A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability

Many different micro and nano sized materials have been used for enzymes immobilization in order to increase their catalytic activity and stability. Generally, immobilized enzymes with conventional immobilization techniques exhibit improved stability while their activity is lowered compared to free enzymes. Recently, an elegant immobilization approach was discovered in synthesis of flower-like organic-inorganic hybrid nanostructures with extraordinary catalytic activity and stability. In this novel immobilization strategy, proteins (enzymes) and metal ions acted as organic and inorganic components, respectively to form hybrid nanoflowers (hNFs). It is demonstrated that the hNFs highly enhanced catalytic activities and stability in a wide range of experimental conditions (pHs, temperatures and salt concentration, etc.) compared to free and conventionally immobilized enzymes. This review mainly discussed the synthesis, characterization, development and applications of organic-inorganic hybrid nanoflowers formed of various enzymes and metal ions and explained potential mechanism underlying enhanced catalytic activity and stability.     

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

Background

Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.

Methods and findings

We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.

Conclusions

Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.     

Aerobic adaptation in yeast. II. Changes in enzyme profiles during a step-down anaerobic-aerobic transfer.

The levels of various enzymes and components of the glycolytic and respiratory pathways of the yeast Saccharomyces cerevisiae have been determined during a step-down, anaerobic-to-aerobic transition. These activities were determined as an adjunct to the respective metabolite data reported in the first paper in this series. It is clear from the data that anaerobic conditions induce an environment conducive to express glycolytic enzyme activities, while manifesting a differential induction/repression effect on oxidative enzymes. An NAD/NADH mediated mechanism is proposed to explain this difference. Of the enzymes assayed only cytochrome c oxidase shows any direct response to oxygen challenge and consequently it is suggested that the assembly of this enzyme is the trigger mechanism and rate-limiting step in aerobic adaptation.     

Immobilized enzymes – valuable tools for the indication of temperature events

Abstract

The enzymes trypsin and urease were covalently tethered to cellulose to utilize their ability to produce colored products as a consequence of enzymatic activity. Therefore, cellulose had to be chemically modified first in order to generate appropriate chemical functionalities. Different approaches including periodate supported oxidation followed by immobilization via reductive amination, insertion of a reactive polymer interface, and cross-linking inside the cellulose matrix were utilized for the immobilization. The success of immobilization was assessed by the quantification of surface-bound protein as well as by recording of enzymatic activities under different conditions. The enzymatic activity of trypsin and urease was maintained best when a hydrophilic intermediate polymer layer was used for immobilization. The applicability of immobilized enzymes as temperature indicators was demonstrated using cross-linked urease.     

Site-specific fluorescent labeling and oriented immobilization of a triple mutant of CYP3A4 via C64

The generation of site-specific bioconjugates of proteins is highly desired for a number of biophysical and nanotechnological applications. To this end, many strategies have been developed that allow the specific modification of certain canonical amino acids and, more recently, noncanonical functional groups. P450 enzymes are heme-dependent monooxygenases involved in xenobiotic metabolism and in the biosynthesis of a variety of secondary metabolites. We became interested in the site-specific modification of these enzymes, CYP3A4 in particular, through our studies of their in vitro biocatalytic properties and our desire to exploit their remarkable ability to oxidize unactivated C-H bonds in a regio- and stereospecific manner. Obtained via a partial cysteine-depletion approach, a functional triple mutant of CYP3A4 (C98S/C239S/C468G) is reported here which is singly modified at C64 by maleimide-containing groups. While cysteine-labeling of the wild-type enzyme abolished >90% of its enzymatic activity, this mutant retained ≥75% of the activity of the unmodified wild-type enzyme with 9 of the 18 maleimides that were tested. These included both fluorescent and solid-supported maleimides. The loss of activity observed after labeling with some maleimides is attributed to direct enzyme inhibition rather than to steric effects. We also demonstrate the functional immobilization of this mutant on maleimide-functionalized agarose resin and silica microspheres.     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Identification of active site residues of the adeno-associated virus type 2 Rep endonuclease

Adeno-associated virus type 2 Rep endonuclease activity is necessary for both viral DNA replication and site-specific integration of the viral genome into human chromosome 19. The biochemical activities required for site-specific endonuclease activity (namely specific DNA binding and transesterification activity) have been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is metal-dependent. To identify this metal-binding site, we have employed a cysteine mutagenesis approach that targets conserved acidic amino acids. By using this technique, we provide functional biochemical data supporting a role for glutamate 83 in the coordination of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved in the coordination of metal ions, is closely associated with the active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published structural studies of the AAV type 5 endonuclease and suggest that although the two enzymes are not highly conserved with respect to the AAV family, their active sites are highly conserved.     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

