环氧化物水解酶产生菌的筛选及其发酵条件和对苯基缩水甘油醚类化合物拆分条件的研究
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摘要
手性环氧化物及其水解产物邻二醇是合成许多生物活性物质和手性目标产物的高价值中间体。
本实验以邻甲氧基苯基缩水甘油醚为唯一碳源进行多轮富集,从土壤中获得380多株能利用该环氧化物的微生物。之后用TLC法定性筛选出25株具有环氧化物水解酶活性的菌株,之后再通过HPLC法筛选出5株具有立体选择性的菌株,从中选取最好的一个菌株进行下一步研究并进行菌株鉴定,经过形态观察及ITS基因序列分析,确定该菌株为Botryosphaeria dothidea,编号为ZJUZQ007。
以单因素方法,对发酵培养基的配方及发酵条件进行了优化,考察了不同的碳源、氮源、金属离子及浓度、发酵培养基初始pH值、菌体培养温度以及底物诱导对菌株的生长及酶活的影响。最终确立了菌株Botryosphaeria d.ZJUZQ007的最优发酵培养基配方:以2%乳糖为碳源,2%玉米粉为氮源,金属离子为2mmol/L的Fe2+,1%的NaCl,最适培养条件为30℃,最适pH在4.0-7.0之间,发酵时间为72h,该酶为非诱导型酶,无需加入底物做诱导剂。
对Botryosphaeria d.环氧化物水解酶拆分邻甲氧基苯醚(EMPP)、邻甲基苯醚(o-GMPE)、间甲基苯醚(m-GMPE)、对甲基苯醚(p-GMPE)四种环氧化物的反应条件进行了优化。确定了最佳反应温度为30℃,最适缓冲液pH为7.6及最适反应时间为40min,在此反应条件下,(R)-EMPP的e.e.s和E值分别达到了99.9%和50.4,(R)-o-GMPE则为99.9%和48.6,而(R)-m-GMPE是91.2%和18.1,(S)-p-GMPE则是98.0%和29.5。同时发现,该菌株环氧化物水解酶对底物的选择性及活性受底物苯环上取代基的类型及取代基位置的影响。用分子模拟对接软件(Discovery Studio 2.1),通过模拟酶与底物的结合情况对这个实验结果成功地进行了解释。
Optically pure epoxides and their corresponding vicinal diols are recognized as important building blocks for the synthesis of various bioactive and chiral products.
More than 380 strains which could use 1,2-Epoxy-3-(2-methoxyphenyloxy)-propane(EMPP) as the sole carbon source were obtained from soil samples. Then 25 strains with epoxide hydrolase were screened by TLC and 5 with enantioselective activity by HPCL. A fungi with best enantioselectivity was further studied and identified as Botryosphaeria dothidea based on morphology and ITS sequence. It’s named ZJUZQ007.
Based on single factor experiments, the fermentation conditions for the strain ZJUZQ007 were investigated, including carbon and nitrogen sources, metal ions and the concentrations, the initial pH of fermentation medium, temperature and inducement. The best fermentation conditions for ZJUZQ007 were: lactose 2.0%, corn flour 2.0%, NaCl 1.0%, FeSO4·7H2O 2mmol/L,temperature 30℃, pH 4.0-7.0, time 30h. The epoxide hydrolase is not inducible.
Furthermore, the conditions for kinetic resolution of racemic EMPP and glycidyl (o, m, p)-methylphenyl ethers by ZJUZQ007 were studied, including reaction temperature, pH of potassium phosphate buffer and reaction time. It showed that the best resolution conditions as follows: racemic epoxides (12 mg) in DMSO (1:10 v/v) was hydrolyzed in 10ml KPB (100 mmol/L, pH 7.6) using wet mycelium of ZJUZQ007 (0.8 g) by shaking the mixture (220 rpm) at 30℃for 40 minutes.
Under this optimized conversion conditions, the e.e.s and E value for (R)-EMPP was 99.9% and 50.4, 99.9% and 48.6 for (R)-o-GMPE, 91.2 and 18.1 for (R)-m-GMPE, 98.0% and 29.5 for (S)-p-GMPE.
We found that by using Botryosphaeria dothidea ZJUZQ007, the sort of substituent group had an effect on the enantioselectivity that the e.e.s and E value for (R)-EMPP were higher than those for (R)-o-GMPE. While the position of the methyl group effected not only on the enantioselectivity but also the configuration of the remained epoxides: the (R)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ethers whereas the (S)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained by enzyme-substrate docking studies.
