGGTCGCGACTGGGTTCCAACGGTGCCGCCGCGCGCTCTTGGTTACCAGCACCCATCTGACTATGTTTGGATCTACCCAGGCAACAGCACGAGCGCGAAGCTTTACCCTGGCCAAGAGAATGTCCACGGTATCCTCACTGTTGCTCGCGAGTTCAACTTTGACGACCACCAAGGTATCTACTTCCACACCCAGATCGGTGCTGTTAGGGGTGAGTGCCCAGCTCAGGTTGGTGCTCAT
实施例2：甘油酯脂肪酶突变体表达菌株的构建、表达及纯化
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。
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。
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，流速4mL/min，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(pH7.4)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。结果显示如图1所示，所有蛋白纯度均在95％以上。
实施例3：甘油酯脂肪酶突变体的DSF筛选
将目的蛋白稀释到相同浓度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的实时荧光定量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，甘油酯脂肪酶突变体G28C-P206C的Tm比野生型提高9.0℃，比突变体S5提高3.0℃。
表2 DSF测定结果
实施例4：二硫键数目测定
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，甘油酯脂肪酶突变体G28C-P206C成功引入了二硫键。
表3二硫键数目测定
实施例5：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比色法测定相对酶活：吸取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10μL的10mM 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，与野生型对比，突变体S5的50℃半衰期提高40.1倍，而突变体G28C-P206C的50℃半衰期提高64.8倍。突变体G28C-P206C的半衰期比突变体S5更长。
实施例6：甘油酯脂肪酶的比活力测定
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
比活测定结果如表4所示，与野生型脂肪酶SMG1对比，突变体S5的比活力无明显变化，而突变体G28C-P206C的比活提高1.1倍。
实施例7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G28C-P206C，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G28C-P206C的DAG含量为72％。可见,本发明所述的甘油酯突变体G28C-P206C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> 华南理工大学
广州永华特医营养科技有限公司
<120> 甘油酯脂肪酶突变体G28C-P206C及其编码基因与应用
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 304
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Leu Phe Ser Arg Phe Val Leu Leu Ala Phe Gly Ser Val Ala Ala
1 5 10 15
Val Ser Ala Ser Ser Ile Tyr Ala Arg Gly Arg Gly Gly Ser Ser Thr
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Asp Gln Pro Val Ala Asn Pro Tyr Asn Thr Lys Glu Ile Ser Leu Ala
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Ala Gly Leu Val Gln Gln Thr Tyr Cys Asp Ser Thr Glu Asn Gly Leu
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Lys Ile Gly Asp Ser Glu Leu Leu Tyr Thr Met Gly Glu Gly Tyr Ala
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Arg Gln Arg Val Asn Ile Tyr His Ser Pro Ser Leu Gly Ile Ala Val
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Ala Ile Glu Gly Thr Asn Leu Phe Ser Leu Asn Ser Asp Leu His Asp
100 105 110
Ala Lys Phe Trp Gln Glu Asp Pro Asn Glu Arg Tyr Ile Gln Tyr Tyr
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Pro Lys Gly Thr Lys Leu Met His Gly Phe Gln Gln Ala Tyr Asn Asp
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Leu Met Asp Asp Ile Phe Thr Ala Val Lys Lys Tyr Lys Lys Glu Lys
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Asn Glu Lys Arg Val Thr Val Ile Gly His Ser Leu Gly Ala Ala Met
165 170 175
Gly 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
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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
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Ala Arg Glu Phe Asn Phe Asp Asp His Gln Gly Ile Tyr Phe His Thr
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Gln Ile Gly Ala Val Met Gly Glu Cys Pro Ala Gln Val Gly Ala His
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<210> 2
<211> 912
