• 제목/요약/키워드: pullulanase

검색결과 51건 처리시간 0.026초

Cloning and Overexpression of Gene Encoding the Pullulanase from Bacillus naganoensis in Pichia pastoris

  • Xu Bo;Yang Yun-Juan;Huang Zun-Xi
    • Journal of Microbiology and Biotechnology
    • /
    • 제16권8호
    • /
    • 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.

Klebsiella pneumoniae NFB-320이 생산하는 Pullulanase를 이용한 전분당화 (Saccharification Using Pullulanase from Klebsiella pneumoniae)

  • 권재민;박경호;백운화;배동훈;유주현
    • 한국미생물·생명공학회지
    • /
    • 제22권5호
    • /
    • 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.

  • PDF

A possible mechanism responsible for translocation and secretion an alkaliphilic bacillus sp. S-1 pullulanase

  • Shim, Jae-Kyoung;Kim, Kyoung-Sook;Kim, Cheorl-Ho
    • Journal of Microbiology
    • /
    • 제35권3호
    • /
    • 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.

  • PDF

토양으로부터 분리한 Klebsiella pneumoniae 의 pullulanase 유전자의 cloning 및 발현

  • 유주현;공인수;정용준;이정기
    • 한국미생물생명공학회:학술대회논문집
    • /
    • 한국미생물생명공학회 1986년도 추계학술대회
    • /
    • 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.

  • PDF

Pullulanase를 생산하는 Aeromonas caviae No.S-76의 특성과 배양조건 (Characterization and Some Cultural Conditions of a Pullulanase Producing Aeromonas caviae No. S-76)

  • 손천배;김명희;이명자
    • 한국미생물·생명공학회지
    • /
    • 제19권4호
    • /
    • pp.315-318
    • /
    • 1991
  • Pullulanase 생산력이 높은 세균 No.S-76을 토양으로부터 분리하였다. 분리된 균은 0.4~$0.6\times 0.8$~1.4 $\mu\textrm{m}$의 크기의 gram음성, 간균으로서 운동성이 있으며, 여러가지 특성을 조사한 결과 Bergey의 세균분류 동종법에 따라 Aeromonas caviae로 동정되었다. 본 균의 pullulanase 생산배지로서는 탄소원은 1 pullulan, soluble strach 또는 corn starch가, 질소원으로는 0.5% yeast extract 또는 peptone이 적당하였으며 initial pH는 9.0의 배지가 가장 좋았으며 $32^{\circ}C$에서 2일간 배양이 적당하였다.

  • PDF

Klebsiella pneumoniae NFB-320의 Pullulanase 유전자의 제한효소 분석과 효소학적 특성 (Restriction Mapping of Cloned Pullulanase Gene and Property of Pullulanase Produced in Escherichia coli (pYKL451) and Klebsiella pneumoniae NFB-320)

  • Yu, Ju-Hyun;Chung, Kun-Sub;Kong, In-Su;Lee, Jung-Kee
    • 한국미생물·생명공학회지
    • /
    • 제15권6호
    • /
    • pp.436-440
    • /
    • 1987
  • 앞서 보고한 바와 같이 토양으로부터 분리한 K. pneumoniae NFB-320의 pullulanase 유전자를 pBR 322을 이용하여 E. coli에 cloning한 결과 약 14.4kb 의 재조합 plasmid DNA pYKL451을 얻었다. 이러한 pullulanase 유전자에 대한 유전적 정보를 얻기 위해 여러 가지 제한효소로 단일 혹은 이중 절단을 행하여 삽입된 pullulanase 유전자의 제한효소 절단지 도를 작성하였으며, E. coli(pYKL451)과 K. pneumoniae NFB-320이 생산하는 pullulanase의 효소적 특성을 조사하였다. 생산되는 두 균주의 효소는 50-55$^{\circ}C$ 부근에서 최적온도를 나타냈으며 최적pH는 모두 6.0이었다. 효소 안정성에 미치는 pH의 영향은 4$0^{\circ}C$에서 90min 간 방치했을 때 pH 5.0-10.0에서 안정하였으며 열안정성은 (pH6.0) 각 온도에서 한시간 처리하였을 때 4$0^{\circ}C$까지는 안정하였으나 5$0^{\circ}C$ 이상에서는 효소의 활성이 급격히 감소하였다.

  • PDF

Amylase분비효모와 alcohol 발효효모의 세포융합에 의한 균주의 개발 - 제4보. S. diastaticus와 C. tropicalis 간의 융합체의 pullulanase생성 및 alcohol발효 - (A study on strain improvement by protoplast fusion between amylase secreting yeast and alcohol fermenting yeast - IV. Alcohol and pullulanase productivities of fusant between S. diastaticus and C. tropicalis -)

  • 서정훈;김영호;홍순덕;권택규
    • 한국미생물·생명공학회지
    • /
    • 제14권5호
    • /
    • pp.365-369
    • /
    • 1986
  • 새로운 alcohol발효성 효모 균주 개발을 목적으로 S. diastaticus 와 C. tropicalis간의 세포 융합을 하여 얻은 fusant의 성질에 대해서는 제3보$^{(10)}$에서 발표한 바이다. 본 보에서 는 fusant의 glucoamylase와 pullulanase activity와 glucoamylase의 성질 및 발효력을 조사하였다. glucoamylase activity는 parent보다 fusant가 1.5배 높은 활성을 나타내었고 pullulanase activity 역시 두배의 높은 활성을 나타내었다. glucoamylase의 성질을 조사한 바 온도, pH에서 비슷한 경향을 나타내었으며 발효력을 조사하기 위하여 정치법에서는 기질을 soluble starch로 한 것 보다 liquefied potato starch에서 alcohol 생성력이 2배 증가되었으며 발효력 또한 더 나았다. 15% 액화한 potato starch를 기질로하여 jar-fermenter에서 발효시켰을 때 당화율 94%에서 생성된 alcohol이 7.8% (v/v)이고 소비된 당에 대한 발효율은 78%였다.

  • PDF

Pullulanase를 생산하는 Bacillus 속 세균의 분리와 효소의 최적 생산조건 및 특성 (Isolation of Bacillus sp. Producing Pullulanase and Culture Conditions for Production and Properties of the Enzyme)

  • 정희경;김병우
    • 생명과학회지
    • /
    • 제6권2호
    • /
    • 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.

  • PDF

Bacillus cereus subsp. mycoides가 생산하는 Pullulanase의 정제와 특성 (Purification and Characteristics of Pullulanase from Bacillus cereus subsp. mycoides)

  • 정만재;우정숙;조대선;이명열;박남규
    • 한국미생물·생명공학회지
    • /
    • 제22권1호
    • /
    • 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.

  • PDF

Purification and Characterization of Pullulanase from Klebsiella pnrumoniae NFB-320

  • Yoo, Seumg-Seouk;Yu, Ju-Hyun
    • Preventive Nutrition and Food Science
    • /
    • 제2권1호
    • /
    • 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.

  • PDF