Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

The self-sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5-hydroxylation of diclofenac.

P450 monooxygenases are able to catalyze the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites. Here we have used the self-sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5-hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5-hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram-scale production of human drug metabolites through recombinant whole cell biocatalysis.

Cultivation and purification of two stereoselective imine reductases from Streptosporangium roseum and Paenibacillus elgii.

The reductive amination is one of the most important reactions in the synthesis of chiral amines. Imine reductases (IREDs) are novel enzymes that catalyze the asymmetric reduction of imines and reductive aminations using NADPH as hydride donor. In this study, we have developed a simple method to produce two enantiocomplementary IREDs from Streptosporangium roseum DSM 43021 (R-IRED-Sr) and Paenibacillus elgii (S-IRED-Pe). The proteins were expressed efficiently in Escherichia coli (E. coli) JW5510 at the 4-L-cultivation scale and were purified to 95% homogeneity in two steps by immobilized metal ion affinity and anion-exchange chromatography. The total protein yield was about 9 g per liter of E. coli culture and resulted in 150-220 mg purified IRED per liter of E. coli culture. The bioactivity of both IREDs was measured by the depletion of the NADPH cofactor in the reduction of model substrates 2-methylpyrroline (R-IRED-Sr) and 3,4-dihydroisoquinoline (S-IRED-Pe). High level reducing activity was found demonstrating the production of correctly folded and active IRED proteins. Specific activities of about 2.58 U/mg and 0.24 U/mg for the R- and S-selective IREDs were obtained, being in agreement with activities reported in the literature.

Production of ω-hydroxy octanoic acid with Escherichia coli.

The present proof-of-concept study reports the construction of a whole-cell biocatalyst for the de novo production of ω-hydroxy octanoic acid. This was achieved by hijacking the natural fatty acid cycle and subsequent hydroxylation using a specific monooxygenase without the need for the additional feed of alkene-like precursors. For this, we used the model organism Escherichia coli and increased primarily the release of the octanoic acid precursors by overexpressing the plant thioesterase FatB2 from Cuphea hookeriana in a ß-oxidation deficient strain, which lead to the production of 2.32mM (8.38mggcww(-1)) octanoic acid in 24h. In order to produce the corresponding ω-hydroxy derivative, we additionally expressed the engineered self-sufficient monooxygenase fusion protein CYP153AMaq(G307A)-CPRBM3 within the octanoic acid producing strain. With this, we finally produced 234µM (0.95mggcww(-1)) ω-hydroxy octanoic acid in a 20h fed-batch set-up.

Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct.

A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq. -CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l(-1) ω-hydroxy dodecanoic from 10 g l(-1) C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l(-1) ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

Risk factors and survival in patients with respiratory failure after cardiac operations.

BACKGROUND: Respiratory failure is a known complication of cardiac operations and contributes to postoperative morbidity and death. This study assessed the relevance of risk factors in the development of respiratory failure, defined as postoperative ventilation exceeding 48 hours, and looked at the effect of respiratory failure on short-term and long-term mortality rates. METHODS: De-identified data for patients who underwent cardiac surgical procedures at The Prince Charles Hospital between January 2002 and December 2007 were collected prospectively and analyzed using logistic regression to identify significant risk factors associated with respiratory failure. Long-term mortality data were analyzed for patients who underwent operations between 1994 and 2005 using Kaplan-Meier survival curves. RESULTS: The risk factor analysis included 7,440 patients. Identified risk factors for respiratory failure included critical preoperative state, neurologic dysfunction, poor left ventricular function, active endocarditis, chronic obstructive pulmonary disease, elevated preoperative creatinine, previous cardiac operation, and age. Survival was assessed in 18,488 patients and demonstrated increased short-term and long-term mortality rates when respiratory failure developed and increased mortality rates with increasing duration of respiratory failure. CONCLUSIONS: Respiratory failure is complication of cardiac operations associated with increased mortality and cost. Identification of patients at risk of respiratory failure may help select surgical candidates and aid resource planning and optimization.

Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs.

The laccase genes lccalpha, lccbeta, lccgamma and lccdelta encoding four isoenzymes from Trametes versicolor have been cloned and expressed in Pichia pastoris. Biochemical characterization allowed classification of these laccases into two distinct groups: Lccalpha and Lccbeta possessed higher thermal stability, but lower catalytic activity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) compared to Lccgamma and Lccdelta. Activities of the laccases were quite different as well. Laccase Lccdelta showed highest phenolic C-C coupling activity with sinapic acid, but lowest oxidizing activity towards polycyclic aromatic hydrocarbons (PAHs). Highest activity towards PAHs was observed with Lccbeta. After 72h, more than 80% of fluorene, anthracene, acenaphthene and acenaphthylene were oxidized by Lccbeta in the presence of ABTS. Investigation of the structural basis of the different activities of the laccases demonstrated the impact of positions 164 and 265 in the substrate binding site on oxidation of PAHs.

Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids.

A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The enzyme has a molecular weight of approximately 65 kDa and demonstrates activity towards canonical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants KM and kcat for ABTS were of 6.5+/-0.2 microM and 83 s(-1), for SGZ of 4.3+/-0.2 microM and 100 s(-1), and for 2,6-DMP of 56.7+/-1.0 microM and 28 s(-1). Highest oxidizing activity towards ABTS was obtained at 85 degrees C. However, after 1 h incubation of CotA at 70 degrees C and 80 degrees C, a residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid by CotA was observed, and highest activity of CotA was found towards sinapic acid.

Development of a fed-batch process for the production of the cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium in E. coli.

A fed-batch process utilizing a pET-based expression system (pET28a+ derivative) and E. coli BL21(DE3) as production strain for the heterologous expression of recombinant cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium was developed. In a first step the expression was optimized during a series of flask experiments testing several parameters for their influence on the expression level, activity and solubility of the recombinant protein. The optimal process parameters found in the flask experiments were transferred to a cultivation process in a 5l (operating volume) bioreactor with a special focus on the feeding strategy and the aeration during expression. Glycerol feeding proved to be superior over glucose as carbon source since the formation of larger amounts of acetate was prevented. Expression levels exceeding 12,500nmoll(-1), corresponding to approximately 1.5gl(-1) of product in culture medium ( approximately 11% of CDW) could be demonstrated. The P450 enzyme showed high activity and high solubility. The findings now can be transferred to other enzyme variants and different P450 monooxygenases to increase production of recombinant proteins.