• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.238-243
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• 2000

An exoinulinase (${\beta}-D-fructofuranosidase$) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. the exoinulinase gene consisted of an ORF of 0,506 bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli $DH5{\alpha}$ strain was also purified to homogeneity as determined by SDS-PAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90 min at $55^{\circ}C$. Taken together, these results indicate that the purified ${\beta}-D-fructofuranosidase$ was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and $55^{\circ}C$, respectively. the enzymehad no apparent requirement for a cofactor, and its activity was completely inactivated by $Ag^{+},{\;}Hg^{2+},{\;}and{\;}Zn^{2+}$. Kinetic experiments gave $K_m,{\;}V_{max},{\;}and{\;}K_{cat}$ values for inulin of 11.5 mM, 18 nM/s, and $72{\;}s^{-1}$, respectively. the exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4 h incubation at $55^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.360-367
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• 2006

The endoinulinase gene (inu1) from Pseudomonas mucidolens was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The inu1 gene of P. mucidolens was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTENIU (8.5kb), was then introduced to S. cerevisiae EBY100 cells and the yeast transformants selected on synthetic defined media lacking uracil and inulin-containing media. The inu1 gene under the control of the GAL1 promoter was successfully expressed in the yeast transformants, and the surface display of endoinulinase confirmed by immunofluorescence microscopy, along with its enzymatic ability to form inulooligosaccharides (IOSs) from inulin. The total endoinulinase activity reached about 2.31 units/ml when the yeast transform ants were cultivated on a YPDG medium. To efficiently hydrolyze the inulin, various reaction conditions were examined, including the pH, temperature, and inulin source. The optimized conditions were then determined as follows: pH, 7.0; temperature, $50^{\circ}C$; inulin source, Jerusalem artichoke. Under the optimized condition and 46 units of endoinulinase per g of inulin, IOSs started to be produced after 10 min of enzymatic reaction. The highest yield, 71.2% of IOSs, was achieved after 30 h of reaction without any significant loss of the initial enzyme activity. As a result of the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) were produced, and F4 was the major product.

• 농업과학연구
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• 제40권1호
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• pp.27-34
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• 2013

본 연구는 원유의 내냉성 미생물의 계절별 분포에 관해 조사하기 위해서 수행하였다. 내냉성 미생물은 최적 생장온도가 $30^{\circ}C$ 근처이지만, $7^{\circ}C$ 이하에서도 생존가능한 균으로 Pseudomonas, Achromobacter, Aeromonas 속 등이 있다. 원유로부터 분리한 총 내냉성 미생물 균수는 다른 계절에 비해 겨울철 원유에서 $3.0{\times}10^4$ CFU/mL로 가장 높은 수준으로 검출되었고, 다른 지역에 비해 B 지역에서 $2.4{\times}10^5$ CFU/mL로 유의적으로 높은 균수가 측정되었다(p<0.05). 분리한 총 706개의 균을 동정한 결과는 Gamma-proteobacteria 강이 전체의 81.02%로 가장 우세하였고, 그 중 Pseudomonas 속이 32.34%로 가장 높은 검출 빈도수를 나타내었으며, 특히 Pseudomonas fluorescens가 39.46%로 가장 우세하였다. 동정된 내냉성 미생물의 지역별 분포는 A 지역은 12개의 Acinetobacter johnsonii, B 지역은 21개의 Pseudomonas fluorescens, C 지역은 13개의 Enterobacter amnigenus, D 지역은 19개의 Psychrobacter maritimus, E 지역은 19개의 Acinetobacter johnsonii, F 지역은 8개의 Acinetobacter haemolyticus, G 지역은 20개의 Pseudomonas fluorescens, H 지역은 9개의 Acinetobacter johnsonii, I 지역은 17개의 Pseudomonas mucidolens가 높은 검출 빈도수를 나타내었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.582-585
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• 2003

Sequence analysis indicated that Bacillus polymyxa MGL21 CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase. Furthemore, CFTase possessed a highly conserved core region. In order to understand the role of the core region on the function of CFTase from B. polymyxa MGL21 CFTase ${\Delta}NC$ was prepared. The molecular weight of the purified wild type CFTase and $CFTase{\Delta}NC$ were 148kDa, 90kDa, respectively.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.921-928
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• 2002

Microorganism producing extracellular CFTase was isolated from soil and designated as Bacillus polymyxa MGL21. The gene encoding the CFTase (cft) from B. polymyxa MGL21 was cloned and sequenced. The ORF of the cf gene was composed of 3,999 nucleotides, encoding a protein (1,333 amino acids) with a predicted molecular mass of 149,375 Da. Sequence analysis indicated that CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase ($50\%$ identity, 259 amino acids). Furthermore, CFTase possessed a highly conserved core region, which is considered to be functional for the hydrolysis reaction of inulin. The cft gene was expressed in a His-tagged form in Escherichia coli cells, and the His-tagged CFTase was purified to homogeneity. The optimal temperature and pH for CFTase activity were found to be $50^{\circ}C$ and 9.0, respectively. The enzyme activity was completely inhibited by 10 mM $Ag^+\;and\;Cu^2+$. Thin-layer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction ut also disproportionation and hydrolysis reactions as well.