Chemical and Biomedical Engineering - Research Publications

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    High-Efficiency Biocatalytic Conversion of Thebaine to Codeine
    Li, X ; Krysiak-Baltyn, K ; Richards, L ; Jarrold, A ; Stevens, GW ; Bowser, T ; Speight, RE ; Gras, SL (American Chemical Society, 2020-04-28)
    An enzymatic biosynthesis approach is described for codeine, the most widely used medicinal opiate, providing a more environmentally sustainable alternative to current chemical conversion, with yields and productivity compatible with industrial production. Escherichia coli strains were engineered to express key enzymes from poppy, including the recently discovered neopinone isomerase, producing codeine from thebaine. We show that compartmentalization of these enzymes in different cells is an effective strategy that allows active spatial and temporal control of reactions, increasing yield and volumetric productivity and reducing byproduct generation. Codeine is produced at a yield of 64% and a volumetric productivity of 0.19 g/(L·h), providing the basis for an industrially applicable aqueous whole-cell biotransformation process. This approach could be used to redirect thebaine-rich feedstocks arising from the U.S. reduction of opioid manufacturing quotas or applied to enable total biosynthesis and may have broader applicability to other medicinal plant compounds.
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    Heat induced denaturation, aggregation and gelation of almond proteins in skim and full fat almond milk
    Devnani, B ; Ong, L ; Kentish, S ; Gras, S (Elsevier BV, 2020-09-30)
    The effect of thermal treatment (45-95 ⁰C for 30 minutes) on the structure of almond milk proteins was assessed, as the unfolding and association of these proteins in response to heat is not well understood. Above 55 ⁰C, protein surface hydrophobicity and particle size increased and alpha helical structure decreased, reducing the stability of skim or full fat milk. Fractal protein clusters were observed at 65-75 ⁰C and weakly flocculated gels with a continuous protein network occurred at 85-95 ⁰C, resulting in gels with high water holding capacity and a strength similar to dairy gels. The presence of almond fat increased gel strength but led to a more heterogenous microstructure, which may be improved by homogenisation. Elasticity could also be increased with protein concentration. This study improves our understanding of the heat stability of almond milk proteins and indicates their potential as a gelling ingredient for vegan and vegetarian products.
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    The role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase
    Karimi Alavijeh, M ; Meyer, AS ; Gras, S ; Kentish, SE (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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    The application of forward osmosis to dairy processing
    Chen, GQ ; Gras, SL ; Kentish, SE (Elsevier, 2020-09-01)
    This work assesses the feasibility for concentrating process streams within dairy processing facilities using commercial forward osmosis membranes; to increase their total solids concentrations before entering energy intensive unit operations including thermal evaporators and spray dryers. These streams include demineralised whey, lactose, whey protein concentrate, sweet whey and skim milk. FTSH2O cellulose acetate (CTA) and Aquaporin flat sheet membranes are used with magnesium chloride concentrations of 1.66 ± 0.12 M as the draw solution. The experimental data are fitted to conventional mathematical models for forward osmosis, further modified by considering the nonlinear relationship between osmotic pressure and solute concentration. The diffusion coefficients of magnesium chloride in 1.6 M solutions at 10 °C, 20 °C and 50 °C are obtained and reported for the first time. Minimal fouling and a significantly smaller degree of concentration polarisation was observed on the membrane surface during lactose concentration compared to the concentration of other dairy solutions, due to the absence of proteins and calcium phosphate salts. The transfer of magnesium into the concentrated products was monitored and shown to be below 100 mg per 100 g dry powder. Acid cleaning alone was not effective in recovering pure water flux, and enzyme cleaners at neutral pH were needed given the limited pH tolerance (3–8) of the CTA membranes. Total solids concentrations of the concentrated dairy streams by forward osmosis (up to 40%) exceed those which can be achieved by nanofiltration and reverse osmosis (i.e., 15–20%). This study shows that forward osmosis is an effective approach to concentrate relevant dairy streams to achieve high concentration factors (e.g. >4 for sweet whey samples) without jeopardising product quality.
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    Formation of Cheddar cheese analogues using canola oil and ultrasonication – a comparison between single and double emulsion systems
    Leong, TSH ; Ong, L ; Gamlath, CJ ; Gras, SL ; Ashokkumar, M ; Martin, GJO (Elsevier BV, 2020-06)
    Cheddar cheese analogues were produced from skim milk in which canola oil was emulsified using ultrasound to form either single (O/W) or double emulsions (W1/O/W2). The double emulsion cheese analogues (DECH) had a distinct microstructure and retained small skim milk droplets, dispersed in the fat phase, for more than 7 months of aging at 4 °C. The single emulsion cheese analogues (SECH), prepared with the same fat content as control cheeses, produced comparable yields of cheese and whey, with similar composition, although the fat droplets were more spherical and showed greater coalescence. The DECH cheese with skim milk encapsulated in the oil droplets was harder, melted less and showed more free fatty acid development over 7 months of aging than the control cheeses. The SECH cheeses were softer than the control and also melted less effectively but did not show greater free fatty acid development.