Search results for: enzyme assay

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: Ezeugwunne Ifeoma Priscilla, Charles Chinedum Onyenekwe, Joseph Eberendu Ahaneku, Rosemary Adanma Analike, Adesuwa Peace Eidangbe

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This study was designed to assess the effect of malaria parasitaemia on serum reproductive hormone levels of asymptomatic HIV adult subjects. A total of 271 participants aged between 17 and 58 ears were conveniently recruited. 135 asymptomatic HIV-infected subjects participated in the study; 67 of them had malaria parasitaemia. 136 HIV seropositive control subjects, 68 of them had malaria parasitaemia. Blood samples were collected from the participants for the determination of HIV status by immunoassay and immunochromatography. Enzyme-linked immunosorbent assay (ELISA) was used to assay for serum LH, FSH, Estrogen, testosterone, progesterone, prolactin, and PSA levels, CD4+T cell counts by Cyflow method, thick and thin films determination of malaria parasitaemia count and density by WHO. Student's t-tests and ANOVA were used to compare means. P<0.05 was considered statistically significant. The results showed significant differences in serum levels of LH, FSH, PSA, estrogen, progesterone, and testosterone amongst the groups at P<0.05, respectively. The serum levels of LH, FSH, and PSA were significantly higher in malaria-infected asymptomatic HIV subjects than in asymptomatic HIV subjects with malaria parasitaemia (P<0.05 in each case). Also, the serum levels of LH, FSH, PSA, estrogen, and progesterone were significantly higher in malaria-infected asymptomatic HIV subjects compared with malaria-infected HIV seronegative subjects (P<0.05, respectively). The mean MP counts and MP density were significantly higher in asymptomatic HIV subjects compared to HIV seronegative subjects (P<0.05, in each case). The mean serum levels of testosterone were significantly lower in both malaria-infected and malaria uninfected HIV seronegative subjects (P<0.05, in each case). In conclusion, Malaria and HIV co-infection might increase the burden of hypogonadism as well as primary testicular failure, hyperprogesteronaemia, elevated levels of estrogen, and PSA in adult males asymptomatic HIV subjects.

Keywords: malaria parasitaemia, HIV, CD4, reproductive hormones

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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: Minh-Phuong Ngoc Bui, Abdennour Abbas

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Microbial spoilage of food/drug has been a constant nuisance and an unavoidable problem throughout history that affects food/drug quality and safety in a variety of ways. A simple and rapid test of fungi and bacteria in food/drugs and environmental clinical samples is essential for proper management of contamination. A number of different techniques have been developed for detection and enumeration of foodborne microorganism including plate counting, enzyme-linked immunosorbent assay (ELISA), polymer chain reaction (PCR), nucleic acid sensor, electrical and microscopy methods. However, the significant drawbacks of these techniques are highly demand of operation skills and the time and cost involved. In this report, we introduce a rapid method for detection of bacteria and fungi in food/drug products using a specific interaction between a reducing agent (tris(2-carboxylethyl)phosphine (TCEP)) and the microbial surface proteins. The chemical reaction was transferred to a transduction system using gold nanoplates-enhanced chemiluminescence. We have optimized our nanoplates synthetic conditions, characterized the chemiluminescence parameters and optimized conditions for the microbial assay. The new detection method was applied for rapid detection of bacteria (E.coli sp. and Lactobacillus sp.) and fungi (Mucor sp.), with limit of detection as low as single digit cells per mL within 10 min using a portable luminometer. We expect our simple and rapid detection method to be a powerful alternative to the conventional plate counting and immunoassay methods for rapid screening of microorganisms in food/drug products.

Keywords: microorganism testing, gold nanoplates, chemiluminescence, reducing agents, luminol

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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: Amir Sasan Mozaffari Nejad

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This study was carried out to detect the presence of aflatoxin B1 (AFB1) in 36 samples of spices from Iran and India that was included of chilli powder (n=12), black pepper powder (n=12) and whole black pepper (n=12). Enzyme-linked immunosorbent assay (ELISA) method was used for analysing the samples. Aflatoxin B1 was found in all the spices samples, the concentration of AFB1 in Iranian samples was ranged from 63.16 to 626.81 ng/kg and in Indian samples was ranged from 31.15 to 245.94 ng/kg. The mean of AFB1 concentration in the chilli powder was significantly higher (P < 0.05) than the whole and powdered black pepper. However, none of the samples exceeded the maximum prescribed limit i.e. 5 µg/kg of European Union regulations for aflatoxin B1. The occurrence of AFB1 in spices samples could be a potential hazard for public health.

