Neuroprotective effect of Lithospermum officinale callus extract on inflamed primary microglial cells.

BACKGROUND: Plants that have therapeutic features for humans or animals are commonly referred to as "medicinal plants". They produce secondary metabolites with antioxidant, antimicrobial and/or anti-cancer effects. Lithospermum officinale, known as European stone seed, is a famous medicinal herb. However, due to the pyrrolizidine alkaloids (PzAl) in the root extract of L.officinal, there are therapeutic limitations to this herb. Objective This research was devoted to the evaluation of the anti-inflammatory capacity of methanolic extracts of L. officinale callus (LoE) (fresh cells) on rat microglial cells, the immune cells of the Central Nervous System, which play an essential role in the responses to neuroinflammation. METHODS: Primary microglia were obtained from neonatal Wistar rats (1 to 3-days old), and then treated with various concentration of CfA and methanolic extracts of 17 and 31-day-old L. officinale callus before LPS-stimulation. In addition to HPLC analysis of the extracts, viability, nitric oxide production, and evaluation of pro-inflammatory genes and cytokines in the inflamed microglia were investigated by MTT, Griess methos, qrt-PCR, and ELISA. RESULTS: Methanolic extract of the 17-day-old callus of L. officinale exhibited anti-inflammatory effects on LPS-stimulated microglial cells much higher than observed for CfA. The data were further supported by the decreased expression of Nos2, Tnf-α, and Cox-2 mRNA and the suppression of TNF-α and IL-1ß release in the activated microglial cells pretreated with the effective dose of LoE (0.8 mg mL-1). CONCLUSION: It was assumed that the better anti-neuroinflammatory performance of LoE than CfA in LPS-activated primary microglia could be a result of the synergism of the components of the extract and the lipophilic nature of RsA as the main phenolic acid of LoE. Considering that LoE shows a high antioxidant capacity and lacks PzAl, it is anticipated that LoE extract might be considered a reliable substitute to play a key role in the preparation of neuroprotective pharmaceutical formulas, which require in vivo research and further experiments.

Structural and inhibitory effects of fulvic and humic acids against tyrosinase.

Inhibition of tyrosinase activity can control fruit browning and preserve the flavor and nutritional value of food. The impacts of fulvic acid (FA) and humic acid (HA) on tyrosinase activity were investigated utilizing circular dichroism (CD) and fluorescence spectroscopy, molecular docking (MD), and molecular dynamics simulations. HA and FA demonstrated a mixed type of inhibition with Ki 2.02 and 5.2 µM, respectively. The thermodynamic parameters displayed that the hydrogen bond and hydrophobic force play a major role in the FA-tyrosinase and HA-tyrosinase interaction, respectively. Fluorescence experiments demonstrated changes in tyrosinase tertiary structures. HA could not destroy the tyrosinase secondary structure significantly, however, FA has a significant influence on the tyrosinase secondary structure. The molecular dynamics findings demonstrated the minimal fluctuations and the lowest flexibility in the complex amino acids in the HA-tyrosinase and FA-tyrosinase interaction. Altogether, HA and FA could be utilized in food industries as an accessible natural source for tyrosinase inhibition. PRACTICAL APPLICATIONS: Recently, the investigation of tyrosinase inhibitors from the biosphere for hindrance of undesired browning in the food industry has increased considerably. Mushroom tyrosinase is a suitable model for kinetic research owing to its availability as well as close conformational similarity to tyrosinase in a mammal. Natural sources and their effective compounds could have wonderful potential on tyrosinase activity and structure, thus, in this study, the interactions between tyrosinase and fulvic acid (FA) and Humic acid (HA) were investigated. Previously, it has been shown that HA and FA have antioxidant properties and they can improve the quality of food via retarding lipid oxidation. Altogether, further investigations are warranted to draw firm conclusions, HA and FA could be utilized in food industries not only as antioxidant agents but also as an accessible natural source for tyrosinase inhibition.

Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01.

