Various type immobilizations of Isocitrate dehydrogenases enzyme on hyaluronic acid modified magnetic nanoparticles as stable biocatalysts.

Magnetic nanoparticles (MNPs) were modified by hyaluronic acid (HA). After the process of functionalization, two different strategies have been used to immobilize isocitrate dehydrogenases (IDH) on MNPs. In the first strategy, cross-linked enzyme aggregates were prepared. For this, firstly hyaluronic acid modified magnetic nanoparticles cross-linked enzyme fine aggregates of isocitrate dehydrogenases (IDH/HA/MNPs-CLEAs) were synthesized, and secondly bovine serum albumin (BSA) as co-feeder was used to synthesize the IDH/BSA/HA/MNPs-CLEAs. In the second strategy, the IDH was effectively immobilized on the HA/MNPs surface. The features of MNPs and its derivatives have been studied by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM), and zeta potential measurements. The activity and stability of IDH in IDH/HA/MNPs, IDH/HA/MNPs-CLEAs, and IDH/BSA/HA/MNPs-CLEAs were enhanced. Besides, the enzyme immobilized was readily separated via external magnet from the reaction medium and reused many times. The acquired findings indicate that HA/MNPs are a novel binder/support system to IDH, and IDH immobilized on this system can become a very important biocatalyst working with high accuracy and sensitivity for the determination of magnesium in drinking water and other biological solutions.

The endothelial nitric oxide synthase gene G894T, glutathione S-transferase (GSTM1 and GSTT1) polymorphisms as a risk factor in the patient with nephrolithiasis.

BACKGROUND: The genetic features indicate a crucial role in nephrolithiasis. The present study was aimed to investigate the role of Glutathione-S-transferase Mu (GSTM1), Glutathione-S- transferase Theta (GSTT1) and endothelial nitric oxide synthase (eNOs) gene polymorphism in nephrolithiasis. METHODS: We involved a case-control study in which 480 individuals were divided into 240 healthy control and 240 patients with nephrolithiasis. For each patient and control, we measured biochemical criteria, levels of glutathione S-transferase, eNOs, GSTM1, GSTT1genes and eNOS genes polymorphism by PCR-RFLP. RESULTS: GSTM1 and GSTT1 null genotypes are not a risk features for nephrolithiasis. The eNOS frequency GG, GT, and TT genotypes by using Ban II enzyme as restriction enzyme were found to be (48.33, 36.67, and 15.00) %. The eNOS frequency TT, GT, and GG genotypes by using the Ban II enzyme as restriction enzyme were found to be 15.84, 25.83, and 58.33%, respectively. The result showed an increase in serum eNOs levels were in the patient's group comparing to control. CONCLUSIONS: This work is the first in the literature to study the relation between eNOs genes polymorphisms and nephrolithiasis. The results conclude that TT genotypes in the eNOs genes are associated with an increase the oxidative stress in patients.

Development of magnetic cross-linked peroxidase aggregates on starch as enhancement template and their application for decolorization.

The cross-linked enzyme aggregates (CLEAs) technology has been considered as an efficient and carrier free immobilizing method. The importance of this method comes from its simplicity and low-cost procedure, along with the utilization probability of the crude enzyme extract. The functionalized magnetic cross linked peroxidase aggregate (MNPs-TA-CLEAs-peroxidase) was prepared successfully using different aggregation agents, followed by crosslinking with glutaraldehyde. Herein, peroxidase enzyme was extracted from cabbage leaves. The beneficial of immobilized peroxidase was confirmed by decolorization studies of different types of dyes. Even the small amount of MNPs-TA-starch-CLEAs-peroxidase was able to remove a higher content of methylene blue, Congo red, indigo carmine, and malachite green (93.18, 90.0, 68.0 and 55.0%), respectively, comparing to the free enzyme. The ability of MNPs-CLEAs-peroxidase to decolorize different types of dyes, may give a suggestion of its possible usage in environmental biotechnology, especially in wastewater treatment.

The novel multi cross-linked enzyme aggregates of protease, lipase, and catalase production from the sunflower seeds, characterization and application.