本实验以邻甲氧基苯基缩水甘油醚为唯一碳源进行多轮富集,从土壤中获得380多株能利用该环氧化物的微生物。之后用TLC法定性筛选出25株具有环氧化物水解酶活性的菌株,之后再通过HPLC法筛选出5株具有立体选择性的菌株,从中选取最好的一个菌株进行下一步研究并进行菌株鉴定,经过形态观察及ITS基因序列分析,确定该菌株为Botryosphaeria dothidea,编号为ZJUZQ007。
以单因素方法,对发酵培养基的配方及发酵条件进行了优化,考察了不同的碳源、氮源、金属离子及浓度、发酵培养基初始pH值、菌体培养温度以及底物诱导对菌株的生长及酶活的影响。最终确立了菌株Botryosphaeria d.ZJUZQ007的最优发酵培养基配方:以2%乳糖为碳源,2%玉米粉为氮源,金属离子为2mmol/L的Fe2+,1%的NaCl,最适培养条件为30℃,最适pH在4.0-7.0之间,发酵时间为72h,该酶为非诱导型酶,无需加入底物做诱导剂。
对Botryosphaeria d.环氧化物水解酶拆分邻甲氧基苯醚(EMPP)、邻甲基苯醚(o-GMPE)、间甲基苯醚(m-GMPE)、对甲基苯醚(p-GMPE)四种环氧化物的反应条件进行了优化。确定了最佳反应温度为30℃,最适缓冲液pH为7.6及最适反应时间为40min,在此反应条件下,(R)-EMPP的e.e.s和E值分别达到了99.9%和50.4,(R)-o-GMPE则为99.9%和48.6,而(R)-m-GMPE是91.2%和18.1,(S)-p-GMPE则是98.0%和29.5。同时发现,该菌株环氧化物水解酶对底物的选择性及活性受底物苯环上取代基的类型及取代基位置的影响。用分子模拟对接软件(Discovery Studio 2.1),通过模拟酶与底物的结合情况对这个实验结果成功地进行了解释。
Optically pure epoxides and their corresponding vicinal diols are recognized as important building blocks for the synthesis of various bioactive and chiral products.
More than 380 strains which could use 1,2-Epoxy-3-(2-methoxyphenyloxy)-propane(EMPP) as the sole carbon source were obtained from soil samples. Then 25 strains with epoxide hydrolase were screened by TLC and 5 with enantioselective activity by HPCL. A fungi with best enantioselectivity was further studied and identified as Botryosphaeria dothidea based on morphology and ITS sequence. It’s named ZJUZQ007.
Based on single factor experiments, the fermentation conditions for the strain ZJUZQ007 were investigated, including carbon and nitrogen sources, metal ions and the concentrations, the initial pH of fermentation medium, temperature and inducement. The best fermentation conditions for ZJUZQ007 were: lactose 2.0%, corn flour 2.0%, NaCl 1.0%, FeSO4·7H2O 2mmol/L,temperature 30℃, pH 4.0-7.0, time 30h. The epoxide hydrolase is not inducible.
Furthermore, the conditions for kinetic resolution of racemic EMPP and glycidyl (o, m, p)-methylphenyl ethers by ZJUZQ007 were studied, including reaction temperature, pH of potassium phosphate buffer and reaction time. It showed that the best resolution conditions as follows: racemic epoxides (12 mg) in DMSO (1:10 v/v) was hydrolyzed in 10ml KPB (100 mmol/L, pH 7.6) using wet mycelium of ZJUZQ007 (0.8 g) by shaking the mixture (220 rpm) at 30℃for 40 minutes.
Under this optimized conversion conditions, the e.e.s and E value for (R)-EMPP was 99.9% and 50.4, 99.9% and 48.6 for (R)-o-GMPE, 91.2 and 18.1 for (R)-m-GMPE, 98.0% and 29.5 for (S)-p-GMPE.
We found that by using Botryosphaeria dothidea ZJUZQ007, the sort of substituent group had an effect on the enantioselectivity that the e.e.s and E value for (R)-EMPP were higher than those for (R)-o-GMPE. While the position of the methyl group effected not only on the enantioselectivity but also the configuration of the remained epoxides: the (R)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ethers whereas the (S)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained by enzyme-substrate docking studies.
引文
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[57] http://www.rcsb.org/pbd
[2]黄量,戴立信.手性药物的化学与生物学[M].化学工业出版社,2002,274.
[3]王磊,李叶芝,黄化民,方唯硕.手性催化的环氧化物立体选择性开环反应[J].有机化学2006,26(9):1208-1216.
[4]唐燕发,新型手性环氧化物水解酶的筛选、特性和应用研究,华东理工大学博士学位论文.
[5]朱长生.普萘洛尔的合成与拆分研究[J].湖南工业职业技术学院学报,2003,3(3):18-19.
