title: 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.
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Jia-wei
authors.initials: JW
authors.lastname: Lou
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Mian-bin
authors.initials: MB
authors.lastname: Wu
authors.forename: Li-rong
authors.initials: LR
authors.lastname: Yang
authors.forename: Jian-ping
authors.initials: JP
authors.lastname: Lin
authors.forename: Pei-lin
authors.initials: PL
authors.lastname: Cen
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000622:Aminolevulinic Acid / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics*; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D000624:5-Aminolevulinate Synthetase
keywords: 5-Aminolevulinic acid; Enzymatic properties; High-level expression; Rhodobacter capsulatus