Presence Of Enzymes Research Articles

Deciphering the structure of a distinctive trimodular cellulosomal licheninase (RfGH16_21), a family 16 glycoside hydrolase from Ruminococcus flavefaciens by computational and experimental methods

In order to know the insights of a unique naturally existing trimodular licheninase from GH16 family, sub-family 21 (RfGH16_21) from Ruminococcus flavefaciens, its structure was modeled to understand its functional relations to reveal information regarding modifying the enzyme for improved properties with enhanced catalytic efficiency. Homology modeling revealed three tandem repeats of β-jelly roll like folds linked by natural linkers. Catalytic pockets and the catalytically important amino acids in each tandem repeat of RfGH16_21 determined by multiple sequence alignment and structure superposition with its homologues indicated that two Glu residues are involved in a retaining-type of catalytic mechanism. Sequential molecular docking revealed maximum binding energy with mixed linked cellotriose showing that cellotriose is the lowest oligomeric hydrolysed product formed by the catalytic action of endo-β-1,3–1,4-glucanase. Molecular dynamic (MD) simulation of RfGH16_21-cellotriose complex confirmed the structural specificity of catalytic residues and increased stability of enzyme in presence of ligand as compared to simulated RfGH16_21 alone. The binding affinity of cellotriose towards the three tandem repeats of RfGH16_21 was also confirmed by calculating total binding Gibbs free energy, i.e. −100.8 ± 2.6 KJ/mol, by using g_mmpbsa tool. The stability of the protein was determined by protein melting analysis that showed Ca2+ and Mg2+ ions imparted structural stability to RfGH16_21. Dynamic light scattering analysis of RfGH16_21 showed monodispersity and hydrodynamic radius of 4.0 nm at 2.0 mg/mL protein concentration, which was comparable with the radius of gyration of 3.2 nm determined by MD simulation showing the protein to be in monomeric form. Communicated by Ramaswamy H. Sarma

Purification and characterization of lipase enzyme from endophytic Bacillus pumilus WSS5 for application in detergent industry

Lipolytic enzymes from endophytic microbes have been the focus of intense and growing research in view of the applicability of such enzymes in various industrial applications and endophytic microorganisms may emerge as a potential source of lipases with distinct characteristics. The current work involved purification and characterization of the lipase from an endophytic-bacteria isolated from the stem of Withania somnifera L. Dunal. Based on the analysis of morphology and 16S rDNA sequence, the endophytic bacteria was identified as Bacillus pumilus and designated as B. pumilus WSS5. Purified lipase from the B. pumilus WSS5 was found to be of 27KDa as revealed by SDS-PAGE with optimum activity at 37 °C and pH 8. The stability of the enzyme in presence of various metal ions, surfactants, inhibitors, and organic solvents suggested that this enzyme could be exploited in leather and detergent industries. In addition, the stain removal potential of the crude and purified lipase was determined on cotton fabric pieces stained with vegetable oil. Lipase added to water along with detergent significantly enhanced the stain removal efficiency of a commercially available detergent. The current findings suggest the potential of B. pumilusWSS5 lipase owing to promising properties such as the alkali-thermostability, organic solvent-tolerance, and broad substrate specificity that makes it a strong candidate for application in detergent formulations, biotransformation, industries, and medicine. To our knowledge, this is the first report on the purification and characterization of lipase from bacterial endophyte isolated from the stem of Withania somnifera L. Dunal.

An in-silico investigation of fluoride ions impact on pancreatic lipase.

