Search results for: enzyme linked immunosorbent assay (ELISA)

Authors: Md. Z. Alam, Devi R. Asih, Md. N. Salleh

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Immobilization of lipase enzyme produced from palm oil mill effluent (POME) by the activated carbon (AC) among the low cost support materials was optimized. The results indicated that immobilization of 94% was achieved by AC as the most suitable support material. A sequential optimization strategy based on a statistical experimental design, including one-factor-at-a-time (OFAT) method was used to determine the equilibrium time. Three components influencing lipase immobilization were optimized by the response surface methodology (RSM) based on the face-centered central composite design (FCCCD). On the statistical analysis of the results, the optimum enzyme concentration loading, agitation rate and carbon active dosage were found to be 30 U/ml, 300 rpm and 8 g/L respectively, with a maximum immobilization activity of 3732.9 U/g-AC after 2 hrs of immobilization. Analysis of variance (ANOVA) showed a high regression coefficient (R2) of 0.999, which indicated a satisfactory fit of the model with the experimental data. The parameters were statistically significant at p<0.05.

Keywords: activated carbon, POME based lipase, immobilization, adsorption

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Authors: Shweta Kumari, Parmjit S. Panesar, Manab B. Bera

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The hydrolysis of lactose using β-galactosidase is one of the most promising biotechnological applications, which has wide range of potential applications in food processing industries. However, due to intracellular location of the yeast enzyme, and expensive extraction methods, the industrial applications of enzymatic hydrolysis processes are being hampered. The use of permeabilization technique can help to overcome the problems associated with enzyme extraction and purification of yeast cells and to develop the economically viable process for the utilization of whole cell biocatalysts in food industries. In the present investigation, standardization of permeabilization process of novel yeast isolate was carried out using a statistical model approach known as Response Surface Methodology (RSM) to achieve maximal b-galactosidase activity. The optimum operating conditions for permeabilization process for optimal β-galactosidase activity obtained by RSM were 1:1 ratio of toluene (25%, v/v) and ethanol (50%, v/v), 25.0 oC temperature and treatment time of 12 min, which displayed enzyme activity of 1.71 IU /mg DW.

Keywords: β-galactosidase, optimization, permeabilization, response surface methodology, yeast

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Authors: Delgado-Meza M., Minor-Pérez H.

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Protease is the generic name of enzymes that hydrolyze proteins. These are classified in the subgroup EC3.4.11-99X of the classification enzymes. In food technology the proteolysis is used to modify functional and nutritional properties of food, and in some cases this proteolysis may cause food spoilage. In general, seafood and rainbow trout have accelerated decomposition process once it has done its capture, due to various factors such as the endogenous enzymatic activity that can result in loss of structure, shape and firmness, besides the release of amino acid precursors of biogenic amines. Some studies suggest the use of protease inhibitors from legume as biological regulators of proteolytic activity. The enzyme inhibitors are any substance that reduces the rate of a reaction catalyzed by an enzyme. The objective of this study was to evaluate the reduction of the proteolytic activity of enzymes in extracts of rainbow trout with protease inhibitors obtained from chickpea flour. Different proportions of rainbow trout enzyme extract (75%, 50% and 25%) and extract chickpea enzyme inhibitors were evaluated. Chickpea inhibitors were obtained by mixing 5 g of flour in 30 mL of pH 7.0 phosphate buffer. The sample was centrifuged at 8000 rpm for 10 min. The supernatant was stored at -15°C. Likewise, 20 g of rainbow trout were ground in 20 mL of phosphate buffer solution at pH 7.0 and the mixture was centrifuged at 5000 rpm for 20 min. The supernatant was used for the study. In each treatment was determined the specific enzymatic activity with the technique of Kunitz, using hemoglobin as substrate for the enzymes acid fraction and casein for basic enzymes. Also biuret protein was quantified for each treatment. The results showed for fraction of basic enzymes in the treatments evaluated, that were inhibition of endogenous enzymatic activity. Inhibition values compared to control were 51.05%, 56.59% and 59.29% when the proportions of endogenous enzymes extract rainbow trout were 75%, 50% and 25% and the remaining volume used was extract with inhibitors. Treatments with acid enzymes showed no reduction in enzyme activity. In conclusion chickpea flour reduced the endogenous enzymatic activity of rainbow trout, which may favor its application to increase the half-life of this food. The authors acknowledge the funding provided by the CONACYT for the project 131998.

Keywords: rainbouw trout, enzyme inhibitors, proteolysis, enzyme activity

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Authors: Sidra Shaheen, Nadir Naveed Siddiqui, Shah Ali Ul Qader

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In recent years, significant progress has been made in discovering and developing new bacterial polysaccharides producing organisms possessing extremely functional properties. Levan is a natural biopolymer of fructose which is produced by transfructosylation reaction in the presence of levansucrase. It is one of the industrially promising enzymes that offer a variety of industrial applications in the field of cosmetics, foods and pharmaceuticals. Although levan has significant applications but the yield of levan produced is not equal to other biopolymers due to the inefficiency of producer microorganism. Among wide range of levansucrase producing microorganisms, Zymomonas mobilis is considered as a potential candidate for large scale production of this natural polysaccharide. The present investigation is concerned with the isolation of levansucrase producing natural isolate having maximum enzyme production. Furthermore, production parameters were optimized to get higher enzyme yield. Levansucrase was partially purified and characterized to study its applicability on industrial scale. The results of this study revealed that the bacterial strain Z. mobilis KIBGE-IB14 was the best producer of levansucrase. Bacterial growth and enzyme production was greatly influenced by physical and chemical parameters. Maximum levansucrase production was achieved after 24 hours of fermentation at 30°C using modified medium of pH-6.5. Contrary to other levansucrases, the one presented in the current study is able to produce high amount of products in relatively short period of time with optimum temperature at 35°C. Due to these advantages, this enzyme can be used on large scale for commercial production of levan and other important metabolites.