The cross-linked enzyme aggregates (CLEAs) have numerous economic advantages in the industrial bio catalysis. In the present study, the multi CLEAs containing protease, catalase, and lipase from the sunflower seeds using starch as a cofeeder as well as bovine serum albumin (BSA) are designed and prepared successfully. After optimization, multi CLEAs of enzyme have been prepared with ammonium sulfate (55% w/v), glutaraldehyde (100 mM), and 8 mg/mL of starch or 20 mg/mL of BSA. The activity recovery of protease, catalase, and lipase multi CLEAs-starch are 87, 61, and 60%, respectively. Whereas, CLEAs prepared with BSA are 74, 61, and 50% activity and multi CLEAs only 60, 44, and 41% of protease, catalase, and lipase, respectively. The multi CLEAs were used to catalyze the reactions for enhanced washing process. After adding multi CLEAs-starch, the stain removal percentage of detergents is enhanced by 83%.The present study reports a high stability, simplicity, low cost, and recyclability of the novel multi CLEAs from the sunflower seeds that make them efficient as a highly active biocatalysts in the biotechnological applications. We believe that these novel multi CLEAs present a new approach to the synthesis of multi enzyme biocatalysts from the cheap and friendly environmental sources.

Lipase-based on starch material as a development matrix with magnetite cross-linked enzyme aggregates and its application.

The Fe3O4 magnetic nanoparticles were prepared by precipitating ferrous ion (Fe2+) and ferric ion (Fe3+) in alkaline solution. The Fe3O4 magnetic nanoparticles were modified by tannic acid. After functionalization process, two methods were used to immobilize Lipase on Fe3O4 magnetic nanoparticles. In the first method, novel tannic acid magnetic cross-linked enzyme aggregates of lipase (TA-MNPs-CLEAs) were synthesized by cross-linking of lipase aggregates and starch as co-feeder with Fe3O4 magnetic nanoparticles improved by tannic acid (TA-MNPs). In the second method, the lipase was successfully immobilized on the surface of TA-MNPs. The properties of Fe3O4 and its modified forms were examined by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), scanning electron microscopy (SEM) and zeta potential measurements. Novel TA-MNPs-lipase and TA-MNPs-CLEAs-starch-lipase were enhanced and provided an effective method to improve the activity and stability of lipase for biodiesel production. Using 1% TA-MNPs-lipase and TA-MNPs-CLEAs-starch (w/w of oil) conversions around 67.87, and 85.88%, respectively, were obtained at 40 °C after 2 h of reaction. Furthermore, the immobilized enzyme was easily recovered from the reaction mixture and reused. The obtained results suggest that TA-MNPs-lipase and TA-MNPs-CLEAs-starch-lipase can become a powerful biocatalyst for biodiesel production.

Evaluation of different saccharides and chitin as eco-friendly additive to improve the magnetic cross-linked enzyme aggregates (CLEAs) activities.

The cross-linked enzyme aggregates (CLEAs) involves formation of a number of covalent bonds between enzyme and the matrix using glutaraldehyde. In general, amino groups of lysine, sulfhydryl groups of cysteine, phenolic OH groups of tyrosine, or imidazol group of histidine are used for enzyme binding under mild conditions. The main advantage of this method is its simplicity, economic advantages in the industrial bio catalysis. The Fe3O4 magnetic nanoparticles were synthesized by coprecipitating Fe2+and Fe3+in alkaline solution. Tannic acid was used to functionalize the Fe3O4 magnetic nanoparticles. After functionalization process, tannic acid magnetic cross-linked enzyme aggregates of enzyme (TA-MNPs-CLEAs) were prepared by cross-linking of enzyme aggregates with different saccharides as additive. The present result reported high stability, simplicity, low cost and recyclability of a saccharide-TA-MNPs-CLEAs-enzyme make it efficient as a highly active biocatalyst in biotechnological applications. The obtained results suggest that disaccharides (maltose, sucrose and lactose) and polysaccharide such as starch are eco-friendly additives to TA-MNPs-lipase and TA-MNPs-CLEAs-peroxidase and can become a powerful biocatalyst in industry applications.

Genetic polymorphisms of human transcription factor-7 like 2 (TCF7L2), ß-defensin (DEFB1) and CD14 genes in nephrolithiasis patients.

BACKGROUND: It is assumed that genetic factors play crucial role in nephrolithiasis. The present study was conducted to explore the role of Human Transcription Factor-7 like-2 (TCF7L2) ß-defensin (DEFB1) and CD14 gene polymorphism in development and progression of nephrolithiasis. METHODS: The genotypes of TCF7L2, DEFB1 and CD14 polymorphism were determined in 240 nephrolithiasis patients and 240 healthy controls by restriction digestion method of PCR. The levels of serum TCF7L2, DEFB1, CD14, uric acid and other biochemical parameters were measured both in nephrolithiasis patients and healthy control. RESULTS: The patients and control groups showed 30% and 50% 1654 AA DEFB1 genotype respectively. The Allele frequency in case of patient's group was 63.67% while in control group it was 36.33%. The mean serum DEFB1 levels of the patients and control groups attained were 115.66 and 239.43 pg/mL respectively. The allele frequency of TCF7L2 in patients and controls were 44.17% and 70.0% for C-allele, 55.83% and 30.00% for T-allele respectively. The mean of serum TCF7L2 levels were significantly decreased in patients compared to control group. CONCLUSIONS: The present findings are first of its class that validates a considerable connection of DEFB1 and TCF7L2 gene polymorphisms with nephrolithiasis and could probably act as indicators to estimate the risk associated to nephrolithiasis.

Characterization and immobilization of protease and lipase on chitin-starch material as a novel matrix.

Protease and lipase were purified from sunflower seeds by frequent purification steps with molecular weights of 72.90 kDa and 27.50 kDa, respectively. The purified lipase and protease were immobilized on various carriers by different methods of immobilization including physical adsorption, ionic binding and covalent binding. The enzymes prepared by covalent binding on a new support materials were made via the combination of chitin and starch had the highest activates. The immobilization was carried out in a simpler way compared with the other immobilization methods which require various chemicals and complicated procedures which is difficult and expensive. The influence of reusability, pH, thermal and storage stability of immobilizing enzymes compared to the free enzyme were studied. The immobilizing protease and lipase with chitin and chitin + starch were used to catalyze reactions through enhanced washing process. After adding immobilizing enzymes with chitin and chitin starch, the stain removal percentage of detergents was enhanced by 78% and 84%, respectively. We approve that these novel immobilizing protease and lipase with chitin + starch present a new approach to the synthesis of multi enzyme biocatalysts from cheap and friendly environmental sources.

Association of MMP-9 gene polymorphisms with nephrolithiasis patients.

BACKGROUND: Nephrolithiasis is one of the causes which lead to chronic kidney disease (CKD). Matrix metalloproteinases (MMPs) are endopeptidases degrading extracellular matrix which correlate with the pathogenesis of atherosclerosis. The current study was designed to analyze the association of (R279Q, C1562T) polymorphism of MMP-9 with nephrolithiasis patients. METHODS: Genotyping of MMP-9/R279Q and of MMP-9/C1562T polymorphism were carried out by PCR-based restriction digestion method. Serum level of MMP-9, oxidative stress marker, MDA, and uric acid were measured in patients and control. RESULTS: Allele frequencies of the MMP-9/C1562T polymorphism for C and T allele were 71.25% and 28.75% in patients, 87.08% and 12.92% in control respectively. The homozygote TT was more frequent in the nephrolithiasis patients group, while T allele frequency was significantly higher in the nephrolithiasis patients group than in the control group. The patients with CT and TT genotype showed a significant increase in serum MMP-9, Total Oxidant Status (TOS), Oxidative Stress Index (OSI), Malondialdehyde (MDA), and uric acid when compared to CC genotype in patients with nephrolithiasis. The R279Q polymorphism site with regard to the relationship with nephrolithiasis was not significant. CONCLUSION: The result indicates that patients with TT genotype had an increased risk of stones. Also, the results demonstrate that TT allele of the C1562T polymorphism in the MMP-9gene is related with an increase of oxidative stress in nephrolithiasis patients and may possibly impose a risk for cardiovascular diseases in patients with TT genotype of MMP-9.

Determination of alpha-2-MRAP gene polymorphisms in nephrolithiasis patients.

BACKGROUND: The intron 5 insertion/deletion polymorphism of Alpha-2-macroglobulin receptor-associated protein gene (Alpha-2-MRAP) has been implicated in numerous diseases. The current study was designed to analyze the association of intron 5 insertion/deletion polymorphism of Alpha-2-MRAP with nephrolithiasis patients. METHODS: PCR was conducted on genomic DNA of patients and control to look for Alpha-2-MRAP insertion/deletion polymorphism. Besides that, serum level of Alpha-2-MRAP, oxidative stress marker myeloperoxidase, Malondialdehyde (MDA), Advanced oxidation protein products (AOPP), and uric acid were determined. RESULTS: The D and I allele frequencies were 57.50% and 42.50% in patients, 77.50% and 22.50% in control, individually. The result showed that II genotype was associated with nephrolithiasis patients group. A significant decrease was observed in serum Alpha-2-MRAP,myeloperoxidase and TAS,while TOS,OSI,MDA,AOPP and uric acid were substantially increased in II and ID when compared to DD genotype in patients with nephrolithiasis. CONCLUSION: Our results demonstrate for the first time that patients with II genotype had an increased risk of stones. Also, the results demonstrate that I allele of the 5 insertion/deletion polymorphism in the Alpha-2-MRAP gene is related with an increase of oxidative stress in nephrolithiasis patients and may possibly impose a risk for cardiovascular diseases in patients with II genotype of Alpha-2-MRAP.

Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals.

OBJECTIVE: Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. METHODS: Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. RESULTS: The result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01). CONCLUSION: This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

Effects of Acute Lymphoblastic Leukemia on Ceruloplasmin Oxidase, Copper and Several Markers of Oxidative Damage, in Children.

BACKGROUND: Acute leukaemia is characterized by fast growth of abnormal clones of haemopoietic precursor cells inside bone marrow leading to undue accumulation in the bone marrow. Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. MATERIALS AND METHODS: The study concerned 50 children diagnosed with ALL (mean age, 8.55±2.54) compared to 40 healthy controls (mean age, 8.00±1.85). The Hb, serum copper, ceruloplasmin oxidase, advanced oxidation protein products (AOPPs), total antioxidant activity (TAA) and protein were measured in all groups. One proteinous component was isolated by gel filtration chromatography from the precipitate produced by polyethylene glycol. RESULTS: Significantly higher levels of AOPP, copper and decrease in total antioxidant activity were noted in the cases. Statistical analysis also showed a significant increase (p<0.01) in the activity of serum ceruloplasmin oxidase in patients with ALL compared to normal subjects. The maximum velocity (Vmax) and Michaelis constant had values of 104.2 U/L and 11.7 mM, respectively. The ΔH* values for ceruloplasmin oxidase in ALL patients were positive, confirming the reaction to be endothermic. CONCLUSIONS: The results from this study showed a significant increase in AOPP, ceruloplasmine oxidase and decrease in total antioxidant activity .These parameters may play a role in development of DNA damage in childhood patients with acute lymphoblastic leukemia (ALL). The ΔS* and ΔG* values were negative, these refer that the reaction of ES formation is spontaneous, but needs energy in a so-called endergonic reaction. Also the negative ΔS* value of ceruloplasmin oxidase indicates that the complex [ES*] is further modulated through increasing structure arrangement.

CD26: A Prognostic Marker of Acute Lymphoblastic Leukemia in Children in the Post Remission Induction Phase.

BACKGROUND: ALL is an irredeemable disease due to the resistance to treatment. There are several influences which are involved in such resistance to chemotherapy, including oxidative stress as a result of the generation of reactive oxygen species (ROS) and presence of hypodiploid cells. Cluster of differentiation 26 (CD26), also known as dipeptidyl peptidase-4, is a 110 kDa, multifunctional, membrane-bound glycoprotein. AIM AND OBJECTIVES: The aim of this study was to evaluate the clinical significance of serum CD26 in patients with acute lymphoblastic leukaemia patients in the post remission induction phase, as well as the relationship between CD26 activity and the oxidative stress status. MATERIALS AND METHODS: CD26, total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI), in addition to activity of related enzymes myeloperoxidase, glutathione- s-transferase and xanthine oxidase, were analysed in sixty children with acute lymphoblastic leukaemia in the post remission induction phase. RESULTS: The study showed significant elevation in CD26, TOS and OSI levels in patients with acute lymphoblastic leukaemia in the post remission induction phase in comparison to healthy control samples. In contrast, myeloperoxidase, glutathione-s-transferase and xanthine oxidase activities were decreased significantly. A significant correlation between CD26 concentration and some oxidative stress parameters was evident in ALL patients. CONCLUSIONS: Serum levels of CD26 appear to be useful as a new biomarker of oxidative stress in children with acute lymphoblastic leukaemia in the post remission induction phase, and levels of antioxidants must be regularly estimated during the treatment of children with ALL.

Correlation of inhibin and several antioxidants in children with acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is most common in childhood. Inhibin (a non-steroidal glycoprotein hormone of gonadal origin) can be used as marker of fertility. The current study was conducted to evaluate inhibin levels in ALL patients and to estimate its correlation with some antioxidants in these in comparison with control subjects. MATERIALS AND METHODS: This study was conducted on sixty patients with ALL and thirty children as controls. Fasting blood samples were taken from each subject and analyzed for haemoglobin, serum protein, vitamin E and C, in addition to glutathione and inhibin. RESULTS: The results of the study showed highly significant decreases (p<0.001) in haemoglobin, glutathione and inhibin levels with significant decreases (p<0.05) in serum protein and vitamin E levels for patients group in comparison with controls while there was no significant differences in vitamin C. Moreover, there were significant correlations between inhibin levels and serum protein, glutathione and both vitamins (E and C) in the ALL patient group (r= 0.81, 0.80, 0.77 and 0.69, respectively). CONCLUSIONS: The present results indicated infertility in patients with ALL demonstrated by low inhibin level as a consequence of abnormality in anti-oxidative metabolism due to the cancer process. So, it can be suggested the need for routine measurement of inhibin for leukemic patients to estimate the action of hormones of gonadal origin.