Yeast Homogenate Research Articles

The antimicrobial effects of silver nanoparticles obtained through the royal jelly on the yeasts Candida guilliermondii NP-4

The effect of silver nanoparticles (Ag NPs) obtained in the presence of royal jelly (RJ) on the growth of yeast Candida guilliermondii NP-4, on the total and H+-ATPase activity, as well as lipid peroxidation process and antioxidant enzymes (superoxide dismutase (SOD), catalase) activity was studied. It has been shown that RJ-mediated Ag NPs have a fungicide and fungistatic effects at the concentrations of 5.4 µg mL−1 and 27 µg mL−1, respectively. Under the influence of RJ-mediated Ag NPs, a decrease in total and H+-ATPase activity in yeast homogenates by ~ 90% and ~ 80% was observed, respectively. In yeast mitochondria total and H+-ATPase activity depression was detected by ~ 80% and ~ 90%, respectively. The amount of malondialdehyde in the Ag NPs exposed yeast homogenate increased ~ 60%, the catalase activity increased ~ 70%, and the SOD activity—~ 30%. The obtained data indicate that the use of RJ-mediated Ag NPs have a diverse range of influence on yeast cells. This approach may be important in the field of biomedical research aimed at evaluating the development of oxidative stress in cells. It may also contribute to a more comprehensive understanding of antimicrobial properties of RJ-mediated Ag NPs and help control the proliferation of pathogenic fungi.

Changes in ATPase activity, antioxidant enzymes and proline biosynthesis in yeast Candida guilliermondii NP-4 under X-irradiation.

This study investigates the effects of X-radiation on ATPase activity and antioxidant enzyme activity, particularly enzymes involved in proline biosynthesis, in yeast C. guilliermondii NP-4. Moreover, the study examined the post-irradiation repair processes in these cells. Results showed that X-irradiation at a dose of 300Gy led to an increase in catalase (CAT) and superoxide dismutase (SOD) activity, as well as, an increase in the CAT/SOD ratio in C. guilliermondii NP-4. The repair of radiation-induced damage requires a substantial amount of energy, resulting in an increased demand for ATP in the irradiated and repaired yeasts. Consequently, the total and FoF1-ATPase activity in yeast homogenates and mitochondria increased after X-irradiation and post-irradiation repair. It was showed an increase in the activity of proline biosynthesis enzymes (ornithine transaminase and proline-5-carboxylate reductase) in X-irradiated C. guilliermondii NP-4, which remained elevated even after post-irradiation repair. As a result, the proline levels in X-irradiated and repaired yeasts were higher than those in non-irradiated cells. These findings suggest that proline may have a radioprotective effect on X-irradiated C. guilliermondii NP-4 yeasts. Taken together this study provides insights into the effects of X-radiation on ATPase activity, antioxidant enzyme activity, and proline biosynthesis in C. guilliermondii NP-4 yeast cells, highlighting the potential radioprotective properties of proline in X-irradiated yeasts.

Detection of Acyl-CoA Derivatized with Butylamide for in vitro Fatty Acid Desaturase Assay.

Membrane-bound fatty acid desaturases acting on acyl-CoA contribute to the biosynthesis of unsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid in higher organisms. We propose a simplified method for measuring the desaturase activity that combines the in vitro reaction by desaturase-expressing yeast cell homogenate and the detection of acyl-CoA product as butylamide derivatives by gas chromatography. To set up the in vitro reaction, we traced the in vivo activity of rat liver ∆6 fatty acid desaturase (D6d) expressed in the yeast, Saccharomyces cerevisiae, and determined the time taken for the D6d activity to reach its maximum level. The cell homogenate of yeast expressing the maximum D6d activity was made to react in vitro with linoleoyl-CoA to generate the D6d product, γlinolenoyl-CoA. This product was successfully detected as a peak corresponding to γ-linolenoyl butylamide on gas chromatography. This procedure, with low background expression, using non-labeled acyl-CoA as substrate, will contribute toward developing a simple in vitro desaturase assay. It will also help in elucidating the functions of membrane-bound fatty acid desaturases with various substrate specificities and regioselectivities.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase.