Keywords: Aflatoxin B1, chilli, black pepper, ELISA, Iran, India

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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: Bipasa Dey, Varsha Panwar, Tanmay Dutta

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Laccase, a multicopper oxidase (EC 1.10.3.2) have been at the forefront as a superior industrial biocatalyst. They are versatile in terms of bestowing sustainable and ecological catalytic reactions such as polymerisation, xenobiotic degradation and bioremediation of phenolic and non-phenolic compounds. Regardless of the wide biotechnological applications, the critical limiting factors viz. reusability, retrieval, and storage stability still prevail. This can cause an impediment in their applicability. Crosslinked enzyme aggregates (CLEAs) have emerged as a promising technique that rehabilitates these essential facets, albeit at the expense of their enzymatic activity. The carrier free crosslinking method prevails over the carrier-bound immobilisation in conferring high productivity, low production cost owing to the absence of additional carrier and circumvent any non-catalytic ballast which could dilute the volumetric activity. To the best of our knowledge, the ε-amino group of lysyl residue is speculated as the best choice for forming Schiff’s base with glutaraldehyde. Despite being most preferrable, excess glutaraldehyde can bring about disproportionate and undesirable crosslinking within the catalytic site and hence could deliver undesirable catalytic losses. Moreover, the surface distribution of lysine residues in Trametes versicolor laccase is significantly less. Thus, to mitigate the adverse effect of glutaraldehyde in conjunction with scaling down the degradation or catalytic loss of the enzyme, crosslinking with inert substances like gelatine, collagen, Bovine serum albumin (BSA) or excess lysine is practiced. Analogous to these molecules, sugars have been well known as a protein stabiliser. It helps to retain the structural integrity, specifically secondary structure of the protein during aggregation by changing the solvent properties. They are comprehended to avert protein denaturation or enzyme deactivation during precipitation. We prepared crosslinked enzyme aggregates (CLEAs) of laccase from T. versicolor with the aid of sugars. The sugar CLEAs were compared with the classic BSA and glutaraldehyde laccase CLEAs concerning physico-chemical properties. The activity recovery for the fructose CLEAs were found to be ~20% higher than the non-sugar CLEA. Moreover, the 𝐾𝑐𝑎𝑡𝐾𝑚⁄ values of the CLEAs were two and three-fold higher than BSA-CLEA and GACLEA, respectively. The half-life (t1/2) deciphered by sugar-CLEA was higher than the t1/2 of GA-CLEAs and free enzyme, portraying more thermal stability. Besides, it demonstrated extraordinarily high pH stability, which was analogous to BSA-CLEA. The promising attributes of increased storage stability and recyclability (>80%) gives more edge to the sugar-CLEAs over conventional CLEAs of their corresponding free enzyme. Thus, sugar-CLEA prevails in furnishing the rudimentary properties required for a biocatalyst and holds many prospects.

Keywords: cross-linked enzyme aggregates, laccase immobilization, enzyme reusability, enzyme stability

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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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: 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: Shimayali Kaushal, Nitesh Priyadarshi, Nitin Kumar Singhal

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Food borne bacterial species have been identified as major pathogens in most of the severe pathogen-related diseases among humans which result in great loss to human health and food industry. Conventional methods like plating and enzyme-linked immune sorbent assay (ELISA) are time-consuming, laborious and require specialized instruments. Nanotechnology has emerged as a great field in case of rapid detection of pathogens in recent years. The AuNRs material has good electro-optical properties due to its larger light absorption band and scattering in surface plasmon resonance wavelength regions. By exploiting the sugar-based adhesion properties of microorganism, we can use the glycoconjugates capped gold nanorods as a potential nanobiosensor to detect the foodborne pathogen. In the present study, polyethylene glycol (PEG) coated gold nanorods (AuNRs) were prepared and functionalized with different types of carbohydrates and further characterized by UV-Visible spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy (TEM). The reactivity of above said nano-biosensor was probed by lectin binding assay and also by different strains of foodborne bacteria by using spectrophotometric and microscopic techniques. Due to the specific interaction of probe with foodborne bacteria (Escherichia coli, Pseudomonas aeruginosa), our nanoprobe has shown significant and selective ablation of targeted bacteria. Our findings suggest that our nanoprobe can be an ideal candidate for selective optical detection of food pathogens and can reduce loss to the food industry.

