High Enzyme Research Articles

Modeling stability and flexibility of α-Chymotrypsin in room temperature ionic liquids

We investigate the structure and dynamics of α-Chymotrypsin in five room temperature ionic liquids (RTILs) sharing a common cation, hydrated with different water percentages (w/w) (weight of water over protein). Results from molecular dynamics simulations are correlated with experimental evidences from studies on the activity of enzymes in RTILs. α-Chymotrypsin protein structure is closer to its native crystallographic structure in RTILs than in aqueous environment. We show that the structural properties of α-Chymotrypsin were affected by the water concentration assayed in a typical bell-shaped profile, which is also frequently reported for organic solvents. The protein structure was more native like at 10–20% of water (w/w) for all RTILs except for [BMIM][Cl]. We found that the fluctuations of the main chain in [BMIM][BF4] and [BMIM][TfO] were not significantly affected by the increasing amount of water. However, we were able to show that the flexible regions were the ones more hydrated, indicating that water is responsible for the flexibility of the protein. The solvation of the enzyme in water-immiscible RTILs, such as [BMIM][PF6] and [BMIM][Tf2N] lead to higher enzyme flexibility at increased water content. Enzyme solvation by [BMIM][Cl] resulted in ion penetration in the core enzyme structure, causing incremented flexibility and destabilization at low water percentages. All RTILs stripped water molecules from the protein surface, following a similar behavior also found in organic solvents. Anions formed structured arrangements around the protein, which allowed non-stripped water molecules to localize on the protein surface.

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as K m = 27.5 ± 4.33 mg/mL, V max = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol.

Insuficiencia pancreática exocrina (ipe) en canina

Exocrine pancreatic insufficiency is a syndrome characterized by bad digestion and poor absorption result of the failure of the secretion of pancreatic enzymes and other substances that facilitate the absorption of dietary nutrients and certain vitamins and minerals necessary for normal digestion food. The goal of treatment is replacement of pancreatic enzymes by oral enzyme extracts when clinical signs appear, which consists of administer enzyme supplements with every meal. In dogs has achieved the highest enzyme activity in the duodenum without enteric coating supplements, including cut crude pancreatic or pulverized enzymes. This review aims to upgrade from exocrine pancreatic insufficiency in dogs, which addresses the etiology, pathophysiology, history, clinical signs, differential diagnosis, sequelae, diagnosis, treatment, prognosis and treatment failures.

Low-temperature acidic amylases from Aspergillus for desizing of cotton fabrics

Aspergillus niger and Aspergillus flavus with strong starch degrading amylase activity were isolated and screened for the production of α-amylases. Production of amylases from both the Aspergillus species was maximum on the fourth day (A. niger – 1780 U/mL/min, A. flavus – 1550 U/mL/min) after inoculation in the basal medium, using submerged fermentation. Higher enzyme activities up to 4000 and 2500 U/mL/min were obtained for A. niger and A. flavus, respectively, with wheat starch as the carbon source compared to the levels obtained in the cases of corn, rice and soluble starches. Maximum amylase activity was observed at 40°C, at pH 5 for A. niger and pH 4.5 for A. flavus. The study revealed that mixed amylases obtained from these sources exhibit higher desizing efficiency of grey cotton fabrics (A. niger – 96%, A. flavus – 90%) with significant improvement in the absorbency, extractable impurities and amylases obtained from these sources, which could be a better alternative for the existing range of desizing agents.

Pengaruh Pemberian Emras Terhadap Pertumbuhan dan Hasil Tanaman Buncis (Phaseolus vulgaris L.) pada Lahan Rawa Lebak

Beans are a kind of edible beans. Beans are rich in protein and vitamins that help lower blood pressure and monitor blood sugar metabolism and are equated with being eaten by those who suffer from diabetes or hypertension. High fiber and enzyme content can help you lose weight. In peanut cultivation it needs to be optimized considering the consumption needs are not comparable with production, One way to increase the productivity of peanut plants is proper fertilization, such as by using organic fertilizers. This study aims (i) to determine the effect of EMRAS on the growth and yield of beans (Phaseolus vulgaris L.) on swampy swamp land. (ii) Getting the best concentration between EMRAS and growth and yield of beans (Phaseolus vulgaris L.) on swamp swamp land. This research was carried out on swamp swamp land in Teluk Buluh Village, Banjang District, Hulu Sungai Utara Regency in May - July 2013, using a Randomized Block Design (RBD), with 6 treatments and 4 replications, resulting in 24 experimental units and each experiment. consists of 2 sample plants, so that the total sample is 48 plants. The factors tested were the provision of EMRAS, e0 = 0% (0 l/beds), e1 = 25% (0,25 l/beds), e2 = 50% (0,5 l/beds), e3 = 75% (0,75 l/beds), e4 = 100% (1 l/beds) and e5 = 125% (1,25 l/beds). The results showed that the observation variables of EMRAS administration affected plant height, number of productive branches, number of pods per plant, and pod wet weight per plant with the best administration at 100% concentration (1 l / bed)

COMPARISON OF THE CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF RETEPLASE IN Escherichia coli

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ∼39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.

