Association between sheath blight resistance and chitinase activity in transgenic rice plants expressing McCHIT1 from bitter melon.

No usable resources with high-level resistance to sheath blight (SB) have yet been found in rice germplasm resources worldwide. Therefore, creating and breeding new disease-resistant rice resources with sheath blight resistance (SBR) are imperative. In this study, we inoculated rice plants with hyphae of the highly pathogenic strain RH-9 of rice SB fungus Rhizoctonia solani to obtain eight stable transgenic rice lines harbouring the chitinase gene (McCHIT1) of bitter melon with good SBR in the T5 generation. The mean disease index for SB of wild-type plants was 92% and 37-44% in transgenic lines. From 24 h before until 120 h after inoculation with R. solani, chitinase activity in stable transgenic plants with increased SBR was 2.0-5.5 and 1.8-2.7 times that of wild-type plants and plants of a disease-susceptible stable transgenic line, respectively. The correlation between SBR and chitinase activity in McCHIT1-transgenic rice line plants was significant. This work stresses how McCHIT1 from bitter melon can be used to protect rice plants from SB infection.

Metabolite profiling and in vitro biological activities of two commercial bitter melon (Momordica charantia Linn.) cultivars.

The current study was designed to characterize the metabolite profile and bioactivity of two commercial bitter melon (Momordica charantia Linn.) genotypes. UPLC-high resolution mass spectrometry (HRMS) was used to identify 15 phenolic and 46 triterpenoids in various bitter melon extracts. Total phenolic levels were the highest (57.28 ±â€¯1.02) in methanolic extract of the inner tissue of Indian Green cultivar, which also correlated to the highest DPPH radical scavenging activity (30.48 ±â€¯2.49 ascorbic acid equivalents (mg of AAE)/g of FD). In addition, highest levels of total saponins were observed in chloroform extract of the Chinese bitter melon pericarp (75.73 mg ±â€¯4.67 diosgenin equivalents (DE)/g of FD). Differential inhibition of α-amylase and α-glucosidase activity was observed in response to polarity of extract, cultivar and tissue type. These results suggest that consumption of whole bitter melon may have potential health benefits to manage diabetes.

Magneto-Priming Improved Nutraceutical Potential and Antimicrobial Activity of Momordica charantia L. Without Affecting Nutritive Value.

The need for some economic strategies for increased growth and nutraceuticals of medicinal plants is well acknowledged now. It was hypothesized that external magnetic field treatment (MFT) of seeds affecting internal magnet of cells may affect growth and metabolism. In this study, seeds were subjected to pre-sowing magnetic field (50 mT at 5 mm for 5 s). At vegetative stage, the leaf growth, chlorophyll content, catalase (CAT), peroxidase (POD), amino acids, proteins, flavonoids, soluble sugars, total soluble phenolics, carotenoids, anthocyanins, phenolic profile (HPLC based), and antimicrobial activity of leaves (in terms of the minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa) were studied. Yield was evaluated for nutritive components in fruit (peel+pulp) and peel. MFT improved germination percentage, growth, leaf chlorophyll, antimicrobial activity, peel amino acids, phenolics, and POD with negligible effect on fruit nutritive value. Moreover, photosynthetic pigments and cinnamic acid exhibited direct correlation with antimicrobial potential against both pathogens. However, sinapic acid showed positive correlation against Staphylococcus aureus only. Cinnamic acid, coumaric acid, syringic acid, and quercetin were in direct correlation against Pseudomonas aeruginosa; it was directly correlated with total flavonoids too. In conclusion, magnetic field can be used to manipulate plant cell metabolism promising improvement of growth, antimicrobial activity, and phenolics of interest.

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis.

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), ß-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits. Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads.

Variation in antioxidant enzyme activities, growth and some physiological parameters of bitter melon (Momordica charantia) under salinity and chromium stress.

In general, salinity and heavy metals interfere with several physiological processes and reduce plant growth. In order to evaluate of three levels of salinity (0, 4 and 8 ds m(-1)) and three concentration of chromium (0, 10 and 20 mg kg(-1) soil) in bitter melon (Momordica charantia), a plot experiment was conducted in greenhouse at university of Shahrood, Iran. The results revealed that chromium treatment had no significant affect on fresh and dry weight, but salinity caused reduction of fresh and dry weight in growth parameter. Salinity and chromium enhanced antioxidant enzymes activities like catalase (CAT), guaiacol peroxidase (GPX) and sodium content in leaves. However salinity and chromium treatments had no effect on potassium, phosphorus in leaves, soluble carbohydrate concentration in leaves and root, but decreased the carotenoid content in leaves. On increasing salinity from control to 8 ds m(-1) chlorophyll a, b and anthocyanin content decreased by 41.6%, 61.1% and 26.5% respectively but chromium treatments had no significant effect on these photosynthetic pigments.

Flexible CDOCKER: Development and application of a pseudo-explicit structure-based docking method within CHARMM.

