Enzyme Activity In Presence Research Articles

Activity-induced diffusion recovery in crowded colloidal suspensions.

We show that the forces generated by active enzyme molecules are strong enough to influence the dynamics of their surroundings under artificial crowded environments. We measured the behavior of polymer microparticles in a quasi-two-dimensional system under aqueous environment, at various area fraction values of particles. In the presence of enzymatic activity, not only was the diffusion of the suspended particles enhanced at shorter time-scales, but the system also showed a transition from subdiffusive to diffusive dynamics at longer time-scale limits. Similar observations were also recorded with enzyme-functionalized microparticles. Brownian dynamics simulations have been performed to support the experimental observations.

THE GLA D313Y MUTATION IN FABRY DISEASE: DIAGNOSTIC DILEMMAS AND THERAPEUTIC CONSIDERATIONS

Abstract Fabry disease is a genetic disorder caused by mutations in the GLA gene, which encodes alpha–galactosidase A, leading to enzyme dysfunction and subsequent accumulation of glycosphingolipids. In the classical form, it occurs in childhood or early adolescence with renal, cardiac, and neurological involvement, resulting in a poor prognosis. The GLA D313Y is a missense mutation resulting in a significant reduction of enzyme activity in plasma, while leukocyte enzyme activity remains within normal limits, leading to a "pseudo–deficiency" of enzyme activity with uncertain pathogenicity. In literature, it has been observed that individuals with this mutation may present predominantly with central neurologic manifestations, characterized by multifocal white matter lesions. Historically they do not receive indications for initiating enzyme replacement or molecular chaperone therapies. We present the case of a man affected by arterial hypertension and history of chronic lower limb pain who experienced a lacunar stroke at the age of 35. The GLA D313Y mutation was identified and enzyme activity assay in plasma was within normal limits, precluding the initiation of enzyme replacement therapy. Over the following years, he suffered two additional ischemic strokes, a progressive decline in renal function to stage 3, and acute coronary syndrome. Transthoracic echocardiography revealed hypertrophic non–obstructive cardiomyopathy (SIVd 20 mm) with normal biventricular ejection fraction. Cardiac magnetic resonance showed late gadolinium enhancement of inferior junctional and inferior wall in middle segment (Fig.1). Considering the patient‘s clinical conditions involving multi–organ impairment, an enzymatic activity test was performed on leukocytes, revealing a reduced activity. This prompted the decision to initiate enzyme replacement therapy. This case report supports the hypothesis that the D313Y mutation may be associated with a later presentation and not only neurological involvement. In our experience, it’s mandatory to proceed with the determination of leukocyte enzyme activity in presence of normal enzyme activity in plasma. As an alternative, the detection of glycosphingolipids’ accumulation could be useful, but myocardial and renal biopsy poses a high bleeding risk, especially in anticoagulated or antiaggregated patients. The authors propose considering the initiation of therapy in patients with the D313Y mutation who exhibit significant organ involvement.

A Novel Variant in the LIPA Gene Associated with Distinct Phenotype.

Deficiency of lysosomal acid lipase (LAL-D) is caused by biallelic pathogenic variants in the LIPA gene. Spectrum of LAL-D ranges from early onset of hepatosplenomegaly and psychomotor regression (Wolman disease) to a more chronic course (cholesteryl ester storage disease - CESD). The diagnosis is based on lipid and biomarker profiles, specific liver histopathology, enzyme deficiency, and identification of causative genetic variants. Biomarker findings are a useful for diagnostics of LAL-D, including high plasma concentration of chitotriosidase as well as elevated oxysterols. Current treatment options include enzyme replacement therapy (sebelipase-alpha), statins, liver transplantation, and stem cell transplantation. We present two pairs of siblings from Serbia with a distinctive phenotype resembling LAL-D with a novel variant of unknown significance (VUS) detected in the LIPA gene and residual LAL activity. All patients presented with hepatosplenomegaly at early childhood. In siblings from family 1, compound heterozygosity for a pathogenic c.419G>A (p.Trp140Ter) variant and a novel VUS c.851C>T (p.Ser284Phe) was detected. Patients from family 2 were homozygous for c.851C>T VUS and both have typical histopathologic findings for LAL-D in the liver. Enzyme activity of LAL was tested in three patients and reported as sufficient, and therefore enzyme replacement therapy could not be approved. When confronted with a challenge of diagnosing an inherited metabolic disorder, several aspects are taken into consideration: clinical manifestations, specific biomarkers, enzyme assay results, and molecular genetic findings. This report brings cases to light which have a considerable discrepancy between those aspects, namely the preserved LAL enzyme activity in presence of clinical manifestations and rare variants in the LIPA gene.