Cellulases are hydrolytic enzymes with wide scientific and industrial applications. We described a novel cellulase, CelC307, from the thermophilic indigenous Cohnella sp. A01. The 3-D structure of the CelC307 was predicted by comparative modeling. Docking of CelC307 with specific inhibitors and molecular dynamic (MD) simulation revealed that these ligands bound in a non-competitive manner. The CelC307 protein was purified and characterized after recombinant expression in Escherichia coli (E. coli) BL21. Using CMC 1% as the substrate, the thermodynamic values were determined as Km 0.46 mM, kcat 104.30 × 10-3 (S-1), and kcat/Km 226.73 (M-1 S-1). The CelC307 was optimally active at 40 °C and pH 7.0. The culture condition was optimized for improved CelC307 expression using Plackett-Burman and Box-Behnken design as follows: temperature 20 °C, pH 7.5, and inoculation concentration with an OD600 = 1. The endoglucanase activity was positively modulated in the presence of Na+, Li+, Ca2+, 2-mercaptoethanol (2-ME), and glycerol. The thermodynamic parameters calculated for CelC307 confirmed its inherent thermostability. The characterized CelC307 may be a suitable candidate for various biotechnological applications.

A novel spray bioreactor for the proliferation of plant callus; Hyoscyamus niger and Arnebia pulchra.

OBJECTIVE: Unlike plant cell suspension culture, the proliferation of callus in bioreactors has received inadequate attention. The magnificent potential of plant callus becomes more appreciated as the research unfolds and promises interesting applications including the production of valuable metabolites, therapeutic antibodies, bioactive extracts with regenerating effects, and the generation of genetically improved plants. Issues such as the lack of 3D-access of the cells to the nutrients, using an interfering gelling substance as the support matrix, and the changes in the medium formulation during the growth phase were discouraging factors for extending research on this topic. Considering the existing drawbacks, a novel open-flow spray bioreactor (OFSB) was configured to circumvent the associated problems with the solid cell culture and promote the applicability of plant callus culture via improving the feeding strategy. METHODS: Applying similar subculture conditions, the proliferation of Arnebia pulchra and Hyoscyamus niger calli as the examples of two important plant families (Boraginaceae and Solanaceae) was studied in the OFSB in comparison with similar calli that grew in Petri dishes and jars. RESULTS: A. pulchra and H. niger calli obtained the weight gains of (%87.3 and %106.7) in the Petri dishes, (%208.7 and %226) in the jars, and (%288.6 and %320.0) in OFSB, respectively, while no significant changes were observed in the productivity indices of the examined calli. CONCLUSION: The simple design of OFSB bypasses most of the notorious problems associated with solid plant callus culture. OFSB technical features allow the bioreactor to be used for growth optimization of various types of plant calli in a cost-effective manner.

Structure and activity of a novel robust peroxidase from Alkanna frigida cell culture.

Demands for peroxidases (POX)s with diverse physicochemical properties have steadily grown as more applications of POXs are demonstrated. Plants are among the best sources of versatile POXs, and plant biotechnology, as an agricultural hassle-free technology, promises to circumvent the limitations of natural resource exploitation and to address the demands. Following this trend, it was shown that POX production steadily increased during the 31-day subculture of Alkanna frigida (from Boraginaceae) callus on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The purified cationic enzyme (POXalf) maintained its optimal activity over pH 4-7 for 2 years. It was resistant to H2O2 high concentrations (IC50 = 543.7 mM) and showed high specific activity in the reaction with phenol (4320.5 AU mg-1 > 20-fold of HRP AU). Furthermore, the specificity constant ratio of guaiacol to phenol indicated a 100 times faster reaction of POXalf with guaiacol. However, in contrast to HRP, it had little effect on diazo derivatives of aniline and meta-diaminobenzene. Based on the resulting primary structure from the tandem mass analysis, the POXalf 3D structure was constructed via homology modelling. Despite the high topological similarity between the HRP and POXalf structures, there were important differences between the active site pockets that could explain the observed differences in the corresponding substrate spectra and the specific activities. Considering the dynamics of POXalf production, its inactivity towards IAA and its high affinity for guaiacol, POXalf may have associated roles with A. frigida cell wall construction and monolignol metabolism.

Biodegradation of cyanide to ammonia and carbon dioxide by an industrially valuable enzyme from the newly isolated Enterobacter zs.

