Unraveling the roles of aromatic cluster side-chain interactions on the structural stability and functional significance of psychrophilic Sphingomonas sp. glutaredoxin 3.

This study investigates the impact of aromatic cluster side-chain interactions in Grx3 (SpGrx3) from the psychrophilic Arctic bacterium Sphingomonas sp. Grx3 is a class I oxidoreductase with a unique parallel arrangement of aromatic residues in its aromatic cluster, unlike the tetrahedral geometry observed in Trxs. Hydrophilic-to-hydrophobic substitutions were made in the aromatic cluster, in ß1 (E5V and Y7F), adjacent ß2 (Y32F and Y32L), both ß1 and ß2 (E5V/Y32L), and short α2 (R47F). The hydrophobic substitutions, particularly those at or near Tyr7 (E5V, Y7F, Y32F, and R47F), increased melting temperatures and conformational stability, whereas disrupting ß1-ß2 interactions (Y32L and E5V/Y32L) led to structural instability of SpGrx3. However, excessive hydrophobic interactions (Y7F and E5V/Y32L) caused protein aggregation at elevated temperatures. All mutations resulted in a reduction in α-helical content and an increase in ß-strand content. The R47F mutant, which formed dimers and exhibited the highest ß-strand content, showed increased conformational flexibility and a significant decrease in catalytic rate due to the disturbance of ß1-α2 interactions. In summary, the configuration of the aromatic cluster, especially Tyr7 in the buried ß1 and Arg47 in the short α2, played crucial roles in maintaining the active conformation of SpGrx3 and preventing its protein aggregation. These modifications, reducing hydrophobicity in the central ß-sheet, distinguish Grx3 from other Trx-fold proteins, highlighting evolutionary divergence within the Trx-fold superfamily and its functional versatility.

C-Terminal ß8-α9 Interaction Modulates Thermal Stability and Enzymatic Activity Differently in Hyperthermophilic Esterase EstE1 and Mesophilic Esterase rPPE.

Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in α/ß-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal ß8-α9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the ß8-α9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the ß8-α9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that α/ß-hydrolases favor the ß8-α9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how α/ß-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal ß8-strand and α9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the ß8-α9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize ß8-α9 strand-helix differently but achieve similar thermal stability. While the ß8-α9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.

Tyr76 is essential for the cold adaptation of a class II glutaredoxin 4 with a heat-labile structure from the Arctic bacterium Sphingomonas sp.

Glutaredoxins (Grxs) are small proteins that share a well-conserved thioredoxin (Trx)-fold and participate in many biological processes. This study examined the cold adaptation mechanism of a Fe-S cluster binding class II Grx4 (SpGrx4) from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621. Three polar residues close to the cis-proline residue (P73) of SpGrx4 form a hydrogen bond network (Q74-S67-Y76) with the cis-proline loop main chain. The hydroxyl group of S67 or Y76 or both is replaced in similar Grxs depending on the temperature of the habitat. Mutants with reduced hydrogen bonds (S67A, Q74A, Y76F, and S67A/Y76W) were more susceptible to urea-induced unfolding and more flexible than the wild-type (WT). By contrast, Y76W, with a bulky indole group, was the most stable. These mutants showed higher melting temperatures than WT as a consequence of increased hydrophobic interactions. These results suggest that the tyrosine residue, Y76, is preferred for the cold adaptation of SpGrx4 with a heat-labile structure despite the rigid cis-proline loop, due to hydrogen bond formation. An aromatic residue on ß3 (cis-proline plus3) modulates the stability-flexibility of the cis-proline loop for temperature adaptation of prokaryotic class II Grx4 members via hydrogen bonds and hydrophobic interactions.

Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central ß-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the ß2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii.

Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized d-sorbitol as its carbon source, among the four polyols examined (d-galactitol, d-mannitol, d-sorbitol and d-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153 and Lys157 as catalytic active site residues and existed as an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4-25°C. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr177. In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.

Purification and characterization of a novel medium-chain ribitol dehydrogenase from a lichen-associated bacterium Sphingomonas sp.

Sugar alcohols (polyols) are abundant carbohydrates in lichen-forming algae and transported to other lichen symbionts, fungi, and bacteria. Particularly, ribitol is an abundant polyol in the lichen Cetraria sp. Polyols have important physiological roles in lichen symbiosis, but polyol utilization in lichen-associated bacteria has been largely unreported. Herein, we purified and characterized a novel ribitol dehydrogenase (RDH) from a Cetraria sp.-associated bacterium Sphingomonas sp. PAMC 26621 grown on a minimal medium containing D-ribitol (the RDH hereafter referred to as SpRDH). SpRDH is present as a trimer in its native form, and the molecular weight of SpRDH was estimated to be 39 kDa by SDS-PAGE and 117 kDa by gel filtration chromatography. SpRDH converted D-ribitol to D-ribulose using NAD+ as a cofactor. As far as we know, SpRDH is the first RDH belonging to the medium-chain dehydrogenase/reductase family. Multiple sequence alignments indicated that the catalytic amino acid residues of SpRDH consist of Cys37, His65, Glu66, and Glu157, whereas those of short-chain RDHs consist of Ser, Tyr, and Lys. Furthermore, unlike other short-chain RDHs, SpRDH did not require divalent metal ions for its catalytic activity. Despite SpRDH originating from a psychrophilic Arctic bacterium, Sphingomonas sp., it had maximum activity at 60°C and exhibited high thermal stability within the 4-50°C range. Further studies on the structure/function relationship and catalytic mechanism of SpRDH will expand our understanding of its role in lichen symbiosis.

Biochemical comparison of two glucose 6-phosphate dehydrogenase isozymes from a cold-adapted Pseudomonas mandelii.

Cold-adapted bacteria primarily have two glucose 6-phosphate dehydrogenase isozymes (G6PD, also known as zwf), zwf-1 for the Entner-Doudoroff pathway and zwf-2 for the oxidative pentose phosphate pathway. Although the roles of zwfs in carbon metabolism and antioxidant defense have been reported, the biochemical properties of zwfs at low and moderate temperatures have not been fully described. In this study, we cloned and characterized zwf-1 (Pmzwf-1) and zwf-2 (Pmzwf-2) from a cold-adapted bacterium Pseudomonas mandelii JR-1. Pmzwf-1 and Pmzwf-2 were expressed in Escherichia coli BL21 (DE3) as soluble tetrameric proteins. Both Pmzwf proteins were active at 4 °C, but Pmzwf-1 exhibited overall better biochemical properties than those of Pmzwf-2, including 10-30% higher specific activity at 4-40 °C as well as consistent conformational flexibility and thermal stability in the 4-40 °C range. Pmzwf-2 showed reduced thermal stability at moderate temperatures. Furthermore, the mRNA expression of Pmzwf-1 was higher than that of Pmzwf-2 at both 4 °C and 25 °C. These results indicate that Pmzwfs are cold-adapted enzymes, but Pmzwf-1 can function at both low to moderate temperatures while Pmzwf-2 is primarily functional at low temperatures. Our results suggest distinct temperature adaptations of two G6PD isozymes in P. mandelii JR-1, adaptations that are metabolic pathway dependent.

Cloning and characterization of a thermostable glutathione reductase from a psychrophilic Arctic bacterium Sphingomonas sp.

Glutathione reductase is an important oxidoreductase that helps maintain redox homeostasis by catalyzing the conversion of glutathione disulfide to glutathione using NADPH as a cofactor. In this study, we cloned and characterized a glutathione reductase (hereafter referred to as SpGR) from Sphingomonas sp. PAMC 26621, an Arctic bacterium. SpGR comprises 449 amino acids, and functions as a dimer. Surprisingly, SpGR exhibits characteristics of thermophilic enzymes, showing optimum activity at 60°C and thermal stability up to 70°C with ∼50% residual activity at 70°C for 2 h. The amino acid composition analysis of SpGR showed a 1.9-fold higher Arg content (6%) and a 2.7-fold lower Lys/Arg ratio (0.75) compared to the Arg content (3.15%) and the Lys/Arg ratio (2.01) of known psychrophilic glutathione reductases. SpGR also exhibits its activity at 4°C, and circular dichroism and fluorescence spectroscopy results indicate that SpGR maintains its secondary and tertiary structures within the temperature range of 4-70°C. Taken together, the results of this study indicate that despite its origin from a psychrophilic bacterium, SpGR has high thermal stability. Our study provides an insight into the role of glutathione reductase in maintaining the reducing power of an Arctic bacterium in a broad range of temperatures.

Distinct roles of an ionic interaction holding an alpha-helix with catalytic Asp and a beta-strand with catalytic His in a hyperthermophilic esterase EstE1 and a mesophilic esterase rPPE.

An ionic interaction that holds an α-helix and a ß-strand on which catalytic Asp and His residues are located, respectively, is conserved in a hyperthermophilic esterase EstE1 (optimum temperature 70 °C) and a mesophilic esterase rPPE (optimum temperature 50 °C). We investigated the role of an ionic interaction between E258 and R275 in EstE1 and that between E263 and R280 in rPPE in active-site stability of serine esterases adapted to different temperatures. Ala substitutions caused a 5-10 °C decrease in the optimum temperature of both EstE1 and rPPE mutants. Surprisingly, disruption of the ionic interaction caused larger effects on the conformational flexibility of EstE1 mutants despite their rigid structures, whereas the disruption had fewer effects on the thermal stability of EstE1 mutants at 60-70 °C, as the structure of EstE1 was adapted to high temperatures. In contrast, mesophilic rPPE mutants showed dramatic decreases in thermal stability at 40-50 °C, but less changes in conformational flexibility because of their inherently flexible structures. The results of this study suggest that the ionic interaction between the α-helix with catalytic Asp and the ß-strand with catalytic His plays an important role in the active-site conformation of EstE1 and rPPE, with larger effects on the conformational flexibility of hyperthermophilic EstE1 and the thermal stability of mesophilic rPPE.

Functional characterization of a SNP (F51S) found in human alpha 1-antitrypsin.

BACKGROUND: Alpha 1-antitrypsin (A1AT) deficiency is related to lung and liver diseases, including pulmonary emphysema and liver cirrhosis in humans. Genetic variations including single nucleotide polymorphisms (SNPs) of SERPINA1 are responsible for A1AT deficiency, but the characteristics of the SNPs are not well-understood. Here, we investigated the features of a rare SNP (F51S) of A1AT, which introduces an additional N-glycosylation site in the N-terminal region of A1AT. METHODS: We evaluated the F51S variant compared with the wild-type (WT) A1AT with regard to expression in CHO-K1 cells, trypsin inhibitory activity, polymerization, and thermal stability. RESULTS: The recombinant F51S protein expressed in CHO-K1 cells was mostly retained inside cells. The F51S variant had trypsin inhibitory activity, but reduced thermal stability compared with the WT A1AT. The native acrylamide gel data showed that F51S tended to prevent polymerization of A1AT. CONCLUSION: The results of this study indicate that Phe51 and the surrounding hydrophobic residue cluster plays an important role in the conformation and secretion of A1AT and suggest the harmful effects of a rare F51S SNP in human health.

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621.

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5⁻8.5 and 30 °C for NADP⁺ and 20 °C for NAD⁺. SpG6PD1 utilized both NADP⁺ and NAD⁺, with the preferential utilization of NADP⁺. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4⁻40 °C, its activity and tertiary structure were better preserved between 4⁻20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.

Organic Solvent-Tolerant Esterase from Sphingomonas glacialis Based on Amino Acid CompositionAnalysis: Cloning and Characterization of EstSP2.

Organic solvent-tolerant (OST) enzymes are widely applied in various industries due to their activity and stability in organic solvents, higher substrate solubility, and increased stereo-selectivity. However, the criteria for identifying OST enzymes largely remain unresolved. In this study, we compared the amino acid composition of 19 OST esterases and 19 non-OST esterases. OST esterases have increased ratio of Ala and Arg residues and decreased ratio of Asn, Ile, Tyr, and Ser residues. Based on the amino acid composition analysis, we cloned acarboxylesterase (EstSP2) from a psychrophilic bacterium, Sphingomonas glacialis PAMC 26605, and characterized its recombinant protein. EstSP2 is substrate specific to p-nitrophenyl acetate and hydrolyzed aspirin, with optimal activityat 40°C; at 4°C, the activity is approximately 50% of its maximum. As expected, EstSP2showstolerance in up to 40% concentration of polar organic solvents, including dimethyl sulfoxide, methanol, and ethanol. The results of this study suggest that selection of OST esterases based on their amino acid composition analysis could be a novel approach to identify OST esterases produced from bacterial genomes.

