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ZJUS-B
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)  2014, Vol. 15 Issue (5): 491-499    DOI: 10.1631/jzus.B1300283
Articles     
High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties
Jia-wei Lou, Li Zhu, Mian-bin Wu, Li-rong Yang, Jian-ping Lin, Pei-lin Cen
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
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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 Co2+, Zn2+, and Cu2+ strongly inhibited the activities of the ALASs, while Mn2+ exerted slight inhibition, and K+, Ca2+, Ba2+, Mg2+, 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.

Key words： 5-Aminolevulinic acid      Rhodobacter capsulatus      High-level expression      Enzymatic properties     
Received: 08 November 2013      Published: 05 May 2014
CLC:  Q819  
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Jia-wei Lou
Li-rong Yang
Li Zhu
Mian-bin Wu
Jian-ping Lin
Pei-lin Cen
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Jia-wei Lou, Li Zhu, Mian-bin Wu, Li-rong Yang, Jian-ping Lin, Pei-lin Cen. High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2014, 15(5): 491-499.

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http://www.zjujournals.com/xueshu/zjus-b/10.1631/jzus.B1300283     OR     http://www.zjujournals.com/xueshu/zjus-b/Y2014/V15/I5/491

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