Keywords: glyco-conjugates, gold nanorods, nanobiosensor, nanoprobe

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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: Hiroshi Nakayama, Yuji Ito

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Synthetic cannabinoids have attracted much public attention recently in Japan. 1-pentyl-3-(1-naphthoyl)-indole (JWH-018), 1-pentyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), 1-(5-fluoropentyl)-3- (1-(2,2,3,3- tetramethylcyclopropyl)) indole (XLR-11) and 1-methyl-3- (1-admantyl) indole (JWH-018 adamantyl analog) are known as synthetic cannabinoids and are also considered dangerous illegal drugs in Japan. It has become necessary to develop sensitive and useful methods for detection of synthetic cannabinoids. We produced two monoclonal antibodies (MAb) against synthetic cannabinoids, named NT1 (IgG1) and NT2 (IgG1), using Hybridoma technology. The cross-reactivity of these produced MAbs was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). In the results, we found both of these antibodies recognize many kinds of synthetic cannabinoids analog. However, neither of these antibodies recognizes naphtoic acid, 1-methyl-indole and indole known as a raw material of synthetic cannabinoid. Thus, the MAbs produced in this study could be a useful tool for the detection of synthetic cannabinoids.

Keywords: ELISA, monoclonal antibody, sensor, synthetic cannabinoid

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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: Hassan Waseem

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Cytomegalovirus (CMV) is ubiquitously distributed viral agent responsible for different clinical manifestations that may vary according to the immunologic status of the patient. CMV can cause morbidity and mortality among fetuses and patients with compromised immune system. A cross-sectional study was carried out in Islamabad to investigate the prevalence and risk factors associated with CMV infection among pregnant women. Blood samples of 172 pregnant women visiting Mother and Child Healthcare, Pakistan Institute of Medical Sciences (PIMS) Islamabad were taken. In present study, serum samples of the women were checked for CMV-specific IgG and IgM antibodies by enzyme linked immunosorbent assay (ELISA). Clinical, obstetrical and socio-demographical characteristics of the women were collected by using structured questionnaires. Out of 172 pregnant women included in the study, 171 (99.4%) were CMV specific IgG positive and 30 (17.4%) were found positive for CMV-IgM antibodies. The CMV has taken an endemic form in Pakistan so, routine screening of CMV among pregnant women is recommended.

Keywords: Cytomegalovirus, blood transfusion, ELISA, seroprevalence

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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: Nattawit Thiapairat

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Multiple diseases have been linked to excessive levels of free radicals, which cause tissue or cell damage as a result of oxidative stress. Many plants are sources of high antioxidant activity. Derris scandens has a high amount of phenolic and flavonoid contents which demonstrated good biological activities. This study focused on the antioxidant activity of polyphenols extracted from D. scandens. This study performs total flavonoids content and various antioxidant assays, which were 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity assays. The total flavonoid content of D. scandens extract was determined and expressed as quercetin equivalents (QE)/g measured by the aluminum chloride colorimetric method. The antioxidant activity of D. scandens extract was also determined by DPPH and ABTS assays. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. The half-maximal inhibitory concentration (IC50) values for D. scandens extract from DPPH and ABTS assays were 41.79 μg/mL ± 0.783 and 29.42 μg/mL ± 0.890, respectively, in the DPPH assay. To conclude, D. scandens extract consists of a high amount of total phenolic content, which exhibits a significant antioxidant activity. However, further investigation regarding antioxidant activity such as SOD, ROS, and RNS scavenging assays and in vivo experiments should be performed.

Keywords: ABTS assay, antioxidant activity, Derris scandens, DPPH assays, total flavonoid content

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Authors: Ibrahim Alshunaibir

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Infection with Helicobacter pylori is common worldwide but few studies focus on the prevalence and spread of the infection in Saudi Arabia. This study was undertaken to observe the epidemiology of Helicobacter pylori infection in patients suffering from gastrointestinal sign and symptoms in one of the largest hospitals in the capital of Saudi Arabia, Riyadh. Methods: Enzyme-linked immunosorbent assay (ELISA) was undertaken for this study with nearly 6000 samples collected and examined for patients suffering from (dyspeptic) symptoms ranging in their age from 5 to 75 years. Results: The prevalence of helicobacter infection was 67% increasing with age. Female shows higher percentage of H. pylori infection than male. Conclusions: The percentage rate was higher in female than male. This study shows a high percentage of helicobacter infection in Saudi Arabia.

