Immobilized Metal Ion Research Articles

Isolation and characterisation of zinc‐chelating peptides from tilapia (Oreochromis niloticus) protein hydrolysate

SummaryThis study aimed to isolate and characterise zinc‐chelating peptides from tilapia (Oreochromis niloticus) protein hydrolysate. The hydrolysate was fractionated using a multi‐ultrafiltration technique, yielding peptide fractions with molecular weights between 0.5 and 5 kDa. These fractions were further purified using immobilised metal ion affinity chromatography (IMAC‐Zn2+) and reverse high‐performance liquid chromatography (RP‐HPLC). Among the fractions obtained, the peptide fraction F211 exhibited a zinc‐binding capacity of 76.26 ± 2.46 mg g−1. Sequence analysis indicated that zinc binding occurred at amino acids on the F21 peptide and at valine or glutamate residues on the F22 peptide fraction. Structural characterisation revealed that amino nitrogen and oxygen atoms from carboxylate groups were the primary binding sites for Zn2+. Additionally, the zinc–peptide complex demonstrated strong thermal stability, which is significant for potential applications in food processing and storage, where maintaining the integrity of nutritional supplements under varying temperature conditions is crucial. These findings provide a feasible approach for the separation and purification of zinc‐binding peptides from tilapia protein hydrolysates and contribute to the understanding of the binding mechanisms between zinc and peptides.

Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris.

Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6-8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries.

Exploitation of porphyrin-based titanium-rich porous organic polymers for targeted phosphopeptide enrichment from the serum of colorectal cancer individuals.

Aporphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol),highselectivity (β-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.

Evaluating Imide-Based Mass Spectrometry-Cleavable Cross-Linkers for Structural Proteomics Studies.

Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas phase. In this study, we further advance the structural capabilities of DSBU by remodeling the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl disuccinic imide (DSSI) as a prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography enrichment. We detail DSSI synthesis and describe its behavior in solution and in the gas phase while cross-linking isolated proteins and human cell lysates. DSSI and DSBU cross-links are compared at the same enrichment depth to bridge these two cross-linker classes. We validate DSSI cross-links by mapping them in high-resolution structures of large protein assemblies. The cross-links observed yield insights into the morphology of intrinsically disordered proteins and their complexes. The DSSI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers.

Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro

BackgroundEnamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent.ResultsIn the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml.ConclusionsThese findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.

Fabrication of 3D aligned porous nanocellulose-based aerogel for enrichment of the histidine-containing polypeptides

The high-throughput immobilized metal affinity chromatography (IMAC) material is urgently required to improve the efficiency of separation and purification of tagged peptides in related biomedical applications. Herein, the amino-thiourea-modified nanocellulose (S-TOCN) -polyvinyl alcohol (PVA) composite aerogel with aligned pores was prepared by a directional freeze-casting technique and then used to immobilize metal ions (M2+) for the selective separation and purification of histidine-containing peptides from protein hydrolysates. The obtained S-TOCN-PVA materials with an oriented porous structure exhibited excellent mechanical properties, good adsorption performance, and reusability under optimal conditions. The adsorption capacity of the S-TOCN-PVA aerogel for metal ions was as follows: Cu2+>Ni2+>Zn2+>Co2+, and the higher metal ion chelating amount of the S-TOCN-PVA composite aerogel for Cu2+ and Ni2+ reached 87.21 mg/g at pH=7 and 83.66 mg/g at pH=6 in the 2 h incubation period, respectively. The enrichment effects of Cu2+/S-TOCN-PVA and Ni2+/S-TOCN-PVA aerogels with the selectivity of His-containing peptides from apricot kernel protein hydrolysates were up to 3.99 folds and 4.81 folds, respectively, for one cycle. Furthermore, after 5cycles, the Ni2+/S-TOCN-PVA aerogels remained at 87.5% of their initial adsorption capacity (848 mg/g), and the concentration increased by 39.05-fold. Molecular docking simulations show that the oxygen of the carboxyl group (-C-O and -CO) and the hydrogen of the imidazole group on the His molecule are mainly bonded to the hydrogen of the amino groups and the oxygen of the carboxyl group (C-O-C) in the structure of M2+/S-TOCN-PVA aerogels. The fabricated metal ion-immobilized 3D-aligned porous nanocellulose-based aerogels are promising IMAC materials for efficient separation and purification of the histidine-containing polypeptides from complicated biological samples.

