Enzymology

This paper examined the effect of several pyridinium and imidazolium-based ionic liquids (ILs) on the protease stability in aqueous solutions. In general, the enzyme was found quite active at low concentrations of hydrophilic ILs. In aqueous environment, the enzyme was stabilized by the kosmotropic anions (such as CF3COO− and CH3COO−) and chaotropic cations (such as [BuPy]+ and [EMIM]+), but was destabilized by chaotropic anions (such as tosylate and BF4−) and kosmotropic cations (such as [BMIM]+).Hydrophilic ionic liquids dissociate into individual ions in water. The effect of individual ions on the enzyme activity follows the Hofmeister series: kosmotropic anions and chaotropic cations stabilize the enzyme.

In this work, we show that adenosine 5'-0-(3-thiotriphosphate) (ATPyS) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1. l) and further, using chirally labeled [y-'70,180]ATPyS, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[ 170,180]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATPyS and the determination of inorganic thio-phosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87,301-3161. We also show in a single turnover experiment performed in

Too intensive training may lead to overreaching or overtraining. To study whether quantitative needle electromyography (QEMG) is more sensitive to detect training (mal)adaptation than muscle enzyme activities, 12 standardbred geldings trained for 32 wk in age-, breed-, and sex-matched fixed pairs. After a habituation and normal training (NT) phase ( phases 1 and 2, 4 and 18 wk, respectively), with increasing intensity and duration and frequency of training sessions, an intensified training (IT) group ( phase 3, 6 wk) and a control group (which continued training as in the last week of phase 2) were formed. Thereafter, all horses entered a reduced training phase ( phase 4, 4 wk). One hour before a standardized exercise test (SET; treadmill), QEMG analysis and biochemical enzyme activity were performed in muscle or in biopsies from vastus lateralis and pectoralis descendens muscle in order to identify causes of changes in exercise performance and eventual (mal)adaptation in skeletal m...

The Phasmatodea (stick insects) have multiple, endogenous, highly expressed copies of glycoside hy-drolase family 9 (GH9) genes. The purpose for retaining so many was unknown. We cloned and expressed the enzymes in transfected insect cell lines, and tested the individual proteins against different plant cell wall component poly-and oligosaccharides. Nearly all isolated enzymes were active against carboxymethylcellulose, however most could also degrade glucomannan, and some also either xylan or xyloglucan. The latter two enzyme groups were each monophyletic, suggesting the evolution of these novel substrate specificities in an early ancestor of the order. Such enzymes are highly unusual for Metazoa, for which no xyloglucanases had been reported. Phasmatodea gut extracts could degrade multiple plant cell wall components fully into sugar monomers, suggesting that enzymatic breakdown of plant cell walls by the entire Phasmatodea digestome may contribute to the Phasmatodea nutritional budget. The duplication and neofunctionalization of GH9s in the ancestral Phasmatodea may have enabled them to specialize as folivores and diverge from their omnivorous ancestors. The structural changes enabling these unprecedented activities in the cellulases require further study.

Candida yeasts are rarely infectious, but frequently cause life-threatening systemic infections in patients immunocompromised by AIDS or by immunosuppressive therapeutics. The secreted aspartic proteases (Saps) are known virulence factors of pernicious Candida species. The most virulent, Candida albicans, possesses at least nine SAP genes, some of which are specifically expressed from cells with morphologies associated with virulence. Only one of these proteases, Sap2, has been previously purified from yeast in sufficient quantities for enzymic studies. The other enzymes are present in low amounts in yeast culture and are difficult to purify. As a consequence, enzyme properties, including the substrate specificities, of all Saps are poorly studied. Therefore, four Saps that are known to be expressed in C. albicans, Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombinant zymogens and purified in large quantities. These proenzymes were autoactivated and purified as active proteases. The enzymic properties including the substrate specificities at the P 1 and P 1 P sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures. All four Saps cleave peptide bonds between larger hydrophobic amino acids, but these somewhat broad specificities differ in detail among the four enzymes at both sites. At the P 1 site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu. Positively charged amino acids are also accommodated, especially by Sap2 and Sap3. The specificities at P 1 P are broader than at P 1 for all four enzymes. Sap6 prefers Ala, whereas other Saps prefer Tyr. Acidic side chains are also accommodated at this site. Analysis of substrates with a hydrophobic amino acid in P 1 P reveals that all the Saps possess a unique preference for Ala at this site. The observed differences of residue preferences among Saps may be utilized for the design of specific substrates and inhibitors.