Modeling of the thermal behaviour of free β-galactosidase from palm weevil, Rhynchophorus palmarum Linn. (Coleoptera: Curculionidae) larvae using Equilibrium model

β-galactosidases are a class of enzyme widely used as biocatalysts in the food, dairy, and fermentation industries. However, due to their biological origin, enhancement of these enzymes is generally necessary. The effect of temperature upon enzymes is a mandatory stage in rational enzyme engineering. The present work was devoted to Rhynchophorus palmarum Linn. β-galactosidase (Rpbgal) as part of the investigation of insect-derived enzymes for biotechnological applications. The thermal behaviour of Rpbgal has been studied in the temperature range 303-353 K by measuring enzymatic activities in presence of oNPG as substrate. Equilibrium model which gives complete and quantitative description of the effect of temperature on enzyme activity has been used to analyze experimental data. A satisfactorily agreement between the calculated results and the experimental data was obtained. The thermodynamic parameters provided by this model were given. Results showed that Rpbgal is relatively stable and active at 323 K. Temperatures over 330 K produce a significant decrease in the enzyme activity. In the temperature range 331 - 339 K, Rpbgal showed the best thermal stability compared to a commercial β-galactosidase from Aspergillus oryzae.

Biochemical disorders in the thyroid gland in rats with hyperhomocysteinemia

Background: In recent years, significant progress has been made in elucidating the mechanisms of development of complications associated with hyperhomocysteinemia (HC), but little is known about the disorders in the thyroid gland. This study aimed to determine the levels of low-molecularweight degradation products and peptides in the thyroid gland of rats with HC. In addition, markers of inflammation and the proteolytic state were also assessed. Methods: HC was induced by intragastric administration of DL-homocysteine thiolactone (100 mg.kg-1 of body weight) to albino non-linear male rats. The levels of low-molecular-weight substances (LMWS) of various nature were detected at wavelengths of 254 and 280 nm. The levels of MMP-2 and cytokine profiles were detected by enzyme-linked immunosorbent assay. The protein composition and the presence of active enzymes were assessed by SDS-polyacrylamide gel electrophoresis and zymography, respectively. Results: Obtained results can indicate the enhancement of catabolic reactions, confirmed by the accumulation of LMWS in the blood and thyroid gland of the rats with HC. The state of inflammation, evidenced by the significantly increased pro-inflammatory cytokines, was also revealed. HC was accompanied by the activation of proteolysis, expressed by an increase in overall proteolytic activity, the level of MMP-2, and changes in protein profile. Proteins accumulated with a molecular weight of less than 30 kDa simultaneously with the decrease in the level of high-molecular-weight proteins.

Physics of self-rolling viruses.

Viruses are right at the interface of inanimate matter and life. However, recent experiments [Sakai et al., J. Virol. 92, e01522-17 (2018)0022-538X10.1128/JVI.01522-17] have shown that some influenza strains can actively roll on glycan-covered surfaces. In a previous letter [Ziebert and Kulić, Phys. Rev. Lett. 126, 218101 (2021)0031-900710.1103/PhysRevLett.126.218101] we suggested this to be a form of viral surface metabolism: a collection of spike proteins that attach to and cut the glycans act as a self-organized mechano-chemical motor. Here we study in more depth the physics of the emergent self-rolling states. We give scaling arguments how the motion arises, substantiated by a detailed analytical theory that yields the full torque-angular velocity relation of the self-organized motor. Stochastic Gillespie simulations are used to validate the theory and to quantify stochastic effects like virus detachment and reversals of its direction. Finally, we also cross-check several approximations made previously and show that the proposed mechanism is very robust. All these results point together to the statistical inevitability of viral rolling in the presence of enzymatic activity.

