Temperature For Maximum Activity Research Articles

Characterization of L-asparaginase from Streptomyces koyangensis SK4 with acrylamide-minimizing potential in potato chips.

Microbial L-asparaginase is well known for its application in food industries to reduce acrylamide content in fried starchy food. L-asparaginase produced by Arctic actinomycetes Streptomyces koyangensis SK4 was purified and studied for biochemical characterization. The L-asparaginase was purified with a yield of 15.49% and final specific activity of 179.77 IU/mg of protein. The enzyme exhibited a molecular weight of 43 kDa. The optimum pH and temperature for maximum activity of the purified enzyme were 8.5 °C and 40 °C, respectively. The enzyme expressed maximum activity at an incubation period of 30 min and a substrate concentration of 0.06 M. The enzyme has a low Km value of 0.041 M and excellent substrate specificity toward L-asparagine. The enzyme activity was inhibited by metal ions Ba2+ and Hg2+, while Mn2+ and Mg2+ enhanced the activity. The study evaluated the acrylamide reduction potential of L-asparaginase from Streptomyces koyangensis SK4 in potato chips. The blanching plus L-asparaginase treatment of potato slices resulted in a 50% reduction in acrylamide content. The study illustrated an effective acrylamide reduction strategy in potato chips using L-asparaginase from a psychrophilic actinomycete. Besides the acrylamide reduction potential, L-asparaginase from Streptomyces koyangensis SK4 also did not exhibit any glutaminase or urease activity which is an outstanding feature of L-asparaginase to be used as a chemotherapeutic agent.

Aluminum Doped Titania as a Support of Copper Catalysts for SCR of Nitrogen Oxides.

Aluminum doped titania samples were synthesized as supports of copper oxide catalysts for NO reduction with ammonia. Samples were prepared by the sol-gel method with various ratios of aluminum to titanium. Their thermal stability was examined by TG/DSC methods which revealed that precursors were decomposed at 450 °C. The XRD measurements showed that aluminum caused the diminishing of titania crystallites and was built into the anatase structure or formed an amorphous phase. The admixture of aluminum in titania resulted in a significant increase in specific surface area of mesoporous supports as determined by low temperature sorption of nitrogen. Results of the catalytic tests over copper/aluminum-titania samples obtained by impregnation pointed out that the addition of aluminum broadened the temperature window of high catalytic activity. The increase in Al concentration shifted the temperature of maximum activity to higher values, and at the same time lowered nitrous oxide formation as well. Better catalytic efficiency could result from high copper dispersion on the catalysts surface, as well as the synergistic interaction between Ti and Cu causing reduction in CuO species as confirmed by XPS measurements. It was shown that copper was present as Cu+ species mainly, forming Cu-O-Ti bonds on the catalysts surface.

Does social thermal regulation constrain individual thermal tolerance in an ant species?

In ants, social thermal regulation is the collective maintenance of a nest temperature that is optimal for individual colony members. In the thermophilic ant Aphaenogaster iberica, two key behaviours regulate nest temperature: seasonal nest relocation and variable nest depth. Outside the nest, foragers must adapt their activity to avoid temperatures that exceed their thermal limits. It has been suggested that social thermal regulation constrains physiological and morphological thermal adaptations at the individual level. We tested this hypothesis by examining the foraging rhythms of six populations of A. iberica, which were found at different elevations (from 100 to 2,000m) in the Sierra Nevada mountain range of southern Spain. We tested the thermal resistance of individuals from these populations under controlled conditions. Janzen's climatic variability hypothesis (CVH) states that greater climatic variability should select for organisms with broader temperature tolerances. We found that the A. iberica population at 1,300m experienced the most extreme temperatures and that ants from this population had the highest heat tolerance (LT50=57.55°C). These results support CVH's validity at microclimatic scales, such as the one represented by the elevational gradient in this study. Aphaenogaster iberica maintains colony food intake levels across different elevations and mean daily temperatures by shifting its rhythm of activity. This efficient colony-level thermal regulation and the significant differences in individual heat tolerance that we observed among the populations suggest that behaviourally controlled thermal regulation does not constrain individual physiological adaptations for coping with extreme temperatures.

