Weak Ion Exchange Resins Research Articles

Simple and efficient method for the extraction, isolation, and purification of peroxidase enzyme from wheat bran

AbstractPeroxidase is an industrially important enzyme with a broad range of applications. In this study, the extraction, isolation, and purification of peroxidase was conducted with wheat bran as a feedstock. Several commercially available buffers (K2HPO4, Na3PO4, CH3COONa, and Tris‐base) were explored for the extraction of peroxidase from wheat bran. Of the tested media, sodium acetate buffer gave an excellent performance and displayed maximum protein (0.44 mg mL−1) and enzyme activity (28 U mL−1). A process optimization study was also conducted to obtain the maximum protein concentration and enzyme activity. The intensified process provided 1.39 mg mL−1 protein concentration with 90 U mL−1 enzyme activity with specific enzyme activity of 135.8 U mg−1. Crude enzyme was purified using a three‐step process: (a) precipitation, (b) ultrafiltration, and (c) chromatographic purification. Commercially available strong and weak ion exchange resins, with different surface properties, were tested for purification. The weak anion exchange resin showed an excellent performance for maximum binding of peroxidase. Adsorption study investigated the best fit model of Freundlich isotherm with maximum binding capacity of 54 mg mL−1. Overall, purification provided peroxidase with a protein concentration of 1.24 mg mL−1 and 727.58 U mg −1 specific enzyme activity with purity increased by around ~6.6 fold. The reported process provides a simple and efficient method for the extraction and purification of peroxidase enzyme from wheat bran for its efficient valorization.

The power of weak ion-exchange resins assisted by amelogenin for natural remineralization of dental enamel: an in vitro study

This study aims to develop an innovative dental product to remineralize dental enamel by a proper combination of ion-exchange resins as controlled release of mineral ions that form dental enamel, in the presence of amelogenin to guide the appropriate crystal growth. The novel product proposed consists of a combination of ion-exchange resins (weak acid and weak base) individually loaded with the remineralizing ions: Ca2+, PO43− and F−, also including Zn2+ in a minor amount as antibacterial, together with the protein amelogenin. Such cocktail provides onsite controlled release of the ions necessary for enamel remineralization due to the weak character of the resins and at the same time, a guiding tool for related crystal growth by the indicated protein. Amelogenin protein is involved in the structural development of natural enamel and takes a key role in controlling the crystal growth morphology and alignment at the enamel surface. Bovine teeth were treated by applying the resins and protein together with artificial saliva. Treated teeth were evaluated with nanoindentation, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The innovative material induces the dental remineralization creating a fluorapatite layer with a hardness equivalent to sound enamel, with the appropriate alignment of corresponding nanocrystals, being the fluorapatite more acid resistant than the original mineral. Our results suggest that the new product shows potential for promoting long-term remineralization leading to the inhibition of caries and protection of dental structures.

Expanding the toolbox of nutrient limitation studies: A novel method of soil microbial in‐growth bags to evaluate nutrient demands in tropical forests

Abstract Ecosystem processes and the organisms involved are generally limited by the availability of one or more elements in soil, an important phenomenon to consider for our understanding of ecosystem functioning and future changes. Especially in tropical forests, typically growing on nutrient depleted soils, nitrogen (N), phosphorus (P) or other limitations are assumed. However, large‐scale nutrient manipulation experiments revealed complex site‐specific patterns and several authors raised the need for novel approaches to reveal deeper mechanistic insights on limitation patterns, especially concerning soil microbial activity. In order to gain such deeper knowledge, based on a short review of previous small‐scale studies focusing on soil micro‐organisms, we developed an experimental approach which controls for common biases, including indirect treatment effects, addition of co‐elements or nutrient pulses. Using this technique, we tested the hypotheses that fungi growing in tropical forest soils are mainly limited by P and that N versus P limitations shift along an altitudinal gradient. Mesh bags of 38 μm filled with sand were amended with weak ion exchange resins loaded with N, P or potassium (K) and buried underneath the litter layer at three altitudinal sites in southern Ecuador. After a period of four months, the in‐growth of fungal hyphae was quantified, phospholipid fatty acids were analysed for a subset of samples, and chemical properties were determined. In line with the first hypothesis, hyphal abundance was increased in P‐amended mesh bags, indicating P limitation. However, this pattern was not significantly shifted along the altitudinal gradient. By contrast, N addition increased hyphal abundance at the lowest site, compared to significant reductions at 2,000 and 3,000 m—not only in fungi but also in bacterial abundance as indicated by PLFA analyses, contradicting common soil‐age hypotheses. Decreased nutrient immobilization and fungal in‐growth at higher elevations suggest slow microbial activity, including nitrification, which may have caused toxic ammonium accumulation. The experimental design offers a promising tool to provide more mechanistic and soil‐focused analyses specifically targeting microbes, which in this system strongly supported the hypothesis of primary fungal P limitation in tropical forest soils. A plain language summary is available for this article.

충진층 반응기에서 고정화 cellulase에 의한 셀룰로스 가수 분해

약 염기성 이온교환 수지에 cellulase를 고정화하였고 고정화 cellulase는 흡착 특성이 Langmuir 흡착 등온선을 잘 따랐다. pH와 온도에 대한 효소 활성은 고정화 효소가 우수한 특성을 보였다. 열에 대한 효소 활성은 1차식으로 감소하였고 고정화 효소가 자유 효소에 비해 열 안정성이 우수하였다. 초기 속도법을 통해서 자유 효소와 고정화 효소의 Michaelis-Menten 속도 상수를 결정하였고 속도상수 Km은 고정화 효소가 큰 값을 나타내었다. 충진층 반응기에서 셀룰로스의 전환 공정을 재순환에 의해 측정하였다. 투입되는 셀룰로스의 유량 변화에 대한 셀룰로스의 변환을 연속 공정에서 조사하였다. 장기 운전의 성능을 평가하기 위해 7일 통한 연속공정을 실시하였고 고정화 효소는 48%의 활성을 유지하였다. Immobilized cellulase on weak ion exchange resin showed a typical Langmuir adsorption isotherm. Immobilized cellulase had better stability with respect to pH and temperature than free cellulase. Kinetics of thermal inactivation on free and immobilized cellulase followed first order rate, and immobilized cellulase had a longer half-life than free cellulase. The initial rate method was used to characterize the kinetic parameters of free and immobilized enzyme. The Michaelis-Menten constant <TEX>$K_m$</TEX> was higher for the immobilized enzyme than it was for the free enzyme. The effect of the recirculation rate on cellulose degradation was studied in a recycling packed-bed reactor. In a continuous packed-bed reactor, the increasing flow rate of cellulose decreased the conversion efficiency of cellulose at different input lactose concentrations. Continuous operation for five days was conducted to investigate the stability of long term operation. The retained activity of the immobilized enzymes was 48% after seven days of operation.

Strong and Weak Ion Exchange Resin in Chemical Desalting Process Combined Application

This paper describes the strong, weak ion exchange resin in combined application of the principle, calculation method, and combined with the actual introduction of the combined application of the equipment and operation mode.

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma.

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non‐enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5–5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.

Mixed-mode anion-cation exchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometry as an alternative to reversed phase for small molecule drug discovery

Within pharmaceutical drug discovery, significant needs currently exist for the analysis and purification of structurally diverse samples prior to or immediately following high-throughput screening. These processes are required to facilitate rapid and accurate biological profiling, structural determination, and resupply of new drug candidates. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) for both analytical and preparative applications has become the small molecule separation/detection tool of choice for meeting many of these needs. However, the separation selectivity provided by RP-HPLC has been limited to the hydrophobicity-based resolution of relatively nonpolar sample components, and for high-throughput drug discovery applications, no sufficient alternative procedures have been identified. In this investigation, a mixed-mode anion-cation exchange/hydrophilic interaction chromatography (ACE-HILIC) method has been developed to provide both direct compatibility with ESI-MS and evaporative light-scattering detection (ELSD) and separation selectivity highly orthogonal to RP-HPLC. The technique employed silica-based small-pore weak ion exchange resins eluted with a combined aqueous and pH gradient. A diverse set of dipeptide probes was employed for the elucidation of the relative contributions of three retention mechanisms. ACE-HILIC-ESI-MS-ELSD should prove useful for the analysis and purification of compounds from both biological (e.g., natural products) and synthetic (e.g., combinatorial chemistry) sources of molecular diversity.

