摘要
巨大芽孢杆菌产胞外青霉素酰化酶发酵液经硫酸铵分级抽提及ＳｅｐｈａｄｅｘＧ－１００、羟基磷灰石、ＤＥＡＥ纤维素ＤＥ５２等层析步骤，提纯了青霉素酰化酶，得到电泳均一的酶制剂。纯酶比活力约为２５Ｕ／ｍｇ蛋白，纯化４９倍，活力回收５８％，经ＰＡＧＥ及ＳＤＳ－ＰＡＧＥ测知该酶不含亚基，其分子量约为１４０ｋＤ。该酶最适ｐＨ为９．０，最适温度４７℃，用底物ＮＩＰＡＢ测活，其Ｋｍ值为６．２×１０^（－４）ｍｏｌ／Ｌ，Ｖｍ值为１．２４×１０４ｍｏｌ／Ｌ。此外还探讨了部分金属离子对该酶的影响。
Penicillin acylase from B. megaterium was purified by the steps including ammonium sulfate fractionation, Sephadex G-100 gel filtration, hydroxyapatite absorption and DEAE-cellulose chromatography. The results showed that the specific activity of the purified enzyme was 25U/mg protein with 58% activity recovery. Only one protein band was detected by 5%-20% gradient PAGE or SDS-PAGE, it indicated that the penicillin acylase was purified to homogeneity and no subunit was found. The molecular weight of the enzyme was about 140kD. The kinetic parameters determined with NIPAB as substrate were Km= 6. 2 ×1 04M and Vm = 1 . 2 × 10￣(-4)M.The purified enzyme was much stable. The optium pH and temperature was 9. 0 and 47 ℃ respectively. The divalent metal ions such as Mn￣(2+), Zn￣(2+), Cd￣(2+), Ni￣(2+) and chelating agent EDTA showed no obvious effect on the enzyme activity.