Fluorine is a halogen beneficial to teeth and bones at a lower concentration. But in excess, it is a toxin and causes adverse effects. Fluoride is toxic to enzymes generally when it inhibits the enzyme activity involved in metabolic pathways. Here we study invitro and invivo findings on the interaction of fluoride on the enzymes Aconitase, Adenylyl cyclase, Arginase, Cytochrome-c-oxidase, Glucose-6-phosphatase, Protein phosphatase, Succinate dehydrogenase from liver and lipase from pancreas by using molecular docking and simulations to gain insights into the mechanism by which fluoride modifies the activity of pancreatic lipase. our molecular modeling and docking studies identified that lipase is the most strongly inhibited enzyme compared to other enzymes mentioned above with -0.42 Kcal/mol binding energy and 495.78 milli molar of predicted IC50 value with interaction with Phe227 residue. To further validate this, we have taken the lipase enzyme in presence of fluoride ions for molecular dynamic simulations of 100 ns. To analyze the impact of fluoride ions on the lipase dynamics, two different simulations of 100 ns each were performed. In one simulation, we have simulated lipase in its apo form in the aqueous environment without any fluoride ions and in another simulation lipase in its apo form was kept in the presence of randomly placed fluoride ions countered with sodium ions to maintain the pH as neutral. The simulation analysis revealed that major fluctuations in lipase was observed between 230 and 300 residues in presence of fluoride ions. Interestingly, this is the exact location of the "lid" like acting loop of residues responsible for the inward/outward movement of the substrate to lipase catalytically active site containing catalytic triad of residues Leu153, His263, and Pro177. His263 residue random flip is believed to be the critical incident that causes the substrate's inward/outward movement at the catalytically active site coordinated by "lid" opening, providing enough space for the substrate.

Enzymatic Biofuel Cells Embedded Polymer-Based Soft Actuators

Enzymatic biofuel cells are presented as an untethered alternative energy source that could power small implantable or wearable medical devices. However, most of these catalytic processes do not provide with enough energy to power common small electronic-mechanical devices. On the other hand, conducting polymer-based actuators are of great interest for their biocompatibility, flexibility, processability, possibility to be miniaturized and low power consumption. So far, these artificial muscles have been driven by external power sources that prevent them for being completely autonomous. There is a need for a novel power source to elaborate actuators that could use physiological processes as a driving force. These soft actuators’ low power consumption matches the electrical power generated by the biocatalysis of some enzymes, such as glucose oxidase and laccase in presence of glucose and oxygen in aqueous media. Here, we present the latest results in the development of polypyrrole-based soft actuators powered by enzymatic biofuel cells. The actuator consists of a tri-layer conductive substrate on which the polypyrrole is electrodeposited in both sides. The polypyrrole layers act as the active part, expanding and contracting upon a redox reaction, resulting in a bending movement. Tetrathiofulvlene-7,7,8,8-tetracyanoquinodimethane (TTF-TCNQ) and 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) electron transfer mediators are cast on the surface of the polypyrrole to help the electron transmission. The glucose oxidase and laccase enzymes are immobilized in the modified-conducting polymer surface, integrating the electrode to the actuator. The bio-catalysis of enzymes in presence of glucose and oxygen in aqueous solution provides the actuator with the electrons needed for the redox reaction, converting the chemical energy into mechanical energy, i.e., movement. The glucose-self-powered soft actuator may contribute to the development of more complex implantable, ingestible, or wearable biomedical devices such as cardio-stimulators, insulin pumps, or muscle implants.

A unique aromatic cluster near the active site of H.pylori CPA is essential for catalytic function

Polyamines are essential for cell growth and proliferation. In plants and many bacteria, including Helicobacter pylori, the parent polyamine putrescine is only produced through the metabolism of N-carbamoylputrescine by N-carbamoylputrescine amidase (CPA). Thus, CPA is a crucial intermediate enzyme. Moreover, the absence of CPA in humans makes its presence in H. pylori a potential target for the development of new therapeutics against this pathogen. Despite this enzyme's presence in plants and bacteria, its function is not completely explored. Using structure-guided biochemical and biophysical studies on H. pylori CPA, we discovered an aromatic cluster containing four conserved tryptophans near the catalytic site and elucidated its role. Mutational studies revealed that they are individually vital to enzyme function. Unlike wild-type, which forms a hexamer, the Trp to Ala mutants only formed dimers. Interestingly, two other conserved residues, Gln155 and Asp278, interact with the tryptophan cluster and perform similar roles. Our results indicate that aromatic-aromatic and H-bonding contacts between the residues (Trp156-Trp273, Trp196-Gln155, and Trp153-Asp278) play a crucial role in stimulating activity through hexamer formation. Additionally, Trp156 is essential to generating a catalytically efficient hexamer. These results suggest dual roles for the tryptophans; in hexamer formation and in generating its functionally active form, thereby providing a mechanistic understanding into the role of the cluster. We also elucidated the catalytic roles of Glu43, Lys115, and Cys152, which are present at the active site. Our findings highlight, for the first time, the importance of a tryptophan cluster in H. pylori CPA that can be exploited to design therapeutic inhibitors.

Aspartic protease-pepstatin A interactions: Structural insights on the thermal inactivation mechanism

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol−1, and from 179 to 359 kcal mol−1 K−1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5 M−1 and ΔGo value was −8.3 kcal mol−1. Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases.

Enzymatically Triggered Jack-in-the-Box-like Hydrogels.

Enzymes can support the synthesis or degradation of biomacromolecules in natural processes. Here, we demonstrate that enzymes can induce a macroscopic-directed movement of microstructured hydrogels following a mechanism that we call a "Jack-in-the-box" effect. The material's design is based on the formation of internal stresses induced by a deformation load on an architectured microscale, which are kinetically frozen by the generation of polyester locking domains, similar to a Jack-in-the-box toy (i.e., a compressed spring stabilized by a closed box lid). To induce the controlled macroscopic movement, the locking domains are equipped with enzyme-specific cleavable bonds (i.e., a box with a lock and key system). As a result of enzymatic reaction, a transformed shape is achieved by the release of internal stresses. There is an increase in entropy in combination with a swelling-supported stretching of polymer chains within the microarchitectured hydrogel (i.e., the encased clown pops-up with a pre-stressed movement when the box is unlocked). This utilization of an enzyme as a physiological stimulus may offer new approaches to create interactive and enzyme-specific materials for different applications such as an optical indicator of the enzyme's presence or actuators and sensors in biotechnology and in fermentation processes.

Biodegradation of textile azo dyes by textile effluent non-adapted and adapted Aeromonas hydrophila

Textile effluent discharged in water and soil have severe effects on living beings as well as environs worldwide. Pioneering, ecologically sustainable and economically viable treatment systems are urgently desired. This study investigated decolorization along with degradation of three azo dyes by non-adapted Aeromonas hydrophila MTCC 1739 and textile-effluent adapted Aeromonas hydrophila SK16 bacteria under optimized physicochemical conditions. UV–visible analysis (for decolorization percentage calculated using absorbance), FTIR (fourier transform infrared spectroscopy for functional group identification), HPLC (high performance liquid chromatography for degraded metabolites analysis based on retention time) and GC-MS (gas chromatography-mass spectrometry for proposing degradative pathways based on mass/charge as well as molecular weight) supported efficient biodegradation of these dyes into simpler metabolites by both cultures. Time taken for decolorization by the adapted strain was less but both were equally efficient in mineralizing dyes, utilizing them as energy source. Enzyme assays revealed over-expression of dye degrading enzymes in presence of toxic azo dyes. Thus, it is suggested that both adapted and non-adapted bacteria act as azo dye degraders with potential use in efficient and lucrative effluent treatment curbing expansive preliminary screening of autochthonous strains for azo dye degradation.

DMSO strengthens chitin deacetylase-chitin interaction: Physicochemical, kinetic, structural and catalytic insights

Chitin deacetylase, an enzyme isolated from Cryptococcus laurentii RY1, catalyzes the hydrolysis of acetamido group of N-acetyl-D-glucosamine unit of chitin. The primary objective of this study was to characterize and comprehend the activation of chitin deacetylase by DMSO. The secondary structure of the protein was determined by circular dichroism(CD).The interaction of protein with DMSO was evaluated by CD and tryptophan fluorescence spectroscopy which revealed that DMSO had no effect on overall secondary structure, but induced change in the tertiary structure of the enzyme. The interaction of chitin deacetylase with chitin in DMSO system when investigated by molecular dynamics simulation revealed stronger chitin deacetylase-chitin interaction involving several amino acid residues. The enhanced activity of the enzyme in presence of DMSO along with the fact that its kcat is highest of all other reported chitin deacetylases makes it a superior candidate in the industrial sector involved in chitosan production from chitin.

Effect of metal ions present in milk on the structure and functional integrity of native and polyaniline chitosan nanocomposites bound β-galactosidase: A multi-spectroscopic approach

β-Galactosidase is vital to dairy industries because it catalyzes the hydrolysis of lactose into glucose and galactose making it useful for lactose intolerant patients to consume milk and its products. The present study demonstrates the effect of metal ions commonly found in milk, namely Zn2+, K+, Ca2+ and Mn2+ on the activity of native and polyaniline chitosan nanocomposites bound Aspergillus oryzae β-galactosidase. In polyaniline chitosan silver nanocomposite adsorbed β-galactosidase, a multi-fold enhancement in catalytic activity was observed in the presence of cocktail of Zn2+, K+, Ca2+ and Mn2+ ions. 3D fluorescence, CD and FTIR studies established significant conformational changes in the secondary structure of polyaniline chitosan silver nanocomposite bound β-galactosidase on addition of metal ions as compared to the native and other bound enzyme. This enhanced catalytic efficiency of the immobilized enzyme in presence of metal ions can promote its economic use for lactose hydrolysis in milk.

Allosteric Site Inhibitor Disrupting Auto-Processing of Malarial Cysteine Proteases

Falcipains are major haemoglobinases of Plasmodium falciparum required for parasite growth and development. They consist of pro- and mature domains that interact via ‘hot-spot’ interactions and maintain the structural integrity of enzyme in zymogen state. Upon sensing the acidic environment, these interactions dissociate and active enzyme is released. For inhibiting falcipains, several active site inhibitors exist, however, compounds that target via allosteric mechanism remains uncharacterized. Therefore, we designed and synthesized six azapeptide compounds, among which, NA-01 & NA-03 arrested parasite growth by specifically blocking the auto-processing of falcipains. Inhibitors showed high affinity for enzymes in presence of the prodomain without affecting the secondary structure. Binding of NA-03 at the interface induced rigidity in the prodomain preventing structural reorganization. We further reported a histidine-dependent activation of falcipain. Collectively, for the first time we provide a framework for blocking the allosteric site of crucial haemoglobinases of the human malaria parasite. Targeting the allosteric site could provide high selectivity and less vulnerable to drug resistance.

Production, Purification and Characterization of Polygalacturonase from Bacillus licheniformis SHG10

Background: Microorganisms are the best source of pectinolytic enzymes as they allow an economical technology with low resource consumption. Objectives: The present study was carried out to isolate, purify and characterize polygalacturonase (PGase) form Bacillus licheniformis SHG10. Methods: The concentrated dialysed cell free extract was loaded on prepacked DEAE-Sepharose Fast-Flow ion exchange chromatography. The active PGase fractions were concentrated and loaded again on sephacryl Fast-Flow High Resolution (FF S-100 HR) chromatography. Molecular weight was determined using slab-gel SDS-Polyacrylamid gel electrophoresis and Characterization of the purified enzyme was performed. Results: The results showed that the enzyme was purified with a purification fold (33.34) and yield (41.73%) with specific activity (8.67 μ moles/min/mg protein). It has a molecular mass of about 68.0 kDa. While, on SDS-PAGE electrophoresis, the purified PGase appeared as single band with molecular mass of about 34.0 kDa, suggesting that the purified PGase is a dimer protein. The purified enzyme exhibited maximal activity at a temperature of 45oC and pH 8.5. The maximum velocity of the enzyme in presence of citrus pectin and pectate as substrates were 10.98 and 14.7 μ mol galacturonate/min/mg protein, respectively. The Michaelis constant (Km) values were 0.085 and 0.039 mM, respectively, indicating that the purified PGase has higher affinity to pectate (non-methylated pectic substance) than citrus pectin as substrate.

Flexible Electrochemical Biochip Array of Patterned Gold on Silver Inkjet Printed Polyimide

Flexible electrodes for bio-electrochemical sensing were fabricated using state-of-the-art inkjet printing followed by two consecutive gold plating steps. Silver nanoparticle printed thin film (1.5μm thick) was sintered at 300°C for 30 minutes and used as seed for the electroless plating of gold (2μm). A further gold electroplating layer was then applied allowing good electrode properties overcoming the printed film roughness and porous morphology which the electroless plating step could not fully cover. The various layers as well as their composite were investigated using electrical, electrochemical and analytical techniques. Eventually, the final electrode was tested with a 3-electrodes setup in a chronoamperometric experiment to detect the Alkaline Phosphatase enzyme in presence of its substrate, ρ-Aminophenyl Phosphate. The electrodes demonstrated a sensitivity of 11.8μA/mM and a limit of detection of 0.9mM.

Molecular cloning, characterization and function of a germinal center kinase MST4 gene from Litopenaeus vannamei in response to Vibrio alginolyticus challenge in TLR-TRAF6 signaling pathway

The serine/threonine protein kinase MST4 plays multiple roles in the regulation of signaling pathways that govern cellular processes including mitosis, migration, homeostasis, polarity, proliferation, differentiation and apoptosis. Here we report the identification and characterization of the full-length sequence of LvMST4 from the shrimp L. vannamei, and investigations into its role in the shrimp's immune response to infection by the pathogenic bacterium Vibrio alginolyticus. Subcellular localization assays demonstrated the enzyme's presence in the shrimp's cytoplasm, and tissue-specific expression analysis revealed that it is expressed ubiquitously but at different levels in different tissues. Infection with V. alginolyticus increased LvMST4 expression and induced a rapid response via the TLR-TRAF6 signaling pathway, causing a decline in the total hemocyte count (THC) and an increase in respiratory burst (RB) activity. In non-infected shrimp, RNAi silencing of LvMST4 with dsRNA had no significant effect on THC but seemed to activate the TRAF6-MKK6-p38 pathway and reduced RB activity. In shrimp challenged with V. alginolyticus, LvMST4 silencing reduced bacterial clearance and increased the initial upregulation of LvTRAF6 while reducing the expression of LvMKK6 and Lvp38. LvMST4 silencing also slightly reduced the THC but caused pronounced increases in RB activity and cumulative mortality. These findings suggest that LvMST4 contributes to antimicrobial responses via the TLR-TRAF6 signal pathway, and helps maintain immunological homeostasis in L. vannamei.

Stabilization of the lipase of Hypocrea pseudokoningii by multipoint covalent immobilization after chemical modification and application of the biocatalyst in oil hydrolysis

Lipase from Hypocrea pseudokoningii was purified using the support Octyl–Sepharose. This adsorption resulted in a 3-fold increase in activity of the immobilized enzyme. Following, still on this support, the lipase was enriched in surface amino groups (by reaction of carboxy groups with ethylendiamine). After amination, the lipase was desorbed from Octyl–Sepharose, while the 2-fold hyper-activation was maintained. The aminated lipase was also successfully immobilized on Glyoxyl–Agarose. The derivative was 45-fold more stable than was the free enzyme at 50 and 60°C. The derivative was also stable in 50% of organic solvents such as methanol, ethanol, propanol and cyclohexane. The multipoint immobilization also increased the enzyme stability in relation to the free enzyme in the presence of ethanol, methanol and cyclohexane for up to 72h. For example, the stabilized derivative was 9-fold more stable than the free enzyme in presence of methanol. The derivatives hydrolyzed fish, cupuaçu (Theobroma grandiflorum), bacuri (Latonia insignis) and murumuru (Astrocaryum murumuru) oils. The multipoint immobilization process increased the hydrolysis of oils up to 15-fold compared with the control, what makes these derivatives attractive for industrial application.

Effect of two series ionic liquids based on non-nutritive sweeteners on catalytic activity and stability of the industrially important lipases from Candida rugosa and Rhizopus delemar

This is the first study on the effect of ionic liquids (ILs) based on non-nutritive sweeteners on the catalytic efficiency and the structural and the thermal stability of lipases from Candida rugosa (CRL) and Rhizopus delemar (RhDL). Initially, we synthesized two series of ILs, namely 1-akyl-3-methylimidazolium saccharinates {[CnC1im][Sac]} and 1-akyl-3-methylimidazolium acesulfamates {[CnC1im][Ace]}, where the alkyl substituent was butyl, hexyl, octyl or decyl. The activities of the two enzymes were tested in two hydrolytic reactions using walnut oil and 4-nitrophenyl acetate as substrates. The activity of CRL toward walnut oil was strongly influenced by the chain-length of the N-alkyl substituents at the imidazolium cation of the compounds. The ILs with short alkyl substituents (butyl or hexyl) activated moderately (up to 30%) or had no effect on CRL activity, while those with medium chain-length substituents strongly inhibited CRL. In the case of CRL, we observed the same behavior pattern for the two tested series of ILs, while for RhDL the same tendency was displayed only for the series of saccharinates. The coating of RhDL with 0.1M [C8C1im][Sac] or [C8C1im][Ace] resulted in up to 1.5-fold increase in the enzyme initial activity. In contrast, CRL was strongly inhibited by these two ILs. We found that the effect of [C8C1im][Ace] on enzyme activity adopted concentration- and time-dependent manner for the two lipases.We observed a moderate increase in hydrolytic activity (by 1.2-fold factor) and good storage stability of RhDL (more than 30 days at 25°C) when small quantities of all ionic liquids (<0.04M), were added to the enzyme solutions. At the same time, by adding of 0.02M [C4C1im][Sac] or [C4C1im][Ace], CRL was also stabilized and its activity was preserved for more than 25 days at room temperature. For comparison, the activities of the stored at 25°C non-treated with ILs soluble CRL and RhDL were reduced by half for 7 days and 5 days, respectively. In contrast, we observed rapid inactivation at higher temperatures for all ionic liquid-coated enzymes. For example, half-lives of 25min and 57.3min at 50°C were estimated for the pre-treated with [C4C1im][Ace]-CRL and for the non-treated-CRL, respectively. In addition, applying Fourier transform infrared spectroscopy, we monitored the changes in the secondary structure of the two enzymes in presence of the tested ILs which explained the altered stability and the activities of the two enzymes. Interestingly, coating with small amounts of ILs prevents aggregation of the two lipases even at high protein concentration.

Influence of exogenous enzymes in presence ofSalix babylonicaextract on digestibility, microbial protein synthesis and performance of lambs fed maize silage

SUMMARYSixteen Suffolk lambs with 29 ± 2·0 kg body weight were housed in individual cages for 60 days and allotted to four treatments in a completely randomized design to determine the effect of administration ofSalix babylonica(SB) extract and/or exogenous enzymes (ZADO®) on lamb performance. Lambs were fed with 300 g/kg concentrate (160 g crude protein (CP)/kg, 13·4 MJ metabolizable energy (ME)/kg dry matter (DM)) and 700 g/kg maize silage (80 g/kg CP, 11·7 MJ ME/kg DM) as a basal diet (control). Another three treatments were tested; the SB extract was administered at 30 ml/day (SB) and exogenous enzymes ZADO®(i.e. an exogenous enzyme cocktail in a powder form) directly fed at 10 g/day (EZ), while the last treatment contained ZADO®at 10 g/day + SB extract at 30 ml/day (EZSB). Lambs of the treatment EZSB had the greatest average daily weight gain (ADG) and feed conversion throughout the period of the experiment. However, during the first 30 days SB was more effective for ADG than EZ andvice versaduring the last 30 days of the experiment. Water consumption was greater for SB, followed by EZ and EZSB compared to the control. Intakes of DM and organic matter (OM) were the highest in EZSB followed by EZ, which had the greatest neutral detergent fibre, acid detergent fibre (ADF) and nitrogen (N) intakes. The EZSB treatment had the greatest DM and OM digestibilities compared to the other treatments; however, SB had the greatest ADF digestibility. Combination of EZ and SB had the best N balance. Allantoin, total purine derivatives (PD), allantoin : -creatinine ratio, and PD : creatinine ratio were increased in EZSB compared to the other treatments. However, EZ supplementation increased uric acid concentration, whereas the microbial N (g N/day) and metabolizable protein (g N/day) were increased in EZSBversusthe other treatments. It can be concluded that addition of 10 g ZADO®in combination withS. babylonicaextract at 30 ml/day in the diet of lambs increased feed intake, nutrient digestibility and daily gain, with a positive impact on the use of N and microbial protein synthesis.

Identification of active site residues of Fenugreek β-amylase: Chemical modification and in silico approach

The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.

Effect of pKa on the kinetics of carbon dioxide absorption in aqueous alkanolamine solutions containing carbonic anhydrase at 298K

Abstract The absorption of carbon dioxide in various aqueous alkanolamine solutions have been studied with and without carbonic anhydrase respectively in a stirred cell reactor at 298 K. The examined alkanolamines were: N,N-diethylethanolamine (DEMEA), N,N-dimethylethanolamine (DMMEA), monoethanolamine (MEA), triethanolamine (TEA) and tri-isopropanolamine (TIPA). This work confirms that the CO2 hydration is catalysed by the enzyme in presence of alkanolamines. The differences in reaction rate between the tested alkanolamines are attributed to the enzyme regeneration step in the mechanism – that is, an acid base reaction. A Langmuir–Hinshelwood-like equation has been postulated to describe the observed overall rate constant of the enzymatic reaction as a function of the enzyme concentration. The two kinetic constants in the postulated equation both depend exponentially on the pKa value of the alkanolamine present in the solution.

Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition

Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40–50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G.stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18h of incubation at 65°C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability.