Purifi cation and characterization of a cytosolic NiFe-hydrogenase from the

Title
Purifi cation and characterization of a cytosolic NiFe-hydrogenase from the
Author(s)
이정현; 전정호; 김민식; 배승섭; 이현숙; 강성균
KIOST Author(s)
Lee, Jung Hyun(이정현)Lee, Hyun Sook(이현숙)Kang, Sung Gyun(강성균)
Publication Year
2013-09-13
Abstract
We have identified a NiFe-hydrognease localized in the cytoplasm of the Thermococcus onnurineus NA1. The enzyme was composed of α, β, and γ subunits and showed similarity to the coenzyme F420-reducing hydrogenase from Methanococcus maripaludis (33%). To understand the functionality of the cytosolic NiFe-hydrogenase in the Thermococcus onnurineus NA1, the hydrogenase was over-expressed in T. onnurineus NA1: the transcription of the three subunit genes was under the control of a strong constitutive promoter, and a strep-tag II was added to the N-terminus of α subunit. The over-expressed hydrogenase was purified by a single affinity chromatography step using the strep-tag II, and followed by characterization. The specific activity of the purified hydrogenase for H2 uptake was 11 fold higher than that for H2 evolution with methyl viologen as the electron carrier, indicating that the enzyme preferentially catalyzed H2 uptake rather than H2 production. Interestingly, a mutant strain overexpressing the cytosolic NiFe-hydrogenase showed a higher growth and H2 production rates than wild type strain in a formate medium as a sole energy source. Additionally, a deletion mutant strain of α subunit showed a lower growth and H2 production rates than wild type strain. This result suggests that the NiFe-hydrogenase might play an important role in the Thermococcus onnurineus NA1 related to cell growth and H2 production.aludis (33%). To understand the functionality of the cytosolic NiFe-hydrogenase in the Thermococcus onnurineus NA1, the hydrogenase was over-expressed in T. onnurineus NA1: the transcription of the three subunit genes was under the control of a strong constitutive promoter, and a strep-tag II was added to the N-terminus of α subunit. The over-expressed hydrogenase was purified by a single affinity chromatography step using the strep-tag II, and followed by characterization. The specific activity of the purified hydrogenase for H2 uptake was 11 fold higher than that for H2 evolution with methyl viologen as the electron carrier, indicating that the enzyme preferentially catalyzed H2 uptake rather than H2 production. Interestingly, a mutant strain overexpressing the cytosolic NiFe-hydrogenase showed a higher growth and H2 production rates than wild type strain in a formate medium as a sole energy source. Additionally, a deletion mutant strain of α subunit showed a lower growth and H2 production rates than wild type strain. This result suggests that the NiFe-hydrogenase might play an important role in the Thermococcus onnurineus NA1 related to cell growth and H2 production.
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/26786
Bibliographic Citation
12th International Thermophiles Conference, pp.81, 2013
Publisher
12th international meeting Thermophiles
Type
Conference
Language
English
Publisher
12th international meeting Thermophiles
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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