NJA-1菌产环氧化物水解酶条件的优化及对脱氧雪腐镰刀菌烯醇生物转化的研究
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摘要
真菌毒素是由真菌或霉菌产生的有毒次级代谢产物,广泛污染各种饲料原料和成品饲料。动物误食被真菌毒素污染的饲料会造成动物的真菌毒素中毒。
脱氧雪腐镰刀菌烯醇(deoxynivalnol,缩写DON)又称呕吐毒素(vomitoxin),属单端孢霉烯族B族化合物,是镰刀菌产生的一种真菌毒素,主要污染谷物及其制品。在自然界中广泛存在,目前被认为是存在于赤霉病小麦中的主要天然毒素之一。
最新研究发现,酶可通过将这些霉菌毒素的分子分解为无害的代谢物而将毒素的毒性去除。而环氧酶则可分解单端孢霉烯族菌毒素分子中的环氧基团。这些酶可以降解真菌毒素的代谢物或改变它们的分子结构来产生更少的毒素或者是那些可以代谢或吸收无害的成分。
本论文主要研究在于优化NJA-1菌产环氧化物水解酶的条件,初步研究环氧化物水解酶的酶学性质并且生物转化DON。
试验ⅠNJA-1菌产环氧化物水解酶条件的优化
本实验室从土壤中分离得到一株NAJ-1菌株。本章实验主要是通过研究碳源、氮源、金属离子等培养基细分,以及pH、温度、装液量、添加诱导物等因素对菌株产酶的影响,优化产环氧化物水解酶的条件。实验结果表明:在本实验条件下此菌培养的最佳碳氮源组成是1%葡萄糖,1%玉米浆,培养基的初始pH为4.0,培养的最佳温度和时间是30℃下培养32h,低浓度底物苯基环氧乙烷对该酶具有诱导作用,培养基中的金属离子Ca~(2+)、Mg~(2+)对产酶有促进作用。
试验Ⅱ环氧化物水解酶性质的初步研究
本试验主要是利用NJA-1菌制备EH初酶液。并且用制备的初酶液进行环氧化物水解酶(EH)酶学性质的初步研究。试验结果表明,EH的最适反应温度是30℃,最适pH是pH 7.0;25-40℃范围内酶具有较好的热稳定性,pH 6.5-8.0范围内酶的稳定性较佳,Mg~(2+)、Ca~(2+)离子可促进酶活,有机溶剂DMSO可轻微促进EH酶活。
试验Ⅲ菌体及初酶液对DON的转化
本实验采用菌体及制备的初酶液和EH酶液,加入不同浓度的DON(浓度分别为:4μg/mL,8μg/mL,20μg/mL,100μg/mL),30℃反应不同时间,用高效液相色谱(HPLC)测定DON的含量。结果表明:相比于未优化菌体,优化菌体其对DON转化率增加11.2%;初酶液可显著生物转化DON,转化率可达70～80%;初酶液和EH酶液都可生物转化DON为几种不同的物质,其出峰时间及数量基本相同。
Mycotoxins were toxic secondary metabolites produced by fungi(molds),which widely contaminated raw material and finished product.It would result in mycotoxins poisoning.
Deoxynivalenol(DON) is a type of harmful mycotoxin produced by Fusarium graminearum Schwabe.DON contamination in harvested grain can significantly lower market grade,and result in significant discount in price to the farmers.
Recent study indicated that the enzyme could removed of the toxicity of toxin by decomposing Mycotoxicoses into innoxious metabolites.Epoxide hydrolase could decompose epoxy groups of Toxin Molecular of Tichothecenes.The enzyme could reduce the production of toxins by degradation of mycotoxins of metabolites or change their molecular structures into innoxious substances.
The aim of the experiment was to optimize of the condition of producing epoxide hydrolase and study the enzymology property of epoxide hydrolase and its biotransformation of DON.
TestⅠThe optimization condition of producing the epoxide hydrolase
A strain was isolated from soil and identified as NJA-1 by our laboratory.The strain showed an activity of epoxide hydrolase and could biotransform DON.The purpose of the test was investigated the optimized conditons for enzyme production.It found that high enzyme activity was achieved when 1.0%corn extract and 1.0%glucose were used as carbon and nitrogen sourced,respectively,at optical initial pH 4.0,optical temperature 30℃for 32h.in addition,the Ca~(2+) and Mg~(2+) colud promote the activity of the epoxide hydrolase.
