Immobilization of Ene Reductase in Polyvinyl Alcohol Hydrogel.

In this study, ene reductase (ER) was entrapped in polyvinyl alcohol hydrogel, adsorbed on montmorillonite and immobilized covalently on glutaraldehyde activated 3-aminopropyl-functionalized silica gel. Although protein recovery yields were at least 85% for adsorption and covalent immobilization, only the encapsulated ER showed activity. The activity of free and entrapped ER preparations was measured by following NADPH-dependent reduction of 2-cyclohexen-1-one. The both protein recovery and activity recovery yields were calculated as 100% when 1 mg protein was used for immobilization. The both free and entrapped ER preparations showed the same optimum pH and temperature as 7.0 and 30 °C, respectively. The entrapped ER showed 34.4-fold more thermal stability than that of the free ER at 30 °C. Michaelis-Menten constant and maximum velocity values were 0.25 mM and 1.2 U/mg protein, respectively for the free ER towards 2-cyclohexen-1-one. The corresponding values were 1.5 mM and 0.9 U/mg protein for the entrapped ER. The results of time-course reduction of 2-cyclohexen-1-one showed that the entrapped ER catalyzed the reaction as effectively as the free ER. The entrapped ER remained 85% of its initial activity after 10 reused cycles.

Effective immobilization of lactate dehydrogenase onto mesoporous silica.

This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica. To develop mesoporous silica as support material for use in l-LDH immobilization, the particle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. The effect of some parameters such as glutaraldehyde concentration, immobilization pH, initial enzyme concentration, and immobilization time was investigated and the optimum conditions for these parameters were determined as 1% (w/v), pH 8.0, 1 mg/ml, and 120 min, respectively. The maximum working pH and temperature for the oxidation of lactate to pyruvate reaction were determined as 10.0 and 35°C for free and 9.0 and 40°C for immobilized l-LDH, respectively. The kinetic parameters (Km and Vmax ) of l-LDH for the oxidation of lactate to pyruvate reaction were examined as 1.02 mM and 7.58 U/mg protein for free and 0.635 mM and 1.7 U/mg protein for immobilized l-LDH, respectively. Moreover, the immobilized l-LDH was 1.3-fold more stable than free l-LDH at 25°C according to calculated t1/2 values. The immobilized l-LDH retained 80% of its initial activity in a batch reactor after 14 reuses.

Modified silicates and carbon nanotubes for immobilization of lipase from Rhizomucor miehei: Effect of support and immobilization technique on the catalytic performance of the immobilized biocatalysts.

Lipase from Rhizomucor miehei (RML) was covalently immobilized on different supports, two silica gels and two carbon nanotube samples, using two different strategies. RML was immobilized on 3-carboxypropyl silica gel (RML@Si-COOH) and multi-wall carbon nanotubes containing carboxylic acid functionalities (RML@MCNT-COOH) using a two-step carbodiimide activation/immobilization reaction. Moreover, the enzyme was also immobilized on 3-aminopropyl silica (RML@Si-Glu) and single-wall carbon nanotubes functionalized with 3-APTES and activated with glutaraldehyde (RML@SCNT-Glu). Before and after RML immobilization, the structurel properties of supports were characterized and compared in detail. After immobilization, the expressed activities were 36.9, 90.2, 16.9, and 26.1 % for RML@Si-COOH, RML@Si-Glu, RML@MCNT-COOH, and RML@SCNT-Glu, respectively. The kinetic parameters of free and immobilized RML samples were determined for three substrates, p-nitrophenyl acetate, p-nitrophenyl butyrate and p-nitrophenyl palmitate, and RML@Si-Glu showed higher catalytic efficiency than the other immobilized RML samples. RML@Si-COOH, RML@Si-Glu, RML@MCNT-COOH, and RML@SCNT-Glu exhibited 5.8, 7.6, 4.2 and 4.6 folds longer half-life values than those of the free enzyme at pH 7.5 and 40 °C. Recyclability studies showed that all the immobilized RML biocatalysts retained over 90 % of their initial activities after ten cycles in the hydrolysis of p-nitrophenyl butyrate.

