A novel β-galactosidase (TsGal48) from Thermus scotoductus was cloned, and the enzyme was biochemically characterized. TsGal48 catalyzed the synthesis of lacto-N-neotetraose (LNnT) from lactose via the transglycosylation reaction with a maximal yield of 20%, which is the highest yield for the synthesis of LNnT so far. To further improve the yield of LNnT, TsGal48 was successfully engineered by directed evolution and site-saturation mutagenesis. A mutated β-galactosidase (mTsGal48) was selected and characterized. mTsGal48 produced LNnT with a yield of 27.7 g/L, which is 1.4-fold higher than that of TsGal48 (19.7 g/L). Then, a developed strategy for LNnT synthesis from chitin powder was provided in a 30 L bioreactor. The reaction process included chitin powder hydrolysis, lacto-N-triose II (LNT2) synthesis, and LNnT synthesis. The reaction time was reduced from 44 to 17 h in chitin powder hydrolysis and LNT2 synthesis. The content of LNnT was up to 25 g/L in the multienzyme system. The green and efficient route may be suitable for large-scale production of LNnT from chitin powder.
Keywords: Thermus scotoductus; chitin; lacto-N-neotetraose; protein engineering; β-galactosidase.