High-sensitivity detection for model organophosphorus and carbamate pesticide with quartz crystal microbalance-precipitation sensor

The effect of acetylcholinesterase (AChE) immobilization over the surface of a quartz crystal microbalance (QCM), by chemisorption of the AChE thiolated with a heterobifunctional cross-linker, sulfo-succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate, and carboxyl-amine coupling of AChE to 3-mercaptopropionic acid self-assembled monolayer, on the responses of a batch-type QCM-precipitation sensor was compared, resulting in a better sensitivity and binding efficiency in the former method. When an inhibition study with the developed sensor was undertaken at the optimized AChE immobilization with varying concentrations of a model organophosphorus pesticide EPN and carbamate one carbofuran, a sensitive detection for them was possible with the limit of detection corresponding to 1.55 x 10(-8) and 1.30 x 10(-9)M, respectively.     

Changes in exopeptidase activities in skeletal muscles during disuse

Some aminopeptidase activities, dipeptidase-, tripeptidase-, and carboxypeptidase activities were measured in two different types of skeletal muscle in rabbit soleus muscle as a slow oxidative, and gastrocnemius muscle as a fast glycolytic type after immobilization in full extension with a plaster cast for 1, 2, 4, 7, 14 or 28 days. In correlation to the higher protein turnover in red muscles, the activities except of leucine and alanine aminopeptidase were higher in the normal soleus muscle than in the gastrocnemius muscle. Much higher activities of the tested enzymes were obtained in the immobilized soleus muscle than in the normal one after 2 weeks of immobilization. In the gastrocnemius muscle the tested enzyme activities generally did not change or decrease. The results demonstrate that the peptidases play a role in the process of protein breakdown in normal and disused skeletal muscles.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Design of a long acting peptide functioning as both a glucagon-like peptide-1 receptor agonist and a glucagon receptor antagonist

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.     

Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and l-N-carbamoylase from Arthrobacter aurescens for the production of optically pure l-amino acids

2The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements.In comparison to the free enzymes, temperature and pH-optima were increased by 10 degrees C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degrees C (60 degrees C).In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.     

Evidence for glycolytic enzyme binding during anaerobiosis of the foot muscle ofPatella caerulea (L.)

Summary The effect of anaerobiosis and aerobic recovery on the degree of binding of glycolytic enzymes to the particulate fraction of the cell was studied in the foot muscle of the marine molluscP. caerulea, in order to assess the role of glycolytic enzyme binding in the metabolic transition between aerobic and anoxic states. Short periods of anoxia (2 h, 4 h) resulted in an increase in enzyme binding in association with the increased glycolytic rate observed; this was particularly pronounced for phosphorylase, phosphofructokinase, aldolase, pyruvate kinase and lactate dehydrogenase. Decreased enzyme binding was observed after prolonged periods of anoxia. These effects were reversed and control values re-established when animals were returned to aerobic conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms inP. caerulea foot muscle. This reversible interaction of glycolytic enzymes with structural proteins may constitute an additional mechanism for metabolic control.     

Differential mechanisms for calcium-dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes

In this work, two different types of supported biomimetic membranes were designed to study the membrane binding properties of two different proteins that both interact with cellular membranes in a calcium-dependent manner. The first one, neurocalcin, is a member of a subfamilly of EF-hand calcium-binding proteins that exhibit a calcium-myristoyl switch. The second protein is a bacterial toxin, the adenylate cyclase produced by Bordetella pertussis, the causative agent of whooping cough. The biomimetic membranes constructed in this study were either hybrid bilayer membranes or polymer-tethered membranes. Hemimembrane formation was obtained in two steps: a monolayer of 1-octadecanethiol or octadecyltrichlorosilane was self-assembled on top of the gold or glass surface, respectively, and then the egg-phosphatidyl choline (PC) vesicle fused on the hydrophobic alkyl layer. Polymer-tethered membranes on solid support were obtained using N-hydroxysuccinimide (NHS)-terminated-poly(ethyleneglycol) (PEG)-phospholipids as anchoring molecules. Egg-PC/1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethyleneglycol)-N-hydroxy-succinimide (DSPE-PEG-NHS) mixture liposomes were injected on the top of an amine grafted surface (cysteamine-coated gold or silanized glass); vesicles were linked to the surface and disrupted, leading to the formation of a bilayer. The biomimetic membrane constructions were followed by surface plasmon spectroscopy, while membrane fluidity and continuity were observed by fluorescence microscopy. Protein/membrane binding properties were determined by resonance surface plasmon measurements. The tethered bilayer, designed here, is very versatile as it can be adapted easily to different types of support. The results demonstrate the potentialities of such polymer-tethered artificial membranes for the study of proteins that insert into biological membranes such as toxins and/or integral membrane proteins.     

