【作者】 钟琦；

【导师】 关怡新；

【作者基本信息】 浙江大学 ， 生物化学工程， 2004， 硕士

【摘要】 氨基酸是构成蛋白质的基础，同时也是一种十分重要的营养物质，它在生命活动中起着举足轻重的作用。 在传统的20种天然氨基酸中，除没有手性中心的甘氨酸外，其他19种氨基酸均为L构型。随着研究的不断深入，越来越多非天然构型—D型氨基酸的作用被发现，它们的需求也随之产生。对于L型和D型都有利用价值的氨基酸，化学合成结合手性拆分的方法，具有低成本、适于大规模生产等优点。而高效、绿色、安全的氨基酰化酶法拆分，是拆分氨基酸的最佳方法之一。 蛋氨酸和苯丙氨酸，都是人体必需的氨基酸。蛋氨酸是一种重要的饲料添加剂。L-苯丙氨酸是合成抗病毒和抗癌药物及人工甜味剂的原料，D-苯丙氨酸能增强人体的免疫功能，具有出色的镇痛作用。 首先，选取了氨基酰化酶含量较为丰富且方便易得的米曲霉3042作为酶源。通过硫酸铵分级沉淀、Sephadex G50凝胶层析和DEAE-Sepharose阴离子交换层析从米曲霉3042中提取到了米曲氨基酰化酶，该酶的纯化倍数为54.29，比活为647.66U／mg，总收率为49.53％。以N-乙酰-DL-蛋氨酸为底物的酶促反应最适pH为7.5-8.0，最适反应温度随催化反应时间的延长而降低，缓冲体系中的离子对酶活有抑制作用，而低浓度的CO²⁺对酶活有激活作用。 将游离酶用于拆分Met和Phe。最佳拆分条件和拆分结果分别为： Met：反应温度为37℃；pH7.5；Co²⁺浓度为5×10^-4mol／L；初始底物浓度为0.3mol／L。产品：L-Met，％O.P=96.2％，收率70.5％；D-Met，％O.P=95.3％，收率50.5％。 Phe：反应温度为37℃；pH7.0；Co²⁺浓度为5×10^-4mol／L；初始底物浓度为0.2mol／L。产品：L-Phe，％O.P=98.6％，收率68.2％；D-Phe，％O.P=97.1％，收率60.1％。 之后，采用了离子交换凝胶吸附法和海藻酸钙微胶囊包埋法对酶进行固定化。 吸附法所选用的固定化介质是国产DEAE-Sepharose凝胶。研究表明，固定化DSA的热稳定性明显优于游离酶。利用DSA柱进行Met的连续拆分，稳定工作30天后，酶活仍保留60％以上。经初步估算，氨基酰化酶固定化后，酶活利用率提高10倍以上，具有良好的工业应用前景。 包埋有氨基酰化酶的海藻酸钙凝胶珠分别与聚阳离子PMCG和PDMDACC形成微胶囊。对于本体系，两种聚阳离子具有相似的效果。两种微胶囊酶的固定化率都较高，同时酶的泄漏问题也得到了较好的解决。但是随着反应次数的增加，浙江大学硕士学位论文摘要微胶囊的操作稳定性随之下降。在应用之前，还需解决微胶囊的易损坏问题。最后，用明胶和戊二醛固定化液态发酵得到的含有胞内酶的米曲霉菌。比较了游离菌体和固定化菌体的性质，结果显示固定化后菌体的稳定性明显提高。更多还原

【Abstract】 Amino acids are the basic building blocks for proteins and nutritionally important key compounds. They play significant roles in the life. Among 20 basic amino acids, glycine is the only one not exhibiting chirality, and other 19 are all L-amino. acids. With the development of amino acids industry, the functions of D-amino acids are being discovered. It is economical to produce amino acids in large scale by chemical synthesis and chiral resolution as both L- and D- amino acids are valuable. And resolution by aminoacylase is one of the best methods in the resolution of amino acids. It is a high efficient, green and safe method.Methionine and Phenylalanine are essential amino acids for human development. Methionine is one of the most important feed supplements. L-phenylalanine can be used to synthesis new medicament and artificial sweetener (aspartame). D-phenylalanine has analgesic activity.Firstly, Aspergillus oryzae 3042 which produces higher aminoacylase was chosen for the production of aminoacylase in this research. The aminoacylase from Aspergillus oryzae 3042 was partially purified by ammonium sulfate fractionation, cloumn chromatography on Sephadex G50 and DEAE-Sepharose. The specific activity of aminoacylase was 647.66U/mg. The purification ratio and recovery was 54.29 and 49.53% respectively. The optimal pH of aminoacylase was 7.5-8.0, and with the increase of catalysis time, the optimal reaction temperature decreased correspondingly. The ions in the buffer lowered the activity of aminoacylase, but the Co2+ in low concentration activated aminoacylase.Optimal conditions and results of optical resolution of Met and Phe by free aminoacylase are as follows respectively:Met:Temperature 37℃; pH7.5; Concentration of Co2+ 5 X 10’4mol/L; Concentration of initial substrate 0.3mol/L. Products: L-Met, %O.P=96.2%, yields 70.5 %; D-Met, %O.P=95.3%, yields 50.5 %.Phe:Temperature 37℃; pH7.0; Concentration of Co2+ 5 X 10’4mol/L; Concentration of initial substrate is 0.2mol/L. Products: L-Phe, %O.P=98.6%, yields 68.2%; D-Met, %O.P=97.1%, yields 60.1 %.Secondly, aminoacylase was immobilized by two methods, ionic binding to DEAE-Sepharose(DSA) and entrapping into Alginate-PMCG/PDMDAAC microcapsules.Domestic DEAE-Sepharose was used to decrease the cost of the production. The stability of DSA was much better than that of the free aminoacylase. Then continuous resolution of Met was performed by DSA column. Over 60% of the aminoacylase activity remained after being operated for 30days. Generally, total amounts of the products obtained by DSA were 10 folds more than that by free aminoacylase. It is suitable to be potentially applied in the industrial production of chiral amino acids.The leakage of aminoacylase., operation stability and properties of Alginate-PMCG microcapsules and Alginate-PDMDAAC microcapsules are quite similar. The operation stability of microcapsules was decreased as the operation times increased. But the breakage of microcapsules should be solved before application.Finally, Aspergillus oryzae 3042 was immobilized in gelatin, then crosslinked by glutaraldehyde. The enzymic characteristics of the immobilized cells were also studied. Optimal pH and temperature of the free and immobilized pellets were determined. The immobilized cells were more stable over broader temperature and pH ranges. In addition, the immobilized cells showed stable activity under operation and storage conditions.更多还原