Christian Nyffenegger

Thermostable β-galactosidases for the synthesis of human milk oligosaccharides

Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk. Using lactose as a cheap donor, some β-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto-N-neotetraose (LNnT; Gal-β(1,4)-GlcNAc-β(1,3)-Gal-β(1,4)-Glc). In order to reduce reaction times and be able to work at temperatures, which are less welcoming to microbial growth, the current study investigates the possibility of using thermostable β-galactosidases for synthesis of LNnT and N-acetyllactosamine (LacNAc; Gal-β(1,4)-GlcNAc), the latter being a core structure in HMOs. Two hyperthermostable GH 1 β-galactosidases, Ttβ-gly from Thermus thermophilus HB27 and CelB from Pyrococcus furiosus, were codon-optimized for expression in Escherichia coli along with BgaD-D, a truncated version of the GH 42 β-galactosidase from Bacillus circulans showing high transgalactosylation activity at low substrate concentrations. The three β-galactosidases were compared in the current study in terms of their transgalactosylation activity in the formation of LacNAc and LNnT. In all cases, BgaD-D was the most potent transgalactosidase, but both thermostable GH 1 β-galactosidases could catalyze formation of LNnT and LacNAc, with Ttβ-gly giving higher yields than CelB. The thermal stability of the three β-galactosidases was elucidated and the results were used to optimize the reaction efficiency in the formation of LacNAc, resulting in 5-6 times higher reaction yields and significantly shorter reaction times.

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3’-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity

A sialidase (EC 3.2.1.18) from the non-pathogenic Trypanosoma rangeli, TrSA, has been shown to exert trans-sialidase activity after mutation of five specific amino acids in the active site (M96V, A98P, S120Y, G249Y, Q284P) to form the so-called TrSA5mut enzyme. By computational and hypothesis driven approaches additional mutations enhancing the trans-sialidase activity have been suggested. In the present work, we made a systematic combination of these mutations leading to seven new variants of the T. rangeli sialidase, having 6–16 targeted amino acid mutations. The resulting enzyme variants were analyzed via kinetics for their ability to carry out trans-sialidase reaction using CGMP and D-lactose as substrates. The sialidase variants with 15 and 16 mutations, respectively, exhibited significantly improved trans-sialidase activity for D-lactose sialylation. Our results corroborate, that computational studies of trans-glycosylation can be a valuable input in the design of novel trans-glycosidases, but also highlight the importance of experimental validation in order to assess the performance. In conclusion, two of the seven mutants displayed a dramatic switch in specificity from hydrolysis towards trans-sialylation and constitute the most potent trans-sialidase mutants of TrSA described in literature to date.

Impact of different alginate lyases on combined cellulase–lyase saccharification of brown seaweed

Two bacterial polysaccharide lyase (PL) family 7 alginate lyases (EC 4.2.2.-) from Sphingomonas sp. (SALy) and Flavobacterium sp. (FALy), respectively, were selected for heterologous, monocomponent expression in Escherichia coli. The thermal stability, pH, and temperature reaction optima and substrate preferences of the enzymes on different alginate polymers were assessed and compared to those of a commercially available microbial alginate lyase (SigmALy). The optimal pH range for SALy was pH 5.5–7.0; for FALy and SigmALy it was pH 7.5. Reaction temperatures of 30–50 °C had no influence on the activity of any of the enzymes, but the thermal stability was reduced above 50 °C. The FALy enzyme preferred poly-mannuronic acid as substrate, but exhibited activity also on poly-guluronic acid, whereas the SALy had highest activity on poly-guluronic acid, and the SigmALy was active only on poly-guluronic acid. When applied together with a fungal cellulase preparation (Cellic®CTec2) at pH 6 and 40 °C on a glucan rich brown seaweed Laminaria digitata the viscosity decreased in the initial minutes while measurable alginate degradation occurred primarily within the first 1–2 hours of reaction. Whereas FALy and SALy addition catalyzed degradation of more alginate in L. digitata than SigmALy addition, only the SigmAly enabled release of 90% of the available glucose within 8 hours of combined enzyme treatment. The level of mannuronic acid moieties released was inversely proportional to the glucose release, indicating that the degradation of mannuronic acid blocks inhibited cellulase catalyzed glucose release from L. digitata. Nevertheless, combined alginate lyase and cellulase treatment for 24 hours released all potential glucose regardless of the applied lyase. The enzymatic treatment moreover induced solubilization of sulfated fucoidan, whereas most of the nitrogen was recovered in the residual seaweed solids.

Performance of microbial phytases for gastric inositol phosphate degradation

Microbial phytases catalyze dephosphorylation of phytic acid, thereby potentially releasing chelated iron and improving human iron absorption from cereal-based diets. For this catalysis to take place in vivo, the phytase must be robust to low pH and proteolysis in the gastric ventricle. This study compares the robustness of five different microbial phytases, evaluating thermal stability, activity retention, and extent of dephosphorylation of phytic acid in a simulated low-pH/pepsin gastric environment and examines secondary protein structural changes at low pH via circular dichroism. The Peniophora lycii phytase was found to be the most thermostable, but the least robust enzyme in gastric conditions, whereas the Aspergillus niger and Escherichia coli phytases proved to be most resistant to gastric conditions. The phytase from Citrobacter braakii showed intermediate robustness. The extent of loss of secondary structure at low pH correlated positively with the extent of activity loss at low pH.