Heterologous expression of a novel linoleic acid isomerase BBI, and effect of fusion tags on its performance.

Functional proteins with the ability to isomerise free linoleic acid (LA) to conjugated linoleic acid (CLA) are termed linoleic acid isomerases (LAI). BBI is a novel LAI from Bifidobacterium breve with unique advantages in the production of a single CLA isomer; however, its complex membrane-bound form hampers over-expression of the protein in its natural host. To overcome this challenge, heterologous expression of BBI in Pichia pastoris was studied. Further, to investigate the influence of His-tags on the heterologous expression of BBI, three P. pastoris recombinant strains carrying either a C-terminal His-tag, an N-terminal His-tag, or none were constructed. The expression of recombinant proteins was analysed by dot and western blotting, and the enzyme activity was determined by GC-MS. The results show that the three P. pastoris recombinant strains successfully expressed the recombinant protein and had LAI activity. Compared with those BBIs without a His-tag or carrying a His-tag on the C-terminus, the BBI carrying an N-terminal His-tag had reduced expression and enzyme activity and that was also explained by the protein modelling analysis. Moreover, this study highlights the advantages of using P. pastoris for BBI expression to achieve efficient production of c9, t11-CLA monomers; the highest conversion rate of the substrate LA was over 80%, resulting in the production of 0.81 mg of c9, t11-CLA per mg of crude enzyme.