果胶酶固定化载体的制备及固定化工艺、酶学特性研究
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摘要
果胶酶是由黑曲霉经发酵精制而得的用于催化植物细胞间质(即果胶质)水解成半乳糖醛酸和果胶酸的一类酶,主要应用于果蔬汁饮料及果酒的榨汁和澄清,对分解果胶物质具有良好的作用。在我国的果汁果酒制造行业,果胶酶的加入可以提高水果蔬菜汁的出汁率及其稳定性,并防止果蔬汁后混浊的发生。但在实际的工业应用中,使用游离酶存在着酶用量大、生产成本高、精制困难等缺点,而固定化酶则可避免这些缺点,扩大应用范围,降低企业的生产成本。
目前,各个研究机构及有关企业对于果胶酶固定化的研究已有较多的报道,但以磁性微球为载体固定果胶酶的还研究比较少。本论文以自制的壳聚糖磁性微球为载体,首先探讨制备载体的工艺参数；在成功获得载体的基础上,将果胶酶固定化,研究最适的固定化工艺条件,并研究固定化果胶酶的酶学特性。主要试验结果如下：
1、本试验采用改良化学共沉淀法通过正交试验设计优化合成磁性Fe304纳米粒子的最佳条件,并将自制的纳米Fe304作为磁核用乳化交联法通过单因素及正交试验制备载体—磁性壳聚糖微球。自制磁核的最佳条件为：Fe2+与Fe3+之比为1：1.7(n／n)、pH值9-10、晶化时间0.5h、晶化温度80℃；自制磁性壳聚糖微球的最佳工艺参数为：壳聚糖浓度1.5%、油水相比例为4：1(mL／mL)、乳化剂用量2 mL、交联剂用量1.0 mL、反应温度50℃-70℃。
2、利用扫描电子显微镜、透射电子显微镜、X-射线衍射仪、傅立叶变换红外光谱仪、激光粒度仪、紫外分光光度计、振动样品磁强计等分别对自制的磁核及微球的外貌形态、颗粒直径及粒度分布、内部结构和磁学特性等进行了表征。试验结果显示,自制的磁核—纳米级Fe304其粒度分布较窄,分散性也较好,实际测得的平均粒径为14.1nm,且具有良好的超顺磁性和晶体结构。自制的果胶酶固定化载体—磁性壳聚糖微球尺寸均匀、成球性较好,实测平均粒径在30μm左右,虽然自制微球具有凹凸面,但微球的骨架结构仍比较完整,也没有团聚现象；自制微球红外光谱特征官能团结构测定结果表明,Fe304粒子成功被壳聚糖材料包覆,且包埋前后的纳米Fe304粒子其晶体结构未改变；试验采用的分光光度法磁性测定结果显示,自制磁性微球磁响应性很强,是酶固定化的优良载体材料。
3、以磁性壳聚糖微球为载体采用单因素试验确定了果胶酶固定化的最佳工艺参数：固定化时间6h、pH值4.0、加酶量1.25 mL、反应温度40℃,在此条件下固定化果胶酶的酶活回收率比较高为83%。
4、对自制固定化果胶酶和游离果胶酶的酶学性质进行了比较,试验结果显示：固定化果胶酶最佳催化温度为60℃,比游离酶(其最佳温度为50℃)高10℃,温度耐受性提高,最佳pH值为4.0,比游离酶的范围稍宽(游离酶最佳pH值为3.5),且固定化酶重复使用六次仍能保持较高活性。
5、以自制固定化果胶酶对苹果汁进行了初步应用研究,试验结果表明：固定化果胶酶用量58mg／L、酶解pH14.0、酶解温度60℃、酶解时间60min的条件下,固定化果胶酶对苹果汁澄清处理的效果较好,透光率可达88.7%以上,且果胶试验呈阴性。
Pectinase is one of enzymes to which degrade pectin into galacturonicacid, it was come from the fermentation of Aspergillus Niger. Pectinolytic enzyme was applicated for juice squeeze and clarification in industry of vegetable, fruit juice, wines and liqueurs to improve the yield and steadiness of juice mainly. Pectinase also be used to avoid the phenomenon of "after haze" in vegetable and fruit juice. But in the industry using of pectinase, dissociated enzymes have so many shortcomings, such as large enzyme's amount, high product cost, difficult refined. Those defects affect pectinase's application in food industry greatly. And then immobilized enzyme may avoid the above weakness in some degree. At the same time, immobilized enzyme can decrease product cost and enlarge the enzyme's utilization.