Atomic force microscopy study of the secretory granule lumen

We have used an atomic force microscope to study the mechanical properties of the matrix found in the lumen of secretory granules isolated from mast cells. The matrices were insoluble and had an average height of 474 +/- 197 nm. The volume of these matrices increased reversibly about tenfold by decreasing the valency of the bathing external cation (La3+ < Ca2+ < Na+). The elastic (Young's) modulus was found to decrease by about 100-fold (4.3 MPa in La3+ to 37 kPa in Na+) upon a tenfold increase in the matrix volume. A swollen granule matrix had an elastic modulus similar to that of gelatin in water. The elastic modulus was inversely related to the change in the volume of the matrix, following a relationship similar to that predicted for the elasticity of weakly cross-linked polymers. Our results show that the matrix of these secretory granules have the mechanical properties of weak ion exchange resins, lending strong support to an ion exchange mechanism for the storage and release of cationic secretory products.

Determination of amino acids from the leaves of evergreen oak (Quercus ilex L.) at four different stages of decomposition

It was established by weak ion exchange resin chromatography that 17 amino acids in proteins and 15 free amino acids account for almost all of the amino acids present in the leaves of evergreen oak (Quercus ilex L.) in four different stages of decomposition. No significant change in the amounts of these amino acids was observed in the course of decomposition. The amino acid content of evergreen oak leaves is comparable to that in pine needles and beech leaves.

Fractionation of mechanically sheared chromatin on ECTHAM-cellulose

Chromatography of chromatin on the weak ion-exchange resin ECTHAM-cellulose was re-examined using the combined salt-pH elution conditions of Stratling, W.H., Van, N.T. and O'Malley, B.W. (1976) Eur. J. Biochem. 66, 423–433. When mechanically sheared rat liver chromatin was chromatographed on ECTHAM-cellulose the histone composition of eluted fractions was very similar, whereas early eluting fractions were enriched in non-histone proteins, including certain high mobility group proteins, and in hnRNP particles, containing newly synthesised RNA. Later eluting fractions were depleted in all of these components. The majority of hnRNP particles in early eluting chromatin were shown to be physically associated with chromatin by centrifugation in metrizamide. Hen erythrocyte chromatin contained no early eluting material. Size of DNA fragments was not a significant factor in determining the elution position of chromatin fragments. Early eluting material was not generated by endogenous nuclease and protease action. The conditions of chromatin preparation, and of elution of early chromatin fractions caused no gross disruption of chromatin structure, or dissociation of chromatin proteins, although some nucleosome sliding may have occurred. The conditions required for elution of some of the later fractions are sufficient to cause dissociation of protein, and alteration of chromatin conformation.

The nature of zinc and copper complexes in the oyster Ostrea edulis

The zinc and copper associated with the soft tissues of the oyster Ostrea edulis Linnaeus have been separated into a soluble component and a tissue-residue, cell-debris bound component. In the case of zinc, the tissue-bound component was found to contain at least two species of complex; a firmly-bound species, exchangeable with 65Zn2+ and a less-firmly, reversibly-bound species, exchangeable with 65Zn2+. The soluble component, which constitutes some 40% of the total zinc and copper, was fractionated on Sephadex G-25 and the zinc and copper shown to be weakly-complexed to the small molecular weight compounds, taurine, lysine, ATP and possibly homarine (N-methyl-α-picolinic acid) and to be fully exchangeable with 65Zn2+. These soluble complexes can act as a freely available mobile reserve of metal to ensure a constant saturation of metal-dependent enzyme systems operating under adverse environments. Sephadex G-25 acts as a weak ion-exchange resin, which can cause a translocation of zinc and copper from its soluble weak complexes and result in the spurious association of the metals with other compounds.

New automated fluorometric methods for estimation of small amounts of adrenaline and noradrenaline

Two automated fluorometric methods are described for estimation of A and NA in plasma, urine, and tissue extracts. By several improvements and the use of a new stabilizing reagent, sodium diethyldithiocarbamate, the sensitivity and efficiency of catecholamine estimation is greatly improved. Preliminary chromatogrphic separation on a weak ion-exchange resin microcolumn can be accomplished in a series of 40 or more samples simultaneously. The subsequent fluorometric estimation by two different procedures includes automatic extraction in one and automatic pH adjustment in the other. The technique presented allows measurement of catecholamine concentrations as low as 0.001 μ/ml, making it possible to perform blood level estimations on a small volume of sample.