200 assays (manual) / 2000 assays (microplate) / 1960 assays (auto-analyser)
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|Content:||200 assays (manual) / 2000 assays (microplate) / 1960 assays (auto-analyser)|
Short term stability: 2-8oC,
Long term stability: See individual component labels
|Stability:||> 2 years under recommended storage conditions|
|Assay Format:||Spectrophotometer, Microplate, Auto-analyser|
|Linear Range:||0.01 to 0.08 U/mL of glucose oxidase per assay|
|Limit of Detection:||10 U/L|
|Reaction Time (min):||~ 20 min|
|Application examples:||Enzyme preparations, and other materials (e.g. biological cultures, samples, etc.).|
|Method recognition:||Novel method|
The Glucose Oxidase assay kit is a simple procedure for the rapid and reliable measurement and analysis of glucose oxidase activity in industrial enzyme preparations and bread improver mixtures.
View more of our assay kits for enzyme activities.
- Very competitive price (cost per test)
- All reagents stable for > 12 months after preparation
- Simple format
- Mega-Calc™ software tool is available from our website for hassle-free raw data processing
- Standard included
- Suitable for manual, microplate and auto-analyser formats
Rinnan, Å., Bruun, S., Lindedam, J., Decker, S. R., Turner, G. B., Felby, C. & Engelsen, S. B. (2017). Analytica Chimica Acta, 962, 15-23.
The combination of NIR spectroscopy and chemometrics is a powerful correlation method for predicting the chemical constituents in biological matrices, such as the glucose and xylose content of straw. However, difficulties arise when it comes to predicting enzymatic glucose and xylose release potential, which is matrix dependent. Further complications are caused by xylose and glucose release potential being highly intercorrelated. This study emphasizes the importance of understanding the causal relationship between the model and the constituent of interest. It investigates the possibility of using near-infrared spectroscopy to evaluate the ethanol potential of wheat straw by analyzing more than 1000 samples from different wheat varieties and growth conditions. During the calibration model development, the prime emphasis was to investigate the correlation structure between the two major quality traits for saccharification of wheat straw: glucose and xylose release. The large sample set enabled a versatile and robust calibration model to be developed, showing that the prediction model for xylose release is based on a causal relationship with the NIR spectral data. In contrast, the prediction of glucose release was found to be highly dependent on the intercorrelation with xylose release. If this correlation is broken, the model performance breaks down. A simple method was devised for avoiding this breakdown and can be applied to any large dataset for investigating the causality or lack of causality of a prediction model.Hide Abstract
Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae.
Yin, H., Bultema, J. B., Dijkhuizen, L. & van Leeuwen, S. S. (2017). Food Chemistry, 225, 230-238.
β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with 13C6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-D-Galp-(1 → 2)-D-Glcp, β-D-Galp-(1 → 3)-D-Glcp and β-D-Galp-(1 → 6)-D-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site.Hide Abstract
Lamberti, F., Salmaso, S., Zambon, A., Brigo, L., Malfanti, A., Gatti, T., Agnoli, S., Granozzi, G., Brusatin, G., Elvassore, N. & Giomo, M. (2017). Advanced Materials Interfaces, In Press.
Surface chemical functionalization of transparent conductive oxides (TCOs) is helpful for a wide range of technological applications, ranging from solar cells to biomedical devices, as it allows to tune the electrical, optical, and morphological properties of TCOs toward the desired goal. The electrochemical grafting technique is a surface modification methodology affording robust coatings with tuneable properties and has the potential to be exploited for modifying TCO surfaces. However, due to technical limitations, like the use of a 3-electrode cell and the need for low pH-solutions, this approach has not been recurrently applied. Here a novel electrochemical-microfluidic combined methodology is used where the use of a microchannel drives the spatially controlled covalent grafting of reagents on a TCO surface. To corroborate the validity of this approach in producing more complex chemical structures localized on selected microscale-sized areas, where a first electrochemical grafting step takes place, an electrochemical glucose biosensor is realized through a layer-by-layer approach that shows a remarkable limit of detection in the micromolar concentration range. The sensing mechanism is based on an efficient electron transfer from glucose to the functionalized TCO surface. Biosensor performance is conveniently tuned by acting on the number of enzymatic units loaded onto the biosensor-tree.Hide Abstract
Maaran, S., Hoover, R., Vamadevan, V., Waduge, R. N. & Liu, Q. (2016). Food Chemistry, 192, 1098-1108.
The objective of this study was to understand the factors underlying the susceptibility of pulse (lablab bean, navy bean, rice bean, tepary bean, velvet bean, and wrinkled pea) and hylon VII starches towards in vitro hydrolysis by the combined action of pancreatin and amyloglucosidase. The time taken to reach an equivalent level of hydrolysis (50%) varied significantly among the starches. Changes to molecular order, crystallinity, double helical content, radial orientation of starch chains (polarized light), enthalpy and apparent amylose content during the progress of hydrolysis showed that rate and extent of hydrolysis were influenced both by the structure of the native starches at different levels (molecular, mesoscopic, microscopic) of granule organization, and by the extent of retrogradation between hydrolysed starch chains.Hide Abstract
Wood, I. P., Cook, N. M., Wilson, D. R., Ryden, P., Robertson, J. A. & Waldron, K. W. (2016). Food Chemistry, 198, 125-131.
Biorefining aims to exploit the full value of plant material by sequentially extracting and valorising its components. Many studies focus on the saccharification of virgin biomass sources, but it may be more efficient to pre-extract high-value components before hydrolysis to fermentable sugars. In the current study, a bran residue from de-starched, protein depleted and xylanase treated wheat bran has been subjected to hydrothermal pretreatment, saccharification and fermentation procedures to convert the residue to ethanol. The most effective pretreatment conditions (>190°C, 10 min) and saccharification conditions were identified following bench-scale liquid hot water pretreatment. Pre-extraction of enzymatically-hydrolysable starch and xylan reduced the release of furfural production, particularly when lower pretreatment severities were used. Pilot-scale steam explosion of the lignocellulosic residue followed by cellulase treatment and conversion to ethanol at a high substrate concentration (19%) gave an ethanol titre of ≈25 g/L or a yield of 93% of the theoretical maximum.Hide Abstract
Sriariyanun, M., Tantayotai, P., Yasurin, P., Pornwongthong, P. & Cheenkachorn, K. (2016). Electronic Journal of Biotechnology, 19, 23-28.
Background: Lignocellulosic biomass is a renewable, abundant, and inexpensive resource for biorefining process to produce biofuel and valuable chemicals. To make the process become feasible, it requires the use of both efficient pretreatment and hydrolysis enzymes to generate fermentable sugars. Ionic liquid (IL) pretreatment has been demonstrated to be a promising method to enhance the saccharification of biomass by cellulase enzyme; however, the remaining IL in the hydrolysis buffer strongly inhibits the function of cellulase. This study aimed to isolate a potential IL-tolerant cellulase producing bacterium to be applied in biorefining process. Result: One Bacillus sp., MSL2 strain, obtained from rice paddy field soil was isolated based on screening of cellulase assay. Its cellulase enzyme was purified and fractionated using a size exclusion chromatography. The molecular weight of purified cellulose was 48 kDa as revealed by SDS-PAGE and zymogram analysis. In the presence of the IL, 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) concentration of 1 M, the cellulase activity retained 77.7% of non-IL condition. In addition, the optimum temperature and pH of the enzyme is 50°C and pH 6.0, respectively. However, this cellulase retained its activity more than 90% at 55°C, and pH 4.0. Kinetic analysis of purified enzyme showed that the Km and Vmax were 0.8 mg/mL and 1000 µM/min, respectively. Conclusion: The characterization of cellulase produced from MSL2 strain was described here. These properties of cellulase made this bacterial strain become potential to be used in the biorefining process.Hide Abstract
Wang, S., Wang, S., Guo, P., Liu, L. & Wang, S. (2016). Journal of Agricultural and Food Chemistry, 65(1), 156-166.
In the present study, the multiscale structures and in vitro digestibility of wheat and yam starches with different water contents after heating at 100°C were investigated. After heating for the same time, the degree of gelatinization of both starches increased with increasing water content, followed by the gradual disruption of multiscale structures of starch granules. At a water content of 37% for wheat and 46% for yam starch, both starches were almost completely gelatinized after heating for 5 min at 100°C. Heat treatment increased greatly in vitro enzymatic digestibility of both starches, especially at a water content of >28%. It is interesting to note that extending heat treatment did not further disrupt the multiscale structures nor increase the in vitro enzymatic digestibility of both starches with the same water content. In contrast to wheat starch, yam starch showed a higher resistance to heat treatment. From this study, we can conclude that water content plays a more important role in determining the gelatinization behavior and in vitro enzymatic digestibility of starch than the duration of heating.Hide Abstract
Ma, Z., Boye, J. I. & Hu, X. (2016). Food Research International, 92, 64-78.
