Molecular cloning, expression and characterisation of glucokinase gene from the mixotrophic green alga Chlorella kessleri
Chen, Jun1,2; Cui, Hongli1,2; Hou, Yanhua3; Cui, Yulin1; Wang, Yifan3; Xu, Yifeng3; Wang, Quanfu3; Qin, Song1; Wang, Quanfu(Harbin Inst Technol, Sch Marine Sci & Technol, Weihai 264200, Shandong, Peoples R China); Qin, Song(Chinese Acad Sci, Yantai Inst Coastal Zone Res, Key Lab Coastal Biol & Biol Resources Utilizat, Yantai 264003, Shandong, Peoples R China)
2017
发表期刊PHYCOLOGIA
ISSN0031-8884
卷号56期号:5页码:477-486
产权排序第1完成单位
摘要Chlorella kessleri is a green alga that grows photoautotrophically, heterotrophically and mixotrophically. To explore the mechanism of glucose metabolism at the molecular level, cDNA (GenBank ID KF011248) encoding glucokinase (named CkeGK) from C. kessleri was cloned and sequenced. The Ckegk cDNA was 1167 base pairs (bp) long, with an open reading frame of 1089 bp encoding 362 amino acids with a calculated molecular mass of 38.98 kDa and an estimated isoelectric point of 4.78. Multiple alignment analysis revealed that the deduced amino acid sequence of Ckegk shared a high identity of 65%-78% with corresponding glucokinase from other eukaryotes. The catalytic motifs of glucokinase were detected in the amino acid sequence of Ckegk. Under different trophic modes, the transcriptional profiles of Ckegk showed that it could be up-regulated under a heterotrophic culture condition but not under a mixotrophic culture condition. Furthermore, the gene was differentially expressed in Escherichia coli, and biochemical characterisation was determined. According to SDS-PAGE, the purified recombinant CkeGK showed a single band about 43.0 kDa, which correlated with the theoretical molecular weight predictions. Biochemical analysis showed that CkeGK was stable at 40 degrees C and 50 degrees C, but it lost 60% of its activity after incubation for 40 min at 60 degrees C. The optimal pH for the activity of CkeGK was 9.0, and the K-m and V-max for ATP were 0.1168 mM and 5.346 nmol ml(-1) min(-1) and for glucose were 0.1465 mM and 6.022 nmol ml(-1) min(-1).
关键词Biochemical Characterisation Chlorella Kessleri Expression Profiles Gene Cloning Glucokinase
DOI10.2216/16-111
项目资助者National Natural Science Foundation of China(41176144, 41376139) ; Public Science and Technology Research Funds Projects of the Ocean(20120527) ; National Science-Technology Support Plan Projects(2013BAB01B00) ; HIT(WH20150206, WH20160206)
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收录类别SCI
关键词[WOS]ATP-DEPENDENT GLUCOKINASE ; THERMOPHILIC ROK GLUCOKINASE ; SYNECHOCYSTIS SP PCC-6803 ; CELL-DENSITY CULTURE ; BIOCHEMICAL-CHARACTERIZATION ; AEROPYRUM-PERNIX ; GLUCOSE ; ASTAXANTHIN ; HEXOKINASE ; MICROALGAE
文章类型Article
语种英语
WOS研究方向Plant Sciences ; Marine & Freshwater Biology
WOS记录号WOS:000412894100001
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文献类型期刊论文
条目标识符http://ir.yic.ac.cn/handle/133337/23458
专题海岸带生物学与生物资源利用所重点实验室
通讯作者Wang, Quanfu; Qin, Song; Wang, Quanfu(Harbin Inst Technol, Sch Marine Sci & Technol, Weihai 264200, Shandong, Peoples R China); Qin, Song(Chinese Acad Sci, Yantai Inst Coastal Zone Res, Key Lab Coastal Biol & Biol Resources Utilizat, Yantai 264003, Shandong, Peoples R China)
作者单位1.Chinese Acad Sci, Yantai Inst Coastal Zone Res, Key Lab Coastal Biol & Biol Resources Utilizat, Yantai 264003, Shandong, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Harbin Inst Technol, Sch Marine Sci & Technol, Weihai 264200, Shandong, Peoples R China
第一作者单位中国科学院烟台海岸带研究所
通讯作者单位中国科学院烟台海岸带研究所
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Chen, Jun,Cui, Hongli,Hou, Yanhua,et al. Molecular cloning, expression and characterisation of glucokinase gene from the mixotrophic green alga Chlorella kessleri[J]. PHYCOLOGIA,2017,56(5):477-486.
APA Chen, Jun.,Cui, Hongli.,Hou, Yanhua.,Cui, Yulin.,Wang, Yifan.,...&Qin, Song.(2017).Molecular cloning, expression and characterisation of glucokinase gene from the mixotrophic green alga Chlorella kessleri.PHYCOLOGIA,56(5),477-486.
MLA Chen, Jun,et al."Molecular cloning, expression and characterisation of glucokinase gene from the mixotrophic green alga Chlorella kessleri".PHYCOLOGIA 56.5(2017):477-486.
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