Wilson's Disease among Children at King Hussein Medical Center

Results: A total of 37 patients diagnosed as Wilson's disease with age ranges between two and 13.5 years were included in this descriptive review. Out of 37 patients, 19 (51%) were males and 18 (49%) were females. Patients with affected siblings were 29 (78%). Central nervous system involvement was found among 9 (24.3%) patients. The commonest presenting symptoms were jaundice (n=16, 43%), abdominal distension (n=13, 35%), fatigue and delayed school performance (n=12, 32.4%). The most common clinical findings were hepatomegaly (n=26, 70%), jaundice (n=16, 43%), splenomegaly (n=14, 37.8%), Kayser-Fleischer ring (n=11, 29.7%), and lower limb edema (n=11, 29.7%) respectively. Low ceruloplasmin level was found in 34 (92%) patients, high liver enzymes in 23 (62%) patients, hemolytic anemia in 13 (35%) patients successively. Twenty-four hour urine collection average copper post Dpenicillamine challenging test was above 230µg/dl. The most common ultrasound findings were hepatomegaly, abnormal echogenecity, splenomegaly and ascitis. Liver biopsies commonly showed liver fibrosis, however fatty liver changes, hepatosteatosis and liver cirrhosis were the least common finding. Conclusion: Family screening is needed once a child in the family is diagnosed. Full investigations to rule out Wilson's disease should be performed in any patient with unexplained elevation of liver enzymes, hepatomegaly, hemolytic anemia, jaundice or neurological/behavioral disturbances.

Response of antioxidant compounds to selenium and salicylic acid in wheat cultivars under dry land conditions

In the present study, three dry land wheat cultivars, Azar 2, Sardary and Rasad, were tested for antioxidant enzyme activity, proline, malondialdehyde (MDA) and dityrosine (DT) content and grain yield after treatment with selenium and salicylic acid (SA). A factorial field experiment was carried out based on a completely randomized block design with three replicates. The experimental factors were three levels of salicylic acid (without SA; seed priming with 0.5 mM SA; seed priming + spraying with 1 mM SA) and two levels of selenium (0 and 20 g/ha). Significant increases in the activity of the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) enzymes and in the proline level were observed after treatment in the leaves of the three genotypes investigated, but this was associated with reduced MDA and DT content. The application of SA as seed priming and the foliar application of Se also increased the grain yield. These results suggest that cultivars exhibiting high antioxidant enzyme activity and proline content under dry land conditions may provide better drought tolerance in wheat.

The Roles of Food Plants on the Dispersion Activities of the Variegated Grasshopper,Zonocerus variegatus(L.) (Orthoptera: Pyrgomorphidae)

Dispersion activities in insects depend on many factors, including diet. The variegated grasshopper, Zonocerus variegatus moves from one place to another in the field when disturbed and in search food and mates. The effect of diets on the tissues (femoral and thoracic muscles) responsible for dispersion in Z. variegatus was investigated in this study. Acalypha wilkesiana, Chromolaena odorata, Carica papaya, Manihot esculenta and a mixture of the four plants were fed to newly emerged adult males (two days after emergence) for eight weeks. After feeding, insect tissues were assayed and activities of five enzymes namely: α-glucosidase, amylase, cellulase, lipase and proteinase were investigated. All the enzymes were detected in the muscles of Z. variegatus. The food plants significantly affected the enzyme activities in these tissues. Z. variegatus fed A. wilkesiana had significantly higher enzyme activities than those insects fed other plants (31.6, 16.2, 5.0, 5.8 absorbance/minute for proteinase, lipase, cellulase and α-glucosidase, respectively, in the femoral muscles). Proteinase and lipase had higher activities than the carbohydrases.

Elastase, Myeloperoxidase, and Alkaline Phosphatase Release and Free Radical Generation in Neutrophils Isolated from Blood of Sows at Different Stages of Oestrous Cycle

Abstract The aim of the study was to assess the influence of the oestrous cycle phase on neutrophil secretory activity and to extrapolate it to susceptibility to uterine infections. The obtained results indicate that the highest enzyme release seen in the late follicular phase (elastase release was 42.18 ±3.11% of maximal release, myeloperoxidase was 45.0 ±5.12%, and alkaline phosphatase was 44.75 ±9.0%) was related to the level of 17β-oestradiol in plasma. Similarly, a free radical generation was also the most enhanced during this phase. Significantly lower values were obtained from sows during the luteal phase in regard to both enzyme release (36.62 ±3.58% for elastase, 27.87 ±8.7% for myeloperoxidase, and 22.12 ±2.4% for alkaline phosphatase), and that of free radicals (2.28 1.6 μM/106 cells for nitric oxide and 2.47 0.6 nM/106 cells for superoxide).