Saskatchewan grown yellow field pea was subjected to different processing conditions including dehulling, micronization, roasting, conventional/microwave cooking, germination, and combined germination and conventional cooking/roasting. Their nutritional and antinutritional compositions, functional properties, microstructure, thermal properties, in vitro protein and starch digestibility, and protein composition were studied. Processed field peas including conventional cooked yellow peas (CCYP), microwave cooked yellow peas (MCYP), germinated-conventional cooked yellow peas (GCCYP), and germinated-roasted yellow peas (GRYP) exhibited the significantly higher in vitro protein digestibility (IVPD), which was in accordance with their significantly lower trypsin inhibitor activity and tannin content. The SDS-PAGE and size exclusion HPLC profiles of untreated pea proteins and their hydrolysates also confirmed the IVPD result that these four treatments facilitated the hydrolysis of pea proteins to a greater extent. The CCYP, MCYP, GCCYP, and GRYP also exhibited significantly higher starch digestibility which was supported by their lower onset (T0), peak (Tp), and conclusion (Tc) temperatures obtained from DSC thermogram, their lower pasting properties and starch damage results, as well as their distinguished amorphous flakes' configuration observed on the scanning electron microscopic image. LC/ESI-MS/MS analysis following in-gel digests of SDS-PAGE separated proteins allowed detailed compositional characterization of pea proteins. The present study would provide fundamental information to help to better understand the functionality of field peas as ingredients, and particularly in regards to agri-food industry to improve the process efficiency of field peas with enhanced nutritional and techno-functional qualities.Hide Abstract
Wallace, J., Brienzo, M., García-Aparicio, M. P. & Görgens, J. F. (2016). New Biotechnology, 33(3), 361-371.
The enzymatic hydrolysis (EH) rate normally decreases during the hydrolysis, leaving unhydrolyzed material as residue. This phenomenon occurs during the hydrolysis of both cellulose (avicel) and lignocellulosic material, in nature or even pretreated. The progression of EH of steam pretreated sugarcane bagasse was associated with an initial (fast), intermediate (slower) and recalcitrant (slowest) phases, at glucan to glucose conversion yields of 61.7, 81.6 and 86%, respectively. Even though the EH of avicel as a simpler material than steam pretreated sugarcane bagasse, EH slowdown was present. The less thermo-stable endo-xylanase lost 58% of initial enzyme activity, followed by β-glucosidase that lost 16%, culminating in FPase activity loss of 30% in the first 24 hours. After 72 hours of EH the total loss of FPase activity was 40% compared to the initial activity. Analysis of the solid residue from EH showed that lignin content, phenolic compounds and ash increased while glucan decreased as hydrolysis progressed. During the initial fast phase of EH, the total solid residue surface area consisted predominantly of internal surface area. Thereafter, in the intermediate and recalcitrant phases of EH, the ratio of external:internal surface area increased. The proposed fiber damage and decrease in internal surface area, probably by EH action, was visualized by scanning electron microscopy imagery. The higher lignin/glucan ratio as EH progressed and enzyme deactivation by thermo instability were the main effects observed, respectively to substrate and enzyme.Hide Abstract
Simsek, S., Ovando-Martinez, M., Marefati, A., Sjӧӧ, M. & Rayner, M. (2015). Food Research International, 75, 41-49.
Octenyl succinate starches are commonly used as emulsifiers and texturizing agents in many food-systems. Rice, tapioca, corn, wheat and potato starches were modified with octenyl succinic anhydride (OSA) at 3% level. Structural characterization, molecular weight, starch digestibility and physical properties of starch granule stabilized emulsions were studied for modified starches. Modified potato (0.022) and wheat (0.018) starches had the highest and lowest degrees of OSA substitution, respectively. For all starches, amylose and amylopectin molecular mass was significantly (P < 0.05) lower for OSA starches. OSA modification may have hydrolyzed the small amylose and amylopectin chains, or caused rearrangement of the starch molecules. Although the starch modification improved emulsification properties, botanical source showed more influence on this parameter. Overall, botanical source had more influence on functional properties than degree of substitution. Further studies on OSA group distribution and fine molecular structure of amylopectin and relationship with functional properties will be important.Hide Abstract
Ogbo, F. C. & Okafor, E. N. (2015). Nigerian Food Journal, 33(1), 29-34.
