Reduced Enzyme Leakage Research Articles

Biodegradation of cyanide in cassava wastewater using a novel thermodynamically-stable immobilized rhodanese

Extracellular rhodanese obtained from Aureobasidium pullulans was employed in both free and immobilized forms for the biodegradation of cyanide present in cassava processing mill effluent (CPME). Crosslinking with glutaraldehyde (at an optimum concentration of 5% v/v) before entrapment in alginate beads resulted in the highest immobilization yield of 94.5% and reduced enzyme leakage of 1.8%. Rhodanese immobilized by cross-linking before entrapment (cbe) retained about 46% of its initial activity after eight cycles of catalysis compared to the entrapment in alginate alone (eaa) which lost more than 79% after the fifth catalytic cycle. A cross-examination of thermodynamic ( ) kinetic () parameters at 30–70 °C showed that cbe displayed a higher resistance to thermal inactivation when compared to the free enzyme (fe) and (eaa). The efficiency of cyanide biodegradation from the CPME by the fe, eaa and cbe were 55, 62, and 74% respectively after 6 h. Rhodanese immobilized via cbe had a higher resistance to thermal denaturation over other enzyme forms. Hence, this makes cbe adaptable for large-scale detoxification of cyanide from CPME.

Bacillus sp. PS35 Lipase-Immobilization on Styrene-Divinyl Benzene Resin and Application in Fatty Acid Methyl Ester Synthesis.

Lipase is an enzyme with immense application potential. Ester synthesis by lipase catalysis in organic media is an area of key industrial relevance. Enzymatic preparations with traits that cater to the needs of this function are hence being intensely researched. The objectives of the study were to immobilize the lipase from Bacillus sp. PS35 by cross-linking and adsorption onto styrene-divinyl benzene (Sty-Dvb) hydrophobic resin and to comparatively characterize the free and immobilized lipase preparations. The work also aimed to apply the immobilized lipase for catalysing the fatty acid methyl ester (FAME) synthesis from palm oil and optimize the process parameters for maximizing the yield. In this study, the purified lipase from Bacillus sp. PS35 was immobilized by adsorption onto styrene-divinyl benzene hydrophobic resin with gluteraldehyde cross-linking. The immobilized enzyme showed better pH and temperature stabilities than the free lipase. Organic solvent stability was also enhanced, with the relative activity in the presence of methanol being shifted from 53% to 81%, thereby facilitating the enzyme's application in fatty acid methyl ester synthesis. It exhibited remarkable storage stability over a 30-day period and after 20 repetitive uses. Cross-linking also reduced enzyme leakage by 49%. The immobilized lipase was then applied for biodiesel production from palm oil. Methanol and oil molar ratio of 5:1, three step methanol additions, and an incubation temperature of 50°C were established to be the ideal conditions favoring the transesterification reaction, resulting in 97% methyl ester yield. These promising results offer scope for further investigation and process scale up, permitting the enzyme's commercial application in a practically feasible and economically agreeable manner.

Role of surface modification in zinc oxide nanoparticles and its toxicity assessment toward human dermal fibroblast cells.

The wide-scale applications of zinc oxide (ZnO) nanoparticles (NPs) in photocatalysts, gas sensors, and cosmetics may cause toxicity to humans and environments. Therefore, the aim of the present study was to reduce the toxicity of ZnO NPs by coating them with a silica (SiO2) layer, which could be used in human applications, such as cosmetic preparations. The sol–gel method was used to synthesize core ZnO with SiO2-shelled NPs (SiO2/ZnO NPs) with varying degrees of coating. Diverse studies were performed to analyze the toxicity of NPs against cells in a dose- and time-dependent manner. To ensure the decreased toxicity of the produced SiO2/ZnO NPs, cytotoxicity in membrane damage and/or intracellular reactive oxygen species (ROS) were assessed by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase, 2′,7′-dichlorofluorescin, and lipid peroxide estimations. The cores of ZnO NPs exhibited cytotoxicity over time, regardless of shell thickness. Nevertheless, the thicker SiO2/ZnO NPs revealed reduced enzyme leakage, decreased peroxide production, and less oxidative stress than their bare ZnO NPs or thinner SiO2/ZnO NPs. Therefore, thicker SiO2/ZnO NPs moderated the toxicity of ZnO NPs by restricting free radical formation and the release of zinc ions, and decreasing surface contact with cells.