Daya simpan buah pepaya potong dengan blanching menggunakan asam askorbat dan natrium benzoat

Consumption of fruit and vegetables in Indonesia is still low. One way to increase fruit consumption is the use of sliced fruit in snack boxes. The preparation of sliced fruit in snack boxes has not been carried out because the durability of cut fruit is very short due to the fruit's physiological characteristics, which is still doing respiration and the presence of enzymatic activity that causes browning. The blanching process can do enzymatic reaction inhibition. This study aims to determine the shelf life of sliced pepaya fruit using the hot water blanching method using ascorbic acid and sodium benzoate immersion. This study used a randomized block design with 1 factor, namely the addition of anti-browning agents in the form of 1% ascorbic acid and 1% sodium benzoate. Storage was carried out for seven days, and parameter observations were carried out on days 0, 1, 3, 5, and 7. The parameters observed were moisture content, vitamin C, pH, texture, and sensory tests (color, aroma, texture, taste, and overall preference). The results showed a decrease in moisture content, vitamin C, pH, and texture of sliced pepaya fruit during storage in all treatments. Blanching by immersion using ascorbic acid can increase vitamin C levels and decrease the pH value of sliced pepaya during storage. Immersion with ascorbic acid and Na benzoate not affected on sliced pepaya’s texture during storage. Sensory testing showed that the immersion of ascorbic acid and Na benzoate could maintain the color, aroma, taste, and overall preference of the panelists for sliced pepaya during storage.

In silico and in vitro assays reveal potential inhibitors against 3CL pro main protease of SARS-CoV-2

The COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is not showing any sign of slowing down even after the ongoing efforts of vaccination. The threats of new strains are concerning, as some of them are more infectious than the original one. A therapeutic against the disease is, therefore, of urgent need. Here, we use the DrugBank database to screen for potential inhibitors against the 3CL pro main protease of SARS-CoV-2. Instead of using the traditional approach of computational screening by docking, we developed a kernel ridge regressor (using a part of the docking data) to predict the binding energy of ligands. We used this model to screen the DrugBank database and shortlist two lead candidates (bromocriptine and avoralstat) for in vitro enzymatic study. Our results show that the 3CL pro enzyme activity in presence of 100 μM concentration of bromocriptine and avoralstat is 9.9% and 15.9%, respectively. Remarkably, bromocriptine exhibited submicromolar IC50 of 130 nM (0.13 μM). Avoralstat showed an IC50 of 2.16 μM. Further, the interactions of both drugs with 3CL pro were analyzed using molecular dynamics simulations of 100 ns. Results indicate that both ligands are stable in the binding pocket of the 3CL pro receptor. In addition, the MM-PBSA analysis revealed that bromocriptine (-29.37 kcal/mol) has a lower binding free energy compared to avoralstat (-6.91 kcal/mol). Further, hydrogen bond analysis also showed that bromocriptine interacts with the two catalytic residues, His41 and Cys145, more frequently than avoralstat. Communicated by Ramaswamy H. Sarma

Etching of AuNPs Through Superoxide Radical Dismutation by Cu-Zn Superoxide Dismutase Resulted in Remarkable Changes of its Localized Surface Plasmon Resonance.

Background: Superoxide dismutases (SODs) are categorized as antioxidant enzymes that are involved in many processes such as stress signalling responses and cell protection against free radical species. The primary function of SOD is the removal of produced radical species like superoxide ions in different physiological processes. There are various isozymes of SODs which are classified according to the metal cofactor in their active sites into four general types of Fe-SOD, Mn-SOD, Cu/Zn-SOD and Ni-SOD. Among metal nanoparticles, gold nanoparticles (AuNPs) are useful for biological purposes as sensing probe for determining critical analysis based on surface plasmon resonance and colorimetric method. In this study, the human Cu-Zn SOD expressed, purified, and its interaction with AuNPs based on a new colorimetric method was investigated.Objectives: In this approach, a colorimetric detection method for SOD activity was developed based on the carboxylic stabilized AuNPs.Material and Methods: The Ni-NTA Sepharose affinity column was performed for the purification process of enzyme. Following SOD purification, the enzyme activity in presence of AuNPs due to the possible etching in the presence of free radicals which are produced by riboflavin, methionine, Na2CO3 and potassium phosphate buffer, have been performed. In addition, Fluorescence spectroscopy analysis toward SOD and gold nanoparticle were performed.Results: Superoxide radicals generated from the enzymatic reaction would preferentially etch AuNPs and resulted in remarkable changes of localized surface plasmon resonance of AuNPs, which is reduced in the presence of SOD. Under the optimized experimental conditions assay (pH~7.8 and 25 ˚C), better selectivity and sensitivity toward SOD activity was shown.Conclusions: In this context, an indirect new colorimetric method for determining of SOD activity based on gold nanoparticles (AuNPs) was evaluated. According to the presented result, it may be concluded that by scavenging of free superoxide radicals in the presence of SOD, the amount of AuNP absorbance can be replenished.