The biodetoxification of cyanide-rich wastewater has been suggested as an appropriate technique due to its environmental friendliness and cost effectiveness. In this research, Enterobacter zs that was newly isolated from cyanide-polluted wastewater was selected to catalyze cyanide via an enzymatic mechanism. Enzyme was purified and its activity was also determined by ammonia assay. Subsequently, the operational procedure was optimized to enhance cyanide biodegradation at variable pH values, temperatures and cyanide concentrations using response surface methodology (RSM). The results revealed that the interactions between pH and temperature, as well as those between pH and cyanide concentration, were significant, and the concentration of cyanide in a 650 mg.L-1 solution was decreased by 73%. According to this study, it can be proposed that due to its higher activity level compared with those of similar enzymes, this enzyme can prove useful in enzymatic biodegradation of cyanide which is a promising approach in the treatment of industrial effluent.

Toward more specific inhibitor for Solanum tuberosum polyphenol oxidase through a structural insight into its activities and inhibition.

To prevent enzymatic browning, applying a polyphenol oxidase (PPO) inhibitor is more desirable, especially when the freshness of the product matters. Most of the inhibition studies were done on mushroom tyrosinase (MT) while the literature indicates that MT and PPO of Solanum tuberosum (PPOsol ) respond differently to the same modulator despite their similar active sites. This research was conducted to deepen our knowledge about PPOsol and introduce a more specific inhibitor for this enzyme to be used in controlling the enzymatic browning of potatoes. A modified procedure was developed for PPOsol purification. The enzyme was subjected to some essential physicochemical and kinetics studies. In parallel to the comparable physicochemical properties, homology modeling revealed high structural similarity between Solanum lycopersicum PPO (PPOsly ) and PPOsol except for their active site pockets. Accordingly, PPOsol showed 5.1- and 34-fold higher affinity toward chlorogenic acid compared with two PPOsly isozymes. Alike PPOsly , PPOsol showed monophenolase activity but it was inactive toward L-tyrosine and p-coumaric acid. Based on structural criteria, phthalic acid, cinnamic acid, ferulic acid, and vanillin were selected and thoroughly examined for inhibition of the catecholase activity of PPOsol . Although all these substances inhibited PPOsol in mixed-inhibition mode, the results were strongly in favor of vanillin with IC50 < 1.37 mM and Ki < 1.2 mM. PRACTICAL APPLICATIONS: There are subtle structural differences in the active site pockets of polyphenol oxidase (PPOs) of various fruits, vegetables, and crops. Consequently, to introduce an efficient inhibitor for hindering enzymatic browning of crop products, it is essential to have detailed knowledge about the structure and activity of its PPO as the main player of this undesirable phenomenon. Results of this study not only shed light on the physicochemical properties of PPOsol but can also be used in making various formulations for safe controlling enzymatic browning of potatoes, especially fresh-cut and minimally processed products, and similar crops products during postharvest and the processes of products preparations.

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol-1, -76.8 kJ.mol-1.°K-1, and 84.1 kJ.mol-1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0-10.0) and temperatures (35-75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

New Insight into the Interactions of Arbutin with Mushroom Tyrosinase.

As a safe substitute for hydroquinone, ß-arbutin, a natural plant substance, and its synthetic counterpart, α-arbutin, are used in depigmentation formulations. However, there are debatable points regarding the impact of arbutin on tyrosinase and the pigmentation process. To shed light on this issue, the effects of Pyrus biossieriana leaves extract (PbLE) and ß-arbutin, extracted from PbLE, on mushroom tyrosinase (MT) were comprehensively examined. The study was focused on cresolase activity as the characteristic reaction of a tyrosinase. Kinetics studies disclosed that ß-arbutin can modulate MT monophenolase activity from inhibition to activation or vice versa. ß-Arbutin inhibited L-tyrosine (LTy) oxidation at concentrations < 0.3 mM but it increased (more than 400%) the enzymatic oxidation of L-tyrosine at the concentrations > 0.3 mM. An opposite pattern (activation then inhibition) was observed when a synthetic substrate was used instead of LTy. Computational studies, focused on the heavy chain of MT, indicated that ß-arbutin effect could be overruled by the enzyme's ability to provide the ligand with a non-specific binding site (MTPc). A plausible mechanism was presented to show the influence of MTPc on the substrate pose in the active site. The possible determinant correlation between the findings of this research and the current studies on human tyrosinase role in the pigmentation process has been presented.

