Chemical and Biomolecular Engineering - Research Publications
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ItemBioactives from Whey: A Sustainable Approach to Enzymatic Production of Sialyl-N-acetyllactosamine(AMER CHEMICAL SOC, 2022-05-16)The use of dairy whey to manufacture pharmaceutical products fosters sustainable environmental and economic development. This study represents a new strategy for upgrading of whey to 3′-sialyl-N-acetyllactosamine (3′-SLN) as an important structural component of glycoproteins and a receptor analog capable of forming complexes with hemagglutinins on influenza viruses. N-Acetyllactosamine (LacNAc) was enzymatically produced and purified directly from whey with no pretreatment required. An engineered and recombinantly produced sialidase with trans-sialylation ability from the non-pathogenic Trypanosoma rangeli was then used to transfer sialic acid from whey-derived, sialylated casein glycomacropeptide (CGMP) to this LacNAc. A maximum of 0.92 mM 3′-SLN was obtained at an equimolar ratio of LacNAc to bound sialic acid in CGMP; on the other hand, a molar ratio of 10 gave a fourfold greater 3′-SLN concentration. The variations in the concentration of 3′-SLN and free sialic acid during the hydrolysis reaction were modeled under different reaction conditions using machine learning and mechanistic approaches. The mechanistic analysis of the reaction indicated that the relative initial trans-sialylation rate to hydrolysis rate is directly proportional to the initial LacNAc concentration, with the ratio of trans-sialylation to hydrolysis rate constants equal to 111 M-1. The maximum 3′-SLN yield obtained was 75% based on α-2,3-sialic acid bound to CGMP. Separation of CGMP and reuse of enzyme were also investigated in an enzymatic membrane reactor.
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ItemSynthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives(AMER CHEMICAL SOC, 2021-07-14)N-Acetyllactosamine (LacNAc) or more specifically β-d-galactopyranosyl-1,4-N-acetyl-d-glucosamine is a unique acyl-amino sugar and a key structural unit in human milk oligosaccharides, an antigen component of many glycoproteins, and an antiviral active component for the development of effective drugs against viruses. LacNAc is useful itself and as a basic building block for producing various bioactive oligosaccharides, notably because this synthesis may be used to add value to dairy lactose. Despite a significant amount of information in the literature on the benefits, structures, and types of different LacNAc-derived oligosaccharides, knowledge about their effective synthesis for large-scale production is still in its infancy. This work provides a comprehensive analysis of existing production strategies for LacNAc and important LacNAc-based structures, including sialylated LacNAc as well as poly- and oligo-LacNAc. We conclude that direct extraction from milk is too complex, while chemical synthesis is also impractical at an industrial scale. Microbial routes have application when multiple step reactions are needed, but the major route to large-scale biochemical production will likely lie with enzymatic routes, particularly those using β-galactosidases (for LacNAc synthesis), sialidases (for sialylated LacNAc synthesis), and β-N-acetylhexosaminidases (for oligo-LacNAc synthesis). Glycosyltransferases, especially for the biosynthesis of extended complex LacNAc structures, could also play a major role in the future. In these cases, immobilization of the enzyme can increase stability and reduce cost. Processing parameters, such as substrate concentration and purity, acceptor/donor ratio, water activity, and temperature, can affect product selectivity and yield. More work is needed to optimize these reaction parameters and in the development of robust, thermally stable enzymes to facilitate commercial production of these important bioactive substances.