Nanofiber adsorbents for high productivity downstream processing

Electrospun polymeric nanofiber adsorbents offer an alternative ligand support surface for bioseparations. Their non-woven fiber structure with diameters in the sub-micron range creates a remarkably high surface area. To improve the purification productivity of biological molecules by chromatography, cellulose nanofiber adsorbents were fabricated and assembled into a cartridge and filter holder format with a volume of 0.15 mL, a bed height of 0.3 mm and diameter of 25 mm. The present study investigated the performance of diethylaminoethyl (DEAE) derivatized regenerated cellulose nanofiber adsorbents based on criteria including mass transfer and flow properties, binding capacity, and fouling effects. Our results show that nanofibers offer higher flow and mass transfer properties. The non-optimized DEAE-nanofiber adsorbents indicate a binding capacity of 10% that of packed bed systems with BSA as a single component system. However, they operate reproducibly at flowrates of a hundred times that of packed beds, resulting in a potential productivity increase of 10-fold. Lifetime studies showed that this novel adsorbent material operated reproducibly with complex feed material (centrifuged and 0.45 µm filtered yeast homogenate) and harsh cleaning-in-place conditions over multiple cycles. DEAE nanofibers showed superior operating performance in permeability and fouling over conventional adsorbents indicating their potential for bioseparation applications.

Heterologous expression of dammarenediol synthase gene in an engineeredSaccharomyces cerevisiae

Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. A dammarenediol-producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT-PCR. Fermentation was carried out in a 5-L GRJY-bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250μgl(-1). However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. This study showed that the high-value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene-derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.

Preparation, characterization, and process performance of composite fibrous adsorbents as cation exchangers for high throughput and high capacity bioseparations

Fibrous materials are proposed as novel chromatographic supports depicting high throughput and high product capacity. In this work, a composite fiber harboring strong cation-exchange moieties has been investigated. Such materials were characterized by a plethora of physical methods including degree of swelling (DS), scanning electron microscope (SEM), confocal laser scanning microscopy (CLSM), and Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR). The composite showed a high degree of grafting (∼30%) and exhibited a high swelling ratio (∼300%). Moreover, homogenous grafting and the development of an internal (functional) hydrogel were observed. The fibrous adsorbent was packed utilizing a designed “double roll” supported-structure and subsequently tested for packing efficiency and chromatography performance. The mentioned system showed similar packing efficiency of height equivalent to a theoretical plate (HETP) value and higher permeability coefficient (0.92×10−7cm2) than commercial resins. Experimentally determined Peclet number (Pe) values were within the range 60–90, suggesting a close-to-plug-flow condition. Total ionic capacity of the fibrous adsorbent was determined by the transition pH method. A capacity of 6.5mequiv./g was obtained. Moreover, a high dynamic binding capacity for lysozyme was found to be 283mg/g. On the other hand, a bed of randomly packed fiber also demonstrated high-resolution ability when a mixture of model protein was utilized to that end. Resolution was maintained at high flow rates (up to 900cm/h) and utilizing shorter gradient development routines. Direct sequestration of a model protein (lysozyme) was also possible from an artificial mixture containing 1.5% yeast homogenate. Summarizing, the composite fibrous adsorbents exhibited superior performance during early protein capture and intermediate-resolution applications.

A monolith purification process for virus-like particles from yeast homogenate

Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11mg/mL column to 0.25mg/mL column.

Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae

An easy and inexpensive method of fractionation of a yeast homogenate was proposed and it is based on differential centrifugation steps of insoluble components and subsequent precipitations of soluble fractions. In this fractionation, the effect of addition of protease inhibitor was studied. The procedure, which was performed in mild conditions in order to minimize protein denaturation, produced four fractions that proceed from distinct parts of the yeast cell and with a different chemical composition: Fr I, Fr II, Fr III and Fr IV. Thermal and surface behavior of these samples was also analyzed. Fr I and Fr II, mainly composed by cell wall debris and membrane cell components, respectively, exhibited an adsorption rate (Δ γ /Δ t 1/2 ) ten-fold higher than Fr III and Fr IV, composed by nucleoproteins and cytoplasmic proteins. All fractions exhibited a unique DSC endotherm with different peak temperature ( T p ) and enthalpy values (Δ H ). Fr IV exhibited the highest T p value (74 °C) and less affected by inhibitor absence. Fr I and Fr II showed the highest Δ H values (27–47 J/g protein) but they were markedly affected reducing their enthalpy values and increasing their surface properties in absence of protease inhibitor.

