铬天青S法结合高效液相色谱法测定甲烷氧化菌素含量
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摘要
目的:建立由M.trichosporium OB3b发酵获得的甲烷氧化菌素(Mb)在发酵过程与发酵成熟阶段的含量测定方法。方法:分别考察了CuSO_4、HDTMA、磷酸缓冲液添加量以及络合时间等因素对铬天青法检测Mb含量的影响;在铬天青S法对发酵后冻干粉Mb含量确定的基础上,为调控Mb的发酵过程,通过高效液相色谱法对发酵前4 d每24 h的Mb含量进行测定。结果:在CuSO_4、HDTMA、磷酸缓冲液添加量分别为1.2、4.0、2.0 mL及络合时间为15 min的条件下,铬天青S法测得纯化的1 L发酵液中Mb的含量为26.91μmoL,最低检测限为0.042μmoL/L。通过与高效液相色谱法的结合,确定在发酵24、48、72、96 h时发酵液中Mb含量分别为1.008、1.336、3.125、7.500μmoL/250 mL。结论:本文所建立的方法稳定、重现性好,可用于Mb在发酵不同阶段含量的测定。
Objective:To establish a determination method of the content of methanobactin(Mb)obtained by fermentation of M. trichosporium OB3b during fermentation process and fermentation ripening stage. Methods:The effects of addition of CuSO_4,HDTMA,phosphate buffer and complexation time on the content of Mb were investigated. Based on the determination of Mb content in freeze-dried powder after fermentation by chrome azurol S method,in order to control the Mb fermentation process,the Mb content of every 24 h in the first 4 days during fermentation was determined by high performance liquid chromatography. Results:The content of CuSO_4,HDTMA and phosphate buffer was 1.2,4.0,2.0 mL and the complexation time was 15 min,the content of Mb in the purified 1 L fermentation broth was 26.91 μmolL,and the minimum detection limit was 0.042 μmol/L.Combining with high performance liquid chromatography,the Mb content in the fermentation broth was determined to be 1.008,1.336,3.125,7.500 μmol/250 mL at 24,48,72 and 96 h,respectively. Conclusion:The method established was stable and had good reproducibility,which could be used for the determination of Mb content in different stages of fermentation.
Objective:To establish a determination method of the content of methanobactin(Mb)obtained by fermentation of M. trichosporium OB3b during fermentation process and fermentation ripening stage. Methods:The effects of addition of CuSO_4,HDTMA,phosphate buffer and complexation time on the content of Mb were investigated. Based on the determination of Mb content in freeze-dried powder after fermentation by chrome azurol S method,in order to control the Mb fermentation process,the Mb content of every 24 h in the first 4 days during fermentation was determined by high performance liquid chromatography. Results:The content of CuSO_4,HDTMA and phosphate buffer was 1.2,4.0,2.0 mL and the complexation time was 15 min,the content of Mb in the purified 1 L fermentation broth was 26.91 μmolL,and the minimum detection limit was 0.042 μmol/L.Combining with high performance liquid chromatography,the Mb content in the fermentation broth was determined to be 1.008,1.336,3.125,7.500 μmol/250 mL at 24,48,72 and 96 h,respectively. Conclusion:The method established was stable and had good reproducibility,which could be used for the determination of Mb content in different stages of fermentation.
引文
[1]唐千,薛校风,王惠,等.湖泊生态系统产甲烷与甲烷氧化微生物研究进展[J].湖泊科学,2018,30(3):597-610
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[11]李春雨,辛嘉英,林惠颖,等.铜离子配位甲烷氧化菌素功能化纳米金模拟过氧化物酶的研究[J].分子催化,2017(5):480-485.
[12]梁洪野.甲烷氧化菌的高密度培养[J].哈尔滨商业大学,2011:9-11.
[13]周琦琼,董静,辛嘉英.利用甲烷氧化菌生产甲烷氧化菌素[J].化工科技市场,2009,10:35-35.
