CN111187792A - 乙基己基甘油的制备方法 - Google Patents
乙基己基甘油的制备方法 Download PDFInfo
- Publication number
- CN111187792A CN111187792A CN201811353198.6A CN201811353198A CN111187792A CN 111187792 A CN111187792 A CN 111187792A CN 201811353198 A CN201811353198 A CN 201811353198A CN 111187792 A CN111187792 A CN 111187792A
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- China
- Prior art keywords
- preparing
- ala
- whole
- ethylhexyl
- gly
- Prior art date
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- ANZUDYZHSVGBRF-UHFFFAOYSA-N 3-ethylnonane-1,2,3-triol Chemical compound CCCCCCC(O)(CC)C(O)CO ANZUDYZHSVGBRF-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims description 30
- 239000003054 catalyst Substances 0.000 claims abstract description 29
- YCUKMYFJDGKQFC-UHFFFAOYSA-N 2-(octan-3-yloxymethyl)oxirane Chemical group CCCCCC(CC)OCC1CO1 YCUKMYFJDGKQFC-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 230000007062 hydrolysis Effects 0.000 claims abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 3
- 230000035484 reaction time Effects 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 36
- 108090000623 proteins and genes Proteins 0.000 claims description 28
- NCZPCONIKBICGS-UHFFFAOYSA-N 3-(2-ethylhexoxy)propane-1,2-diol Chemical compound CCCCC(CC)COCC(O)CO NCZPCONIKBICGS-UHFFFAOYSA-N 0.000 claims description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- 102000005486 Epoxide hydrolase Human genes 0.000 claims description 14
- 108020002908 Epoxide hydrolase Proteins 0.000 claims description 14
- 229940100524 ethylhexylglycerin Drugs 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 3
- 241000186216 Corynebacterium Species 0.000 claims description 3
- 241000588722 Escherichia Species 0.000 claims description 3
- 241000223218 Fusarium Species 0.000 claims description 3
- 241000589516 Pseudomonas Species 0.000 claims description 3
- 241000235070 Saccharomyces Species 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 239000000411 inducer Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 2
- 239000012535 impurity Substances 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 2
- 230000009467 reduction Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 230000006698 induction Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 238000010353 genetic engineering Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000003755 preservative agent Substances 0.000 description 6
- 241000233540 Novosphingobium aromaticivorans Species 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- GZYNMZQXFRWDFH-YTWAJWBKSA-N Thr-Arg-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O GZYNMZQXFRWDFH-YTWAJWBKSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- 108010036413 histidylglycine Proteins 0.000 description 4
- 229910001868 water Inorganic materials 0.000 description 4
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- DKJPOZOEBONHFS-ZLUOBGJFSA-N Ala-Ala-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O DKJPOZOEBONHFS-ZLUOBGJFSA-N 0.000 description 2
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 2
- IFTVANMRTIHKML-WDSKDSINSA-N Ala-Gln-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O IFTVANMRTIHKML-WDSKDSINSA-N 0.000 description 2
- OSRZOHXQCUFIQG-FPMFFAJLSA-N Ala-Phe-Pro Chemical compound C([C@H](NC(=O)[C@@H]([NH3+])C)C(=O)N1[C@H](CCC1)C([O-])=O)C1=CC=CC=C1 OSRZOHXQCUFIQG-FPMFFAJLSA-N 0.000 description 2
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 2
- GMGWOTQMUKYZIE-UBHSHLNASA-N Ala-Pro-Phe Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 GMGWOTQMUKYZIE-UBHSHLNASA-N 0.000 description 2
- KJGNDQCYBNBXDA-GUBZILKMSA-N Arg-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N KJGNDQCYBNBXDA-GUBZILKMSA-N 0.000 description 2
- BHSYMWWMVRPCPA-CYDGBPFRSA-N Arg-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCN=C(N)N BHSYMWWMVRPCPA-CYDGBPFRSA-N 0.000 description 2
- NGYHSXDNNOFHNE-AVGNSLFASA-N Arg-Pro-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O NGYHSXDNNOFHNE-AVGNSLFASA-N 0.000 description 2
- PYDIIVKGTBRIEL-SZMVWBNQSA-N Arg-Trp-Pro Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N1CCC[C@H]1C(O)=O PYDIIVKGTBRIEL-SZMVWBNQSA-N 0.000 description 2
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 2
- VHWNKSJHQFZJTH-FXQIFTODSA-N Asp-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N VHWNKSJHQFZJTH-FXQIFTODSA-N 0.000 description 2
- SPKCGKRUYKMDHP-GUDRVLHUSA-N Asp-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N SPKCGKRUYKMDHP-GUDRVLHUSA-N 0.000 description 2
- OEDJQRXNDRUGEU-SRVKXCTJSA-N Asp-Leu-His Chemical compound N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O OEDJQRXNDRUGEU-SRVKXCTJSA-N 0.000 description 2
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Abstract
本发明公开了一种乙基己基甘油的制备方法，涉及生物催化技术领域，解决了减少杂质，节约成本的问题，其技术方案要点是：全细胞催化剂催化水解的底物是乙基己基缩水甘油醚，底物浓度范围5‑50%(W/V)；用缓冲液为50 mM的磷酸盐缓冲液重悬，pH范围6.0～9.0，加入乙基己基缩水甘油醚；每毫升反应体系中全细胞催化剂的用量为0.3～0.5g；转化反应温度为30～50℃；反应时间为4～8小时，生成乙基己基甘油。
Description
技术领域
本发明涉及生物催化技术领域,更具体地说，它涉及一种乙基己基甘油的制备方法。
背景技术
2-乙基己基甘油（Cas No：70445-33-9）也叫异辛基甘油醚或辛氧基甘油，是一个两性分子，具有润滑、抗菌、保湿和除臭等效果。乙基己基甘油是一种全世界广泛使用的日化产品，用于各种沐浴产品，清洁产品，除臭剂，眼妆，粉底，护发产品和防晒产品等。乙基己基甘油能够在提高润肤剂、保湿剂配方滋润效果的同时增加柔滑的肤感，添加到膏霜体系中，可以提高皮肤对膏霜的吸收效率，同时克服发粘及涂白等肤感上的缺点。乙基己基甘油的抑菌活性可以抑制皮肤表面引发异味的细菌的滋生和繁殖，具有除臭效果。此外，乙基己基甘油与传统的防腐剂如苯氧乙醇等混合使用具有显著的防腐增效功能。这一特性可有效地降低传统防腐剂的加入量，显著降低防腐体系的毒害性，让消费者在使用化妆品的时候更加安全放心。乙基己基甘油已成为绿色防腐剂重要品种，用途广泛，用量巨大。
乙基己基甘油虽然是一种绿色防腐剂，公告号为CN108191614A的中国专利公开了一种制备乙基己基甘油的方法，包括以下步骤：(1)以三氟化硼***溶液作为催化剂，在搅拌条件下，使异辛醇和缩水甘油酯类在室温下搅拌反应，生成中间体；(2)在步骤(1)得到的中间体中加入酸作催化剂，再加入水或者醇类，在常压或者减压条件下反应，使步骤(1)得到的中间体水解或者醇解；(3)将步骤(2)所得产物水洗分液后，蒸馏得到乙基己基甘油。
但该方法反应合成过程中有副反应发生，杂质多。化学合成过程中杂质的存在会导致生产的乙基己基甘油色泽深，有异味等现象产生，有些杂质还引发毒性反应。为了去除这些杂质，需要在后处理过程中增加脱色除杂等步骤，进一步增加了生产成本。
发明内容
本发明的目的是提供一种乙基己基甘油的制备方法，达到减少杂质，节约成本的目的。
本发明的上述技术目的是通过以下技术方案得以实现的：一种乙基己基甘油的制备方法，包括：
（1）全细胞催化剂催化水解的底物是乙基己基缩水甘油醚，底物浓度范围5-50% (W/V)；
（2）用缓冲液为50 mM的磷酸盐缓冲液重悬，pH范围6.0～9.0，加入乙基己基缩水甘油醚；
（3）每毫升反应体系中全细胞催化剂的用量为0.3～0.5g；
（4）转化反应温度为30～50℃；反应时间为4～8小时，生成乙基己基甘油。
通过采用上述技术方案，可以将500g/L乙基己基缩水甘油醚8小时内全部转化乙基己基甘油，且无任何副产物生成，显示了良好的工业利用潜力，达到减少杂质，节约成本的目的。
本发明的另一个目的是提供一种制备如上述所述乙基己基甘油的制备方法中的全细胞催化剂的方法，包括：将高产环氧化物水解酶的基因工程菌经种子培养基37℃过夜活化后，按10% 比例接种到发酵培养基，37℃、200rpm 培养至对数中期，加入诱导剂乳糖至终浓度1mM，25 ～ 30℃、180rpm 诱导培养4 ～ 6 小时左右，离心收集菌体。
通过采用上述技术方案，全细胞催化剂具有高活性和耐受高底物浓度的特点，使得底物转化反应效率高，底物转化率为100%。
本发明进一步设置为，所述种子培养基是常规的LB培养基，配方如下：蛋白胨10g/L、酵母膏5g/L、NaCl 10g/L ；NH3·H2O 调节至pH 7.0，平板培养基为LB 培养基再加入15g/L 琼脂粉，在121℃灭菌20min。
本发明进一步设置为，所述发酵培养基是常规的TB培养基，配方如下：蛋白胨12g/L、酵母膏24g/L、甘油 4ml/L、定容到900mL，同时配制100ml 0.17M的磷酸二氢钾-磷酸氢二钾溶液，115℃灭菌20min，灭完菌两种溶液混合后待用。
本发明的另一个目的是提供一种制备如上述所述的制备全细胞催化剂的方法中高产环氧化物水解酶的基因工程菌的方法，通过基因工程PCR的方法得到Novosphingobium aromaticivorans的NaEH基因，基因产物序列号为ABD26703.1，并在基因两端加上NdeI和BamHI酶切位点，并将基因构建到pET30a中，获得基因的高表达载体pET30NaEH，然后将高表达载体转化入受体菌大肠杆菌BL21(DE3)中，即得到所述的高产环氧化物水解酶的基因工程菌。
通过采用上述技术方案，高产环氧化物水解酶的基因工程菌表达出的目的蛋白能达到全蛋白的20～40％，实现了目的蛋白的高效异源表达。
本发明进一步设置为，所述的供体菌为含有NaEH基因的埃希氏菌属、棒杆菌属、芽孢杆菌属、假单胞菌属、酵母菌属，镰刀霉菌属。
本发明的另一个目的是提供一种如上述所述的高产环氧化物水解酶的基因工程菌，在全细胞转化生产合成乙基己基甘油的方法中的应用。
综上所述，本发明具有以下有益效果：全细胞催化剂催化底物转化率为100%，与目前工业上通过化学法制备乙基己基甘油的方法相比，工艺简单，绿色环保，无副产物生成，可大幅降低酸碱和有机溶剂的使用，生产效率高。
附图说明
图1为NaEH的纯度；
图2为利用工程菌转化浓度为20%的乙基己基缩水甘油醚制备的乙基己基甘油的气相分析图；
图3为利用工程菌转化浓度为30%的乙基己基缩水甘油醚制备的乙基己基甘油的气相分析图；
图4为利用工程菌转化浓度为40%的乙基己基缩水甘油醚制备的乙基己基甘油的气相分析图；
图5为利用工程菌转化浓度为50%的乙基己基缩水甘油醚制备的乙基己基甘油的气相分析图。
具体实施方式
以下结合实施例对本发明作进一步详细说明。
本发明中所使用的试剂和耗材都为市购获得的常规产品。
实施例1：一种制备高产环氧化物水解酶的基因工程菌的方法
根据Novosphingobium aromaticivorans的NaEH基因的序列设计引物：
正向引物：CGCCATATGAATGTTGCGCCTTTCGT，下划线序列为酶切位点NdeI。
反向引物：CCGGATCCgcacatcagggaaaacgcggccc，下划线序列为HindIII酶切位点。基因产物序列号为ABD26703.1。
PCR 得到的基因序列两端带有NdeI、HindIII两个位点，将基因***到pET30a载体中，得到的高表达的基因工程载体质粒命名为pET30NaEH，然后将高表达载体转化入受体菌大肠杆菌BL21(DE3)中，得到高产环氧化物水解酶的基因工程菌。得到的基因工程菌表达出的蛋白在C端带有His-Tag标签蛋白。
此外供体菌为含有NaEH基因的埃希氏菌属、棒杆菌属、芽孢杆菌属、假单胞菌属、酵母菌属，镰刀霉菌属等菌属。
高表达菌株的获得：将制备得到的重组载体用常规方法导入大肠杆菌BL21(DE3)以构建重组酶以可溶形式存在于菌体内的基因工程菌，筛选出组建成功的基因工程菌，其中以大肠杆菌BL21(DE3)为宿主菌的重组菌目的蛋白表达相对较好。以目的蛋白表达量不低于30％的工程菌，作为生产用工程菌菌种，并以甘油菌或牛奶冻干菌种形式保存。
具体转化方法如下：从冰箱中取出100μL 的感受态细胞。细胞在冰上融化2-5分钟。融解后轻弹管壁1-2 次以重悬细胞。将1μL 的pET30NaEH质粒加入感受态细胞中。轻微摇动混和随后将管重置于冰中。冰浴30min。42℃水浴90秒。然后迅速将管转移到冰上放置2分钟，使细胞冷却。向每个离心管中加入500μ L 无菌无抗的LB 培养基，混匀后置于37℃摇床振荡培养45min(150rpm/min)，使菌体复苏。混匀吸取100μL加到含有卡那霉素（Kan 100μg/mL）的LB 固体培养基上，用玻璃刮轻轻涂匀，将平板放置于室温直至液体被吸收，倒置平板，37℃培养12-16 小时。获得高表达的基因工程菌：大肠杆菌BL21(DE3)（pET30NaEH）。
实施例2：实施例1中的高产环氧化物水解酶的基因工程菌，在全细胞转化生产合成乙基己基甘油的方法中的应用。
实施例3：环氧化物水解酶NaEH的表达和纯化
(1) 种子培养基活化挑取培养皿上的单菌落于5mL LB(Kan100μg/mL) 培养基中，37℃、200rpm 过夜活化。
(2) 发酵培养将过夜活化的种子液分别按10%比例接种到800mL LB发酵培养基(kan100g/mL)，37℃、200rpm 培养至对数中期OD600 ＝ 0.6左右。
(3) 诱导培养对数中期时加入乳糖进行诱导，使其终浓度为1mM，25 ～ 30℃、180rpm 诱导培养4 ～ 6 小时。
(4)SDS-PAGE 取将诱导完成的发酵液4mL，12000rpm 离心90sec，加入800μL 去离子水，超声破碎5min。12000rpm 离心10min。取离心后上清20μL 加入5μL 上样缓冲液；离心后沉淀用去离子水洗剂1 次，加入800μL 去离子水后取20μL 加入5μL 上样缓冲液。SDS-PAGE 分离胶浓度为12.5％，每孔上样10μL。
(5) 纯化工作在AKTApurifier 10 仪器中进行，纯化条件如下：用缓冲液A（50mMTris-HCl，0.5M NaCl，5%甘油，20mM咪唑，pH7.5）平衡柱子HisTrap HP-5，将（4）中制备的含酶的上清液上柱，洗脱除去杂蛋白，目标蛋白用缓冲液B（50mM Tris-HCl，0.5M NaCl，5%甘油，0.5M咪唑，pH7.5）洗脱，透析脱盐，酶液经过超滤膜浓缩，保存于-80℃备用。
（6）NaEH的纯度见附图1。
实施例4 ：一种制备全细胞催化剂的方法以及利用全细胞催化剂转化乙基己基缩水甘油醚产生乙基己基甘油实验：
(1) 种子培养基活化挑取培养皿上的单菌落于5mL LB (Kan 100μg/mL) 培养基中，37℃、200rpm 过夜活化。种子培养基是常规的LB培养基，配方如下：蛋白胨10g/L、酵母膏5g/L、NaCl 10g/L ；NH3·H2O 调节至pH 7.0，平板培养基为LB 培养基再加入15g/L 琼脂粉，在121℃灭菌20min。
(2) 发酵培养将过夜活化的种子液分别按10%比例接种到1L TB 发酵培养基(Kan100μg/mL)，37℃、200rpm 培养至对数中期OD600 ＝ 0.6 ～ 1 左右。发酵培养基是常规的TB培养基，配方如下：蛋白胨12g/L、酵母膏24g/L、甘油 4ml/L、定容到900mL，同时配制100ml 0.17M的磷酸二氢钾-磷酸氢二钾溶液，115℃灭菌20min，灭完菌两种溶液混合后待用。
(3) 诱导培养对数中期时加入乳糖进行诱导，使其终浓度为1mM，25 ～ 30℃、180rpm 诱导培养4 ～ 6 小时。8000rpm，10min 离心收菌。
