交联纤维素酶聚集体的酶学性质及其作用机理
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摘要
传统的酶固定化方法存在着酶活回收率不高、环境适应性较差以及结构不太稳定等问题,开发高酶活和高稳定性的固定化酶成为酶应用研究的重要课题,交联酶聚集体(Cross-linked enzyme aggregates,CLEAs)技术以其制备技术简单、酶活回收率高、良好环境适应性等独特优势,日益引起人们的关注和研究。
本研究以纤维素酶为研究对象,主要研究了交联纤维素酶聚集体(Cross-linked cellulase aggregates,CLEAs-C)的制备工艺和酶学性质,从酶聚集体的颗粒尺度以及微观结构对CLEAs-C的催化作用机理进行了研究和探讨。主要研究内容和结果如下:
以硫酸铵为沉淀剂,戊二醛为交联剂制备CLEAs-C。最优沉淀工艺参数为:硫酸铵饱和度、硫酸铵pH以及酶浓度分别为95%、6.0和50mg/mL;最佳的交联参数为戊二醛浓度、交联温度、交联时间为3%、30℃和10h,获得的CLEAs-C相对酶活回收率较高,约为80%。
对CLEAs-C的酶学性质进行系统的研究,与游离酶相比,CLEAs-C的最适温度为60℃,提高了10℃,最适pH和离子浓度没有变化,分别为pH3.0和0.2mol/L;CLEAs-C对温度和pH稳定性均优于游离酶,并且储存28d之后仍有80%以上的储存酶活回收率;在最佳制备条件和酶解条件下,CLEAs-C的酶活回收率比游离酶提高了约2.5倍。
采用傅立叶红外光谱仪、原子力显微镜等对CLEAs-C的特征及结构进行了分析。结果表明,CLEAs-C的刚性二级结构有所增加,具有更好的环境稳定性;并推测在CLEAs-C内部具有一定的空隙,有利于CLEAs-C内部的活性中心伸展以及底物分子与活性部分的充分接触,从而实现提高CLEAs-C的催化能力。
运用马尔文激光粒度仪对不同搅拌速度制备的CLEAs-C的颗粒尺度进行分析,并结合其相对酶活回收率,初步研究并探讨了CLEAs-C的酶解机理,即CLEAs-C的酶活力随其颗粒尺度的增加呈现先增加,达到最高酶活力后再减低的趋势。当聚集体粒径小时,一方面被交联的纤维素酶少,另一方面聚集体内形成的空间结构小,不利于底物与活性部位接触。而当粒径过大时,聚集体的比表面小,也不利于底物与活性部位接触。只有适当的颗粒尺度时,聚集体有适当的比表面,适当的内部空间结构以及适当的聚集程度,CLEAs-C才呈现最好的酶催化作用。
Cross-linked enzyme aggregates(CLEAs) exhibit high active recovery, steady structure and stronger environment adaptability compared to the immobilized enzymes prepared by traditional methods. In this study, cross-linked cellulase aggregates were prepared by cellulase Trichoderma viride. The preparation process and properties of cross-linked cellulase aggregates (CLEAs-C) were investigated. The reaction mechanism of CLEAs-C was studied by analyzing the structure and characteristics. The main results listed as followed:
CLEAs-C were prepared by ammonium sulfate used as the precipitant and glutaraldehyde used as and cross-linker. The results showed the optimum parameters of preparing CLEAs-C were: ammonium sulfate saturation of 95%, pH value of 6.0, the amount of cellulases of 50mg/mL, glutaraldehyde concentration of 3%, immobilization temperature of 30℃, immobilization time of 10h. Under the optimum parameters, the maximal relatively active recovery of CLEAs-C was about 80%.
The enzymatic properties of CLEAs-C were studied in this paper. Compared with free cellulase, the optimal temperature of CLEAs-C was improved from 50℃to 60℃, the optimal pH value and ion concentration were 3.0 and 0.2mol/L respectively, which was the same as those of free cellulases. The stability of temperature and pH of CLEAs-C were superior to free cellulase, CLEAs-C had above 80% store activity recovery after 28 days compared with initial activity. Under the optimal conditions of enzymic preparation and hydrolysis, the highest activity recovery of CLEAs-C was increased by 250% compared with free cellulases.
The characters and stutruct structure of CLEAs-C were analyzed using FTIR, and Atomic Force Microscope. The results showed the rigid structures of secondary structure were increased, exhibiting a better stability. It was speculated that there were some interspace in CLEAs-C, which make made the active sites of cellulases extend and allow the substrate molecule entering to contact with active sites, improving the enzymatic hydrolysis could be happened.
The partical size of CLEAs-C prepared by different stirring rate was analyzed by Malvern Laser Particle Sizer, and the reaction mechanism of CLEAs-C was studied in terms of relatively active recovery of CLEAs-C. The results showed the activity of CLEAs-C increased firstly and reached the maximum, and then decreased with particle size. When the particle size of CLEAs-C was small, it meant less cellulases were cross-linked or less steric space inside the CLEAs-C, resulting into lower catalysis capacity. However, in the case of large particle size, the specific surface area of aggregates was small, decreasing the possibility of contacting between cellulase and substrate. Therefore, when CLEAs-C had proper particle sizes, proper specific surface area, proper steric space inside and proper degree of aggregation, CLEAs-C inhibited high catalysis capacity.