Keywords: levansucrase, metabolites, polysaccharides, transfructosylation

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Authors: Ndidi F. Amulu, Calistus N. Ude, Patrick E. Amulu, Nneka N. Uchegbu

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The effects of temperature and enzyme concentration on the quality of mixed pineapple and pawpaw blended fruits juice were studied. Extracts of the two fruit juices were separately treated at 70  for 15 min each so as to inactivate micro-organisms. They were analyzed and blended in different proportions of 70% pawpaw and 30% pineapple, 60% pawpaw and 40% pineapple, 50% pineapple and 50% pawpaw, 40% pawpaw and 60% pineapple. The characterization of the fresh pawpaw and pineapple juice before blending showed that the juices have good quality. The high water content of the product may have affected the viscosity, vitamin C content and total soluble solid of the blended juice to be low. The effects of the process parameters on the quality showed that better quality of the blended juice can be obtained within the optimum temperature range of (50-70 °C) and enzyme concentration range (0.12-0.18 w/v). The ratio of mix 60% pineapple juice: 40% pawpaw juice has better quality. This showed that pawpaw and pineapple juices can blend effectively to produce a quality juice.

Keywords: clarification, pawpaw, pineapple, viscosity, vitamin C

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Authors: Jinyoung Park, Eun Joo Song

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Introduction: Deubiquitinating enzymes (DUBs) are proteases that cleave ubiquitin or ubiquitin-like modifications on substrates. Deubiquitination could regulate cellular physiology, such as signal transduction, DNA damage and repair, and cell cycle progression. Although more than 100 DUBs are encoded in the human and the importance of DUBs has been realized, the functions of most DUBs are unknown. This study aims to identify the molecular mechanism by which deubiquitinating enzyme USP35 regulates cell cycle progression for the first time. Methods: USP35 RNAi was mainly used to identify the function of USP35 in cell cycle progression. To find substrates of USP35, we analyzed protein-protein interaction using LC-MS. Several biological methods, such as ubiquitination assay, cell synchronization, immunofluorescence, and immunoprecipitation assay were used to investigate the exact mechanism by which USP35 affects successful completion of mitosis. Results: USP35 knockdown caused not only reduction of mitotic cell number but also induction of mitotic cells with abnormal spindle formation. Actually, cell proliferation was decreased by USP35 knockdown. Interestingly, we found that loss of USP35 decreased the stability and expression of Aurora B, a member of chromosomal passenger complex (CPC), and the phosphorylation of its substrate. Indeed, USP35 interacted with Aurora B and deubiquitinated it. In addition, USP35 knockdown induced abnormal localization of Aurora B in mitotic cells. Finally, CDH1-mediated ubiquitination of Aurora B level was rescued by USP35 overexpression, but not inactive form of USP35, USP35 C450A. Discussion: Our findings suggest that USP35 regulates Aurora B-mediated mitotic spindle assembly and G2-M transition by blocking CDH1-induced degradation of Aurora B.

Keywords: USP35, HSP90, Aurora B, cell cycle progression

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Authors: Çiğdem Sezer, Aksem Aksoy, Leyla Vatansever

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This study has been done for the purpose of evaluation of public health and identifying of enterotoxigenic Staphyloccocus aureus in kitchen of catering. In the kitchen of catering, samples have been taken by swabs from surface of equipments which are in the salad section, meat section and bakery section. Samples have been investigated with classical cultural methods in terms of Staphyloccocus aureus. Therefore, as a 10x10 cm area was identified (salad, cutting and chopping surfaces, knives, meat grinder, meat chopping surface) samples have been taken with sterile swabs with helping FTS from this area. In total, 50 samples were obtained. In aseptic conditions, Baird-Parker agar (with egg yolk tellurite) surface was seeded with swabs. After 24-48 hours of incubation at 37°C, the black colonies with 1-1.5 mm diameter and which are surrounded by a zone indicating lecithinase activity were identified as S. aureus after applying Gram staining, catalase, coagulase, glucose and mannitol fermentation and termonuclease tests. Genotypic characterization (Staphylococcus genus and S.aureus species spesific) of isolates was performed by PCR. The ELISA test was applied to the isolates for the identification of staphylococcal enterotoxins (SET) A, B, C, D, E in bacterial cultures. Measurements were taken at 450 nm in an ELISA reader using an Ridascreen-Total set ELISA test kit (r-biopharm R4105-Enterotoxin A, B, C, D, E). The results were calculated according to the manufacturer’s instructions. A total of 50 samples of 97 S. aureus was isolated. This number has been identified as 60 with PCR analysis. According to ELISA test, only 1 of 60 isolates were found to be enterotoxigenic. Enterotoxigenic strains were identified from the surface of salad chopping and cutting. In the kitchen of catering, S. aureus identification indicates a significant source of contamination. Especially, in raw consumed salad preparation phase of contamination is very important. This food can be a potential source of food-borne poisoning their terms, and they pose a significant risk to consumers have been identified.

Keywords: Staphylococcus aureus, enterotoxin, catering, kitchen, health

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Authors: Ankita Kushwaha, M. P. Singh

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Background: In the present investigation the efficiency of laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) from Pleurotus citrinopileatus was assessed for the decolorization of azo dyes. Aim: Enzyme production, characterization and kinetics of a partially purified laccase from Pleurotus citrinopileatus were determined for its application in bioremediation of azo dyes. Methods & Results: Laccase has been partially purified by using 80% ammonium sulphate solution. Total activity, total protein, specific activity and purification fold for partially purified laccase were found to be 40.38U, 293.33mg/100ml, 0.91U/mg and 2.84, respectively. The pH and temperature optima of laccase were 5.0 and 50ºC, respectively, while the enzyme was most stable at pH 4.0 and temperature 30ºC when exposed for one hour. The Km of the partially purified laccase for substrates guaiacol, DMP (2,6-dimethoxyphenol) and syringaldazine (3,5-dimethoxy-4-hydroxybenzaldehyde azine) were 60, 95 and 26, respectively. This laccase has been tested for the use in the bioremediation of azo dyes in the absence of mediator molecules. Two dyes namely congo red and bromophenol blue were tested. Discussion: It was observed that laccase enzyme was very effective in the decolorization of these two dyes. More than 80% decolorization was observed within half an hour even in the absence of mediator and their lower Km value indicates that efficiency of the enzyme is very high. The results were promising due to quicker decolorization in the absence of mediators showing that it can be used as a valuable biocatalyst for quick bioremediation of azo dyes. Conclusion: The enzymatic properties of laccase from P. citrinopileatus should be considered for a potential environmental (biodegradation and bioremediation) or industrial applications.

