Immobilized lipase from potential lipolytic microbes for catalyzing biodiesel production using palm oil as feedstock

Biodiesel has been regarded as a biodegradable and non-polluting fuel. Enzymatic transesterification reaction for manufacturing biodiesel from vegetable oils with alcohol is an attractive approach. However, the cost of enzyme remains a barrier for its industrial implementation. The aim of this research was the screening of lipase-producing microorganisms and the studies of potential lipase-mediated biodiesel production using palm oil as substrate. A total of 360 strains of bacteria, yeasts and fungi were isolated and screened from the samples of oil-contaminated soil and waste water. Among all the screened microbes, the potential lipolytic bacterium,  Staphylococcus warneri, unicellular yeast, Candida rugosa and filamentous fungus, Fusarium solani were selected because of their high specific  activities. The lipase-producing conditions were subsequently optimized by using palm oil as an inducer and lipase activities were compared for both hydrolytic and synthetic catalysis. C. rugosa lipase, which exhibited the highest potential for catalyzing the biodiesel production, was further  purified and immobilized on various hydrophobic supports. The catalysis of transesterification between methanol and palm oil by the C. rugosa immobilized lipases revealed that immobilized lipase from C. rugosa on Sepabeads EC-OD was the most promising for further development as a biocatalyst for the application of enzyme-catalyzed biodiesel synthesis.Key words: Screening, lipases, immobilization, biodiesel

Effects of green tea extract treatment on erythropoiesis and iron parameters in iron-overloaded β-thalassemic mice

β-Thalassemia is characterized by ineffective erythropoiesis leading to chronic anemia. Thus, increased iron absorption from the duodenum and via blood transfusions is required to maintain normal blood hemoglobin (Hb) levels and iron chelators in the removal of excessive iron. Certain agents are also needed for the improvement of stress erythropoiesis and iron dysregulation. Green tea extract (GTE), which is rich in epigallocatechin-3-gallate (EGCG), is known to possess radical scavenging and iron-chelating activities. We aimed to assess the effects of green tea extract on erythroid regulators, iron mobilization and anti–lipid peroxidation in the liver, spleen, and kidneys of iron-loaded β-globin gene knockout thalassemic (BKO) mice. Our results indicate that treatments of green tea extract and/or deferiprone (DFP) diminished levels of plasma erythropoietin (EPO) and erythroferrone (ERFE), and consistently suppressed kidney Epo and spleen Erfe mRNA expressions (p <.05) in iron- loaded BKO mice when compared with untreated mice. Coincidently, the treatments decreased plasma ferritin (Ft) levels, iron content levels in the liver (p <.05), spleen (p <.05), and kidney tissues of iron–loaded BKO mice. Furthermore, lipid-peroxidation products in the tissues and plasma were also decreased when compared with untreated mice. This is the first evidence of the orchestral role of green tea extract abundant with epigallocatechin-3-gallate in improving ineffective erythropoiesis, iron dysregulation and oxidative stress in iron-overloaded β-thalassemic mice

A free weekly iron-folic acid supplementation and regular deworming program is associated with improved hemoglobin and iron status indicators in Vietnamese women