Characterization of cross linked Burkholderia cepacia lipase in alginate and κ-carrageenan hybrid matrix

Immobilization of lipases is gaining importance due to a broad variety of industrial applications for their catalytic efficiency. In this study, lipase from Burkholderia cepacia was first cross linked with glutaraldehyde followed by entrapment into hybrid matrix of equal amounts of alginate and κ-carrageenan natural polymers. Alginate and κ-carrageenan can be easily extracted from the local seaweeds of Malaysia thus making the process less costly and more environmental friendly. The effect of pH, temperature, reusability, enzyme leakage, solvent and storage stability on immobilized lipase were studied. An activity yield of 89.26% was observed after immobilization. The immobilized lipase also retained 84.02% of its initial activity following two weeks of storage at 4°C. After 10 cycles of reuse, the immobilized lipase showed 75.54% of residual activity. Comparative kinetic parameters Km and Vmax values were found to be 3.15μM and 12.5μmol/min for free lipase and 4.17μM and 11.11μmol/min for immobilized lipase respectively. A significant reduction of 65.76% enzyme leakage was observed with this hybrid matrix. Higher thermal stability, good storage stability, reduced enzyme leakage and better hydrolysis with olive oil were the salient features achieved by this method of enzyme immobilization.

Endoplasmic and Vascular Surface Activation During Organ Preservation: Refining Upon the Benefits of Machine Perfusion

The endoplasmic reticulum (ER) represents a subcellular target reactive to various cytosolic impairments. The involvement of ER-stress in organ preservation was investigated, comparing machine preservation, cold storage (CS) and a novel concept of only temporary perfusion after procurement. Rat livers were retrieved 30 min after cardiac arrest and preserved for 18 h by CS, oxygenated machine perfusion for 18 h (18 h MP) or for 2 h with subsequent CS for 16 h (2 h MP + 16 h CS). Upon reperfusion, 18 h MP significantly improved enzyme leakage (ALT, LDH) and promoted a 2-fold increase of metabolic recovery compared to CS. However, vascular stress, evaluated by endothelin-release, was significantly elevated after 18 h MP. Interestingly, better viability was obtained using the short-term perfusion protocol (2 h MP + 16 h CS), which further reduced enzyme leakage, maintained energetic recovery and mitigated endothelin-release compared to 18 h MP. Caspase 12-mRNA was upregulated in the 18 h MP-group but unchanged after CS or 2 h MP + 16 h CS. Activation/cleavage of caspase 12 protein was significantly enhanced after 18 h MP and very low in the 2 h MP + 16 h CS-group. Correspondingly, electron microscopy showed ultrastructural alterations of ER after CS and especially after 18 h MP but not after 2 h MP + 16 h CS. At this time mitochondrial appearance was unaffected in all groups, suggesting the ER to be an early subcellular target of preservation injury. In our model, ER and vascular endothelium were best protected by only temporary machine perfusion, which also maintained overall graft viability.

Effect of Melatonin Implantation to Sperm Donor Rams on Post-thaw Viability and Acrosomal Integrity of Sperm Cells in the Breeding and Non-breeding Season

The influence of melatonin administration to sperm donors on the freezability of ram semen and enzyme leakage through sperm cells during different steps of the cryopreservation process were evaluated in the breeding and non-breeding season. Melatonin implantation to rams in the breeding season improved post-thaw sperm viability and intact acrosome rates without influencing the motility rate (p < 0.05). Likewise, the post-thaw alkaline phosphatase release through sperm cells was significantly lower in the melatonin-treated group in comparison with untreated controls (p < 0.05). In the non-breeding season, melatonin administration enhanced intact acrosome rates (p < 0.05) and reduced aspartate aminotransferase activity (p < 0.05) post-thaw in the offseason ejaculates. Melatonin implantation twice in the breeding and non-breeding season did not produce any further improvement in the post-thaw sperm parameters in the non-breeding season ejaculates. It was concluded that melatonin administration to sperm donors improved freezability of ram semen collected from these rams and reduced enzyme leakage through sperm cells during cryopreservation.