Effective treatment of celiac disease is an unmet medical need. A glutenase that destroys immunogenic gluten peptides may be clinically valuable. Twenty patients with celiac disease were randomly assigned to ingest a large gluten meal (16 g daily for 3 days) pre-treated with ALV003, a mixture of highly specific glutenases (n = 10), or pre-treated with placebo (n = 10). Peripheral blood T-cell IFN-γ ELISpot responses to gliadin and an immunogenic 33mer and symptoms were assessed. While baseline IFN-γ ELISpot responses to gliadin and the 33mer were negative in all patients, a significant ELISpot response to gliadin or the 33mer was observed in 6 of 10 patients consuming placebo-treated gluten and 0 of 10 consuming ALV003 pre-treated gluten (p = 0.011). Symptoms typically associated with gluten ingestion occurred in both groups and were not significantly reduced by ALV003 pre-treatment. ALV003 pre-treatment can abolish immune responses induced by gluten in patients with celiac disease.

Metal cofactors and arginine-166 residue are active site participants in alkaline phosphatase catalysis. However, the mechanism by which the metal cofactors coordinate with arginine-166 residue during alkaline phosphatase catalysis is elusive. This study investigated the effects of Mg 2+ and Zn 2+ on monoesterase activity of wild-type and mutant E. coli alkaline phosphatases (ECAPs). The intact arginine-166 residue of wild-type ECAP was replaced by alanine and serine in the mutant ECAPs, R166A and R166S, respectively. Monoesterase activity of ECAP was measured by monitoring the rate of hydrolysis of para-nitrophenyl phosphate (pNPP). The monoesterase activity of wild-type ECAP was approximately 2-fold higher than the mutant ECAPs. Mg 2+ (0.1-10mM) increased the activities of wild-type and mutant enzymes in a concentration-dependent manner. Zn 2+ (0.05-0.1mM) slightly increased the activities of wild-type and mutants ECAPs. In the absence and in the presence of 10mM Mg 2+ or 0.1mM Zn 2+ , the maximum reaction rate of wild-type ECAP was higher than those of the mutant ECAPs while its Michaelis constant was lower than those of the mutant ECAPs. Findings in this study revealed that monoesterase activity of ECAP was greatly reduced by the loss of arginine-166 residue but its modulation by Mg 2+ and Zn 2+ ions was independent of arginine-166 residue.

c-Glutamyl transpeptidase (E.C. 2.3.2.2; GGT) catalyses hydrolysis of c-glutamyl linkages in c-glutamyl peptides and transfer of the c-glutamyl group to amino acids and peptides. Although plant c-glutamyl peptide metabolism is important in biosynthesis and metabolism of secondary products and xenobiotics, plant GGTs are poorly characterised. We purified a membrane-associated GGT from sprouting onion bulbs that catalyses transpeptidation of methionine by the synthetic substrate c-glutamyl-p-nitroanilide (GGPNA) and obtained N-terminal peptide sequence. We also cloned the full-length coding region of an onion GGT by homology with the Arabidopsis enzyme and confirmed that this shared the same N-terminal sequence. Enzyme kinetic studies show that the enzyme has high affinity for glutathione and glutathione conjugates, and that affinity for S-substituted glutathione analogs decreases as the substituted chain length increases. The major onion c-glutamyl peptide, c-glutamyl trans-S-1-propenyl cysteine sulfoxide (GGPrCSO) exhibited uncompetitive inhibition of transpeptidation by GGPNA. This suggests that GGPrCSO is a poor glutamyl donor and therefore unlikely to be an in vivo substrate for peptidase activity by this enzyme. (J.A. McCallum). www.elsevier.com/locate/phytochem Phytochemistry 66 (2005) 515-522 PHYTOCHEMISTRY

