鞘氨醇单胞菌产3-苯氧基苯甲酸降解酶固定化条件及其酶学性质研究
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摘要
以海藻酸钠为载体,对鞘氨醇单胞菌SC-1产3-苯氧基苯甲酸(3-PBA)降解酶进行固定化,探讨酶的固定化条件及固定化酶的酶学性质。结果表明,当酶液与海藻酸钠体积比为1∶5、海藻酸钠质量分数2%、氯化钙质量分数1%时,固定化酶的酶活较高,固定化时间对酶活影响不显著,而固定化酶凝珠直径对其酶活影响较大,在最优条件下固定化酶的酶活达223.1 U/g,酶活回收率为86%。在30℃,p H 7.0时固定化酶的酶活达到最高值;Mg2+,Mn2+及较高浓度Ba2+,低浓度Cu2+,10-2~10-6mol/L EDTA,0.01%BSA,10-6mol/L邻菲罗啉,0.1%VC对固定化酶的酶活有促进作用。固定化酶的米氏常数(Km)为46.63×106nmol/L,最大反应速率(Vmax)为3.70×103U/g。固定化3-PBA降解酶在最适条件下连续反应900 min,对5μg/m L的3-PBA降解率为26.14%。
Sodium alginate was used for immobilization of 3-phenoxyzoic acid(3-PBA)-degradation enzyme which was produced by Sphingomonas sp. SC-1. The effects of immobilized enzyme and the immobilized enzyme properties were studied. The results showed that the optimal immobilized condition of 3-PBA-degradation enzyme was: a ratio of enzyme liquid and sodium alginate 1 ∶5(V/V), sodium alginate and calcium chloride concentration at 2% and 1% respectively.Immobilization time had no obvious effect on enzyme activity, but immobilized enzyme particle diameter had great influence on it. Under the optimum conditions. The activity of immobilized enzyme reached 223.1 U/g and the activity recovery reached 86%. The highest enzyme activity appeared while reaction temperature was 30 ℃, and p H was 7.0; Mg2+, Mn2+and high concentrations of Ba2+, low concentration of Cu2+can promote the immobilized enzyme activity; 10-2-10-6mol/L ethylene diaminetetraacetic acid, 0.01% bovine serum albumin, 0.1% vitamin C, 10-6mol/L phenanthroline had promoting effect on enzyme activity. The michaelis constant(Km) of immobilized enzyme was 46.63 ×106nmol/L, maximum reaction rate(Vmax) was 3.70×103U/g. The degradation rate of 3-PBA immobilized enzyme to 5 μg/m L 3-PBA was 26.14% after900 minutes continuous reaction under the optimum conditions.
Sodium alginate was used for immobilization of 3-phenoxyzoic acid(3-PBA)-degradation enzyme which was produced by Sphingomonas sp. SC-1. The effects of immobilized enzyme and the immobilized enzyme properties were studied. The results showed that the optimal immobilized condition of 3-PBA-degradation enzyme was: a ratio of enzyme liquid and sodium alginate 1 ∶5(V/V), sodium alginate and calcium chloride concentration at 2% and 1% respectively.Immobilization time had no obvious effect on enzyme activity, but immobilized enzyme particle diameter had great influence on it. Under the optimum conditions. The activity of immobilized enzyme reached 223.1 U/g and the activity recovery reached 86%. The highest enzyme activity appeared while reaction temperature was 30 ℃, and p H was 7.0; Mg2+, Mn2+and high concentrations of Ba2+, low concentration of Cu2+can promote the immobilized enzyme activity; 10-2-10-6mol/L ethylene diaminetetraacetic acid, 0.01% bovine serum albumin, 0.1% vitamin C, 10-6mol/L phenanthroline had promoting effect on enzyme activity. The michaelis constant(Km) of immobilized enzyme was 46.63 ×106nmol/L, maximum reaction rate(Vmax) was 3.70×103U/g. The degradation rate of 3-PBA immobilized enzyme to 5 μg/m L 3-PBA was 26.14% after900 minutes continuous reaction under the optimum conditions.
引文
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[26]曹晓星,田蕴,胡忠,等.PAHs降解基因及降解酶研究进展[J].生态学杂志,2007,26(6):917-924.
[27]苟敏,唐溪,孔春雷,等.环境微生物中芳环加氧酶的获取策略[J].应用与环境生物学报,2012,18(5):880-887.
[28]周鑫淼,陈洁君,耿立召,等.邻苯二酚2,3-双加氧酶的结构和功能研究进展[J].生物技术通报,2007,(4):51-54.
[2]Raghavendra K,Barik TK,Sharma P,et al.Chlorfenapyr:a new insecticide with novel mode of action can control pyrethroid resistant malaria vectors[J].Malaria Journal,2011,10(1):16.
