• Journal of Microbiology
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• 제35권3호
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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.

• 생명과학회지
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• 제7권2호
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• pp.95-106
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• 1997

전분의 효율적인 이용을 목적으로 토양으로부터 균체외 플루탄아제효소를 대량분비하는 약알칼리성균주 Bacillus sp. S-1를 분리하였다. 본 균주의 풀루란아제효소 생샨의 최적 pH는 6-10 사이였으며, 조효소의 가용성전분과 플루란에 대한 주요반응 최종산물은 말토트리오스로, 본 효소가 ${\alpha}$-1,6-glycosidic 결합에 특이적임을 알 수 있었다. 본 균주는 지금까지 알려진 알칼리성균주들의 플루란아제 생산성보다 월등히 높은 7.0U/ml를 생산하였으며, 정제효소의 최적 pH와 온도는 각각 8.0-10.0와 50-60$^{\circ}$C로서 알칼리성 및 호열성의 특성을 나타났었다. 또한 정제효소는 pH12에서도 약 10%의 활성을 유지하며, 넓은 pH범위에서도 안정하였다. 이러한 결과들은 본 균주가 플루란아제 생산균주로서의 이용가능한 잠재력을 보유하고 있음을 시사하였다.

• BMB Reports
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• 제30권1호
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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.