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: Soil enzyme activities in Kasuga-yama Hill Primeval Forest (Nara, Japan) were examined to determine levels of mineralization and metabolism. Samples were selected from the soil surrounding laurel-leaved (BB-1) and Carpinus japonica (BB-2 and Pw) trees for analysis. Cellulase, β-xylosidase, and protease activities were higher in BB-1 samples those in BB-2 samples. These activity levels corresponded to the distribution of cellulose and hemicellulose in the soil horizons. Cellulase, β-xylosidase, and chymotrypsin activities were higher in soil from the Pw forest than in that from the BB-2 forest. The relationships between the soil enzymes calculated by Spearman’s rank correlation indicate that the interactions between enzymes in BB-2 samples were more complex than those in Pw samples.
Abstract: Meat Tenderness is one of the most important factors affecting consumers' assessment of meat quality. Variation in meat tenderness is genetically controlled and varies among breeds, and it is also influenced by environmental factors that can affect its creation during rigor mortis and postmortem. The final postmortem meat tenderization relies on the extent of proteolysis of myofibrillar proteins caused by the endogenous activity of the proteolytic calpain system. This calpain system includes different calcium-dependent cysteine proteases, and an inhibitor, calpastatin. It is widely accepted that in farm animals including chickens, the μ-calpain gene (CAPN1) is a physiological candidate gene for meat tenderness. This study aimed to identify the association of single nucleotide polymorphism (SNP) markers in the CAPN1 gene with the tenderness of chicken breast meat from two Malaysian native and commercial broiler breed crosses. Ten, five months old native chickens and ten, 42 days commercial broilers were collected from the local market and breast muscles were removed two hours after slaughter, packed separately in plastic bags and kept at -20ºC for 24 h. The tenderness phenotype for all chickens’ breast meats was determined by Warner-Bratzler Shear Force (WBSF). Thawing and cooking losses were also measured in the same breast samples before using in WBSF determination. Polymerase chain reaction (PCR) was used to identify the previously reported C7198A and G9950A SNPs in the CAPN1 gene and assess their associations with meat tenderness in the two breeds. The broiler breast meat showed lower shear force values and lower thawing loss rates than the native chickens (p
Abstract: Food contamination occurs during post process
handling. This leads to spoilage and growth of pathogenic
microorganisms in the food, thereby reducing its shelf life or
spreading of food borne diseases. Several methods are tried and one
of which is use of antimicrobial packaging. Here, papain, a protease
enzyme, is covalently immobilized with the help of glutarldehyde on
polyurethane and used as a food wrap to protect food from microbial
contamination. Covalent immobilization of papain was achieved at a
pH of 7.4; temperature of 4°C; glutaraldehyde concentration of 0.5%;
incubation time of 24h; and 50mg of papain. The formation of -C=Nobserved
in the Fourier transform infrared spectrum confirmed the
immobilization of the enzyme on the polymer. Immobilized enzyme
retained higher activity than the native free enzyme. The modified
polyurethane showed better reduction of Staphylococcus aureus
biofilm than bare polymer film (eight folds reduction in live colonies,
two times reduction in protein and 6 times reduction in
carbohydrates). The efficacy of this was studied by wrapping it over
S. aureus contaminated cottage cheese (paneer) and cheese and
stored at a temperature of 4°C for 7days. The modified film reduced
the bacterial contamination by eight folds when compared to the bare
film. FTIR also indicated reduction in lipids, sugars and proteins in
the biofilm.
Abstract: Fibrin degradation is an important part in prevention
or treatment of intravascular thrombosis and cardiovascular diseases.
Plasmin like fibrinolytic enzymes has given new hope to patient with
cardiovascular diseases by treating fibrin aggregation related diseases
with traditional plasminogen activator which have many side effects.
Various researches involving wide range of sources for production of
fibrinolytic proteases, from bacteria, fungi, insects and fermented
foods. But few have looked into endophytic fungi as a potential
source. Sixteen (16) endophytic fungi were isolated from Hibiscus sp.
leaves from six different locations in Shah Alam, Selangor. Only two
endophytic fungi, FH3 and S13 showed positive fibrinolytic protease
activities. FH3 produced 5.78cm and S13 produced 4.48cm on Skim
Milk Agar after 4 days of incubation at 27°C. Fibrinolytic activity
was observed; 3.87cm and 1.82cm diameter clear zone on fibrin plate
of FH3 and S13 respectively. 18srRNA was done for identification of
the isolated fungi with positive fibrinolytic protease. S13 had the
highest similarity (100%) to that of Penicillium citrinum strain TG2
and FH3 had the highest similarity (99%) to that of Fusarium sp.
