Kim Do-Yeon
(Department of Biology, University of lncheon)
,
Cha Choon-Hwan
(Department of Biology, University of lncheon)
,
Oh Wan-Seok
(Department of Biology, University of lncheon)
,
Yoon Young-Jun
(Department of Biology, University of lncheon)
,
Kim Jung-Wan
(Department of Biology, University of lncheon)
An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B...
An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.
An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.
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제안 방법
Since the yvdF gene was highly homologous to bbmA, the catalytic properties of the enzyme encoded by yvdF might resemble those of BbmA. Therefore, genetic analysis of the gene for MAase became possible by using the well-established genetic system of B, subtilis 168 to investigate the physiological roles of the enzyme in the cell. In this study, the expression pattern of the bbmA gene under various growth conditions was monitored by following the p-galactosidase activity that was produced from the bbmA promoter fused to the lacZ gene in B.
이론/모형
Transformation into E. coli was carried out using the CaCl2 method (Sambrook et al., 1986). For B.
성능/효과
Based on the results obtained in this study, one possible role of MAase in the cytoplasm of Bacillus could be hydrolysis of linear maltodextrins, which were produced by extracellular amylolytic enzymes including a-amylase, pullulanase, and/or CGTase, and taken up via a binding protein-dependent ABC transporter(s), to mainly maltose. Since the yvdF gene was highly homologous to bbmA, the catalytic properties of the enzyme encoded by yvdF might resemble those of BBMA.
Therefore, genetic analysis of the gene for MAase became possible by using the well-established genetic system of B, subtilis 168 to investigate the physiological roles of the enzyme in the cell. In this study, the expression pattern of the bbmA gene under various growth conditions was monitored by following the p-galactosidase activity that was produced from the bbmA promoter fused to the lacZ gene in B. subtilis 168 wild-type and a sporulation mutant (spoOA). The effects of the yvdF mutation on the (3-CD hydrolyzing activity and bacterial growth were also investigated in defined media containing various carbohydrates.
1). The results correlated well with the sequence comparison data, which showed that yvdF of B. subtilis 168 was 99% identical to bbmA at the nucleotide sequence level, the B. licheniformis MAase gene 70%, and the B. stearothermophilus MAase gene only 53%. These resuIts suggested that MAase was ubiquitous among Bacillus spp.
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