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgctcttca gtcgctttgt tcttcttgcg ttcggttcgg tggccgccgt ctcggccagc 60
agtatttacg cccgtggccg tggtggtagc tctaccgacc agccagtggc aaacccttac 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 ccgctatggg 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 tgggtgagtg cccagctcag 900
gttggtgctc at 912
<210> 3
<211> 304
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Leu Phe Ser Arg Phe Val Leu Leu Ala Phe Gly Ser Val Ala Ala
1 5 10 15
Val Ser Ala Ser Ser Ile Tyr Ala Arg Gly Arg Cys Gly Ser Ser Thr
20 25 30
Asp Gln Pro Val Ala Asn Pro Tyr Asn Thr Lys Glu Ile Ser Leu Ala
35 40 45
Ala Gly Leu Val Gln Gln Thr Tyr Cys Asp Ser Thr Glu Asn Gly Leu
50 55 60
Lys Ile Gly Asp Ser Glu Leu Leu Tyr Thr Met Gly Glu Gly Tyr Ala
65 70 75 80
Arg Gln Arg Val Asn Ile Tyr His Ser Pro Ser Leu Gly Ile Ala Val
85 90 95
Ala Ile Glu Gly Thr Asn Leu Phe Ser Leu Asn Ser Asp Leu His Asp
100 105 110
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 Met
165 170 175
Gly 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 Cys 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 Met Gly Glu Cys Pro Ala Gln Val Gly Ala His
290 295 300
<210> 4
<211> 912
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgctcttca gtcgctttgt tcttcttgcg ttcggttcgg tggccgccgt ctcggccagc 60
agtatttacg cccgtggccg ttgtggtagc tctaccgacc agccagtggc aaacccttac 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 ccgctatggg tttgctttgc 540
gctatggaca ttgagctgcg tatggatggt ggtctgtaca agacgtacct gtttggactt 600
ccccgtcttg gtaactgcac 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 tgggtgagtg cccagctcag 900
gttggtgctc at 912
<210> 5
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
taaccctctc ccatgcagta aaggagctcg ctgtc 35
<210> 6
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gacagcgagc tcctttactg catgggagag ggtta 35
<210> 7
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aggcgagtga tagatgcaga cacgctgacg agcg 34
<210> 8
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgctcgtcag cgtgtctgca tctatcactc gcct 34
<210> 9
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgtacccttc gggtagcact ggatgtaacg ctc 33
<210> 10
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gagcgttaca tccagtgcta cccgaagggt aca 33
<210> 11
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gtacagacca ccatccatac ggcactcaat gtccatagcg caaag 45
<210> 12
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ctttgcgcta tggacattga gtgccgtatg gatggtggtc tgtac 45
<210> 13
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggtgttgtaa gggcatgcca ctggctggtc ggtag 35
<210> 14
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ctaccgacca gccagtggca tgcccttaca acacc 35
<210> 15
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggccagggta aagcttgcag ctcgtgctgt tgcct 35
<210> 16
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aggcaacagc acgagctgca agctttaccc tggcc 35
<210> 17
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gtagagctac cacaacggcc acgggcg 27
<210> 18
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cgcccgtggc cgttgtggta gctctac 27
<210> 19
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tcaacgaacg aagcaaatgt gcagttacca agacggggaa gtcc 44
<210> 20
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ggacttcccc gtcttggtaa ctgcacattt gcttcgttcg ttga 44
<210> 21
