• Journal of Microbiology and Biotechnology
• /
• v.16 no.8
• /
• pp.1185-1191
• /
• 2006

The expression of a pullulanase gene in Pichia pastoris was investigated. The gene encoding pullulanase was cloned by PCR using the chromosomal DNA of Bacillus naganoensis as the template. The expression vector pPIC9K-Pu was constructed by inserting the pullulanase gene into plasmid pPIC9K and then transformed into Pichia pastoris SMD 1168 by electroporation. Activity determination, SDS-PAGE, and PCR amplification indicated that the gene of the pullulanase from B. naganoensis had successfully been expressed in SMD 1168 and the molecular size of the expressed recombinant product was about 119.9 kDa. This is the first report on the successful expression of the pullulanase from B. naganoensis in P. pastoris. The transformant secreted recombinant pullulanase with the activity of 350.8 IU/ml in shake-flask culture. The properties of the recombinant pullulanase were characterized.

• Microbiology and Biotechnology Letters
• /
• v.22 no.5
• /
• pp.499-506
• /
• 1994

In order to convert starch to the fermentative sugar, the effect of pullulanase on the saccharification of starch and pullulanase was investigated. The optimum pH and temperature for the enzyme activity of the glucoamylase and the crude pullulanase from Klebsiella pneumoniae NFB-320 were shown to be identical as pH 6.0 and 60$\circC, respectively. The crude pullulanase was stable between pH 5.0~6.5, and up to 40$\circC, whereas the glucoamylase was stable between pH 4.0~6.5, and up to 40$\circC. When pullulanase and glucoamylase were engaged together in the sacchrification of starch, saccharification yield was increased by 3.2% than the yield obtained by glucoamylase, alone. And the two enzymes produced sugar from pulltulan 18 times much higher than the single use of pullulanase.

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

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 1986.12a
• /
• pp.518.2-519
• /
• 1986

토양으로부터 분리한 질소고정균인 Klebsiella pneumoniae NFB320의 chromosomal DNA를 BamHI으로 절단하여 동일한 제한효소로 절단한 pBR322에 ligation시켜 E. coli HB101에 형질전환을 행하여 pullulanase activity를 나타내는 clone을 얻어내었다. 이 형질 전환체로부터 분리한 pullulanase 유전자가 재조합된 plasmid DNA는 약 10kb의 DNA단편을 가지고 있었으며, 재조합된 plasmid로부터 생산되는 pullulanase의 특성은 최적 활성 pH가 6.0이며, 효소의 pH안정성은 5-10이었다. 또한 형질 전환체로부터 생산되는 pullulanase의 localization,효소활성에 영향을 미치는 온도안정성 둥을 조사하였다.

• Microbiology and Biotechnology Letters
• /
• v.19 no.4
• /
• pp.315-318
• /
• 1991

- A bacterial strain No. S-76 which produced pullulanase powerfully was isolated frorn soil. The isolated bacterium was 0.4~$0.6\times 0.8$~1.4 $\mu\textrm{m}$ in size, gram negative, rods, motile and was identified as Aerornonas caviae by Bergey's manual of determinative bacteriology with various characteristics investigated. The highest yield of pullulanase of the strain was obtained by using the following medium: 1% pullulan, soluble starch or corn starch as a carbon sources and 0.5% yeast extract, peptone as nitrogen sources with an initial pH of 9.0. The optimal cutture conditions for production of pullulanase were at $32^{\circ}C$ for 2 days.

• Microbiology and Biotechnology Letters
• /
• v.15 no.6
• /
• pp.436-440
• /
• 1987

Pullulanase gene (pul) of Klebsiella pneumoniae NFB-320 which was cloned previously in Escherichia coli with plasmid pBR322. The gene was analyzed with various restriction enzymes. The cloned gene was contained within n 10 kb BamHI DNA fragment. We constructed the restriction map of the hybrid plasmid pYKL451. The optimum temperatures for pullulanases produced in E. coli (pYKL451) and K. pneumoniae NFB-320 were almost the same, 50-55 $^{\circ}C$. The optimum pHs for the reaction of the enzymes produced by E. coli (pYKL451) and K. pneumoniae NFB-320 was 6.0. Both enzyme preparations were stable under the range of pH 5.0 to 10.0 when those were kept at 40 $^{\circ}C$ for 90 min and were stable until 40 $^{\circ}C$ when allowed to stand for 1hr at various temperatures.