Simple Categorization of Human Figure Drawings at 5 Years of Age as an Indicator of Developmental Delay.

Purpose: To elucidate the association between developmental stage of human figure drawing(HFD) and fine motor control, visual perception, and further investigate its potential to be used for screening developmental delay. Methods: Participants were 301 children at 5½ years of age, 176 born preterm and 125 at term, whose HFDs were categorized into six developmental stages. Motor-Free-Visual-Perception Test, Movement-ABC, Performance Intelligence Quotient (PIQ: Wechsler Scale), and the Visual-Motor Integration test were used. Fine motor functions were explored using ImageJ. Results: Age-expected HFDs were drawn by 87% of the children, while 13%, mostly preterm boys, drew immature ones. Stages of HFD were related to both PIQ and Movement-ABC. Visuomotor control and visual perception significantly explained the HFD. The sensitivity and specificity of HFD as a screening tool was moderate to good. Conclusions: HFD is influenced by visual perception and visuomotor control and can be used for screening developmental delay at preschool age.

Covalent Immobilization and Characterization of a Novel Pullulanase from Fontibacillus sp. Strain DSHK 107 onto Florisil® and Nano-silica for Pullulan Hydrolysis.

A novel pullulanase partially purified from Fontibacillus sp. was covalently immobilized on Florisil® and nano-silica through both glutaraldehyde and (3-glycidyloxypropyl)trimethoxysilane spacer arms. The pullulanase immobilized on Florisil® and nano-silica through glutaraldehyde spacer arm showed 85 and 190 % activity of its free form, respectively, whereas no activity was observed when it was immobilized on the same supports through (3-glycidyloxypropyl)trimethoxysilane spacer arm. The maximum working pHs of both the immobilized pullulanases on Florisil® and nano-silica through glutaraldehyde spacer arm were determined as 5.0; however, the maximum working pH of the free pullulanase was pH 6.0. The maximum temperatures of all the pullulanase preparations were determined as 35 °C. The apparent K m values were 1.49, 1.54, and 0.59 mg/mL pullunan, respectively, for the free and immobilized pullulanases on Florisil® and nano-silica. The corresponding apparent V max values were 0.59, 1.53, and 1.57 U mg prot.(-1) min.(-1). Thermal stability of pullulanases immobilized on Florisil® and nano-silica was enhanced 6.5- and 15.6-folds, respectively at 35 °C and 6.6- and 16.0-folds, respectively, at 50 °C. The pullulanases immobilized on Florisil® and nano-silica protected 71 and 90 % of their initial activities after 10 reuses.

Immobilization of pectinase on silica-based supports: Impacts of particle size and spacer arm on the activity.

The pectinase was separately immobilized onto Florisil and nano silica supports through both glutaraldehyde and 3-glyoxypropyltrietoxysilane spacer arms. The effects of spacer arm, particle size of support and ionic liquids on the activities of pectinase preparations were investigated. The immobilization of pectinase onto Florisil and nano silica through 3-glyoxypropyltrietoxysilane spacer arm completely led to inactivation of enzyme; however, 10 and 75% pectinase activity were retained when it was immobilized through glutaraldehyde spacer arm onto Florisil and nano silica, respectively. The pectinase immobilized onto nano silica through glutaraldehyde spacer arm showed 6.3-fold higher catalytic efficiency than that of the pectinase immobilized onto Florisil through same spacer arm. A 2.3-fold increase in thermal stability of pectinase was provided upon immobilization onto nano silica at 35°C. The effects of IL/buffer mixture and volume ratio of IL/buffer mixture on the catalytic activities of free and immobilized pectinase preparations were also tested. All the pectinase preparations showed highest activity in 10% (v/v) 1-butyl-3-methylimidazolium hexafluorophosphate containing medium and their activities significantly affected from the concentration of 1-butyl-3-methylimidazolium hexafluorophosphate.

Highly stable and reusable immobilized formate dehydrogenases: Promising biocatalysts for in situ regeneration of NADH.

