One-pot Purification Research Articles

Facile purification and immobilization of organophosphorus hydrolase on protein-inorganic hybrid phosphate nanosheets

Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands. However, the supporting materials used for orientated immobilization are usually costly or complicated in preparation, affecting their practical applications. In this work, a facile purification and immobilization method was proposed for enzyme immobilization based on organic-inorganic hybrid calcium phosphate nanocrystal (CaPs) induced by Cu2+ modified bovine serum albumin (BSA-Cu). Then, the as-prepared hybrid calcium phosphate nanosheet, [email protected], was utilized for one-pot purification and immobilization of His-tagged organophosphorus hydrolase (OPH) by metal-affinity binding to the incorporated BSA. [email protected] exhibited enhanced pH stability and thermal stability compared to the free enzyme. Moreover, [email protected] could retain more than 75% and 56% of initial activity after reuse 5 and 10 times, respectively. The results demonstrated that this facile strategy was promising for the effective biodegradation of organophosphorus pesticides with the immobilized enzyme.

One-Pot Purification and Immobilization of Phenylalanine Dehydrogenase from Bacillus nanhaiensi by Functional Reduced Graphene Oxide.

The one-pot immobilization of halophilic phenylalanine dehydrogenase from marine microorganism with metal ions modified reduced graphene oxide (CRGO) material was studied. Phenylalanine dehydrogenase was from Bacillus nanhaiensi and expressed with a C-terminal His-tag. Investigation of CRGO, CRGO-PEI, CRCO-Mn, and CRGO-PEI-Mn for one-pot purification and immobilization of phenylalanine dehydrogenase from crude enzyme solution was carried out. Enzyme activity yield rate achieved 80.0% by immobilization with CRCO-Mn, and the loading capacity was 6.7mg/mg. Manganese ion coordination greatly improved the selectivity of the CRGO for the target His-tagged enzyme. Furthermore, the effect of NaCl concentration on the immobilization was investigated, which the loading capacity of CRGO-PEI and CRGO-Mn-PEI was increased by 10.7% and 30.6% with 1M NaCl, respectively. The adsorption curves of crude enzyme one-pot immobilized by CRGO-Mn and purified enzyme immobilized by CRGO-Mn were similar. Therefore, one-pot immobilization strategy is promising for industrial application with advantages such as high efficiency and low cost, which shorten the pipelines for enzyme discovery towards industrial applications through the establishing of marine enzyme collections.

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

Protecting Carbon Nanotubes from Oxidation for Selective Carbon Impurity Elimination

Purity of carbon nanotubes (CNTs) is essential to avoid a dramatic decrease in their performances. In addition to metallic impurities, carbonaceous impurities have been shown to be responsible for pronounced effects. However, they are highly difficult to be selectively removed from CNT samples because of the similar chemical reactivity of these two kinds of carbon species. The existing purification methods often lead to high CNT consumption (>90 wt %). The proposed method consists of a one-pot gas-phase treatment combining chlorine and oxygen. The CNT powder maintained in a chlorine stream is submitted to oxygen at moderate temperature [350 and 500 °C for single-walled CNTs (SWCNTs) and double-walled CNTs (DWCNTs), respectively], and the thermal treatment is then pursued at 900–1000 °C under chlorine alone. Our work reveals that this approach is able to significantly improve the selectivity of elimination of carbonaceous impurities. Thanks to the proposed purification treatment, only 19 and 11 wt % of carbon species (mainly carbon impurities) are lost for DWCNTs and SWCNTs, respectively. The mechanism proposed involves a protective effect by grafting of chlorine favored to the CNT walls. Because our simple one-pot purification method is also versatile and scalable, it opens new perspectives for CNT applications in high-added value fields.

Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.

A Simple One Pot Purification of Bacterial Amylase From Fermented Broth Based on Affinity Toward Starch-Functionalized Magnetic Nanoparticle

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet–visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

Simultaneous Analysis of Heparan Sulfate, Chondroitin/Dermatan Sulfates, and Hyaluronan Disaccharides by Glycoblotting-Assisted Sample Preparation Followed by Single-Step Zwitter-Ionic-Hydrophilic Interaction Chromatography

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Synthesis of carbon-encapsulated iron nanoparticles by pyrolysis of iron citrate andpoly(vinyl alcohol): a critical evaluation of yield and selectivity

Carbon-encapsulated iron nanoparticles were synthesized by pyrolysis at1000 °C of two solid precursors: poly(vinyl alcohol) and iron citrate. The weight ratio between theprecursors controlled the reaction yield, crystallinity, morphological features and magneticproperties of the products. The encapsulation yield of iron nanoparticles in carbon shellswas strongly influenced by the iron-to-carbon ratio and depended on the iron citratecontent in the initial reactant mixtures. Despite the inherent simplicity of theprocess and the use of low cost starting materials the demonstrated route possesseslimited selectivity, especially at high iron-to-carbon ratios. At these experimentalconditions the as-obtained products contained non-encapsulated Fe particles andgraphite in addition to magnetic carbon encapsulates. These by-products wereeffectively removed by a one-pot purification procedure that included acid treatment.

