Intracellular and Extracellular Forms of Alkaline Pullulanase from an AlkaliphilicBacillussp. S-1

Bacillussp. S-1 alkaline pullulanase (AP) exists in two forms: a precursor form (PUL-Ia,Mr180,000) and a processed form (PUL-Ib,Mr140,000). PUL-Ia was accumulated intracellularly in large amounts, and PUL-Ib was detected in both the membrane fraction and the fraction trapped between the cytoplasmic membrane and the cell wall. Two forms of AP were purified to homogeneity and their properties were compared with previously purified PUL-E (140 kDa). PUL-Ib showed similar properties, such as theMrvalue, the pIvalue (5.7), specific activity, substrate specificity, the NH2-terminal amino acid sequence (Phe–Leu–Asn–Met–Ser), and biophysical characters. However, in the case of PUL-Ia, even though the patterns of optimum pH and temperature, substrate specificity, and enzyme inhibition and activation were similar to those for PUL-Ib and PUL-E, theMrvalue and the pIvalue (5.97) were different. Furthermore, the NH2-terminal amino acid sequence of PUL-Ia was completely blocked, and the stabilities over pH and temperature ranges were decreased. The catalytic activities of PUL-Ia were distinguishable in theKmandVmaxvalues for various substrates and in the specific activity (71.4 U/mg) for pullulan hydrolysis. PUL-Ib and PUL-E showed 10-fold higher specific activities (744.6 for PUL-E and 736 for PUL-Ib) than PUL-Ia. However, PUL-Ia was immunologically identical to PUL-E and PUL-Ib. Therefore, it was concluded that PUL-Ib and PUL-E are the same form of the enzyme, suggesting that PUL-Ia is initially synthesized and proteolytically processed to the mature form of PUL-E. On the other hand, the translocation of AP required processing of the AP protein and the processing facilitated enzymatic activation and stabilization through a complete conformational change, resulting in an increase in affinity for substrates of PUL-E.