Tonya L. Peeples

Lipase-mediated epoxidation utilizing urea–hydrogen peroxide in ethyl acetate

A green method for alkene epoxidation based on the chemo-enzymatic perhydrolysis of carboxylic acids and esters has been optimized using Novozyme 435, the immobilized form of Candida antarctica lipase B, and the complex urea–hydrogen peroxide (UHP). UHP, an anhydrous form of hydrogen peroxide, has the potential of releasing hydrogen peroxide in a controlled manner and thus avoids the need to add the aqueous hydrogen peroxide slowly to the reaction mixture. The absence of water in the reaction media was also beneficial, because it minimized undesired reactions of the oxidized products. A minimum amount of enzyme was necessary to show the catalytic effect. On recycling, the enzyme maintained its activity up to six rounds of epoxidations. A range of alkenes was epoxidized by this method providing yields ranging from 75 to 100 percent.

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.

Archaeal tetraether lipids: unique structures and applications.

The extremely stable biomolecules manufactured by organisms from extreme environments are of great scientific and engineering interest in the development of robust and stable industrial biocatalysts. Identification of molecules that impart stability under extremes will also have a profound impact on our understanding of cellular survival. This review discusses isolation and characterization of archaeal tetraethers as well as target technologies for tetraether lipid application. The isolation and characterization of archaeal tetraether lipids has led to some interesting applications improving on ester lipid technologies. Potential applications include novel lubricants, gene-delivery systems, monolayer lipid matrices for sensor devices, and protein stabilization. Following this review, patent abstracts and additional literature pertaining to the isolation, characterization, and application of archaeal membrane lipids are listed.

Phospholipid FA of piezophilic bacteria from the deep sea.

Phospholipid FA (PLFA) profiles were determined on four piezophilic bacteria from the deep sea: Moritella japonica DSK1, Shewanella violacea DSS12, S. benthica DB6705, and S. benthica DB21MT-2. The total concentrations of PLFA were higher in strains grown at low pressure (DSK1, 10 MPa, 27.0 mg/g dry wt cells; DSS12, 50 MPa, 24.0 mg/g), and lower in strains grown at high pressure (DB6705, 85 MPa, 1.9 mg/g; DB21MT-2, 100 MPa, 3.0 mg/g). The piezophilic bacteria were characterized by a high abundance of unsaturated FA (62–73% of total FA). In particular, PUFA were detected in all piezophiles examined. Moritella japonica DSK 1 produced 22∶6n−3 (DHA), whereas the three Shewanella strains produced 20∶5n−3 (EPA) with trace amounts of DHA. The detection of low levels of the medium-chain-length PUFA 18∶2n−6 and 18∶3 (DSK1) and 20∶2 (DB6705 and DB21MT-2) suggests that the biosynthesis of EPA and DHA may be regulated by the formation and desaturation of di-and tri-unsaturated FA.

Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-Liquid Biphasic Systems

ABSTRACT Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was performed. Escherichia coli expressing TOM-Green, a variant of toluene ortho-monooxygenase (TOM), was used for this oxidation. Three different solvents, dodecane, dioctyl phthalate, and lauryl acetate, were screened for biotransformations in biphasic media. Of the solvents tested, lauryl acetate gave the best results, producing 0.72 ± 0.03 g/liter 1-naphthol with a productivity of 0.46 ± 0.02 g/g (dry weight) cells after 48 h. The effects of the organic phase ratio and the naphthalene concentration in the organic phase were investigated. The highest 1-naphthol concentration (1.43 g/liter) and the highest 1-naphthol productivity (0.55 g/g [dry weight] cells) were achieved by optimization of the organic phase. The ability to recycle both free cells and cells immobilized in calcium alginate was tested. Both free and immobilized cells lost more than ∼60% of their activity after the first run, which could be attributed to product toxicity. On a constant-volume basis, an eightfold improvement in 1-naphthol production was achieved using biphasic media compared to biotransformation in aqueous media.

Microbial oxidation of naphthalene to cis-1,2-naphthalene dihydrodiol using naphthalene dioxygenase in biphasic media.

