Browse > Article
http://dx.doi.org/10.5352/JLS.2007.17.9.1197

Cloning, Sequencing and Expression of the Gene Encoding a Thermostable β-Xylosidase from Paenibacillus sp. DG-22  

Lee, Tae-Hyeong (Department of Biotechnology, Dongguk University)
Lee, Yong-Eok (Department of Biotechnology, Dongguk University)
Publication Information
Journal of Life Science / v.17, no.9, 2007 , pp. 1197-1203 More about this Journal
Abstract
A genomic DNA library of the bacterium Paenibacillus sp. DG-22 was constructed and the ${\beta}-xylosi-dase-positive$ clones were identified using the fluorogenic substrate $4-methylumbelliferyl-{\beta}-D-xylopyr-anoside$ $({\beta}MUX)$. A recombinant plasmid was isolated from the clone and 4.3-kb inserted DNA was sequenced. The ${\beta}-xylosidase$ gene (xylA) was comprised of a 2,106 bp open reading frame (ORF) en-coding 701 amino acids with a molecular weight of 78,710 dalton and a pI of 5.0. The deduced amino acid sequence of the xylA gene product had significant similarity with ${\beta}-xylosidases$ classified into family 52 of glycosyl hydrolases. The xylA gene was subcloned into the pQE60 expression vector to fuse with six histidine-tag. The recombinant ${\beta}-xylosidase$ $(XylA-H_6)$ was purified to homogeneity by heat-treatment and immobilized metal affinity chromatography. The pH and temperature optima of the $XylA-H_6$ enzyme were pH 5.5-6.0 and $60^{\circ}C$, respectively.
Keywords
Paenibacillus sp. ${\beta}-xylosidase$; cloning; expression;
Citations & Related Records
Times Cited By KSCI : 4  (Citation Analysis)
연도 인용수 순위
1 Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, U.S.A
2 Sanger, F., S. Nicklen and A. R. Coulson. 1977. DNA sequencing with chain- terminating inhibitors. Pro. Natl. Acad. Sci. USA 74, 5463-5467   DOI   ScienceOn
3 Takami, H., K. Nakasone, Y. Takaki, G. Maeno, R. Sasaki, N. Masui, F. Fuji, C. Hirama, Y. Nakamura, N. Ogasawara, S. Kuhara and K. Horikoshi. 2000. Complete genome sequence of the alkaliphilic bacterium Bacillus holodurans and sequence comparison with Bacillus subtilis. Nucleic Acids Res. 28, 4317-4331   DOI   ScienceOn
4 Ward, O. P. and M. Moo-Young. 1989. Enzymatic degradation of cell wall and related plant polysaccharides. Crit. Rev. Biotechnol. 8, 237-274   DOI   ScienceOn
5 Watson, M. E. E. 1984. Compilation of published signal sequences. Nucleic Acids Res. 12, 5145-5164   DOI   ScienceOn
6 Henrissat, B. and A. Bairoch. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695-696   DOI
7 Suzuki, T., E. Kitagawa, F. Sakakibara, K. Ibata, K. Usui and K. Kawai. 2001. Cloning, expression, and characterization of a family 52 $\beta$-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1. Biosci. Biotechnol. Biochem. 65, 487-494   DOI   ScienceOn
8 Wong, K. K. Y. and J. N. Saddler. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52, 305-317
9 Lee, T. H., P. O. Lim and Y.-E. Lee. 2007. Regulation of $\beta$-xylosidase biosynthesis in Paenibacillus sp. DG-22. J. Life Sci. 17, 407-411   과학기술학회마을   DOI   ScienceOn
10 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254   DOI   ScienceOn
11 Bravman, T., G. Zolotnitsky, S. Shulami, V. Belakhov, D. Solomon, T. Baasov, G. Shoham and Y. Shoham. 2001. Stereochemistry of family 52 glycosyl hydrolases: a $\beta$-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme. FEBS Lett. 495, 39-43   DOI   ScienceOn
12 Henrissat, B. and A. Bairoch. 1993. New family in the classification of glycosyl hydrolases based on amino acid sequence similarities, Biochem. J. 293, 781-788   DOI
13 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685   DOI   ScienceOn
14 Lee, Y-E. and P. O. Lim. 2004. Purification and characterization of two thermostable xylanases from Paenibacillus sp. DG-22. J. Microbiol. Biotechnol. 14, 1014-1021
15 Lee, T. H., P. O. Lim and Y.-E. Lee. 2007. Cloning, characterization, and expression of xylanase A gene from Paenibacillus sp. DG-22 in Escherichia coli. J. Microbiol. Biotechnol. 17, 29-36   과학기술학회마을
16 Lee, Y-E. 2004. Isolation and characterization of thermostable xylanase-producing Paenibacillus sp. DG-22. Kor. J. Microbiol. Biotechnol. 32, 22-28
17 Lee, Y-E. and J. G. Zeikus. 1993. Genetic organization, sequence and biochemical characterization of recombinant $\beta$-xylosidase from Thermoanuercoacterium saccharolyticum strain B6A-RI. J. Gen. Microbiol. 139, 1235-1243   DOI   ScienceOn
18 Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208-218   DOI
19 Poutanen, K. and J. Puls. 1988. Characteristics of Trichoderma reesei $\beta$-xylosidase and its use in the hydrolysis of solubilized xylans. Appl. Microbiol. Biotechnol. 28, 425-432   DOI
20 Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402   DOI   ScienceOn
21 Baba, T., R. Shinke and T. Nanmori. 1994. Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, $\beta$-xylosidase and xylanase, of Bacillus stearothermophilus 21. Appl. Environ. Microbiol. 60, 2252-2258
22 Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3, 286-290   DOI   ScienceOn