Expression and biochemical characterization of a novel thermostable alkaline β-1,3–1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1

New thermostable β-1,3–1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 μmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

Infectious bronchitis virus (IBV) is considered as one of the main causes of economic loss in chicken farms worldwide, especially in poultry producing nations. This virus, a member of the Coronaviridae family, is classified into different genotypes based on its surface spike glycoproteins which also play important roles in cell attachment and immune response. Due to continual genetic mutation of IBV, attenuated and inactivated vaccines show decreased effectiveness or lack of cross – protection; thus ongoing studies focus on the development of new generation vaccines to prevent new IBV outbreaks. This paper describes our study on expression of the antigenic region in S1 subunit of the spike in Nicotiana benthamiana. Two expression vectors carrying either S1 or receptor binding domain (RBD) coding gene (pCB301-S1 and pCB301-RBD) were constructed and transformed into tobacco leaves by agroinfiltration method. The RBD showed a clearly higher level of expression compared to the whole S1. Purification of RBD by immobilized metal ion chromatography and size exclusion chromatography represented a mixture of monomer, dimer and trimer glycoprotein with expected sizes in Western blot. In summary, this study demonstrated our primary success in establishing an expression model that could be used to investigate plant-based IBV recombinant vaccine.

Cu(II)-Loaded Polydopamine-Coated Urchin-like Titanate Microspheres as a High-Performance IMAC Adsorbent for Hemoglobin Separation.

Immobilized metal ion affinity chromatography (IMAC) adsorbents generally have excellent affinity for histidine-rich proteins. However, the leaching of metal ions from the adsorbent usually affects its adsorption performance, which greatly affects the reusable performance of the adsorbent, resulting in many limitations in practical applications. Herein, a novel IMAC adsorbent, i.e., Cu(II)-loaded polydopamine-coated urchin-like titanate microspheres (Cu-PDA-UTMS), was prepared via metal coordination to make Cu ions uniformly decorate polydopamine-coated titanate microspheres. The as-synthesized microspheres exhibit an urchin-like structure, providing more binding sites for hemoglobin. Cu-PDA-UTMS exhibit favorable selectivity for hemoglobin adsorption and have a desirable adsorption capacity towards hemoglobin up to 2704.6 mg g-1. Using 0.1% CTAB as eluent, the adsorbed hemoglobin was easily eluted with a recovery rate of 86.8%. In addition, Cu-PDA-UTMS shows good reusability up to six cycles. In the end, the adsorption properties by Cu-PDA-UTMS towards hemoglobin from human blood samples were analyzed by SDS-PAGE. The results showed that Cu-PDA-UTMS are a high-performance IMAC adsorbent for hemoglobin separation, which provides a new method for the effective separation and purification of hemoglobin from complex biological samples.

Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.

Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered as a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of immobilized metal Ion affinity chromatography (IMAC) for producing 3Cpro. We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.

Light-mediated intracellular polymerization.

The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques.

Simulation of Ni2+ Chelating Peptides Separation in IMAC: Prediction of Langmuir Isotherm Parameters from SPR Affinity Data

Chromatography modeling for simulation is a tool that can help to predict the separation of molecules inside the column. Knowledge of sorption isotherms in chromatography modeling is a crucial step and methods such as frontal analysis or batch are used to obtain sorption isotherm parameters, but they require a significant quantity of samples. This study aims to predict Langmuir isotherm parameters from Surface Plasmon Resonance (SPR) affinity data (requiring less quantity of sample) to simulate metal chelating peptides (MCPs) separation in Immobilized Metal ion Affinity Chromatography (IMAC), thanks to the analogy between both techniques. The validity of simulation was evaluated by comparing the peptide’s simulated retention time with its experimental retention time obtained by IMAC. Results showed that the peptide affinity constant (KA) can be conserved between SPR and IMAC. However, the maximal capacity (qmax) must be adjusted by a correction factor to overcome the geometry differences between IMAC (spherical particles) and SPR (plane sensor ship). Therefore, three approaches were studied; the best one was to use qmax,IMAC imidazole determined experimentally while a correction factor was applied on qmax,SPR to obtain the qmax,IMAC of the peptide, thus minimizing the discrepancy between the experimental and simulated retention times of a peptide.

