Experimental and in silico studies of competitive inhibition of family GH10 Aspergillus fumigatus xylanase A by Oryza sativa xylanase inhibitor protein.

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn2+ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (Ki) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was -30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239OsXIP stably interacted with the catalytic site E140AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104AfXylA10/V99AfXylA10-Q5OsXIP, R256AfXylA10-E235OsXIP, D155AfXylA10-Y243OsXIP and D145AfXylA10-R194OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP.

Comparative study of covalent and hydrophobic interactions for α-amylase immobilization on cellulose derivatives.

Indispensability of enzymes in living systems, their unique characteristics and simultaneous focus on development of greener methods have led to substitution of various chemical reactions by enzyme catalyzed reactions. One of the aspects in enzyme research is immobilization of enzymes. Immobilization provides a platform for reusability of significant enzymes. Varieties of methods have been explored for enzyme immobilization such as entrapment, adsorption, ionic interactions etc. Keeping in view the industrial utility of α-Amylase in leather, paper and other industries related to starch hydrolysis, we immobilized α-Amylase on cellulose isolated from banana peel. In present study, two different methods of immobilization - covalent bonding (Cellulose Dialdehyde as a support) and hydrophobic interactions (Nano Cellulose- Cetyl Trimethyl Ammonium Bromide) were used. Cellulose obtained from bio-waste has been characterized using Fourier transform Infrared Spectroscopy (FT-IR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD). In this comparative study, Cellulose Dialdehyde (CDA) immobilized enzyme depicts high reusability, good enzyme loading, storage capacity up to 49 days, optimum pH 6, optimum temperature 95 °C, good pH and thermal stability as compared to native enzyme having optimum pH and temperature of 7 and 37 °C. On the contrary, nanocellulose - Cetyl Trimethyl Ammonium Bromide (NC-CTAB) matrix shows good enzyme loading and optimum pH shift of about 3 units but poor recyclability. Outcome of this study presents the promising nature of covalent mode of immobilization for industrial use.

Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5.

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.

The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop.

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold-adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B-factors of the R336L crystal structure to that of the wild-type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibility resulted in a reduced catalytic rate. The large loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl group affinity (inactive).

Enzyme Stability in Nanoparticle Preparations Part 1: Bovine Serum Albumin Improves Enzyme Function.

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments.

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25.

Exo-inulinases are members of the glycoside hydrolase family 32 and function by hydrolyzing inulin into fructose with yields up to 90-95%. The N-terminal tail contributes to enzyme thermotolerance, which plays an important role in enzyme applications. However, the role of N-terminal amino acid residues in the thermal performance and structural properties of exo-inulinases remains to be elucidated. In this study, three and six residues of the N-terminus starting from Gln23 of the exo-inulinase InuAGN25 were deleted and expressed in Escherichia coli. After digestion with human rhinovirus 3 C protease to remove the N-terminal amino acid fusion sequence that may affect the thermolability of enzymes, wild-type RfsMInuAGN25 and its mutants RfsMutNGln23Δ3 and RfsMutNGln23Δ6 were produced. Compared with RfsMInuAGN25, thermostability of RfsMutNGln23Δ3 was enhanced while that of RfsMutNGln23Δ6 was slightly reduced. Compared with the N-terminal structures of RfsMInuAGN25 and RfsMutNGln23Δ6, RfsMutNGln23Δ3 had a higher content of (1) the helix structure, (2) salt bridges (three of which were organized in a network), (3) cation-π interactions (one of which anchored the N-terminal tail). These structural properties may account for the improved thermostability of RfsMutNGln23Δ3. The study provides a better understanding of the N-terminus-function relationships that are useful for rational design of thermostability of exo-inulinases.

A novel metagenome-derived thermostable and poultry feed compatible α-amylase with enhanced biodegradation properties.

According to the numerous applications of feed processing by enzymatic conversion can be a fantastic tool to extreme its industrial usages. In this study, a novel acidic-thermostable α-amylase (PersiAmy3) was in-silico screened from the sheep rumen microbiota by computationally guided experiments instead of costly functional screening. At first, an in-silico screening approach was utilized to find primary candidate enzymes with superior properties. Among the selected candidates, PersiAmy3 was cloned, expressed, purified, and characterized. The PersiAmy3 was able to retain 65% of its maximum activity after 14 days of storage and exhibited optimal activity at pH 6-7 and 50 °C. The enzyme had excellent activity in the presence of various chemicals, it showed an excellent ability to hydrolyze different substrates, and was Ca2+ independent. Due to the high stability and activity of the PersiAmy3 on the corn powder as substrate, its ability to degrade the corn-based poultry feed at three high temperatures (50°C, 70°C, and 85°C), followed by the structural analysis was investigated. The result of this study indicated the power of computational selected candidates to discover novel acidic thermostable α-amylases. The selection method was very accurate, effective biodegradation of the poultry feed for industry was achieved using the selected candidate PersiAmy3.

Bioinformatics and experimental studies on the structural roles of a surface-exposed α-helix at the C-terminal domain of Chondroitinase ABC I.

A series of single and double mutants generated on residues of a surfaced-exposed helix at the C-terminal domain of chondroitinase ABC I (cABC I) from proteus vulgaris. M886A, G887E, and their respective double mutant, MA/GE were inspired by the sequence of a similar helix segment in 30S ribosomal protein S1. Additionally, M889I, Q891K, and the corresponding double mutant, MI/QK, were made regarding the sequence of a similar helix in chondroitin lyase from Proteus mirabilis. Circular dichroism spectra in the far-UV region, demonstrate that the ordered structure of wild-type (WT), and double mutants are the same; however, the helicity of the ordered structures in MI/QK is higher than that of the WT enzyme. When compared with the single mutants, the double mutants showed higher activity, and that the activity of MI/QK is higher than that of the WT enzyme. Heat-induced denaturation experiments showed that the stability of the tertiary structure of double mutants at moderate temperatures is higher compared with the WT, and single mutants. It concluded that this helix can be considered as one of the hot spots region that can be more manipulated to obtain improved variants of cABC I.

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops.

Protein stability is a widely studied topic, there are still aspects however that need addressing. In this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VPR, using the thermostable structural homologue AQUI as a template. Four locations for proline substitutions were chosen to imitate the structure of AQUI. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPRΔC_N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. In addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.

Thermostability of Lactate Dehydrogenase in Rat Brain under Conditions of Short-Term Moderate Hypothermia.

Thermostability of rat brain lactate dehydrogenase (LDH) was studied in intact animals and animals subjected to moderate short-term hypothermia. Two exponential stages, rapid and slow, were distinguished in the thermodenaturation kinetics. The contribution of the rapid phase to the lactate dehydrogenase denaturation kinetics was more significant: the energy of activation for this phase was 2.33 times lower than that for the slow phase. Moderate shortterm hypothermia led to a significant decrease of lactate dehydrogenase thermostability: thermodenaturation rate constants for the rapid (k1) and slow (k2) phases increased. Significant changes in parameters a and b reflecting the initial proportion of the two native forms of the enzyme developed only at 40°C. As hypothermia caused no appreciable changes in the energy of activation of lactate dehydrogenase denaturation, a significant contribution of the entropic factor to the decrease of free energy of enzyme denaturation was hypothesized. The data indicated significant labilization of lactate dehydrogenase structure under conditions of moderate hypothermia.

Coimmobilization of different lipases: Simple layer by layer enzyme spatial ordering.

This paper shows the step by step coimmobilization of up to five different enzymes following two different orders in the coimmobilization to alter the effect of substrate diffusion limitations. The enzymes were the lipases A and B from Candida antarctica, the lipases from Rhizomocur miehei and, Themomyces lanuginosus and the phospholipase Lecitase Ultra. The utilized strategy was a layer by layer immobilization, coating the immobilized enzymes with polyethylenimine followed by the crosslinking of the enzyme and PEI with glutaraldehyde to prevent enzyme release, and them adding a new lipase layer. The use of previously inactivated biocatalysts (using diethyl p-nitrophenylphosphate) permitted to visualize the immobilization of each enzyme layer, which was later confirmed by SDS-PAGE. This also confirmed the successful and complete covalent crosslinking of the glutaraldehyde treated enzyme layers. Activity of the combibiocatalysts was followed using diverse substrates. The protocol was successful and permitted to immobilize in an ordered way the 5 different enzymes in a down-up distribution.

Expression and characterization of a thermostable citrate synthase from Microcystis aeruginosa PCC7806.

Citrate synthase (CS) is an important enzyme in energy conversion and material circulation, participating in many important biochemical processes. In the present study, CS from Microcystis aeruginosa PCC7806 (MaCS) was cloned and expressed in Escherichia coli Rosetta (DE3). The recombinant MaCS was purified and its enzymological properties were characterized. The results showed that MaCS formed dimers in native status. The optimum temperature and pH of MaCS was 30°C and 8.2, respectively. MaCS displayed relative high thermal stability. Treatment at 50°C for 20 min only decreased 11.30% activity of MaCS and the half-life of MaCS was approximately 35 min at 55°C. The kcat and Km of acetyl-CoA and oxaloacetic acid were 17.133 s-1 (kcat) and 11.62 µM (Km), 24.502 s-1 and 103.00 µM, respectively. MaCS activity was not drastically inhibited by monovalent ions and NADH but depressed by divalent ions and some small molecular compounds, especially Mg2+, Zn2+, Co2+ and DTT. Overall, these data contributed to further understanding of energy metabolism in cyanobacteria and also provided basic information for industrial application of CS.

A highly thermostable crude endoglucanase produced by a newly isolated Thermobifida fusca strain UPMC 901.

A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24 hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9 U/mL was achieved at pH 5 and 60 °C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70 °C for 24 hours. Furthermore, the activity was found to be completely maintained without any loss at 50 °C and 60 °C for 144 hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.

Multimerization of an Alcohol Dehydrogenase by Fusion to a Designed Self-Assembling Protein Results in Enhanced Bioelectrocatalytic Operational Stability.

Proteins designed for supramolecular assembly provide a simple means to immobilize and organize enzymes for biotechnology applications. We have genetically fused the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus to a computationally designed cage-forming protein (O3-33). The trimeric form of the O3-33-AdhD fusion protein was most active in solution. The immobilization of the fusion protein on bioelectrodes leads to a doubling of the electrochemical operational stability as compared to the unfused control proteins. Thus, the fusion of enzymes to the designed self-assembling domains offers a simple strategy to increase the stability in biocatalytic systems.

Substrate inhibition imposes fitness penalty at high protein stability.

Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift toward lower stability or purifying selection against excess stability-for which no experimental evidence was found so far-is also at work. Here, we show that mutations outside the active site in the essential Escherichia coli enzyme adenylate kinase (Adk) result in a stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition-prone mutants. Furthermore, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may be an important factor determining stability observed for modern-day Adk.

New Recombinant Cold-Adapted and Organic Solvent Tolerant Lipase from Psychrophilic Pseudomonas sp. LSK25, Isolated from Signy Island Antarctica.

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.

Monomeric Camelus dromedarius GSTM1 at low pH is structurally more thermostable than its native dimeric form.

Glutathione S‒transferases (GSTs) are multifunctional enzymes that play an important role in detoxification, cellular signalling, and the stress response. Camelus dromedarius is well-adapted to survive in extreme desert climate and it has GSTs, for which limited information is available. This study investigated the structure-function and thermodynamic properties of a mu-class camel GST (CdGSTM1) at different pH. Recombinant CdGSTM1 (25.7 kDa) was expressed in E. coli and purified to homogeneity. Dimeric CdGSTM1 dissociated into stable but inactive monomeric subunits at low pH. Conformational and thermodynamic changes during the thermal unfolding pathway of dimeric and monomeric CdGSTM1 were characterised via a thermal shift assay and dynamic multimode spectroscopy (DMS). The thermal shift assay based on intrinsic tryptophan fluorescence revealed that CdGSTM1 underwent a two-state unfolding pathway at pH 1.0-10.0. Its Tm value varied with varying pH. Another orthogonal technique based on far-UV CD also exhibited two-state unfolding in the dimeric and monomeric states. Generally, proteins tend to lose structural integrity and stability at low pH; however, monomeric CdGSTM1 at pH 2.0 was thermally more stable and unfolded with lower van't Hoff enthalpy. The present findings provide essential information regarding the structural, functional, and thermodynamic properties of CdGSTM1 at pH 1.0-10.0.

Production of thermotolerant, detergent stable alkaline protease using the gut waste of Sardinella longiceps as a substrate: Optimization and characterization.

The gut wastes of Sardinella longiceps were used as substrate for protease production. The gut waste has 61.6% proteins, 21.8% lipids, 8.5% carbohydrates on dry weight basis and trace elements. The significant factors of protease fermentation were screened by Plackett-Burman design. A protease activity of 68.56 U/ml was predicted at 46.31 °C, incubation time 71.11 h, inoculum 4.86% (v/v) and substrate concentration 2.66% (w/v), using response surface methodology. However, the validation experiment showed 73.52 U/ml activity. The artificial neural network was found as a better tool to predict the experimental results. The partially purified protease showed higher activity at pH 9 and 10 and retained 90% activity after 120 h at pH 9. It showed maximum activity at 50 °C and retained 88% residual activity until 90 min at 50 °C. Zn++ enhanced the protease activity by 40%. The protease retained an activity of 93, 103, 90 and 98% against urea, ß-mercaptoethanol, SDS and tween 80 respectively. The alkaline protease was compatible with all the commercial detergents tested with the residual activity above 90%. The alkaline protease exhibited 22% higher activity on the tryptone soya substrate. The gut waste of S. longiceps is a worthy low cost substrate for the production of industrially important alkaline protease.

Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase.

A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley ß-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0-6.0 and 60-65 °C with barley ß-glucan and CMC-Na as substrates and had a melting temperature (Tm) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and Tm values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.

Glu571 of PheT plays a pivotal role in the thermal stability of Escherichia coli PheRS enzyme.

As of date the two temperature sensitive mutations isolated in pheST operon include pheS5 (G293 →A293 ) and pheT354. Recently, we reported that G673 of pheS defines a hot spot for intragenic suppressors of pheS5. In this investigation, in 13 independent experiments, a collection of temperature sensitive mutants were isolated by localized mutagenesis. Complementation using clones bearing pheS+ , pheT+ , and pheS+ T+ indicated that 34 mutants could harbor lesion(s) in pheS and four could be in pheT and one mutant might be a double mutant. Surprisingly, all the 34 pheS mutants harbored the very same (G293 →A293 ) transition mutation as present in the classical pheS5 mutant. Most unexpectedly, the four pheT mutants isolated harbored the same G1711 →A1711 transition, a mutation which is hitherto unreported. Since all the four pheT mutants were defined by the same G1711 →A1711 base change, we believe that getting other mutations could be hard hitting and therefore it is proposed that G1711 itself could be a "hot spot" for emergence of Ts mutations in pheT and similarly G293 itself could be a "hot spot" for Ts lesions in pheS. These results clearly imply a vital role for Glutamic acid571 (Glu571 ) of PheT and reinforce criticality of Glycine98 (Gly98 ) of PheS in the thermal stability of PheRS enzyme.