Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger

Several variants of glucoamylase 1 (GA1) from Aspergillus roger were create d in which the highly O-glycosylated peptide (aa 468-508) connecting the (a lpha/alpha)(6)-barrel catalytic domain and the starch binding domain was su bstituted at the gene level by equivalent segments of glucoamylases from Ho rmoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae l inker was elongated by proline-rich sequences as this linker itself apparen tly was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated po lypeptide chain molecular masses for wild-type GA1, a nonnatural proline-ri ch linker variant, H. grisea, and R. oryzae linker variants were 65 784, 63 777, 63 912, and 65 614 Da, respectively, MALDI-TOF-MS gave values of 82 0 42, 73 800, 73 413, and 90 793 Da, respectively, where the latter value cou ld partly be explained by an N-glycosylation site introduced near the linke r C-terminus. The kg and K-m for hydrolysis of maltooligodextrins and solub le starch, and the rate of hydrolysis of barley starch granules were essent ially the same for the variants as for wild-type GA1. beta -Cyclodextrin, a carbose, and two heterobidentate inhibitors were found by isothermal titrat ion calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the st arch binding site was maintained. The stability of GA1 linker variants towa rd GdnHCl and heat, however, was reduced compared to wild-type.