Ester Synthesis by a Recombinant Cutinase in Reversed Micelles of a Natural Phospholipid

Fusarium solani pisi recombinant cutinase solubilized in reversed micelles of a nonionic surfactant (phosphatidylcholine) in isooctane was used to catalyze the esterification of fatty acids with 2-butanol. Various parameters affecting the catalytic activity of the microencapsulated cutinase, such as pH, w_o (molar ratio water/surfactant), temperature and substrate concentration were investigated. Maximal specific activity were obtained with w_o=13, at pH 10.7 and 35d`C. The cutinase showed a higher specific activity for short length fatty acids, namely butyric acid. Calculation of the apparent kinetic parameters (k_m and V_max) for the synthesis of butyl butyrate, showed a low apparent affinity of the cutinase in phosphatidylcholine reversed micelles for both substrates.     

The interaction of alcohol radicals with human hemoglobin

Aqueous deoxyhemoglobin solutions (2 mg/ml) were gamma-irradiated by a⁶⁰Co source in the presence of methanol, ethanol, 1-butanol andt-butanol under N₂O or argon. The effects of the interaction of the particular alcohol radical species with hemoglobin were determined according to the detected spectral alterations in the visible range. The amounts of stable final products in the form of methemoglobin (MetHb) and the sum of hemichromes and cholehemichromes (Hemichr) were estimated in irradiated preparations. For preparations irradiated under N₂O, the radiation yield for MetHb formation was threefold lower in the presence of ethanol and 1-butanol [G(MetHb)=0.33] compared with preparations irradiated in the presence oft-butanol or without alcohol [G(MetHb)=1.00]. The yield of hemichromes and cholehemichromes in preparations irradiated under N2O increased in the order: ethanol (G=0.38), 1-butanol (G=0.52),t-butanol (G=0.59), and in the absence of alcohol (G=0.72). The high effectivity oft-butanol radicals for iron oxidation and Hb destruction is apparently due to their oxidative properties, compared with the other radicals. It was also shown that ethanol radicals reduce MetHb 10 times more effectively [G(Fe(II)) = 2.5] compared witht-butanol radicals [G(Fe(II)) = 0.24]. For samples irradiated under argon all the observed changes were similar, regardless of the presence of alcohols. This effect can be attributed to reconstruction reactions of Hb molecules in the presence of both oxidizing (OH ort-but·) and reducing agents (e _aq ^/– ). The following sequence of effectivities of water radiolysis products and secondary alcohol radicals for hemoglobin destruction has been identified: meth·, eth·1-but·e _aq ^/– t-but··OH.This work was supported by State Committee for Scientific Research (Poland), grant no. 44509203     

The synthesis and turnover of spermidine and spermine in mouse brain

Following the administration to mice of radiolabeled putrescine by intraventricular injection, changes in the specific radioactivity of putrescine, spermidine, and spermine have been measured. Putrescine decline was biphasic, being more rapid over the first 12 hr(t _1/2=5 hr) than over the remainder of the 48-hr period (t _1/2=11 hr) that significant labeling was detected. Spermidine was rapidly labeled during the decline in putrescine radioactivity and maximum incòrporation of label occurred at 18 hr. Subsequently, spermidine specific activity declined with a half-life of 22 days. Spermine synthesis was slower, with maximum labeling occurring after 4 days. Spermine turnover, measured at a time when spermidine radioactivity had substantially declined, was extremely slow (t _1/2=92 days). The data supports the view that putrescine is a precursor of spermidine which in turn is required for spermine synthesis.     

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')₂ (t_1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')₂, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t_1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t_1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.     

Changes in the Activity of Superoxide Dismutase in Rauwolfia serpentina Benth. Callus Cultures Grown under Standard Conditions and Heat Shock

The rate of superoxide dismutase (SOD) accumulation inRauwolfia serpentinaBenth. cell culture under heat shock conditions (3 h, 45°C) decreased insignificantly (by 4%), whereas low positive temperature (24 h, 7°C) caused a drastic drop (by 48%). The observed decrease in the level of SOD activity resulted from a slowdown of the biosynthesis rate of the enzyme and a decrease in its concentration in the cultivated cells. In addition, a compensatory decrease in degradation of the active protein (K _d) was observed at low positive temperatures and, consequently, an increase in its half-life (tt _1/2), compensating partially for a deficiency in de novo synthesized SOD molecules. The parameters studied were restored to normal after a 24-h adaptation of cells under standard temperature conditions.     

A Semi-Rational Approach to Engineering Laccase Enzymes

In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t _1/2 at pH 5 = 35 days), neutral (t _1/2 at pH 7 = 38 days) and alkaline (t _1/2 at pH 10 = 62 days) pH values.     

