A Photobacterium sp α2-6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens

In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 alpha 2-6-sialyl-transferase Psp2,6ST(15-501)-His(6) in sialylating alpha-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15-501)-His(6) toward alpha-GalNAc-containing acceptors by 21-115% without significantly affecting its sialylation activity to beta-galactosides. Furthermore, the expression level was improved from 18-40 mg L-1 for the wild-type enzyme to 72-110 mg L-1 for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15-501)-His(6) for synthesizing Neu5Ac alpha 2-6GalNAc alpha OSer/Thr STn antigens. (C) 2014 Elsevier Ltd. All rights reserved.