Protection induced by a brief ischemic episode in the langendorff perfused rat heart

Ischemic episodes lasting approximately 1 min may be associated with coronary angioplasty. We explored whether such episodes could induce myocardial protection against prolonged ischemic episodes in an ex vivo model. Protection afforded by pretreatment with a 1-min ischemic episode (ischemic preconditioning) against prolonged ischemia was investigated in the isolated rat heart. Left ventricular developed pressure (LVDP; LV systolic pressure-LV end-diastolic pressure), heart rate (HR), and enzyme leakage (lactate dehydrogenase) were indexes of protection. An increased recovery of LVDP x HR after 16 and 19 min of ischemia of 37% and 28%, respectively, paralleled by reduced enzyme leakage, was observed in preconditioned hearts after 10 min of reperfusion. However, the difference between preconditioned and control hearts was lost after 30 min of reperfusion. Ischemic episodes lasting approximately 1 min are not sufficient to initiate stable protection even if initial functional and metabolic indexes suggest a protective effect.

Alanine Protects Liver From Injury Caused by D–Galactosamine and Ccl4

The liver is the main organ involved in amino acid metabolism, and it utilizes glucogenic amino acids as substrates for glucose or adenosine triphosphate (ATP), but this process is impaired in clinical and experimental liver diseases. In this study, we administered high doses of amino acids in rats or cultured hepatocytes with experimental models of liver injury to examine whether such supplementation could attenuate liver damage. We found that the addition of alanine reduced enzyme leakage from primary cultured rat hepatocytes treated with D-galactosamine (D-gal), while other amino acids did not. A significant decrease of lactate dehydrogenase (LDH) leakage was observed when cells were cultured with >6 from mmol/L alanine. Alanine also reduced enzyme leakage from normal hepatocytes that were not treated with D-gal. In D-gal-treated rats, constant infusion of a high dose of alanine significantly reduced the plasma transaminase and total bilirubin levels when compared with infusion of an amino acid mixture. Bolus administration of alanine significantly prevented the elevation of plasma transaminase levels and histological liver damage in CCl4-treated rats, while fructose-1,6 bisphosphate (FDP) had little effect. Alanine might promote the restoration of damaged liver in hepatotoxicant-treated rats, because significant effect was found after the elevation in plasma transaminase levels. Alanine also prevented the decrease of cellular ATP caused by D-gal and appeared to promote ATP production in primary cultured rat hepatocytes. These results indicate that alanine reduces experimental liver damage by a direct effect on hepatocytes.

Alanine protects liver from injury caused by D-galactosamine and CCl4

The liver is the main organ involved in amino acid metabolism, and it utilizes glucogenic amino acids as substrates for glucose or adenosine triphosphate (ATP), but this process is impaired in clinical and experimental liver diseases. In this study, we administered high doses of amino acids in rats or cultured hepatocytes with experimental models of liver injury to examine whether such supplementation could attenuate liver damage. We found that the addition of alanine reduced enzyme leakage from primary cultured rat hepatocytes treated with D- galactosamine (D-gal), while other amino acids did not. A significant decrease of lactate dehydrogenase (LDH) leakage was observed when cells were cultured with >6 from mmol/L alanine. Alanine also reduced enzyme leakage from normal hepatocytes that were not treated withD-gal. In D-gal-treated rats, constant infusion of a high dose of alanine significantly reduced the plasma transaminase and total bilirubin levels when compared with infusion of an amino acid mixture. Bolus administration of alanine significantly prevented the elevation of plasma transaminase levels and histological liver damage in CCl4- treated rats, while fructose-1,6 bisphosphate (FDP) had little effect. Alanine might promote the restoration of damaged liver in hepatotoxicant-treated rats, because significant effect was found after the elevation in plasma transaminase levels. Alanine also prevented the decrease of cellular ATP caused by D-gal and appeared to promote ATP production in primary cultured rat hepatocytes. These results indicate that alanine reduces experimental liver damage by a direct effect on hepatocytes. (Hepatology 1996 Jul;24(1):185-91)

