Molecular cloning and characterization of the gene encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4.
The gene bgaP encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the BgaP gene revealed an open reading frame of 2031 bp encoding for a protein of 677 amino acid residues. The BgaP was heterologously expressed in E. coli and purified followed by Ni2+ affinity chromatography. The molecular mass of the native enzyme was approximately 156 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the BgaP indicated molecular masses of 78 and 77.311 kDa, respectively, suggesting that the BgaP is a dimer. The purified BgaP had an isoelectric point of 4.8 and exhibited maximal activity at 20 degrees C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 degrees C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG); the Km values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 degrees C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 degrees C had 14 and 47 times more than that of E. coli beta-galactosidase at 20 degrees C, respectively. Therefore, cold-active beta-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4 could conceivably be developed to fulfill the practical requirements to enable its use for lactose removal in milk and dairy products at low temperature or a reporter enzyme for psychrophilic genetic systems.
Hu
Ji M
JM
State Key Laboratory of Biocontrol, School of Life science, Zhongshan University, Guangzhou, 510275, People's Republic of China.
Li
He
H
Cao
Li X
LX
Wu
Pei C
PC
Zhang
Chen T
CT
Sang
Shu L
SL
Zhang
Xiao Y
XY
Chen
Min J
MJ
Lu
Jia Q
JQ
Liu
Yu H
YH
D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C089138:beta-galactanase; D007785:Lactose