Keywords: azo dyes, decolorization, laccase, P.citrinopileatus

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Authors: Abdur Rahman, Saima, T. N. Pasha, Muhammad Younus, Yassar Abbas, Shahid Jaleel

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Non-starch polysaccharides (NSPs) are not completely digested by broiler endogenous enzymes and consequently the soluble NSPs in feed results in high digesta viscosity and poor retention of nutrients. Supplementation of NSPs digesting enzymes may release the nutrients from feed and reduce the anti-nutritional effects of NSP’s. The present study was conducted to determine the effects of NSPs digesting enzymes (Zympex) in broiler chicks. A total of 120 day old broiler chicks (Hubbard) were categorized into 3 treatments and each treatment was having four replicates with 10 birds in each. Dietary treatments comprised of Basal diet (2740 KCal/Kg) as control-1 (T1), low energy diet (2630 KCal/kg) control-2 (T2) and low energy diet with 0.5 gm/Kg enzyme as T3. Multi-enzymes supplementation showed significant (P < 0.05) positive effect on weight gain (last three weeks), feed intake (last two weeks), FCR (1st, 2nd, 4th and 5th) and nutrient retention in T3 when compared with control-2. Weight gain was lower (P < 0.05) in low caloric feed group C when compared with control-1 in all weeks except last week (P > 0.05), feed consumption was significantly lower (P < 0.05) in 5th week and results showed significantly poor FCR (P < 0.05) in 2nd, 3rd and 4th week but non-significant effect in 1st and 5th week when compared with control-1 group, which revealed the positive effect of enzyme supplementation in low energy diet. These results revealed that enzyme supplementation releases more energy from low energy diets and results in equal performance to normal diet.

Keywords: body weight, FCR, feed intake, enzyme, non-starch polysaccharides

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Authors: A. H. Shaikat, M. B. Hossain, S. K. M. A. Islam, M. M. Hassan, S. A. Khan, A. K. M. Saifuddin, M. N. Islam, M. A. Hoque

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A total of eighteen (18) sex reversed chickens with unusual phenotypic characteristics of male birds were identified over 2000 Hyline layer chickens at Motaher Poultry Farm, Ramu, Cox’s Bazar. Chickens were subdivided into two groups (case = 18, control = 20) based on the appearance of sex-reversed secondary sexual characteristics. Phenotypic traits of studied chickens were measured with farm management details. Hormone assay using ELISA, autopsy followed by gross examination of viscera was performed. The study found higher body weight (gm) (1579.3; 95% CI: 1561.7-1596.8), comb length (cm) (12.2; 11.5-12.8), comb width (cm) (7.9; 7.7-8.2), wattle length (cm) (4.9; 4.8-5.1) distinct spur, and shortened pubic bones distance, suggesting decrease oviposition in sex-reversed chickens. Testosterone concentration (ng/ml) (8.5; 6.4-10.6) was significantly higher (p<0.001) along with decrease estrogen (pg/ml) (5.1; 4.9-5.5) and progesterone concentration (pg/ml) (310.9; 289.4-332.5) in sex-reversed chickens. Mass abdominal fat deposition with atrophied ovary was found upon exploration of viscera.

Keywords: ovary, phenotypic traits, sex hormone, sex reversal

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Authors: V. Viswanathan, Md. Irshad Ahmad, Prashant K. Singh, Nayeem Ahmad, Pradeep Sharma, Sujata Sharma, Tej P Singh

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Lactoperoxidase (LPO) is a heme containing mammalian enzyme which uses hydrogen peroxide (H2O2) to catalyze the conversion of substrates into oxidized products. LPO is found in body fluids and tissues such as milk, saliva, tears, mucosa and other body secretions. The previous structural studies have shown that LPO converts substrates, thiocyanate (SCN‾) and iodide (I‾) ions into oxidized products, hypothiocyanite (OSCN‾) and hypoiodite (IO‾) ions, respectively. We report here a new structure of the complex of LPO with an oxidized product, nitrite (NO2‾). This product was generated from NO using the two step reaction of LPO by adding hydrogen peroxide (H2O2) in the solution of LPO in 0.1M phosphate buffer at pH 6.8 as the first step. In the second step, NO gas was added to the above mixture. This was crystallized using 20% (w/v) PEG-3350 and 0.2M ammonium iodide at pH 6.8. The structure determination showed the presence of NO2‾ ion in the distal heme cavity of the substrate binding site of LPO. The structure also showed that the propionate group, which is linked to pyrrole ring D of the heme moiety, was disordered. Similarly, the side chain of Asp108, which is covalently linked to heme moiety, was also split into two components. As a result of these changes, the conformation of the side chain of Arg255 was altered, allowing it to form new interactions with the disordered carboxylic group of propionate moiety. These structural changes are indicative of an intermediate state in the catalytic reaction pathway of LPO.

