Molecular Cloning and Characterization of the Gene Encoding Cold-Active β-Galactosidase from a Psychrotrophic and Halotolerant Planococcus sp. L4

• Published in: Journal of agricultural and food chemistry 2007-03, Vol.55 (6), p.2217-2224
• Main Authors: Hu, Ji M, Li, He, Cao, Li X, Wu, Pei C, Zhang, Chen T, Sang, Shu L, Zhang, Xiao Y, Chen, Min J, Lu, Jia Q, Liu, Yu H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Animals; Bacillus - enzymology; Base Sequence; beta-glucosidase; bgaP gene; Biological and medical sciences; Cloning, Molecular; Cold Temperature; enzyme activity; Escherichia coli - genetics; food biotechnology; Food industries; Fundamental and applied biological sciences. Psychology; Gene Expression; genes; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Lactose - metabolism; Milk - chemistry; molecular cloning; Molecular Sequence Data; molecular weight; nucleotide sequences; Planococcus (bacteria); psychrotrophic bacteria; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; temperature
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf062910r

The gene bgaP encoding cold-active β-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 °C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 °C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-β-d-galactopyranoside (ONPG); the K m values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 °C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 °C had 14 and 47 times more than that of E. coli β-galactosidase at 20 °C, respectively. Therefore, cold-active β-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. Keywords: Cold-active β-galactosidase; gene cloning; properties; psychrotrophic and halotolerant Planococcus sp. L4