[6] Iliyas A, Sayyed VV. Asymmetric synthesis of aryloxypropanolamines via OsO4-catalyzed asymmetric dihydroxylation. Tetrahedron. 2005,61:2831–2838
[7]崔艳霞,姜卫国,李淑芬.阿替洛尔的合成[J].中国药物化学杂志,1996,6(1):62-63.
[8]王世玉,王普善.合成药物与中间体手册[M].北京:化学工业出版社, 2004.293-294
[9]朱宝泉,李安良.新编药物合成手册[M].北京:化学工业出版社, 2003.8
[10] Alexander AB, Elena IS, Dmitry VZ, et al. Solid state properties of 1,2-epoxide-3-(2-methoxyphenyloxy)-prepane-valuable intermediate in non-racemic drug synthesis[J]. Tetrahedron: Asymmetry. 2005,16:3361-3366.
[11] Bisi A, Rampa A, Budriesi R. Cardiovascular Hybrid Drugs: New Benzazepinone Derivatives as Bradycardic Agents Endowed with Selectiveβ1-Non-competitive Antagonism [J]. Bioorganic & Medicinal Chemistry. 2003,11:1353–1361.
[12] John WN, Christophe M, Bruce DH. Epoxide hydrolase: their roles and interactions with lipid metabolism[J]. Progress in lipid research. 2005,44:1-51.
[13] Summerer S, Hanano A, Utsumi S, et al. Stereochemical features of the hydrolysis of 9,10-epoxystearic acid catalysed by plant and mammalian epoxide hydrolases[J]. Biochem J,2002,366:471–80.
[14] Beetham JK, Tian T, Hammock BD. cDNA cloning and expression of a soluble epoxide hydrolase from human liver[J]. Arch Biochem Biophys,1993,305:197–201.
[15] Pace-Asciak CR, Lee WS. Purification of hepoxilin epoxide hydrolase from rat liver[J]. JBiol Chem. 1989;264:9310–3.
[16] Orning L, Gierse JK, Fitzpatrick FA. The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity [J]. J Biol Chem 1994;269:11269–73.
[17] Schladt L, Thomas H, Hartmann R, Oesch F. Human liver cytosolic epoxide hydrolases[J]. Eur J Biochem 1988;176:715–23.
[18] Friedberg T, Becker R, Oesch F, Glatt H. Studies on the importance of microsomal epoxide hydrolase in the detoxification of arene oxides using the heterologous expression of the enzyme in mammalian cells[J].Carcinogenesis 1994;15:171–5.
[19] Oesch F, Timms CW, Walker CH, Guenthner TM, Sparrow A, Watabe T, et al. Existence of multiple forms of microsomal epoxide hydrolases with radically different substrate specificities [J]. Carcinogenesis 1984;5:7–9.
[20] Debernard S, Morisseau C, Severson TF, et al. Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta [J]. Insect Biochem Mol Biol. 1998,28:409-419.
[21] Zou J,Hallberg BM,Bergfors T,et al. Structure of Aspergillus niger epoxide hydrolase at 1.8 A resolution: implication for the structure and function of the mammalian microsomal class of epoxide hydrolases[J]. Structure. 2000,8:111-122.
[22] Armstrong RN, Cassidy CS. New structural and chemical insight into the catalytic mechanism of epoxide hydrolases [J].Drug Metab Rev. 2000,32:327-338.
[23] Yamada T,Morisseau C,Maxwell JE,et al. Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase [J]. J Biol Chem. 2000,275:23082-23088.
[24] Steinreiber A, Faber K. Microbial epoxide hydrolases for preparative biotransformations [J].Curr Opin Biotechnol.,2001,12:552-558.
[25] Pedragosa-Moreau S, Morisseau C, et al. Microbiological transformations.33.Fungal epoxide hydrolases applied to the snthesis of enantiopure para-substituted styrene oxides.A mechanistic approach [J]. J Org Chem. 1996,61:7402-7407.
[26] Tokunaga M, Larrow JF, Kakiuchi F, et al.Asymmetric catalysis with water: efficient kinetic resolution of terminal epoxides by means of catalytic hydrolysis [J]. Science.1997, 277:937-938
[27] Katsuki T, Sharpless K B. The first practical method for asymmetric epoxidation [J]. J Am Chem Soc.1980,102(18):5974-5976.
[28] Zhang W, Leobach J L, Wilson S R,et al. J Am Chem Soc.1990,112:2801.
[29] Irie R, Noda K, Tto Y, et al. Catalytic asymmetric epoxidation of unfunctionalized olefins[J]. Tetrahedron Lett. 1990,31:7345-7348.
[30] Jacobsen EN. Asymmetric catalysis of epoxide ring-opening reactions [J]. Acc Chem Res, 2000,33:421-431.
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