海洋芽孢杆菌酯酶BSE-1的分离纯化和生化性质研究
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摘要
海洋芽孢杆菌酯酶BSE-1,是一株海洋芽孢杆菌(Bacillus sp.)分泌的碱性嗜热酶,本文主要针对海洋细菌酯酶产生菌的菌种筛选和鉴定、酯酶分离纯化、酶学性质、酶的催化动力学以及热失活动力学进行了系统研究。以期为进一步研究海洋酯酶的酶学特性、结构与功能的关系创造条件,同时也为海洋细菌酯酶的工业化生产和应用奠定良好的基础。
菌种筛选过程中,根据酯酶的作用特性和机理,在培养基中添加三丁酸甘油酯和橄榄油等特异底物和Rhodamine B等指示剂,建立了一种简便有效的筛选模型,最终筛选出酯酶的高产菌株( EB-1),产酶活力达到22.8 U/ mL。在此基础上对菌株EB-1进行了形态结构、培养特征、生理生化特征等常规分类鉴定和16S rDNA的分子分类鉴定研究,最终将该菌株鉴定为Bacillus subtilis var. niger。
在发酵培养基中添加0.5%的橄榄油和0.5%的甘油后,成功将胞内酯酶诱导到胞外。胞外酯酶发酵液通过低温高速离心(10 000r/min,30min,4℃)、乙醇-硫酸铵双水相沉淀和Q-Sepharose HR阴离子交换层析等技术进行纯化,最终得到电泳纯的酯酶BSE-1。SDS-PAGE结果表明,该酶表观分子量为30 kDa。以对硝基苯磷酸酯(PNPP)为底物对BSE-1进行理化性质研究,结果表明,BSE-1的最适反应温度为60℃,最适pH=10.0,为嗜热碱性酯酶;BSE-1在60℃以下、pH 7~11范围内具有良好的热稳定性;与常见酸根离子、有机溶剂的配伍性较好,但金属离子对酶活影响较大,Li+、Mg2+、Ca2+有激活作用,Ba2+、Fe3+、Ag+、Sr2+有抑制作用。EDTA对BSE-1的抑制作用明显,在反应体系中添加EDTA的浓度≥0.5mmol/L时, BSE-1的酯酶活性完全被抑制。
以PNPP为底物,对电泳纯BSE-1的催化性质、催化动力学和热失活动力学进行了研究。BSE-1的酶促反应动力学符合米氏方程,其中Km=8.15 mmol·L?1, Vmax=0.97 mmol·mg?1·min?1。金属离子动力学研究表明,Ca2+为混合型激活作用,Ba2+为非竞争性抑制作用。构建BSE-1热失活的连续模型,并对BSE-1在70℃的热失活过程进行模拟,求得酶的失活速率常数k1=1.41,k2=0.28。
Marine Bacillus sp. esterase BSE-1 was secreted by marine Bacillus sp.. The dissertation focused on the screening and identification of the strains, separation and purification of BSE-1, and its properties, catalytic kinetic, thermal inactivation dynamics etc. The research will make a basis to its further structure-functional study, its industrialization and application.
Based on the fundamental principle and speciality of the microbial esterase, proper substrates (Tributyrin) and indicators (Rhodamine B) were used to screen the esterase-producing bacteria in the preliminary and secondary screening. A precise and rapid screening method was developed. The strain of EB-1, which was isolated from related marine soil samples, showed the highest esterase-producing ability (22.8 U/ mL). The strain EB-1 was identified as Bacillus subtilis var. niger based on the analysis of the morphological, culture, physiological biochemical characteristics and the sequence of 16S rDNA. BSE-1 was induced from intracellular enzyme to extracellular enzyme by adding 0.5% olive oil and 0.5% glycerol in the culture medium.
SDS-PAGE homogeneity BSE-1 was purified from the fermentation liquid by high speed freezing centrifugation (10000r/min, 30min, 4℃), ethanol-ammonium sulfate aqueous two-phase precipitation and Q-Sepharose HR anion exchange chromatography. SDS-PAGE showed its molecular weight was 30 kDa. In further studies, with PNPP as substrate,the results showed that the optimum pH of this esterase was 10.0 and its optimum reaction temperature was 60℃. BSE-1 was stable when the temperature was below 60℃and pH among 7~11. This character gave BSE-1 good property to apply under higher temperature. BSE-1 also showed high activity at rather higher alkaline pH (higher than 10). BSE-1 was compatible with some acid radical ions and showed good resistance to general organic solvent. The effects of metal ions on BSE-1 showed that Li+,Mg2+,Ca2+ could increase its activity. On the other hand, Ba2+,Fe3+,Ag+,Sr2+ inhibited the activity of BSE-1. The inhibition of EDTA on BSE-1 was remarkable, which suggested there were metal ions in the active centre of BSE-1.
The catalytic kinetics and thermal inactivation kinetics of a novel alkaline esterase BSE-1 were studied with the PNPP as substrate. The behavior of the BSE-1 on PNPP hydrolization followed Michaelis- Menten kinetics,with Km= 8.15 mmol·L-1 and Vmax=0.97 mmol·mg-1·min-1. The effect of Ca2+ on the enzyme was found to be of mixed activation type. The inhibition of Ba2+ on the enzyme appeared to be partial noncompetitive type. In addition,thermal deactivation kinetics of BSE-1 was analyzed by continuous model under the temperature of 70℃. Suitable mechanism formula was proposed to describe the inactivation process. And the constants of thermal inactivation rate were k1=1.41 and k2=0.28.
