牛玻璃酸酶分离纯化及其部分酶学特性研究

发布时间：2018-03-31 22:18

  本文选题：牛睾丸　切入点：玻璃酸酶　出处：《甘肃农业大学》2014年硕士论文

【摘要】：玻璃酸酶是一类主要分解透明质酸的糖苷酶。在自然界中广泛存在，将其用于眼科、外科及牙科等手术麻醉时，可加速局部麻醉剂的扩散；对于手术及创伤后局部水肿或血肿有消散的功能。本研究以牛睾丸为原料，采用单因素试验和Plackett-Burman试验筛选对玻璃酸酶提取影响显著的因素；在此基础上，采用响应面试验对显著因素进行优化，获得玻璃酸酶提取的最佳参数；通过对填料、盐浓度、洗脱方式及流速等参数进行筛选，获得最佳纯化工艺；对纯化后的酶采用紫外全波长扫描和SDS-PAGE对酶及分子量进行初步鉴定；最后对酶进行部分特性的研究。本试验提高了牛副产物的利用率，且为牛玻璃酸酶的深入研究提供理论依据及参考。主要研究结果如下： 1.牛玻璃酸酶提取：采用单因素试验、Plackett-Burman试验及响应面试验对玻璃酸酶提取工艺条件进行研究，试验结果表明：浸提缓冲液pH值、NaCl浓度、料液比三个因素对提取工艺影响显著，且各因素对酶活力影响程度为：浸提缓冲液pH值NaCl浓度料液比；最佳提取条件为：浸提缓冲液pH值5.71、NaCl浓度0.14mol/L、料液比1:1.74。在此条件下，牛睾丸玻璃酸酶的比活力为118mU/mg，提取率为0.938%。 2.牛玻璃酸酶纯化：采用离子交换层析法初步纯化玻璃酸酶，最佳工艺条件为：选择SP Sepharose Fast Flow层析柱，采用分步洗脱方式，NaCl浓度为0、0.1、0.2、0.3、0.4、0.5mol/L。在此条件下，，玻璃酸酶的纯化倍数为7.53，回收率为67.87%。采用凝胶过滤层析法对玻璃酸酶进一步纯化，最佳工艺条件为：选择Sephacryl S-100层析柱，以0.5mL/min的流速收集玻璃酸酶，其纯化倍数为26.11，回收率为12.93%。 3.牛玻璃酸酶鉴定：采用紫外波长扫描，对N-乙酰-D-氨基葡萄糖标品及酶与底物的生成物进行扫描，结果显示：两条曲线基本吻合，均在544nm和585nm波长左右出现两个最高值点，故确定纯化出的酶是牛睾丸玻璃酸酶。采用SDS-PAGE对酶进行分子量的初步鉴定，试验结果显示出两条接近的条带，分子量分别约为60kDa和67kDa。 4.牛玻璃酸酶酶学特性：对酶的部分酶学特性进行研究，试验结果表明：最适反应温度和pH值分别为40℃、4.5，酶在20~40℃及pH值为4~5的范围内具有较好的稳定性；NaCl浓度为0.3mol/L时，酶活力相对较高；金属离子Fe3+、Ca2+、Mn2+、Mg2+、Zn2+、Al3+、K+对酶均有不同程度的抑制作用；该酶的Km值为0.106mg/mL。
[Abstract]:Hyaluronidase is a kind of glycosidase which mainly decomposes hyaluronic acid. It is widely found in nature and can accelerate the diffusion of local anesthetics when used in ophthalmic, surgical and dental anesthesia. In this study, bovine testis were used as raw materials, univariate test and Plackett-Burman test were used to screen the significant factors affecting the extraction of hyaluronidase. The parameters of hyaluronidase extraction were optimized by response surface test, and the optimal purification process was obtained by screening the parameters such as packing, salt concentration, elution mode and flow rate. The purified enzyme was preliminarily identified by UV full-wavelength scanning and SDS-PAGE. Finally, some properties of the enzyme were studied. It also provides theoretical basis and reference for further study of bovine hyaluronidase. The main results are as follows:. 1. Bovine hyaluronidase extraction: single factor test Plackett-Burman test and response surface test were used to study the extraction conditions of hyaluronidase. The results showed that the pH value of extraction buffer and the ratio of material to liquid had significant effects on the extraction process. The optimum extraction conditions were as follows: the pH value of the extraction buffer solution was 5.71 mol / L NaCl concentration, and the ratio of material to liquid was 1: 1.74. Under these conditions, the specific activity of bovine testis hyaluronidase was 118mUmgand the extraction rate was 0.938wt%. 2. Purification of bovine hyaluronidase: hyaluronidase was preliminarily purified by ion exchange chromatography. The optimum conditions were as follows: the SP Sepharose Fast Flow column was selected, and the concentration of 0 0. 1 ~ 0. 2 ~ 0. 2 ~ 0. 2 ~ 0. 3 ~ 0. 3 ~ 0. 4 ~ 0. 5 mol 路L ~ (-1) Flow was used as a stepwise elution method. The purification multiple of hyaluronidase was 7.53 and the recovery rate was 67.87.The optimum conditions for further purification of hyaluronidase by gel filtration chromatography were as follows: Sephacryl S-100 chromatographic column was selected and 0.5mL/min flow rate was used to collect hyaluronidase. The purification multiple was 26.11 and the recovery was 12.93%. 3. Identification of bovine hyaluronidase: UV wavelength scanning was used to scan N-acetyl-Dglucosamine standard and the products of enzyme and substrate. The results showed that the two curves were basically consistent, and there were two highest points in the wavelength of 544nm and 585nm. Therefore, the purified enzyme was bovine testis hyaluronidase. The molecular weight of the enzyme was preliminarily identified by SDS-PAGE. The results showed that there were two similar bands, the molecular weight of which was about 60kDa and 67kDa, respectively. 4. Enzymatic properties of bovine hyaluronidase. The results showed that the optimum reaction temperature and pH value were 40 鈩

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