Background Anemia due to iron deficiency is recognized as one of the major nutritional deficiencies in women and children in developing countries. Daily iron supplementation for pregnant women is recommended in many countries although there are few reports of these programs working efficiently or effectively. Weekly iron-folic acid supplementation (WIFS) and regular deworming treatment is recommended for non-pregnant women living in areas with high rates of anemia. Following a baseline survey to assess the prevalence of anemia, iron deficiency and soil transmitted helminth infections, we implemented a program to make WIFS and regular deworming treatment freely and universally available for all women of reproductive age in two districts of a province in northern Vietnam over a 12 month period. The impact of the program at the population level was assessed in terms of: i) change in mean hemoglobin and iron status indicators, and ii) change in the prevalence of anemia, iron deficiency and hookworm infections. Method Distribution of WIFS and deworming were integrated with routine health services and made available to 52,000 women. Demographic data and blood and stool samples were collected in baseline, and three and 12-month post-implementation surveys using a population-based, stratified multi-stage cluster sampling design. Results The mean Hb increased by 9.6 g/L (95% CI, 5.7, 13.5, p < 0.001) during the study period. Anemia (Hb<120 g/L) was present in 131/349 (37.5%, 95% CI 31.3, 44.8) subjects at baseline, and in 70/363 (19.3%, 95% CI 14.0, 24.6) after twelve months. Iron deficiency reduced from 75/329 (22.8%, 95% CI 16.9, 28.6) to 33/353 (9.3%, 95% CI 5.7, 13.0) by the 12-mnth survey, and hookworm infection from 279/366 (76.2%,, 95% CI 68.6, 83.8) to 66/287 (23.0%, 95% CI 17.5, 28.5) over the same period. Conclusion A free, universal WIFS program with regular deworming was associated with reduced prevalence and severity of anemia, iron deficiency and h

Detection of cathepsin B up-regulation in neoplastic thyroid tissues by proteomic analysis

Nodular or multinodular goiter is the most common non-neoplastic thyroid disease and may be difficult to distinguish from true neoplastic thyroid diseases using microscopic criteria. We have used two-dimensional gel electrophoresis to study the protein patterns of thyroid tissues including normal thyroid, multinodular goiter, diffuse hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma. Specific proteins, in the region of molecular mass 15-30 kDa and isoelectric point 4.5-6.5, were identified by electrospray tandem mass spectrometry and protein sequencing. The most distinctive protein found is cathepsin B, which could be detected as four spots, with differential expression in different thyroid diseases. In particular, two of these cathepsin B spots CB2 and CB3 are strongly up-regulated in neoplastic diseases, compared to non-neoplastic diseases. In addition, overexpression of ATP synthase D chain and prohibitin were observed in papillary carcinoma, which should allow it to be differentiated from follicular carcinoma. Changes in expression of other proteins were also observed in disease states compared to normal tissues, namely translationally controlled tumor protein, thioredoxin peroxidase 1, glutathione-S-transferase P, DJ-1 protein, superoxide dismutase (Cu, Zn), and heat shock protein 27, but these changes are less characteristic, so they do not allow the differentiation between neoplastic and non-neoplastic tissues. Thus, the proteomic approach is a useful diagnostic tool for studying diseases involving the thyroid nodule

Rapid visual recovery following intravenous tocilizumab in glucocorticoid resistant refractory giant cell arteritis.

A 72-year-old man presented with a short history of headache, jaw claudication, double vision, amaurosis fugax and distended temporal arteries. A diagnosis of giant cell arteritis (GCA) was confirmed on temporal artery ultrasound and temporal artery biopsy. Despite treatment with high-dose oral glucocorticoid (GC) and multiple pulses of intravenous methylprednisolone, his vision deteriorated to hand movements in one eye. 8 mg/kg intravenous tocilizumab, a humanised, recombinant anti-IL-6 receptor antibody, was administered within 48 hours of vision loss and continued monthly, resulting in marked visual improvement within days, as well as sustained remission of GCA. This case suggests a possible role for tocilizumab as a rescue therapy to prevent or recover visual loss in patients with GCA resistant to GC treatment, termed refractory GCA. Further research is required to elucidate the role of intravenous administration of tocilizumab in this setting

