CN102286593B - 一种提高八氢番茄红素脱氢酶体外反应速率的方法 - Google Patents
一种提高八氢番茄红素脱氢酶体外反应速率的方法 Download PDFInfo
- Publication number
- CN102286593B CN102286593B CN 201110190940 CN201110190940A CN102286593B CN 102286593 B CN102286593 B CN 102286593B CN 201110190940 CN201110190940 CN 201110190940 CN 201110190940 A CN201110190940 A CN 201110190940A CN 102286593 B CN102286593 B CN 102286593B
- Authority
- CN
- China
- Prior art keywords
- reaction
- phytoene
- phytoene dehydrogenase
- product
- petroleum ether
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 53
- 108010001545 phytoene dehydrogenase Proteins 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000000338 in vitro Methods 0.000 title abstract description 18
- YVLPJIGOMTXXLP-UHFFFAOYSA-N 15-cis-phytoene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-N 0.000 claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000000047 product Substances 0.000 claims abstract description 19
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 16
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 claims abstract description 13
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 claims abstract description 13
- 235000011765 phytoene Nutrition 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
- 229940088598 enzyme Drugs 0.000 claims abstract description 12
- 239000003208 petroleum Substances 0.000 claims abstract description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 8
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 8
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 8
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 6
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 6
- 238000009835 boiling Methods 0.000 claims abstract description 3
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 3
- 241000588724 Escherichia coli Species 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 102000016938 Catalase Human genes 0.000 claims description 7
- 108010053835 Catalase Proteins 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 4
- 230000004151 fermentation Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims 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 claims description 2
- 238000013016 damping Methods 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 2
- 210000001082 somatic cell Anatomy 0.000 claims 2
- 230000000844 anti-bacterial effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 claims 1
- 239000012467 final product Substances 0.000 claims 1
- 230000010355 oscillation Effects 0.000 claims 1
- 230000003534 oscillatory effect Effects 0.000 claims 1
- 238000005215 recombination Methods 0.000 claims 1
- 230000006798 recombination Effects 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 16
- 230000035484 reaction time Effects 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000006911 enzymatic reaction Methods 0.000 abstract description 4
- 239000000203 mixture Substances 0.000 abstract description 3
- 230000003647 oxidation Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 239000006185 dispersion Substances 0.000 abstract description 2
- 238000004945 emulsification Methods 0.000 abstract description 2
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 11
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 description 11
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 11
- 229960004999 lycopene Drugs 0.000 description 11
- 235000012661 lycopene Nutrition 0.000 description 11
- 239000001751 lycopene Substances 0.000 description 11
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 description 11
- 235000021466 carotenoid Nutrition 0.000 description 6
- 150000001747 carotenoids Chemical class 0.000 description 6
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940083466 soybean lecithin Drugs 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 241000191025 Rhodobacter Species 0.000 description 2
- 241000191043 Rhodobacter sphaeroides Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- BIWLELKAFXRPDE-UHFFFAOYSA-N zeta-Carotene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)CCC=C(C)CCC=C(C)C BIWLELKAFXRPDE-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- VKICVXOZINUTEB-YLKQRWOGSA-N Bisdehydrolycopene/ Tetradehydrolycopene Chemical compound CC(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(C)C VKICVXOZINUTEB-YLKQRWOGSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000192091 Deinococcus radiodurans Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010066605 Geranylgeranyl-Diphosphate Geranylgeranyltransferase Proteins 0.000 description 1
- YTZIWAULTIDEEY-UHFFFAOYSA-N Isomeres zeta-Carotin Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)C=CC=C(C)C=CC=C(C)CCC=C(C)C YTZIWAULTIDEEY-UHFFFAOYSA-N 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000588696 Pantoea ananatis Species 0.000 description 1
- 101710173432 Phytoene synthase Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 101100114901 Streptomyces griseus crtI gene Proteins 0.000 description 1
- JLPULHDHAOZNQI-JLOPVYAASA-N [(2r)-3-hexadecanoyloxy-2-[(9e,12e)-octadeca-9,12-dienoyl]oxypropyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC JLPULHDHAOZNQI-JLOPVYAASA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007697 cis-trans-isomerization reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 101150081158 crtB gene Proteins 0.000 description 1
- 101150000046 crtE gene Proteins 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- BIWLELKAFXRPDE-PCYOLSTGSA-N di-cis-zeta-carotene Natural products CC(C)=CCCC(C)=CCCC(C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(\C)CCC=C(C)CCC=C(C)C BIWLELKAFXRPDE-PCYOLSTGSA-N 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000001782 photodegradation Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- -1 streptoerythrin Chemical compound 0.000 description 1
- 229930195735 unsaturated hydrocarbon Natural products 0.000 description 1
- BIWLELKAFXRPDE-XXKNMTJFSA-N zeta-Carotene Natural products C(=C\C=C\C=C(/C=C/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)\C)(\C=C\C=C(/CC/C=C(\CC/C=C(\C)/C)/C)\C)/C BIWLELKAFXRPDE-XXKNMTJFSA-N 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种提高水-有机两相酶促反应速率的方法，尤其涉及一种提高八氢番茄红素脱氢酶体外反应速率的方法，属于生物工程技术领域。它包括如下步骤：(1)取破碎粗酶液，加入八氢番茄红素丙酮溶液、葡萄糖、葡萄糖氧化酶和过氧化氢酶，超声波混匀，28℃～30℃避光密闭振荡反应，然后加甲醇，55℃～60℃温浴15～20分钟，加入沸程30℃～60℃的石油醚振荡萃取反应产物，收集上层石油醚层，经真空旋转蒸发，得产物。本发明利用超声波的均质、分散和乳化等力学效应，对八氢番茄红素脱氢酶体外反应体系进行优化，增大了底物和酶接触的机会，显著提高了酶促反应速率，缩短了反应时间，减少产物的氧化损失，提高了数据的准确度。
Description
技术领域
本发明涉及一种提高水-有机两相酶促反应速率的方法，尤其涉及一种提高八氢番茄红素脱氢酶体外反应速率的方法，属于生物工程技术领域。
发明背景
八氢番茄红素脱氢酶(phytoene desaturase 1.14.99.-)是类胡萝卜素合成代谢的关键酶，能催化无色的八氢番茄红素(phytoene)生成有颜色的类胡萝卜素，该类酶广泛存在于植物和微生物中。根据脱氢的数目不同，八氢番茄红素脱氢酶的产物最多有6种，依次为六氢番茄红素、ζ-胡萝卜素、链孢红素、番茄红素、3，4-二脱氢番茄红素、3，4，3’，4’-四脱氢番茄红素，其中以链孢红素和番茄红素为产物的八氢番茄红素脱氢酶最为常见。自然界天然存在的类胡萝卜素几乎都是以链孢红素或番茄红素为中间产物生成。
番茄红素是一种非常重要的类胡萝卜素，不但是许多类胡萝卜素生物合成的中间体，而且是目前自然界中发现的最强抗氧化剂，具有防癌、抗老化、提高免疫力等生理功能。微生物发酵法生产番茄红素相比于传统的从番茄中提取的方法，具有周期短、不受气候产地限制，成本低的特点，因而受到广泛重视。相应地，八氢番茄红素脱氢酶催化番茄红素的合成反应也成为近年来的研究热点。
虽然近年来关于八氢番茄红素脱氢酶的研究越来越多，但一直没有一个高效快速的酶活测定方法，主要是因为八氢番茄红素脱氢酶体外反应涉及水-有机两相反应，反应速率慢；而且反应底物和产物都是不饱和烃，含有不同数量的共轭双键，稳定性很差，容易发生顺反异构化、氧化和光降解，反应时间过长容易导致产物的减少。目前主要是通过在反应液中加入L-α-卵磷脂乳浊液和振荡来促进反应进行，但底物八氢番茄红素很难有效与酶接触，反应速率仍然很慢。因此，改进八氢番茄红素脱氢酶体外反应体系，提高两相反应的速率对八氢番茄红素脱氢酶的研究极为重要。
发明内容
本发明针对现有酶体外反应体系反应速率慢的缺陷，提供一种有效提高八氢番茄红素脱氢酶体外反应速率的方法。
本发明的目的通过以下技术方案得以实现：
一种提高番茄红素脱氢酶体外反应速率的方法，包括如下步骤：
(1)取400μL破碎粗酶液，加入八氢番茄红素丙酮溶液、葡萄糖、葡萄糖氧化酶和过氧化氢酶，使终浓度分别为2～20μmol/L(八氢番茄红素)、2mmol/L(葡萄糖)、20U/mL(葡萄糖氧化酶)和20000U/ml(过氧化氢酶)，反应体系用缓冲液定容至500μL，得反应液；
(2)将步骤(1)制得的反应液，在20～23kHz、50～150W超声波混匀10～60秒，28℃～30℃避光密闭振荡反应1～5小时，得反应后溶液；
(3)向步骤(2)制得的反应后溶液中加2.5～3mL甲醇，55℃～60℃温浴15～20分钟，终止反应，加入沸程30℃～60℃的石油醚200～500μL，振荡萃取反应产物，收集上层石油醚层，经真空旋转蒸发，得产物。
所述步骤(1)中破碎粗酶液的制备方法为：将表达八氢番茄红素脱氢酶的重组大肠杆菌发酵液离心，收集菌体细胞，按体积比10～30∶1的比例将发酵液与pH7.9的100mmol/LTris·HCl缓冲液混合重悬菌体细胞，超声波破碎，即得。
上述表达八氢番茄红素脱氢酶的重组大肠杆菌，是通过将球形红细菌(Rhodobactersphearoides)菌种保藏号为ATCC No.17025的八氢番茄红素脱氢酶基因(GenBank No.CP000661)连接到大肠杆菌表达载体pET-22b(Novagen，Germany)上构建得到重组质粒，重组质粒转化大肠杆菌BL21(DE3)感受态细胞得到的。
所述步骤(1)中八氢番茄红素丙酮溶液，是用丙酮在包含质粒pACCRT-EB的重组大肠杆菌BL21(DE3)菌体细胞中提取得到的。具体步骤可参见[Zhenjian Xu，Bing Tian，ZongtaoSun.Jun Lin and Yuejin Hua.Identification and Functional analysis of a phytoenedesaturase gene from the extremely radioresistant bacterium Deinococcus radiodurans.Microbiology，2007，153：1642-1652]。
上述质粒pACCRT-EB，是通过将菠萝泛菌(Pan toea ananatis)的牻牛儿牻牛儿焦磷酸合成酶基因(crtE)(GenBank No.D90087)和八氢番茄红素合成酶基因(crtB)(GenBank No.D90087)分别通过BamHI-SacI，NdeI-KpnI酶切位点连接到大肠杆菌表达载体pACYCDuet-1(Novagen，Germany)上构建得到。
所述步骤(3)中收集上层石油醚层后，还包括如下检测步骤：
将上层石油醚层采用HPLC检测，色谱柱为TC-C18，流动相甲醇/乙腈的体积比为4/6，流速1mL/分钟，柱温30℃，检测波长474nm。
以上操作步骤、实验条件及试剂如无特别说明，均采用本领域常规操作和常用试剂。
本发明的有益效果如下：
本发明利用超声波的均质、分散和乳化等力学效应，对八氢番茄红素脱氢酶体外反应体系进行优化，增大了底物和酶接触的机会，显著提高了酶促反应速率，缩短了反应时间，减少产物的氧化损失，提高了数据的准确度。
附图说明
图1是本发明所述应用超声波技术提高球形红细菌八氢番茄红素脱氢酶体外反应的产物HPLC图谱；
其中，1、番茄红素；2、链孢红素。
图2是本发明实验例所述没有应用超声波技术的对照体外反应产物HPLC图谱；
其中，1、番茄红素；2、链孢红素。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明，但本发明并不受这些内容所限制。
实施例中HPLC流动相所用化学试剂为色谱纯，其他化学试剂为分析纯。
实施例1：超声波混匀法建立八氢番茄红素脱氢酶体外反应
(1)表达八氢番茄红素脱氢酶的重组大肠杆菌的构建
将球形红细菌(Rhodobacter sphearoides)菌种保藏号为ATCC No.17025的八氢番茄红素脱氢酶基因(GenBank No.CP000661)进行PCR扩增，通过NdeI和SalI酶切位点连接到大肠杆菌表达载体pET-22b(Novagen，Germany)上构建得到重组质粒，重组质粒转化大肠杆菌BL21(DE3)感受态细胞得到表达八氢番茄红素脱氢酶的重组大肠杆菌；
(2)细胞破碎液的制备
将步骤(1)制得的200mL表达八氢番茄红素脱氢酶的重组大肠杆菌培养液12000rpm离心5分钟，收集菌体细胞；将收集到的大肠杆菌细胞重悬于10mL、pH7.9的100mmol/LTris·HCl缓冲液中，超声波破碎仪(Sonics，USA)破碎30分钟(22kHZ，150W)；
(3)八氢番茄红素脱氢酶体外反应
取细胞破碎液400μL于1.5mL离心管中，加入八氢番茄红素丙酮溶液、葡萄糖、葡萄糖氧化酶和过氧化氢酶，使终浓度分别为10μmol/L(八氢番茄红素)、2mmol/L(葡萄糖)、20U/mL(葡萄糖氧化酶)和20000U/ml(过氧化氢酶)，反应体系用缓冲液定容至500μL；用超声波破碎仪(Sonics，USA)混匀30秒(22kHZ，100W)；30℃避光密闭振荡反应1小时；加2.5mL甲醇55℃温浴15分钟终止反应，加入300μL石油醚，振荡萃取反应产物，收集上层石油醚层；反应产物采用HPLC检测(Agilent，USA)，色谱柱为TC-C18(Agilent，USA)，流动相甲醇/乙腈(体积比为4/6)，流速1mL/分钟，柱温30℃，检测波长474nm。
上述涉及到底物八氢番茄红素和产物的所有操作没有特殊说明要求弱光条件下进行。上述球形红细菌八氢番茄红素脱氢酶体外反应主要产物为链孢红素，有少量番茄红素生成(附图1)，体外反应体系中产物总产率为1.520±0.148μg·mL-1·h-1。
实验例1：反应体系不用超声波混匀对照
取实施例中制备的重组大肠杆菌破碎液400μL，加入与实施例中相同量的八氢番茄红素丙酮溶液、葡萄糖、葡萄糖氧化酶和过氧化氢酶，用缓冲液定容至500μL；混匀后30℃避光密闭振荡反应，反应条件、反应时间、反应终止和产物的提取与检测同实施例1。
上述反应体系不用超声波混匀，体外反应体系中主产物为链孢红素，有微量番茄红素生成(附图2)，类胡萝卜素产物总产率0.557±0.143μg·mL-1·h-1。
实验例2：对照反应体系加入大豆卵磷脂乳浊液
取实施例中制备的重组大肠杆菌破碎液400μL，加入与实施例中相同量的八氢番茄红素丙酮溶液、葡萄糖、葡萄糖氧化酶和过氧化氢酶，加入40mg/mL乳化大豆卵磷脂10μL，用缓冲液定容至500μL；30℃避光密闭振荡反应，反应条件、反应时间、反应终止和产物的提取与检测同实施例1。
上述对照反应体系加入大豆卵磷脂乳浊液体外反应，产物总产率0.540±0.225μg·mL-1·h-1，与实验例1相似，在粗酶液中大豆卵磷脂对提高反应速率没有明显效果。
上述乳化卵磷脂配制方法为：称取40mg大豆卵磷脂，加入100mmol/L Tris·HCl(pH7.9)缓冲液1mL，超声波破碎仪(Sonics，USA)乳化1分钟(22kHZ，150W)。
结果分析
经实施例与试验例的实验结果对比见表1。从表中可以看出，超声波的应用大大提高了八氢番茄红素脱氢酶的体外反应速率，1小时内产物量为不采用超声波的2.7倍。
表1
Claims (2)
1.一种提高番茄红素脱氢酶体外反应速率的方法，包括如下步骤：
（1）取400μL破碎粗酶液，加入八氢番茄红素丙酮溶液、葡萄糖、葡萄糖氧化酶和过氧化氢酶，使终浓度分别为10μmol/L、2mmol/L、20U/mL和20000U/ml，反应体系用缓冲液定容至500μL，得反应液；
（2）将步骤（1）制得的反应液，在22kHz、100W超声波混匀30秒， 30℃避光密闭振荡反应1小时，得反应后溶液；
（3）向步骤（2）制得的反应后溶液中加2.5～3 mL甲醇，55℃～60℃温浴15～20分钟，终止反应，加入沸程30℃～60℃的石油醚300μL，振荡萃取反应产物，收集上层石油醚层，经真空旋转蒸发，得产物。
