Park Jeong-Won
(Division of Life Sciences, College of Natural Sciences and Biotechnology Research Institute, Chungbuk National University)
,
Oh Yong-Sik
(Division of Life Sciences, College of Natural Sciences and Biotechnology Research Institute, Chungbuk National University)
,
Lim Jai-Yun
(Division of Life Sciences, College of Natural Sciences and Biotechnology Research Institute, Chungbuk National University)
,
Roh Dong-Hyun
(Division of Life Sciences, College of Natural Sciences and Biotechnology Research Institute, Chungbuk National University)
[ ${\beta}-Galactosidase$ ] is extensively employed in the manufacture of dairy products, including lactose-reduced milk. Here, we have isolated two gram-negative and rod-shaped coldadapted bacteria, BS 1 and HS 39. These strains were able to break down lactose at low temperatures. Althou...
[ ${\beta}-Galactosidase$ ] is extensively employed in the manufacture of dairy products, including lactose-reduced milk. Here, we have isolated two gram-negative and rod-shaped coldadapted bacteria, BS 1 and HS 39. These strains were able to break down lactose at low temperatures. Although two isolates were found to grow well at $10^{\circ}C$, the BS 1 strain was unable to grow at $37^{\circ}C$. Another strain, HS-39, evidenced retarded growth at $37^{\circ}C$. The biochemical characteristics and the results of 16S rDNA sequencing identified the BS 1 isolate as Rahnella aquatilis, and showed that the HS 39 strain belonged to genus Buttiauxella. Whereas the R. aquatilis BS 1 strain generated maximal quantities of ${\beta}-galactosidase$ when incubated for 60h at $10^{\circ}C$, Buttiauxella sp. HS-39 generated ${\beta}-galactosidase$ earlier, and at slightly lower levels, than R. aquatilis BS 1. The optimum temperature for ${\beta}-galactosidase$ was $30^{\circ}C$ for R. aquatilis BS-1, and was $45^{\circ}C$ for Buttiauxella sp. HS-39, thereby indicating that R. aquatilis BS-1 was able to generate a cold-adaptive enzyme. These two cold-adapted strains, and most notably the ${\beta}-galactosidase$ from each isolate, might prove useful in some biotechnological applications.
[ ${\beta}-Galactosidase$ ] is extensively employed in the manufacture of dairy products, including lactose-reduced milk. Here, we have isolated two gram-negative and rod-shaped coldadapted bacteria, BS 1 and HS 39. These strains were able to break down lactose at low temperatures. Although two isolates were found to grow well at $10^{\circ}C$, the BS 1 strain was unable to grow at $37^{\circ}C$. Another strain, HS-39, evidenced retarded growth at $37^{\circ}C$. The biochemical characteristics and the results of 16S rDNA sequencing identified the BS 1 isolate as Rahnella aquatilis, and showed that the HS 39 strain belonged to genus Buttiauxella. Whereas the R. aquatilis BS 1 strain generated maximal quantities of ${\beta}-galactosidase$ when incubated for 60h at $10^{\circ}C$, Buttiauxella sp. HS-39 generated ${\beta}-galactosidase$ earlier, and at slightly lower levels, than R. aquatilis BS 1. The optimum temperature for ${\beta}-galactosidase$ was $30^{\circ}C$ for R. aquatilis BS-1, and was $45^{\circ}C$ for Buttiauxella sp. HS-39, thereby indicating that R. aquatilis BS-1 was able to generate a cold-adaptive enzyme. These two cold-adapted strains, and most notably the ${\beta}-galactosidase$ from each isolate, might prove useful in some biotechnological applications.
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제안 방법
In this study, we attempted to isolate psychrophilic or psychrotrophic microorganisms which were able to hydrolyze lactose at low temperatures, and to monitor growth behavior at a variety of temperatures, and to characterize the production and properties of P- galactosidase.
P-Galactosidase production was evaluated via measurements of the rate of hydrolysis of 2-nitrophenyl- P-D-galactopyranoside (ONPG; Duchefa Biochemie, Holland) in Z buffer (60 mM NaHPOWHQ 40 mM Na2HPO4H2O, 10 mM KC1, 1 mM MgSO4-7H2O, 50 mM P-mercaptoethanol; pH 7.0) for 10 min at 15℃, using the crude cell lysates from cells which were time-course incubated at 10, 20, and 30℃. The optimal enzyme activity temperatures were determined via 10 min of incubation of the cell lysates in 1 ml of Z buffer with 0.
The 16S rDNA gene was amplified from the chromosomal DNA of the isolated strains as a template via the polymerase chain reaction (PCR). PCR was conducted in a 50 ul reaction mixture containing 50 ng of template DNA, 2 pmol of each primer, 2.5 mM each dNTP, 1.25 U of Takara Ex Taq DNA polymerase (Takara Biomedicals, Japan) using a 2720 Thermal cyber (Applied Biosystems, USA) with the following program; 30 cycles of denatu!'ation at 94℃ for 30 sec, annealing at 50℃ for 30 sec, and extension at 72℃ for 1 min and 20 sec. The primers used were as follows: forward, 27F (E.
The 16S rDNA sequences obtained in this study were assembled with SeqMan software (DNAStar, USA), and utilized for phylogenetic analysis. The 16S rDNA gene sequences of the related taxa were acquired from the GenBank database at the National Center for Biotechnology Information (USA).
Due to our interest in two isolated microorganisms, we have attempted to determine their phylogenetic relationships. We amplified nearly the complete sequence (1, 465 nucleotides) of the 16S rDNA gene via PCR, determined its sequence, and then examined the relevant phylogenetic relationships via the Neighborjoining method. The isolated BS 1 was clustered to a type strain, Rahnella aquatilis, and type strains in Yersinia spp.
대상 데이터
25 U of Takara Ex Taq DNA polymerase (Takara Biomedicals, Japan) using a 2720 Thermal cyber (Applied Biosystems, USA) with the following program; 30 cycles of denatu!'ation at 94℃ for 30 sec, annealing at 50℃ for 30 sec, and extension at 72℃ for 1 min and 20 sec. The primers used were as follows: forward, 27F (E. coli; 5'-AG AGTTTGATCMTGGCTCAG-31) and reverse, 1492R (K coli; 5'-TACGGYTACCTTGTTACGACTT・3'). The products were purified using a cleaning kit (Bioneer, Korea), and sequenced at SolGent Co.
이론/모형
Genomic DNA was acquired from the is이ates via the bead beating method. 1 ml of harvested cells were suspended in 0.
, 1997), and gaps were edited using the BioEdit program (Hall, 1999). The evolutionary distances were calculated with the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree for the data set was created via the Neighbor-joining method (Saitou and Nei, 1987) using the Mega 3 program (Kumar et al.
The evolutionary distances were calculated with the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree for the data set was created via the Neighbor-joining method (Saitou and Nei, 1987) using the Mega 3 program (Kumar et al., 2004). The stability of relationships was evaluated via bootstrap analyses of the neighbor-joining data, on the basis of 1,000 replications (Felsenstein, 1985).
성능/효과
4). Taken together, R. aquatilis BS 1 evidenced adaptive growth at lower temperatures than did HS 39 (Fig. 2), and produced P-galactosidase that was more cold-adaptive than HS 39. Ohgiya et al.
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