title: Microbial β-Galactosidase ofPediococcus pentosaceusID-7: Isolation, Cloning, and Molecular Characterization.
abstract: Pediococcus pentosaceusID-7 was isolated from kimchi, a Korean fermented food, and it showed high activity for lactose hydrolysis. The β-galactosidase ofP. pentosaceusID-7 belongs to the GH2 group, which is composed of two distinct proteins. The heterodimeric LacLM type of β-galactosidase found inP. pentosaceusID-7 consists of two genes partially overlapped,lacLandlacMencoding LacL (72.2 kDa) and LacM (35.4 kDa). In this study,Escherichia coliMM294 was used for the production of LacL, LacM, and LacLM. These three types of recombinant proteins were expressed, purified, and characterized. The specific activities of LacLM and LacL were 339 and 31 U/mg, respectively. However, activity was not detected with LacM alone. The optimal pH of LacLM and LacL was pH 7.5 and pH 7.0, and the optimal temperature of LacLM and LacL was 40°C and 50°C, respectively. The optimal temperature changes indicate that LacLM is able to achieve higher activity at a relatively lower temperature. LacLM was strongly activated by Mg2+, Mn2+, and Zn2+, which was not true for LacL. Consistent with this, EDTA strongly inactivated LacLM and LacL, but the presence of reducing agents did not dramatically alter the activity. Taken together, multiple alignment of amino acid sequences and phylogenetic analysis results of LacL and LacM ofP. pentosaceusID-7 suggest the evolution of LacL into LacLM and that the use of divalent metal ions results in higher activity.
authors.affiliation: Department of Bio and Fermentation Convergence Technology, BK21 PLUS Project, Kookmin University, Seoul 02707, Republic of Korea.
authors.forename: Ji-Yeong
authors.initials: JY
authors.lastname: Lee
authors.affiliation: BioLeaders Corporation, Daejeon 34028, Republic of Korea.
authors.forename: Mi-Sun
authors.initials: MS
authors.lastname: Kwak
authors.affiliation: BioLeaders Corporation, Daejeon 34028, Republic of Korea.
authors.forename: Jong-Bok
authors.initials: JB
authors.lastname: Roh
authors.affiliation: BioLeaders Corporation, Daejeon 34028, Republic of Korea.
authors.forename: Kwang
authors.initials: K
authors.lastname: Kim
authors.affiliation: Department of Bio and Fermentation Convergence Technology, BK21 PLUS Project, Kookmin University, Seoul 02707, Republic of Korea.
authors.forename: Moon-Hee
authors.initials: MH
authors.lastname: Sung
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D000070006:Pediococcus pentosaceus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001616:beta-Galactosidase
keywords: Heterodimeric β-galactosidase; LacLM; LacZ; Pediococcus pentosaceus