[3]Zhang ZY,Liu XJ,Hong XY,et al.Effects of home preparation on pesticide residues in cabbage[J].Food Con trol,2007,18(12):1484-1487.
[4]Charizopoulos E,Papadopoulou-Mourkidou E.Occurrence of pesticides in rain of the Axios River Basin,Greece[J].Environmental Science and Technology,1999,33(14):2363-2368.
[5]廖文军,李中华.惠州市饮食服务行业茶叶农药残留调查[J].中国热带医学,2007,7(8):1318,1479.
[6]华纯.拟除虫菊酷类农药的进展和剂型[J].世界农药,2009,31(5):39-44.
[7]Jin MQ,Li L,Xu C,et al.Estrogenic activities of two synthetic pyrethroids and their metabolites[J].Journal of Environmental Sciences,2010,22(2):290-296.
[8]李少南.农药对土壤生物群落的副作用的研究方法[J].生态毒力学报,2010,5(2):18-24.
[9]Halden RU,Tepp SM,Halden BG,et al.Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(p POB)and two modified Pseudomonas strains[J].Applied and Environmental Microbiology,1999,65(8):3354-3359.
[10]张锡辉.高等环境化学与微生物学原理及应用[M].北京:化学工业出版社,2001:50-70.
[11]Topp E,Zhu H,Nour SM,et al.Characterization of an atrazine-degrading Pseudaminobacter sp.isolated from canadian and french agricultural soils[J].Applied and Environmental Micr-obiology,2000,66(7):2773-2782.
[12]虞云龙,陈鹤鑫,樊德方,等.拟除虫菊酯类杀虫剂的酶促降解[J].环境科学,1998,19(3):67-68.
[13]刘建立.微生物降解有机磷农药酶促机制[J].生物学通报,2010,45(5):1-4.
[14]许育新,李晓慧,秦华,等.3-苯氧基苯甲酸降解菌的分离及降解特性研究[J].微生物学通报,2005,32(5):62-66.
[15]Chen SH,Yang L,Hu MY,et al.Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp.strain ZS-S-01 and its use in bioremediation of contaminated soils[J].Environmental Biotechnology,2011,90(2):755-767.
[16]段晓芹,郑金伟,张隽,等.3-PBA降解菌BA3的降解特性及基因工程菌构建[J].环境科学,2011,32(1):240-246.
[17]Chen SH,Hu W,Xiao Y,et al.Degradation of 3-phenoxybenzoic acid by a bacillus sp[J].Plos One,2012,7(11):1-12.
[18]Chen SH,Hu MY,Yang L,et al.Biodegradation of beta-cypermethrin and 3-phenoxybenzo-ic acid by a novel Ochrobactrum lupini DG-S-01[J].Journal of Hazardous Materials,2011,187(1/2/3):433-440.
[19]赵楠.3-苯氧基苯甲酸降解菌M4的分离及降解特性、降解途径的初步研究[D].雅安:四川农业大学,2011.
[20]袁怀瑜,刘书亮,王志龙,等.3-苯氧基苯甲酸降解真菌的筛选及其降解特性研究[J].食品科学,2012,34(7):152-156.
[21]Chen SH,Lai KP,Li YN,et al.Biodegradation of deltamethrin and its hydrolysis product 3-phenoxybenzaldehyde by a newly isolated Streptomyces aureus strain HP-S-01[J].Applied Microbiology Biotechnology,2011,90(4):1471-1483.
[22]Tang J,Yao K,Liu SL,et al.Biodegradation of 3-phenoxybenzoic acid by a novel Sphingomonas sp.SC-1.Fresenius Environmental Bulletin,2013,22(5):1564-1572.
[23]李建龙,王志龙,刘书亮,等.鞘氨醇单胞菌的3-苯氧基苯甲酸降解酶定位及分子质量的确定[J].食品与发酵工业,2014,40(1):24-29.
[24]周蕊,余蓉,杨继虞.固定化胰蛋白酶的快速制备[J].生物技术,2004,14(3):26-27.
[25]郑文爽,赵敏.海藻酸钠包埋固定胆红素氧化酶的优化[J].东北林业大学学报,2011,39(3):127-128.
[26]曹晓星,田蕴,胡忠,等.PAHs降解基因及降解酶研究进展[J].生态学杂志,2007,26(6):917-924.
[27]苟敏,唐溪,孔春雷,等.环境微生物中芳环加氧酶的获取策略[J].应用与环境生物学报,2012,18(5):880-887.
[28]周鑫淼,陈洁君,耿立召,等.邻苯二酚2,3-双加氧酶的结构和功能研究进展[J].生物技术通报,2007,(4):51-54.