New Provisional Function of OmpA from Acinetobacter sp. Strain SA01 Based on Environmental Challenges.

An outer membrane protein A (OmpA) from Acinetobacter sp. strain SA01 was identified and characterized in-depth based on the structural and functional characteristics already known of its homologues. In silico structural studies showed that this protein can be a slow porin, binds to peptidoglycan, and exhibits emulsifying properties. Characterization of the recombinant SA01-OmpA, based on its emulsifying properties, represented its promising potentials in biotechnology. Also, the presence of SA01-OmpA in outer membrane vesicles (OMV) and biofilm showed that this protein, like its homologues in Acinetobacter baumannii, can be secreted into the extracellular environment through OMVs and play a role in the formation of biofilm. After ensuring the correct selection of the protein of interest, the role of oxidative stress induced by cell nutritional parameters (utilization of specific carbon sources) on the expression level of OmpA was carefully studied. For this purpose, the oxidative stress level of SA01 cell cultures in the presence of three nonrelevant carbon sources (sodium acetate, ethanol, and phenol) was examined under each condition. High expression of SA01-OmpA in ethanol- and phenol-fed cells with higher levels of oxidative stress than acetate suggested that oxidative stress could be a substantial factor in the regulation of SA01-OmpA expression. The significant association of SA01-OmpA expression with the levels of oxidative stress induced by cadmium and H2O2, with oxidative stress-inducing properties and lack of nutritional value, confirmed that the cells tend to harness their capacities with a possible increase in OmpA production. Collectively, this study suggests a homeostasis role for OmpA in Acinetobacter sp. SA01 under oxidative stress besides assuming many other roles hitherto attributed to this protein.IMPORTANCE Acinetobacter OmpA is known as a multifaceted protein with multiple functions, including emulsifying properties. Bioemulsifiers are surface-active compounds that can disperse hydrophobic compounds in water and help increase the bioavailability of hydrophobic hydrocarbons to be used by degrading microorganisms. In this study, an OmpA from Acinetobacter sp. SA01 was identified and introduced as an emulsifier with a higher emulsifying capacity than Pseudomonas aeruginosa rhamnolipid. We also showed that the expression of this protein is not dependent on the nutritional requirements but is more influenced by the oxidative stress caused by stressors. This finding, along with the structural role of this protein as a slow porin or its role in OMV biogenesis and biofilm formation, suggests that this protein can play an important role in maintaining cellular homeostasis under oxidative stress conditions. Altogether, the present study provides a new perspective on the functional performance of Acinetobacter OmpA, which can be used both to optimize its production as an emulsifier and a target in the treatment of multidrug-resistant strains.

Withdrawal Notice: Neuroprotective Effect of Lithospermum officinale callus Extract on Inflamed Primary Microglial Cells

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Production of phenolic acids in hairy root cultures of medicinal plant Mentha spicata L. in response to elicitors.

In this study, hairy root induction in leaf and stem explants of Mentha spicata using various Agrobacterium rhizogenes strains was established for the first time. Although inoculation of explants by immersion method resulted in tissue necrosis, direct injection of explants by all examined strains (A13,R318,A4,GMI 9534 and ATCC15834) was effective. All different parts of the stem were susceptible to A. rhizogenes infection. However, the middle and lower internodes showed a higher rate of transformation. Among the different strains, the strain A13 exhibited the highest infection efficiency (almost 75% of the explants). A13 and R318-infected hairy roots showed the highest biomass production (close to 60 mg/flask), while infection with GMI 9534 produced the highest content of phenolic acids. Finally, the effect of phytohormone elicitation on hairy root growth and phenolic acid biosynthesis was investigated. A substantial increase in root growth and phenolic acids accumulation was obtained followed by 0.3 mg L-1 IBA and 100 µM MeJA treatment, respectively.

A new sensitive spectrophotometric method for determination of saliva and blood glucose.

There is an increasing need for accurate and inexpensive glucometers as the world moves toward personalized medicine. Among the existing technologies, photometric based devices are more desired due to the cost-effectiveness, ease-of-use and the potential to be adopted in the smart-phone technology for remote sensing and self-monitoring purposes. However, the accuracy, precision, and reproducibility of the results of these devices are heavily dependent on the details of the chosen glucose measuring method. Considering the delicate problems with the current spectrophotometric methods, a new method was developed for more precise, accurate, and fast measurement of blood glucose via the coupled reactions of glucose oxidase and peroxidase using 4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid (GASA) as the substrate. Stability of GASA and its oxidized products along with its direct and fast consumption by peroxidase, made it possible to determine blood glucose concentration in <20 s with high reproducibility. The low detection limit of GASA method (0.36 mg dL-1) with a linear range from 0.36 to 399.6 mg.dL-1 also allowed determination of salivary glucose concentration (SGC). As compared with the blood samples, the SGC results were more dispersed, especially for the diabetic participants, assumingly due to the diverse nature of salivary samples. However, a good correlation coefficient of 0.81 for non-diabetic individuals showed that it is accurate enough to recognize non-diabetic from diabetic condition. Results of this study disclose the potential application of GASA method as a reliable alternative for the current spectrophotometric methods with the ability to be adopted in miniaturized glucometers.

Cohnella sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme.

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O2 to H2O. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the Cohnella sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to E. coli BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including Km, Vmax, turn over number (kcat), and catalytic efficiency (kcat/Km) of the enzyme were 106 min at 90 °C and 686 µM, 10.69 U/ml, 20.3 S-, and 0.029 s-1 µM-1, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu2+, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of Cohnella sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.

Pyrrolizidine Alkaloids-Free Extract from the Cell Culture of Lithospermum officinale with High Antioxidant Capacity.

The benefits of Lithospermum officinale has encouraged people to continue using its extract (CAS 90063-58-4) in both medicinal and cosmetic industries despite the fact that chemical analysis confirms the presence of pyrrolizidine alkaloids (PAs) in the extract. While the cultivation of L. officinale takes, at least, 2 years to produce usable crops, its callus culture proliferated 8.3 times with 4.9-fold biomass in less than 30 days under the applied conditions in this study. Under the applied conditions, the cell extract contained no toxic PAs while phenylpropanoid pathway was active toward phenolic acids formation not toward naphthoquinone derivatives. Rosmarinic acid was produced as the main constituent. Total phenolic content and antioxidant capacity of the proliferated cell extracts were similar to those of the extracts of the natural plant tissues, in particular from the root. These results support the idea that the extract of L. officinale cells can be a reliable substitute for the extract of the natural plant tissues.

High phenol degradation capacity of a newly characterized Acinetobacter sp. SA01: Bacterial cell viability and membrane impairment in respect to the phenol toxicity.

An efficient phenol-degrading bacterial strain, belonging to Acinetobacter genus, was isolated and selected to study the impact of different environmentally relevant phenol concentrations on the degradation process. The bacterial isolate, labeled as Acinetobacter sp. SA01 was able to degrade the maximum phenol concentration of 1 g/l during 60 h at optimum condition of pH 7, 30 °C and 180 rpm. Aeration and initial cell density, the two important factors, were carefully examined in the optimal growth conditions. The results showed that these two variables related proportionally with phenol degradation rate. Further investigations showed no effect of inoculum size on the enhancement of degradation of phenol at over 1 g/l. Flow cytometry (FCM) study was performed to find out the relationship between phenol-induced damages and phenol degradation process. Single staining using propidium iodide (PI) showed increased cell membrane permeability with an increase of phenol concentration, while single staining with carboxyfluorescein diacetate (cFDA) demonstrated a considerable reduction in esterase activity of the cells treated with phenol at more than 1 g/l. A detailed investigation of cellular viability using concurrent double staining of cFDA/PI revealed that the cell death increases in cells exposed to phenol at more than 1 g/l. The rate of cell death was low but noticeable in the presence of phenol concentration of 2 g/l, over time. Phenol at concentrations of 3 and 4 g/l caused strong toxicity in living cells of Acinetobacter sp. SA01. The plate count method and microscopy analysis of the cells treated with phenol at 1.5 and 2 g/l confirmed an apparent reduction in cell number over time. It was assumed that the phenol concentrations higher than 1 g/l have destructive effects on membrane integrity of Acinetobacter sp. SA01. Our results also revealed that the toxicity did not reduce by increasing initial cell density. Scanning electron microscopy (SEM) examination of bacterial cells revealed the surface morphological changes following exposure to phenol. The bacterial cells, with wizened appearance and wrinkled surface, were observed by exposing to phenol (1 g/l) at lag phase. A morphological change occurred in the mid-logarithmic phase as the bacterial cells demonstrated coccobacilli form as well as elongated filamentous shape. The wrinkled cell surface were totally disappeared in mid-stationary phase, suggesting that the complete degradation of phenol relieve the stress and direct bacterial cells toward possessing smoother cell membrane.