This study aimed to prepare robust immobilized formate dehydrogenase (FDH) preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150), Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU), and Immobead 150 support functionalized with aldehyde groups (FDHIALD). The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t 1/2) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.

Enantioselective Synthesis of Various Cyanohydrins Using Covalently Immobilized Preparations of Hydroxynitrile Lyase from Prunus dulcis.

The carrier-based and carrier-free (cross-linked enzyme aggregate) covalent immobilizations of Prunus dulcis hydroxynitrile lyase were investigated. The immobilized preparations were tested for enantioselective carbon-carbon bond formation activity in the biphasic medium. Of the tested preparations, only cross-linked enzyme aggregate of P. dulcis hydroxynitrile lyase (PdHNL-CLEA) achieved the synthesis of (R)-mandelonitrile with 93% yield and 99% enantiopurity. PdHNL-CLEA was also used in the synthesis of various (R)-cyanohydrins from corresponding aldehydes/ketones and hydrocyanic acid. When 4-methoxybenzaldehyde, 4-methyl benzaldehyde, and 4-hydroxybenzaldehyde were used as substrates, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were obtained as 95-95, 85-79, and 2-25%, respectively, after 96 h at pH 4.0 and 5 °C. For acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, and 4-iodoacetophenone, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were 1-99, 20-84, 11-95, 5-99, and 3-24%, respectively at the same conditions. The results demonstrate PdHNL-CLEA can be effectively used in the synthesis of (R)-mandelonitrile.

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins.

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100-99, 100-21, 100-99, 83-91, 100-99, 100-72, and 100-14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity.

Covalent immobilization of catalase onto spacer-arm attached modified florisil: characterization and application to batch and plug-flow type reactor systems.

Catalase was covalently immobilized onto florisil via glutaraldehyde (GA) and glutaraldehyde+6-amino hexanoic acid (6-AHA) (as a spacer arm). Immobilizations of catalase onto modified supports were optimized to improve the efficiency of the overall immobilization procedures. The V(max) values of catalase immobilized via glutaraldehyde (CIG) and catalase immobilized via glutaraldehyde+6-amino hexanoic acid (CIG-6-AHA) were about 0.6 and 3.4% of free catalase, respectively. The usage of 6-AHA as a spacer arm caused about 40 folds increase in catalytic efficiency of CIG-6-AHA (8.3 × 105 M⁻¹ s⁻¹) as compared to that of CIG (2.1 × 104 M⁻¹ s⁻¹). CIG and CIG-6-AHA retained 67 and 35% of their initial activities at 5 °C and 71 and 18% of their initial activities, respectively at room temperature at the end of 6 days. Operational stabilities of CIG and CIG-6-AHA were investigated in batch and plug-flow type reactors. The highest total amount of decomposed hydrogen peroxide (TAD-H2O2) was determined as 219.5 µmol for CIG-6-AHA in plug-flow type reactor.

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase.

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 µmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.

Simultaneous and sequential co-immobilization of glucose oxidase and catalase onto florisil.

The co-immobilization of Aspergillus niger glucose oxidase (GOD) with bovine liver catalase (CAT) onto florisil (magnesium silicate-based porous carrier) was investigated to improve the catalytic efficiency of GOD against H2O2 inactivation. The effect of the amount of bound CAT on the GOD activity was also studied for 12 different initial combinations of GOD and CAT, using simultaneous and sequential coupling. The sequentially co-immobilized GOD-CAT showed a higher efficiency than the simultaneously co-immobilized GOD-CAT in terms of the GOD activity and economic costs. The highest activity was shown by the sequentially co-immobilized GOD-CAT when the initial amounts of GOD and CAT were 10 mg and 5 mg per gram of carrier. The optimum pH, buffer concentration, and temperature for GOD activity for the same co-immobilized GOD-CAT sample were then determined as pH 6.5, 50 mM, and 30 degrees C, respectively. When compared with the individually immobilized GOD, the catalytic activity of the co-immobilized GOD-CAT was 70% higher, plus the reusability was more than two-fold. The storage stability of the co-immobilized GOD-CAT was also found to be higher than that of the free form at both 5 degrees C and 25 degrees C. The increased GOD activity and reusability resulting from the co-immobilization process may have been due to CAT protecting GOD from inactivation by H2O2 and supplying additional O2 to the reaction system.