Turnover of the fatty acyl and glycerol moieties of microsomal membrane lipids from liver, gill and muscle tissue of thermally acclimated rainbow trout,Salmo gairdneri

Summary Rainbow trout (Salmo gairdneri) acclimated to 5°C or 20°C were administered 2-³H-glycerol and 1-¹⁴C-acetate (63 Ci of each isotope/100 g body weight) via intraperitoneal injection, and subsequently maintained at their respective acclimation temperatures. Total lipid extracts (>80% phospholipid) were prepared from isolated microsomes of liver, gill and muscle tissue at various times over a three week period. Half-lives were determined independently for the fatty acyl and glycerol moieties from slopes of regression lines relating dpm/nmole phospholipidP _i vs time. In liver tissue, rates of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) turnover were also determined. Membrane turnover was most rapid in liver followed by gill and muscle. In liver, membrane fatty acids turned over more rapidly in warm-(t _1/2=3.4 days) than in cold-(t _1/2=6.8 days) acclimated fish, whereas in gill, rates of fatty acid turnover, did not differ significantly between acclimation groups. In contrast, rates of glycerol turnover were independent of acclimation temperature in liver, but faster (t _1/2=6.7 days) in warm- than cold- (t _1/2=15.1 days) acclimated fish in gill. In total lipid extracts, rates of fatty acid and glycerol turnover were equivalent in warm-acclimated fish, however, in cold-acclimated trout, there was a tendency for fatty acids (t _1/2=9.1 days) to turnover more rapidly than glycerol (t _1/2=15.1 days) in gill tissue, but more slowly (t _1/2=6.82 days) than glycerol (t _1/2=4.1 days) in liver. Although rates of glycerol turnover were equivalent in PC and PE of liver microsomes, the fatty acyl component turned over significantly more rapidly in PC at both acclimation temperatures. In cold-acclimated trout, rates of fatty acid and glycerol turnover were equivalent in PE, but the fatty acyl moiety of PC (t _1/2=4.7 days) turned over significantly more rapidly than glycerol (t _1/2=7.5 days). These results were interpreted as indicating that: (1) acclimation temperature independently influenced rates of fatty acid and glycerol turnover in a tissue specific manner, (2) a deacylation-reacylation pathway was activated in both liver and gill as a consequence of cold acclimation, but that liver tissue was more effective than gill in reutilizing the fatty acids released by phospholipase activity, and (3), in liver microsomes, patterns of turnover were phospholipid specific, with PC and PE differing either in the susceptibility of their acyl groups to degradation, or in their ability to reutilize fatty acids cleaved during membrane turnover at cold temperatures.     

Nucleic acid-solvent interactions: temperature dependence of the heat of solution of thymine in water and ethanol

The heats of solution of thymine in water and ethanol have been determined calorimetrically as a function of temperature. These data, along with solubility data, have been used to calculate the thermodynamic quantities (ΔG_t, ΔH_t, ΔS_t and ΔC_p,t) associated with the transfer of thymine from ethanol to water. Since ΔS_t = ?2 cal/mole deg and ΔC_p,t = 0, it has been concluded that hydrophobic bonding does not play an important role in the thermocynamic stability of nucleic acids. However, large heat capacities of solution of thymine are observed in both solvents (ΔC°_p2 = 45 ± 4 cal/mole deg). This is explained in terms of temperature variation in the degree of solvent–solute hydrogen bonding. It is our proposal that the components of macromolecules (i.e., nucleic acid bases and amino acids) do not make all possible hydrogen bonds with the solvent in the vicinity of room temperature. Thus the thermodynamic contribution of hydrogen bonding to the stability of macromolecules in aqueous solution must be reassessed.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Synthesis of fatty acid esters by a recombinant cutinase in reversed micelles

Fusarium solani pisi recombinant cutinase, solubilized in AOT/isooctane-reversed micelles, was used to catalyze the esterification of fatty acids with aliphatic alcohols. Some relevant parameters for the enzyme activity such as pH, W(o) (water/surfactant molar ratio), temperature, and substrate concentration were optimized. Maximal specific activity was obtained for hexanol. The cutinase showed selectivity for short-chain fatty acids. The stability of the microencapsulated cutinase was investigated at various concentrations of water and different values of pH. Oleic acid had a negative effect on the cutinase stability, while hexanol proved to be a strong stabilizer increasing the half-life of the enzyme about 45 times. (c) 1993 John Wiley & Sons, Inc.     

Enzymatic preparation of a carbohydrate ester of medium-chain fatty acid in supercritical carbon dioxide

Acylation of methyl--d-fructofuranoside and caprylic acid with an immobilized lipase, Novozym 435, was carried out in supercritical carbon dioxide (SCCO₂) with tert-butanol as a co-solvent. Initial rate of acylation was 12-fold higher in SCCO₂ than in tert-butanol. The equilibrium conversion was increased up to 70% with an increase in the molar ratio up to a maximum of 1:20 (methyl--d-fructofuranoside:caprylic acid) at 16 MPa and 70 °C.     

Different glycosylation of cadherins from human bladder non-malignant and cancer cell lines

Background  

We describe an alternative method to determine mRNA half-life (t_1/2) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability.     