There have been many attempts to understand the mechanisms that determine the activity and stability of enzymes at extreme temperatures. Previous studies often compared enzymes from mesophiles and thermophiles and speculated that the observed differences were factors involved in adaptation to a certain temperature range. Most of these studies concluded that gross structural changes such as the quantity of certain amino acids or the total number of hydrogen bonds are responsible for the adaptation of optimal activity to temperature extremes. However, these studies have compared similar enzymes from distantly related organisms leaving one to wonder if the differences noted are from an adaptation to temperature or merely the result of genetic drift.
In this dissertation, I present an analysis of the cold-active beta-galactosidase (BgaS) from the Arthrobacter sp. SB. A study of the primary sequence and modeled structure of BgaS showed that many of the proposed adaptations for optimal activity in the cold did not hold true for this enzyme. Consequently, I decided to alter the BgaS enzyme to determine the contributions of specific amino acids to activity and stability at low temperatures.
I first examined the area of BgaS that aligned with the domain five mobile loop of the LacZ beta-galactosidase of Escherichia coli. In LacZ this area (residues 794-803) aids the binding of substrate and alterations at residue 794 increased the catalytic efficiency of the enzyme with lactose. However, when similar mutations were made in bgaS, they caused either a complete loss or a decrease of activity showing that although this area is also important for BgaS function the alterations affect the enzymes differently.
To further explore the low temperature activity of BgaS, I screened for second site revertants of a null mutant resulting from a G803D change. Restoration of activity was accomplished with the addition of only two mutations (E229D and V405A). Separation of these two mutations into a wild type background yielded an enzyme with a three fold increase in catalytic efficiency with little effect on the thermostability. This shows that small and subtle changes to the enzyme can further increase the activity at low temperatures.
I also explored the thermostability of BgaS through directed mutagenesis and directed evolution studies. The rational mutagenesis targeted the C-terminal portion of BgaS, an area in LacZ known to affect thermostability. From this study, I discovered that a single cysteine to glycine or cysteine to serine mutation resulted in an increase in thermal optimum for BgaS and in another closely related enzyme. The directed evolution study primarily targeted the active site in an attempt to create a more active version of BgaS. One mutant obtained from this screen, with a single alteration in amino acid sequence, created an enzyme with more activity and a 3.3 fold increase in the time it remained active at 30C, compared to BgaS.
Through the combination of mutational analysis and biochemical characterization, I have shown that the introduction of a limited number of amino acid changes are sufficient to alter the activity and/or thermal properties of an enzyme whereas previous studies have suggested that multiple alterations would be required. I have also increased the thermal optimum of three closely related enzymes by altering one amino acid. Considering the large size of the beta-galactosidase subunit, the finding that one change in the C-terminal 25 residues has any effect on the enzyme, not to mention an up to 20C increase in temperature optimum, is quite interesting. Taken as a whole, this work illustrates that small, unpredicted changes in the amino acid sequence of even large enzymes can have dramatic effects on their thermostability and/or activity.

Thermophilic microorganisms are of special interest as a source of novel thermostable enzymes. A total of 62 bacterial strains, isolated from water hot springs and desert soil in Egypt were screened for the extracellular enzyme, pectinase. The results showed that, the two bacterial isolates: Wh20 and Wh24 were found to exhibit a higher pectinase production. The two most potent bacterial isolates were identified based on morphological and biochemical characters as a species belonging to the genus Bacillus and identified as Anoxybacillus contaminans Wh20 & Anoxybacillus flavithermus Wh24. Identification was confirmed by 16S r RNA sequencing. Maximum pectinases productivities for Anoxybacillus contaminans Wh20 & Anoxybacillus flavithermus Wh24 were (43.54 and 46.72 U/ml respectively) at pH 8.0 incubated for 18h at 55ºC under static condition. In presence of lactose and peptone as additional carbon and nitrogen source respectively and 0.4% pectin substrate concentration. Pectinolytic activity exhibited high thermostability over broad range of pH 6.0-10 and temperature 50-90 ºC. The crude pectinase was purified by salt precipitation, dialysis and gel filtration chromatography. The specific activities of the purified pectinases for Anoxybacillus contaminans Wh20 & Anoxybacillus flavithermus Wh24 were estimated to be 2250 and 2087.46 U/mg of enzyme protein respectively corresponding to (19.27 and 16.93 time) purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),for Anoxybacillus contaminans Wh20 showed that, the molecular weight of purified enzyme was determined at two bands, 30 kDa and 35 kDa, while Anoxybacillus flavithermus Wh24, showed that one band at 32 kDa. This pectinase may be industrially used in extraction and clarification processes.

AAP Alanyl aminopeptidase ADA Azodicarbonamide ADP Adenosine Diphosphate ATP Adenosine triphosphate DEAE Diethylaminoethyl DIFP Diisopropyl fluorophosphates DPP Dipeptidyl peptidases EC Enzyme Commission GM Genetically Modified (Organnisms) LAP Leucyl amino peptidase MAP Methionyl aminopeptidase PGAP Pyroglutamyl aminopeptidase RAP Arginyl aminopeptidase ROS Reactive-oxygen species