(4) 称取0.6g 菌体，用2mL 50mM PBS缓冲液（pH 7.0）重悬，加入终浓度为50g/L的底物乙基己基缩水甘油醚，转化反应温度为37℃，200rpm 下转化3 小时，生成乙基己基甘油。每毫升反应体系中全细胞催化剂的用量为0.3～0.5g。
实施例5 ：利用全细胞催化剂转化乙基己基缩水甘油醚产生乙基己基甘油实验：
（1）全细胞催化剂的培养及制备同实施例4。
（2）称取0.6g 菌体，用2mL 50mM PBS缓冲液（pH 7.0）重悬，加入终浓度为100g/L的底物乙基己基缩水甘油醚，37℃，200rpm 下转化4 小时，生成乙基己基甘油。
实施例6 ：利用全细胞催化剂转化乙基己基缩水甘油醚产生乙基己基甘油实验：
（1）全细胞催化剂的培养及制备同实施例4。
（2）称取0.6g 菌体，用2mL 50mM PBS缓冲液（pH 7.0）重悬，加入终浓度为200g/L的底物乙基己基缩水甘油醚，37℃，200rpm 下转化8 小时，生成乙基己基甘油。
使用气相色谱分析检测转化率和生成的产品乙基己基甘油的纯度，结果见附图2。
实施例7 ：利用全细胞催化剂转化乙基己基缩水甘油醚产生乙基己基甘油实验：
（1）全细胞催化剂的培养及制备同实施例4。
（2）称取1.0g 菌体，用2mL 50mM PBS缓冲液（pH 7.0）重悬，加入终浓度为300g/L的底物乙基己基缩水甘油醚，37℃，200rpm 下转化6小时，生成乙基己基甘油。
使用气相色谱分析检测转化率和生成的产品乙基己基甘油的纯度，结果见附图3。
实施例8 ：利用全细胞催化剂转化乙基己基缩水甘油醚产生乙基己基甘油实验：
（1）全细胞催化剂的培养及制备同实施例4。
（2）称取1.0g 菌体，用2mL 50mM PBS缓冲液（pH 7.0）重悬，加入终浓度为400g/L的底物乙基己基缩水甘油醚，37℃，200rpm 下转化8 小时，生成乙基己基甘油。
使用气相色谱分析检测转化率和生成的产品乙基己基甘油的纯度，结果见附图4。
实施例9 ：利用全细胞催化剂转化乙基己基缩水甘油醚产生乙基己基甘油实验：
（1）全细胞催化剂的培养及制备同实施例4。
（2）称取1.0g 菌体，用2mL 50mM PBS缓冲液（pH 7.0）重悬，加入终浓度为500g/L的底物乙基己基缩水甘油醚，37℃，200rpm 下转化8 小时，生成乙基己基甘油。
使用气相色谱分析检测转化率和生成的产品乙基己基甘油的纯度，结果见附图5。
所述气相色谱分析所用的色谱柱为Agilent HP-5（0.2mm×30m），起始温度100℃，升温速度25℃/min，进样量2μL。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
核苷酸序列表
SEQUENCE LISTING
<110> 南京盛德生物科技研究院有限公司
<120> 乙基己基甘油的制备方法
<130> 000
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1134
<212> DNA
<213> Novosphingobium aromaticivorans
<400> 1
atgaatgttg cgcctttcgt tgtcgacata cctcgcggcg agatcgagga cctgcatcgt 60
cgcatcgaca tgacacgctg gcccgagaaa gagacggtcg acgactggtc gcagggcacg 120
ccgcttggcg cgttgcagga tttcgtgagc tactggcgcg gcggctatga ctggtacgcg 180
tgccagagga tgctgaacga ctggggcatg ttcgagaccg agatcgacgg agtggcgatc 240
cgcttcctcc acgtgcgctc tgcgcaagca gatgcgcggc ccttgctgct gacccacggc 300
gggccgggat cgattctcga gttccgccgc tgcatcgcgc cgctgacccg cccagaggag 360
catggtggta ctgccgccga cgcgttccat ctcgtgatcc cttgcctgcc gggatacggc 420
ttttccggaa agcccacgcg caagggctgg agcgtgcaga agatcgcgca ggcctggggc 480
gaactgatga agcggctggg ctacgaaagc tggcttgcac agggcggggg ctggcgttcc 540
gccgttacca ctgccatcgg ggcgctgaag gtggagggct gcgcgggcat ccatctcaac 600
atgccgatcg cccggcccct gccggaggac ctggccgctc cgacgccgga ggagctaagg 660
gcgctgaccg cgctacagca ctatcaggat tgggattcgg ggtattccaa ggagcaggcg 720
acccggcccc agacggtagg ttacgggctg gtcgattcgc cggtcgggct ggctggctgg 780
atctacgaga agatgtgggc ctggaccgac aacgagggcg cgcccgagga cgcgctgagc 840
cgcgacgaca tgctcgacaa catcatgctg tactggctga cggcggcagg ggcatcgtcg 900
