The rapidly growing interest in the area of proteomics induces intensive efforts to find robust, automated and sensitive high-throughput analytical tools. In this context, the concept of solid-phase digestion (ex. trypsin immobilization on a solid support) has received great attention in the last years. Trypsin (EC 220.127.116.11) has been covalently immobilized on different monolithic supports and resulting bioreactors used as immobilized enzyme reactors (IMERs) for on-line digestion, peptide separation and peptide mapping. Bioreactors efficiencies were evaluated with different recombinant proteins after on-line digestion. The technique used for the separation and identification of peptides was high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS).
Viruses have proven to be useful vectors for gene therapy purposes. As therapeutics for human use they must be pure and contaminant free. Traditionally, viruses are purified by complicated and time consuming methods such as CsCl density gradient centrifugation or similar. In recent years liquid chromatography has became interesting method for virus purification. It provides high level of purity required for human use and increases productivity. Traditional chromatographic supports were mostly designed for purification of proteins and as such are commonly inappropriate for viruses. Alternative to traditional chromatographic support are methacrylate monoliths (CIM monoliths), characterized by large channel diameter, high surface accessibility and convective mass transport.
The aim of this work was to characterize CIM supports for separation and possible purification of a model virus Tomato mosaic virus (ToMV) from crude plant material.
Traditionally, viruses are purified by time consuming methods such as CsCl density gradient centrifugation or similar. These methods are often inefficient and limited to small scale. In recent years different methods for virus purification, based on ion exchange, gel filtration and affinity chromatography have became popular. Recently, CIM® disk monolithic columns were used for successful concentration of two plant viruses (1) and for improved detection of two human viruses (2). Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) were concentrated and subsequently detected from extremely diluted samples in which they were initially undetectable. Successful concentrations of both viruses encourage us to explore the possibilities of CIM® supports for virus purification. As a model virus ToMV was selected. ToMV is a rod shaped plant virus with a typical size of 300 x 18 nm and isoelectric point at pH 4.6.
The only four drugs approved for the clinical treatment of Alzheirner’s Disease (tacrine. rivastigmine, donepezil and galantamine) are acetylcholinesterase inhibitors which act by maintaining high levels of acetylcholine at the muscarinic and nicotinic receptors in the central nervous system. Human acetylcholinesterase (HuAChE) represents a widely studied target enzyme and it is still object of research for the development of new drugs as enzyme inhibitors.
In a previous paper we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm 1.0.) The yeld of immobilization and the stability of the AChE-IMER were considered satisfactory, but some problems arose. The length of the IMER and the large amount of enzyme covalently bound to the chromatographic support resulted in catalysis product long elution times and some inhibitors aspecific matrix absorption with delayed enzyme activity recovery. In order to avoid these complications and considering the high rate of AChE enzymatic reaction. we decided to reduce the dimension of the solid support for immobilization, hence the amount of immobilized enzyme, by selecting a monolithic matrix disk (12 x 3 mm I.D.).
CIM® (Convective Interaction Media) monolithic supports (Biaseparations. Lubiana) represent a novel generation of stationary phases used for liquid chromatography, bioconversions, and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, CIM® supports are cast as continuous homogeneous phases and provide high rates of mass transfer at lower back pressure.
In the present work a CIMK disk with immobilised human recombinant acetylcholinesterase (HuAChE-ClM® Disk) was developed. The activity of immohilised enzyme, the long term stability and reproducibility were tested. HuAChE-CIM® disk was applied as an immobilised enzyme micro-reactor (micro-IMER) in on-line HPLC system for inhibitory potency determination of known AChE inhibitors.
Traces of DNA in RNA samples represent impurities that could affect results of mRNA quantification and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which specifically breaks down DNA. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the DNA size, very few supports available on the market enable efficient interaction between immobilized enzyme and DNA.
In recent years a new group of supports named monoliths was introduced. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are significantly accelerated. Therefore also the efficiency of DNA removal using immobilised enzyme might be competitive to the degradation with free enzyme.