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw

Threonine dehydratase (EC 4.3.1.19, TDH) catalyzing the degradation of Thr to α-ketobutyrate, is a rate-limiting enzyme in L-Ile pathway. The tdcB gene encoding TDH was obtained from Escherichia coli K12 by PCR and expressed at E. coli BL21 (DE3). Then the tdcB gene was inserted into the shuttle expression vector pXMJ19 and the recombinant plasmid was electroporated into the L-isoleucine-producing strain of Corynebacterium glutamicum YILW. Crude extracts of the microbial strain containing the plasmid pXMJ19tdcB retained 60% of the original TDH activity even in the presence of 300 mM L-Ile. The recombinant strain of bacteria showed 7.5% higher enzyme activity and 11.3% higher L-Ile production compared to the original strain.

Assessing the Blood–brain barrier permeability with high dose enzyme replacement therapy in mucopolysaccharidosis type I mice

Assessing the Blood–brain barrier permeability with high dose enzyme replacement therapy in mucopolysaccharidosis type I mice

Liver disease spectrum in hospitalized type 2 diabetes and related risk factors analysis of non-alcoholic fatty liver disease

To explore the liver disease spectrum in patients with type 2 diabetes (T2DM) and the risk factors of non-alcoholic fatty liver disease (NAFLD). From September 2009 to October 2011, 1069 hospitalized patients with T2DM in Department of Endocrinology and Metabolism were involved in the study. The history informations, results of laboratory examination, hepatic ultrasound and hepatic proton magnetic resonance spectrum ((1)H MRS) of all patients were collected to analysis. (1) The detectable rate of raised liver enzymes in T2DM patients was 28.7% (307/1069), composed mainly of NAFLD (39.4%, 121/307). After excluding the factors such as alcoholic abuse, viral hepatitis, the detect rate of raised liver enzymes in T2DM patients was 26.9% (185/688). (2) The detectable rate of fatty liver by ultrasound in T2DM patients was 56.7% (500/882), composed mainly of NAFLD (72.6%, 363/500), and the detectable rate of NAFLD was 58.0% (363/626). (3) The detectable rate of fatty liver by hepatic (1)H MRS was 72.8% (227/312), composed mainly of NAFLD (69.6%, 158/227). The detectable rate of NAFLD was 69.6% (158/227). (4) Of the three methods for diagnosing NAFLD, (1)H MRS had the highest detectable rate, followed by ultrasound, and the hepatic enzymes was the lowest. Set the hepatic (1)H MRS as gold diagnosing standard of NAFLD, the combination of hepatic enzymes and ultrasound increase the sensitivity. The optional cut-off points of ALT were 19.7 U/L (male, ROCAUC = 0.689, P < 0.01) and 17.0 U/L (female, ROCAUC = 0.727, P < 0.01). (5) Logistic stepwise regression analysis showed sex, BMI, hemoglobin, fasting C-peptide and uric acid (OR = 3.803, 1.195, 1.037, 2.896, 1.011, all P < 0.05) were positively correlated with NAFLD, and diabetes duration (OR = 0.948, P < 0.05) was positively correlated with NAFLD independently. The detectable rate of fatty liver was high in T2DM which was composed mainly of NAFLD. High abnormal liver enzymes detectable rate indicated that NAFLD with T2DM are prone to NASH.

High Affinity Membranes for Cellulase Enzyme Detection in Subterranean Termites

ABSTRACTThe United States dependence on fossil fuels has become mandatory over the past few decades. The fuel shortage during the 1970s and after Hurricane Katrina has catalyzed a need for creating alternative energy sources, improving the efficacy of these alternative energy sources, and enhancing energy sustainability. The U.S. Department of Energy has set goals to replace 30% of the liquid petroleum transportation fuel with biofuels and to replace 25% of industrial organic chemicals with biomass-derived chemicals by 2025. In the southeast United States, subterranean termites are prevalent and microbes in their gut degrade wood based materials such as cellulose which produce simple sugars that can be used to produce bioethanol. Upon seasonal change, subterranean termites undergo less enzymatic activity and wood-eating capability limiting the amount of sugars that may be produced. This limited activity sparks an interest to investigate this poorly understood phenomenon of how temperature may affect the enzymatic activity in subterranean termites’ guts. In this study, we report the development thermoresponsive biomaterial nanofiber mats containing cellulose to model cellulase activity. Using electrospinning techniques, poly(N-vinylcaprolactam) celluose fiber mats have been prepared via alkaline hydrolysis and labeled with fluorescent tags. Subterranean termites (reticulitermes species) were feed fiber mats for 10 consecutive days to assess enzyme mapping and kinetics. Fluorescent microscopy images confirmed spatial and temporal localization of cellulase enzyme throughout the termite gut upon time and temperature change. These novel high affinity enzyme detection membranes show promise towards future biofuel production.

