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Abstract: A novel transglycosylating β-galactosidase was purified from Enterobacter agglomerans B1. It was a homodimer of ∼248kDa. The optimal pH and temperature for oNPGal hydrolysis were 7.5–8.0 and 37–40°C, respectively. The K m values for oNPGal and lactose were 0.06 and 114mM, respectively. The enzyme produced galacto-oligosaccharides in a 38% yield at the lactose concentration of 12.5% (w/v). When using oNPGal as donor, the enzyme was able to catalyze glycosyl transfer to a series of acceptors, including hexose, pentose, β- or α-disaccharides, hexahydroxy alcohol, cyclitol, and aromatic glycosides. This suggested the enzyme to be a potential synthetic tool for preparing galactose-containing chemicals. The gene encoding this enzyme was cloned by degenerate PCR and TAIL-PCR. It revealed an ORF of 3090 nucleotides encoding a 1029 amino-acid protein, which had been expressed in Escherichia coli. Transferase activities in both recombinant and natural enzymes were similar. [Copyright &y& Elsevier]

Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1) and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo . [ABSTRACT FROM AUTHOR]