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co 2+ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD + with a representative synthesis of ( S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS–PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for ( S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.

Purification and Functionalization of Diamond Nanopowders

AbstractPurification of diamond nanopowder (DNP) was conducted in a less-destructive mild polyphosphoric acid (PPA)/phosphorous pentoxide (P2O5). The wide-angle X-ray diffraction (XRD) showed that the intensity of the characteristic diamond d-spacing (111) at 2.07 Å from purified DNP (PDNP) was fairly increased compared to pristine DNP, indicating that significant amount of carbonaceous impurities were removed. Chemical modification of pristine DNP and PDNP with 4-ethylbenzoic acid was carried out to afford 4-ethylbenzoyl-functionalized DNP (EBA-g-DNP) and PDNP (EBA-g-PDNP). The morphologies of EBA-g-DNP and EBA-g-PDNP from scanning electron microscopy (SEM) were further affirmed the feasibility of chemical modification. The results suggested that the reaction condition was indeed viable for the one-pot purification and functionalization of DNP. The resultant functionalized DNP could be useful for nanoscale additives. Hence, EBA-g-DNP and EBA-g-PDNP was brominated by using N-bromosuccinimide (NBS). The resultant N-brominated DNP and PDNP could be used as initiator for the atom transfer radical polymerization (ATRP) to introduce many polymers onto the surface of functionalized DNP and PDNP.

One-pot purification and functionalization of single-walled carbon nanotubes in less-corrosive poly(phosphoric acid)

As-received commercial single-walled carbon nanotubes (SWCNTs) were treated in mild, inorganic polyacid, viz. polyphosphoric acid (PPA) with or without additional phosphorous pentoxide (P 2O 5) at 130, 160, and 190 °C. Unlike the treatment in strong acids such as nitric acid/sulfuric acid mixtures, nitric acid and hydrochloric acid, PPA with or without additional P 2O 5 could selectively remove the tenacious carbonaceous and metallic impurities with little or no damage to the basic frameworks of SWCNTs and crystalline carbon materials. Since the medium PPA/P 2O 5 is known for an efficient “direct” Friedel-Crafts acylation using a carboxylic acid instead of a carboxylic acid chloride, it provides the advantage of combining both purification and functionalization steps into a one-pot process in manufacturing of functionalized SWCNTs.

Targeted removal of bioavailable metal as a detoxification strategy for carbon nanotubes

There is substantial evidence for toxicity and/or carcinogenicity upon inhalation of pure transition metals in fine particulate form. Carbon nanotube catalyst residues may trigger similar metal-mediated toxicity, but only if the metal is bioavailable and not fully encapsulated within fluid-protective carbon shells. Recent studies have documented the presence of bioavailable iron and nickel in a variety of commercial as-produced and vendor “purified” nanotubes, and the present article examines techniques to avoid or remove this bioavailable metal. First, data are presented on the mechanisms potentially responsible for free metal in “purified” samples, including kinetic limitations during metal dissolution, the re-deposition or adsorption of metal on nanotube outer surfaces, and carbon shell damage during last-step oxidation or one-pot purification. Optimized acid treatment protocols are presented for targeting the free metal, considering the effects of acid strength, composition, time, and conditions for post-treatment water washing. Finally, after optimized acid treatment, it is shown that the remaining, non-bioavailable (encapsulated) metal persists in a stable and biologically unavailable form up to two months in an in vitro biopersistence assay, suggesting that simple removal of bioavailable (free) metal is a promising strategy for reducing nanotube health risks.

A Highly Selective, One-Pot Purification Method for Single-Walled Carbon Nanotubes

We report on a one-pot, highly selective chemistry to remove residual catalysts from single-walled carbon nanotubes (SWNTs). The impurities, initially present at approximately 35 wt % and mostly as carbon-coated iron nanoparticles, can be driven below 5 wt % with nearly no loss of SWNTs. The carbon-coated iron impurities are dissolved simply by reacting with an aqueous mixture of H2O2 and HCl at 40-70 degrees C for 4-8 h. This purification combines two known reactions involving H2O2 and HCl, respectively; however, by combining these two typically inefficient reactions into a one-pot reaction, the new process is surprisingly selective toward the removal of the metal impurities. This high selectivity derives from the proximity effect of the iron-catalyzed Fenton chemistry. At pH approximately 1-3, iron is dissolved upon exposure, avoiding the otherwise aggressive iron-catalyzed digestion of SWNTs by H2O2. This extremely simple and selective chemistry offers a "green" and scalable process to purify carbon nanotube materials.