Biochemical and molecular characterization of the hyaluronidase from Bothrops atrox Peruvian snake venom

Snake venoms are a rich source of enzymes such as metalloproteinases, serine proteinases phospholipases A2 and myotoxins, that have been well characterized structurally and functionally. However, hyaluronidases (E.C.3.2.1.35) have not been studied extensively. In this study, we describe the biochemical and molecular features of a hyaluronidase (Hyal-Ba) isolated from the venom of the Peruvian snake Bothrops atrox. Hyal-Ba was purified by a combination of ion-exchange and gel filtration chromatography. Purified Hyal-Ba is a 69-kDa (SDS-PAGE) monomeric glycoprotein with an N-terminal amino acid sequence sharing high identity with homologous snake venom hyaluronidases. Detected associated carbohydrates were hexoses (16.38%), hexosamines (2.7%) and sialic acid (0.69%). Hyal-Ba selectively hydrolyzed only hyaluronic acid (HA; specific activity = 437.5 U/mg) but it did not hydrolyze chondroitin sulfate or heparin. The optimal pH and temperature for maximum activity were 6.0 and 40 °C, respectively, and its Km was 0.31 μM. Its activity was inhibited by EDTA, iodoacetate, 2-mercaptoethanol, TLCK and dexamethasone. Na+ and K+ (0.2 M) positively affect hyaluronidase activity; while Mg2+, Br2+, Ba2+, Cu2+, Zn2+, and Cd2+ reduced catalytic activity. Hyal-Ba potentiates the hemorrhagic and hemolytic activity of whole venom, but decreased subplantar edema caused by an l-amino acid oxidase (LAAO). The Hyal-Ba cDNA sequence (2020 bp) encodes 449 amino acid residues, including the catalytic site residues (Glu135, Asp133, Tyr206, Tyr253 and Trp328) and three functional motifs for N-linked glycosylation, which are conserved with other snake hyaluronidases. Spatial modeling of Hyal-Ba displayed a TIM-Barrel (α/β) fold and an EGF-like domain in the C-terminal portion. The phylogenetic analysis of Hyal-Ba with other homologous Hyals showed the monophyly of viperids. Further, Hyal-Ba studies may extend our knowledge of B. atrox toxinology and provides insight to improve the neutralizing strategies of therapeutic antivenoms.

Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity.

The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of - 64.964KJ/mol for wild-type Cel5E and - 176.148, - 200.921, and - 120.038KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better β-glucanase activity on barley β-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and β-glucanase activity and shifted toward higher temperature of maximum activity.

Molecular cloning and characterization of a halotolerant α-amylase from marine metagenomic library derived from Arabian Sea sediments.

Functional screening of a metagenomic library of marine sediment revealed an amylolytic clone BTM109. This report states the purification and characterization of a moderately halotolerant α-amylase, with more than 51% activity in 2.5M NaCl. The molecular mass of purified protein was determined to be 55.7kDa by MALDI-TOF MS. The optimum pH for enzyme activity was pH 7 and temperature for maximal activity was 40°C. At 5mM concentration, Ca2+ enhanced the enzyme activity indicating that the enzyme is a Ca2+ dependent α-amylase which was confirmed by the starch hydrolysis pattern using TLC. These physico-chemical properties support the suitability of this enzyme for various industrial applications.

Modification and characterization of natural aluminosilicates, expanded perlite, and its application to immobilise α – amylase from A. oryzae

The aim of this study was to modify natural silicoaluminates as expanded perlite (EP) through simple and inexpensive treatments in order to get a greater reactivity of surface and immobilise α-amylase from A. oryzae by adsorption and covalent binding. The materials obtained (expanded perlite treated with HCl, rehydroxylated expanded perlite, zeolite, and the incorporation of polystyrene in expanded perlite) were analysed by infrared spectroscopy, thermal analysis, zeta potential, adsorption of N2, and Scanning Electron Microscopy. The analysis allowed us to confirm that the modifications on the materials studied were effective. This is perceived by an increase in bands corresponding to OH groups, which is beneficial when considering functional groups that interact directly with the enzyme. It also allowed us to determine that the materials tested exhibit good thermal stability at the temperature range in which enzymatic studies are conducted. The conditions for immobilisation (pH, concentration of glutaraldehyde, time of contact between the enzyme and the substrate, enzyme concentration) and some properties of the immobilised derivatives (optimum pH, temperature of maximum activity, and reuse) were analysed. The suitable range for the immobilisation of α-amylase from A. oryzae by covalent binding was pH between 5 and 6, and for immobilisation by adsorption the suitable pH range was between 5 and 5.5. These values ​​are consistent with the zeta potential values ​​previously determined for the different media studied. The reuse of the immobilised enzyme by covalent binding resulted in a residual activity of 90% up to the sixth reuse. Immobilisation by covalent binding was more effective than immobilisation by adsorption.

Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE. A total of eight single-site mutants were designed, and five of them showed prolonged half-life of inactivation at 80°C. Combinatorial mutations were subsequently introduced, and the superior mutant was double mutant E161D/N365P. The half-life was approximately 4-fold higher than that of the wild type enzyme. In addition, the reaction temperature for maximum activity increased from 80°C to 87.5°C, and catalytic efficiency (kcat/Km) for lactulose production was increased 29%. Moreover, this mutant E161D/N365P was more stable against chemical denaturation and showed also a broader pH profile. The second most stable variant were mutant E161D/S180P/S351G with a 3.3-fold increase in half-life. These results provided new insights into the thermostability of CsCE and suggested further potential industrial applications.

English

Lipases hydrolyze lipids to yield fatty acids and glycerol which are used as raw materials in industries. The lipase from raphia mesocarp was extracted, purified and characterized. Assay was carried out using p- nitrophenyl palmitate as substrate. The lipase was subjected to 80% ammonium sulphate precipitation, purified on ion exchange and gel filtration chromatography. Native and subunit molecular weights were determined on a gel filtration column and 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis respectively. Kinetic parameters (Km and Vmax), effect of temperature, pH, sensitivity to metal ions and substrate specificities of the lipase enzyme were studied. Specific activity of 17.40µmol/min/mg and purification fold of 2.68% were obtained.Native and subunitmolecular weightwas 35 kDa. The Km and Vmax values were0.01mM and 20.5µmol/min/ml respectively. The lipase enzyme was inhibited by all metallic salts used and enhanced by CaCl2. Optimal pH was 7.0 and temperature for maximal activity was 40C. The lipase hydrolyzed coconut oil at about twice the rate of palm kernel oil and palm oil, and at a higher rate than olive oil and Raphia oil. The study reveals that raphia mesocarp isa possible source of lipase which can be of industrial use. Key words: Lipase, raphia palm fruit, mesocarp

Synthesis of a hybrid polymer-inorganic biomimetic support incorporating in situ pectinase from Aspergillus niger ATCC 9642.

The hybrid alginate/gelatin/calcium oxalate (AGOCa) support was successfully synthesized through the biomimetic mineralization method for immobilization in situ of a pectinolytic extract from Aspergillus niger ATCC 9642 via entrapment technique. The efficiency of immobilization reached 72.7%. Sodium oxalate buffer (100 mM, pH 5.5) was selected as adjuvant of the immobilization process by allowing the formation of a calcified shell around the calcium alginate capsule, significantly increasing the stability to storage, thermal and recycling of the enzymatic immobilized pectinolytic extract. The pH and temperature for maximum activity were from 5.0 to 6.0 and 60 to 80 °C, respectively. The new hybrid support can be a potential alternative to obtain immobilized pectinases with properties for advantageous industrial applications.

Urease-carrying electrospun polyacrylonitrile mat for urea hydrolysis

Electrospinning was used to fabricate beadless microfibrous polyacrylonitrile (ePAN) mats with an average fiber diameter of 1448±380nm from a 10wt.% PAN in dimethylformamide (DMF) dope solution at applied voltage of 18kV and 20cm fiber collection distance. Urease (EC 3.5.1.5) was then covalently immobilized on dispersed microfibrous ePAN mats following the chemical treatment of fibers with ethylenediamine (EDA) and glutaraldehyde (GA). The optimal concentration of GA for immobilization was 5%. The amount of loaded urease reached 157μg/mg mat, exhibiting 54% of the free urease activity. The surface chemistry of as-spun and chemically treated fibers was examined with Fourier transform infrared (FTIR) spectroscopy. Field emission scanning electron microscopy (FESEM) was used to study the morphology and diameter of the pristine, chemically treated, and urease-immobilized microfibrous mats. Immobilized urease showed increased temperature for maximum activity (from 37 to 50°C for free and immobilized urease, respectively) and improved storage stability (20days). The immobilized urease was also less sensitive to the changes in pH, especially in acid conditions. In addition, nearly 70% of initial activity of the immobilized urease was retained after 15 cycles of reuse, which proved the applicability of the electrospun fibers as successful enzyme carriers.