Rhizosphere priming regulates soil organic carbon and nitrogen mineralization: The significance of abiotic mechanisms

The effect of living roots on the co-mineralization of soil organic carbon (SOC) and nitrogen (SON) and driving mechanisms of the rhizosphere priming effect (RPE) remains unclear. Moreover, it is still poorly understood whether the abiotic mechanisms, whereby roots accelerate soil organic matter (SOM) loss by destabilizing organo-mineral associations, are involved. Biotic and abiotic processes involved in the RPE and gross N mineralization (GNM) were investigated using three paddy soils (C3) under maize (C4 plant) cultivation. The soils had high and low total N (1.79 and 3.3 g kg−1 soil) as well as iron- (Fe-) (hydr-) oxide (1.1 and 2.2 g kg−1 soil) contents, which gave the following combinations: high-Fe/low-N, low-Fe/low-N, and low-Fe/high-N. The RPEs and GNM were measured using a 13C-natural abundance approach and 15N-pool dilution technique, respectively, on day 86 of maize cultivation. Living roots enhanced native SOC mineralization by 70.4–204% and GNM by 118–382%. As expected, biotic mechanisms contributed to RPEs (‘microbial activation’ and ‘microbial N-mining’) that was supported by the increase of soil microbial biomass C and extracellular enzyme activities in presence of plant, and the lower C/N ratio of primed SOM than the original SOM in unplanted soils. Higher RPEs and relative primed N mineralization via stronger ‘microbial N-mining’ were found in low-Fe/low-N vs low-Fe/high-N soils. In contrast to expectations, the RPEs and relative primed N mineralization were greater in high-Fe/low-N versus low-Fe oxide soils. The strongest decrease in SOM-derived Fe-bound C and increase in root-derived Fe-bound C were observed in the high-Fe soil, because root exudates liberated more C from Fe-bound SOM and co-precipitated with Fe oxides. This shows that the abiotic process was involved in RPEs whereby root exudates promoted soil C loss by releasing it from Fe-organic complexes. Thus, this study demonstrated that coupled biotic-abiotic processes could regulate RPEs.

Pluronic® F127-mediated control of insulin release rates from NPH microcrystals and blood glucose depression in STZ-induced diabetic rats.

Introduction: Neutral protamine Hagedorn (NPH) insulin is an intermediate-acting basal insulin with a long history of clinical use, consisting native human insulin. Its rather undesirable action profile, characterized by a peak release within a few hours, followed by insufficient insulin delivery upon a single subcutaneous (s.c.) dose, is well-documented. This may have been caused by the inherent microcrystal structure involving the basic peptide protamine, as well as the presence of tissue enzyme activities that readily act on protamine at the injection site. This issue may be circumvented by utilizing thermosensitive, erodible Pluronic F127 (PF127) to modulate the kinetics of insulin release from NPH over a period of 24 hours in which the hydrogel is completely eroded. Methods: Previously, we have shown that insulin release rates in vitro from NPH/PF127 formulations (0-25% PF127) markedly reduced the initial insulin release, especially in the presence of enzyme activity that selectively degraded protamine at 1-5 U/mL. Insulin release over the course of 20 hours was better modulated in the presence of increasing PF127 content. In this study, the insulin formulations (0, 20, and 25% PF127) were administered s.c. (4 U/kg) to streptozotocin (STZ)-induced diabetic rats and blood glucose levels were monitored over 24 hours. Results: In vivo blood glucose depression profiles in STZ-induced diabetic rats exhibited a similar pattern of control to in vitro data at the single s.c. dose of 4 U/kg, apparently extending the duration of action of NPH over a 24-hour period in the presence of PF127. Conclusion: Our findings suggest that the undesirable kinetics of insulin release from NPH is significantly influenced by tissue enzyme activity and that the presence of PF127 provided a timely modulation of insulin release from NPH microcrystals in the STZ-induced diabetic rat model.

Modification in durability of mango wood through reactive reinforcement of polyacrylonitrile

Reactive reinforcement of polyacrylonitrile (PAN) into mango wood (MW) has afforded a series of wood polymer composites (WPCs) with improved mechanical properties, resistance against solvents, chemicals and biodegradation. The process of reactive reinforcement of PAN into MW was conducted through treatment of leached MW panels with a composition of acrylonitrile (AN, 20–60 v/v) and 2,2-azobisisobutyronitrile (1 wt%) in dimethyl formamide over 12 h under ambient conditions followed by curing at 95 ± 1 °C over additional 6 h. This has afforded a series of WPCs with PAN loading in the range of 5.5 to 15.0. Scanning electron microscopy in combination with wood content data reveals the loading (wt%) of PAN into the matrix of MW. PAN loading of 8.85 wt% was found optimum towards enhancing the compressive, impact and static bending strength of WPCs. PAN loading has raised the resistance of WPCs in organic solvents, hot water and aqueous NaOH. WPC with PAN loading of 15 wt% has shown enhanced resistance against biodegradation in presence of a decay fungus Coriolous versicolor. This has been revealed through the controlled release of protein, reducing sugar contents and enzyme activities in presence of a decay fungus under incubation over 168 hr. The present investigation reveals a viable method of reinforcement of PAN into MW to achieve the WPCs with improved strength, stability in degrading chemicals, controlled biodegragation.

Expression of Cell Wall-Modifying Enzymes in Aspen for Improved Lignocellulose Processing.

Wood is an important source of biomass for materials and chemicals, and a target for genetic engineering of its properties for different applications or for research. Wood properties can be altered by using different enzymes acting on cell wall polymers postsynthetically in cell walls. This approach allows for a precise polymer structure modification thanks to the specificity of enzymes used. Such enzymes can originate from all kinds of organisms, or even be modified in a desired way for novel attributes. Here we present a general strategy for expressing a microbial enzyme in aspen and targeting it to cell wall, using an example of fungal glucuronoyl esterase. We describe methods of vector cloning, plant transformation, transgenic line selection and multiplication, testing for the presence of enzymatic activity in different cell compartments, and finally the method of plant transferring from sterile culture to the greenhouse conditions.

Role of carbohydrate binding module (CBM3c) of GH9 β-1,4 endoglucanase (Cel9W) from Hungateiclostridium thermocellum ATCC 27405 in catalysis

The function of CBM3c in the enzymatic catalysis varies among the members of family 9 Glycoside Hydrolases (GH). A new member of family 9 GH (Cel9W) from thermophilic anaerobic bacterium, Hungateiclostridium thermocellum was explored for elucidation of the role of CBM3c in catalysis by GH9. Cel9W is a multimodular theme B1, cellulase enzyme comprising a catalytic module of family 9 glycoside hydrolase (HtGH9t) at N-terminal, a family 3c carbohydrate binding module (HtCBM3c) and a dockerin domain at the C-terminal. The ORF of Cel9W encoding full length β-1,4-glucanase, HtGH9 (containing both GH9 and CBM3c modules), the truncated GH9 catalytic module (HtGH9t) and module CBM3c (HtCBM3c) were cloned and over-expressed using E. coli BL21 cells. HtGH9 showed maximum activity at pH 6.5 and 90 °C. It displayed highest activity of 64 U/mg against lichenan followed by 44.6 U/mg (β-glucan) and 22.3 U/mg (Carboxymethyl cellulose). HtGH9 showed stability in the pH ranging from 5.0 to 9.0 and thermal stability up to 70 °C for 1.0 h. The presence of EDTA and EGTA decreased the activity of HtGH9 and also shifted the melting curve peak from 93 °C to 88 °C indicating that the enzyme inherently possesses metal ions, which play role in catalysis and structural stability. The TLC analysis of HtGH9 hydrolysed Avicel showed the presence cellotetraose indicating the processive endoglucanase activity in HtGH9. The module, HtGH9t alone exhibited very low level of activity (1.22 U/mg) against lichenan. It partially recovered (51%) the enzyme activity in presence of equimolar concentration of HtCBM3c. Non-denaturing gel electrophoresis revealed a non-covalent binding interaction between the catalytic HtGH9t module with HtCBM3c module and their physical association showed the partial recovery of its endoglucanase activity. This study confirmed that the physical association of the catalytic module HtGH9t with HtCBM3c is necessary for HtGH9 to efficiently hydrolyze the cellulosic substrates.

Redox potential changes during ATP-dependent corrinoid reduction determined by redox titrations with europium(II)-DTPA.

Corrinoids are essential cofactors of enzymes involved in the C1 metabolism of anaerobes. The active, super‐reduced [CoI] state of the corrinoid cofactor is highly sensitive to autoxidation. In O‐demethylases, the oxidation to inactive [CoII] is reversed by an ATP‐dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)–diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [CoII]/[CoI] couple of the protein‐bound corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the corrinoid as the electron‐accepting site is achieved by increasing the potential of the corrinoid cofactor from −530 ± 15 mV to −250 ± 10 mV (E SHE, pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide‐bound AE with the corrinoid protein or its cofactor. The remaining 150–200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)–DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low‐potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.

Active site gate of M32 carboxypeptidases illuminated by crystal structure and molecular dynamics simulations

Enzyme gates are important dynamic features that regulate function. Study of these features is critical for understanding of enzyme mechanism. In this study, the active-site gate of M32 carboxypeptidases (M32CP) is illuminated. Only a handful of members of this family have been structurally and functionally characterized and various aspects of their activity and mechanism are yet not clarified. Here, crystal structure of putative M32CP from Deinococcus radiodurans (M32dr) was solved to 2.4Å resolution. Enzymatic assays confirmed its identity as a carboxypeptidase. Open and relatively closed conformations observed in the structure provided supporting evidence for previously hypothesized hinge motion in this family of enzymes. Molecular dynamics simulations of 1.5μs displayed distinct open and closed conformations revealing amplitude of the motion to be beyond what was observed in the crystal structure. Hinge region and anchoring region of this shell-type gate were identified. A small displacement of 3Å and a helical tilt of 9° propagated by the hinge region translates into a 10Å motion at the top of the gate. The dynamics of the gate was supported by our mutagenesis experiment involving formation of disulphide bond across helices of the gate. The nearly inactive mutant enzyme showed 65-fold increase in the enzymatic activity in presence of reducing agent. Further, while a previously proposed structural basis would have led to its classification in subfamily II, experimentally observed substrate length restriction places M32dr in subfamily I of M32CPs.

Ultrasound-assisted improvements in biocatalytic activity and production of organic-solvent stable protease from Bacillus circulans MTCC 7942

This study reports the ultrasound-assisted improvements in biocatalytic activity and production of protease from organic solvent-tolerant, alkaliphilic Bacillus circulans MTCC 7942. The strain withstands 10min ultrasound treatment (33kHz); this treatment promotes the growth of bacterium. The protease production was improved by 1.5-fold on multiple ultrasound treatment at 0, 6, 12h intervals. Although the substrate casein remains unaffected by ultrasound treatment (33kHz, 10min); the treatment had boosted the enzyme activity of purified protease. Also, the ultrasound treated purified protease showed enhancement in enzyme activity in presence of n-dodecane, n-octane, n-hexadecane, n-decane, xylene, butanol, toluene, pyridine and chloroform.

Controlled enzymatic stability and release characteristics of supramolecular chiral peptide amphiphile nanofiber gels

Supramolecular bioarchitectures formed by assembly of achiral or chiral building blocks play important roles in various biochemical processes. Stereochemistry of amino acids is important for structural organization of peptide and protein assemblies and structure-microenvironment interactions. In this study, oppositely charged peptide amphiphile (PA) molecules with L-, D- and mixture of l- and d-amino acid conformations are coassembled into supramolecular nanofibers and formed self-supporting gels at pH 7.4 in water. The enzymatic stability of the PA nanofiber gels was studied in the presence of proteinase K enzyme, which digest a broad spectrum of proteins and peptides. The structural changes on the chiral PA nanofibers were also analyzed at different time periods in the presence of enzymatic activity. Controlled release of a model cargo molecule through the chiral PA nanofiber gels was monitored. The diffusivity parameters were measured for all gel systems. Release characteristics and the enzymatic stability of the peptide nanofiber gels were modulated depending on organization of the chiral PA molecules within the supramolecular assemblies.

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg−1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0–14.0 pH and 30–80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

Curious Cases of the Enzymes.

SummaryLife as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts… Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This paper is dedicated to these early moments of this remarkable science that touched our lives in the past and will make life a lot more efficient for humanity in the future. There was almost always a delicate, fundamentally essential relationship between mankind and the enzymes. Challenged by a very alien and hostile Nature full of predators, prehistoric men soon discovered the medicinal properties of the plants, through trial and error. In fact, they accidently discovered the enzyme inhibitors and thus, in crude terms, kindled a sparkling area of research. These plant-derivatives that acted as enzyme inhibitors helped prehistoric men in their pursuit of survival and protection from predators; in hunting and fishing… Later in history, while the underlying purposes of survival and increasing the quality of life stayed intact, the ways and means of enzymology experienced a massive transformation, as the ‘trial and error’ methodology of the ancients is now replaced with rational scientific theories.