Chemical and Biomedical Engineering - Research Publications
Permanent URI for this collection
Search Results
1 - 10 of 17
-
ItemStructure-Dependent Interfacial Properties of Chaplin F from Streptomyces coelicolor(MDPI, 2017-09)Chaplin F (Chp F) is a secreted surface-active peptide involved in the aerial growth of Streptomyces. While Chp E demonstrates a pH-responsive surface activity, the relationship between Chp F structure, function and the effect of solution pH is unknown. Chp F peptides were found to self-assemble into amyloid fibrils at acidic pH (3.0 or the isoelectric point (pI) of 4.2), with ~99% of peptides converted into insoluble fibrils. In contrast, Chp F formed short assemblies containing a mixture of random coil and β-sheet structure at a basic pH of 10.0, where only 40% of the peptides converted to fibrils. The cysteine residues in Chp F did not appear to play a role in fibril assembly. The interfacial properties of Chp F at the air/water interface were altered by the structures adopted at different pH, with Chp F molecules forming a higher surface-active film at pH 10.0 with a lower area per molecule compared to Chp F fibrils at pH 3.0. These data show that the pH responsiveness of Chp F surface activity is the reverse of that observed for Chp E, which could prove useful in potential applications where surface activity is desired over a wide range of solution pH.
-
ItemThe Assembly of Individual Chaplin Peptides from Streptomyces coelicolor into Functional Amyloid Fibrils(PUBLIC LIBRARY SCIENCE, 2011-04-19)The self-association of proteins into amyloid fibrils offers an alternative to the natively folded state of many polypeptides. Although commonly associated with disease, amyloid fibrils represent the natural functional state of some proteins, such as the chaplins from the soil-dwelling bacterium Streptomyces coelicolor, which coat the aerial mycelium and spores rendering them hydrophobic. We have undertaken a biophysical characterisation of the five short chaplin peptides ChpD-H to probe the mechanism by which these peptides self-assemble in solution to form fibrils. Each of the five chaplin peptides produced synthetically or isolated from the cell wall is individually surface-active and capable of forming fibrils under a range of solution conditions in vitro. These fibrils contain a highly similar cross-β core structure and a secondary structure that resembles fibrils formed in vivo on the spore and mycelium surface. They can also restore the growth of aerial hyphae to a chaplin mutant strain. We show that cysteine residues are not required for fibril formation in vitro and propose a role for the cysteine residues conserved in four of the five short chaplin peptides.
-
ItemThe propensity of the bacterial rodlin protein RdlB to form amyloid fibrils determines its function in Streptomyces coelicolor(NATURE PORTFOLIO, 2017-02-17)Streptomyces bacteria form reproductive aerial hyphae that are covered with a pattern of pairwise aligned fibrils called rodlets. The presence of the rodlet layer requires two homologous rodlin proteins, RdlA and RdlB, and the functional amyloid chaplin proteins, ChpA-H. In contrast to the redundancy shared among the eight chaplins, both RdlA and RdlB are indispensable for the establishment of this rodlet structure. By using a comprehensive biophysical approach combined with in vivo characterization we found that RdlB, but not RdlA, readily assembles into amyloid fibrils. The marked difference in amyloid propensity between these highly similar proteins could be largely attributed to a difference in amino acid sequence at just three sites. Further, an engineered RdlA protein in which these three key amino acids were replaced with the corresponding residues from RdlB could compensate for loss of RdlB and restore formation of the surface-exposed amyloid layer in bacteria. Our data reveal that RdlB is a new functional amyloid and provide a biophysical basis for the functional differences between the two rodlin proteins. This study enhances our understanding of how rodlin proteins contribute to formation of an outer fibrillar layer during spore morphogenesis in streptomycetes.
-
ItemHigh-Efficiency Biocatalytic Conversion of Thebaine to Codeine(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.
-
ItemHeat induced denaturation, aggregation and gelation of almond proteins in skim and full fat almond milk(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.
-
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.
-
ItemThe application of forward osmosis to dairy processing(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.
-
ItemFormation of Cheddar cheese analogues using canola oil and ultrasonication – a comparison between single and double emulsion systems(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.
-
ItemThe microstructure and textural properties of Australian cream cheese with differing composition(Elsevier, 2019-12-01)Confocal laser scanning microscopy was used to compare the microstructure of six Australian commercial cream cheese products. The optimal conditions for cryo scanning electron microscopy (cryo SEM) analysis of cream cheese microstructure were also examined. These complementary techniques revealed a typical cream cheese microstructure of homogenised fat globules embedded in a non-continuous protein network. The association between fat and protein within the microstructure was influenced by product composition (fat:protein ratio, moisture content) and ingredients. The addition of emulsifier led to a softer product with distinct microstructure. Cryo SEM also revealed a “honeycomb”-like structure, which was interpreted as a eutectic artefact formed by the addition of gum(s). Product hardness and gel strength generally correlated with high fat, low moisture content and a compact microstructure. Overall, this study shows how product composition affects the microstructure, texture and rheological properties of cream cheese.
-
ItemProduction of metabolites of the anti-cancer drug noscapine using a P450BM3 mutant library.(Elsevier, 2019-12)Cytochrome P450 enzymes are a promising tool for the late-stage diversification of lead drug candidates and can provide an alternative route to structural modifications that are difficult to achieve with synthetic chemistry. In this study, a library of P450BM3 mutants was produced using site-directed mutagenesis and the enzymes screened for metabolism of the opium poppy alkaloid noscapine, a drug with anticancer activity. Of the 18 enzyme mutants screened, 12 showed an ability to metabolise noscapine that was not present in the wild-type enzyme. Five noscapine metabolites were detected by LC-MS/MS, with the major metabolite for all mutants being N-demethylated noscapine. The highest observed regioselectivity for N-demethylation was 88%. Two hydroxylated metabolites, a catechol and two C-C cleavage products were also detected. P450-mediated production of hydroxylated and N-demethylated noscapine structures may be useful for the development of noscapine analogues with improved biological activity. The variation in substrate turnover, coupling efficiency and product distribution between the active mutants was considered alongside in silico docking experiments to gain insight into structural and functional effects of the introduced mutations. Selected mutants were identified as targets for further mutagenesis to improve activity and when coupled with an optimised process may provide a route for the preparative-scale production of noscapine metabolites.