TestⅡPreliminariy study on the enzymatic properities of epoxide hydrolase
The aim of the test was to produce extract EH using the NJA-1 stain,and to study the character of the epoxide hydrolase.Reaserch of enzymatic properties showed that the optimal temperature and pH were 30℃and 7.0.The epoxide hydratase activity is stable between temperature 25～40℃and between pH 6.0～9.0.The Mg~(2+) and Ca~(2+) could promote the activity of the epoxide hydrolase and the organic solvent DMSO also colud slightly promote the activity of the epoxide hydrolase.
TestⅢThe transformation of DON by thallus and extracted enzyme
We added thalli and extraction enzyme and EH enzyme into different concentration of DON(4μg/ml,8μg/ml,20μg/ml,100μg/ml).After reaction at 30℃,the content of DON is detected by high performance liquid Chromatogram(HPLC).
The results of HPLC showes that the product was absorbent at wave length 218nm. The detection of product of the deoxynivalenol biotransformation by the thalli,extract enzyme and EH enzyme.Compared with the thalli before optimization,the thalli after optimization increase the conversion of DON by 11.2%.The extract enzyme and EH enzyme also could significantly biotransform DON to several products.Compared extraction enzyme and EH enzyme,the time and number of the UV absorbing peak of the products is basically identical showed by HPLC.
脱氧雪腐镰刀菌烯醇(deoxynivalnol,缩写DON)又称呕吐毒素(vomitoxin),属单端孢霉烯族B族化合物,是镰刀菌产生的一种真菌毒素,主要污染谷物及其制品。在自然界中广泛存在,目前被认为是存在于赤霉病小麦中的主要天然毒素之一。
最新研究发现,酶可通过将这些霉菌毒素的分子分解为无害的代谢物而将毒素的毒性去除。而环氧酶则可分解单端孢霉烯族菌毒素分子中的环氧基团。这些酶可以降解真菌毒素的代谢物或改变它们的分子结构来产生更少的毒素或者是那些可以代谢或吸收无害的成分。
本论文主要研究在于优化NJA-1菌产环氧化物水解酶的条件,初步研究环氧化物水解酶的酶学性质并且生物转化DON。
试验ⅠNJA-1菌产环氧化物水解酶条件的优化
本实验室从土壤中分离得到一株NAJ-1菌株。本章实验主要是通过研究碳源、氮源、金属离子等培养基细分,以及pH、温度、装液量、添加诱导物等因素对菌株产酶的影响,优化产环氧化物水解酶的条件。实验结果表明:在本实验条件下此菌培养的最佳碳氮源组成是1%葡萄糖,1%玉米浆,培养基的初始pH为4.0,培养的最佳温度和时间是30℃下培养32h,低浓度底物苯基环氧乙烷对该酶具有诱导作用,培养基中的金属离子Ca~(2+)、Mg~(2+)对产酶有促进作用。
试验Ⅱ环氧化物水解酶性质的初步研究
本试验主要是利用NJA-1菌制备EH初酶液。并且用制备的初酶液进行环氧化物水解酶(EH)酶学性质的初步研究。试验结果表明,EH的最适反应温度是30℃,最适pH是pH 7.0;25-40℃范围内酶具有较好的热稳定性,pH 6.5-8.0范围内酶的稳定性较佳,Mg~(2+)、Ca~(2+)离子可促进酶活,有机溶剂DMSO可轻微促进EH酶活。
试验Ⅲ菌体及初酶液对DON的转化
本实验采用菌体及制备的初酶液和EH酶液,加入不同浓度的DON(浓度分别为:4μg/mL,8μg/mL,20μg/mL,100μg/mL),30℃反应不同时间,用高效液相色谱(HPLC)测定DON的含量。结果表明:相比于未优化菌体,优化菌体其对DON转化率增加11.2%;初酶液可显著生物转化DON,转化率可达70～80%;初酶液和EH酶液都可生物转化DON为几种不同的物质,其出峰时间及数量基本相同。
Mycotoxins were toxic secondary metabolites produced by fungi(molds),which widely contaminated raw material and finished product.It would result in mycotoxins poisoning.
Deoxynivalenol(DON) is a type of harmful mycotoxin produced by Fusarium graminearum Schwabe.DON contamination in harvested grain can significantly lower market grade,and result in significant discount in price to the farmers.
Recent study indicated that the enzyme could removed of the toxicity of toxin by decomposing Mycotoxicoses into innoxious metabolites.Epoxide hydrolase could decompose epoxy groups of Toxin Molecular of Tichothecenes.The enzyme could reduce the production of toxins by degradation of mycotoxins of metabolites or change their molecular structures into innoxious substances.
