• Journal of Microbiology
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• v.35 no.3
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• pp.213-221
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• 1997

The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly bue quantitatively released into the medium during growth of the cells in medium contianing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M$\_$r/ pullulanase to a 140,000M$\_$r/ polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M$\_$r/ extracellular form. Processing of the 180,000 M$\_$r/ protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

• Journal of Life Science
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• v.7 no.2
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• pp.95-106
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• 1997

The moderately alkalophilic bacterium, identified as Bacillus sp. S-1 , was isolated from soils and effectively secrete extracellular pullulanase. The isolate was moderately alkalophilic since enzyme production occurred at pHs from 6.0 to 10.0. Extracellular crude enzymes of the isolate gave maltotriose as the major product from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes, this isolate secreted extremely high concentration(7.0 units/ml) of pullulanase. The purified pullulanase was moderately alkalophilic and thermoactive; optimal activity was detected at pH 8.0-10.0 and between 50-60$^{\circ}$C. Even at pH 12.0, 10% of S-1 pulluanase activity remained and the strain had broad pH ranges and moderate thermo-stability for their enzyme activities. These results indicate that the new isolate have potential as producer of pullulanase for use in the starch industry.

• BMB Reports
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• v.30 no.1
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• pp.46-54
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• 1997

Alkaliphilic Bacillus sp. S-1 secretes a large amount (approximately 80% of total pullulanase activity) of an extracellular pullulanase (PUL-E). The pullulanase exists in two forms: a precursor form (PUL-I: $M_r$ 180,000), and a processed form (PUL-E: $M_r$ 140,000). Two forms were purified to homogeneity and their properties were compared. PUL-I was different in molecular weight, isoelectric point, $NH_2$-terminal amino acid sequence, and stabilities over pH and temperature ranges. The catalytic activities of PUL-I were also distinguishable in the $K_m$ and $V_{max}$ values for various substrates, and in the specific activity for pullulan hydrolysis. PUL-E showed 10-fold higher specific activities than PUL-I. However. PUL-I is immunologically identical to PUL-E, suggesting that PUL-I is initially synthesized and proteolytically processed to the mature form of PUL-E. Processing was inhibited by PMSF, but not by pepstatin, suggesting that some intracellular serine proteases could be responsible for processing of the PUL-I. PUL-I has a different conformational structure for antibody recognition from that of PUL-E. It is also postulated that the translocation of alkaline pullulanase(AP) in the bacterium possibly requires processing of the $NH_2$-terminal region of the AP protein. Processing of the precursor involves a conformational shift. resulting in a mature form. Therefore. precursor processing not only cleaves the signal peptide, but also induces conformational shift. allowing development of active form of the enzyme.