Protein-ligand docking is a commonly used method for lead identification and refinement. While traditional structure-based docking methods represent the receptor as a rigid body, recent developments have been moving toward the inclusion of protein flexibility. Proteins exist in an interconverting ensemble of conformational states, but effectively and efficiently searching the conformational space available to both the receptor and ligand remains a well-appreciated computational challenge. To this end, we have developed the Flexible CDOCKER method as an extension of the family of complete docking solutions available within CHARMM. This method integrates atomically detailed side chain flexibility with grid-based docking methods, maintaining efficiency while allowing the protein and ligand configurations to explore their conformational space simultaneously. This is in contrast to existing approaches that use induced-fit like sampling, such as Glide or Autodock, where the protein or the ligand space is sampled independently in an iterative fashion. Presented here are developments to the CHARMM docking methodology to incorporate receptor flexibility and improvements to the sampling protocol as demonstrated with re-docking trials on a subset of the CCDC/Astex set. These developments within CDOCKER achieve docking accuracy competitive with or exceeding the performance of other widely utilized docking programs.

Constitutive expression of McCHIT1-PAT enhances resistance to rice blast and herbicide, but does not affect grain yield in transgenic glutinous rice.

To produce new rice blast- and herbicide-resistant transgenic rice lines, the McCHIT1 gene encoding the class I chitinase from Momordica charantia and the herbicide resistance gene PAT were introduced into Lailong (Oryza sativa L. ssp. Japonica), a glutinous local rice variety from Guizhou Province, People's Republic of China. Transgenic lines were identified by ß-glucuronidase (GUS) histochemical staining, PCR, and Southern blot analyses. Agronomic traits, resistance to rice blast and herbicide, chitinase activities, and transcript levels of McCHIT1 were assessed in the T2 progeny of three transgenic lines (L1, L8, and L10). The results showed that the introduction of McCHIT1-PAT into Lailong significantly enhanced herbicide and blast resistance. After infection with the blast fungus Magnaporthe oryzae, all of the T2 progeny exhibited less severe lesion symptoms than those of wild type. The disease indices were 100% for wild type, 65.66% for T2 transgenic line L1, 59.69% for T2 transgenic line L8, and 79.80% for T2 transgenic line L10. Transgenic lines expressing McCHIT1-PAT did not show a significant difference from wild type in terms of malondialdehyde (MDA) content, polyphenol oxidase (PPO) activity, and superoxide dismutase (SOD) activity in the leaves. However, after inoculation with M. oryzae, transgenic plants showed significantly higher SOD and PPO activities and lower MDA contents in leaves, compared with those in wild-type leaves. The transgenic and the wild-type plants did not show significant differences in grain yield parameters including plant height, panicles per plant, seeds per panicle, and 1000-grain weight. Therefore, the transgenic plants showed increased herbicide and blast resistance, with no yield penalty.

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia.

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA(Tyr I). Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5'-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.

Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.

Honokiol trimers and dimers via biotransformation catalyzed by Momordica charantia peroxidase: novel and potent α-glucosidase inhibitors.

Ten honokiol oligomers (1-10), including four novel trimers (1-4) and four novel dimers (5-8), were obtained by means of biotransformation of honokiol catalyzed by Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of spectroscopic methods. The biological results demonstrated that most of the oligomers were capable of inhibiting α-glucosidase with significant abilities, which were one to two orders of magnitude more potent than the substrate, honokiol. In particular, compound 2, the honokiol trimer, displayed the greatest inhibitory activity against α-glucosidase with an IC50 value of 1.38µM. Kinetic and CD studies indicated that 2 inhibited α-glucosidase in a reversible, mixed-type manner and caused conformational changes in the secondary structure of the enzyme protein. These findings suggested that 2 might be exploited as a promising drug candidate for the treatment of diabetes.

α-Glucosidase inhibitors via green pathway: biotransformation for bicoumarins catalyzed by Momordica charantia peroxidase.

Peroxidase extracted from Momordica charantia catalyzed the H(2)O(2)-dependent oxidative coupling of 7-hydroxy-4-methylcoumarin to form four new dimers (1-4) and two known ones (5, 6). The structures, including the absolute configurations of axially chiral compounds, were unambiguously characterized by NMR spectroscopy, online HPLC-CD, and a variety of computational methods. Bioactive experiments demonstrated that compounds 1 and 2 had significant inhibitory effects on yeast α-glucosidase, much better than the controls. Noncompetitive binding mode was found by the graphical analysis of steady-state inhibition data. The mechanism of enzymatic inhibition confirmed in some depth that the inhibitors altered the secondary structure of α-glucosidase by decreasing the α-helix and increasing the ß-sheet content. In summary, bicoumarins 1 and 2 might be exploited as the lead compounds for further research of antidiabetic agents, and this research provided a "green" method to synthesize compounds with the chiral biaryl axis generally calling for multistep reactions in organic chemistry.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress.

Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

In vitro and in vivo anticarcinogenic effects of RNase MC2, a ribonuclease isolated from dietary bitter gourd, toward human liver cancer cells.