[14]Dunfield P F,Liesack W,Henckel T,et al.High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph[J].Applied & Environmental Microbiology,1999,65(3):1009.
[15]王建坤,范新宇,张昊,等.基于EDTA络合滴定法测定重金属离子浓度[J].天津工业大学学报,2018,1(2):48-52.
[16]苏瑶,孔娇艳,张萱,等.甲烷氧化过程中铜的作用研究进展[J].应用生态学报,2014,25(4):1221-1230.
[17]辛嘉英,董静,闫超泽,等.甲烷氧化菌素的产生和铜捕获作用[J].中国生物工程杂志,2011,31(8):40-46.
[18]Long Y,Lei Y B,Zhang Y Z,et al.Determination of free calcium oxide in steel slag by EDTA complexometric titration[J].Metallurgical Analysis,2010,30(7):65-68.
[2]Kim H J,Graham D W,Dispirito A A,et al.Methanobactin,a copper-acquisition compound from methane-oxidizing bacteria[J].Science,2004,305(5690):1612-1615.
[3]张秀凤.甲烷氧化菌素介导纳米金合成[D].哈尔滨:哈尔滨商业大学,2013:1-2.
[4]曹淑贞,沈媛媛,王风芹,等.土壤甲烷氧化菌群落结构研究进展[J].生物学杂志,2017,34(6):78-82.
[5]魏聪,刘国生.甲烷氧化菌素的筛选与生理特性研究[J].安徽农业科学,2013,41(7):2851-2852.
[6]Choi D W,Semrau J D,Antholine W E,et al.Oxidase,superoxide dismutase,and hyd-rogen peroxide reductase activities of methanobactin from types I and II methanotrophs[J].Journal of Inorganic Biochemistry,2008,102(8):1571-1572.
[7]陈鸿鹏,谭晓凤.超氧化物歧化酶.研究综述[J].经济林研究,2007,25(1):59-65.
[8]闫超泽,张帅,辛嘉英,等.铬天青S分光光度法测定甲烷氧化菌素的研究[J].分析测试技术与仪器,2011,17(2):69-73.
[9]Fujita Y,Mori I,Kitano S.Determination of proteins by using the color reaction with pyrocatechol violet-molybdenum(VI)complex.[J].Chemical & Pharmaceutical Bulletin,1984,32(10):4161-4164.
[10]Saito Y,Kanetsuna A,Okuda H,et al.A sensitive spectrophotometric method for the determination of human serum albumin with chrome-azurol S aluminium chelate[J].Chemical & Pharmaceutical Bulletin,1986,34(2):746-750.
[11]李春雨,辛嘉英,林惠颖,等.铜离子配位甲烷氧化菌素功能化纳米金模拟过氧化物酶的研究[J].分子催化,2017(5):480-485.
[12]梁洪野.甲烷氧化菌的高密度培养[J].哈尔滨商业大学,2011:9-11.
[13]周琦琼,董静,辛嘉英.利用甲烷氧化菌生产甲烷氧化菌素[J].化工科技市场,2009,10:35-35.
[14]Dunfield P F,Liesack W,Henckel T,et al.High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph[J].Applied & Environmental Microbiology,1999,65(3):1009.
[15]王建坤,范新宇,张昊,等.基于EDTA络合滴定法测定重金属离子浓度[J].天津工业大学学报,2018,1(2):48-52.
[16]苏瑶,孔娇艳,张萱,等.甲烷氧化过程中铜的作用研究进展[J].应用生态学报,2014,25(4):1221-1230.
[17]辛嘉英,董静,闫超泽,等.甲烷氧化菌素的产生和铜捕获作用[J].中国生物工程杂志,2011,31(8):40-46.
[18]Long Y,Lei Y B,Zhang Y Z,et al.Determination of free calcium oxide in steel slag by EDTA complexometric titration[J].Metallurgical Analysis,2010,30(7):65-68.
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