The selective oxidation of aryl substrates to chiral cis‐1,2‐dihydrodiols is an industrially important reaction for the production of intermediates that can be used to produce fine chemicals, pharmaceuticals, and many other bioactive natural products. More specifically, the oxidation of naphthalene to produce optically pure (+)‐ cis‐(1 R,2 S)‐1,2‐napthalene dihydrodiol (NDHD) to be used as a chiral synthon for specialty chemicals has gained much interest. Escherichia coli JM109(DE3) pDTG141 expresses naphthalene dioxygenase which catalyzes this reaction. Poor substrate solubility and substrate toxicity are barriers to using the power of these enzymes in large‐scale aqueous whole cell systems. A biphasic reaction system was chosen to overcome these barriers. The optimal biphasic conditions for E. coli JM109(DE3) pDTG141 were determined to be 20% dodecane as the organic solvent containing 40 g/L naphthalene. The productivity of the biotransformation using resting cells was 1.75 g‐diol/g‐cdw/h for the first 6 h with 20% organic phase, which was increased from 0.59 g‐diol/g‐cdw/h for growing cells with 40% organic phase. The biocatalytic activity was retained for at least 12 h. The biocatalyst could be recycled for at least four runs in both suspended and immobilized form. The stability of the 12 h recycle was improved by immobilization in calcium alginate beads. The process has been improved both environmentally and economically by reducing the amount of solvent used and by recycling the biocatalyst.

Microbial C-hydroxylation and β-4-O-methylglucosidation of methyl-benzamide 7-azanorbornane ethers with Beauveria bassiana

Abstract N -Substituted 7-azanorbornanes were prepared by acylation of easily accessible 7-azanorbornane hydrochloride. Derivatives possessing an electron-withdrawing docking/protecting group and bearing an aryl methylether were subjected to biotransformation with the fungus Beauveria bassiana ATCC 7159. O -Demethylation and β-4- O -methylglucosidation reactions were observed for the major metabolite in this biotransformation (isolation yields: 6 , 30%; 11a , 44%; 11b , 47%; 11c , 14%). C -Hydroxylation on an unfunctionalized carbon was also observed in most of the cases.

Amyloglucosidase enzymatic reactivity inside lipid vesicles

Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmaxand Km, were determined by initial velocity measurements, and Kiwas obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.

Screening Extremophiles for Bioconversion Potentials

A screening protocol was developed and implemented to evaluate extremophiles, in particular hyperthermophiles and thermoacidophiles, for their capacity to transform starch‐based feedstocks to high‐value organic acids and solvents. Screening results of 14 extremophiles showed promising growth and biotransformation potentials. In particular, Hyperthermus butylicus, Thermococcus litoralis, and Sulfolobus acidocaldarius were identified as producers of both organic acids and solvents under the screening protocol. The screening effort presented here represents an important step toward realization of biotransformation potentials of extremophiles, potentially improving upon biomass‐based processes.

Determining Design and Scale‐up Parameters for Degradation of Atrazine with Suspended Pseudomonas sp. ADP in Aqueous Bioreactors

This work investigated the kinetic parameters of atrazine mineralization by suspended cells of Pseudomonas sp. ADP in both shake flasks and spherical stirred tank batch reactors (SSTR). The degradation of atrazine and growth of Pseudomonas sp. ADP were studied. Experiments were performed at different temperatures and stirring speeds in both reactors at varying initial concentrations of atrazine. Cell growth and atrazine concentration were monitored over time, and a Monod model with one limiting substrate was used to characterize the kinetic behavior. Temperature, stirring speed, and reactor type were all found to significantly affect the regressed Monod parameters. At 27 °C and 200 rpm, for the shaker flask experiments, μmax and Ks were determined to be 0.14 (±0.01) h−1 and 1.88 (±1.80) mg/L, respectively. At 37 °C, μmax and Ks increased to 0.25 (±0.05) h−1 and 9.59 (±6.55) mg/L, respectively. As expected, stirrer speed was also found to significantly alter the kinetic parameters. At 27 °C and 125 rpm, μmax and Ks were 0.04 (±0.002) h−1 and 3.72 (±1.05) mg/L, respectively, whereas at 37 °C and 125 rpm, μmax and Ks were 0.07 (±0.008) h−1 and 1.65 (±2.06) mg/L. In the SSTR the kinetic parameters μmax and Ks at room temperature were determined to be 0.12 (±0.009) h−1 and 2.18 (±0.47) mg/L, respectively. Although the μmax values for both types of reactors were similar, the shaker flask experiments resulted in considerable error. Error analysis on calculated values of Ks were found to impact estimates in atrazine concentration by as much as two orders of magnitude, depending on the reactor design, illustrating the importance of these factors in reactor scale‐up.