Keywords: lactoperoxidase, structure, nitric oxide, nitrite ion, intermediate, complex

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Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

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Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity

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Authors: Moon Jung Kim, Guil Rhim

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The clinical sensitivity of the “VEUPLEXTM TBI assay”, a clinical trial medical device, in mild traumatic brain injury was 28.6% (95% CI, 19.7%-37.5%), and the clinical specificity was 94.0% (95% CI, 89.3%). -98.7%). In addition, when the results analyzed by marker were put together, the sensitivity was higher when interpreting the two tests together than the two tests, UCHL1 and GFAP alone. Additionally, when sensitivity and specificity were analyzed based on CT results for the mild traumatic brain injury patient group, the clinical sensitivity for 2 CT-positive cases was 50.0% (95% CI: 1.3%-98.7%), and 19 CT-negative cases. The clinical specificity for cases was 68.4% (95% CI: 43.5% - 87.4%). Since the low clinical sensitivity for the two CT-positive cases was not statistically significant due to the small number of samples analyzed, it was judged necessary to secure and analyze more samples in the future. Regarding the clinical specificity analysis results for 19 CT-negative cases, there were a large number of patients who were actually clinically diagnosed with mild traumatic brain injury but actually received a CT-negative result, and about 31.6% of them showed abnormal results on VEUPLEXTM TBI assay. Although traumatic brain injury could not be detected in 31.6% of the CT scans, the possibility of actually suffering a mild brain injury could not be ruled out, so it was judged that this could be confirmed through follow-up observation of the patient. In addition, among patients with mild traumatic brain injury, CT examinations were not performed in many cases because the symptoms were very mild, but among these patients, about 25% or more showed abnormal results in the VEUPLEXTM TBI assay. In fact, no damage is observed with the naked eye immediately after traumatic brain injury, and traumatic brain injury is not observed even on CT. But in some cases, brain hemorrhage may occur (delayed cerebral hemorrhage) after a certain period of time, so the patients who did show abnormal results on VEUPLEXTM TBI assay should be followed up for the delayed cerebral hemorrhage. In conclusion, it was judged that it was difficult to judge mild traumatic brain injury with the VEUPLEXTM TBI assay only through clinical findings without CT results, that is, based on the GCS value. Even in the case of CT, it does not detect all mild traumatic brain injury, so it is difficult to necessarily judge that there is no traumatic brain injury, even if there is no evidence of traumatic brain injury in CT. And in the long term, more patients should be included to evaluate the usefulness of the VEUPLEXTM TBI assay in the detection of microscopic traumatic brain injuries without using CT.

Keywords: brain injury, traumatic brain injury, GFAP, UCHL1

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Authors: Adam Abate, Elham Rasti, Philip Romero

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Beta-glucosidase is the key enzyme component present in cellulase and completes the final step during cellulose hydrolysis by converting the cellobiose to glucose. The regulatory properties of beta-glucosidases are most commonly found for the retaining and inverting enzymes. Hydrolysis of a glycoside typically occurs with general acid and general base assistance from two amino acid side chains, normally glutamic or aspartic acids. In order to obtain more detailed information on the dynamic events origination from the interaction with enzyme active site, we carried out molecular dynamics simulations of beta-glycosidase in protonated state (Glu-H178) and deprotonated state (Glu178). The theoretical models generated from our molecular dynamics simulations complement and advance the structural information currently available, leading to a more detailed understanding of Beta-glycosidase structure and function. This article presents the important role of Asn307 in enzyme activity of beta-glucosidase

Keywords: Beta-glucosidase, GROMACS, molecular dynamics simulation, structural parameters

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Authors: Waranya Imprasittichai, Patsarawadee Paojinda, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. In this study, we constructed gene fusions of Plasmodium falciparum OMPDC-OPRT (1,836 bp) in pTrcHisA vector and expressed as an 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21, Rosetta, TOP10) at 18 °C, 25 °C and 37 °C. The recombinant bifunctional protein was partially purified by Ni-Nitrilotriacetic acid-affinity chromatography. Specific activities of OPRT and OMPDC domains in the bifunctional enzyme expressed in E. coli TOP10 cells were approximately 3-4-fold higher than those in BL21 cells. There were no enzymatic activities when the construct vector expressed in Rosetta cells. Maximal expression of the fused gene was observed at 18 °C and the bifunctional enzyme had specific activities of OPRT and OMPDC domains in a ratio of 1:2. These results provide greater yields and better catalytic activities of the bifunctional OMPDC-OPRT enzyme for further purification and kinetic study.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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Authors: Vanessa Roulet, Marc Turini, Juliane Hey, Stéphanie Bord-Romeu, Emilie Bonzom, Mahmoud Badawi, Mohammed-Amine Chakir, Valérie Simon, Vanessa Viotti, Jérémie Gautier, Françoise Le Boulaire, Catherine Coignard, Claire Vincent, Sandrine Greaume, Isabelle Voisin

