Mutation Hotspot for Changing the Substrate Specificity of ß-N-Acetylhexosaminidase: A Library of GlcNAcases.

ß-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal ß-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides.

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galß4GlcNAcß3Galß4Glc), employing sequential cascade reactions catalyzed by ß3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, ß4-galactosidase BgaD-B from Bacillus circulans, ß4-N-acetylgalactosaminidase from Talaromyces flavus, and ß3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.

Reprint of: Advanced glycosidases as ingenious biosynthetic instruments.

Until recently, glycosidases, naturally hydrolyzing carbohydrate-active enzymes, have found few synthetic applications in industry, being primarily used for cleaving unwanted carbohydrates. With the establishment of glycosynthase and transglycosidase technology by genetic engineering, the view of glycosidases as industrial biotechnology tools has started to change. Their easy production, affordability, robustness, and substrate versatility, added to the possibility of controlling undesired side hydrolysis by enzyme engineering, have made glycosidases competitive synthetic tools. Current promising applications of engineered glycosidases include the production of well-defined chitooligomers, precious galactooligosaccharides or specialty chemicals such as glycosylated flavonoids. Other synthetic pathways leading to human milk oligosaccharides or remodeled antibodies are on the horizon. This work provides an overview of the synthetic achievements to date for glycosidases, emphasizing the latest trends and outlining possible developments in the field.

Advanced glycosidases as ingenious biosynthetic instruments.

Until recently, glycosidases, naturally hydrolyzing carbohydrate-active enzymes, have found few synthetic applications in industry, being primarily used for cleaving unwanted carbohydrates. With the establishment of glycosynthase and transglycosidase technology by genetic engineering, the view of glycosidases as industrial biotechnology tools has started to change. Their easy production, affordability, robustness, and substrate versatility, added to the possibility of controlling undesired side hydrolysis by enzyme engineering, have made glycosidases competitive synthetic tools. Current promising applications of engineered glycosidases include the production of well-defined chitooligomers, precious galactooligosaccharides or specialty chemicals such as glycosylated flavonoids. Other synthetic pathways leading to human milk oligosaccharides or remodeled antibodies are on the horizon. This work provides an overview of the synthetic achievements to date for glycosidases, emphasizing the latest trends and outlining possible developments in the field.

A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors.

The ß-N-acetylhexosaminidase from Penicillium oxalicum (PoHex; EC 3.2.1.52) is a fungal glycosidase with an outstandingly high GalNAcase/GlcNAcase activity ratio. It has a remarkable synthetic capability and can process carbohydrates functionalized at various positions. However, the production in the native fungal host is lengthy, unselective and purification from the fungal medium is complicated and low yielding. We present here a novel production method of this enzyme in the eukaryotic host of Pichia pastoris, followed by elegant one-step purification to homogeneity. The resulting recombinant enzyme has improved biochemical and catalytic properties compared to the fungal wild type. Its good production yield (11 mg/400 mL cultivation medium) greatly expands the scope of synthetic applications. We further demonstrate the synthetic utility and broad acceptor specificity of recombinant PoHex in the glycosylation of a series of challenging acceptors with varying structural architectures, namely secondary and tertiary hydroxyl, aldoxime and a poly-hydroxylated compound.

Applicability of low-flow atmospheric pressure chemical ionization and photoionization mass spectrometry with a microfabricated nebulizer for neutral lipids.

RATIONALE: Mass spectrometry with atmospheric pressure chemical ionization (APCI) or photoionization (APPI) is widely used for neutral lipids involved in many fundamental processes in living organisms. Commercial APCI and APPI sources operate at high flow rates compatible with conventional high-performance liquid chromatography (HPLC). However, lipid analysis is often limited by a small amount of sample, which requires low flow rate separations like capillary or micro-HPLC. Therefore, APCI and APPI suitable for microliter-per-minute flow rates need to be developed and applied for neutral lipids. METHODS: A micro-APCI/APPI source with a heated chip nebulizer was assembled and mounted on a Thermo ion trap instrument. The ion source operated in APCI, APPI or dual mode was optimized for low microliter-per-minute sample flow rates. The source performance was investigated for squalene, wax esters, fatty acid methyl esters, triacylglycerols, and cholesterol. RESULTS: The ion source behaved as a mass-flow-sensitive detector. Direct infusion of methyl oleate showed superior analytical figures of merit when compared with high-flow ion sources. A detection limit of 200 pmol/mL and a linear dynamic range spanning three orders of magnitude were measured for micro-APCI. The mass spectra of most lipids differed from high flow rate spectra. Unlike micro-APCI, micro-APPI spectra were complicated by odd-electron species. Dual APCI/APPI mode did not show any benefits for neutral lipids. Applications for lipid samples were demonstrated. CONCLUSIONS: Micro-APCI-MS is a useful detection technique for neutral lipids at microliter-per-minute flow rates. It offers high sensitivity and high quality of spectra in direct infusion mode and promises successful utilization in capillary and micro-HPLC applications.