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1.

The aromatic amino acids, Tyr or Trp, which line the active-site walls of esterases, stabilize the catalytic His loop via hydrogen bonding. A Tyr residue is preferred in extremophilic esterases (psychrophilic or hyperthermophilic esterases), whereas a Trp residue is preferred in moderate-temperature esterases. Here, we provide evidence that Tyr and Trp play distinct roles in cold adaptation of the psychrophilic esterase EstSP1 isolated from an Arctic bacterium Sphingomonas glacialis PAMC 26605. Stern-Volmer plots showed that the mutation of Tyr191 to Ala, Phe, Trp, and His resulted in reduced conformational flexibility of the overall protein structure. Interestingly, the Y191W and Y191H mutants showed increased thermal stability at moderate temperatures. All Tyr191 mutants showed reduced catalytic activity relative to wild-type EstSP1. Our results indicate that Tyr with its phenyl hydroxyl group is favored for increased conformational flexibility and high catalytic activity of EstSP1 at low temperatures at the expense of thermal stability. The results of this study suggest that, in the permanently cold Arctic zone, enzyme activity has been selected for psychrophilic enzymes over thermal stability. The results presented herein provide novel insight into the roles of Tyr and Trp residues for temperature adaptation of enzymes that function at low, moderate, and high temperatures.

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures.

Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation.

Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W(208) was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W(208) to Tyr (W(208)Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W(208)Y suggested that Y(208) could form a hydrogen bond with D(308), which is located next to catalytic residue H(307), stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.

Nonspecific cleavage of proteins using graphene oxide.

In this article, we report the intrinsic catalytic activity of graphene oxide (GO) for the nonspecific cleavage of proteins. We used bovine serum albumin (BSA) and a recombinant esterase (rEstKp) from the cold-adapted bacterium Pseudomonas mandelii as test proteins. Cleavage of BSA and rEstKp was nonspecific regarding amino acid sequence, but it exhibited dependence on temperature, time, and the amount of GO. However, cleavage of the proteins did not result in complete hydrolysis into their constituent amino acids. GO also invoked hydrolysis of p-nitrophenyl esters at moderate temperatures lower than those required for peptide hydrolysis regardless of chain length of the fatty acyl esters. Based on the results, the functional groups of GO, including alcohols, phenols, and carboxylates, can be considered as crucial roles in the GO-mediated hydrolysis of peptides and esters via general acid-base catalysis. Our findings provide novel insights into the role of GO as a carbocatalyst with nonspecific endopeptidase activity in biochemical reactions.

Increased thermal stability of cold-adapted esterase at ambient temperatures by immobilization on graphene oxide.

In this study, the effect of graphene oxide (GO) on the thermal stability of a recombinant esterase from cold-adapted Pseudomonas mandelii, rEstKp, was investigated. The complex GO-rEstKp was formed by cross-linking. Both free rEstKp and GO-rEstKp complex showed similar optimum pH and temperatures. GO-rEstKp complex exhibited enhanced thermal stability at ambient temperatures than rEstKp, which prevented the denaturation of the enzyme by hydrophilic interactions. However, the catalytic efficiency of GO-rEstKp complex was lowered to approximately 40% of that of free rEstKp. This study provides an insight into the addition of GO for industrial applications of cold-adapted enzymes at ambient temperatures.

An organic solvent-tolerant alkaline lipase from cold-adapted Pseudomonas mandelii: cloning, expression, and characterization.

A gene encoding a novel organic solvent-tolerant alkaline lipase, lipS (GenBank ID JQ071496), was cloned from cold-adapted Pseudomonas mandelii. Recombinant LipS was expressed in Escherichia coli as a 32-kDa soluble protein and was purified by standard procedures. It maintained more than 80% of its activity under alkaline conditions, pH 8-10.5, with an apparent optimum temperature range of 40-50 °C. It maintained thermal stability from 4 to 50 °C. After 1 h of incubation at 60 °C, approximately 50% of its activity remained. It retained its activity in organic solvents, and activity increased in the presence of ethanol and of DMSO. Our data indicate that LipS is an alkaline lipase with relatively high thermal stability and notable tolerance of organic solvents.