2.如权利要求1所述的方法，其特征在于，所述步骤（1）中破碎粗酶液的制备方法为：将表达八氢番茄红素脱氢酶的重组大肠杆菌发酵液离心，收集菌体细胞，按体积比10～30：1的比例将发酵液与pH7.9的100mmol/L Tris·HCl缓冲液混合重悬菌体细胞，超声波破碎，即得。
3、如权利要求2所述的方法，其特征在于，上述表达八氢番茄红素脱氢酶的重组大肠杆菌，是通过将菌种保藏号为ATCC No.17025的球形红细菌中八氢番茄红素脱氢酶基因连接到大肠杆菌表达载体pET-22b上构建得到重组质粒，重组质粒转化大肠杆菌BL21（DE3）感受态细胞得到的。
4、如权利要求1所述的方法，其特征在于，所述步骤（3）中收集上层石油醚层后，还包括如下检测步骤：
将上层石油醚层采用HPLC检测，色谱柱为TC-C18，流动相甲醇/乙腈的体积比为4/6，流速1mL/分钟，柱温30℃，检测波长474nm。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110190940 CN102286593B (zh) | 2011-07-08 | 2011-07-08 | 一种提高八氢番茄红素脱氢酶体外反应速率的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110190940 CN102286593B (zh) | 2011-07-08 | 2011-07-08 | 一种提高八氢番茄红素脱氢酶体外反应速率的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102286593A CN102286593A (zh) | 2011-12-21 |
| CN102286593B true CN102286593B (zh) | 2013-03-13 |
Family
ID=45333347
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110190940 Expired - Fee Related CN102286593B (zh) | 2011-07-08 | 2011-07-08 | 一种提高八氢番茄红素脱氢酶体外反应速率的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102286593B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218352A (zh) * | 2005-03-18 | 2008-07-09 | 米克罗比亚精确工程公司 | 产油酵母和真菌中类胡萝卜素的产生 |
| CN101432436A (zh) * | 2004-08-16 | 2009-05-13 | 纳幕尔杜邦公司 | 类胡萝卜素羟化酶 |
| CN101979587A (zh) * | 2010-10-14 | 2011-02-23 | 浙江大学 | 少动鞘氨醇单胞菌的八氢番茄红素脱氢酶基因及其应用 |
-
2011
- 2011-07-08 CN CN 201110190940 patent/CN102286593B/zh not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101432436A (zh) * | 2004-08-16 | 2009-05-13 | 纳幕尔杜邦公司 | 类胡萝卜素羟化酶 |
| CN101218352A (zh) * | 2005-03-18 | 2008-07-09 | 米克罗比亚精确工程公司 | 产油酵母和真菌中类胡萝卜素的产生 |
| CN101979587A (zh) * | 2010-10-14 | 2011-02-23 | 浙江大学 | 少动鞘氨醇单胞菌的八氢番茄红素脱氢酶基因及其应用 |
Non-Patent Citations (3)
| Title |
|---|
| Identification and functional analysis of a phytoene desaturase gene from the extremely radioresistant bacterium Deinococcus radiodurans;Zhenjian xu et al;《Microbiology》;20071231;第153卷;全文 * |
| Zhenjian xu et al.Identification and functional analysis of a phytoene desaturase gene from the extremely radioresistant bacterium Deinococcus radiodurans.《Microbiology》.2007,第153卷全文. |
| 薛蕾等.白菜型油菜八氢番茄红素脱氢酶基因（PDS3）的克隆与序列分析.《2009年中国作物学会学术年会》.2009,全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102286593A (zh) | 2011-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Srivastava et al. | Improved production of reducing sugars from rice straw using crude cellulase activated with Fe3O4/Alginate nanocomposite | |
| Zhang et al. | Characterization of a D-psicose 3-epimerase from Dorea sp. CAG317 with an acidic pH optimum and a high specific activity | |
| CN104774889B (zh) | 一种利用果糖基转移酶制备蔗糖-6-乙酸酯的方法 | |
