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On-line Access: 2014-05-05

Received: 2013-11-08

Revision Accepted: 2014-03-26

Crosschecked: 2014-04-09

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Journal of Zhejiang University SCIENCE B 2014 Vol.15 No.5 P.491-499

http://doi.org/10.1631/jzus.B1300283


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


Author(s):  Jia-wei Lou, Li Zhu, Mian-bin Wu, Li-rong Yang, Jian-ping Lin, Pei-lin Cen

Affiliation(s):  . Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

Corresponding email(s):   wumb@zju.edu.cn

Key Words:  5-Aminolevulinic acid, Rhodobacter capsulatus , High-level expression, Enzymatic properties


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[J]. Journal of Zhejiang University Science B, 2014, 15(5): 491-499.

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author="Jia-wei Lou, Li Zhu, Mian-bin Wu, Li-rong Yang, Jian-ping Lin, Pei-lin Cen",
journal="Journal of Zhejiang University Science B",
volume="15",
number="5",
pages="491-499",
year="2014",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1300283"
}

%0 Journal Article
%T High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties
%A Jia-wei Lou
%A Li Zhu
%A Mian-bin Wu
%A Li-rong Yang
%A Jian-ping Lin
%A Pei-lin Cen
%J Journal of Zhejiang University SCIENCE B
%V 15
%N 5
%P 491-499
%@ 1673-1581
%D 2014
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1300283

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T1 - High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties
A1 - Jia-wei Lou
A1 - Li Zhu
A1 - Mian-bin Wu
A1 - Li-rong Yang
A1 - Jian-ping Lin
A1 - Pei-lin Cen
J0 - Journal of Zhejiang University Science B
VL - 15
IS - 5
SP - 491
EP - 499
%@ 1673-1581
Y1 - 2014
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1300283


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 [(k cat/K m)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.

荚膜红细菌hemA基因的高效可溶表达及其酶学性质对比研究

研究目的:提供一种酶学性质优良的,可在大肠杆菌体内高活性可溶表达的,具有工业应用前景的5-氨基乙酰丙酸合成酶。
创新要点:利用E. coli Rosetta (DE3)表达编码荚膜红细菌5-氨基乙酰丙酸合成酶(RC-ALAS)的hemA基因,实现其在大肠杆菌体内高活性可溶表达。以放射形土壤杆菌和类球红细菌hemA基因表达的5-氨基乙酰丙酸合成酶(AR-ALAS和RS-ALAS)为参照,对分离纯化后的RC-ALAS进行酶学性质对比研究。
研究方法:工程菌的构建如图1所示,酶的分离纯化采用镍柱亲和层析和凝胶过滤层析,酶学性质的测定采用对比研究法。
重要结论:(1)RC-ALAS酶活性高达198.2 U/mg,比AR-ALAS(151.1 U/mg)和RS-ALAS(116.9 U/mg)分别提高31.2%和69.5%;(2)RC-ALAS对底物琥珀酰辅酶A的专一性常数((kcat/Km)S-CoA)为1.4989,高于AR-ALAS(0.7456)和RS-ALAS(1.1699),具有较高的催化效率;(3)对含有荚膜红细菌hemA基因的工程菌进行补料分批发酵,发酵结束时,ALA的产量高达8.8 g/L(67 mmol/L)。

关键词:5-氨基乙酰丙酸;荚膜红细菌(Rhodobacter capsulatus);高效表达;酶学性质

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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