There are some studying reports of pectinase immobilization at present in some enterprises and academic institution. But the study for magnetic chitosan microspheres support immobilized pectinase is few. In this paper, the magnetic chitosan microspheres support was prepared, and the conditions of preparation were discussed. On the basis of first studying, the pectinase was immobilized on the magnetic chitosan microspheres support. The optimal conditions of immobilization, characteristics of immobilized enzyme were mainly investigated. The experimental results were as follows:
1、With better coprecipitation method, magnetic nanometer Fe3O4 particle was composed, and then using suspension cross-linking technique, the support (magnetic chitosan microspheres) were prepared. The optimal preparation conditions of nanometer Fe3O4 particle were as follow:Fe3+:Fe2+ is 1.7:1, pH 9-10, the temperature of crystallization 80℃, the crystallization time 0.5 h. The optimal preparation conditions of magnetic chitosan microspheres support through orthogonal experiment were as follow:chitosan concentration 1.5%, oil-water ratio 4:1, the amount of emulsifier 4 mL, the amount of crosslinker 1.0 mL, reaction temperature from 50℃to 70℃.
2、The support and magnetic core appearance, diameter and size distribution, scrystal strueture and magnetie response properties were characterized by means of Scanning Electron Microscopy,Transmission Electron Microscopy, X-Ray Diffractometer, FT-IR Spectrometers, Ultraviolet Spectrophotometer, Laser Particle Sizer, Vibrating Sample Magnetometer. The results indicate that the magnetic Fe3O4 nanopartieles have narrow size-distribution and fine dispersion stability. Fe3O4 nanopartieles also have fine scrystal strueture and superparamagnetic, their average sizes achieve 14.1nm. The supports (magnetic chitosan microspheres) have fine dispersion and narrow size distribution, average size is about 30μm. Although the surface of magnetic chitosan microspheres presents concavo-convex and the hollows, the support has intact skeleton construeture and has no agglomeration. The FT-IR results of support indicate that the Fe3O4 particle was covered by the chitosan utterly and there is no change in Fe3O4 particle structure. The spectrophotometry results show that magnetic chitosan microspheres have fine magnetic response properties. It is a kind of good carrier of pectinolytic enzyme.immobilization.
3、The optimal conditions of pectinase immobilization were investigated through single factor experiment using magnetic chitosan microspheres as supports. The optimum immobilizd conditions were:time 6 h, pH 4.0,40℃,1.25 mL pectniase. Using those optimal conditions, the immobilized pectinase was prepared and its activity recovery will get to the highest.
4、The properties of immobilized pectinase and free pectinase were compared. The test results show that immobilized pectinase optimum reacting temperature was 60℃(the free enzyme's 50℃), the immobilization enzyme's thermal stability has increased. The optimum reacting pH of immobilized pectinase was 4.0(the free enzyme's pH 3.5). It indicated that immobilized pectinolytic enzyme had better pH tolerance, its reused times was six and its function is better than free pectinase.
5、Self-made immobilized pectinase was applied in apple juice for clarity. The test results show that immobilized pectinase 58mg/L, enzymatic pH4.0, hydrolysis temperature 60℃, time 60min. Under those conditions, the light transmission rate of apple juice get up to 88.7%, and the pectin test was negative.
目前,各个研究机构及有关企业对于果胶酶固定化的研究已有较多的报道,但以磁性微球为载体固定果胶酶的还研究比较少。本论文以自制的壳聚糖磁性微球为载体,首先探讨制备载体的工艺参数；在成功获得载体的基础上,将果胶酶固定化,研究最适的固定化工艺条件,并研究固定化果胶酶的酶学特性。主要试验结果如下：
1、本试验采用改良化学共沉淀法通过正交试验设计优化合成磁性Fe304纳米粒子的最佳条件,并将自制的纳米Fe304作为磁核用乳化交联法通过单因素及正交试验制备载体—磁性壳聚糖微球。自制磁核的最佳条件为：Fe2+与Fe3+之比为1：1.7(n／n)、pH值9-10、晶化时间0.5h、晶化温度80℃；自制磁性壳聚糖微球的最佳工艺参数为：壳聚糖浓度1.5%、油水相比例为4：1(mL／mL)、乳化剂用量2 mL、交联剂用量1.0 mL、反应温度50℃-70℃。
2、利用扫描电子显微镜、透射电子显微镜、X-射线衍射仪、傅立叶变换红外光谱仪、激光粒度仪、紫外分光光度计、振动样品磁强计等分别对自制的磁核及微球的外貌形态、颗粒直径及粒度分布、内部结构和磁学特性等进行了表征。试验结果显示,自制的磁核—纳米级Fe304其粒度分布较窄,分散性也较好,实际测得的平均粒径为14.1nm,且具有良好的超顺磁性和晶体结构。自制的果胶酶固定化载体—磁性壳聚糖微球尺寸均匀、成球性较好,实测平均粒径在30μm左右,虽然自制微球具有凹凸面,但微球的骨架结构仍比较完整,也没有团聚现象；自制微球红外光谱特征官能团结构测定结果表明,Fe304粒子成功被壳聚糖材料包覆,且包埋前后的纳米Fe304粒子其晶体结构未改变；试验采用的分光光度法磁性测定结果显示,自制磁性微球磁响应性很强,是酶固定化的优良载体材料。
3、以磁性壳聚糖微球为载体采用单因素试验确定了果胶酶固定化的最佳工艺参数：固定化时间6h、pH值4.0、加酶量1.25 mL、反应温度40℃,在此条件下固定化果胶酶的酶活回收率比较高为83%。
4、对自制固定化果胶酶和游离果胶酶的酶学性质进行了比较,试验结果显示：固定化果胶酶最佳催化温度为60℃,比游离酶(其最佳温度为50℃)高10℃,温度耐受性提高,最佳pH值为4.0,比游离酶的范围稍宽(游离酶最佳pH值为3.5),且固定化酶重复使用六次仍能保持较高活性。
