Centre for Research and Graduate Studies, University of Cyberjaya, Persiaran Bestari, Cyberjaya, 63000 Selangor, Malaysia.
Biorefinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li 32003, Taiwan.
J Biosci Bioeng. 2023 Oct;136(4):312-319. doi: 10.1016/j.jbiosc.2023.07.001. Epub 2023 Jul 26.
Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E. coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4 h, induction time of 12 h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 U mg protein and total protein concentration of 0.518 mg mg of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 U mg) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.
聚 3-羟基丁酸酯 (P(3HB)) 是聚羟基烷酸酯 (PHA) 家族的一员，是一种具有多种工业应用的可生物降解聚酯。NADPH 依赖性乙酰乙酰辅酶 A 还原酶 (phaB) 是一种酶，通过催化中间产物的转化，在 P(3HB) 合成中发挥着重要作用。研究了使用重组大肠杆菌 BL-21(DE3)表达 phaB 酶和合成酶的纯化。含有源自 Ralstonia eutropha H16 的 phaB 基因的 pET-B3 质粒在大肠杆菌 BL-21(DE3)中由 lac 启动子驱动。该酶的表达受到不同诱导时间、温度和细胞龄的影响。结果表明，细胞龄为 4 h、37°C 下诱导 12 h 是酶还原酶表达的最佳条件。获得了 0.151 U·mg 蛋白的比酶活和 0.518 mg·mg 干细胞重量（DCW）的总蛋白浓度。亲和层析用于纯化 His 标记的 phaB 酶，尽管酶的得率相对较低（44.6%），但比酶活（14.44 U·mg）和纯化倍数（38 倍）得到了提高。纯化的 phaB 在 30°C 和 pH 8.0 下表现出最佳的酶活性。研究结果为可用于工业规模生产可生物降解聚合物的还原酶的合成提供了一种替代方法。