FW2PhC1, Fusarium sp. 13002, Fusarium sp. 08006, Fusarium
equiseti strain Salicorn 8 and Fungal sp. FCASAn-2. Media
composition variation showed the effects of carbon nitrogen on
protein concentration, where the decrement of 50% of media
composition caused drastic decrease in protease of FH3 from 1.081 to
0.056 and also S13 from 2.946 to 0.198.
Abstract: Using an enzyme of known specificity the hydrolysis of protein was carried out in a controlled manner. The aim was to obtain oligopeptides being the so-called active peptides or their direct precursors. An original way of expression of the protein hydrolysis kinetics was introduced. Peptide bonds contained in the protein were recognized as a diverse-quality substrate for hydrolysis by the applied protease. This assumption was positively verified taking as an example the hydrolysis of albumin by thermolysin. Peptide linkages for this system should be divided into at least four groups. One of them is a group of bonds non-hydrolyzable by this enzyme. These that are broken are hydrolyzed at a rate that differs even by tens of thousands of times. Designated kinetic constants were k'F = 10991.4 L/g.h, k'M = 14.83L/g.h, k'S about 10-1 L/g.h for fast, medium and slow bonds, respectively. Moreover, a procedure for unfolding of the protein, conducive to the improved susceptibility to enzymatic hydrolysis (approximately three-fold increase in the rate) was proposed.
Abstract: This study was designed for assessment of 3 types of Cysteine protease inhibitors (CPIs) fluromethylketone (FMK), vinyl sulfone (VS) and sodium nitro prussid (SNP), to define which of them is the best for curing S. mansoni infection in mice? In vitro, treated S. mansoni adult worms recorded a mortality rate after 1 hr of exposure to 500 ppm of FMK, VS and SNP as 75, 70 and 60%, respectively. FMK+PZQ treatment recorded the maximum reduction in worm burden (97.2% at 5 wk PI). VS treatment alone or combined with PZQ increases IgM, total IgG, IgG2 and IgG4 levels. In EM study, the completely implanted spines were reported in the degenerated tegument of adult worms in all groups treated with CPIs. VS+PZQ Treatment increased Igs levels but, its effect was different on worm reduction. So, it is not enough to eliminate the infection and FMK+PZQ considered the antischistosomicidal drug of choice.
Abstract: Strain M was isolated from the latex of Hevea brasiliensis that grow in the rubber farm area of Malaysia Rubber Board. Strain M was tentatively identified as Bacillus sp. Strain M demonstrated high protease production at pH 9, and this was suitable to be applied in rubber processing that was in alkaline conditions. The right and suitable proportion to be used in applying supernatant into the latex was two parts of latex and one part of enzyme. In this proportion, the latex was stable throughout the 72 hours of treatment. The potential of strain M to degrade protein in the natural rubber latex was proven with the reduction of 79.3% nitrogen in 24 hours treatment. Centrifugation process of the latex before undergoing the treatment had increased the protein degradation in latex. Although the centrifugation process did not achieve zero nitrogen content, it had improved the performance of protein denaturing in the natural rubber.
Abstract: The present study describes the biosynthesis of a milkclotting
protease by solid state fermentation (SSF) of a locally
isolated mould, Rhizopus stolonifer. The production medium was
prepared using wheat bran at 50% (w/v). The production conditions
are optimized by varying 7 parameters: carbon and nitrogen sources,
medium moisture, temperature, pH, fermentation time and
inoculum-s size. The maximum enzyme synthesis was measured after
96 h of incubation time at temperature of 28°C. The optimum pH
determined was 6 and the inoculum size was 3.106spores/ml. The
optimum initial moisture content is comprised between 50 to 70%.
The formation of milk clotting protease is enhanced when galactose
and peptone are used at 10% (w/v) and 1% (w/v) concentrations
respectively. The maximum production of milk clotting protease is
120 US/ml.
Abstract: Chikungunya virus (CHICKV) is an arboviruses belonging to family Tagoviridae and is transmitted to human through by mosquito (Aedes aegypti and Aedes albopictus) bite. A large outbreak of chikungunya has been reported in India between 2006 and 2007, along with several other countries from South-East Asia and for the first time in Europe. It was for the first time that the CHICKV outbreak has been reported with mortality from Reunion Island and increased mortality from Asian countries. CHICKV affects all age groups, and currently there are no specific drugs or vaccine to cure the disease. The need of antiviral agents for the treatment of CHICKV infection and the success of virtual screening against many therapeutically valuable targets led us to carry out the structure based drug design against Chikungunya nSP2 protease (PDB: 3TRK). Highthroughput virtual screening of publicly available databases, ZINC12 and BindingDB, has been carried out using the Openeye tools and Schrodinger LLC software packages. Openeye Filter program has been used to filter the database and the filtered outputs were docked using HTVS protocol implemented in GLIDE package of Schrodinger LLC. The top HITS were further used for enriching the similar molecules from the database through vROCS; a shape based screening protocol implemented in Openeye. The approach adopted has provided different scaffolds as HITS against CHICKV protease. Three scaffolds: Indole, Pyrazole and Sulphone derivatives were selected based on the docking score and synthetic feasibility. Derivatives of Pyrazole were synthesized and submitted for antiviral screening against CHICKV.
Abstract: Rhizopus oligosporus was used in the present study
for the production of protease enzyme under SSF. Sunflower meal
was used as by-product of oil industry incorporated with organic salts
was employed for the production of protease enzyme. The main
purpose of the present was to study different parameters of protease
productivity, its yields and to optimize basal fermentation conditions.
The optimal conditions found for protease production using
sunflower meal as a substrate in the present study were inoculum size
(1%), substrate (Sunflower meal), substrate concentration (20 g), pH
(3), cultivation period (72 h), incubation temperature (35oC),
substrate to diluent-s ratio (1:2) and tween 81 (1 mL). The maximum
production of protease in the presence of cheaper substrate at low
concentration and stability at acidic pH, these characteristics make
the strain and its enzymes useful in different industry.
Abstract: Two commercial proteases from Bacillus
licheniformis (Alcalase 2.4 L FG and Alcalase 2.5 L, Type DX) were
screened for the production of Z-Ala-Phe-NH2 in batch reaction.
Alcalase 2.4 L FG was the most efficient enzyme for the C-terminal
amidation of Z-Ala-Phe-OMe using ammonium carbamate as
ammonium source. Immobilization of protease has been achieved by
the sol-gel method, using dimethyldimethoxysilane (DMDMOS) and
tetramethoxysilane (TMOS) as precursors (unpublished results). In
batch production, about 95% of Z-Ala-Phe-NH2 was obtained at
30°C after 24 hours of incubation. Reproducibility of different
batches of commercial Alcalase 2.4 L FG preparations was also
investigated by evaluating the amidation activity and the entrapment
yields in the case of immobilization. A packed-bed reactor (0.68 cm
ID, 15.0 cm long) was operated successfully for the continuous
synthesis of peptide amides. The immobilized enzyme retained the
initial activity over 10 cycles of repeated use in continuous reactor at
ambient temperature. At 0.75 mL/min flow rate of the substrate
mixture, the total conversion of Z-Ala-Phe-OMe was achieved after 5
hours of substrate recycling. The product contained about 90%
peptide amide and 10% hydrolysis byproduct.
Abstract: A rare phenomenon of SDS-induced activation of a latent protease activity associated with the purified silkworm excretory red fluorescent protein (SE-RFP) was noticed. SE-RFP aliquots incubated with SDS for different time intervals indicated that the protein undergoes an obligatory breakdown into a number of subunits which exhibit autoproteolytic (acting upon themselves) and/or heteroproteolytic (acting on other proteins) activities. A strong serine protease activity of SE-RFP subunits on Bombyx mori nucleopolyhedrovirus (BmNPV) polyhedral protein was detected by zymography technique. A complete inhibition of BmNPV infection to silkworms was observed by the oral administration assay of the SE-RFP. Here, it is proposed that the SE-RFP prevents the initial infection of BmNPV to silkworms by obliterating the polyhedral protein. This is the first report on a silkworm red fluorescent protein that exhibits a protease activity on exposure to SDS. The present studies would help in understanding the antiviral mechanism of silkworm red fluorescent proteins.
Abstract: The overall objective of this research is a strain
improvement technology for efficient pectinase production. A novel
cells cultivation technology by immobilization of fungal cells has
been studied in long time continuous fermentations. Immobilization
was achieved by using of new material for absorption of stores of
immobilized cultures which was for the first time used for
immobilization of microorganisms. Effects of various conditions of
nitrogen and carbon nutrition on the biosynthesis of pectolytic
enzymes in Aspergillus awamori 1-8 strain were studied. Proposed
cultivation technology along with optimization of media components
for pectinase overproduction led to increased pectinase productivity
in Aspergillus awamori 1-8 from 7 to 8 times. Proposed technology
can be applied successfully for production of major industrial
enzymes such as α-amylase, protease, collagenase etc.
Abstract: The enzyme alkaline protease production was determined under
solid state fermentation using the soil bacteria Serratia marcescens
sp7. The maximum production was obtained from wheat bran
medium than ground nut shell and chemically defined medium. The
physiological fermentation factors such as pH of the medium (pH 8),
Temperature (40oC) and incubation time (48 hrs) played a vital role
in alkaline protease production in all the above. 100Mm NaCl has
given better resolution during elution of the enzymes. The enzyme
production was found to be associated with growth of the bacterial
culture.