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agccagagaa atctctttgc agttgtaagg gtttgccact 40
<210> 22
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agtggcaaac ccttacaact gcaaagagat ttctctggct 40
<210> 23
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tctgggtgtg gcagtagata ccttggtggt cgt 33
<210> 24
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
acgaccacca aggtatctac tgccacaccc aga 33
<210> 25
<211> 304
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Leu Phe Ser Arg Phe Val Leu Leu Ala Phe Gly Ser Val Ala Ala
1 5 10 15
Val Ser Ala Ser Ser Ile Tyr Ala Arg Gly Arg Gly Gly Ser Ser Thr
20 25 30
Asp Pro Pro Val Pro Asn Pro Tyr Asn Thr Lys Glu Ile Ser Leu Ala
35 40 45
Ala Gly Leu Val Gln Gln Thr Tyr Cys Asp Ser Thr Glu Asn Gly Leu
50 55 60
Lys Ile Gly Asp Ser Glu Leu Leu Tyr Thr Met Gly Glu Gly Tyr Ala
65 70 75 80
Arg Gln Arg Val Asn Ile Tyr His Ser Pro Ser Leu Gly Ile Ala Val
85 90 95
Ala Ile Glu Gly Thr Asn Leu Phe Ser Leu Asn Ser Asp Leu His Asp
100 105 110
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突变体G28C-P206C，其特征是，其氨基酸构成包括如SEQ ID NO.3所示序列；或其编码基因包括如SEQ ID NO.4所示序列或其反向互补序列。
2.一种甘油酯脂肪酶突变体G28C-P206C，其特征是，其氨基酸构成包括如SEQ ID NO.3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。
3.一种编码权利要求1或2所述甘油酯脂肪酶突变体G28C-P206C的核苷酸序列。
4.根据权利要求3所述的核苷酸序列，其特征是，其如SEQ ID NO.4或其反向互补序列所示。
5.一种插入有包括权利要求3或4所述的编码甘油酯脂肪酶突变体G28C-P206C的核苷酸序列的重组表达载体。
6.一种转入有权利要求5所述重组表达载体的重组工程菌株。
7.权利要求1或2所述的甘油酯脂肪酶突变体G28C-P206C、和/或权利要求3或4所述的核苷酸序列在油脂制备中的应用。
8.权利要求5所述的重组表达载体、和/或权利要求6所述的重组工程菌株在油脂制备中的应用。
9.根据权利要求7或8所述的应用，其特征是，所述油脂是甘油二酯或甘油单酯。
10.一种制备油脂的方法，其特征是，所用脂肪酶是权利要求1或2所述的甘油酯脂肪酶突变体G28C-P206C，优选地，所述油脂是甘油二酯或甘油单酯。
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| CN112574975B (zh) * | 2020-09-30 | 2022-04-01 | 华南理工大学 | 甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用 |
Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02174676A (ja) * | 1988-12-26 | 1990-07-06 | Amano Pharmaceut Co Ltd | 新規リパーゼ |
| JPH0670764A (ja) * | 1992-08-28 | 1994-03-15 | Daiwa Kasei Kk | 新規モノグリセリドリパーゼ |
| CN101052702A (zh) * | 2004-07-16 | 2007-10-10 | 丹尼斯科公司 | 脂肪分解酶及其在食品工业中的应用 |
| WO2011033039A1 (en) * | 2009-09-16 | 2011-03-24 | Basf Se | An enzymatically catalyzed method of preparing mono-acylated polyols |
| CN102839164A (zh) * | 2012-09-06 | 2012-12-26 | 江南大学 | 基于二硫键强化折叠的热稳定性脂肪酶突变体及其构建方法 |
| CN102965404A (zh) * | 2012-11-13 | 2013-03-13 | 华南理工大学 | 一种高纯度甘油二酯的制备方法 |
| CN103361326A (zh) * | 2013-07-08 | 2013-10-23 | 华南理工大学 | 一种耐热性提高的偏甘油酯脂肪酶突变体及突变质粒、重组菌株和制备方法 |
| 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 |
| CN103966187A (zh) * | 2014-04-30 | 2014-08-06 | 华南理工大学 | 一种海洋微生物来源的低温偏甘油酯脂肪酶及其应用 |
| CN104688744A (zh) * | 2015-01-29 | 2015-06-10 | 华南理工大学 | 一种球形马拉色菌的偏甘油脂肪酶的抑制剂和应用 |
| CN106566658A (zh) * | 2016-10-10 | 2017-04-19 | 华南理工大学 | 一种高酸价油脂的酶法脱酸方法 |
| CN106906194A (zh) * | 2017-03-09 | 2017-06-30 | 华南理工大学 | 一种偏甘油酯脂肪酶及富含pufa的油脂的酶法脱酸方法 |
| CN107254494A (zh) * | 2017-06-22 | 2017-10-17 | 华南理工大学 | 一种甘油酯型pufa的制备方法 |
| CN108239667A (zh) * | 2016-12-27 | 2018-07-03 | 丰益(上海)生物技术研发中心有限公司 | 一种磷脂酶c活力的检测方法 |
| CN110904073A (zh) * | 2019-05-31 | 2020-03-24 | 江南大学 | 一种脂肪酶突变体及其在去污方面的应用 |
| CN111328287A (zh) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | 新型核酸分子 |
| CN112322670A (zh) * | 2020-11-06 | 2021-02-05 | 华南理工大学 | 甘油二酯的合成方法 |
| CN112592909A (zh) * | 2020-09-30 | 2021-04-02 | 广州永华特医营养科技有限公司 | 一种甘油酯脂肪酶smg1突变体及其编码基因与应用 |