The liver slice system: An in vitro acute toxicity test for assessment of hepatotoxins and their antidotes

A simple method for rapid and reliable assessment of hepatotoxic agents is described. Liver slices from rats and mice of two age groups were incubated with the test hepatotoxins. Exposure of liver slices from 3-month-old mice to acetaminophen (6.8 mg/ml) resulted in 80% leakage of lactate dehydrogenase into the incubation medium, whereas liver slices from one-day-old mice showed only 12% leakage. Similar results were obtained with rat liver slices. The relative lack of response by livers of newborn rats was also demonstrated with carbon tetrachloride. The in vitro liver slice system has also been used to test the potency of N-acetylcysteine (NAC) as an antidote to acetaminophen toxicity. NAC protected mouse liver slices against acetaminophen toxicity in a dose-dependent manner. Addition of the antidote (10 m m) 20 min following hepatotoxin application reduced enzyme leakage by 75% as compared with the system with acetaminophen only. These findings demonstrate that the liver slice system provides the same type of information about hepatotoxins that is usually obtained by the use of acute in vivo tests on a large number of animals. It can be used for testing potential antidotes against hepatotoxins as well as for demonstration of species and age differences in the toxicity of various substances.

Beta-blockade and ischaemic injury: effects of partial agonist activity.

In this study we have attempted: firstly to assess the extent to which the presence of partial agonist activity in a beta-blocking compound and/or sympathetic drive, influence ischaemic injury, and secondly to determine if any of the observed differences are due to changes in contractile activity. In the isolated, globally ischaemic (low flow) rat heart, oxprenolol (which possesses partial agonist activity) did not reduce creatine kinase leakage to the same extent as propranolol (no partial agonist activity) regardless of the background level of catecholamine drive. However, increasing background catecholamine drive increased the relative protection against enzyme leakage, of both beta-blockers. Under conditions of zero contractile activity (high K+ - medium) neither beta-blocker reduced enzyme leakage. Thus at equi-blocking doses oxprenolol and propranolol reduce enzyme leakage to different extents and the lesser protection afforded by oxprenolol is likely to be attributed to the presence of partial agonist activity. In addition, the reduction in enzyme leakage caused by both these compounds is due to reductions in contractile activity.

The effects of incubation conditions on enzyme release from anoxic heart cell cultures

Cultured myocytes from neonate rats were subjected to varying concentrations of glucose and serum under normoxic and anoxic conditions. Cellular metabolism was measured in terms of glucose utilization and lactate production. Leakage of cytoplasmic lactate dehydrogenase (LDH) into the medium was measured to give an indication of cell injury. The effect of variations in temperature on these parameters was evaluated. Myocytes cultured under anoxic conditions for 16 h utilized more glucose than normoxic cells with a concomitant increase in lactate production. When the initial glucose concentration in the medium was below 3 m m, anoxic myocytes released large amounts of LDH. Also, at low initial glucose concentrations there was a surge of anoxic LDH release between 12 and 15 h of incubation. The addition of exogeneous lactate to anoxic cultures had no influence on enzyme leakage. However, reduced enzyme leakage was observed when medium lactate was increased under normoxic conditions. An increased concentration of serum in the medium stimulated glucose metabolism in both normoxic and anoxic myocytes. Despite this enhancement of glycolysis, anoxic cells released significant amounts of LDH into the medium. LDH release by anoxic myocytes is minimal in medium containing 1 to 2% v/v serum. Maximum glucose metabolism and lactate production at 5 m m glucose occurred between 37 to 40°C. The relation between LDH release and temperature was not marked. Significant release of LDH did occur in anoxic cultures incubated between 37°C and 40°C when compared with normoxic cultures at the same temperature. At 4°C, in the absence of glucose metabolism, LDH release occurred from both normoxic and anoxic cells. These data demonstrate that significant cellular enzyme release occurs during anoxia: (a) below a threshold glucose concentration and (b) in proportion to the serum content of the medium.