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates.

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (florisil) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide which is the substrate of CAT was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in presence of both their substrate: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in presence of their substrates highly improved the activity and reusability of both enzymes.

Kinetic properties and storage stability of catalase immobilized on to florisil.

The covalent immobilization of bovine liver catalase (CAT) on to florisil via glutaraldehyde was investigated. Optimum immobilization pH and temperature were determined as pH 6.0, 10 degrees C respectively, while the amount of initial CAT per g of carrier and immobilization time was determined as 5 mg g(-1) and 120 min, respectively. The Vmax values for free and immobilized CAT were found to be 1.7 x 10(5) and 2.0 x 10(4) micromol H2O2 min(-1) mg protein(-1), respectively, whereas KM values were 33.3 mM and 1722.0 mM respectively. Operational stability was determined by using a stirred batch-type column reactor. Immobilized CAT retained about 40% of its initial activity after 50 uses. It showed higher storage stability than free CAT at 4 degrees C and 25 degrees C. Its storage stability increased with increasing relative humidity (RH) from 0 to 20% of the medium. The highest storage stability was obtained in 20% RH, however, further increase in RH from 40 to 100% significantly decreased the storage stability.

Radiological findings in the diagnosis of genitourinary candidiasis.

The presence of fungus balls within the collecting system is an important clue to the radiological diagnosis of genitourinary candidiasis. In this report, an 8-month-old infant with this opportunistic infection is described. Emphasis is placed on the radiological findings of renal candidiasis, including previously unreported MR appearances. Sonographic and Doppler findings of accompanying Candida epididymitis are also described.

Effects of sulfhydryl compounds on the inhibition of erythrocyte membrane Na+(-)K+ ATPase by ozone.

Exposure of human erythrocyte membranes to ozone (5 mumol/10 min) resulted in the inhibition of erythrocyte membrane Na+(-)K+ ATPase (EC.3.6.1.39). It was determined that, the degree of enzyme inhibition in the directly ozone exposed membranes was greater than that of membranes obtained from ozone exposed intact erythrocytes. In the presence of varying concentrations (0-1.0 mM) of dithiotrethiol or mercaptoethanol Na+(-)K+ ATPase activities of both types of ozone exposed membranes were increased almost proportionally with the concentration of dithiotrethiol or mercaptoethanol however, the activities were still lower than the normal Na+(-)K+ ATPase value. The results indicate that, dithiotrethiol or mercaptoethanol prevent the enzyme inhibition by ozone in vitro. This suggests that the membrane thiol groups are primary targets for ozone and thereby preventing the oxidation of essential functional groups of enzyme protein.

Radial scar and tubular carcinoma of the breast.

A case of tubular carcinoma within a radial scar pattern is reported. The aim of this study is to support the opinion of the authors who believe that there is a relationship between tubular carcinoma and radial scar. We think that surgical biopsy should be recommended in all cases of stellate lesions detected at mammography.

Mammography in men with breast cancer: review of the mammographic findings in five cases.

Male breast carcinoma is an uncommon malignancy. The age at diagnosis and tumour stage are more advanced in males than in females. Poor alertness of both men and physicians for this infrequent disease may account for the delay in diagnosis. In this study, we reviewed male breast carcinomas seen within a 3 year period and assessed the mammographic findings. There is limited experience in the diagnosis of breast carcinoma in male patients and this has been considerably influenced by the knowledge of female breast cancer. The diagnostic criteria for the female counterpart cannot always be safely applied to male breast cancer. Mammograms of men with breast cancer usually show an uncalcified subareolar mass, which may mimic or be obscured by gynaecomastia. Occasionally, punctate calcifications may indicate malignant disease in the male breast. Early detection of male breast cancer may prolong the survival rate as in cases of female breast cancer.