Keywords: Helicobacter pylori, percentage, dyspeptic, Saudi Arabia

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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

Abstract:

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: 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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Authors: C. Asha Poorna, N. S. Pradeep

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The biological function of lipase is to catalyze the hydrolysis of triacylglycerols to give free fatty acid, diacylglycerols, mono-acylglycerols and glycerol. They constitute the most important group of biocatalysts for biotechnological applications. The aim of the present study was to identify the lipolytic activity of Streptomyces sp. From soil sample collected from the sacred groves of southern Kerala. The culture conditions of the isolate were optimised and the enzyme was purified and characterised. The purification was attempted with acetone precipitation. The isolate observed to have high lipolytic activity and identified to be of Streptomyces strain. The purification was attempted with acetone precipitation. The purified enzyme observed to have an apparent molecular mass of ~60kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed maximum activity at 60oC and pH-8. The lipase showed tolerance towards different organic solvents like ethanol and methanol that are commonly used in transesterification reactions to displace alcohol from triglycerides contained in renewable resources to yield fatty acid alkyl esters known as biodiesel.

Keywords: lipase, Streptomyces, biodiesel, fatty acid, transesterification

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Authors: Michelle Belinda S. Weerawardana, Gobika Thiripuranathar, Priyani A. Paranagama

Abstract:

Most of fresh vegetables and fruits available in the retail markets undergo a physiological disorder in its appearance and coloration, which indeed discourages consumer purchase. A loss of millions of dollars yearly to the food industry had been due to this pronounced color reaction called Enzymatic Browning which is driven due to the catalytic activity by an oxidoreductase enzyme, polyphenol oxidase (PPO). The enzyme oxidizes the phenolic compounds which are abundantly available in fruits and vegetables as substrates into quinones, which could react with proteins in its surrounding to generate black pigments, called melanins, which are highly UV-active compounds. Annona muricata (Katu anoda) and Musa acuminata (Ash plantains) is a fruit and a vegetable consumed by Sri Lankans widely due to their high nutritional values, medicinal properties and economical importance. The objective of the present study was to evaluate and determine the effective natural anti-browning inhibitors that could prevent PPO activity in the selected fruit and vegetable. Enzyme extracts from Annona muricata (Katu anoda) and Musa acuminata (Ash plantains), were prepared by homogenizing with analytical grade acetone, and pH of each enzyme extract was maintained at 7.0 using a phosphate buffer. The extracts of inhibitors were prepared using powdered ginger rhizomes and essential oil from the bark of Cinnamomum zeylanicum. Water extracts of ginger were prepared and the essential oil from Ceylon cinnamon bark was extracted using steam distillation method. Since the essential oil is not soluble in water, 0.1µl of cinnamon bark oil was mixed with 0.1µl of Triton X-100 emulsifier and 5.00 ml of water. The effect of each inhibitor on the PPO activity was investigated using catechol (0.1 mol dm-3) as the substrate and two samples of enzyme extracts prepared. The dosages of the prepared Cinnamon bark oil, and ginger (2 samples) which were used to measure the activity were 0.0035 g/ml, 0.091 g/ml and 0.087 g/ml respectively. The measurements of the inhibitory activity were obtained at a wavelength of 525 nm using the UV-visible spectrophotometer. The results evaluated thus revealed that % inhibition observed with cinnamon bark oil, and ginger for Annona muricata was 51.97%, and 60.90% respectively. The effects of cinnamon bark oil, and ginger extract on PPO activity of Musa acuminata were 49.51%, and 48.10%. The experimental findings thus revealed that Cinnamomum zeylanicum bark oil was a more effective inhibitor for PPO enzyme present in Musa acuminata and ginger was effective for PPO enzyme present in Annona muricata. Overall both the inhibitors were proven to be more effective towards the activities of PPO enzyme present in both samples. These inhibitors can thus be corroborated as effective, natural, non-toxic, anti-browning extracts, which when added to the above fruit and vegetable will increase the shelf life and also the acceptance of the product by the consumers.

Keywords: anti-browning agent, enzymatic browning, inhibitory activity, polyphenol oxidase

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