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Mol Biotechnol. 2010 Jun;45(2):121-8. doi: 10.1007/s12033-010-9255-8.

Molecular cloning and characterization of a malic enzyme gene from the oleaginous yeast Lipomyces starkeyi.

Author information

1
Dalian Institute of Chemical Physics, CAS, Dalian 116023, People's Republic of China.

Abstract

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5' untranslated sequence and a 92-bp 3' untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD(+) or NADP(+) as the coenzyme but preferred NAD(+). The K (m) values for malic acid, NAD(+) and NADP(+) were 0.85 +/- 0.05 mM, 0.34 +/- 0.08 mM, and 7.4 +/- 0.32 mM, respectively, at pH 7.3.

PMID:
20217282
DOI:
10.1007/s12033-010-9255-8
[Indexed for MEDLINE]

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