gcccggcttt attgggagag ttttgcgagc ttcgggccat cgcagatcga cattccggcc 960
gcggccagcg cctttccgaa ggagataatt cccgcgccgc gcaagtggtt cgagcgcaac 1020
tgttcgaagc tggtctactg gggcgagctg gaaaagggcg gccactttgc cgcgtgggag 1080
cagcccgaag ccttcgtgaa ggaacttcgg gccgcgtttt ccctgatgtg ctag 1134
<210> 2
<211> 377
<212> PRT
<213> Novosphingobium aromaticivorans
<400> 2
Met Asn Val Ala Pro Phe Val Val Asp Ile Pro Arg Gly Glu Ile Glu
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Asp Leu His Arg Arg Ile Asp Met Thr Arg Trp Pro Glu Lys Glu Thr
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Val Asp Asp Trp Ser Gln Gly Thr Pro Leu Gly Ala Leu Gln Asp Phe
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Val Ser Tyr Trp Arg Gly Gly Tyr Asp Trp Tyr Ala Cys Gln Arg Met
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Leu Asn Asp Trp Gly Met Phe Glu Thr Glu Ile Asp Gly Val Ala Ile
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Arg Phe Leu His Val Arg Ser Ala Gln Ala Asp Ala Arg Pro Leu Leu
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Leu Thr His Gly Gly Pro Gly Ser Ile Leu Glu Phe Arg Arg Cys Ile
100 105 110
Ala Pro Leu Thr Arg Pro Glu Glu His Gly Gly Thr Ala Ala Asp Ala
115 120 125
Phe His Leu Val Ile Pro Cys Leu Pro Gly Tyr Gly Phe Ser Gly Lys
130 135 140
Pro Thr Arg Lys Gly Trp Ser Val Gln Lys Ile Ala Gln Ala Trp Gly
145 150 155 160
Glu Leu Met Lys Arg Leu Gly Tyr Glu Ser Trp Leu Ala Gln Gly Gly
165 170 175
Gly Trp Arg Ser Ala Val Thr Thr Ala Ile Gly Ala Leu Lys Val Glu
180 185 190
Gly Cys Ala Gly Ile His Leu Asn Met Pro Ile Ala Arg Pro Leu Pro
195 200 205
Glu Asp Leu Ala Ala Pro Thr Pro Glu Glu Leu Arg Ala Leu Thr Ala
210 215 220
Leu Gln His Tyr Gln Asp Trp Asp Ser Gly Tyr Ser Lys Glu Gln Ala
225 230 235 240
Thr Arg Pro Gln Thr Val Gly Tyr Gly Leu Val Asp Ser Pro Val Gly
245 250 255
Leu Ala Gly Trp Ile Tyr Glu Lys Met Trp Ala Trp Thr Asp Asn Glu
260 265 270
Gly Ala Pro Glu Asp Ala Leu Ser Arg Asp Asp Met Leu Asp Asn Ile
275 280 285
Met Leu Tyr Trp Leu Thr Ala Ala Gly Ala Ser Ser Ala Arg Leu Tyr
290 295 300
Trp Glu Ser Phe Ala Ser Phe Gly Pro Ser Gln Ile Asp Ile Pro Ala
305 310 315 320
Ala Ala Ser Ala Phe Pro Lys Glu Ile Ile Pro Ala Pro Arg Lys Trp
325 330 335
Phe Glu Arg Asn Cys Ser Lys Leu Val Tyr Trp Gly Glu Leu Glu Lys
340 345 350
Gly Gly His Phe Ala Ala Trp Glu Gln Pro Glu Ala Phe Val Lys Glu
355 360 365
Leu Arg Ala Ala Phe Ser Leu Met Cys
370 375
核苷酸序列表
SEQUENCE LISTING
<110> 南京盛德生物科技研究院有限公司
<120> 乙基己基甘油的制备方法
<130> 000
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1134
<212> DNA
<213> Novosphingobium aromaticivorans
<400> 1
atgaatgttg cgcctttcgt tgtcgacata cctcgcggcg agatcgagga cctgcatcgt 60
cgcatcgaca tgacacgctg gcccgagaaa gagacggtcg acgactggtc gcagggcacg 120
ccgcttggcg cgttgcagga tttcgtgagc tactggcgcg gcggctatga ctggtacgcg 180
tgccagagga tgctgaacga ctggggcatg ttcgagaccg agatcgacgg agtggcgatc 240
cgcttcctcc acgtgcgctc tgcgcaagca gatgcgcggc ccttgctgct gacccacggc 300
gggccgggat cgattctcga gttccgccgc tgcatcgcgc cgctgacccg cccagaggag 360
catggtggta ctgccgccga cgcgttccat ctcgtgatcc cttgcctgcc gggatacggc 420
ttttccggaa agcccacgcg caagggctgg agcgtgcaga agatcgcgca ggcctggggc 480
gaactgatga agcggctggg ctacgaaagc tggcttgcac agggcggggg ctggcgttcc 540
gccgttacca ctgccatcgg ggcgctgaag gtggagggct gcgcgggcat ccatctcaac 600
atgccgatcg cccggcccct gccggaggac ctggccgctc cgacgccgga ggagctaagg 660
gcgctgaccg cgctacagca ctatcaggat tgggattcgg ggtattccaa ggagcaggcg 720
acccggcccc agacggtagg ttacgggctg gtcgattcgc cggtcgggct ggctggctgg 780
atctacgaga agatgtgggc ctggaccgac aacgagggcg cgcccgagga cgcgctgagc 840
cgcgacgaca tgctcgacaa catcatgctg tactggctga cggcggcagg ggcatcgtcg 900
gcccggcttt attgggagag ttttgcgagc ttcgggccat cgcagatcga cattccggcc 960
gcggccagcg cctttccgaa ggagataatt cccgcgccgc gcaagtggtt cgagcgcaac 1020
tgttcgaagc tggtctactg gggcgagctg gaaaagggcg gccactttgc cgcgtgggag 1080
cagcccgaag ccttcgtgaa ggaacttcgg gccgcgtttt ccctgatgtg ctag 1134
<210> 2
<211> 377
<212> PRT
<213> Novosphingobium aromaticivorans
<400> 2
Met Asn Val Ala Pro Phe Val Val Asp Ile Pro Arg Gly Glu Ile Glu
1 5 10 15
Asp Leu His Arg Arg Ile Asp Met Thr Arg Trp Pro Glu Lys Glu Thr
20 25 30
Val Asp Asp Trp Ser Gln Gly Thr Pro Leu Gly Ala Leu Gln Asp Phe
35 40 45
Val Ser Tyr Trp Arg Gly Gly Tyr Asp Trp Tyr Ala Cys Gln Arg Met
50 55 60
Leu Asn Asp Trp Gly Met Phe Glu Thr Glu Ile Asp Gly Val Ala Ile
65 70 75 80
Arg Phe Leu His Val Arg Ser Ala Gln Ala Asp Ala Arg Pro Leu Leu
85 90 95
Leu Thr His Gly Gly Pro Gly Ser Ile Leu Glu Phe Arg Arg Cys Ile
100 105 110
Ala Pro Leu Thr Arg Pro Glu Glu His Gly Gly Thr Ala Ala Asp Ala
115 120 125
Phe His Leu Val Ile Pro Cys Leu Pro Gly Tyr Gly Phe Ser Gly Lys
130 135 140
Pro Thr Arg Lys Gly Trp Ser Val Gln Lys Ile Ala Gln Ala Trp Gly
145 150 155 160
Glu Leu Met Lys Arg Leu Gly Tyr Glu Ser Trp Leu Ala Gln Gly Gly
165 170 175
Gly Trp Arg Ser Ala Val Thr Thr Ala Ile Gly Ala Leu Lys Val Glu
180 185 190
Gly Cys Ala Gly Ile His Leu Asn Met Pro Ile Ala Arg Pro Leu Pro
195 200 205
Glu Asp Leu Ala Ala Pro Thr Pro Glu Glu Leu Arg Ala Leu Thr Ala
210 215 220
Leu Gln His Tyr Gln Asp Trp Asp Ser Gly Tyr Ser Lys Glu Gln Ala
225 230 235 240
Thr Arg Pro Gln Thr Val Gly Tyr Gly Leu Val Asp Ser Pro Val Gly
245 250 255
Leu Ala Gly Trp Ile Tyr Glu Lys Met Trp Ala Trp Thr Asp Asn Glu
260 265 270
Gly Ala Pro Glu Asp Ala Leu Ser Arg Asp Asp Met Leu Asp Asn Ile
275 280 285
Met Leu Tyr Trp Leu Thr Ala Ala Gly Ala Ser Ser Ala Arg Leu Tyr
290 295 300
Trp Glu Ser Phe Ala Ser Phe Gly Pro Ser Gln Ile Asp Ile Pro Ala
305 310 315 320
Ala Ala Ser Ala Phe Pro Lys Glu Ile Ile Pro Ala Pro Arg Lys Trp
325 330 335
Phe Glu Arg Asn Cys Ser Lys Leu Val Tyr Trp Gly Glu Leu Glu Lys
340 345 350
Gly Gly His Phe Ala Ala Trp Glu Gln Pro Glu Ala Phe Val Lys Glu
355 360 365
Leu Arg Ala Ala Phe Ser Leu Met Cys
370 375
Claims (7)
1.一种乙基己基甘油的制备方法，其特征在于，包括：
（1）全细胞催化剂催化水解的底物是乙基己基缩水甘油醚，底物浓度范围5-50% (W/V)；
（2）用缓冲液为50 mM的磷酸盐缓冲液重悬，pH范围6.0～9.0，加入乙基己基缩水甘油醚；
（3）每毫升反应体系中全细胞催化剂的用量为0.3～0.5g；
（4）转化反应温度为30～50℃；反应时间为4～8小时，生成乙基己基甘油。
2.一种制备如权利要求1中所述乙基己基甘油的制备方法中的全细胞催化剂的方法，其特征在于，包括：将高产环氧化物水解酶的基因工程菌经种子培养基37℃过夜活化后，按10% 比例接种到发酵培养基，37℃、200rpm 培养至对数中期，加入诱导剂乳糖至终浓度1mM，25 ～ 30℃、180rpm 诱导培养4 ～ 6 小时左右，离心收集菌体。
3.根据权利要求2所述的制备全细胞催化剂的方法，其特征在于，所述种子培养基是常规的LB培养基，配方如下：蛋白胨10g/L、酵母膏5g/L、NaCl 10g/L ；NH3·H2O 调节至pH7.0，平板培养基为LB 培养基再加入15g/L 琼脂粉，在121℃灭菌20min。
4.根据权利要求2所述的制备全细胞催化剂的方法，其特征在于，所述发酵培养基是常规的TB培养基，配方如下：蛋白胨12g/L、酵母膏24g/L、甘油 4ml/L、定容到900mL，同时配制100ml 0.17M的磷酸二氢钾-磷酸氢二钾溶液，115℃灭菌20min，灭完菌两种溶液混合后待用。
5.一种制备如权利要求2中所述的制备全细胞催化剂的方法中高产环氧化物水解酶的基因工程菌的方法，其特征在于，通过基因工程PCR的方法得到Novosphingobium aromaticivorans的NaEH基因，基因产物序列号为ABD26703.1，并在基因两端加上NdeI和BamHI酶切位点，并将基因构建到pET30a中，获得基因的高表达载体pET30NaEH，然后将高表达载体转化入受体菌大肠杆菌BL21(DE3)中，即得到所述的高产环氧化物水解酶的基因工程菌。
6.根据权利要求5所述的制备高产环氧化物水解酶的基因工程菌的方法，其特征在于，所述的供体菌为含有NaEH基因的埃希氏菌属、棒杆菌属、芽孢杆菌属、假单胞菌属、酵母菌属，镰刀霉菌属。
7.一种如权利要求5-6中所述的高产环氧化物水解酶的基因工程菌，在全细胞转化生产合成乙基己基甘油的方法中的应用。
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| WO2024000620A1 (zh) * | 2022-06-27 | 2024-01-04 | 南京华狮新材料有限公司 | 一种固定化酶及其用于制备乙基己基甘油的用途及方法 |
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