Understanding Enzyme Molecular Evolution toward Thermal Adaptation using Multistate Computational Protein Design

A careful balance between structural-stability and flexibility (allostery) are hallmarks of enzymatic function and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Here we describe the rational design of 100 temperature-adapted adenylate kinase (AdK) enzymes via a novel multistate design strategy that takes into consideration the impact of conformational changes to the backbone structure and stability, based on boundaries set by molecular evolution. In turn, each AdK protein variant was expressed and experimentally characterized for thermostability and temperature-adapted function. Comparison of the experimental temperature of maximum activity to the melting-temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data was used to produce accurate predictions of thermostability via multistage nodes, providing the requisite complement for de novo temperature-adapted enzyme design. To the best of our knowledge the rational design of temperature adaptive enzyme function has not been realized, prior to this work. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of enzymatic allostery as it relates to the thermodynamic and structural limits of function.

Improving the thermostability and activity of lipoxygenase from Anabaena sp. PCC 7120 by directed evolution and site-directed mutagenesis

Abstract A sensitive and reliable high-throughput approach to screen lipoxygenase (LOX) mutants was developed to permit a directed evolution study in an attempt to increase the activity and thermostability of LOX from Anabaena sp. PCC 7120 using error-prone polymerase chain reaction (PCR) and DNA shuffling. The best mutant, named D22, contained one key amino acid change (N305D) and its half-life of inactivation at 50 °C was increased by approximately twofold in comparison to the wild-type enzyme, while specific activity 1.87-fold higher than the wild-type enzyme. The N305D substitution was confirmed to be critical for the thermostability and catalytic activity of LOX by site-saturation mutagenesis. Indeed, the reaction temperature for maximum activity increased from 45 °C to 50 °C, while catalytic efficiency (kcat/Km) was increased by 83%. This study reports for the first time on the directed evolution of the LOX from Anabaena sp. for improving thermostability and catalytic activity, and the mutant is of potential importance in food processing and flavor biosynthesis.

The influence of bridge type on the activity of supported Metallocene catalysts in ethylene polymerization

Ethylene polymerization was carried out by immobilization of rac-ethylenebis(1-indenyl)zirconium dichloride (Et(Ind)2ZrCl2) and rac-dimethylsilylbis(1-indenyl)zirconium dichloride (Me2Si(Ind)2ZrCl2) preactivated with methylaluminoxane (MAO) on calcinated silica at different temperatures. Polymerizations of ethylene were conducted at different temperatures to find the optimized polymerization temperature for maximum activity of the catalyst. The Me2Si bridge catalyst showed higher activity at the lower polymerization temperature compared to the Et bridge catalyst. The highest catalytic activities were obtained at temperatures about 50 °C and 70 °C for Me2Si(Ind)2ZrCl2/MAO and Et(Ind)2ZrCl2/MAO catalysts systems, respectively. Inductively coupled plasma-atomic emission spectroscopy results and polymerization activity results confirmed that the best temperature for calcinating silica was about 450 °C for both catalysts systems. The melting points of the produced polyethylene were about 130 °C, which could be attributed to the linear structure of HDPE.

α-Amylase Aspergillus oryzae Immobilized on Modified Expanded Perlite

Abstract The α-amylase from Aspergillus oryzae was immobilized covalently onto expanded perlite (EP) and modified EP by treatment with TiO2 (EP-TiO2), dye HE3B (EP-HE3B) polyethylene terephthalate (PET)-hydrazide (EP-PET) and magnetite (EP-magnetite). The modified EP was characterized using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The supports were functionalized with aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA). The optimum pH for free and immobilized α-amylase was 5.5. Temperature of maximum activity for free enzyme and immobilized enzyme on EP-HE3B was 50°C. The immobilized enzyme in EP-APTES this value was 55°C. The immobilized α-amylase in EP-APTES and EP-HE3B-APTES exhibited better thermostability than free enzyme. The immobilized derivatives showed moderate operational stability by retaining 50% of initial activity after seven successive reuses.