Selection and Classification of Bacterial Strains Using Standardization and Cluster Analysis

This study utilized a standardization and cluster analysis technique for the selection and classification of beneficial bacteria. A set of synthetic data consisting of 100 individual variables with three characteristics was created for analysis. The three characteristics assigned to each independent variable were designated to have different numeric scales, averages, and standard deviations. The variables were bacterial isolates at random, and the three characteristics were fermentation products, including cell yield, antioxidant activity of culture, and enzyme production. A standardization method utilizing a standard normal distribution equation to record fermentation yields of each isolate was employed to weight their different numeric scales and deviations. Following transformation, the data set was analyzed by cluster analysis. The Manhattan method for dissimilarity matrix construction along with complete linkage technique, an agglomerative method for hierarchical cluster analysis, was employed using statistical computing program R. A total of 100 isolates were classified into groups A, B, and C. In a comparison of the characteristics of each group, all characteristics in groups A and C were higher than those of group B. Isolates displaying higher cell yield were classified as group A, whereas those isolates showing high antioxidant activity and enzyme production were assigned to group C. The results of the cluster analysis can be useful for the classification of numerous isolates and the preparation of an isolation pool using numerical or statistical tools. The present study suggests that a simple technique can be applied to screen and select beneficial microbes using the freely downloadable statistical computing program R.

Fermentação de farelo de cacau por Aspergillus niger para obtenção de lipase

The aim of this study was to use cocoameal as substrate for obtaining fungal lipase producedby solid state fermentation by Aspergillus niger .The spore concentration used was 107 spores/gsubstrate. Fermentations were conducted in a bacteriologicalincubator at 35°C with an initial moisturecontent of 60% and the fermentation time was 96hours. The determination of enzyme activity, pH,water activity and moisture were taken every 24hours. The higher enzyme activity was quantifi ed11.67 U/g aft er 48 hours of fermentation. Aft er thistime there was reduction in enzyme production.The moisture content and water activity decreasedduring the fermentation process and the pH remainedconstant. Key words: water activity, fermentation time, solidstatefermentation.

Effect of environmental salinity and dopamine injections on key digestive enzymes in hepatopancreas of the euryhaline crab &lt;i&gt;Cyrtograpsus angulatus&lt;/i&gt; (Decapoda: Brachyura: Varunidae)

Fil: Michiels, Maria Soledad. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico - CONICET - Mar del Plata. Instituto de Investigaciones Marinas y Costeras; Argentina; Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina;

An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains

A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows: adding 50 μL of diluted enzyme sample and 50 μL substrate, incubating at 45 °C for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid (DNS) reagent, maintaining at boiling point for 15 min, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200 μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.

In vitro gastric digestion of heat-induced aggregates of β-lactoglobulin

In vitro gastric digestion of heat-induced aggregates of β-lactoglobulin (β-LG) in simulated gastric fluid was investigated using sodium dodecyl sulfate-PAGE (under nonreducing and reducing conditions), native PAGE, 2-dimensional electrophoresis, and size exclusion chromatography. Heating at 90°C significantly increased the digestibility of β-LG, with a high initial digestion rate followed by a relatively constant rate of digestion at a high enzyme:substrate (E:S) ratio of 3:1. At a low E:S ratio (1:6), the rate of digestion of β-LG was slower, and intermediate- and low-molecular-weight species could be seen. The high-molecular-weight nonnative aggregates (e.g., pentamers, tetramers, and trimers) were digested relatively rapidly, whereas some of the nonnative dimers were resistant to digestion and others were digested rapidly. The intermediate-molecular-weight species (21 to 23kDa) were digested slowly. The digestibility of nonnative β-LG aggregates varied significantly depending on the E:S ratio and the types of aggregate. Further investigation is necessary to identify and characterize slowly digested dimers and species of intermediate molecular weight.

Association of Aspirin Intake and Myocardial Infarction Size

Myocardial infarction (MI) is one of the most important health burdens worldwide. Aspirin as an non- Steroid Anti—inflammatory drug, has been proven to be a protective factor to decrease the incidence, however its effect of MI size is still unknown. We designed this study to compare the biomarkers after MI in patients with and without aspirin intake. 378 patients were enrolled and the results showed lower cardiac troponin T and Creatine Kinases in patients with protective dose of aspirin intake. In addition, Creatine Kinases were significantly higher in patients with no history of MI. We conclude that aspirin can reduce the size of the infraction. Also, higher enzymes can be due to higher muscle content in patient without MI history.[GMJ. 2012;1(1):35-37]