本研究以纤维素酶为研究对象,主要研究了交联纤维素酶聚集体(Cross-linked cellulase aggregates,CLEAs-C)的制备工艺和酶学性质,从酶聚集体的颗粒尺度以及微观结构对CLEAs-C的催化作用机理进行了研究和探讨。主要研究内容和结果如下:
以硫酸铵为沉淀剂,戊二醛为交联剂制备CLEAs-C。最优沉淀工艺参数为:硫酸铵饱和度、硫酸铵pH以及酶浓度分别为95%、6.0和50mg/mL;最佳的交联参数为戊二醛浓度、交联温度、交联时间为3%、30℃和10h,获得的CLEAs-C相对酶活回收率较高,约为80%。
对CLEAs-C的酶学性质进行系统的研究,与游离酶相比,CLEAs-C的最适温度为60℃,提高了10℃,最适pH和离子浓度没有变化,分别为pH3.0和0.2mol/L;CLEAs-C对温度和pH稳定性均优于游离酶,并且储存28d之后仍有80%以上的储存酶活回收率;在最佳制备条件和酶解条件下,CLEAs-C的酶活回收率比游离酶提高了约2.5倍。
采用傅立叶红外光谱仪、原子力显微镜等对CLEAs-C的特征及结构进行了分析。结果表明,CLEAs-C的刚性二级结构有所增加,具有更好的环境稳定性;并推测在CLEAs-C内部具有一定的空隙,有利于CLEAs-C内部的活性中心伸展以及底物分子与活性部分的充分接触,从而实现提高CLEAs-C的催化能力。
运用马尔文激光粒度仪对不同搅拌速度制备的CLEAs-C的颗粒尺度进行分析,并结合其相对酶活回收率,初步研究并探讨了CLEAs-C的酶解机理,即CLEAs-C的酶活力随其颗粒尺度的增加呈现先增加,达到最高酶活力后再减低的趋势。当聚集体粒径小时,一方面被交联的纤维素酶少,另一方面聚集体内形成的空间结构小,不利于底物与活性部位接触。而当粒径过大时,聚集体的比表面小,也不利于底物与活性部位接触。只有适当的颗粒尺度时,聚集体有适当的比表面,适当的内部空间结构以及适当的聚集程度,CLEAs-C才呈现最好的酶催化作用。
Cross-linked enzyme aggregates(CLEAs) exhibit high active recovery, steady structure and stronger environment adaptability compared to the immobilized enzymes prepared by traditional methods. In this study, cross-linked cellulase aggregates were prepared by cellulase Trichoderma viride. The preparation process and properties of cross-linked cellulase aggregates (CLEAs-C) were investigated. The reaction mechanism of CLEAs-C was studied by analyzing the structure and characteristics. The main results listed as followed:
CLEAs-C were prepared by ammonium sulfate used as the precipitant and glutaraldehyde used as and cross-linker. The results showed the optimum parameters of preparing CLEAs-C were: ammonium sulfate saturation of 95%, pH value of 6.0, the amount of cellulases of 50mg/mL, glutaraldehyde concentration of 3%, immobilization temperature of 30℃, immobilization time of 10h. Under the optimum parameters, the maximal relatively active recovery of CLEAs-C was about 80%.
The enzymatic properties of CLEAs-C were studied in this paper. Compared with free cellulase, the optimal temperature of CLEAs-C was improved from 50℃to 60℃, the optimal pH value and ion concentration were 3.0 and 0.2mol/L respectively, which was the same as those of free cellulases. The stability of temperature and pH of CLEAs-C were superior to free cellulase, CLEAs-C had above 80% store activity recovery after 28 days compared with initial activity. Under the optimal conditions of enzymic preparation and hydrolysis, the highest activity recovery of CLEAs-C was increased by 250% compared with free cellulases.
The characters and stutruct structure of CLEAs-C were analyzed using FTIR, and Atomic Force Microscope. The results showed the rigid structures of secondary structure were increased, exhibiting a better stability. It was speculated that there were some interspace in CLEAs-C, which make made the active sites of cellulases extend and allow the substrate molecule entering to contact with active sites, improving the enzymatic hydrolysis could be happened.
The partical size of CLEAs-C prepared by different stirring rate was analyzed by Malvern Laser Particle Sizer, and the reaction mechanism of CLEAs-C was studied in terms of relatively active recovery of CLEAs-C. The results showed the activity of CLEAs-C increased firstly and reached the maximum, and then decreased with particle size. When the particle size of CLEAs-C was small, it meant less cellulases were cross-linked or less steric space inside the CLEAs-C, resulting into lower catalysis capacity. However, in the case of large particle size, the specific surface area of aggregates was small, decreasing the possibility of contacting between cellulase and substrate. Therefore, when CLEAs-C had proper particle sizes, proper specific surface area, proper steric space inside and proper degree of aggregation, CLEAs-C inhibited high catalysis capacity.
引文
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