Characterization of the cross-linked enzyme aggregates of a novel ß-galactosidase, a potential catalyst for the synthesis of galacto-oligosaccharides.

A novel ß-galactosidase (Bgal1-3) was isolated from a marine metagenomic library and then its cross-linked enzyme aggregates (CLEAs) were prepared. The enzymatic properties of Bgal1-3-CLEAs were studied and compared with that of the free enzyme. The thermostability and storage stability of Bgal1-3 were significantly improved after it was immobilized as CLEAs. The galactose-tolerance of the enzyme was also enhanced after the immobilization, which could relieve the inhibitory effect and then tends to be beneficial for the galacto-oligosaccharides (GOS) synthesis. Moreover, higher GOS yield was achieved (59.4 ± 1.5%) by Bgal1-3-CLEAs compared to the free counterpart (57.1 ± 1.7%) in an organic-aqueous biphasic system. The GOS content and composition of the syrups synthesized by the free enzyme and Bgal1-3-CLEAs were similar and they both contained at least seven different oligosaccharides with the degree of polymerization (DP) ranging between 3 and 9. Furthermore, Bgal1-3-CLEAs maintained 82.1 ± 2.1% activity after ten cycles of reuse; the GOS yield of the tenth batch was 52.3 ± 0.3%, which was still higher than that of the most former reports. To the best of our knowledge, this is the first report on the GOS synthesis using CLEAs of ß-galactosidase in an organic-aqueous biphasic system. The study not only further expands the application scope of CLEA, but also provides a potential catalyst for the synthesis of GOS with low cost.

Identification and characterization of an unusual glycosyltransferase-like enzyme with ß-galactosidase activity from a soil metagenomic library.

Glycosyltransferases and glycoside hydrolases are two diversified groups of carbohydrate-active enzymes (CAZymes) in existence, they serve to build and break down the glycosidic bonds, respectively, and both categories have formed many sequence-based families. In this study, a novel gene (glyt110) conferring ß-galactosidase activity was obtained from a metagenomic library of Turpan Basin soil. Sequence analysis revealed that glyt110 encoded a protein of 369 amino acids that, rather than belonging to a family typically known for ß-galactosidase activity, belonged to glycosyltransferase family 4. Because of this unusual sequence information, the novel gene glyt110 was subsequently expressed in Escherichia coli BL21(DE3), and the recombinant enzyme (Glyt110) was purified and characterized. Biochemical characterization revealed that the ß-galactosidase activity of Glyt110 toward o-nitrophenyl-ß-D-galactopyranoside (ONPG) and lactose were identified to be 314±18.3 and 32±2.7 U/mg, correspondingly. In addition, Glyt110 can synthesize galacto-oligosaccharides (GOS) using lactose as substrate. A GOS yield of 47.2% (w/w) was achieved from 30% lactose solution at 50 °Ð¡, pH 8.0 after 10 h reaction. However, Glyt110 was unable to glycosylate either N-acetylated saccharides or lactose and galactose using UDP-gal as sugar donor, and its glycosyltransferase activity needs further investigation. These results indicated that Glyt110 is an unusual enzyme with ß-galactosidase activity but phylogenetically related to glycosyltransferase. Our findings may provide opportunities to improve the insight into the relationship between glycosyltransferases and glycoside hydrolases and the sequence-based classification.

Enzymatic synthesis of galacto-oligosaccharides in an organic-aqueous biphasic system by a novel ß-galactosidase from a metagenomic library.

Prebiotic galacto-oligosaccharides (GOS) were effectively synthesized from lactose in organic-aqueous biphasic media by a novel metagenome-derived ß-galactosidase BgaP412. A maximum GOS yield of 46.6% (w/w) was achieved with 75.4% lactose conversion rate in the cyclohexane/buffer system [95:5 (v/v) cyclohexane/buffer] under the optimum reaction conditions (initial lactose concentration = 30% (w/v), T = 50 °C, pH 7.0, and t = 8 h). The corresponding productivity of GOS was approximately 17.5 g L(-1) h(-1). The GOS mixture consisted of tri-, tetra-, and pentasaccharides. Trisaccharides were the chief component of reaction products. These experimental results showed that a low water content, a high initial lactose concentration, and an elevated reaction temperature could significantly promote the transgalactosylation activity of ß-galactosidase BgaP412; at the same time, the enhanced GOS yield in an organic-aqueous biphasic system is because of the fact that thermodynamic equilibrium can be shifted to the synthetic direction by reversing the normal hydrolysis.