枯草芽孢杆菌FB123产生的枯草菌素的研究
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摘要
本文主要是对枯草芽孢杆菌(Bacillus subtilis)FB123产生的枯草菌素进行分离纯化、表征以及初步结构的研究。
对Bacillus subtilis FB123所产粗枯草菌素的理化性质及抑菌谱的研究表明粗枯草菌素最适合作用pH为6,具有较好的酸碱稳定性;最适作用温度为40℃,热稳定性较好。多种蛋白酶可使其部分失活。Na~+、Fe~(2+)、Zn~(2+)和Ca~(2+)离子对其具有增效作用,而Mg~(2+)、Al~(3+)、K~+和Cu~(2+)离子会部分抑制其抗菌活性。有机溶剂能明显抑制其活性。粗枯草菌素的抑菌谱较宽,对多种细菌以及真菌都具有良好的抗菌作用。
利用硫酸铵沉淀、Sephacryl S-200层析、DEAE-Sephamse Fast Flow层析从粗枯草菌素中分离出主要活性组分P2、P3-2、P4、P5。
P3-2经SDA-PAGE电泳鉴定为单亚基蛋白,分子量为14.8kDa,等电点为3.64,采用Edman降解法测得N端15个氨基酸序列为N-DREISEGSVDQSSWC-15。采用过碘酸-Schiff试剂染色法PAS和苏丹黑B脂染色法对P3-2进行糖基、脂基研究,结果表明该枯草菌素可能为一种不含糖的脂蛋白;P3-2对蛋白酶不敏感;具有广泛的pH活性范围;30℃为其最适作用温度,具有很好的热稳定性;有机溶剂能明显降低P3-2的抑菌活性;K~+、Zn~(2+)、Ca~(2+)、Na~+能部分抑制P3-2的活性,而Al~(3+)、Mg~(2+)、Fe~(2+)、Cu~(2+)对P3-2具有增强作用;P3-2对其亲缘相近的革兰氏阳性菌有强的抑制作用,对革兰氏阴性菌大肠杆菌无抑菌作用,但对部分真菌也有抗菌作用。
研究P2活性组分:1.测得P2 N端8个氨基酸序列为N-(Q、M)KPINISD-8。2.P2活性组分对部分真菌具有较强抑制作用,表明在相同浓度下P2的抑制真菌活性要比粗细菌素以及P3-2组分活性要高。
In the present study,subtilin produced by Bacillus subtilis FB123 were purified and characterized,and their primary structures were studied.
The characteristics and the antimicrobial spectrum of the crude subtilin were analyzed.The results showed that the crude subtilin kept its activity in a broad range of pH value and had the optimum pH of 6.0.The crude subtilin also showed a stronger thermostability and had the optimal culture temperature at 40℃.It can be partly inactivated by proteinase.The activity of crude subtilin could be partly improved by Na~+、Fe~(2+)、Zn~(2+)and Ca~(2+),partly inhibited by Mg~(2+)、Al~(3+)、K~+and Cu~(2+)Organic Solvents can strongly inhibit its activity.The antimicrobial spectrum tests showed that the crude subtilin exhibited stronger inhibition to both gram negative and positive bacteria used and had a weak inhibition to part fungi tested.
The crude subtilin was purified by ammonium sulfate precipitation,Sephacryl S-200,DEAE Sepharose Fast Flow chromatography and four components of subtilin(P2、P3-2、P4、P5)was obtained.
P3-2 was identified to be homogenous with SDS-PAGE,and its molecular weight and pI value were of 14.8 kDa and 3.64;15 amino acids residues of its N-terminal was measured by Edman degradation method,the secquence was N-DREISEGSVDQSSWC-15;The analysis based on periodic acid schiff reaction(PAS) and Sudan B lipid staining suggested P3-2 might be a lipoprotein,but not a glycoprotein; P3-2 showed insensitive to proteinase;and kept activity within wide pH value;it showed stronge thermal stability and with the optimal temperature of 30℃.The activity of P3-2 could be partly inhibited by K~+、Zn~(2+)、Ca~(2+)、Na~+and partly improved by Al~(3+)、Mg~(2+)、Fe~(2+)、Cu~(2+);P3-2showed broad ranges of anti-microbial activities not only to some gram-positive bacteria but also some fungi;but it showed no inhibition to Ecoli.
Supplementary study on the P2 component:1.the 8 amino acid residues in the N-terminal of P2 were measured,it was N-(Q、M)KPINISD-8;2.the anti-fungi activity of P2 component was studied,the result showed that the P2 showed stronger anti-fungi activity than the crude subtilin and P3-2 components in the same concentration.
