Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

We cloned and expressed a new recombinant β-galactosidase（TN0949） from Thermotoga naphthophila RKU-10 with the pET28a（＋） vector system in Escherichia coli BL21（DE3）, and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni2＋-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. TN0949 can hydrolyze o-nitrophenylβ-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The Km values for the hydrolyses of o-nitrophenyl fl-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH（3.0 to 7.0） after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 ℃ were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.

High yield synthesis of nigerooligosaccharides by transglycosylation catalyzed by α-glucosidase TaAglA from Thermoplasma acidophilum

Nigerooligosaccharides (NOS) is a new functional oligosaccharide containing α-1,3 glucosidic bond with good anti-digestive properties and intestinal probiotics.Transglycosylation catalyzed by α-glucosidase is an effective method for the preparation of oligosaccharides.However,there are few reports on the enzymatic synthesis of nigerooligosaccharides by α-glucosidase at present.This study was aimed to investigate the transglycosylation property of the GH31 α-glucosidase from Thermoplasma acidophilum,TaAglA,and also evaluate its application performance in the preparation of NOS.It was found that TaAglA exhibited selectivity for α-1,3 and α-1,4 linkages when catalyzing hydrolysis,but for α-1,3 and α-1,6 linkages when catalyzing transglycosylation.Using 10% glucose and 20% maltose as substrates,TaAglA yielded 88.5 g/L NOS under the condition of pH 6.0,80 ℃ and 1 U/mL enzyme addition,which was the highest level to our knowledge.In addition,components with higher polymerization degrees,i.e.nigerotriose and nigerosyl-glucose,occupied 53.6% proportion of the total NOS products,giving it better probiotic functions.Futhermore,after purification by glucoamylase digestion and yeast culture,the final yield of NOS was 26.1%,and the purity of the product was 93%.These findings on TaAglA were expected to provide a new candidate for large-scale enzymatic synthesis of NOS,and also have important theoretical significance for the study of GH31 α-glucosidase.

Glycosylation engineering of therapeutic IgG antibodies： challenges for the safety, functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex.type oligosaccharide attached to Asn297 of the Fc is essen- tial for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that gen- erate structural heterogeneity （glycoforms） exhibit unique biological activities. Hence, efficient and quan- titative glycan analysis techniques have been increas- ingly important for the development and quality control of therapeutic antibodies, and g｜ycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosy- lation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibili- ties for the design of novel antibody therapeutics. Fur- thermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosyn- thases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next- generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety,functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10：an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides

A novel thermostable β-glucosidase（Tnap0602） with β-galactosidase activity was cloned from Thermotoga naphthophila RUK-10 and overexpressed in Escherichia coli BL21（DE3） with the aid of pET28b（＋） vector. The recombinant β-glucosidase was purified to homogeneity by heat precipitation and Ni^2＋-affinity chromatography. The molecular weight of the recombinant enzyme was estimated to be 51 kDa by SDS-PAGE analysis. The optimum temperature for the hydrolyses of p-nitrophenyl-β-D-glucopyranoside and o-nitrophenyl-β-D-galactopyranoside by the recombinant β-glucosidase were both above 95 ℃, and the corresponding optimum pH value was found to be the same as 7.0. Thermostability studies show that the half-lives of the recombinant enzyme at 75, 80, 85 and 90℃ are respectively 84, 32, 14, and 3 h, and it is quite stable in a pH range of 5.0-10.0. The Km and Vmax values of the recombinant β-glucosidase for the hydrolysis of pNPGlu at 80 ℃ are 0.127 mmol/L and 18389.1 μmol·min^1·mg^-1, the corresponding values are 0.625 mmol/L and 6250 μmol·min^1·mg^-1 for the hydrolysis of oNPGal, respectively, The enzyme also display the hydrolysis activity for lactose and cellobiose. Galacto-oligosaccharide and alkyl galactopyranosides could be synthesized from Tnap0602 when lactose was used as the transglycosylation substrate, indicating that the thermostable β-glucosidase could be a candidate for industrial application.