绮丽刺毛霉的一种新型甘氨酸氨肽酶的研究  

Study on the Properties of a Novel Glycine Amino Peptidase from Actinomucor elegans

作　　者：马晓航[1] 孙桂芹[1] 赵宇华[1] 贾小明[1] 

机构地区：[1]浙江大学生命科学学院,杭州310029

出　　处：《生物工程学报》2004年第4期578-583,共6页Chinese Journal of Biotechnology

摘　　要：研究了产自于绮丽刺毛霉 (Actinomucorelegans)的一种甘氨酸氨肽酶。分子筛层析表明该酶的天然分子的分子量为 32 0kD ,SDS PAGE分析表明蛋白质的亚基分子量为 5 6 5kD。该酶水解含有甘氨酸残基的底物 (如glycine naphthylamine)的效率要较其它氨基酸残基高得多。该酶的最佳反应温度为 30℃ ,最佳pH为 8 0。酶的Km和Kcat值分别为 0 2 4mmol L与 1 0 0 8s- 1 。 1 0mmol LZn2 + ,Cu2 + 和Cd2 + 可完全抑制该酶的活性。作用于酶巯基的化学物质对酶活性都有抑制作用。根据络合剂反应的实验结果表明该酶是一种含有金属的酶。当与蛋白酶共同作用时该酶除了甘氨酸外还能提高脯氨酸。The glycine amino peptidase of Actinomucor elegans was studied in this work. For the enzyme production Actinomucor elegans was cultured with an enzyme producing medium. Then the cells were collected and subjected to enzyme purification. The glycine aminopeptidase was purified 592 times by a DEAE Toyopearl column, a Toyopearl HW 65 C column and a Superdex 200 column subsequently and the purified enzyme had a specific activity of 14 2 u/mg. The enzyme was estimated to have molecular mass of 320kD by gel filtration and a subunit size of 56 5kD by SDS PAGE. It hydrolyzes glycine residue containing substrates such as glycine βnaphthylamine more efficiently than those containing other amino acid residue. Addition to Gly βNA, the en zyme could also hydrolyze Ala βNA, Met βNA, Leu βNA, Arg βNA and Ser βNA but it had no activity on the substrates such as Trp βNA, Pyr βNA, Pro βNA, Asp βNA, Lys βNA, Val βNA. It was also observed when the glycine βnaphthylamine concentration was higher than 2mmol/L the enzyme showed a substrate inhibition, and at the 20 mmol/L the enzyme only showed about 55% activity as it showed at the 2mmol/L. Whereas no such phenomenon was observed on the other substrate such as alanine βnaphthylamine. The optimal temperature and pH for the reaction of this enzyme is 30℃ and pH 8 0, respectively. The K m and K cat of the enzyme for glycine βnaphthylamine is 0 24 mmol/L and 100 8 s -1 , respectively. Zn 2+ , Cu 2+ and Cd 2+ suppress almost all activities of the enzyme at the concentration of 1 0 mmol/L. Based on the study of chelating reagents, GAP belongs to the metalloenzyme. When a gelatin solution was hydrolyzed with 0 5% of alkaline proteinase together with glycine aminopeptidase at 50℃ for 18 hours, the glycine aminopeptidase could improve the hydrolysis degree of the protease. The total free amino acid was improved about 13% and although the enzyme mainly had the activity to hydrolyze the glycine residue, individual

关 键 词：肽酶 甘氨酸 

分 类 号：Q55[生物学—生物化学]