• Microbiology and Biotechnology Letters
• /
• v.14 no.5
• /
• pp.365-369
• /
• 1986

The activity of glucoamylase and pullulanase, properties of glucoamylase and ethanol productivities of fusants were studied. Glucoamylase and pullulanase activity of fusants were higher than parents. The optimal pH and temperature of glucoamylase of fusants were very similar to the those produced by S. diastaticus. In alcohol fermentation. fermenting ability and fermentation rate of fusants were higher and faster than either of its parental strain. The maximum of alcohol yield in 15% of liquefied potato starch was 7.8% (v/v)

• Journal of Life Science
• /
• v.6 no.2
• /
• pp.79-86
• /
• 1996

A bacterium producing pullulanase was from soil, and was identified Bacillus cereus and named as Bacillus cereus JK36. The optimal culture conditions for the efficident production of pullulanase from B. cereus JK36 was obtained by cultivating with the medium composed of 1% pullulan, 1% teast extract, 1% bactopeptone, 0.1% NaH$_{2}$PO$_{4}$, 2H$_{2}$O, 0.02% MgSO$_{4}$\ulcorner7H$_{2}$O at 40$\circ$C, initial pH 6.5 for 70 hours. Using the culture supernatant as crude enzyme, the optimal pH and temperature of the pullulanase of this strain were 6.5 and 50$\circ$C. In effect of pH and temperature on the stability of the enzyme, the enzyme was stable in the range of pH6.0$\sim$9.5 and up to 40$\circ$C, respectively. The hydrolysis product on pullulan was mainly maltotriose.

• Microbiology and Biotechnology Letters
• /
• v.22 no.1
• /
• pp.73-79
• /
• 1994

The optimum cultural temperature and time for the pullulanase production by Bacillus cereus subsp. mycoides were 35$\circ $C and 48 hrs, respectively. The addition of egg albumin and casein to the basal medium increased the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. specific activity of the purified enzyme was 82.37 U/mg protein and yield of theenzyume activity was 62.1%. The purified enzuyme showed a single band on ployacrylamide disc gel electrophoresis and its molecular weight was estimated to be 66.,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelcular point for the purified enzyme was pH 5.0. The optimum temperature and pH were 50$\circ $C and pH 6.5, respectively. The purified enzyme was stable below 40$\circ $C and in the pH range of 6.5~10.0 The pullulanase activity was greatly inhited by Ag$^{+}$, Hg$^{2+}$ and EDTA, and its heat stability was increased by the addition of Ca$^{2+}$. The tydrolysis product with the enzyme on pullulan was maltotriose.

• Preventive Nutrition and Food Science
• /
• v.2 no.1
• /
• pp.71-76
• /
• 1997

Pullulanase was produced from the Klebisella pneumonias NFB_320 with the conmposition of 0.1% pullualn 1.5% yeast extract, 0.2% $K_2$HPO$_4$ and 0.02% MgSO$_4$.7$H_2O$(pH5.5). The optimum temperature for activity of the pulluanase was 3$0^{\circ}C$ and the highest yield of the enzyme was obtained after cell growth at 3$0^{\circ}C$ for 18hr, and maintained until 24hr cultivation. The pullulanase was successively purified 52.6 folds with 7.8% yield by acetone precipitation. DEAE-cellulose column chromatography and gel fitrations. The purified enzyme hydrolyzed pullulan into maltotriose exclusively. Chemical and physical properties of purified pullulanase from Klebisella pneumonias NFB-320 were examined. The optimum pH and temperature for enzyme activity were 5.0 and 6$0^{\circ}C$, respectively. The enzyme was stable between pH4 and 7, and up 5$0^{\circ}C$. The effect of mo-dification on the rate of enzyme reaction was studies with various chemicals and metal ions. The enzyme has been found to be inactivated by I$_2$ and N-bromosussinimide(NBS), which probably indicated the involve- ment of tryptophan residues in the active center of the enzyme.