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Ion-Exchange Sample Displacement Chromatography as a Method for Fast and Simple Isolation of Low- and High-Abundance Proteins from Complex Biological Mixtures

Martina Srajer Gajdosik1, Spomenka Kovac1, Nela Malatesti2, Egbert Müller3
and Djuro Josic2,4*

1
Department of Chemistry, Josip Juraj Strossmayer University, Cara Hadrijana 8/A,
HR-31000 Osijek, Croatia

2Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, HR-51000 Rijeka, Croatia
3Tosoh Bioscience GmbH, Zettachring 6, DE-70567 Stuttgart, Germany
4Warren Alpert Medical School, Brown University, Richmond Street 222, RI-02903 Providence, USA

Article history:

Received April 18, 2013

Accepted December 18, 2013

Key words:

sample displacement chromatography, ion-exchange mode, plasma proteins

Summary:

Sample displacement chromatography (SDC) in reversed phase and ion-exchange
modes was introduced at the end of 1980s. This chromatographic method was first used for preparative purification of synthetic peptides, and subsequently adapted for protein fractionation, mainly in anion-exchange mode. In the past few years, SDC has been successfully used for enrichment of low- and medium-abundance proteins from complex biological fluids on both monolithic and bulk chromatographic supports. If aqueous mobile phase is used with the application of mild chromatographic conditions, isolated proteins are not denatured and can also keep their biological activity. In this paper, the use of SDC in anion-exchange mode on a high-capacity chromatographic resin for separation of proteins from complex biological mixtures such as human plasma is demonstrated. By use of three and more columns coupled in series during sample application, and subsequent parallel elution of detached columns, additional separation of bound proteins was achieved. Highly enriched human serum albumin fraction and a number of physiologically active medium- and low-abundance proteins could be fractionated and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix-assisted laser desorption/ ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS). The use of the aforementioned columns that can be sanitized with 1 M sodium hydroxide for further application of SDC in biotechnology and food technology was discussed.


 

*Corresponding author:        djuro_josic@brown.edu                                        
                                       
   +385 51 584 560
                                            +385 51 584 599

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Generation of New Genotypic and Phenotypic Features in Artificial and Natural Yeast Hybrids

Walter P. Pfliegler1, Lea Atanasova2, Edina Karanyicz1, Matthias Sipiczki1,
Ursula Bond3, Irina S. Druzhinina2, Katja Sterflinger4 and Ksenija Lopandic4*


1
Department of Genetics and Applied Microbiology, University of Debrecen, Egyetem tér 1,
H-4032 Debrecen, Hungary

2
Research Area Biotechnology and Microbiology, Institute of Chemical Engineering,
Vienna University of Technology, Gumpendorferstrasse 1a, A-1060 Vienna, Austria

3
The School of Genetics and Microbiology, Trinity College, College Green, Dublin 2, Ireland

4VIBT-BOKU, University of Natural Resources and Applied Life Sciences,
Department of Biotechnology, Muthgasse 11, A-1190 Vienna, Austria


Article history:

Received March 29, 2013

Accepted September 23, 2013


Key words:

Saccharomyces cerevisiae, S. uvarum, S. kudriavzevii, yeast interspecies hybrids, AFLP, karyotyping


Summary:

Evolution and genome stabilization have mostly been studied on the Saccharomyces hybrids isolated from natural and alcoholic fermentation environments. Genetic and phenotypic properties have usually been compared to the laboratory and reference strains, as the true ancestors of the natural hybrid yeasts are unknown. In this way the exact impact of different parental fractions on the genome organization or metabolic activity of the hybrid yeasts is difficult to resolve completely. In the present work the evolution of geno- and phenotypic properties is studied in the interspecies hybrids created by the cross-breeding of S. cerevisiae with S. uvarum or S. kudriavzevii auxotrophic mutants. We hypothesized that the extent of genomic alterations in S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii should affect the physiology of their F1 offspring in different ways. Our results, obtained by amplified fragment length polymorphism (AFLP) genotyping and karyotyping analyses, showed that both subgenomes of the S. cerevisiae x S. uvarum and of S. cerevisiae × S. kudriavzevii hybrids experienced various modifications. However, the S. cerevisiae × S. kudriavzevii F1 hybrids underwent more severe genomic alterations than the S. cerevisiae × S. uvarum ones. Generation of the new genotypes also influenced the physiological performances of the hybrids and the occurrence of novel phenotypes. Significant differences in carbohydrate utilization and distinct growth dynamics at increasing concentrations of sodium chloride, urea and miconazole were observed within and between the S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii hybrids. Parental strains also demonstrated different contributions to the final metabolic outcomes of the hybrid yeasts. A comparison of the genotypic properties of the artificial hybrids with several hybrid isolates from the wine-related environments and wastewater demonstrated a greater genetic variability of the S. cerevisiae × S. kudriavzevii hybrids. Saccharomyces cerevisiae × S. uvarum artificial and natural hybrids showed considerable differences in osmolyte tolerance and sensitivity to miconazole, whereas the S. cerevisiae × S. kudriavzevii hybrids exhibited differences also in maltotriose utilization. The results of this study suggest that chromosomal rearrangements and genomic reorganizations as post-hybridization processes may affect the phenotypic properties of the hybrid progeny substantially. Relative to their ancestors, the F1 segregants may generate different phenotypes, indicating novel routes of evolution in response to environmental growth conditions.
 