| CN112592907A (zh) * | 2019-06-17 | 2021-04-02 | 云南师范大学 | 一种催化活性提高的脂肪酶突变体d259y及其应用 |
| CN113005111A (zh) * | 2019-12-20 | 2021-06-22 | 丰益(上海)生物技术研发中心有限公司 | 新型的甘油单-二酰酯脂肪酶 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112574975B (zh) * | 2020-09-30 | 2022-04-01 | 华南理工大学 | 甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用 |
-
2021
- 2021-01-04 CN CN202110004098.8A patent/CN112574975B/zh active Active
Patent Citations (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02174676A (ja) * | 1988-12-26 | 1990-07-06 | Amano Pharmaceut Co Ltd | 新規リパーゼ |
| JPH0670764A (ja) * | 1992-08-28 | 1994-03-15 | Daiwa Kasei Kk | 新規モノグリセリドリパーゼ |
| CN101052702A (zh) * | 2004-07-16 | 2007-10-10 | 丹尼斯科公司 | 脂肪分解酶及其在食品工业中的应用 |
| CN101818177A (zh) * | 2004-07-16 | 2010-09-01 | 丹尼斯科公司 | 脂肪分解酶及其在食品工业中的应用 |
| WO2011033039A1 (en) * | 2009-09-16 | 2011-03-24 | Basf Se | An enzymatically catalyzed method of preparing mono-acylated polyols |
| CN102839164A (zh) * | 2012-09-06 | 2012-12-26 | 江南大学 | 基于二硫键强化折叠的热稳定性脂肪酶突变体及其构建方法 |
| CN102965404A (zh) * | 2012-11-13 | 2013-03-13 | 华南理工大学 | 一种高纯度甘油二酯的制备方法 |
| CN103361326A (zh) * | 2013-07-08 | 2013-10-23 | 华南理工大学 | 一种耐热性提高的偏甘油酯脂肪酶突变体及突变质粒、重组菌株和制备方法 |
| 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 |
| CN103966187A (zh) * | 2014-04-30 | 2014-08-06 | 华南理工大学 | 一种海洋微生物来源的低温偏甘油酯脂肪酶及其应用 |
| CN104688744A (zh) * | 2015-01-29 | 2015-06-10 | 华南理工大学 | 一种球形马拉色菌的偏甘油脂肪酶的抑制剂和应用 |
| CN106566658A (zh) * | 2016-10-10 | 2017-04-19 | 华南理工大学 | 一种高酸价油脂的酶法脱酸方法 |
| CN108239667A (zh) * | 2016-12-27 | 2018-07-03 | 丰益(上海)生物技术研发中心有限公司 | 一种磷脂酶c活力的检测方法 |
| CN106906194A (zh) * | 2017-03-09 | 2017-06-30 | 华南理工大学 | 一种偏甘油酯脂肪酶及富含pufa的油脂的酶法脱酸方法 |
| US20210130860A1 (en) * | 2017-03-09 | 2021-05-06 | South China University Of Technology | Partial glyceride lipase and method for enzymatic deacidification of pufa-rich lipid |
| CN107254494A (zh) * | 2017-06-22 | 2017-10-17 | 华南理工大学 | 一种甘油酯型pufa的制备方法 |
| CN111328287A (zh) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | 新型核酸分子 |
| CN110904073A (zh) * | 2019-05-31 | 2020-03-24 | 江南大学 | 一种脂肪酶突变体及其在去污方面的应用 |
| CN112592907A (zh) * | 2019-06-17 | 2021-04-02 | 云南师范大学 | 一种催化活性提高的脂肪酶突变体d259y及其应用 |
| CN112608912A (zh) * | 2019-06-17 | 2021-04-06 | 云南师范大学 | 一种催化活性提高的脂肪酶突变体d170a及其应用 |
| CN113005111A (zh) * | 2019-12-20 | 2021-06-22 | 丰益(上海)生物技术研发中心有限公司 | 新型的甘油单-二酰酯脂肪酶 |
| CN112592909A (zh) * | 2020-09-30 | 2021-04-02 | 广州永华特医营养科技有限公司 | 一种甘油酯脂肪酶smg1突变体及其编码基因与应用 |
| CN112322670A (zh) * | 2020-11-06 | 2021-02-05 | 华南理工大学 | 甘油二酯的合成方法 |
Non-Patent Citations (7)
| Title |
|---|
| TINGTING XU等: ""Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity"", 《JOURNAL OF STRUCTURAL BIOLOGY》 * |
| XU,J.等: ""LIP1, secretory lipase (family 3) [Malassezia globosa CBS 7966]"", 《GENBANK DATABASE》 * |
| XU,J.等: ""Malassezia globosa CBS 7966 LIP1, secretory lipase (family 3) partial mRNA"", 《GENBANK DATABASE》 * |
| 刘晓慧等: ""偏甘油酯脂肪酶PrLip的重组表达及酶学性质表征"", 《食品科学技术学报》 * |
| 姜占宝等: ""定点突变引入二硫键提高华根霉脂肪酶热稳定性的研究"", 《工业微生物》 * |
| 徐碧林等: ""微生物脂肪酶稳定性研究进展"", 《微生物学通报》 * |
| 高重亮: ""球形马拉色菌脂肪酶SMG1的定点突变研究及生物信息学分析"", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112574975B (zh) * | 2020-09-30 | 2022-04-01 | 华南理工大学 | 甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用 |
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