本文選題：牛睪丸　切入點：玻璃酸酶　出處：《甘肅農(nóng)業(yè)大學》2014年碩士論文

【摘要】：玻璃酸酶是一類主要分解透明質(zhì)酸的糖苷酶。在自然界中廣泛存在，將其用于眼科、外科及牙科等手術麻醉時，可加速局部麻醉劑的擴散；對于手術及創(chuàng)傷后局部水腫或血腫有消散的功能。本研究以牛睪丸為原料，采用單因素試驗和Plackett-Burman試驗篩選對玻璃酸酶提取影響顯著的因素；在此基礎上，采用響應面試驗對顯著因素進行優(yōu)化，獲得玻璃酸酶提取的最佳參數(shù)；通過對填料、鹽濃度、洗脫方式及流速等參數(shù)進行篩選，獲得最佳純化工藝；對純化后的酶采用紫外全波長掃描和SDS-PAGE對酶及分子量進行初步鑒定；最后對酶進行部分特性的研究。本試驗提高了牛副產(chǎn)物的利用率，且為牛玻璃酸酶的深入研究提供理論依據(jù)及參考。主要研究結果如下： 1.牛玻璃酸酶提�。翰捎脝我蛩卦囼�、Plackett-Burman試驗及響應面試驗對玻璃酸酶提取工藝條件進行研究，試驗結果表明：浸提緩沖液pH值、NaCl濃度、料液比三個因素對提取工藝影響顯著，且各因素對酶活力影響程度為：浸提緩沖液pH值NaCl濃度料液比；最佳提取條件為：浸提緩沖液pH值5.71、NaCl濃度0.14mol/L、料液比1:1.74。在此條件下，牛睪丸玻璃酸酶的比活力為118mU/mg，提取率為0.938%。 2.牛玻璃酸酶純化：采用離子交換層析法初步純化玻璃酸酶，最佳工藝條件為：選擇SP Sepharose Fast Flow層析柱，采用分步洗脫方式，NaCl濃度為0、0.1、0.2、0.3、0.4、0.5mol/L。在此條件下，，玻璃酸酶的純化倍數(shù)為7.53，回收率為67.87%。采用凝膠過濾層析法對玻璃酸酶進一步純化，最佳工藝條件為：選擇Sephacryl S-100層析柱，以0.5mL/min的流速收集玻璃酸酶，其純化倍數(shù)為26.11，回收率為12.93%。 3.牛玻璃酸酶鑒定：采用紫外波長掃描，對N-乙酰-D-氨基葡萄糖標品及酶與底物的生成物進行掃描，結果顯示：兩條曲線基本吻合，均在544nm和585nm波長左右出現(xiàn)兩個最高值點，故確定純化出的酶是牛睪丸玻璃酸酶。采用SDS-PAGE對酶進行分子量的初步鑒定，試驗結果顯示出兩條接近的條帶，分子量分別約為60kDa和67kDa。 4.牛玻璃酸酶酶學特性：對酶的部分酶學特性進行研究，試驗結果表明：最適反應溫度和pH值分別為40℃、4.5，酶在20~40℃及pH值為4~5的范圍內(nèi)具有較好的穩(wěn)定性；NaCl濃度為0.3mol/L時，酶活力相對較高；金屬離子Fe3+、Ca2+、Mn2+、Mg2+、Zn2+、Al3+、K+對酶均有不同程度的抑制作用；該酶的Km值為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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