Browse > Article
http://dx.doi.org/10.4014/jmb.1606.06010

Molecular Characterization of the α-Galactosidase SCO0284 from Streptomyces coelicolor A3(2), a Family 27 Glycosyl Hydrolase  

Temuujin, Uyangaa (Department of Bioscience and Bioinformatics, Myongji University)
Park, Jae Seon (Department of Bioscience and Bioinformatics, Myongji University)
Hong, Soon-Kwang (Department of Bioscience and Bioinformatics, Myongji University)
Publication Information
Journal of Microbiology and Biotechnology / v.26, no.9, 2016 , pp. 1650-1656 More about this Journal
Abstract
The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters Vmax and Km for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.
Keywords
Streptomyces coelicolor; SCO0284; α-galactosidase; GH 27 family; NPCBM;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Fernández-Leiro R, Pereira-Rodríguez A, Cerdán ME, Becerra M, Sanz-Aparicio J. 2010. Structural analysis of Saccharomyces cerevisiae α-galactosidase and its complex with natural substrates reveals new insights into substrate specificity of GH 27 glycosidases. J. Biol. Chem. 285: 28020-28033.   DOI
2 Green MR, Sambrook J. 2012. Molecular Cloning. A Laboratory Manual, 4th Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
3 Katrolia P, Jia H, Yan Q, Song S, Jiang Z, Xu H. 2012. Characterization of a protease resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides. Bioresour. Technol. 110: 578-586.   DOI
4 Katrolia P, Rajashekhara E, Yan Q, Jiang Z. 2014. Biotechnological potential of microbial α-galactosidases. Crit. Rev. Biotechnol. 34: 307-317.   DOI
5 Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. 2000. Practical Streptomyces Genetics. John Innes Foundation, Norwich, England.
6 Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.   DOI
7 Kim WD, Kaneko S, Park GG, Tanaka I, Kusakabe I, Kobayashi H. 2003. Purification and characterization of α-galactosidase from sunflower seeds. Biotechnol. Lett. 25: 353-358.   DOI
8 Kondoh K, Morisaki K, Kim WD, Parkm GG, Kaneko S, Kobayashi H. 2005. Cloning and expression of the gene encoding Streptomyces coelicolor A3(2) alpha-galactosidase belonging to family 36. Biotechnol. Lett. 27: 641-647.   DOI
9 Kumar SKP, Mulimani VH. 2010. Continuous hydrolysis of raffinose family oligosaccharides in soymilk by fluidized bed reactor. LWT Food Sci. Technol. 43: 220-225.   DOI
10 Lim JH, Lee CR, Dhakshnamoorthy V, Park JS, Hong SK. 2016. Molecular characterization of Streptomyces coelicolor A(3) SCO6548 as a cellulose 1,4-β-cellobiosidase. FEMS Microbiol. Lett. 363(3). DOI: 10.1093/femsle/fnv245.   DOI
11 Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 658-666.   DOI
12 Liu QP, Sulzenbacher G, Yuan H, Bennett EP, Pietz G, Saunders K, et al. 2007. Bacterial glycosidases for the production of universal red blood cells. Nat. Biotechnol. 25: 454-464.   DOI
13 Murphy RA, Power RFG. 2002. Expression of an a-galactosidase from Saccharomyces cerevisiae in Aspergillus awamori and Aspergillus oryzae. J. Ind. Microbiol. Biotechnol. 28: 97-102.   DOI
14 Naumoff DG. 2004. Phylogenetic analysis of alpha-galactosidases of the GH27 family. Mol. Biol. (Engl. Transl.) 38: 388-399.   DOI
15 Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.   DOI
16 Temuujin U, Chi WJ, Lee SY, Chang YK, Hong SK. 2011. Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose. Appl. Microbiol. Biotechnol. 92: 749-759.   DOI
17 Post DA, Luebke VE. 2005. Purification, cloning and properties of α-galactosidase from Saccharopolyspora erythraea and its use as a reporter system. Appl. Microbiol. Biotechnol. 67: 91-96.   DOI
18 Shibuya H, Nagasaki H, Kaneko S, Yoshida S, Park GG, Kusakabe I, Kobayashi H. 1998. Cloning and high level expression of α-galactosidase cDNA from Penicillium purpurogenum. Appl. Environ. Microbiol. 64: 4489-4494.
19 Temuujin U, Chi WJ, Chang YK, Hong SK. 2012. Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose. J. Bacteriol. 194: 142-149.   DOI
20 Wang H, Luo H, Li J, Bai Y, Huang H, Shi P, et al. 2010. An α-galactosidase from an acidophilic Bispora sp. MEY-1 strain acts synergistically with β-mannanase. Bioresour. Technol. 101: 8376-8382.   DOI
21 Chen Y, Jin M, Egborg T, Coppla G, Andre J, Calhoun DH. 2000. Expression and characterization of glycosylated and catalytically active recombinant human α-galactosidase A produced in Pichia pastoris. Protein Expr. Purif. 20: 472-484.   DOI
22 Balabanova LA, Bakunina IY, Nedashkovskaya OI, Makarenkova ID, Zaporozhets TS, Besednova NN, et al. 2010. Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 alpha-galactosidase. Mar. Biotechnol. (NY) 12: 111-120.   DOI
23 Bentley SD, Chater KF, Cerdeño-Tárraga AM, Challis GL, Thomson NR, James KD, et al. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417: 141-147.   DOI
24 Bradford MM. 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
25 Doumith M, Weingarten P, Wehmeier UF, Salah-Bey K, Benhamou B, Capdevila C, et al. 2000. Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea. Mol. Genet. Genomics 264: 477-485.   DOI
26 Gherardini F, Babcock M, Salyers AA. 1985. Purification and characterization of two α-galactosidases associated with catabolism of guar gum and other α-galactosides by Bacteroides ovatus. J. Bacteriol. 161: 500-506.
27 Enkhbaatar B, Lee CR, Hong YS, Hong SK. 2016. Molecular characterization of xylobiose- and xylopentaose-producing β-1,4-endoxylanase SCO5931 from Streptomyces coelicolor A3(2). Appl. Biochem. Biotechnol. 180: 349-360.   DOI
28 Enkhbaatar B, Temuujin U, Lim JH, Chi WJ, Chang YK, Hong SK. 2012. Identification and characterization of a xyloglucan-specific family 74 glycosyl hydrolase from Streptomyces coelicolor A3(2). Appl. Environ. Microbiol. 78: 607-611.   DOI