1887

Abstract

The β-hexosyltransferase (BHT) from is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1–110), which included two predicted endogenous structural domains. The first domain (amino acids 1–22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23–110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant strains carrying combinations of the predicted structural domains and the α mating factor (MFα) from as positive control. Upon replacement of the leader domain (amino acids 1–22) by MFα (α- ), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53- and 14-fold, respectively. Leader interference was demonstrated when MFα preceded the putative classical rBHT leader (amino acids 1–22), explaining the limited secretion of soluble protein by (GS115 : : α- ). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-β-galactosidase single-chain variable antibody fragment scFv13R4. The recombinants carrying chimeras of the N-terminal 1–110 regions of rBHT preceding correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : α- -HIS and GS115 : : α- ) showed comparable activity rates of GOS generation.

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2016-01-01
2019-10-15
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