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ItemImproving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization(AMER CHEMICAL SOC, 2020-11-02)The biotransformation of lactose into gut-bioactive glycans catalyzed by β-galactosidase can give economic value to lactose-rich side streams generated in the food or the dairy industry. Herein, we study the immobilization of the commercially used β-galactosidase from Bacillus circulans onto silica particles using an enzyme immobilization technology involving a cross-linked layer-by-layer encapsulation method. The immobilized β-galactosidase was used for the synthesis of N-acetyllactosamine (LacNAc) as an important precursor for numerous bioactive compounds and a prebiotic in itself. Techniques including molecular analysis, enzyme activity determination, secondary structure analysis, thermodynamic characterization, and the determination of thermal and operational stability were conducted to characterize the immobilized enzyme. Changes in the activity of the enzyme after immobilization were attributed to possible changes in electrostatic, covalent, and protein-protein interactions. Immobilization significantly improved the enzymatic LacNAc yield compared to the free enzyme. In turn, this improved the economics and the sustainability of the process. The immobilized enzyme encapsulated in multilayer films was significantly more stable in the presence of divalent cations and its thermostability also substantially increased with the thermal denaturation activation energy increasing from 53 to 294 kJ mol-1. The immobilized enzyme was successfully reused in eight consecutive reaction cycles with no significant reduction in the LacNAc yield. The improved transgalactosylation yield and productivity, higher stability, and reusability obtained with this immobilization method provide new opportunities for industrial applications.
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ItemThe role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase(Elsevier, 2020-09-01)A β-galactosidase (EC 3.2.1.23) from Bacillus circulans (Biolacta FN5) can catalyse transgalactosylation reactions with lactose as a donor. In addition to their function as cofactors and structural stabilisers in biocatalytic reactions, cations can play a role in salt-bridge interactions and electrostatic charge screening of proteins. In this work, we investigated the impact of calcium, magnesium, sodium and potassium, commonly found in dairy whey systems, on the transgalactosylation kinetics of the β-galactosidase from Bacillus circulans. Both molecular modeling and quantitative experimental methods were used to assess enzyme aggregation and resulting loss in enzyme activity that is initiated by high concentrations of these cations. The effect of this loss in activity with time was studied during the transgalactosylation of N-acetylglucosamine (GlcNAc) to N-acetyllactosamine (LacNAc) using lactose as the donor. No significant change in hydrolysis or transgalactosylation reaction kinetics was observed at low concentrations of divalent cations (Ca2+ or Mg2+) or up to 100 mM of monovalent cations (Na+ or K+). The enzymatic yield and selectivity, however, were significantly affected at concentrations of 100 mM of Ca2+ or Mg2+. These changes were the result of both the loss in enzyme activity and a reduction in the reaction rate constant for hydrolysis and formation of the undesired isomer, Allo-LacNAc. In particular, addition of magnesium enhanced the selectivity for LacNAc over Allo-LacNAc, with no significant reduction in the LacNAc yield. These findings suggest that cations can be employed to regulate the action of β-galactosidase during transgalactosylation through the formation of protein aggregates.
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ItemSimulation and economic assessment of large-scale enzymatic N-acetyllactosamine manufacture(Elsevier BV, 2020-02-15)N-acetyllactosamine (LacNAc) is an important lactose-derived molecule which can act as an effective prebiotic. In this study a process for the enzymatic synthesis and downstream purification of LacNAc was designed based on the use of thermostable β-galactosidases from Bacillus circulans (BgaD-D), Thermus thermophilus HB27 or Pyrococcus furiosus (CelB) respectively. Four configurations for the purification stage were simulated; anion-exchange chromatography, an activated charcoal-Celite column, N-acetylglucosamine (GlcNAc) crystallization and an activated charcoal-Celite column, as well as selective crystallization. While the enzyme CelB has greater stability at higher temperatures, this enzyme gives a lower LacNAc yield, leading to significant capital investment. For the design based on the BgaD-D biocatalyst and anion exchange chromatography, recovery of GlcNAc improved the project profitability when the GlcNAc price was greater than $10 per kg. GlcNAc was the main contributor to the raw material costs for most processes, although methanol contributed 72% of these costs for the process based on an activated charcoal column. The use of a crystallizer for GlcNAc separation before this column, reduced this methanol consumption by 73%. The use of selective crystallization proved the best approach, reducing the minimum LacNAc sales price to $2 per gram. The plant was more economic when the acceptor to donor ratio was reduced from 10 to 4 and the lactose concentration increased from 50 mM to 550 mM.