However,

there are few reports on β-galactosidases obtained via metagenomic strategies up to now. Recently, a novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library [18]. Nevertheless, this enzyme was regarded as a cold-adapted β-galactosidase due to its optimal temperature of 38°C and 54% residual activity at 20°C. Thus, identification of novel β-galactosidases Akt inhibitor with high thermostability and low inhibition of reaction product via metagenomic strategy is still urgently in demand. In the present study, a metagenomic library from soil samples of Turpan Basin, the hottest and driest area in China, was constructed, and a novel β-galactosidase (Gal308) was identified and expressed in Escherichia coli (E. coli). The enzymatic properties of Gal308 with MS-275 mouse N-terminal fusion tag were investigated after purification, and this enzyme displayed several novel properties including high thermostability, high tolerance of galactose and glucose, as well as high enzymatic activity for lactose. These properties JSH-23 purchase make it a good candidate in the production of low-lactose milk and dairy products after further study. Results Screening for β-galactosidase from a metagenomic library

To discover novel thermostable β-galactosidases, a metagenomic library consisting of approximately 8,000 clones was constructed using DNA extracted from soil samples of the Mountain of Flames of the Turpan Basin in China. Restriction analysis of 20 randomly selected clones from

metagenomic library indicated that 95% of clones contained inserts of 2.5 to 7.5 kb in size, with an average size of 4.2 kb. Thus, the metagenomic library covered theoretically about 33.6 MB of soil microbial community DNA. One positive clone with bright blue color was finally identified from the metagenomic library. The activity of the positive clone was reconfirmed after retransformation, and then the plasmid of this clone was extracted and an insert of 5215 bp was sequenced. The ORF-finder and blastX analysis revealed the presence of an open reading frame of 1977 bp, which encoded a glycoside GNAT2 hydrolase family 42 (GH family 42) protein (Gal308) of 658 amino acids. A protein blast (blastp) search in the databases of NCBI indicated that the protein had the highest identity of 49% (291/599) with the β-galactosidase from one thermophilic microbe Geobacillus thermocatenulatus, as well as a low identity of only 38% (224/593) with the β-galactosidase from the other thermophilic microbe Thermoanaerobacterium thermosaccharolyticum DSM 571, suggesting that Gal308 is probably a novel thermostable β-galactosidase from unculturable microorganisms. In addition, multiple sequence alignment of Gal308 and other five homologous β-galactosidases from GH family 42 allowed the identification of the active site residues of Gal308 (Figure 1).