Improving the thermostability of Serratia marcescens B4A chitinase via G191V site-directed mutagenesis.

Chitinases with high thermostability are important for many industrial and biotechnological applications. This study was conducted to enhance the stability of Serratia marcescens B4A chitinase by site directed mutagenesis of G191 V. Further characterization showed that the thermal stability of the mutant showed marked increase of about 5 and 15 fold at 50 and 60 °C respectively, while the optimum temperature and pH was retained. Kinetic analysis showed decreased Km and Vmax of the mutant in comparison with the wild type chitinase of about 1.3 and 3 fold, respectively. Based on structural prediction, it was speculated that this replacement shortened an important loop concomitant with the extension of adjacent ß sheets. Accordingly, a higher thermostability of G191 V up to 90 °C supporting the decreased flexibility of unfolded state was also indicated. Finally, a practical proof of kinetic and thermal stabilization of chitinase was provided through decreased flexibility and entropic stabilization of its surface loops.

New insight into the allosteric effect of L-tyrosine on mushroom tyrosinase during L-dopa production.

Kinetics studies of L-tyrosine (LTy) ortho-hydroxylation by mushroom tyrosinase (MT) confirmed that MT was severely, but not completely, inhibited at higher concentrations of LTy. Despite the availability of the crystal structure reports, no allosteric site has been identified on MT. To examine the assumption that a non-specific binding site works as a regulatory site, docking simulations were run for the second molecule of L-tyrosine (LTy2) on the complexes of the first L-tyrosine molecule (LTy1) with the heavy chain (H) of MT (LTy1/HMT) and its dimer with the light chain (Ty1/LHMT). In both, LTy2 occupied a non-specific binding site (MTPc). MD simulations revealed LTy2/HMT/LTy1 and LTy2/LHMT/LTy1 were stable. Binding free-energy analysis supported the formation of LTy2/HMT/LTy1 and LTy2/LHMT/LTy1 at higher concentrations of LTy and disclosed the importance of ΔEelec and ΔGpolar during binding of LTy2 to MTPc. Upon LTy2 binding to MTPc, the Cu-Cu distance remained unchanged while the spatial position of LTy1 in the active site (MTPa) changed so that it would not be able to participate in ortho-hydroxylation. This study suggests a tuning role for L chain during binding of the ligands to MTPa and MTPc. Given these results, a plausible mechanism was proposed for the MT substrate inhibition.

A cold-adapted endoglucanase from camel rumen with high catalytic activity at moderate and low temperatures: an anomaly of truly cold-adapted evolution in a mesophilic environment.

Endoglucanases are important enzymes in plant biomass degradation. They have current and potential applications in various industrial sectors including human and animal food processing, textile, paper, and renewable biofuel production. It is assumed that the cold-active endoglucanases, with high catalytic rates in moderate and cold temperatures, can improve the cost-effectiveness of industrial processes by lowering the need for heating and, thus, energy consumption. In this study, the endoglucanase CelCM3 was procured from a camel rumen metagenome via gene cloning and expression in Escherichia coli BL21 (DE3). The maximum activity of the enzyme on carboxymethyl cellulose (CMC) was obtained at pH 5 and 30 °C with a Vmax and Km of 339 U/mg and 2.57 mg/ml, respectively. The enzyme with an estimated low melting temperature of 45 °C and about 50% activity at 4 °C was identified to be cold-adapted. A thermodynamic analysis corroborated that CelCM3 with an activation energy (Ea), enthalpy of activation (ΔH), and Gibb's free energy (ΔG) of, respectively, 18.47 kJ mol-1, 16.12 kJ mol-1, and 56.09 kJ mol-1 is a cold-active endoglucanase. In addition, CelCM3 was tolerant of metal ions, non-ionic detergents, urea, and organic solvents. Given these interesting characteristics, CelCM3 shows promise to meet the requirements of industrial applications.

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.

Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.