Enrichment of Phosphopeptides Based on Zirconium Phthalocyanine-Modified Magnetic Nanoparticles.

Highly selective extraction of phosphopeptides is necessary before mass spectrometry (MS) analysis. Herein, zirconium phthalocyanine-modified magnetic nanoparticles were prepared through a simple method. The Fe-O groups on Fe3O4 and the zirconium ions on phthalocyanine had a strong affinity for phosphopeptides based on immobilized metal ion affinity chromatography (IMAC). The enrichment platform exhibited low detection limit (0.01 fmol), high selectivity (α-/β-casein/bovine serum albumin, 1/1/5000), good reusability (10 circles), and recovery (91.1 ± 1.1%) toward phosphopeptides. Nonfat milk, human serum, saliva, and A549 cell lysate were employed as actual samples to assess the applicability of the enrichment protocol. Metallo-phthalocyanine will be a competitive compound for designing highly efficient adsorbents and offers a new approach to phosphopeptide analysis.

Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35–40 deg and 40–45 deg in Ni-M/A, but only one nickel peak at 35–40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Combination of click chemistry and Schiff base reaction: Post-synthesis of covalent organic frameworks as an immobilized metal ion affinity chromatography platform for efficient capture of global phosphopeptides in serum with chronic obstructive pulmonary disease.

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti4+ functionalized hydrophilic covalent organic frameworks by introducing glutathione (GSH) and 2,3,4-trihydroxy benzaldehyde (THBA) via click chemistry and Schiff base reaction (COF-V@GSH-THBA-Ti4+ ) was constructed and applied for selective enrichment of phosphopeptides in serum. Benefit from the high surface area, excellent hydrophilicity as well as regular mesoporous structure, COF-V@GSH-THBA-Ti4+ displayed high selectivity (molar ratio of 2000:1), low limit of detection (0.5 fmol), high load capacity (100.0mg/g) and excellent size-exclusion effect (1:10000) for enrichment of phosphopeptides. For actual bio-sample analysis, 15 phosphopeptides assigned to 10 phosphoproteins with 16 phosphorylated sites and 33 phosphopeptides assigned to 25 phosphoproteins with 34 phosphorylated sites were detected from the serum of patients with chronic obstructive pulmonary disease (COPD), and normal controls. Biological processes and molecular functions analysis further disclosed the difference of serums with phosphoproteomics between COPD and normal controls.

Purification of pineapple bromelain by IMAC chromatography using chlorophyll-activated macroporous matrices

This study investigated the purification of bromelain obtained from pineapple fruit using a new adsorbent for immobilized metal ion affinity chromatography (IMAC), with chlorophyll obtained from plant leaves as a chelating agent. The purification of bromelain was evaluated in batches from the crude extract of pineapple pulp (EXT), and the extract precipitated with 50 % ammonium sulfate (EXT.PR), the imidazole buffer (200 mM, pH 7.2) being analyzed and sodium acetate buffer, pH 5.0 + 1.0 NaCl as elution solutions. All methods tested could separate forms of bromelain with molecular weights between ±21 to 25 kDa. Although the technique using EXT.PR stood out in terms of purity, presenting a purification factor of around 3.09 ± 0.31 for elution with imidazole and 4.23 ± 0.12 for acetate buffer solution. In contrast, the EXT methods obtained values between 2.44 ± 0.23 and 3.21 ± 0.74 for elution with imidazole and acetate buffer, respectively, for purification from EXT.PR has lower yield values (around 5 %) than EXT (around 15 %). The number of steps tends to reduce yield and increase process costs, so the purification process in a monolithic bed coupled to the chromatographic system using the crude extract was evaluated. The final product obtained had a purification factor of 6, with a specific enzymatic activity of 59.61 ± 0.00 U·mg−1 and a yield of around 39 %, with only one band observed in the SDS-PAGE electrophoresis analysis, indicating that the matrix produced can separate specific proteins from the total fraction in the raw material. The IMAC matrix immobilized with chlorophyll proved promising and viable for application in protease purification processes.

