Amino acid/water interactions study: a new amino acid scale

Partition ratios of 8 free l-amino acids (Gln, Glu, His, Lys, Met, Ser, Thr, and Tyr) were measured in 10 different polymer/polymer aqueous two-phase systems containing 0.15?M NaCl in 0.01?M phosphate buffer, pH 7.4. The solute-specific coefficients representing the solute dipole/dipole, hydrogen-bonding and electrostatic interactions with the aqueous environment of the amino acids were determined by multiple linear regression analysis using a modified linear solvation energy relationship. The solute-specific coefficients determined in this study together with the solute-specific coefficients reported previously for amino acids with non-polar side-chains where used in a Quantitative Structure/Property Relationship analysis. It is shown that linear combinations of these solute-specific coefficients are correlated well with various physicochemical, structural, and biological properties of amino acids.     

Continuous aqueous two-phase extraction of human antibodies using a packed column

The performance of a pilot scale packed differential contactor was evaluated for the continuous counter-current aqueous two-phase extraction (ATPE) of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant (CS) enriched with pure protein. Preliminary studies have been firstly performed in order to select the dispersed phase (phosphate-rich or polyethylene glycol 3350 Da (PEG)-rich phase) and the column packing material. The PEG-rich phase has been selected as the dispersed phase and the stainless steel as the preferred material for the column packing bed since it was not wetted preferentially by the selected dispersed phase. Hydrodynamic studies have been also performed, and the experimental results were successfully adjusted to the Richardson-Zaki and Mísek equations, typically used for the conventional organic-aqueous two-phase systems. An experimental set-up combining the packed column with a pump mixer-settler stage showed to have the best performance and to be advantageous when compared to the IgG batch extraction. An IgG recovery yield of 85% could be obtained with about 50% of total contaminants and more than 85% of contaminant proteins removal. Mass transfer studies have revealed that the mass transfer was controlled by the PEG-rich phase. A higher efficiency could be obtained when using an extra pump mixer-settler stage and higher flow rates.     

Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Introduction

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

Methods

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients'' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

Results

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

Conclusions

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus , microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

Analysis of non-TIR NBS-LRR resistance gene analogs in <Emphasis Type="Italic">Musa acuminata</Emphasis> Colla: Isolation,RFLP marker development,and physical mapping

Background  

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.     

A continuous membrane bioreactor for ester synthesis in organic media: I. Operational characterization and stability

The feasibility of continuous ester synthesis in a membrane bioreactor (MBR) by a recombinant cutinase from Fusarium solani pisi was investigated, using the optimal conditions previously attained by medium engineering. The objective was to analyze the MBR behavior as a differential or an integral reactor. The main component of the reactor was an anisotropic ceramic membrane with 15,000 NMWCO. The operating variables included the influence of substrates ratio and flow rate on the conversion degree and on the productivity. The highest conversion degree was obtained using 1M of hexanol and 0.1M of butyl acetate as acyl donor. The use of these substrate concentrations led to a conversion degree of 79.3% and a specific productivity of 41 g hexyl acetate/(d x g cutinase), when the permeate flow rate was 0.025 mL/min. The increase of flow rate to 0.4 mL/min decreased the conversion to 35.6%, although the productivity was enhanced to 294 g product/day x g enzyme. The MBR characterization involved the calculations of mass balance, recirculation rate, conversion per pass, number of cycles, and hydraulic residence time. The operational stability was also evaluated in a longterm experiment over 900 hours and the enzyme half-life was estimated to be approximately 2 years.     

Cutinase: from molecular level to bioprocess development

This review analyzes the role of cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal cutinase, Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three-dimensional structure of this cutinase is also analyzed and its function as a lipase is discussed and compared with other lipases. The biocatalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media in which the different types of reactions have been performed, namely hydrolysis, esterification, transesterification, and resolution of racemic mixtures. The stability of cutinase preparations is discussed and, in particular, the cutinase stability in anionic reversed micelles is analyzed considering the role of hexanol as a substrate, a cosurfactant, and a stabilizer. Process development, based on the operation of cutinase reactors, is also reviewed.     

Heme and pH-dependent stability of an anionic horseradish peroxidase

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.