Isolation and identification of bacteria degrading soy lecithin and dimethoate and comparision of enzyme activities
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摘要:目的
明确2株有机磷降解菌Yj2和Yj3对大豆卵磷脂和乐果有机磷的降解特性及酶活性。
方法利用16S rDNA鉴定从大豆土壤中分离得到的Yj2和Yj3菌株，在菌株最佳生长条件下，测定了不同磷源时菌体的酶活性并分级纯化了有机磷降解酶。
结果Yj2为醋酸钙不动杆菌Acinetobacter sp.，其最佳生长碳源为葡萄糖，氮源为硫酸铵，pH为8；Yj3为芽孢杆菌Bacillus sp.，其最佳生长碳源为葡萄糖，氮源为蛋白胨，pH为9。大豆卵磷脂为磷源时，Yj3的菌体生长情况稍优于Yj2。乐果为磷源时，Yj2的菌体生长情况稍优于Yj3；72 h内Yj2酸性和碱性磷酸酶活性整体高于Yj3，而有机磷降解酶活性低于Yj3。硫酸铵沉淀法+阳离子交换层析分别从Yj2和Yj3菌体中成功分离纯化了有机磷降解酶，SDS-PAGE结果显示纯化的蛋白均为单一条带。Yj2硫酸铵沉淀法+阳离子交换层析的提纯倍数是硫酸铵沉淀的7.77倍，硫酸铵沉淀为粗酶的1.35倍。Yj3硫酸铵沉淀法+阳离子交换层析的提纯倍数是硫酸铵沉淀的5.07倍，硫酸铵沉淀为粗酶的1.53倍。
结论菌株Acinetobacter sp. Yj2和Bacillus sp. Yj3都具有降解大豆卵磷脂及乐果有机磷的特性，对有机磷降解起主要作用的是酸性磷酸酶、碱性磷酸酶及有机磷降解酶，它们在2株菌株对大豆卵磷脂和乐果降解过程中所起的作用有明显差异。可从Yj2和Yj3菌体分离纯化获得提纯倍数较高的有机磷降解酶蛋白。
Abstract:ObjectiveIn order to clarify degradation characteristics and enzyme activities of two organic phosphorus degrading bacteria in soy lecithin and dimethoate.
MethodBacterium strains Yj2 and Yj3 were isolated from soy soil and identified by 16S rDNA identification method. The growth conditions of Yj2 and Yj3 were optimized by orthogonal test. The enzyme activities from two strains were determined under different phosphorus sources, and organophosphate degrading enzymes were classified and purified.
ResultYj2 was identified as Acinetobacter sp., and Yj3 was identified as Bacillus sp.. The result of growth condition optimization showed that the best carbon and nitrogen source and pH values for strain Yj2 and Yj3 were glucose, ammonium sulfate, pH 8 and glucose, peptone, pH 9, respectively. The growth of Yj3 was slightly better than that of Yj2 when the phosphorus source was soy lecithin. However, the growth trend was the opposite with dimethoate as the phosphorus source. Within 72 h of dimethoate being the phosphorus source, acid phosphatase activity and alkaline phosphatase activity of Yj2 were generally higher than that of Yj3, but organophosphate degradation enzyme activiy of Yj2 was lower than those of Yj3. The organophosphorus degradation enzymes were isolated and purified respectively from strains Yj2 and Yj3 by ammonium sulfate precipitation followed with cation exchange chromatography. SDS-PAGE results showed that the purified proteins were both a single band. The purification ratio of ammonium sulfate precipitation followed with cation exchange was 7.77 times higher than that of the ammonium sulphate precipitation for Yj2, and the latter was 1.35 times higher than crude enzyme for Yj2. The corresponding purification ratios for Yj3 were 5.07 and 1.53 times respectively.
ConclusionAcinetobacter sp. Yj2 and Bacillus sp. Yj3 both can degrade soy lecithin and dimethoate. The activities of acid phosphatase, alkaline phosphatase and organophosphate degradation enzymes, which play the main roles on degrading organic phosphate, have obvious differences between two strains in soy lecithin and dimethoate degrading process. The organophosphate degradation enzymes with relatively high purity can be isolated and purified from YJ2 and Yj3, respectively.
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图 1 Yj2和Yj3溶磷效果和菌株形态
A、B分别为以大豆卵磷脂和乐果为磷源时Yj2的溶磷效果；C:Yj2的菌株形态(100×油镜)；D、E分别为以大豆卵磷脂和乐果为磷源时Yj3的溶磷效果；F:Yj3的菌株形态(100×油镜)。
Figure 1. Effects on phosphorus-solubilizing and morphology of strains Yj2 and Yj3
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图 2 Yj2和Yj3 16S rDNA基因的克隆及重组质粒的酶切结果
M: DL 2000 Marker, 1、3为Yj2，2、4为Yj3；A：PCR产物，B：酶切结果。
Figure 2. Amplification of 16S rDNA gene and restriction enzyme digestion of recombinant plasmids of Yj2 and Yj3
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图 3 Yj2和Yj3基于16S rDNA序列构建的系统发育树
Figure 3. Phylogeny tree of 16S rDNA sequence from Yj2 and Yj3
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图 4 以大豆卵磷脂和乐果为磷源时Yj2和Yj3的生长曲线
Figure 4. Growth curve of Yj2 andYj3 using soy lecithin dimethoate as phosphorus source
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图 5 乐果为磷源时Yj2和Yj3的酸性磷酸酶活性、碱性磷酸酶活性和有机磷降解酶活性测定
Figure 5. The enzyme activity of acid phosphatase, alkaline phosphatase and organophosphorus degradation of Yj2 and Yj3 using dimethoate as phosphorus source
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图 6 Yj2和Yj3有机磷降解酶纯化结果的SDS-PAGE
M为蛋白Marker，1为流川液，2~4分别表示0.1、0.2、0.5 mol·L-1 NaCl; A:Yj2, B:Yj3。
Figure 6. SDS-PAGE of purified organophosphorus degradation enzyme of Yj2 and Yj3
表 1 有机磷降解酶分级纯化的酶活力分析
Table 1 Classification and purification of organophosphate degradation enzyme activity analysis
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