The resistant starch (RS) content of some Nigerian cassava varieties and staples, “fufu”, “garri” and “abacha” processed from them were determined. Tubers of six varieties studied contained different concentrations of resistant starch, ranging from 5.70% in TMS 4(2)1425 to 7.07% in the TMS 30,572. Processing using traditional methods reduced the RS content in all cassava based foods compared with tubers from which they were processed. RS concentration was reduced by an average of 70.4% in “fufu”, 52.8% in “garri” and 35.85% in “abacha” for the four varieties of cassava tested. Cassava processing steps involving fermentation were responsible for the major reductions in concentration of RS. Steps involving cooking or frying, resulted in increase in concentrations of RS relative to other processing methods. Modifications of traditional methods of processing such as the addition of bitter leaf during retting or the addition of oil to mash during dewatering of “garri” affected RS concentrations in foods studied. Results of this work suggest that manipulation of processing methods and conditions employed during cassava processing can be used to improve RS concentration in cassava based foods, thus making them more functional.Hide Abstract
Kong, X., Chen, Y., Zhu, P., Sui, Z., Corke, H. & Bao, J. (2015). Journal of Agricultural and Food Chemistry, 63(27), 6241-6248.
We determined the relationships among the structural properties, in vitro digestibility, and genetic factors in starches of 14 rice cultivars. Weight-based chain-length distributions in amylopectin ranged from 18.07% to 24.71% (fa, DP 6-12), 45.01% to 55.67% (fb1, DP 13-24), 12.72% to 14.05% (fb2, DP 25-36), and 10.80 to 20.72% (fb3, DP > 36), respectively. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) ranged from 78.5% to 87.5%, 1.2% to 6.0%, and 10.1% to 18.0%, respectively. AAC was negatively correlated with RDS content but positively correlated with RS content in rice starch. The proportion of short chains in amylopectin, i.e. the amount of fraction IIa (FrIIa) fractionated by gel permeation chromatography (GPC), was positively correlated with RDS. Starch synthase IIa (SSIIa) gene controlled the degree of crystallinity, the amount of fa chains of amylopectin. SSIIIa gene controlled the amount of fb1 chains. Wx gene controlled the FrI, FrIIa, RDS, and RS. Starch debranching enzyme isoamylase II (ISA2) gene also controlled the RDS, which may suggest that RDS was also affected by amylopectin structure, although no correlation between them was found. This study indicated that genetics (i.e., starch biosynthesis related genes) controlled the structural properties of starch, and both amylose content and amylopectin fine structure determined functional properties of rice starch (i.e., the digestion), each in a different way. Understanding the “genetics-structure-function” relationships in rice starches will assist plant breeders and food processors in developing new rice varieties and functional foods.Hide Abstract
Simsek, S., Herken, E. N. & Ovando‐Martinez, M. (2015). Journal of the Science of Food and Agriculture, 96(8), 2896-2905.
BACKGROUND: Chickpea is considered a wholesome and nutritious food due to its nutritional properties and glycemic response. Such properties can be influenced by the thermal treatment used to cook this legume and produce a snack named leblebi. From the consumers' point of view, it is desirable to improve texture and palatability of the chickpea by the processing steps used to make leblebi. However, consumers are increasingly concerned with the nutritional value of snack foods. RESULTS: Nutritional components and digestibility properties of single and double heat-treated chickpea, single and double roasted leblebi and white leblebi were studied. High sodium, starch damage and soluble dietary fiber content were observed in white leblebi; while the other samples showed significantly (P < 0.05) higher insoluble dietary fiber content. Heat treatment and processing significantly (P < 0.05) altered the viscosity and starch properties of the samples. High resistant starch content (28.28% to 30.20%) and low estimated glycemic index (38.67 to 41.28) in heat-treated chickpeas and roasted leblebi were observed. CONCLUSION: The results indicate that heat-treated chickpea and roasted leblebi have good nutritional quality and low glycemic response. White leblebi had relatively high sodium content and glycemic response.Hide Abstract
Hanušová, K., Vápenka, L., Dobiáš, J. & Mišková, L. (2013). Central European Journal of Chemistry, 11(7), 1066-1078.