Self-assembly and photocatalytic activity of branched silicatein/silintaphin filaments decorated with silicatein-synthesized TiO<Subscript>2</Subscript> nanoparticles

The fundamental mechanisms of biomineralization and their translation into innovative synthetic approaches have yielded promising perspectives for the fabrication of biomimetic and bioinspired organic–inorganic hybrid materials. In siliceous sponges, the enzyme silicatein catalyzes the polycondensation of molecular precursors to nano-structured SiO₂ that is deposited on self-assembled filaments consisting of the two silicatein isoforms (silicatein-α and -β) and the scaffold protein silintaphin-1. Due to its broad substrate specificity silicatein is also able to convert in vitro various other precursors to non-biogenic materials (e.g., hydrolysis of titanium bis(ammonium lactato)-dihydroxide [TiBALDH] and subsequent polycondensation to titania [TiO₂]). In the present approach, silicatein was bioengineered to carry a protein tag (Arg-tag) that confers binding affinity to TiO₂. Then, by combining Arg-tagged silicatein-α with silicatein-β and silintaphin-1, self-assembled branched hybrid protein microfilaments were fabricated. Upon subsequent incubation with TiBALDH the filaments were decorated with TiO₂ and assayed for photocatalytic activity through photodegradation of the dye methylene blue. This is the first approach that considers concomitant application of two silicatein isoforms for the synthesis of bioinspired organic–inorganic hybrid materials. It is also the first time that the biocatalytic activity of the enzymes has been combined with both the structure-providing properties of silintaphin-1 and a TiO₂ affinity protein tag to fabricate self-assembled branched protein filaments as template for a silicatein-synthesized TiO₂ photocatalyst. The TiO₂-decorated filaments might be explored as a practical alternative to approaches where biotemplates have to be laboriously isolated from their original biological source prior to TiO₂ immobilization.     

Site-specific immobilization of a (His)6-tagged acetylcholinesterase on nickel nanoparticles for highly sensitive toxicity biosensors

This paper reports site-specific affinity immobilization of (His)6-tagged acetylcholinesterase (AChE) onto Ni/NiO nanoparticles for the development of an electrochemical screen-printed biosensor for the detection of organophosphate pesticides. The method is based on the specific affinity binding of the His-tagged enzyme to oxidized nickel nanoparticle surfaces in the absence of metal chelators. This approach allows stable and oriented attachment of the enzyme onto the oxidized nickel through the external His residue in one-step procedure, allowing for fast and sensitive detection of paraoxon in the concentration range from 10⁻⁸ to 10⁻¹³ M. A detection limit of 10⁻¹² M for paraoxon was obtained after 20 min incubation. This method can be used as a generic approach for the immobilization of other His-tagged enzymes for the development of biosensors.     

Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis.

Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3.     

Enzymes of intermediary carbohydrate metabolism in Ureaplasma urealyticum and Mycoplasma mycoides subsp. mycoides

Cell extracts of Ureaplasma urealyticum and Mycoplasma mycoides were examined for enzymes of intermediary carbohydrate metabolism using a sensitive radiochemical assay procedure. For M. mycoides, the enzyme activities detected were supporting evidence for the existence of a glycolytic pathway giving lactate anaerobically and acetate aerobically. U. urealyticum also had activities of many glycolytic enzymes. Enzymes of the pentose phosphate pathway occurred in both M. mycoides and U. urealyticum. Their presence allowed the proposal of a sequence for the synthesis from glycolytic pathway intermediates of ribose 5-phosphate, and hence phosphoribosyl diphosphate, for the synthesis of nucleotides. Pathways for the further metabolism of deoxyribose 1-phosphate and ribose 1-phosphate produced from nucleoside phosphorylase reactions operated in extracts from both organisms.