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Background: Beckman Coulter, Inc. has recently developed fully automated assays for the detection of HBsAg on a new immunoassay platform. The objective of this European multicenter study was to evaluate the performance of the ACCESS HBsAg and ACCESS HBsAg Confirmatory assays† on the recently CE-marked DxI 9000 ACCESS Immunoassay Analyzer. Methods: The clinical specificity of the ACCESS HBsAg and HBsAg Confirmatory assays was determined using HBsAg-negative samples from blood donors and hospitalized patients. The clinical sensitivity was determined using presumed HBsAg-positive samples. Sample HBsAg status was determined using a CE-marked HBsAg assay (Abbott ARCHITECT HBsAg Qualitative II, Roche Elecsys HBsAg II, or Abbott PRISM HBsAg assay) and a CE-marked HBsAg confirmatory assay (Abbott ARCHITECT HBsAg Qualitative II Confirmatory or Abbott PRISM HBsAg Confirmatory assay) according to manufacturer package inserts and pre-determined testing algorithms. False initial reactive rate was determined on fresh hospitalized patient samples. The sensitivity for the early detection of HBV infection was assessed internally on thirty (30) seroconversion panels. Results: Clinical specificity was 99.95% (95% CI, 99.86 – 99.99%) on 6047 blood donors and 99.71% (95%CI, 99.15 – 99.94%) on 1023 hospitalized patient samples. A total of six (6) samples were found false positive with the ACCESS HBsAg assay. None were confirmed for the presence of HBsAg with the ACCESS HBsAg Confirmatory assay. Clinical sensitivity on 455 HBsAg-positive samples was 100.00% (95% CI, 99.19 – 100.00%) for the ACCESS HBsAg assay alone and for the ACCESS HBsAg Confirmatory assay. The false initial reactive rate on 821 fresh hospitalized patient samples was 0.24% (95% CI, 0.03 – 0.87%). Results obtained on 30 seroconversion panels demonstrated that the ACCESS HBsAg assay had equivalent sensitivity performances compared to the Abbott ARCHITECT HBsAg Qualitative II assay with an average bleed difference since first reactive bleed of 0.13. All bleeds found reactive in ACCESS HBsAg assay were confirmed in ACCESS HBsAg Confirmatory assay. Conclusion: The newly developed ACCESS HBsAg and ACCESS HBsAg Confirmatory assays from Beckman Coulter have demonstrated high clinical sensitivity and specificity, equivalent to currently marketed HBsAg assays, as well as a low false initial reactive rate. †Pending achievement of CE compliance; not yet available for in vitro diagnostic use. 2023-11317 Beckman Coulter and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

Keywords: dxi 9000 access immunoassay analyzer, hbsag, hbv, hepatitis b surface antigen, hepatitis b virus, immunoassay

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Authors: Patricia M. B. Saint-Vincent, Anna Furches, Stephanie Galanie, Erica Teixeira Prates, Piet Jones, Nancy Engle, David Kainer, Wellington Muchero, Daniel Jacobson, Timothy J. Tschaplinski

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Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that catalyze sugar transfer to a variety of plant metabolites. UGT substrates, which include plant secondary metabolites involved in lignification, demonstrate new activities and incorporation when glycosylated. Knowledge of UGT function, substrate specificity, and enzyme products is important for plant engineering efforts, especially related to increasing plant biomass through lignification. UGTs in Populus trichocarpa, a biofuel feedstock, and model woody plant, were selected from a pool of gene candidates using rapid prioritization strategies. A functional genomics workflow, consisting of a metabolite genome-wide association study (mGWAS), expression of synthetic codon-optimized genes, and high-throughput biochemical assays with mass spectrometry-based analysis, was developed for determining the substrates and products of previously-uncharacterized enzymes. A total of 40 UGTs from P. trichocarpa were profiled, and the biochemical assay results were compared to predicted mGWAS connections. Assay results confirmed seven of 11 leaf mGWAS associations and demonstrated varying levels of substrate specificity among candidate UGTs. P. trichocarpa UGT substrate processing confirms the role of these newly-characterized enzymes in lignan, flavonoid, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress responses.

Keywords: Populus, metabolite-gene associations, GWAS, bio feedstocks, glycosyltransferase

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Authors: Michio Kurosu

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Alterations in glycosylation not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Identification of cell type-specific glycoconjugates (tumor markers) has led to the discovery of new assay systems for certain cancers via immunodetection reagents. N- and O-linked glycans are the most abundant forms of glycoproteins. Recent studies of cancer immunotherapy are based on the immunogenicity of truncated O-glycan chains (e.g., Tn, sTn, T, and sLea/x). The prevalence of N-linked glycan changes in the development of tumor cells is known; however, therapeutic antibodies against N-glycans have not yet been developed. This is due to the lack of specificity of N-linked glycans between normal/healthy and cancer cells. Abnormal branching of N-linked glycans has been observed, particularly in solid cancer cells. While the discovery of drug-like glycosyltransferase inhibitors that block the biosynthesis of specific branching has a very low likelihood of success, altered glycosylation levels can be exploited by suppressing N-glycan biosynthesis through the inhibition of dolichyl-phosphate N-acetylglucosaminephosphotransferase1 (DPAGT1) activity. Inhibition of DPAGT1 function leads to changes of O-glycosylation on proteins associated with mitochondria and zinc finger binding proteins (indirect effects). On the basis of dynamic crosstalk between DPAGT1 and Snail/Slung/ZEB1 (a family of transcription factors that promote the repression of the adhesion molecules), we have developed pharmacologically acceptable selective DPAGT1 inhibitors. Tunicamycin kills a wide range of cancer and healthy cells in a non-selective manner. In sharp contrast, our DPAGT1 inhibitors display strong cytostatic effects against 16 solid cancers, which require the overexpression of DPAGT1 in their progression but do not affect the cell viability of healthy cells. The identified DPAGT1 inhibitors possess impressive anti-metastatic ability in various solid cancer cell lines and induce their mitochondrial structural changes, resulting in apoptosis. A prototype DPAGT1 inhibitor, APPB has already been proven to shrink solid tumors (e.g., pancreatic cancers, triple-negative breast cancers) in vivo while suppressing metastases and has strong synergistic effects when combined with current cytotoxic drugs (e.g., paclitaxel). At this conference, our discovery of selective DPAGT1 inhibitors with drug-like properties and proof-of-pharmaceutical concept studies of a novel DPAGT1 inhibitor are presented.