Hepatocellular carcinoma (HCC) constitutes a predominant part of primary liver cancer which ranks as the fifth most common cancer as well as the third most common cause of cancer mortality. In view of the poor prognosis of unresectable liver cancers, it is of pivotal importance to develop novel chemotherapeutical regimens. RNase MC2 is a 14-kDa ribonuclease isolated from dietary bitter gourd (Momordica charantia) that manifested antitumor potential against breast cancers. In this study, we investigated the potential application of RNase MC2 on Hep G2 cells. We showed that RNase MC2 inhibited cell proliferation and induced cell apoptosis in both in vitro and in vivo studies. RNase MC2 treatment caused cell cycle arrest predominantly at the S-phase and apoptosis, which is associated with the activation of both caspase-8 and caspase-9 regulated caspase pathways. Our further investigation disclosed that RNase MC2 down-regulated the anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bak. Moreover, the phosphorylation of ERK and JNK was involved in the apoptosis process. Importantly, RNase MC2 significantly suppressed the growth of Hep G2 xenograft-bearing nude mice by inducing apoptosis. This notion is supported by data indicating an increased number of caspase-3- and PARP-positive cells, and TUNEL-positive cells in RNase MC2-treated tumor tissues. In summary, we have revealed the antitumor potential of RNase MC2 toward Hep G2 cells. Considering that bitter gourd is a common dietary component in many countries, this study may help to prompt the clinical application of RNase MC2.

Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase.

Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways.

Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker's yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.

Catalyzed degradation of disperse dyes by calcium alginate-pectin entrapped bitter gourd (Momordica charantia) peroxidase.

Calcium-alginate pectin entrapped bitter gourd peroxidase (BGP) has been employed for the treatment of disperse dyes: Disperse Brown 1 (DB 1) and Disperse Red 17 (DR 17). Peroxidase alone was unable to decolorize DR 17 and DB 1. However, the investigated dyes were decolorized maximally by BGP in the presence of 0.2 mmol/L redox mediator, violuric acid (VA). A slow decrease in percent decolorization was observed when VA concentration was higher than 0.2 mmol/L which could likely be due to the high reactivity of its aminoxyl radical (> N-O*) intermediate, that might undergo chemical reactions with aromatic amino acid side chains of the enzyme thereby inactivating it. Maximum decolorization of the dyes was observed at pH 3.0 and 40 degrees C within 2 hr of incubation. Immobilized peroxidase decolorized 98% DR 17 and 71% DB 1 using 35 U of BGP in batch process in 90 min. Immobilized enzyme decolorized 85% DR 17 and 51% DB 1 whereas soluble enzyme decolorized DR 17 to 48% and DB 1 to 30% at 60 degrees C. UV-visible spectral analysis was used to evaluate the degradation of these dyes and their toxicity was tested by Allium cepa test. The generally observed higher stability of the bioaffinity bound enzymes against various forms of inactivation may be related to the specific and strong binding of enzyme with bioaffinity support which prevents the unfolding/denaturation of enzyme. Thus entrapped peroxidase was found to be effective in the decolorization of the investigated dyes.

Dimerization of piceatannol by Momordica charantia peroxidase and α-glucosidase inhibitory activity of the biotransformation products.

Stilbenes, especially those oligomers, have great potential to be antihyperglycemic agents. In this study, eight stilbene dimers, including five new ones, were obtained by biotransformation of piceatannol using Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of spectroscopic evidences. These piceatannol dimers displayed potential α-glucosidase inhibitory activities, and trans double bond, tetrahydrofuran ring, and free adjacent phenolic dihydroxyls were found to be important for their activities. Enzymatic biotransformation of stilbenes by M.charantia peroxidase (MCP) was showed to be a prominent way to produce oligomeric stilbenes for antihyperglycemic development.

Anticlastogenic and anticarcinogenic potential of Thai bitter gourd fruits.

Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possess phase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activating enzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)- induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined the anticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, in mice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesis in rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing 6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end of experiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. For anticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% ground freeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kg once a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleated peripheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstrated that rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as the multiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruits possesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potential against AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm when giving during the initiation stage.

Removal of benzidine from polluted water by soluble and immobilized peroxidase in batch processes and continuous horizontal bed reactor.

Bitter gourd peroxidase entrapped on calcium-alginate-starch beads was employed for the treatment of water, polluted with benzidine, in a batch process as well as in a continuous reactor. The immobilized enzyme had the same pH and temperature optima as the soluble enzyme. The immobilized enzyme could effectively remove more than 70% of benzidine in a stirred batch process after three hours. After its sixth repeated use, the immobilized enzyme was able to oxidatively degrade and polymerize 58% of benzidine. The horizontal bed reactor, containing the peroxidase entrapped on calcium-alginate-starch beads, retained more than 40% benzidine removal efficiency after one month of its continuous operation. The absorption spectra of the treated benzidine exhibited a marked difference in the absorption at various wavelengths, compared with untreated compound. The horizontal bed reactor containing immobilized peroxidase would be significantly successful for the large-scale treatment of water polluted with aromatic compounds.