对Bacillus subtilis FB123所产粗枯草菌素的理化性质及抑菌谱的研究表明粗枯草菌素最适合作用pH为6,具有较好的酸碱稳定性;最适作用温度为40℃,热稳定性较好。多种蛋白酶可使其部分失活。Na~+、Fe~(2+)、Zn~(2+)和Ca~(2+)离子对其具有增效作用,而Mg~(2+)、Al~(3+)、K~+和Cu~(2+)离子会部分抑制其抗菌活性。有机溶剂能明显抑制其活性。粗枯草菌素的抑菌谱较宽,对多种细菌以及真菌都具有良好的抗菌作用。
利用硫酸铵沉淀、Sephacryl S-200层析、DEAE-Sephamse Fast Flow层析从粗枯草菌素中分离出主要活性组分P2、P3-2、P4、P5。
P3-2经SDA-PAGE电泳鉴定为单亚基蛋白,分子量为14.8kDa,等电点为3.64,采用Edman降解法测得N端15个氨基酸序列为N-DREISEGSVDQSSWC-15。采用过碘酸-Schiff试剂染色法PAS和苏丹黑B脂染色法对P3-2进行糖基、脂基研究,结果表明该枯草菌素可能为一种不含糖的脂蛋白;P3-2对蛋白酶不敏感;具有广泛的pH活性范围;30℃为其最适作用温度,具有很好的热稳定性;有机溶剂能明显降低P3-2的抑菌活性;K~+、Zn~(2+)、Ca~(2+)、Na~+能部分抑制P3-2的活性,而Al~(3+)、Mg~(2+)、Fe~(2+)、Cu~(2+)对P3-2具有增强作用;P3-2对其亲缘相近的革兰氏阳性菌有强的抑制作用,对革兰氏阴性菌大肠杆菌无抑菌作用,但对部分真菌也有抗菌作用。
研究P2活性组分:1.测得P2 N端8个氨基酸序列为N-(Q、M)KPINISD-8。2.P2活性组分对部分真菌具有较强抑制作用,表明在相同浓度下P2的抑制真菌活性要比粗细菌素以及P3-2组分活性要高。
In the present study,subtilin produced by Bacillus subtilis FB123 were purified and characterized,and their primary structures were studied.
The characteristics and the antimicrobial spectrum of the crude subtilin were analyzed.The results showed that the crude subtilin kept its activity in a broad range of pH value and had the optimum pH of 6.0.The crude subtilin also showed a stronger thermostability and had the optimal culture temperature at 40℃.It can be partly inactivated by proteinase.The activity of crude subtilin could be partly improved by Na~+、Fe~(2+)、Zn~(2+)and Ca~(2+),partly inhibited by Mg~(2+)、Al~(3+)、K~+and Cu~(2+)Organic Solvents can strongly inhibit its activity.The antimicrobial spectrum tests showed that the crude subtilin exhibited stronger inhibition to both gram negative and positive bacteria used and had a weak inhibition to part fungi tested.
The crude subtilin was purified by ammonium sulfate precipitation,Sephacryl S-200,DEAE Sepharose Fast Flow chromatography and four components of subtilin(P2、P3-2、P4、P5)was obtained.
P3-2 was identified to be homogenous with SDS-PAGE,and its molecular weight and pI value were of 14.8 kDa and 3.64;15 amino acids residues of its N-terminal was measured by Edman degradation method,the secquence was N-DREISEGSVDQSSWC-15;The analysis based on periodic acid schiff reaction(PAS) and Sudan B lipid staining suggested P3-2 might be a lipoprotein,but not a glycoprotein; P3-2 showed insensitive to proteinase;and kept activity within wide pH value;it showed stronge thermal stability and with the optimal temperature of 30℃.The activity of P3-2 could be partly inhibited by K~+、Zn~(2+)、Ca~(2+)、Na~+and partly improved by Al~(3+)、Mg~(2+)、Fe~(2+)、Cu~(2+);P3-2showed broad ranges of anti-microbial activities not only to some gram-positive bacteria but also some fungi;but it showed no inhibition to Ecoli.
Supplementary study on the P2 component:1.the 8 amino acid residues in the N-terminal of P2 were measured,it was N-(Q、M)KPINISD-8;2.the anti-fungi activity of P2 component was studied,the result showed that the P2 showed stronger anti-fungi activity than the crude subtilin and P3-2 components in the same concentration.
引文
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