5、以自制固定化果胶酶对苹果汁进行了初步应用研究,试验结果表明：固定化果胶酶用量58mg／L、酶解pH14.0、酶解温度60℃、酶解时间60min的条件下,固定化果胶酶对苹果汁澄清处理的效果较好,透光率可达88.7%以上,且果胶试验呈阴性。
Pectinase is one of enzymes to which degrade pectin into galacturonicacid, it was come from the fermentation of Aspergillus Niger. Pectinolytic enzyme was applicated for juice squeeze and clarification in industry of vegetable, fruit juice, wines and liqueurs to improve the yield and steadiness of juice mainly. Pectinase also be used to avoid the phenomenon of "after haze" in vegetable and fruit juice. But in the industry using of pectinase, dissociated enzymes have so many shortcomings, such as large enzyme's amount, high product cost, difficult refined. Those defects affect pectinase's application in food industry greatly. And then immobilized enzyme may avoid the above weakness in some degree. At the same time, immobilized enzyme can decrease product cost and enlarge the enzyme's utilization.
There are some studying reports of pectinase immobilization at present in some enterprises and academic institution. But the study for magnetic chitosan microspheres support immobilized pectinase is few. In this paper, the magnetic chitosan microspheres support was prepared, and the conditions of preparation were discussed. On the basis of first studying, the pectinase was immobilized on the magnetic chitosan microspheres support. The optimal conditions of immobilization, characteristics of immobilized enzyme were mainly investigated. The experimental results were as follows:
1、With better coprecipitation method, magnetic nanometer Fe3O4 particle was composed, and then using suspension cross-linking technique, the support (magnetic chitosan microspheres) were prepared. The optimal preparation conditions of nanometer Fe3O4 particle were as follow:Fe3+:Fe2+ is 1.7:1, pH 9-10, the temperature of crystallization 80℃, the crystallization time 0.5 h. The optimal preparation conditions of magnetic chitosan microspheres support through orthogonal experiment were as follow:chitosan concentration 1.5%, oil-water ratio 4:1, the amount of emulsifier 4 mL, the amount of crosslinker 1.0 mL, reaction temperature from 50℃to 70℃.
2、The support and magnetic core appearance, diameter and size distribution, scrystal strueture and magnetie response properties were characterized by means of Scanning Electron Microscopy,Transmission Electron Microscopy, X-Ray Diffractometer, FT-IR Spectrometers, Ultraviolet Spectrophotometer, Laser Particle Sizer, Vibrating Sample Magnetometer. The results indicate that the magnetic Fe3O4 nanopartieles have narrow size-distribution and fine dispersion stability. Fe3O4 nanopartieles also have fine scrystal strueture and superparamagnetic, their average sizes achieve 14.1nm. The supports (magnetic chitosan microspheres) have fine dispersion and narrow size distribution, average size is about 30μm. Although the surface of magnetic chitosan microspheres presents concavo-convex and the hollows, the support has intact skeleton construeture and has no agglomeration. The FT-IR results of support indicate that the Fe3O4 particle was covered by the chitosan utterly and there is no change in Fe3O4 particle structure. The spectrophotometry results show that magnetic chitosan microspheres have fine magnetic response properties. It is a kind of good carrier of pectinolytic enzyme.immobilization.
3、The optimal conditions of pectinase immobilization were investigated through single factor experiment using magnetic chitosan microspheres as supports. The optimum immobilizd conditions were:time 6 h, pH 4.0,40℃,1.25 mL pectniase. Using those optimal conditions, the immobilized pectinase was prepared and its activity recovery will get to the highest.
4、The properties of immobilized pectinase and free pectinase were compared. The test results show that immobilized pectinase optimum reacting temperature was 60℃(the free enzyme's 50℃), the immobilization enzyme's thermal stability has increased. The optimum reacting pH of immobilized pectinase was 4.0(the free enzyme's pH 3.5). It indicated that immobilized pectinolytic enzyme had better pH tolerance, its reused times was six and its function is better than free pectinase.
5、Self-made immobilized pectinase was applied in apple juice for clarity. The test results show that immobilized pectinase 58mg/L, enzymatic pH4.0, hydrolysis temperature 60℃, time 60min. Under those conditions, the light transmission rate of apple juice get up to 88.7%, and the pectin test was negative.
引文
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