Kinetics of a Two-Component p-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the Oxygenase

p-Hydroxyphenylacetate hydroxylase (HPAH) from 'Acinetobacter baumannii' catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins:  a flavin mononucleotide (FMN) reductase (C₁) and an oxygenase (C₂). C₁ catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C₂ in the hydroxylation reaction. C₁ is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH- from the enzyme. This study quantitatively shows the kinetics of how the C₁-bound FMN can be reduced and released to be used efficiently as the substrate for the C₂ reaction; additional FMN is not necessary. Reactions in which O₂ is rapidly mixed with solutions containing C₁-FMNH- and C₂ are very similar to those in which solutions containing O₂ are mixed with one containing the C₂-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C₂ and has already been completely transferred to C₂ before it reacts with oxygen. Rate constants for the transfer of FMNH- from C₁ to C₂ were found to be 0.35 and ≥74 s⁻¹ in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH- was also used to measure the dissociation rate of FMNH- from C₁. In the absence of HPA, FMNH- dissociates from C1 at 0.35 s⁻¹, while with HPA present it dissociates at 80 s⁻¹; these are the same rates as those for the transfer from C₁ to C₂. Therefore, the dissociation of FMNH- from C₁ is rate-limiting in the intermolecular transfer of FMNH- from C₁ to C₂, and this process is regulated by the presence of HPA. This regulation avoids the production of H₂O₂ in the absence of HPA. Our findings indicate that no protein-protein interactions between C₁ and C₂ are necessary for efficient transfer of FMNH- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH- from C₁

Luciferase from 'Vibrio campbellii' is more thermostable and binds reduced FMN better than its homologues

A new luciferase from 'V. campbellii' (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH-). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 4°C and pH 8. The measured Kd at 4°C for the binding of FMNH- to Lux_Vc was 1.8 μM whereas to Lux_Vh, it was 11 μM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t1/2 of 1020 min while Lux_Vh has t1/2 of 201 min at 37°C. The superior thermostability and tighter binding of FMNH- make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before

Use of 8-Substituted-FAD Analogues To Investigate the Hydroxylation Mechanism of the Flavoprotein 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) is a flavoprotein that catalyzes the oxygenation of MHPC to form α-(N-acetylaminomethylene)-succinic acid. Although formally similar to the oxygenation reactions catalyzed by phenol hydroxylases, MHPCO catalyzes the oxygenation of a pyridyl derivative rather than a simple phenol. Therefore, in this study, the mechanism of the reaction was investigated by replacing the natural cofactor FAD with FAD analogues having various substituents (-Cl, -CN, -NH2, -OCH3) at the C8-position of the isoalloxazine. Thermodynamic and catalytic properties of the reconstituted enzyme were investigated and found to be similar to those of the native enzyme, validating that these FAD analogues are reasonable to be used as mechanistic probes. Dissociation constants for the binding of MHPC or the substrate analogue 5-hydroxynicotinate (5HN) to the reconstituted enzymes indicate that the reconstituted enzymes bind well with ligands. Redox potential values of the reconstituted enzymes were measured and found to be more positive than the values of free FAD analogues, which correlated well with the electronic effects of the 8-substituents. Studies of the reductive half-reaction of MHPCO have shown that the rates of flavin reduction by NADH could be described as a parabolic relationship with the redox potential values of the reconstituted enzymes, which is consistent with the Marcus electron transfer theory. Studies of the oxidative half-reaction of MHPCO revealed that the rate of hydroxylation depended upon the different analogues employed. The rate constants for the hydroxylation step correlated with the calculated pKa values of the 8-substituted C(4a)-hydroxyflavin intermediates, which are the leaving groups in the oxygen transfer step. It was observed that the rates of hydroxylation were greater when the pKa values of C(4a)-hydroxyflavins were lower. Although these results are not as dramatic as those from analogous studies with parahydroxybenzoate hydroxylase (Ortiz- Maldonado et al., (1999) Biochemistry 38, 8124-8137), they are consistent with the model that the oxygenation reaction of MHPCO occurs via an electrophilic aromatic substitution mechanism analogous to the mechanisms for parahydroxybenzoate and phenol hydroxylases

Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells

Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-glucosidase (GBA2), which may have different isoforms because of alternative splicing. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and Western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide, suggesting that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks