title: Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11.
abstract: A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27,528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50 degrees C, although the enzyme displayed stability at temperatures up to 60 degrees C.
authors.affiliation: CSIR-Bio/Chemtek, P.O. Box 395, Pretoria 0001, South Africa.
authors.forename: Stephens M
authors.initials: SM
authors.lastname: Mnisi
authors.forename: Maureen E
authors.initials: ME
authors.lastname: Louw
authors.forename: Jacques
authors.initials: J
authors.lastname: Theron
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004795:Enzyme Stability; D016680:Genome, Bacterial; D008969:Molecular Sequence Data
chemical_list: D004269:DNA, Bacterial; D043182:Carboxylesterase