【作者】 曹治云；

【导师】 谢必峰； 郑腾；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 酸性蛋白酶是在酸性条件下分解蛋白质的酶类的总称,黑曲霉和米曲霉是酸性蛋白酶工业化生产的主要菌种。为进一步研究酸性蛋白酶的分子作用机制,克服工业生产中由于酸性蛋白酶的不稳定性造成的应用难题,本论文研究了黑曲霉(Aspergillus niger)SL2-111菌株酸性蛋白酶的分离纯化,在此基础上探讨了该酶的分子活性基团和针对不同底物的反应动力学并研究了液体粗酶的稳定剂,主要结果如下: (1)利用硫酸铵盐析、Sephadex G-25柱脱盐、DEAE-Sepharose Fast Flow离子交换层析、Sephacryl S-200分子筛层析和高效液相色谱方法提取了酸性蛋白酶,并经SDS-PAGE验证该酶纯化水平己达到电泳纯。该酶的表观分子量约为47.0Kd,N端氨基酸序列为SKGSAVTTPQ。 (2)采用NBS、DTT、Acetyl acetone、MA、BrAc、N-AI、CDC、PCMB 8种化学修饰剂处理纯化的酸性蛋白酶,结果表明色氨酸、组氨酸、酪氨酸残基以及二硫键是酶活力所必需的基团。对牛血红蛋白、牛血清白蛋白、酪蛋白和鸡卵清蛋白四种不同底物的水解差异性为酪蛋白的水解性能最强,而鸡卵清蛋白的水解性能最弱。动力学研究结果为酪蛋白的Km最小为0.22g/L,牛血红蛋白的Km最大为0.83g/L,酪蛋白为其最适底物。 (3)探讨了防腐剂、醇类、糖类及金属离子作为稳定剂,试验结果表明氯化钠,山梨酸钾,丁醇,明胶,黄原胶,淀粉及7%氯化钠、8%丁醇、8%山梨酸钾、1%淀粉的复合稳定剂都能够使液体粗酶提高酶活保存率,其中效果最明显的是黄明胶,在相当于25℃下三个月的时间内可以使酶活提高一倍。更多还原

【Abstract】 Acid protease is a general call for any enzyme which can decompose protein under condition of acidity, Aspergillus niger and Aspergillus oryzae was the major mold producing acid protease in industry. In order to exploit the capacity of Aspergillus niger in molecular acting mechanism and conquer the problems in industry application , it is of more necessary to carry out this paper.In this study, the production conditions of three components of acid protease from Aspergillus niger SL2-111 were investigated. Major results were as follows:(1) Acid Protease was purified to elecrophoretic homogeneity by the steps of ammonium sulfate precipitation.DEAE-Sepharose Fast Flow column chromatography.Sephacryl S-200 column chromatography and high performance liquid chromatography.Its purity was checked to be about a single band by SDS-PAGE. the molecular weight was about 47 Kda, .The N-terminal partial sequence was SKGSAVTTPQ ,which indicated a high homology with other protease produced by aspergillus.(2) Acid protease was not affected by PCMB, Acetyl acetone, MA .. CDC modification,indicating that ε-amino group, arginine residues , Mercapto and carboxyl were non-essential to the enzyme activity. The enzyme activity was significantly decreased after NBS,N-AI and DTT modification,and was slightly decreased after BrAc modification.These results indicated that the histidineresidues.tryptophane residues,tyrosine residues and disulfides groups seemed to be essential to catalytic activity or located at the active site of acid protease. The otherness of Hydrolyze of Bovine haemoglobin ,Bovine serum albumin, Egg albumin and casein which casein was the most obvious Hydrolyzing , the result of dynamics was that the casine was the optimum substrate.更多还原