菌种筛选过程中,根据酯酶的作用特性和机理,在培养基中添加三丁酸甘油酯和橄榄油等特异底物和Rhodamine B等指示剂,建立了一种简便有效的筛选模型,最终筛选出酯酶的高产菌株( EB-1),产酶活力达到22.8 U/ mL。在此基础上对菌株EB-1进行了形态结构、培养特征、生理生化特征等常规分类鉴定和16S rDNA的分子分类鉴定研究,最终将该菌株鉴定为Bacillus subtilis var. niger。
在发酵培养基中添加0.5%的橄榄油和0.5%的甘油后,成功将胞内酯酶诱导到胞外。胞外酯酶发酵液通过低温高速离心(10 000r/min,30min,4℃)、乙醇-硫酸铵双水相沉淀和Q-Sepharose HR阴离子交换层析等技术进行纯化,最终得到电泳纯的酯酶BSE-1。SDS-PAGE结果表明,该酶表观分子量为30 kDa。以对硝基苯磷酸酯(PNPP)为底物对BSE-1进行理化性质研究,结果表明,BSE-1的最适反应温度为60℃,最适pH=10.0,为嗜热碱性酯酶;BSE-1在60℃以下、pH 7~11范围内具有良好的热稳定性;与常见酸根离子、有机溶剂的配伍性较好,但金属离子对酶活影响较大,Li+、Mg2+、Ca2+有激活作用,Ba2+、Fe3+、Ag+、Sr2+有抑制作用。EDTA对BSE-1的抑制作用明显,在反应体系中添加EDTA的浓度≥0.5mmol/L时, BSE-1的酯酶活性完全被抑制。
以PNPP为底物,对电泳纯BSE-1的催化性质、催化动力学和热失活动力学进行了研究。BSE-1的酶促反应动力学符合米氏方程,其中Km=8.15 mmol·L?1, Vmax=0.97 mmol·mg?1·min?1。金属离子动力学研究表明,Ca2+为混合型激活作用,Ba2+为非竞争性抑制作用。构建BSE-1热失活的连续模型,并对BSE-1在70℃的热失活过程进行模拟,求得酶的失活速率常数k1=1.41,k2=0.28。
Marine Bacillus sp. esterase BSE-1 was secreted by marine Bacillus sp.. The dissertation focused on the screening and identification of the strains, separation and purification of BSE-1, and its properties, catalytic kinetic, thermal inactivation dynamics etc. The research will make a basis to its further structure-functional study, its industrialization and application.
Based on the fundamental principle and speciality of the microbial esterase, proper substrates (Tributyrin) and indicators (Rhodamine B) were used to screen the esterase-producing bacteria in the preliminary and secondary screening. A precise and rapid screening method was developed. The strain of EB-1, which was isolated from related marine soil samples, showed the highest esterase-producing ability (22.8 U/ mL). The strain EB-1 was identified as Bacillus subtilis var. niger based on the analysis of the morphological, culture, physiological biochemical characteristics and the sequence of 16S rDNA. BSE-1 was induced from intracellular enzyme to extracellular enzyme by adding 0.5% olive oil and 0.5% glycerol in the culture medium.
SDS-PAGE homogeneity BSE-1 was purified from the fermentation liquid by high speed freezing centrifugation (10000r/min, 30min, 4℃), ethanol-ammonium sulfate aqueous two-phase precipitation and Q-Sepharose HR anion exchange chromatography. SDS-PAGE showed its molecular weight was 30 kDa. In further studies, with PNPP as substrate,the results showed that the optimum pH of this esterase was 10.0 and its optimum reaction temperature was 60℃. BSE-1 was stable when the temperature was below 60℃and pH among 7~11. This character gave BSE-1 good property to apply under higher temperature. BSE-1 also showed high activity at rather higher alkaline pH (higher than 10). BSE-1 was compatible with some acid radical ions and showed good resistance to general organic solvent. The effects of metal ions on BSE-1 showed that Li+,Mg2+,Ca2+ could increase its activity. On the other hand, Ba2+,Fe3+,Ag+,Sr2+ inhibited the activity of BSE-1. The inhibition of EDTA on BSE-1 was remarkable, which suggested there were metal ions in the active centre of BSE-1.
The catalytic kinetics and thermal inactivation kinetics of a novel alkaline esterase BSE-1 were studied with the PNPP as substrate. The behavior of the BSE-1 on PNPP hydrolization followed Michaelis- Menten kinetics,with Km= 8.15 mmol·L-1 and Vmax=0.97 mmol·mg-1·min-1. The effect of Ca2+ on the enzyme was found to be of mixed activation type. The inhibition of Ba2+ on the enzyme appeared to be partial noncompetitive type. In addition,thermal deactivation kinetics of BSE-1 was analyzed by continuous model under the temperature of 70℃. Suitable mechanism formula was proposed to describe the inactivation process. And the constants of thermal inactivation rate were k1=1.41 and k2=0.28.
引文
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