The aim of the experiment was to optimize of the condition of producing epoxide hydrolase and study the enzymology property of epoxide hydrolase and its biotransformation of DON.
TestⅠThe optimization condition of producing the epoxide hydrolase
A strain was isolated from soil and identified as NJA-1 by our laboratory.The strain showed an activity of epoxide hydrolase and could biotransform DON.The purpose of the test was investigated the optimized conditons for enzyme production.It found that high enzyme activity was achieved when 1.0%corn extract and 1.0%glucose were used as carbon and nitrogen sourced,respectively,at optical initial pH 4.0,optical temperature 30℃for 32h.in addition,the Ca~(2+) and Mg~(2+) colud promote the activity of the epoxide hydrolase.
TestⅡPreliminariy study on the enzymatic properities of epoxide hydrolase
The aim of the test was to produce extract EH using the NJA-1 stain,and to study the character of the epoxide hydrolase.Reaserch of enzymatic properties showed that the optimal temperature and pH were 30℃and 7.0.The epoxide hydratase activity is stable between temperature 25～40℃and between pH 6.0～9.0.The Mg~(2+) and Ca~(2+) could promote the activity of the epoxide hydrolase and the organic solvent DMSO also colud slightly promote the activity of the epoxide hydrolase.
TestⅢThe transformation of DON by thallus and extracted enzyme
We added thalli and extraction enzyme and EH enzyme into different concentration of DON(4μg/ml,8μg/ml,20μg/ml,100μg/ml).After reaction at 30℃,the content of DON is detected by high performance liquid Chromatogram(HPLC).
The results of HPLC showes that the product was absorbent at wave length 218nm. The detection of product of the deoxynivalenol biotransformation by the thalli,extract enzyme and EH enzyme.Compared with the thalli before optimization,the thalli after optimization increase the conversion of DON by 11.2%.The extract enzyme and EH enzyme also could significantly biotransform DON to several products.Compared extraction enzyme and EH enzyme,the time and number of the UV absorbing peak of the products is basically identical showed by HPLC.
引文
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[1]Arahira M,Nong VH,Udaka K,et al.Purification,molecμLar ethylene-inducible expression of a soluble-type epoxide hydrolase(Glycine max[L]Merr.)[J].Eur J Biochem 2000,267:2649-2657.
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[5]李从发.新型环氯化物水解酶筛选及其在手性合成中的应用[J].山东大学博士学位论文.2005.
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[1]尹珺,张洪英,张海彬等.脱氧雪腐镰刀菌烯醇(DON)理化检测方法的研究[J].畜牧与兽医.2006(10).
[2]何成华.脱氧雪腐镰刀菌烯醇毒性和生物转化的研究.南京农业大学博士学位论文.2007.
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[4]何成华 樊彦红 刘田芳 张海彬等.一株脱氧雪腐镰刀苗烯醇生物转化苗的分离,鉴定和转化效果的初步研究.中国科技论文在线.2007.
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[1]尹珺,张洪英,张海彬等.脱氧雪腐镰刀菌烯醇(DON)理化检测方法的研究[J].畜牧与兽医.2006(10).
[2]何成华.脱氧雪腐镰刀菌烯醇毒性和生物转化的研究.南京农业大学博士学位论文.2007.
[3]庞炜,王治伦,吕社民,等,真菌毒素DON研究进展[J].中国地方病防治杂志1996,11(6):349-351.
[4]何成华 樊彦红 刘田芳 张海彬等.一株脱氧雪腐镰刀苗烯醇生物转化苗的分离,鉴定和转化效果的初步研究.中国科技论文在线.2007.
[5]He P,Young LG,Forsberg C.Microbial transformation ofdeoxynivalenol(vomitoxin)[J].Appl.Environ.Microbiol,1992,58(12):3857-3863.
[6]隋凯,李军,卫峰,等.谷物中脱氧署腐镰刀菌烯醇的高效液相色谱检测及质谱确证[J].分析化学.2005.(33)11:1643-1646.
[7]Shima,J.Takase S.,Takahashi Y.,et al.Novel detoxification of the trichothecene mycotoxin deoxyninalenol by a soil bacterium isolated by enrichment culture[J].Appl Environm Microbiol,1997,63:3825-3830.
[8]Poppenberger B,Berthiller F,Lucyshyn D,et al.Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana[J].J Biol Chem,2003,278(48):47905-47914.