Understanding Thermal Adaptation of Enzymes through the Multistate Rational Design and Stability Prediction of 100 Adenylate Kinases

Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.

A novel ∼34‐kDa α‐amylase from psychrotroph Exiguobacterium sp. SH3: Production, purification, and characterization

An amylase-producing psychrotroph bacterium was isolated from soil and identified as belonging to the genus Exiguobacterium. A novel cold-adapted α-amylase, Amy SH3, was purified from culture medium of this bacterium using acetone precipitation and DEAE-Sepharose anion-exchange chromatography. The molecular mass of the enzyme was estimated about 34 kDa using SDS-PAGE. Biochemical characterization of Amy SH3 revealed that the optimum temperature for maximum activity of Amy SH3 was 37°C. However, Amy SH3 was also active at cold temperatures, showing 13% and 39% activity at 0 and 10°C, respectively. The optimum pH for maximum activity of Amy SH3 was pH 7, whereas the amylase was active over a pH range of 5 to 10. The activity of Amy SH3 was enhanced by Co²⁺ but decreased by Mg²⁺, Mn²⁺, Zn²⁺, Fe²⁺, and Ca²⁺. Amy SH3 was able to retain 76% of its activity in the presence of 0.5% SDS. The K(m) and V(max) of the enzyme were calculated to be 0.06 mg/mL and 4,010 U/mL, respectively. The cold-adapted Amy SH3 seems very promising for applications at ambient temperature.

A novel cold‐adapted pullulanase from Exiguobacterium sp. SH3: Production optimization, purification, and characterization

Cold‐adapted enzymes, elaborated by psychrophiles and psychrotrophs, seem to have potential for current and future applications. In the current study, pullulanase production by the cold‐adapted Exiguobacterium sp. SH3 was investigated. The Plackett–Burman design and the response surface methodology were applied to identify and optimize the significant variables affecting the pullulanase production of Exiguobacterium sp. SH3. The results showed that temperature, time, and CaCl2 concentration were significant variables while shaking, starch, yeast extract, tryptone, pH, MnCl2, MgCl2, and KH2PO4 were not significant. Using statistical analyses and optimizations, the pullulanase production was significantly elevated from 200 ± 18 to 950 ± 27 U/mL (4.75 times) as compared to non‐optimized conditions. A pullulanase of about 70 kDa, designated as Pul‐SH3, was purified 16.2‐folds from the optimized culture, and identified to be an amylopullulanase. The Km and Vmax of the enzyme were 0.069 mg/mL and 967 U/mL, respectively. The optimum pH and temperature for maximum activity of Pul‐SH3 were 7.5 and 30°C, respectively. As a cold‐adapted enzyme, Pul‐SH3 retained 23% of the maximum activity at 0°C. The biochemical characteristics and N‐terminal amino acid sequence of Pul‐SH3 suggest that the enzyme is a novel cold‐adapted amylopullulanase with remarkably high specific activity at moderate ambient temperature.

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5°C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔHbinding) crosses zero (TH) and becomes favorable (negative). This TH/activity upshift temperature is 15°C for Klenow and 30°C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG‡, ΔH‡, and TΔS‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH‡) switches from positive to negative.

THE ABILITY OF Trichoderma harzianum TO PRODUCE CHITINASE

The result showed that optimum temperature for maximum activity of Chitinase was 25 Cº with 2.74 unit/µm/min. while optimum pH for maximum activity Chitinase was 5 with 2.25 unit/µm/min . Significant activities of chitinase was produced by T. harzianum in culture media amended with fresh mycelium of M. phaseolina , where the releasing of reducing sugar N-acetylglucosamin was 280 µg/ml which Significantly differ from T. harzianum culture media amended with fresh and dry mycelium of R.solani and F.solani where the releasing of reducing sugar N-acetylglucosamin was 200 µg/ml. Production of chitinase using a medium containing chitin reached 40 µg/ml . Maximium chitinase inhibition activity, purified by ammonium sulphate, was recorded for M. phaseolina mycelium growth 47.03% , which Significantly differ with of R.solani and F.solani where the inhibition activity was 35.18 and 33.33% respectively