Keywords: DPAGT1 inhibitors, anti-metastatic drugs, natural product based drug designs, cytostatic effects

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Authors: Ali Aydin, Mert Sudagidan, Aysen Coban, Alparslan Kadir Devrim

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Pseudomonas spp. (specifically, P. fluorescens and P. fragi) are considered the principal spoilage microorganisms of refrigerated poultry meats. The higher the level psychrophilic spoilage Pseudomonas spp. on carcasses at the end of processing lead to decrease the shelf life of the refrigerated product. The aim of the study was the identification of psychrophilic Pseudomonas spp. having proteolytic and lipolytic activities from poultry meats by 16S rRNA and rpoB gene sequencing, investigation of protease and lipase related genes and determination of proteolytic activity of Pseudomonas spp. In the of isolation procedure, collected chicken meat samples from local markets and slaughterhouses were homogenized and the lysates were incubated on Standard method agar and Skim Milk agar for selection of proteolytic bacteria and tributyrin agar for selection of lipolytic bacteria at +4 °C for 7 days. After detection of proteolytic and lipolytic colonies, the isolates were firstly analyzed by biochemical tests such as Gram staining, catalase and oxidase tests. DNA gene sequencing analysis and comparison with GenBank revealed that 126 strong enzyme Pseudomonas spp. were identified as predominantly P. fluorescens (n=55), P. fragi (n=42), Pseudomonas spp. (n=24), P. cedrina (n=2), P. poae (n=1), P. koreensis (n=1), and P. gessardi (n=1). Additionally, protease related aprX gene was screened in the strains and it was detected in 69/126 strains, whereas, lipase related lipA gene was found in 9 Pseudomonas strains. Protease activity was determined using commercially available protease assay kit and 5 strains showed high protease activity. The results showed that psychrophilic Pseudomonas strains were present in chicken meat samples and they can produce important levels of proteases and lipases for food spoilage to decrease food quality and safety.

Keywords: Pseudomonas, chicken meat, protease, lipase

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Authors: J. B. Minari, O. B. Oloyede

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Polymorphonuclear neutrophils (PMNs) play a crucial role in a variety of infections caused by bacteria, fungi, and parasites. Indeed, the involvement of PMNs in host defence against Plasmodium falciparum is well documented both in vitro and in vivo. Many of the antimalarial drugs such as chloroquine used in the treatment of human malaria significantly reduce the immune response of the host in vitro and in vivo. Myeloperoxidase is the most abundant enzyme found in the polymorphonuclear neutrophil which plays a crucial role in its function. This study was carried out to investigate the effect of chloroquine on the enzyme. In investigating the effects of the drug on myeloperoxidase, the influence of concentration, pH, partition ratio estimation and kinetics of inhibition were studied. This study showed that chloroquine is concentration-dependent inhibitor of myeloperoxidase with an IC50 of 0.03 mM. Partition ratio estimation showed that 40 enzymatic turnover cycles are required for complete inhibition of myeloperoxidase in the presence of chloroquine. The influence of pH on the effect of chloroquine on the enzyme showed significant inhibition of myeloperoxidase at physiological pH. The kinetic inhibition studies showed that chloroquine caused a non-competitive inhibition with an inhibition constant Ki of 0.27mM. The results obtained from this study shows that chloroquine is a potent inhibitor of myeloperoxidase and it is capable of inactivating the enzyme. It is therefore considered that the inhibition of myeloperoxidase in the presence of chloroquine as revealed in this study may partly explain the impairment of polymorphonuclear neutrophil and consequent immunosuppression of the host defence system against secondary infections.

Keywords: myeloperoxidase, chloroquine, inhibition, neutrophil, immune

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Authors: Hye Jin Kim, Hwa Sung Shin

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Ischemic stroke means the caused brain damage due to neurological defects, occurring occlusion of cerebral vascular resulting in thrombus or embolism. t-PA (tissue Plasminogen Activator) is the only thrombolytic agent passed the FDA (Food and Drug Administration). However, t-PA directly dissolves the thrombus (direct activity) through fibrinolysis, showing side effects such as re-occlusion. In this study, we evaluated the thrombolytic activities of the serine protease extracted from lugworms inhabiting tidal flats. The new serine protease identified as 38 kDa by SDS-PAGE was not toxic to brain endothelial cells line (hCMEC/D3). Also, the plasmin synthesis inhibition activity (indirect activity) of the new serine protease was confirmed through fibrin zymography assay and fibrin plate assay. It was higher than direct activity as compared to u-PA (urokinase Plasminogen Activator). The activities were found to be maintained at a wide range of temperature (4-70 ℃) and pH 7-10 compared to previous thrombolytic agents from the azocasein assay. In addition, the new serine protease has shown anticoagulant activity from fibrinogenolytic activity assay. In conclusion, the serine protease in lug worms inhabiting the tidal flats could be considered a promising thrombolytic candidate for the treatment of ischemic stroke.

Keywords: alkaline serine protease, bifunctional thrombolytic activity, fibrinolytic activity, ischemic stroke, lug worms

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Authors: Kyung Jin Kim, Jiwon Kwak, Jae-Hoon Lee, Soo Suk Lee

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MicroRNAs (miRNA) are a class of endogenous, single-stranded, small, and non-protein coding RNA molecules typically 20-25 nucleotides long. They are thought to regulate the expression of other genes in a broad range by binding to 3’- untranslated regions (3’-UTRs) of specific mRNAs. The detection of miRNAs is very important for understanding of the function of these molecules and in the diagnosis of variety of human diseases. However, detection of miRNAs is very challenging because of their short length and high sequence similarities within miRNA families. So, a simple-to-use, low-cost, and highly sensitive method for the detection of miRNAs is desirable. In this study, we demonstrate a novel bi-directional extension (BDE) assay. In the first step, a specific linear RT primer is hybridized to 6-10 base pairs from the 3’-end of a target miRNA molecule and then reverse transcribed to generate a cDNA strand. After reverse transcription, the cDNA was hybridized to the 3’-end which is BDE sequence; it played role as the PCR template. The PCR template was amplified in an SYBR green-based quantitative real-time PCR. To prove the concept, we used human brain total RNA. It could be detected quantitatively in the range of seven orders of magnitude with excellent linearity and reproducibility. To evaluate the performance of BDE assay, we contrasted sensitivity and specificity of the BDE assay against a commercially available poly (A) tailing method using miRNAs for let-7e extracted from A549 human epithelial lung cancer cells. The BDE assay displayed good performance compared with a poly (A) tailing method in terms of specificity and sensitivity; the CT values differed by 2.5 and the melting curve showed a sharper than poly (A) tailing methods. We have demonstrated an innovative, cost-effective BDE assay that allows improved sensitivity and specificity in detection of miRNAs. Dynamic range of the SYBR green-based RT-qPCR for miR-145 could be represented quantitatively over a range of 7 orders of magnitude from 0.1 pg to 1.0 μg of human brain total RNA. Finally, the BDE assay for detection of miRNA species such as let-7e shows good performance compared with a poly (A) tailing method in terms of specificity and sensitivity. Thus BDE proves a simple, low cost, and highly sensitive assay for various miRNAs and should provide significant contributions in research on miRNA biology and application of disease diagnostics with miRNAs as targets.

Keywords: bi-directional extension (BDE), microRNA (miRNA), poly (A) tailing assay, reverse transcription, RT-qPCR

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Authors: Sergejs Kolesovs, Armands Vigants

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The use of dairy industry by-products and wastewater as a cheap substrate for microalgal growth is gaining recognition. However, the mechanisms of lactose utilization remain understudied, limiting the potential of successful microalgal biomass production using various dairy by-products, such as whey and permeate. The necessity for microalgae to produce a specific enzyme, β-galactosidase, requires the selection of suitable strains. This study focuses on a freshwater microalgal isolate's ability to grow on a semi-synthetic medium supplemented with lactose. After 10 days of agitated cultivation, an axenic microalgal isolate achieved significantly higher biomass production under mixotrophic growth conditions (0.86 ± 0.07 g/L, dry weight) than heterotrophic growth (0.46 ± 0.04 g/L). Moreover, mixotrophic cultivation had significantly higher biomass production compared to photoautotrophic growth (0.67 ± 0.05 g/L). The activity of β-galactosidase was detected in both supernatant and microalgal biomass under mixotrophic and heterotrophic growth conditions, showing the potential of extracellular and intracellular mechanisms of enzyme production. However, the main limiting factor in this study was the increase of pH values during the cultivation, significantly reducing the activity of the β-galactosidase enzyme after 3rd day of cultivation. It highlights the need for stricter control of growth parameters to ensure the enzyme's activity. Further research will assess the isolate's suitability for dairy by-product bioconversion and biomass composition.

Keywords: microalgae, lactose, whey, permeate, beta-galactosidase, mixotrophy, heterotrophy

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Authors: Hatem H.Saleh

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The aim of the study was to examine the effect of different concentrations of crude actinidin enzyme extract from kiwifruit juice and distilled water on some quality attributes of Awassi rams meats. Twelve Awassi rams were divided into four groups, After exsanguinations of rams carcasses they were infused (10% body weight) with crude of actinidin enzyme extract of kiwifruit juice with 10 and 15% of extract, and other group was infused with distilled water and were compared with other groups a non infusion treatment which were acted as a control. Thereafter samples from two main muscles, namely longissimus dorsi (LD) and Semimembranosus (SM) of the carcasses was chilled then stored in freezing, until testing time . The results showed a decrease in the rate pH decline on LD and SM muscle which was measured at time (0, 3, 6, 9, 12, 24 hours) postmortem among different treatments, It also reported lower values of the rate pH on the LD and SM muscle during the first of 12 hrs postmortem. No significant differences of the rate internal meat temperature in LD and SM muscle were observed among treatments postmortem except decreased of internal meat temperature during 3 hours postmortem when treated with enzyme extract. The results recorded higher values of glycolysis rate (R-value) in LD and SM muscle when treated with enzyme extract. Treated LD and LM muscle samples with 10 and 15% of crude actinidin enzyme extract of kiwifruit juice led to improve water holding capacity and higher significant differences in total tyrosine/ tryptophan index (T.T/T) in LD and SM muscles comparison with treatment control. It could be concluded that extract of kiwifruit juice infusion is could be used to improve of meat tenderization.

Keywords: extract of kiwifruit, decline of pH and Temperature , R-value, tyrosine / tryptophan index, sheep meat

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Authors: Shima Mahmoudi, Setareh Mamishi, Babak Pourakbari, Majid Marjani

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In the last years, the potential role of distinct T-cell subsets as biomarkers of active tuberculosis TB and/or latent tuberculosis infection (LTBI) has been studied. The aim of this study was to investigate the potential role of interleukin-2 (IL-2) in whole blood stimulated with M. tuberculosis-specific antigens in the QuantiFERON-TB Gold In Tube (QFT-G-IT) for the discrimination of active and latent tuberculosis. After 72-h of stimulation by antigens from the QFT-G-IT assay, IL-2 secretion was quantitated in supernatants by using ELISA (Mabtech AB, Sweden). Observing the level of IL-2 released after 72-h of incubation, we found that the level of IL-2 were significantly higher in LTBI group than in patients with active TB infection or control group (P value=0.019, Kruskal–Wallis test). The discrimination performance (assessed by the area under ROC curve) between LTBI and patients with active TB was 0.816 (95%CI: 0.72-0.97). Maximum discrimination was reached at a cut-off of 13.9 pg/mL for IL-2 following stimulation with 82% sensitivity and 86% specificity. In conclusion, although cytokine analysis has greatly contributed to the understanding of TB pathogenesis, data on cytokine profiles that might distinguish progression from latency of TB infection are scarce and even controversial. Our data indicate that the concomitant evaluation of IFN- γ and IL-2 could be instrumental in discriminating of active and latent TB infection.

Keywords: interleukin-2, discrimination, active TB, latent TB

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Authors: Ehsan Khorshidian, Afshin Farahbakhsh, Sina Aghili

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Enzymes are promising biocatalysts for many organic reactions. They have excellent features like high activity, specificity and selectivity, and can catalyze under mild and environment friendly conditions. Epoxy-activated supports are almost-ideal ones to perform very easy immobilization of proteins and enzymes at both laboratory and industrial scale. The activated epoxy supports (chitosan/alginate, Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. The enzyme is firstly covalently immobilized under conditions pH 7.0 and 10.0. The remaining groups of the support are blocked to stop additional interaction between the enzyme and support by mercaptoethanol or Triton X-100. The results show support allowed obtaining biocatalysts with high immobilized protein amount and hydrolytic activity. The immobilization of lipases on epoxy support may be considered as attractive tool for obtaining highly active biocatalysts to be used in both aqueous and anhydrous aqueous media.

Keywords: immobilization of enzymes, epoxy supports, enzyme multipoint covalent attachment, microbial lipases

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Authors: Masauso Moses Phiri

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Frequent glucose monitoring is essential to the management of diabetes. Plasmonic enzyme-based glucose biosensors have the advantages of greater specificity, simplicity and rapidity. The aim of this study was to develop a rapid plasmonic colorimetric glucose biosensor based on biocatalytic enlargement of AuNS guided by GOx. Gold nanoparticles of 18 nm in diameter were synthesized using the citrate method. Using these as seeds, a modified seeded method for the synthesis of monodispersed gold nanostars was followed. Both the spherical and star-shaped nanoparticles were characterized using ultra-violet visible spectroscopy, agarose gel electrophoresis, dynamic light scattering, high-resolution transmission electron microscopy and energy-dispersive X-ray spectroscopy. The feasibility of a plasmonic colorimetric assay through growth of AuNS by silver coating in the presence of hydrogen peroxide was investigated by several control and optimization experiments. Conditions for excellent sensing such as the concentration of the detection solution in the presence of 20 µL AuNS, 10 mM of 2-(N-morpholino) ethanesulfonic acid (MES), ammonia and hydrogen peroxide were optimized. Using the optimized conditions, the glucose assay was developed by adding 5mM of GOx to the solution and varying concentrations of glucose to it. Kinetic readings, as well as color changes, were observed. The results showed that the absorbance values of the AuNS were blue shifting and increasing as the concentration of glucose was elevated. Control experiments indicated no growth of AuNS in the absence of GOx, glucose or molecular O₂. Increased glucose concentration led to an enhanced growth of AuNS. The detection of glucose was also done by naked-eye. The color development was near complete in ± 10 minutes. The kinetic readings which were monitored at 450 and 560 nm showed that the assay could discriminate between different concentrations of glucose by ± 50 seconds and near complete at ± 120 seconds. A calibration curve for the qualitative measurement of glucose was derived. The magnitude of wavelength shifts and absorbance values increased concomitantly with glucose concentrations until 90 µg/mL. Beyond that, it leveled off. The lowest amount of glucose that could produce a blue shift in the localized surface plasmon resonance (LSPR) absorption maxima was found to be 10 – 90 µg/mL. The limit of detection was 0.12 µg/mL. This enabled the construction of a direct sensitivity plasmonic colorimetric detection of glucose using AuNS that was rapid, sensitive and cost-effective with naked-eye detection. It has great potential for transfer of technology for point-of-care devices.

Keywords: colorimetric, gold nanostars, glucose, glucose oxidase, plasmonic

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Authors: Pantip Kayee

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17α-ethinylestradiol (EE2) is a recalcitrant micropollutant which is found in small amounts in municipal wastewater. But these small amounts still adversely affect for the reproductive function of aquatic organisms. Evidence in the past suggested that full-scale WWTPs equipped with nitrification process enhanced the removal of EE2 in the municipal wastewater. EE2 has been proven to be able to be transformed by ammonia oxidizing bacteria (AOB) via co-metabolism. This research aims to clarify the EE2 degradation pattern by different consortium of ammonia oxidizing microorganism (AOM) including AOA (ammonia oxidizing archaea) and investigate contribution between the existing ammonia monooxygenase (AMO) and new synthesized AOM. The result showed that AOA or AOB of N. oligotropha cluster in enriched nitrifying activated sludge (NAS) from 2mM and 5mM, commonly found in municipal WWTPs, could degrade EE2 in wastewater via co-metabolism. Moreover, the investigation of the contribution between the existing ammonia monooxygenase (AMO) and new synthesized AOM demonstrated that the new synthesized AMO enzyme may perform ammonia oxidation rather than the existing AMO enzyme or the existing AMO enzyme may has a small amount to oxidize ammonia.

Keywords: 17α-ethinylestradiol, nitrification, ammonia oxidizing bacteria, ammonia oxidizing archaea

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Authors: Belgheis Ebrahimi, Jun Lu

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In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: combination, enhancement effect, perna canaliculus, undaria pinnatifida

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Authors: David D. Adams, Ibrahim B. Bello

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Spent oil contaminated Soil from ten selected mechanic workshops were investigated for their bacteria and bioremediation potentials. The bacterial isolates were morphologically and molecularly identified as Enterobacter hormaechei, Escherichia coli, Klebsiella pneumoniae, Shigella flexneri , Wesiella cibaria, Lactobacillus planetarium. The singles and a consortium of these bacteria incubated in the minimal salt medium incorporated with 1% engine oil exhibited various biodegradation rates, with the mixed consortium exhibiting the highest for this oil. The gene for the hydrocarbon enzyme Catechol 2, 3 dioxygenase (C2,30) was detected and amplified in Enterobacter hormaechei, Escherichia coli and Shigella flexneri using PCR and Agarose gel electrophoresis. The detection of the (C2,30) enzyme gene in, and the spent oil biodegradation activity exhibited by these bacteria suggest their possible possession of bioremediating potentials for the spent engine oil. It is therefore suggested that a pilot study on the field application of these bacteria for bioremediation and restoration of spent oil polluted environment should be done in mechanic workshops.

Keywords: spent engine oil, pollution, bacteria, enzyme, bioremediation, mechanic workshop

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