A systematic approach for modeling chromatographic processes—Application to protein purification

AbstractIn this article, a systematic approach is presented which allows the estimation of model parameters including isotherm parameters for equilibrium‐dispersive and general rate models including a procedure for how to subsequently select the most appropriate isotherm and model for a given application. The approach consists of three parts: determination of feed concentration, parameter estimation, and model selection. Accurate modeling of any separation process depends on the starting point, i.e. the feed and the feed concentration, however, for many chromatographic separations, this concentration is unknown. The first part of this work therefore presents a systematic procedure for feed concentration determination. The estimation of isotherm or other model parameters is done using the simulation and optimization tool gPROMS. Finally, the predictions from each model alternative are then compared using fractionation and maximum purification diagrams. The estimation approach is successfully applied to three case studies, including purification of alcohol dehydrogenase from a yeast homogenate supernatant and gradient elution ion‐exchange of myoglobin, which illustrate the approach and the relative suitability and range of application of the models in describing the behavior of complex chromatographic separations. It is found that, while the equilibrium‐dispersive model is a good approximation in most cases, there are cases when the more detailed general rate model is required. © 2008 American Institute of Chemical Engineers AIChE J, 2008

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine - 186%, L-leucine - 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ss-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ss-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ss-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Assessing adsorbent–biomass interactions during expanded bed adsorption onto ion exchangers utilizing surface energetics

Biomass adhesion onto an adsorbent matrix or “interaction” as well as biological particle co-adhesion or “aggregation” can severely affect the overall performance of many direct-contact methods for downstream processing of bioproducts. Studies to quantitatively describe this biomass–adsorbent interaction were developed utilizing surface energetics. An indirect thermodynamic approach via contact angle and zeta potential measurements was utilized. Intact yeast cells, yeast homogenates, and disrupted bacterial paste were employed as model system. Various surfaces that are relevant to biochemical and environmental applications were characterized. The extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) theory was found to appropriately predict biomass adhesion behaviour. It was observed that cell attachment onto anion-exchange supports is promoted by strong and close interaction within a secondary energy minimum followed by moderate multilayer cell aggregation. On the other hand, cell interaction with cation-exchange materials can take place within a reversible secondary energy minimum and at longer separation distance. The influence of particle charge and size, as well as the influence of the nature of the material under study were summarized in the form of energy vs. distance profiles. These investigations lead to many process-related conclusions: (a) process buffer conductivity windows can be recommended for anion-exchange chromatography (AEX) vs. cation-exchange chromatography (CEX) systems, (b) increased hydrodynamic shear is required to prevent biomass attachment onto AEX as compared to CEX, and (c) aggregation phenomena is a function of contact time and biomass concentration. Understanding biomass–adsorbent interaction at the particle (local) level is opening the pave for optimized operation of expanded bed adsorption methods at the process (macro) scale. A universal methodological approach is presented to guide both process and material design.

A study of the influence of yeast cell debris on protein and α‐glucosidase adsorption at various zones within the expanded bed using In‐Bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.

Assessment of 4-nitro-1,8-naphthalic anhydride reductase activity in homogenates of bakers’ yeast by reversed-phase high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous determination of yield and conversion ratio of 4-nitro-1,8-naphthalic anhydride to 4-amino-1,8-naphthalic anhydride following incubation with a crude bakers’ yeast homogenate. The analytes were separated on a C18 column in gradient mode. The detection limit of 4-amino-1,8-naphthalic anhydride is 10 ng/μl when using a 10 μl sample injection volume. The nitroreductase activity in the homogenate system can be assessed during the bioconversion process. The method can be used for the simultaneous detection of 4-hydroxylamino-1,8-naphthalic anhydride, an intermediate with limited stability.

The performance of anion exchange expanded bed adsorption chromatography on the recovery of G6PDH from unclarified feedstock with high biomass concentration

The bed stability of Streamline DEAE (p=1.2 g/mL) in a 20 mm (i.d.) glass expanded bed contactor, and its performance on the recovery of glucose 6-phosphate dehydrogenase (G6PDH) from unclarified yeast homogenate were investigated. A residence time distribution study showed that a stable expanded bed was achieved. The theoretical plate and Bodenstein numbers determined were 25 and 53, respectively. A recovery yield of 87% and purification factor of 4.1 were achieved in the operation using 5% (w/v) biomass concentration feedstock. The performance of the anion exchange EBAC was still considerable good at a biomass concentration as high as 15% (w/v).

