Isolation and Identification of Serratia marcescens Yj1，and Separation and Purification of Organophosphate Degrading Enzymes

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.027

Abstract

Abstract: The strain Yj1 isolated and purified from the soybean soil could degrade soy lecithin and dimethoate. The 16S rDNA identification of the strain, optimization of growth conditions and examination of enzyme activities were performed and organophosphate degradation enzyme from Yj1 was separated and purified. The results revealed that the similarity of 16S rDNA in Yj1 and Serratia marcescens WW4(CP003959. 1)was 99%. The orthogonal experiment for the culture medium demonstrated that the optimal growth condition of Yj1 was the combination of mannose, peptone and pH8. The growth rate of Yj1 was poor in either of 2 phosphorus sources；however, the growth rate in soy lecithin was better than that in the dimethoate within 72 h. Moreover, the activities of acid phosphatase, alkaline phosphatase and organophosphate degrading enzyme(ODE)were higher when the soybean lecithin as the sole phosphorus source than those of dimethoate, and the activity of alkaline phosphatase was obviously greater than that of acid phosphatase and ODE within 72 h. ODE was successfully purified from Yj1 by combining ammonium sulfate precipitation and SP Sepharose Fast Flow. SDS-PAGE results showed that the purified protein was in a single band. The purified multiple by SP Sepharose Fast Flow was 5. 303 times as many by ammonium sulfate precipitation, and by the latter 1. 416 times as many by crude enzyme.

Key words: Serratia marcescens, Yj1, dimethoate, soy lecithin, organophosphate degrading enzyme

Yu Ting, Wang Chunhong, Zhang Tingting, Ji Tian, Wu Zhihai, Yang Meiying. Isolation and Identification of Serratia marcescens Yj1，and Separation and Purification of Organophosphate Degrading Enzymes[J]. Biotechnology Bulletin, 2015, 31(7): 180-187.