Strains of the anaerobic bacterial genus are thought to play an important role in fiber degradation. sp. Mz5 was previously isolated from the rumen of a black and white Friesian cow and its xylanolytic activity was proved to be at least 1,65 times higher than the activities of all of the compared well known xylan-degrading rumen bacterial species and strains (1). High xylanolytic activity was the reason for partial isolation of its xylanases in order to study their special characteristics and possible biotechnological applications later.
CIM® supports are novel monolithic chromatographic supports. In contrast to conventional particle based chromatographic supports they consist of a single porous polymer. The pores form a highly interconnected network, which enables the flow of the mobile phase through the monolith. Molecules to be separated are transported to the surface by the convection. Since the diffusion is not a bottleneck any more, also the resolution and the dynamic capacity of the monolith are flow independent and an average analysis time is typically below one minute. Furthermore, CIM® columns were successfully applied for the purification of proteins directly from the fermentation broth.
Manganese peroxidases (MnP) and lignin peroxidases (LiP) are a family of glicosilated hemo-proteins, which are excreted into the growth medium during the idiophasic growth of the white rot fungus Phanerochaete chrysosporium. They are both involved in the lignin degradation. For their analysis and separation from the growth medium, HPLC is commonly applied. Besides the separation by Na-acetate concentration gradient (2), also the chromatofocusing can be used (3). A fast method for LiP isoenzyme separation from the growth medium of P. chrysosporium using CIM™ QA disk monolithic columns has been recently developed (1). A modified method was tested on the growth medium containing MnP isoenzymes.
The aim of our work was to study the direct monitoring and purification of proteins from the fermentation broth using ion-exchange CIM® supports. Therefore, we studied the possibility of monitoring and purifying lignin peroxidase extracelular protein isoforms produced by the fungus Phanerochaete chrysosporium. These isoenzymes which also differ in their catalytic properties are able to partially depolymerize lignin and to oxidise several xenobiotics.
The white rot fungus Phanerochaete chrysosporium under nitrogen or carbon limitation produces extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidize several xenobiotics (DDT, PCB, PAH, etc.). By HPLC separation and isoelectric focusing multiple molecular forms of LiP have been isolated from the culture filtrate. For the isolation of LiP from the growth medium, mostly the HPLC technique with ion exchange Mono-Q or DEAE columns is used. The medium should be dialyzed before separation and usually also concentrated. Medium freezing is used to remove mucilaginous polysaccharides which disturb separation. The whole procedure is time consuming and information about isoenzyme content and their relative amounts in the growth medium is delayed for at least 1 day. HPLC separation itself lasts nearly an hour. For the separation of LiP isoenzymes from the culture filtrate, we used the monolithic stationary phase with weak (DEAE-diethylamine) and strong (QA-quaternary amine) ion exchange groups commercially available under trademark CIM (Convective Interaction Media). CIM supports are glycidyl methacrylate based monolithic porous polymer supports. As such they differ from conventional particle shaped chromatographic supports. The liquid is forced to flow through the support channels. Molecules to be separated are transported mainly by convection resulting in travelling times shorter for at least an order of magnitude. As a consequence the resolution as well as the binding capacity remain unaffected with the flow rate and a shorter analysis time can be achieved. This effect is even more pronounced in the case of large molecules such as proteins, which have a low diffusion coefficient. As such, CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.
White rot fungus Phanerochaete chrysosporium produces under nitrogen limitation extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidise several xenobiotics (DDT, PCB, PAH, ) and synthetic dyes. Trough HPLC separation and isoelectric focusing multiple molecular forms of LiP have been determined and isolated from the culture filtrate. Depending on growth conditions, separation technique, strain employed and culture age 2-15 different LiP izoenzymes were observed in culture media of Phanerochaete chrysosporium. They are structurally similar but differ in stability, quantity and in catalytic properties. For the isolation of LiP from growth medium, mostly the procedure employing HPLC ionexchange columns as shown on Scheme 1 is used. For the separation of LiP isoenzymes from the culture filtrate, we used CIM (Convective Interaction Media) units. Their advantage is very fast separation of macromolecules due to their particular threedimensional structure. In contrast to particle supports containing closed pores, CIM units consist of monolith porous material containing flow through pores. Therefore, macromolecules to be separated are transported to the active site by convection rather than by diffusion. As a consequence, the separation resolution and dynamic binding capacity are flow independent. As such CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.