*Corresponding author:
       
ksenija.lopandic@boku.ac.at                                         
                                      
   +43 1 47654 6943

                                          +43 1 47654 6675

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Microbial Anchoring Systems for Cell-Surface Display of Lipolytic Enzymes


Ana Bielen, Renata Teparić, Dušica Vujaklija2* and Vladimir Mrša1*


1
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6,
HR-10000 Zagreb, Croatia
2Division of Molecular Biology, Institute Ruđer Bošković, Bijenička 54, HR-10000 Zagreb, Croatia


Article history
:
Received January 17, 2014
Accepted March 3, 2014

Key words:

surface display, genetic immobilization, lipolytic enzymes, bacterial envelope, yeast cell wall

Summary
:                                                                                                                                                                                
Studies of microbial cell envelopes and particularly cell surface proteins and mechanisms
of their localization brought about new biotechnological applications of the gained knowledge in surface display of homologous and heterologous proteins. By fusing surface proteins or their anchoring domains with different proteins of interest, their so-called genetic immobilization is achieved. Hybrid proteins are engineered in a way that they are expressed in the host cells, secreted to the cell surface and incorporated into the wall/ envelope moiety. In this way, laborious and often detrimental procedure of chemical immobilization of the protein is avoided by letting the cells do the whole procedure. Both bacterial and yeast cells have been used for this purpose and a number of potential biotechnological applications of surface-displayed proteins have been reported. Among the most frequently used passenger proteins are lipolytic enzymes, due to their great technological significance and numerous important applications. In this review, our current knowledge on mechanisms and molecular systems for surface display of lipolytic enzymes on bacterial and yeast cell surfaces is summarized.

 

 

*Corresponding author:      vujaklij@irb.hr, vmrsa@pbf.hr

§
Both authors contributed equally to this paper


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Factors Affecting Volatile Phenol Production During Fermentations with Pure and Mixed Cultures of Dekkera bruxellensis and Saccharomyces cerevisiae

Janez Kosel, Neža Čadež and Peter Raspor1*


University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

1
Current address: University of Primorska, Faculty of Health Sciences, Polje 42, SI-6310 Izola, Slovenia


Article history
:
Received July 17, 2013

Accepted February 4, 2014

Key words:

Saccharomyces cerevisiae
, Dekkera bruxellensis, wine fermentation, volatile phenols


Summary:

The paper examines the impact of ethanol, and hidroxycinnamic and vinylphenol precursors
on the production of volatile phenols in fermentations of mixed and pure cultures of yeasts Saccharomyces cerevisiae and Dekkera bruxellensis. Three different D. bruxellensis strains were examined and they all showed a unique volatile phenol production pattern in the fermentations of pure and mixed cultures. Generally, the results showed that in mixed culture fermentations less vinylphenols and more ethylphenols were produced in comparison with D. bruxellensis pure culture fermentations. Vinylphenol precursors significantly inhibited the growth of S. cerevisiae and the production of ethylphenols. Nevertheless, it was found that D. bruxellensis genes encoding for enzymes coumaric acid decarboxylase (CAD) and vinylphenol reductase (VPR) are more responsive to vinylphenol precursors in  comparison with hidroxycinnamic acids. Consequently, higher concentrations of vinylphenols in the cell were found to be more cytotoxic than hidroxycinnamic acids. In general, low ethanol concentrations induced higher production of volatile phenols by S. cerevisiae and D. bruxellensis. This was confirmed with the expression pattern of gene encoding for CAD of D. bruxellensis.


*Corresponding author:      peter.raspor@fvz.upr.si

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Brief Note on the Development of Biotechnology

Karl Bayer

Department of Biotechnology, University of Natural Resources and Life Sciences,
Muthgasse 18, A-1190 Vienna, Austria

Article history:

Received April 10, 2013

Accepted September 23, 2013

Key words:
molecular biotechnology, biopharmaceuticals, PAT, QbD

Summary:

Biotechnology, with the main applications in food and nutrition, dates back to the early
times of mankind. In the recent decades the progress in natural sciences, mathematics and computer science has led to a new branch termed molecular biotechnology, which finally developed as an autonomous scientific discipline. The field of biotechnology, in the past generally empirically driven, now largely benefits from molecular biotechnology by improved systems, knowledge and understanding. Thereby, compliance with the recently published initiatives of the regulatory authorities to accelerate the approval process for the manufacturing of biopharmaceuticals can be gained.


*Corresponding author:        karl.bayer@boku.ac.at

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