Modulation of alkaline phosphatases from Saccharomyces cerevisiae

We aimed at characterizing Saccharomyces cerevisiae alkaline phosphatases (ALPs) activity modulation by ALP activity classic inhibitors, cholesterol and steroid hormones as well as at identifying catalytic similarities between yeast and mammals ALPs. Saccharomyces cerevisiae expresses two ALPs, coded for PHO8 and PHO13 genes. PHO8 product is repressible by Pi in the medium. ALP activity from yeast (either grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). L-Aminoacids and levamisole activated but EDTA and vanadate inhibited hPiALP, although with different patterns of effects. L-Homoarginine and L-tryptophan had a lower activatory effect on lPiALP. Corticosterone inhibited hPiALP but had the effect cancelled by growth on low phosphate medium. Cholesterol had a significantly different effect on hPiALP and lPiALP. beta-Estradiol and progesterone effects were different on hPiALP and lPiALP, showing a concentration-dependent activation of lPiALP (especially beta-estradiol). L-Homoarginine 5 mM was the stronger activator of hPiALP. L-Homoarginine 5 mM and beta-estradiol 5 mM may be used to discriminate between lPiALP and hPiALP. Our set of results does not allow identification of similarities between the behaviour of 2 Saccharomyces cerevisiae ALPs and any of the 4 human ALPs but allow us to raise the hypothesis of in vivo differential regulation of Saccharomyces cerevisiae ALPs by L-tryptophan, L-homoarginine and beta-estradiol.

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column

This study examined the impact of fouling with yeast homogenate on capacity and breakthrough performance of an ion exchange packed bed column. Column performance was assessed by analysis of breakthrough curves obtained with BSA as a test protein. The overall impact of fouling on breakthrough performance depended heavily on the level of clarification of the feed stream. Challenging the column with particulate-free homogenate caused no change in column performance. Loading successive small volumes of poorly clarified homogenate, interspersed with frequent column salt washes, did not alter significantly the column capacity. By contrast, when the column was challenged with an equivalent cumulative volume of poorly clarified homogenate, dynamic binding capacity decreased significantly and changes in breakthrough curves suggested increased intraparticle and external mass transfer limitations. These changes were ascribed to deposition of solid particulates in void spaces in the bed and colloidal contaminants in the bead pores.

Application of rotary microfiltration in debittering process of spent brewer’s yeast

This study concerns the production of yeast extract from spent brewer’s yeast using rotary microfiltration as a means to combine debittering and cell debris separation into a single step, without using a toxic alkali wash. The pH of yeast homogenate was found to affect protein yield and bitterness of the product. Rotary filtration of yeast homogenate at various pHs resulted in different percent protein transmissions. These were found to be 5.05%, 9.83%, and 30.83% for pH 5, 6, and 7.5, respectively. The bitterness concentration in the permeate was also found to be higher at higher pHs. Autolysis of the cell homogenate prior to filtration increased protein yield and decreased bitterness considerably. At pH 5.5, the protein transmission was increased to 60% and debittering efficiency was increased from 59% to 86%. The permeate flux and protein productivity could be further increased by increasing the rotational speed, but this resulted in a decrease in debittering efficiency. Thus, the rotational speed should be carefully selected to compromise between the yield and product quality. Furthermore, for the tested rotational speeds of 600 and 1000 rpm, the change in feed flow rate from 11 to 35 L h −1 changes the flow behavior from turbulent vortex flow to laminar vortex flow, thus decreasing the flux and protein productivity.

Expanded Bed Adsorption for Purification of Alcohol Dehydrogenase Using a Dye-Iminodiacetic Acid Matrix

Expanded bed adsorption (EBA) was successfully applied for the purification of alcohol dehydrogenase (ADH) from unclarified bakers' yeast homogenate using a new dye-iminodiacetic acid (IDA) matrix carrying immobilized metal ions. The existence of cells and cell debris do not have a significant effect on the adsorption of ADH on the matrix. The presence of imidazole in adsorption buffer was found to be an important factor in reducing the level of the adsorbed contaminant proteins. The optimal imidazole concentration was 5 mM. The dynamic binding capacity of the dye-IDA matrix in EBA was found to be 569 U/ml matrix. A chromatography of one-step elution with imidazole was developed, in which ADH was successfully adsorbed in the presence of 5 mM imidazole; subsequently, the column was washed with 5 mM imidazole containing 1 M NaCl and eluted with 150 mM imidazole containing 1.5 M NaCl. The purification factor and ADH recovery were 8.8 and 93.5%, respectively.

A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.