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).
Abstract: Wheat gluten hydrolyzates (WGHs) and anchovy fine
powder hydrolyzates (AFPHs) were produced at 300 MPa using
combinations of Flavourzyme 500MG (F), Alcalase 2.4L (A),
Marugoto E (M) and Protamex (P), and then were compared to those
produced at ambient pressure concerning the contents of soluble solid
(SS), soluble nitrogen and electrophoretic profiles. The contents of SS
in the WGHs and AFPHs increased up to 87.2% according to the
increase in enzyme number both at high and ambient pressure. Based
on SS content, the optimum enzyme combinations for one-, two-,
three- and four-enzyme hydrolysis were determined as F, FA, FAM
and FAMP, respectively. Similar trends were found for the contents of
total soluble nitrogen (TSN) and TCA-soluble nitrogen (TCASN). The
contents of SS, TSN and TCASN in the hydrolyzates together with
electrophoretic mobility maps indicates that the high-pressure
treatment of this study accelerated protein hydrolysis compared to
ambient-pressure treatment.
Abstract: In this paper, a mathematical model of human immunodeficiency
virus (HIV) is utilized and an optimization problem is
proposed, with the final goal of implementing an optimal 900-day
structured treatment interruption (STI) protocol. Two type of commonly
used drugs in highly active antiretroviral therapy (HAART),
reverse transcriptase inhibitors (RTI) and protease inhibitors (PI), are
considered. In order to solving the proposed optimization problem an
adaptive memetic algorithm with population management (AMAPM)
is proposed. The AMAPM uses a distance measure to control the
diversity of population in genotype space and thus preventing the
stagnation and premature convergence. Moreover, the AMAPM uses
diversity parameter in phenotype space to dynamically set the population
size and the number of crossovers during the search process.
Three crossover operators diversify the population, simultaneously.
The progresses of crossover operators are utilized to set the number
of each crossover per generation. In order to escaping the local optima
and introducing the new search directions toward the global optima,
two local searchers assist the evolutionary process. In contrast to
traditional memetic algorithms, the activation of these local searchers
is not random and depends on both the diversity parameters in
genotype space and phenotype space. The capability of AMAPM in
finding optimal solutions compared with three popular metaheurestics
is introduced.
Abstract: Urinary Tract Infections (UTI) account for an estimated 25-40% nosocomial infection, out of which 90% are associated with urinary catheter, called Catheter associated urinary tract infection (CAUTI). The microbial populations within CAUTI frequently develop as biofilms. In the present study, microbial contamination of indwelling urinary catheters was investigated. Biofilm forming ability of the isolates was determined by tissue culture plate method. Prevention of biofilm formation in the urinary catheter by Pseudomonas aeruginosa was also determined by coating the catheter with some enzymes, gentamycin and EDTA. It was found that 64% of the urinary catheters get contaminated during the course of catheterization. Of the total 6 isolates, biofilm formation was seen in 100% Pseudomonas aeruginosa and E. coli, 90% in Enterococci, 80% in Klebsiella and 66% in S. aureus. It was noted that the biofilm production by Pseudomonas was prolonged by 7 days in amylase, 8 days in protease, 6 days in lysozyme, 7days in gentamycin and 5 days in EDTA treated catheter.
Abstract: Attachment of the circulating monocytes to the
endothelium is the earliest detectable events during formation of
atherosclerosis. The adhesion molecules, chemokines and matrix
proteases genes were identified to be expressed in atherogenesis.
Expressions of these genes may influence structural integrity of the
luminal endothelium. The aim of this study is to relate changes in the
ultrastructural morphology of the aortic luminal surface and gene
expressions of the endothelial surface, chemokine and MMP-12 in
normal and hypercholesterolemic rabbits. Luminal endothelial
surface from rabbit aortic tissue was examined by scanning electron
microscopy (SEM) using low vacuum mode to ascertain
ultrastructural changes in development of atherosclerotic lesion. Gene
expression of adhesion molecules, MCP-1 and MMP-12 were studied
by Real-time PCR. Ultrastructural observations of the aortic luminal
surface exhibited changes from normal regular smooth intact
endothelium to irregular luminal surface including marked globular
appearance and ruptures of the membrane layer. Real-time PCR
demonstrated differentially expressed of studied genes in
atherosclerotic tissues. The appearance of ultrastructural changes in
aortic tissue of hypercholesterolemic rabbits is suggested to have
relation with underlying changes of endothelial surface molecules,
chemokine and MMP-12 gene expressions.