Deciphering Interactions Involved in Immobilized Metal Ion Affinity Chromatography and Surface Plasmon Resonance for Validating the Analogy between Both Technologies

Various peptides can be obtained through protein enzymatic hydrolysis. Immobilized metal ion affinity chromatography (IMAC) is one of the methods which can be used to separate metal chelating peptides (MCPs) in a hydrolysate mixture. In this context, this work aims to understand deeply the interactions in IMAC and surface plasmon resonance (SPR) in order to validate experimentally the analogy between both technologies and to be further able to perform IMAC modeling in the next work using peptide sorption isotherm parameters obtained from SPR. Indeed, chromatography modeling can be used to predict separation of MCPs in IMAC and the knowledge of peptide sorption isotherm obtained from SPR is a crucial step. For this purpose, 22 peptides were selected and investigated in IMAC using HisTrap X-Ni2+ and HiFliQ NTA-Ni2+ columns and were also studied in SPR as well. Results showed that peptides with histidine residues had good affinity to Ni2+, while the high positive charge of peptides was responsible of ionic interactions. Further, most of the peptides with good retention time in IMAC showed a good affinity in SPR as well, which validated experimentally the SPR-IMAC analogy.

Heterologous expression and characterization of a novel thermostable and alkali stable recombinant lipase enzyme from Bacillus thuringensis into E. coliBL21(DE3) for detergent formulation

AbstractPresent study concerned with expression and biochemical characterization of lipase enzyme for potential use in detergent formulation. Lipase gene (1242 bp) of Bacillus thuringensis was cloned and expressed in Escherichia coli BL21 strain using pET‐21a(+) expression vector. Maximum expression of cloned lipase gene was obtained at 37°C with an induction of 0.4 mM IPTG (Isopropyl ß‐D‐1‐thiogalactopyranoside) after 4 h of induction. Recombinant lipase was purified to homogeneity using immobilized metal ion affinity chromatography carrying 109.80 U/mg specific activity with 38.79 purification folds. Molecular mass of purified lipase was determined as 45 kDa using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Purified recombinant lipase showed stability up to 90°C and retained significant activity (52%) after 4 h at 90°C. It was also found to be stable at a wide range of pH and in the presence of higher concentrations of several inhibitors (sodium dodecyl sulfate, dimethylsulfoxide, sodium azide, β‐mercaptoethanol, polysorbate 80, dithiothreitol) as well as organic solvents (acetone, methanol, ethanol, isopropanol, n‐butanol). The activity of recombinant lipase was enhanced in the presence of various metal ions and activated up to 200% by Ca2+. The compatibility of recombinant lipase with commercial detergents and other additives as well as its broad substrate specificity endorse the potential application of this enzyme in detergent formulations.

Label-free detection of biomolecules using inductively coupled plasma mass spectrometry (ICP-MS).

Bioassays using inductively coupled plasma mass spectrometry (ICP-MS) have gained increasing attention because of the high sensitivity of ICP-MS and the various strategies of labeling biomolecules with detectable metal tags. The classic strategy to tag the target biomolecules is through direct antibody-antigen interaction and DNA hybridization, and requires the separation of the bound from the unbound tags. Label-free ICP-MS techniques for biomolecular assays do not require direct labeling: they generate detectable metal ions indirectly from specific biomolecular reactions, such as enzymatic cleavage. Here, we highlight the development of three main strategies of label-free ICP-MS assays for biomolecules: (1) enzymatic cleavage of metal-labeled substrates, (2) release of immobilized metal ions from the DNA backbone, and (3) nucleic acid amplification-assisted aggregation and release of metal tags to achieve amplified detection. We briefly describe the fundamental basis of these label-free ICP-MS assays and discuss the benefits and drawbacks of various designs. Future research is needed to reduce non-specific adsorption and minimize background and interference. Analytical innovations are also required to confront challenges faced by in vivo applications.