Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
J Zhejiang Univ Sci B. 2014 May;15(5):491-9. doi: 10.1631/jzus.B1300283.
The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS), was expressed in Escherichia coli Rosetta (DE3) and the enzymatic properties of the purified recombinant ALAS (RC-ALAS) were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter (AR-ALAS) and Rhodobacter sphaeroides (RS-ALAS), the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS (151.1 U/mg) and RS-ALAS (116.9 U/mg), respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 °C, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co(2+), Zn(2+), and Cu(2+) strongly inhibited the activities of the ALASs, while Mn(2+) exerted slight inhibition, and K(+), Ca(2+), Ba(2+), Mg(2+), or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [(kcat/Km)(S-CoA)] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS (0.7456) and RS-ALAS (1.1699), showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid (ALA) achieved was 8.8 g/L (67 mmol/L) under the appropriate conditions.
类球红细菌 hemA 基因编码 5-氨基酮戊酸合酶（ALAS），在大肠杆菌 Rosetta（DE3）中表达，并研究了纯化的重组 ALAS（RC-ALAS）的酶学性质。与农杆菌（AR-ALAS）和球形红杆菌（RS-ALAS）hemA 基因编码的 ALAS 相比，RC-ALAS 的比活达到 198.2 U/mg，分别比 AR-ALAS（151.1 U/mg）和 RS-ALAS（116.9 U/mg）高约 31.2%和 69.5%。三种酶的最适 pH 值和温度均为 pH 7.5 和 37°C。此外，RC-ALAS 对 pH 更敏感，而其他两种酶对温度更敏感。还研究了金属、乙二胺四乙酸（EDTA）和十二烷基硫酸钠（SDS）对三种 ALAS 的影响。结果表明，它们对三种 ALAS 的活性具有相同的影响。SDS 和金属离子如 Co(2+)、Zn(2+)和 Cu(2+)强烈抑制 ALASs 的活性，而 Mn(2+)则有轻微抑制作用，K(+)、Ca(2+)、Ba(2+)、Mg(2+)或 EDTA 则没有显著影响。RC-ALAS 的琥珀酰辅酶 A 特异性常数 [(kcat/Km)(S-CoA)] 为 1.4989，高于 AR-ALAS（0.7456）和 RS-ALAS（1.1699），表明其具有较高的催化效率。在适当的条件下，通过含有类球红细菌 hemA 基因的重组菌株进行分批补料发酵，5-氨基酮戊酸（ALA）的产量达到 8.8 g/L（67 mmol/L）。
