Abstract: Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA4404. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.
Abstract: Cocoa beans (Theobroma cocoa L.) are the main components for chocolate manufacturing. The beans must be correctly fermented at first. Traditional process to perform the first fermentation (lactic fermentation) often consists in confining cacao beans using banana leaves or a fermentation basket, both of them leading to a poor product thermal insulation and to an inability to mix the product. Box fermenter reduces this loss by using a wood with large thickness (e>3cm), but mixing to homogenize the product is still hard to perform. Automatic fermenters are not rentable for most of producers. Heat (T>45°C) and acidity produced during the fermentation by microbiology activity of yeasts and bacteria are enabling the emergence of potential flavor and taste of future chocolate. In this study, a cylindro-rotative fermenter (FCR-V1) has been built and coconut fibers were used in its structure to confine heat. An axis of rotation (360°) has been integrated to facilitate the turning and homogenization of beans in the fermenter. This axis permits to put fermenter in a vertical position during the anaerobic alcoholic phase of fermentation, and horizontally during acetic phase to take advantage of the mid height filling. For circulation of air flow during turning in acetic phase, two woven rattan with grid have been made, one for the top and second for the bottom of the fermenter. In order to reduce air flow during acetic phase, two airtight covers are put on each grid cover. The efficiency of the turning by this kind of rotation, coupled with homogenization of the temperature, caused by the horizontal position in the acetic phase of the fermenter, contribute to having a good proportion of well-fermented beans (83.23%). In addition, beans’pH values ranged between 4.5 and 5.5. These values are ideal for enzymatic activity in the production of the aromatic compounds inside beans. The regularity of mass loss during all fermentation makes it possible to predict the drying surface corresponding to the amount being fermented.
Abstract: Beta-glucosidase, chitinase, leucine-aminopeptidase, acid phosphomonoesterase and acetate-esterase enzyme activities in
the soils under the impact of metallurgical industrial activity in Lori
marz (district) were investigated. The results of the study showed that
the activities of the investigated enzymes in the soils decreased with increasing distance from the Shamlugh copper mine, the Chochkan
tailings storage facility and the ore transportation road. Statistical
analysis revealed that the activities of the enzymes were positively
correlated (significant) to each other according to the observation
sites which indicated that enzyme activities were affected by the
same anthropogenic factor. The investigations showed that the soils
were polluted with heavy metals (Cu, Pb, As, Co, Ni, Zn) due to
copper mining activity in this territory. The results of Pearson
correlation analysis revealed a significant negative correlation
between heavy metal pollution degree (Nemerow integrated pollution
index) and soil enzyme activity. All of this indicated that copper
mining activity in this territory causing the heavy metal pollution of
the soils resulted in the inhabitation of the activities of the enzymes
which are considered as biological catalysts to decompose organic
materials and facilitate the cycling of nutrients.
Abstract: In order to detect and quantify the phenolic contents
of a wastewater with biosensors, two working electrodes based on
modified Poly(Pyrrole) films were fabricated. Enzyme horseradish
peroxidase was used as biomolecule of the prepared electrodes.
Various phenolics were tested at the biosensor. Phenol detection was
realized by electrochemical reduction of quinones produced by
enzymatic activity. Analytical parameters were calculated and the
results were compared with each other.
Abstract: This research was undertaken to study enzymatic activity in the shoots, roots, and rhizosphere of alfalfa (Medicago sativa L.) grown in quartz sand that was uncontaminated and
contaminated with phenanthrene at concentrations of 10 and 100 mg kg-1. The higher concentration of phehanthrene had a distinct
phytotoxic effect on alfalfa, inhibiting seed germination energy, plant survival, and biomass accumulation. The plant stress response to the
environmental pollution was an increase in peroxidase activity. Peroxidases were the predominant enzymes in the alfalfa shoots and
roots. The peroxidase profile in the shoots differed from that in the roots and had different isoenzyme numbers. 2,2'-Azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS) peroxidase was
predominant in the shoots, and 2,7-diaminofluorene (2,2-DAF)
peroxidase was predominant in the roots. Under the influence of
phenanthrene, the activity of 2,7-DAF peroxidase increased in the
shoots, and the activity of ABTS peroxidase increased in the roots.
Alfalfa root peroxidases were the prevalent enzyme systems in the
rhizosphere sand. Examination of the activity of alfalfa root
peroxidase toward phenanthrene revealed the possibility of
involvement of the plant enzyme in rhizosphere degradation of the
PAH.
Abstract: Sol-gel immobilization of enzymes, which can improve considerably their properties, is now one of the most used techniques. By deposition of the entrapped lipase on a solid support, a new and improved biocatalyst was obtained, which can be used with excellent results in acylation reactions. In this paper, lipase B from Candida antarctica was double immobilized on different adsorbents. These biocatalysts were employed in the kinetic resolution of several aliphatic secondary alcohols in organic medium. High total recovery yields of enzymatic activity, up to 560%, were obtained. For all the studied alcohols the enantiomeric ratios E were over 200. The influence of the reaction medium was studied for the kinetic resolution of 2-pentanol.
Abstract: Characterization and evaluation of the activity of Vespa basalis DPP-IV, which expressed in Spodoptera frugiperda 21 cells. The expression of rDPP-IV was confirmed by SDS–PAGE, Western blot analyses, LC-MS/MS and measurement of its peptidase specificity. One-step purification by Ni-NTA affinity chromatography and the total amount of rDPP-IV recovered was approximately 6.4mg per liter from infected culture medium; an equivalent amount would be produced by 1x109 infected Sf21 insect cells. Through the affinity purification led to highly stable rDPP-IV enzyme was recovered and with significant peptidase activity. The rDPP-IV exhibited classical Michaelis–Menten kinetics, with kcat/Km in the range of 10-500 mM-1×S-1 for the five synthetic substrates and optimum substrate is Ala-Pro-pNA. As expected in inhibition assay, the enzymatic activity of rDPP-IV was significantly reduced by 80 or 60% in the presence of sitagliptin (a DPP-IV inhibitor) or PMSF (a serine protease inhibitor), but was not apparently affected by iodoacetamide (a cysteine protease inhibitor).