Effects of Commercial Fatty Acids on Cutinase Release by Ascochyta pisi

The effect of a range of commercially available C₁₆ and C₁₈ fatty acids, on the release of cutinase by Ascochyta pisi, was studied. When juniperic acid was used as sole carbon source, cutinase activity was released in the culture medium, and was markedly enhanced by post-treatment of A. pisi cultures with acetone used as enzyme extractor. Without acetone, less cutinase was naturally released in culture medium containing juniperic acid at 0.5 %, than at 0.0l % or 0.05 %. Upon post-treatment with acetone, the same level of cutinase was released with all three concentrations, thus suggesting that the enzyme was induced, but not completely released in the presence of 0.5 % juniperic acid. When ricinelaidic or ricinoleic acids were supplemented at 0.5 % to cutin in the culture medium, they strongly inhibited the release of cutinase, even with acetone post-treatment. Comparable inhibition by ricinoleic acid was also observed when juniperic acid was used as cutinase inducer, thus suggesting that not only the release, but also the production of cutinase were inhibited.     

Stability of 2'-deoxyxanthosine in DNA

The deamination of nucleobases in DNA occurs by a variety of mechanisms and results in the formation of hypoxanthine from adenine, uracil from cytosine, and xanthine and oxanine from guanine. 2′-Deoxyxanthosine (dX) has been assumed to be an unstable lesion in cells, yet no study has been performed under biological conditions. We now report that dX is a relatively stable lesion at pH 7, 37°C and 110 mM ionic strength, with a half-life (t_1/2) of 2.4 years in double-stranded DNA. The stability of dX as a 2′-deoxynucleoside (t_1/2 = 3.7 min at pH 2; 1104 h at pH 6) was increased substantially upon incorporation into a single-stranded oligodeoxynucleotide, in which the half-life of dX at different pH values was found to range from 7.7 h at pH 2 to 17 700 h at pH 7. Incorporation of dX into a double-stranded oligodeoxynucleotide resulted in a statistically insignificant increase in the half-life to 20 900 h at pH 7. Data for the pH dependence of the stability of dX in single-stranded DNA were used to determine the rate constants for the acid-catalyzed (2.6 × 10^–5 s^–1) and pH-independent (1.4 × 10^–8 s^–1) depurination reactions for dX as well as the dissociation constant for the N⁷ position of dX (6.1 × 10^–4 M). We conclude that dX is a relatively stable lesion that could play a role in deamination-induced mutagenesis.     

Synthesis of alkyl esters by cutinase in miniemulsion and organic solvent media

The main objective of this work was studying and testing the nature and influence of reaction media (organic solvent vs. miniemulsion system) on the synthesis of alkyl esters catalyzed by Fusarium solani pisi cutinase. Ester synthesis and cutinase selectivity for different chain length of acids and alcohols (ethyl and hexyl) were evaluated. In iso-octane, after 1 h of reaction, cutinase exhibits rates of esterification between 0.24 μmol x mg^–1 x min^–1 for ethyl oleate and 1.15 μmol x mg^–1 x min^–1 for ethyl butyrate, while in a miniemulsion system the rates were from 0.05 for ethyl heptanoate to 0.76 μmol x mg^–1 x min^–1 for ethyl decanoate. The reaction rate for the synthesis of hexyl esters in a miniemulsion system was from 0.19 for hexyl heptanoate to 1.07 μmol x mg^–1 x min^–1 for hexyl decanoate. High conversion yields of 95% at equilibrium after 8 h of reaction in iso-octane for pentanoic acid (C₅) with ethanol at equimolar concentration (0.1 M) was achieved. Additionally, this work showed that a significant and unexpected shift in cutinase selectivity occurred towards longer chain length carboxylic acids (C₈–C₁₀) in miniemulsion system as compared to organic solvent (iso-octane) and previous studies in reverse micellar systems. The possibility of working with higher concentration of substrates, without inhibitory effect on the enzyme, was another advantage of the miniemulsion system.     

Protein stabilization with a dipeptide‐mimic triazine‐scaffolded synthetic affinity ligand

Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity‐like interactions with a biomimetic triazine‐scaffolded ligand. A synthetic lead compound (ligand 3′/11, K_a ≈ 10⁴ M^?1) was selected from a previously screened solid‐phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well‐established solid‐phase synthesis method, was able to strongly bind cutinase and led to impressive half‐lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25‐ and 57‐fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability. Copyright © 2013 John Wiley & Sons, Ltd.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis

A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and k_cat values of 2.72 and 6.03 s^?1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.     

Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E?I complex and first-order rate constant for inactivation were determined by incubation (K_i* = 26.6?±?2.8 µM, k₂ = 12.2?±?0.6 min–1) or progress curve (K_i* = 6.5?±?1.5 µM, k₂ = 0.11?±?0.01 min–1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t_1/2 = 11.3?±?0.2?h), matrix-assisted laser desorption/ionization (MALDI, t_1/2 = 13.5?±?0.4?h), and electro-spray ionization (ESI, t_1/2 = 12.2?±?0.5?h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.