Christine Kelly

A novel enzymatic microreactor with Aspergillus oryzae β-galactosidase immobilized on silicon dioxide nanosprings.

The use of silicon dioxide (SiO2) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm2 × 60 μm thick) was functionalized with γ‐aminopropyltriethoxysilane, then treated with N‐succinimidyl‐3‐(2‐pyridyldithio)‐propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (SH) groups. SPDP‐modified β‐galactosidase from Aspergillus oryzae was immobilized on the thiolated nanosprings by reversible disulfide linkages. The enzyme‐coated nanospring mat was placed into a 175‐μm high microchannel, with the mat partially occluding the channel. The kinetics and steady‐state conversion of hydrolysis of o‐nitrophenyl β‐D‐galactosylpyranoside at various substrate flow rates and concentrations were measured. Substantial flow was observed through the nanosprings, for which the Darcy permeability κ ≈ 3 × 10−6 cm2. A simple, one‐parameter numerical model coupling Navier‐Stokes and Darcy flow with a pseudo‐first‐order reaction was used to fit the experimental data. Simulated reactor performance was sensitive to changes in κ and the height of the nanospring mat. Permeabilities lower than 10−8 cm2 practically eliminated convective flow through the nanosprings, and substantially decreased conversion. Increasing the height of the mat increased conversion in simulations, but requires more enzymes and could cause sealing issues if grown above channel walls. Preliminary results indicate that in situ regeneration by reduction with DTT and incubation with SPDP‐modified β‐galactosidase is possible. Nanosprings provide high solvent‐accessible surface area with good permeability and mechanical stability, can be patterned into existing microdevices, and are amenable to immobilization of biomolecules. Nanosprings offer a novel and useful support for enzymatic microreactors, biosensors, and lab‐on‐chip devices.

Recombinant manganese peroxidase (rMnP) from Pichia pastoris. Part 1: Kraft pulp delignification.

Abstract Manganese peroxidase (MnP) is the main enzyme implicated in the biobleaching of kraft pulps by white-rot fungi. However, potential commercial applications of this enzyme have been limited by its availability in large quantities. Advances have been made to produce high-yield concentrated recombinant MnP (rMnP). The objective of this study was to evaluate the ability of rMnP to delignify and brighten kraft pulps. The rMnP, produced from the yeast Pichia pastoris – in high-cell density and in fed-batch fermentations – was found to be effective in lignin removal in both hardwood and softwood unbleached kraft pulps. The rMnP applied at 30 U g-1 pulp for 24 h followed by alkali extraction caused significant kappa number reductions for all the pulps tested with different initial lignin contents and structures. Softwood and hardwood pulps showed similar delignification rates during rMnP treatments. Highly delignified pulps with kappa number less than 10 are less susceptible to delignification by rMnP compared with the pulps with higher lignin content. The rMnP-treated pulp was also shown to be more susceptible to subsequent peroxide bleaching compared with the control pulp. More than 60% of the kappa number reduction was achieved by sequential rMnP treatments combined with alkaline extraction. Sequential treatment with xylanase and rMnP also resulted in more extensive delignification than in each enzyme treatment alone or in the case of simultaneous application of the enzymes.

Activation of immobilized lipase in non‐aqueous systems by hydrophobic poly‐DL‐tryptophan tethers

Many industrially important reactions use immobilized enzymes in non‐aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule (“tether”) between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most “long” linkers (e.g., polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker–enzyme conjugate in polar environments, but this provides little benefit in non‐polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non‐porous, functionalized 1‐µm silica microspheres, with and without an intervening hydrophobic poly‐DL‐tryptophan tether (n ≈ 78). The polytryptophan‐tethered enzyme exhibited 35 times greater esterification of n‐propanol with lauric acid in the organic phase and five times the hydrolytic activity against p‐nitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface‐immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed. Biotechnol. Biotechnol. Bioeng. 2008;101: 9–18.

Cloning, Expression and Characterization of Xylose Isomerase from the Marine Bacterium Fulvimarina pelagi in Escherichia coli.

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a Vmax of 0.142 U/mg purified rXI, and a KM for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol KI values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low KM of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca2+ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher Vmax at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production.

Recombinant manganese peroxidase (rMnP) from Pichia pastoris. Part 2: Application in TCF and ECF bleaching.

Abstract The recombinant manganese peroxidase (rMnP) produced from the yeast Pichia pastoris has been investigated in totally chlorine free (TCF) and elemental chlorine free (ECF) bleaching sequences for improving the bleachability of kraft pulps. In TCF bleaching, oxygen delignified hardwood kraft pulp was treated with rMnP, followed by a sequence combining a chelating and alkaline peroxide bleaching stage. The inclusion of the enzymatic treatment significantly improved the pulp brightness to a level that is difficult to obtain by chemical bleaching alone. Furthermore, the treatment with rMnP resulted in energy savings during pulp refining with PFI mill with a slight improvement in pulp strength properties such as tensile index and burst index. In ECF bleaching, a significant reduction in chlorine dioxide consumption was obtained. A three-stage rMnP treatment combined with alkaline extraction, followed by DED bleaching sequence for hardwood kraft pulp (HWKP) or DEDED bleaching sequence for softwood kraft pulp (SWKP), reduced the total effective chlorine by 41% and 32% for HWKP and SWKP, respectively, compared with the conventional bleaching sequences without enzymatic treatment. The strength properties of the enzyme-treated pulp were also slightly better than that of the control pulp. Further reductions in the consumption of total effective chlorine were obtained when a xylanase pretreatment was incorporated into the bleaching sequence before the repeated rMnP treatment.

Furfural and 5-hydroxymethyl-furfural degradation using recombinant manganese peroxidase

Biomass pretreatment-derived degradation compounds, such as furfural and 5-hydroxymethyl-furfural (HMF), inhibit the growth of fermentation microorganisms that utilize biomass to produce fuels and chemicals. Here we report that recombinant manganese peroxidase (rMnP) produced from the yeast Pichia pastoris can degrade furfural and HMF making them less toxic to microorganisms. Treatment with rMnP or manganese(III) acetate reduced furfural and HMF concentrations in a dose-dependent manner. Furfural disappearance was accompanied by malonate disappearance and accumulation of four distinct degradation products. Furfural was more readily degraded by rMnP and manganese(III) acetate than HMF. Growth assays using Saccharomyces cerevisiae indicated that rMnP treatment reduced the toxicity of furfural and HMF. This work provides an avenue to use rMnP to increase the growth of fermentation microorganisms that are inhibited by toxic compounds derived from pretreatment of biomass.

Techno-economic analysis of glucosamine and lipid production from marine diatom Cyclotella sp.

A techno-economic analysis (TEA) was performed on glucosamine and lipid production from a marine diatom Cyclotella sp. in raceway open pond (RWP) and tubular photobioreactor (PBR) cultivation systems. Two PBR operating schemes were assessed: one to produce high lipid (HL) content, and another to produce high chitin (HC) content. In order to generate 1kg of glucosamine, 9700kg (RWP)/1050kg (PBR HL) freshwater, 40kg CO2, 0.70kg nitrogen, 0.18kg phosphorus, and 1.2kg silicon nutrients are required for algae cultivation with water and nutrient recovery. With a price of

Methylene blue as a lignin surrogate in manganese peroxidase reaction systems

1.5 for lipid as coproduct, the projected selling price of glucosamine were

Student Perceptions of Learning in the Laboratory: Comparison of Industrially Situated Virtual Laboratories to Capstone Physical Laboratories

35/kg,

Effect of Furfural, Vanillin and Syringaldehyde on Candida guilliermondii Growth and Xylitol Biosynthesis

106/kg and