EXTRACELLULAR SECRETION OF PULLULANASE IS UNAFFECTED BY MINOR SEQUENCE CHANGES BUT IS USUALLY PREVENTED BY ADDING REPORTER PROTEINS TO ITS N-TERMINAL OR C-TERMINAL END

Linker insertions in the pullulanase structural gene (pulA) were exami ned for their effects on pullulanase activity and cell surface localiz ation in Escherichia coli carrying the cognate secretion genes from Kl ebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activ ity but none were found to prevent secretion. To see whether more dras tic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose -binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular le vansucrase) to three different positions in the pullulanase polypeptid e: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids w ere efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which malto se-binding protein was fused close to the N terminus of pullulanase. A ffinity-purified endoglucanase-pullulanase and pullulanase-endoglucana se hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hy brid had not been dramatically altered by the presence of the reporter . However, pullulanase endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-te rminal cellulose-binding domain, suggesting that at least one factor l imiting hybrid protein secretion might be the size of the reporter.