Abstract
ABSTRACTConjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. CLA is an intermediate product of the biohydrogenation pathway of linoleic acid (LA) in bacteria. Several bacterial species capable of efficiently converting LA into CLA have been widely reported in the literature, among them Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230. Over the last few years, a multicomponent enzymatic system consisting of three enzymes involved in the biohydrogenation process of LA has been proposed. Sequencing the genome of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. The OleH was cloned, expressed, purified, and characterized. Fatty acid measurements were made by an internal standard method using a gas chromatography-coupled flame ionization detector (GC-FID) system. It was found that the enzyme is a hydratase/dehydratase, leading to a reversible transformation between LA and RA. In addition, the results showed that L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. In conclusion, the OleH of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 catalyzes the initial stage of saturation metabolism of LA, although it has not converted the substrates directly into CLA.IMPORTANCE This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c9, t11 CLA isomer and stress tolerance by E. coli has been assisted. The results provide an understanding of the factors which influence OleH activity. L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH presented two putative fatty acid-binding sites. Recombinant OleH catalyzed both LA hydration and RA dehydration. OleH was shown to play a role in bacterial growth performance in the presence of LA.
Highlights
Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits
The genome sequencing of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 suggested that only one gene capable of encoding an oleate hydratase enzyme [14] based on homology testing
Our group identified that the production of the CLA isomers in a coculture of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 and Streptococcus thermophilus strain 360 is conditioned to the predominant bacterial species in the starter culture
Summary
Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. Bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. Bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. Bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. Bulgaricus LBP UFSC 2230 (BioProject accession number PRJNA615231; SRA accession number SRR11741240) confirmed the presence of a single gene (oleH) capable of expressing an enzyme which was identified as an oleate hydratase and not the presence of other genes which are typically found in other such lactobacilli strains, generally involved in this multienzyme process [14]. The current study aimed to characterize the oleate hydratase (EC 4.2.1.53) enzyme from L. delbrueckii subsp. bulgaricus LBP UFSC 2230 by analyzing the enzymatic activity (in vivo and in vitro) of the expressed protein in a heterologous host
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