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Cloning and expression of fumarylacetoacetate hydrolase derived from marine yeast Rhodosporidium diobovatum
Author(s) -
Liu Yuxuan,
Xia Weiwei,
Yang Pucheng,
Zhang Shuo,
Shi Zhihui,
Tang Hui,
Zhang Liping
Publication year - 2015
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201400908
Subject(s) - biochemistry , chemistry , complementary dna , hydrolase , escherichia coli , tyrosine , microbiology and biotechnology , enzyme , biology , gene
Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon–carbon bond in fumarylacetoacetate to yield acetoacetate and fumarate as the final step in tyrosine degradation. In this study, the FAH genomic DNA and cDNA of Rhodosporidium. diobovatum were cloned by using the methods of degenerate polymerase chain reaction and RACE method. The results showed that the gene contained six exons, and that the encoded polypeptide held a sequence of 308 amino acid residues, containing a 75 residue N‐terminal prosequence, a 216 residue proteinase domain, and a 17 residue C‐terminal domain. Then, the expression vectors pET28a‐ FAH were constructed and expressed in Escherichia coli . The enzymatic activity of FAH protein was determined by monitoring the decrease in optical density at 330 nm of the substrate fumarylacetoacetate. The values for K m and V max of the purified enzyme was 1.4 µM and 2.3 µmol · min −1 · mg −1 , respectively.