| Gao et al. | The production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest | |
| Zhang et al. | Overexpression of secreted sucrose isomerase in Yarrowia lipolytica and its application in isomaltulose production after immobilization | |
| Mól et al. | β-glucosidase: An overview on immobilization and some aspects of structure, function, applications and cost | |
| Fang et al. | Characterization of a novel β-glucosidase from Gongronella sp. W5 and its application in the hydrolysis of soybean isoflavone glycosides | |
| Ma et al. | Transformation of ginsenoside via deep eutectic solvents based on choline chloride as an enzymatic reaction medium | |
| Martins et al. | Exploring magnetic and imprinted cross-linked enzyme aggregates of rhamnopyranosidase in microbioreactors | |
| Gabardo et al. | Performance of different immobilized‐cell systems to efficiently produce ethanol from whey: fluidized batch, packed‐bed and fluidized continuous bioreactors | |
| Narayanasamy et al. | Biobutanol production from sugarcane bagasse by Clostridium beijerinckii strains | |
| Wang et al. | Preparation of gentiooligosaccharides using Trichoderma viride β-glucosidase | |
| CN105219756A (zh) | 一种l-鼠李树胶糖-1-磷酸醛缩酶在催化合成稀有糖中的应用 | |
| Sharma et al. | Structure and biochemical characterization of glucose tolerant β-1, 4 glucosidase (HtBgl) of family 1 glycoside hydrolase from Hungateiclostridium thermocellum | |
| Zhai et al. | Structure and Function Insight of the α-Glucosidase QsGH13 from Qipengyuania seohaensis sp. SW-135 | |
| Chunhua et al. | Enzyme-catalyzed synthesis of vitamin E succinate using a chemically modified Novozym-435 | |
| Jin et al. | Immobilization of recombinant Escherichia coli cells expressing glucose isomerase using modified diatomite as a carrier for effective production of high fructose corn syrup in packed bed reactor | |
| Zhang et al. | Immobilization of sucrose isomerase from Erwinia sp. with graphene oxide and its application in synthesizing isomaltulose | |
| CN103937765A (zh) | 一种耐热α-葡萄糖苷酶的制备与固定化的方法 | |
| CN102250958A (zh) | 一种体外酶反应生成的类胡萝卜素的快速提取方法 | |
| Aydogan et al. | Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61 | |
| Guo et al. | Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides | |
| CN102286593B (zh) | 一种提高八氢番茄红素脱氢酶体外反应速率的方法 | |
| Kovaleva et al. | Characteristics of inulinases: methods for regulation and stabilization of their activity | |
| Wang et al. | In vivo multienzyme complex coconstruction of N-acetylneuraminic acid lyase and N-acetylglucosamine-2-epimerase for biosynthesis of N-acetylneuraminic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130313 Termination date: 20180708 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |