[Cloning, expression and characterization of a heterodimeric beta-galactosidase from Lactobacillus acidophilus ATCC 4356].
Heterodimeric beta-galactosidase of Lactobacillus acidophilus belongs to glycoside hydrolase family 2, encoded by two overlapping and translational coupling genes, lacL and lacM. The lacL and lacM genes of the sequenced strain L. acidophilus NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively.


OBJECTIVE
To clone, overexpress and characterize the enzyme in Escherichia coli.


METHODS
We cloned the fragment (2834 bp) containing ribose-binding site (RBS) and coding regions of the lacLM genes from L. acidophilus ATCC 4356 into expression vector pQE31. RBS and HIS-Tag of pQE31 were substituted by inserted fragment. Recombinant plasmid was electrotransformed into E. coli JM109. Expression product was purified by ammonium sulphate fractionation, anion-exchange, affinity chromatography and gel permeation. Native molecular mass of homogenous enzyme was measured by gel permeation, and beta-galactosidase activity was determined by using o-nitrophenyl-beta-D-galactopyranoside (ONPG) as the substrate.


RESULTS
Overexpression of the soluble enzyme in E. coli was achieved. Amino acid residue 512 of recombinant LacL was different from that of L. acidophilus NCFM. Homogenous enzyme was obtained by purification. The homogenous enzyme had a specific activity of 226 U/mg protein, a native molecular mass of 96.3+/-4.6 kDa, an optimum temperature at 49 degrees C and an optimum pH of 7. The Km and Vmax values of the enzyme were 2.18+/-0.12 mmol/L, 273+/-5 U/mg protein respectively.
Pan
Qu
Q
Department of Microbiology, Chengdu Medical College, Chengdu 610083, China. ppqlove2006@yahoo.com.cn
Li
Jinchuan
J
Cong
Yanguang
Y
Liu
Lina
L
Zhu
Junmin
J
Hu
Fuquan
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D007779:Lactobacillus acidophilus / Q000737:chemistry / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D001616:beta-Galactosidase