High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties

Abstract

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.

Keywords: 5-Aminolevulinic acid; Enzymatic properties; High-level expression; Rhodobacter capsulatus.

Fig. 1

Construction of expression vector

Fig. 2

Agarose gel electrophoresis analysis of hemA from R. capsulatus Lane 1: marker; Lanes 2–4: hemA

Fig. 3

Expression of the R. capsulatus hemA gene inE. coli Rosetta (DE3) Lane 1: marker; Lane 2: crude RC-ALAS

Fig. 4

SDS-PAGE analysis of purified recombinant ALASs Lane 1: marker; Lane 2: RC-ALAS; Lane 3: AR-ALAS; Lane 4: RS-ALAS

Fig. 5

Effects of various temperatures on the activity of recombinant ALAS The activity of the sample assayed at 37 °C was set as 100%. Data are expressed as mean±SD (n=9)

Fig. 6

Effects of various pH values on the activity of recombinant ALAS The activity of the sample assayed at pH 7.5 was set as 100%. Data are expressed as mean±SD (n=9)

Fig. 7

Temperature stability of recombinant ALAS Data are expressed as mean±SD (n=3)

Fig. 8

pH stability of recombinant ALAS Data are expressed as mean±SD (n=3)

Fig. 9

Effects of various metal ions, EDTA, and SDS on the activity of recombinant ALAS The activity of the untreated sample was set as 100%. Data are expressed as mean±SD (n=3)

Fig. 10

Production of ALA using E. coli Rosetta (DE3)/pET28a-R.C.hemA Mixture of glycine and succinate acid was added continuously by the pH feed-back control system. The pH value was controlled at 6.2 initially and at 6.5 after 6 h of incubation. The temperature was controlled at 28 °C after 2 h of incubation and increased to 37 °C after 22 h. Data are expressed as mean±SD (n=3)