Packaging based on immobilization of antimicrobial enzymes provides a promising form of active packaging systems applicable in food processing. Glucose oxidase and lysozyme were immobilized by the Ugi reaction with cyclohexyl isocyanide and glutaraldehyde on polyamide and ionomer films partially hydrolysed by hydrochloric acid. The immobilization of the enzymes on the surface of films was confirmed by FT-IR spectroscopy and the films were characterized by the specific activity of the immobilized enzymes. The enzyme migration into model solutions and the effect of pH, temperature and storage time on the activity of immobilized enzyme were also evaluated. Immobilization of lysozyme onto polyamide and ionomer films resulted in the loss of enzyme activity. The polyamide and ionomer films with immobilized glucose oxidase inhibited the growth of bacteria Escherichia coli CNCTC 6859, Pseudomonas fluorescens CNCTC 5793, Lactobacillus helveticus CH-1, Listeria ivanovii CCM 5884 and Listeria innocua CCM 4030 on agar media.Hide Abstract
Odenigbo, A. M., Ngadi, M., Manful, J. & Danbaba, N. (2013). International Journal of Food Science & Technology, 48(12), 2535-2540.
This study determined the levels of nutritionally important starch fractions in selected milled, nonparboiled rice cultivated in Nigeria. Five improved varieties (FARO 52, FARO 57, FARO 44, FARO 60 and FARO 61) and four local varieties (Kwandala, Yardass, Jeep and Jamila) were evaluated. There were significant differences in starch fractions among varieties. Resistant starch (RS) ranged between 1.43% and 3.13%. Rapidly digestible starch (RDS) was lowest in Jamila (27.70%) and highest in FARO 61 (39.26%). Generally, the local varieties had significant higher RS (2.71%) with a lower RDS (32.82%) compared with improved varieties (RS; 1.88% and RDS; 36.07%). RS was inversely related to RDS and starch digestion index (SDI). The SDI had a highly significant positive correlation with RDS (r = 0.879, P < 0.01). These results highlight the need for further work in the identification of milled, nonparboiled rice varieties with less rapid digestion for its associated health benefits to consumers.Hide Abstract
Odenigbo, A., Rahimi, J., Ngadi, M., Amer, S. & Mustafa, A. (2012). Functional Foods in Health and Disease, 2(7), 280-289.
Background: Sweet potato (Ipomoea batatas L.) is a very rich source of starch. There is increased interest in starch digestibility and the prevention and management of metabolic diseases. Objective: The aim of this study was to evaluate the levels of starch fractions and predicted glycemic index of different cultivars of sweet potato. Material and Method: French fries produced from five cultivars of sweet potato (‘Ginseng Red’, ‘Beauregard’, ‘White Travis’, ‘Georgia Jet clone #2010’ and ‘Georgia Jet’) were used. The level of total starch (TS), resistant starch (RS), digestible starch (DS), and starch digestion index starch digestion index in the samples were evaluated. In vitro starch hydrolysis at 30, 90, and 120 min were determined enzymatically for calculation of rapidly digestible starch (RDS), predicted glycemic index (pGI) and slowly digestible starch (SDS) respectively. Results: The RS content in all samples had an inversely significant correlation with pGI (-0.52; P<0.05) while RDS had positive and significant influence on both pGI (r=0.55; P<0.05) and SDI (r= 0.94; P<0.01). ‘White Travis’ and ‘Ginseng Red’ had higher levels of beneficial starch fractions (RS and SDS) with low pGI and starch digestion Index (SDI), despite their higher TS content. Generally, all the cultivars had products with low to moderate GI values. Conclusion: The glycemic index of these food products highlights the health promoting characteristics of sweet potato cultivars.Hide Abstract
Park, H. J., McConnell, J. T., Boddohi, S., Kipper, M. J. & Johnson, P. A. (2011). Colloids and Surfaces B: Biointerfaces, 83(2), 198-203.
