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Research Support, Non-U.S. Gov't
- Identification and Characterization of a Novel β-Galactosidase from Victivallis vadensis ATCC BAA-548, an Anaerobic Fecal Bacterium
- Uyangaa Temuujin , Won-Jae Chi , Jae-Sun Park , Yong-Keun Chang , Jae Yang Song , Soon-Kwang Hong
- J. Microbiol. 2012;50(6):1034-1040. Published online December 30, 2012
- DOI: https://doi.org/10.1007/s12275-012-2478-6
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- 13 Citations
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Abstract
- Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T7 promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni2+-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His6-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The Km and Vmax of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.
Journal Article
- A Selected Probiotic Strain of Lactobacillus fermentum CM33 Isolated from Breast-Fed Infants as a Potential Source of β-Galactosidase for Prebiotic Oligosaccharide Synthesis
- Wattana Sriphannam , Saisamorn Lumyong , Piyanuch Niumsap , Hisashi Ashida , Kenji Yamamoto , Chartchai Khanongnuch
- J. Microbiol. 2012;50(1):119-126. Published online February 27, 2012
- DOI: https://doi.org/10.1007/s12275-012-1108-7
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- 20 Citations
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Abstract
- Lactic acid bacteria from healthy breast-fed infants were isolated and screened for β-galactosidase production in MRS broth. Among 49 isolates that exhibited the yellow clear zone on MRS agar supplemented with bromocresol blue, the isolate CM33 was selected as being the highest β-galactosidase producer and was identified as Lactobacillus fermentum based on its morphological characteristics and 16S rDNA nucleotide sequence. L. fermentum CM33 exhibited a good survival rate under the simulated stomach passage model, comparable to known probiotic strains L. gallinarum JCM2011 and L. agilis JCM1187. L. fermentum CM33 was antagonistic to pathogenic bacteria Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella typhi, and Salmonella enteriditis, using the well diffusion method. In addition, the selected lactobacilli exhibited a high growth rate when cultivated in modified MRS containing commercial galactooligosaccharide (GOS) as a sole carbon source, as well as in glucose. A preliminary study on the enzymatic synthesis of oligosaccharide using crude β-galactosidase revealed the capability for oligosaccharide synthesis by the transgalactosylation activity.
Research Support, Non-U.S. Gov't
- Isolation and Characterization of Cold-adapted Strains Producing β-Galactosidase
- Jeong-Won Park , Yong-Sik Oh , Jai-Yun Lim , Dong-Hyun Roh
- J. Microbiol. 2006;44(4):396-402.
- DOI: https://doi.org/2414 [pii]
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Abstract
- β-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°C, the BS 1 strain was unable to grow at 37°C. Another strain, HS-39, evidenced retarded growth at 37°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 β-galactosidase when incubated for 60 h at 10°C, Buttiauxella sp. HS-39 generated β-galactosidase earlier, and at slightly lower levels, than R. aquatilis BS 1. The optimum temperature for β-galactosidase was 30°C for R. aquatilis BS-1, and was 45°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 β-galactosidase from each isolate, might prove useful in some biotechnological applications.
- Identification and characterization of pH-regulated genes in saccharomyces cerevisiae
- Hong, Sung Ki , Choi, Eui Yul
- J. Microbiol. 1996;34(4):327-333.
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Abstract
- Yeast, like many other microbes, encounters large variations in ambient pH in their natural environments. Microorganisms capable of growing over a wide pH range require a versatile, efficient pH homeostatic mechanism protecting intracellular processes against extremes of pH. In several organisms, fusions to the bacterial lacZ gene have been extremely useful for the identification of genes expressed at different time during the life cycle or under different growth conditions. In this study, using the lacZ gene screening system, we surveyed a large number of yeast strains with lacZ insertion to identify genes regulated by pH. A yeast genomic library was constructed and inserted with lacZ by a shuttle mutagenesis procedure. The yeast transformants were individually picked up with a toothpick, replica-plated, and grown in alkaline pH medium. Among the 35,000 colonies screened, 10 candidate strains were identified initially by the β-gal assay. We finally confirmed two yeast strains carrying the genes whose expression are strictly dependent on pH of growth medium. One of the fusions showing a 10-fodl induction in expression level in response to alkali pH was selected and further characterized. The pH-regulated gene was cloned by inverse PCR and a partial sequence of the gene was determined. Identification and characterization of the gene is currently under investigation.
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