References

[1] Abd-Alla MH. Phosphatases and the utilization of organic phosphorus by Rhizobium legum inosarum biovarviceae[J]. Letters in Applied Microbiology, 1994, 18：294-296 .
[2]王光华, 赵英, 周德瑞, 等. 解磷菌的研究现状与展望[J]. 生态环境, 2003, 12(1)：96-100.
[3]吉蓉. 土壤解磷微生物及其解磷机制综述[J]. 甘肃农业科技, 2013, 8：42-44.
[4]Tao GC, Tian SJ, Cai MY, et al. Phosphorus-solubilizing and mineralizing abilities of bacteria isolated from soils[J]. Pedosphe-re, 2008, 18(4)：51.
[5]Xie LH, Lu JL, Zhang YP, et al. Influence of long term fertilization on phosphorus fertility of calcareous soilⅡ, Inorganic and organic phosphorus1[J]. Chinese Journal of Applied Ecology, 2004, 15(5)：790-794.
[6]王继雯. 有机磷降解菌的分离筛选及初步鉴定[J]. 河南科学, 2011, 29(1)：31-34.
[7]杨小蓉, 宗浩, 郑鸽, 等. 一株降解氧乐果的高效菌的分离和鉴定[J]. 四川师范大学学报：自然科学版, 2001, 24(4)：392-394.
[8]王堃, 王志春, 李荣, 等. 乐果降解菌L3 的分离、鉴定及降解性能[J]. 中国环境科学, 2008, 28(11)：994-998.
[9]Whitel MA, Harden TJ, Bender GL. Phosphorus solubilization in solution culture by the soil fungus Penicillium radicum[J]. Australian Journal of Soil Research, 1997, 35：291-300.
[10]马春浩. 解磷微生物及其应用研究综述[J]. 安徽农学通报, 2007, 13(4)：34-36.
[11]Sperber JI. Solution of mineral phosphoruss by soil bacteria[J]. Nature, 1957, 180：994-995.
[12]Reddy MS, Kumar S, Babita K, et al. Biosolubilization of poorly soluble rock phosphoruss by Aspergillus tubingensis and Aspergillus niger[J]. Bioresource Technology, 2002, 84：187-189.
[13]Kobus J. The distribution of microorganisms mobilizing phosphorus in different soils[J]. Scta Microbiologia Plolonica, 1962, 11：255-264.
[14]黄雅, 李政一, 赵博生. 有机磷农药乐果降解的研究现状与进展[J]. 环境科学与管理, 2009, 43(4)：20-24.
[15]邓晓, 李勤奋, 侯宪文, 等. 乐果降解菌LGX1的筛选及其降解特性研究[J]. 生态环境学报, 2010, 19(5)：1034-1039.
[16]王华, 艾涛, 温晓芳, 等. 土壤中乐果降解菌的筛选及其特性研究[J]. 农业环境科学学报, 2006, 25(5)：1255-1259.
[17]王永杰, 李顺鹏, 沈标. 有机磷农药乐果降解菌的分离及其活性研究[J]. 南京农业大学学报, 2001, 24(2)：71- 74.
[18]马丽娜, 官雪芳, 朱育菁, 等. 乐果降解菌的分离、筛选和鉴定[J]. 中国农学通报, 2008, 24(7)：441-444.
[19]赵小蓉, 林启美, 李保国. 微生物溶解磷矿粉能力与 pH 及分泌有机酸的关系[J]. 微生物学杂志, 2003, 23(3)：5-7.
[20] Yi YM, Huang WY, Ge Y. Exopolysaccharide：a novel importantfactor in the microbial dissolution of tricalcium phosphorus[J]. World J Microbiol Biotech, 2008, 24：1059-10651.
[21]孙嘉, 郎朗. 有机磷农药降解酶的研究进展[J]. 资源节约与环保, 2014, 5：149-154.
[22] 萨姆布鲁克 J, 拉塞尔 DW, 黄培堂, 译. 分子克隆实验指南[M]. 第3版. 北京：科学出版社, 2008.
[23]伊鋆. 高效解磷细菌的筛选及解磷机理的研究[D]. 大连：大连理工大学, 2011.
[24]金彬明, 刘佳明. 海洋微生物有机磷降解酶的纯化与性质研究[J]. 中国微生态学杂志, 2007, 19(1)：37-39.
[25]伍宁丰, 梁果义, 邓敏捷, 等. 有机磷农药降解酶及其基因工程研究进展[J]. 生物技术通报, 2003(5)：9-12.
[26]肖佳沐, 袁海平, 楼紫阳, 等. 一株乐果降解菌的筛选、鉴定及其降解特性研究[J]. 环境污染与防治, 2010, 32(12)：67-71.
[27]史延华. CP1 菌株的分离、筛选及其对毒死蜱的降解[J]. 微生物学通报, 2011, 38(9)：1331-1338.
[28]王莉. Hyphomicrobium sp. MAP-1菌株修复甲胺磷乙酰甲胺磷和水胺硫磷污染土壤的实验研究[J]. 农业环境科学学报, 2013, 32(1)：81-87.
[29]许育新, 冯昭中, 陆鹏, 等. 甲基对硫磷降解菌PF32的分离鉴定及其降解特性研究[J]. 农药学学报, 2009, 11(3)：329-334.
[30]Cycona M, Zmijowska A, Wójcik M, et al. Biodegradation and bioremediation potential of diazinon-degrading Serratia marcescens to remove other organophosphorus pesticides from soils[J]. Journal of Environmental Management, 2013, 117：7-16.
[31]赵小蓉, 林启美, 李保国. C、N源及C/N比对微生物溶磷的影响[J]. 植物营养与肥料学报, 2002, 8(2)：197-204.
[32]陈燕飞. pH 对微生物的影响[J]. 太原师范学院学报：自然科学版, 2009, 8(3)：121-124.
[33]钟传青, 黄为一. 不同种类解磷微生物的溶磷效果及其磷酸酶活性的变化[J]. 土壤学报, 2005, 42(2)：286-293.
[34]于群英. 土壤磷酸酶活性及其影响因素研究[J]. 安徽技术师范学院学报, 2001, 15(4)：5-8.
[35]刘玉焕, 钟英长. 华丽曲霉Z58有机磷农药降解酶的纯化和性质[J]. 微生物学报, 2000, 40(4)：430-434.
[36]Munnecke DM. Enzymatic detoxification of waste organophosphorus pesticides[J]. Agric Food Chem, 1980, 28：105-111.