Azura Azami Nor, Ira Aryani Wirjon, Aik-Hong Teh, Amirul A A
Centre for Chemical Biology, Penang, Malaysia.
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; Centre for Chemical Biology, Penang, Malaysia; Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM, Malaysia.
Protein Expr Purif. 2019 Mar;155:35-42. doi: 10.1016/j.pep.2018.10.008. Epub 2018 Oct 21.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg, Ca, K, Na, Fe) and organic solvents (methanol, ethanol, and acetone). The apparent K values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The V values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
解聚酶是一种在聚羟基脂肪酸酯（PHA）水解过程中发挥重要作用的酶。在当前研究中，洋葱伯克霍尔德菌DP1分离自马来西亚槟城，其中的酶通过离子交换和凝胶过滤（Superdex - 75）柱色谱法进行纯化。使用SDS - PAGE估计该酶的分子量为53.3 kDa。纯化后酶活性提高到36.8倍，回收率为16.3%。在pH 6.0 - 10以及35 - 55°C之间检测到酶活性，pH 6.0和45°C时活性最高。吐温 - 20、吐温 - 80、SDS和PMSF可使解聚酶失活，但该酶对金属离子（Mg、Ca、K、Na、Fe）和有机溶剂（甲醇、乙醇和丙酮）不敏感。纯化的聚（3 - 羟基丁酸酯）解聚酶对聚（3 - 羟基丁酸酯）[P(3HB)]和聚（3 - 羟基丁酸酯 - 共 - 14% 3 - 羟基戊酸酯）[P(3HB - co - 14% 3HV)]的表观K值分别为0.7 mg/ml和0.8 mg/ml。纯化酶对P(3HB)和P(3HB - co - 14% 3HV)的V值分别为10 mg/min和8.89 mg/min。当前研究从洋葱伯克霍尔德菌DP1的培养上清液中分离并纯化出一种新的胞外聚（3 - 羟基丁酸酯）[P(3HB)]解聚酶，且纯化至同质。据我们所知，这是首次报道对来自伯克霍尔德菌属的P(3HB)解聚酶进行纯化及生化特性研究。
