title: Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.
abstract: Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.
authors.affiliation: The School of Food Engineering and Biological Technology, Tianjin University of Science and Technology, Tianjin 300547, P.R. China.|Shandong Provincial Key Laboratory of Microbial Engineering, Qilu University of Technology, Jinan 250353, P.R. China.
authors.forename: Xi
authors.initials: X
authors.lastname: He
authors.affiliation: Shandong Provincial Key Laboratory of Microbial Engineering, Qilu University of Technology, Jinan 250353, P.R. China.
authors.forename: Ning
authors.initials: N
authors.lastname: Han
authors.affiliation: The School of Food Engineering and Biological Technology, Tianjin University of Science and Technology, Tianjin 300547, P.R. China.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D007778:Lactobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase
keywords: Heterodimeric β-galactosidase activity; Lactobacillus kefiranofaciens ZW3; Recombinant expression; large subunit