The influence of particle size on the activity and recycling capabilities of enzyme conjugated magnetic nanoparticles was studied. Co-precipitation and oxidation of Fe(OH)2 methods were used to fabricate three different sizes of magnetic nanoparticles (5 nm, 26 nm and 51 nm). Glucose oxidase was covalently bound to the magnetic nanoparticles by modifying the surfaces with 3-(aminopropyl)triethoxysilane (APTES) and a common protein crosslinking agent, glutaraldehyde. Analysis by Transmission Electron Microscopy (TEM) showed that the morphology of the magnetic nanoparticles to be spherical and sizes agreed with results of the Brunauer, Emmett, and Teller (BET) method. Magnetic strength of the nanoparticles was analyzed by magnetometry and found to be 49 emu g-1 (5 nm), 73 emu g-1 (26 nm), and 85 emu g-1 (51 nm). X-ray photoelectron spectroscopy (XPS) confirmed each step of the magnetic nanoparticle surface modification and successful glucose oxidase binding. The immobilized enzymes retained 15–23% of the native GOx activity. Recycling stability studies showed approximately 20% of activity loss for the large (51 nm) and medium (26 nm) size glucose oxidase-magnetic nanoparticle (GOx-MNP) bioconjugate and about 96% activity loss for the smallest GOx-MNP bioconjugate (5 nm) after ten cycles. The bioconjugates demonstrated equivalent total product conversions as a single reaction of an equivalent amount of the native enzyme after the 5th cycle for the 26 nm nanoparticles and the 7th cycle for the 51 nm nanoparticles.Hide Abstract
Arrabito, G., Musumeci, C., Aiello, V., Libertino, S., Compagnini, G. & Pignataro, B. (2009). Langmuir, 25(11), 6312-6318.
Arrays of circular spots of glucose oxidase have been obtained on functionalized silicon oxide by piezoelectric inkjet printing and the enzymatic activity toward glucose recognition has been monitored. The addition of glycerol to the molecular ink allows to obtain high spot definition and resolution (tens of micrometers wide; one molecule tall), but in spite of its well-known structural stabilizing properties, in dynamic conditions it may lead to increased protein stresses. The jetting voltage and pulse length have been found to be critical factors for both activity retention and pattern definition. High voltages and pulse lengths results in stress effects along with the loss of activity, which, at least in our experimental conditions, has been found to be recovered in time.Hide Abstract
Smith, A. M. & Zeeman, S. C. (2006). Nature Protocols, 1(3), 1342-1345.
This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted to remove free glucose. Starch is solubilized by heating, then digested to glucose by adding glucan hydrolases. Glucose is assayed enzymatically. The method is more sensitive and accurate than iodine-based protocols, and is suitable for tissues that have a wide range of starch contents. Measurements on multiple samples can be completed within a day.Hide Abstract
Patchornik, G. & Albeck, A. (2005). Bioconjugate Chemistry, 16(5), 1310-1315.
Purification of proteins on a large scale is a complex multistep process, and alternative economic strategies are required. This study presents a novel approach (Affinity Sinking, AS) for purification of native proteins utilizing nonimmobilized modified ligands. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports. Therefore, purification from large volumes can be accomplished without the use of industrial-scale affinity columns. The mechanism of product capture is formation and precipitation of a specific [target-protein/modified-ligand] complex by using a soluble interconnecting entity that generates an insoluble [target-protein/modified-ligand/interconnecting entity] sediment containing the target protein. Rabbit IgG and two glycoproteins were purified accordingly, utilizing free avidin (as the interconnecting entity) and either desthiobiotinylated-protein A (DB-ProA) or desthiobiotinylated-concanavalin A (DB-ConA) as the modified ligand. The recovery yields for the IgG and the two glycoproteins were 80−86% and 70−75%, respectively. Target proteins are eluted from the generated pellet nearly without disrupting the [modified-ligand/interconnecting entity] macro-complex, thus enabling a practical procedure of recovering target proteins. Leaching of the DB-ProA ligand under eluting conditions (pH 3) was found to be lower than 1%. The two modified ligands, DB-ProA and DB-ConA, were regenerated without any chromatographic procedure in 80% and 85%-89% yield, respectively. The advantage of excluding the polymeric component from the purification process and obtaining highly purified proteins has been demonstrated, and it implies that other contaminants (e.g. endotoxins, prions, host DNA) could be excluded as well, thereby reducing the number of purification steps in a typical downstream process.Hide Abstract