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

作     者：Jia-wei LOU Li ZHU Mian-bin WU Li-rong YANG Jian-ping LIN Pei-lin CEN 

作者机构：Key Laboratory of Biomass Chemical Engineering of Ministry of EducationDepartment of Chemical and Biological EngineeringZhejiang University 

出 版 物：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 (浙江大学学报（英文版）B辑（生物医学与生物技术）)

年 卷 期：2014年第15卷第5期

页      面：491-499页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基　　金：Project supported by the National Natural Science Foundation of China (No.20306026) the National Basic Research Program (973) of China (No.2007CB707805) 

主　　题：5-Aminolevulinic acid Rhodobacter capsulatus High-level expression Enzymatic properties 

摘      要：The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil 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 ℃, 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 [（kcatlKm）^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.