Nitrate, the most oxidized form of nitrogen, is regulated to protect people and animals from harmful levels as there is a large over abundance due to anthropogenic factors. Widespread field testing for nitrate could begin to address the nitrate pollution problem, however, the Cadmium Reduction Method, the leading certified method to detect and quantify nitrate, demands the use of a toxic heavy metal. An alternative, the recently proposed Environmental Protection Agency Nitrate Reductase Nitrate-Nitrogen Analysis Method, eliminates this problem but requires an expensive proprietary spectrophotometer. The development of an inexpensive portable, handheld photometer will greatly expedite field nitrate analysis to combat pollution. To accomplish this goal, a methodology for the design, development, and technical validation of an improved open-source water testing platform capable of performing Nitrate Reductase Nitrate-Nitrogen Analysis Method. This approach is evaluated for its potential to i) eliminate the need for toxic chemicals in water testing for nitrate and nitrite, ii) reduce the cost of equipment to perform this method for measurement for water quality, and iii) make the method easier to carryout in the field. The device is able to perform as well as commercial proprietary systems for less than 15% of the cost for materials. This allows for greater access to the technology and the new, safer nitrate testing technique.

Aim: To Find the Effect of Concentration of Pectinase on Apple (Malus domestica) Juice Production
Research Question
How does an increase in the concentration of the enzyme pectinase affect the production of apple juice from apple (Malus domestica) pulp?

Las enzimas son macro-moléculas biológicas que actúan como catalizadores, es decir, que alteran la velocidad de una reacción favorablemente. Este texto surge tras la necesidad de estudiar los efectos de la concentración de la enzima catalasa en la descomposición de peróxido de hidrógeno y etanol como inhibidor. A través del apoyo de la interfaz Logger Pro ® se midió la variación de presión en un tubo de ensayo con soluciones preparadas y con dicha información se pudo obtener el comportamiento de la reacciones a temperatura constante. Se linealizaron los comportamiento y se obtuvieron parámetros importantes en el campo de cinética química como las velocidades inciales de reacción y el orden de la misma.

BACKGROUND: Serrapeptase is a proteolytic enzyme with many favorable biological properties like anti-inflammatory, analgesic, anti-bacterial, fibrinolytic properties and hence, is widely used in clinical practice for the treatment of many diseases. Although Serrapeptase is widely used, there are very few published papers and the information available about the enzyme is very meagre. Hence this review article compiles all the information about this important enzyme Serrapeptase. METHODS: A literature search against various databases and search engines like PubMed, SpringerLink, Scopus etc. was performed. RESULTS: We gathered and highlight all the published information regarding the molecular aspects, properties, sources, production, purification, detection, optimizing yield, immobilization, clinical studies, pharmacology, interaction studies, formulation, dosage and safety of the enzyme Serrapeptase. CONCLUSION: Serrapeptase is used in many clinical studies against various diseases for its anti-inflammatory, fibrinolytic and analgesic effects. There is insufficient data regarding the safety of the enzyme as a health supplement. Data about the anti-atherosclerotic activity, safety, tolerability, efficacy and mechanism of action of the Serrapeptase are still required.

Twenty eight pre-identified fungal isolates and strains were tested qualitatively for their cellulolytic activities, among which 12 superior producers were quantitatively tested. Three Aspergillus spp. namely A5, A6 and A7 were found to be the best cellulolytic fungi, while both Aspergillus terreus NRRL260 and Phanerochaete chrysosporium NRRL6361 were found to be the best xylanase producers. On solid state fermentation (SSF) using pretreated rice straw (RS), the Aspergillus spp. A5, A6 and A7 gave total cellulases (as FPase), exo-cellulases (as CMCase) and cellobiase (as β-glucosidase) activities of ≥ 16, 130 and 21U.g-1 ds, respectively. On the other hand, Aspergillus terreus NRRL260 showed maximum xylanases activity of 804 U.g-1 ds. The recovered crude enzymes forming the crude enzyme mixture (CEM) was concentrated (LCEM), ammonium sulfate fractionated (PPE1), were semi-purified by gel permeation on Sephadex G100 and finally lyophilized to powder form (PPE2). The resulting cellulases specific activities were 0.50, 0.68, 1.01 and 1.47 U.mg-1 , respectively. Those purification and concentration steps resulted in purification fold increase by 1.28, 2.08 and 2.78 for LCEM, PPE1 and PPE2, respectively. PPE2 achieved maximum degree of saccharification (DoS) at enzyme load ≥ 10 FPU, substrate concentration ≥ 5% and incubation period after 48 hr to be 85%, 87% and 88%, respectively.

Proteins and reducing sugars are well known because of their capability of reducing compounds like DNS and elements like copper. When they join with these compounds, they manifest certain coloration that can be measured with a spectrophotometer; therefore with solutions of different protein and sugar concentrations, we constructed a calibration curve. Alfa amylase degrade starch, and its enzyme activity was determined by staining the product with DNS and measuring its absorbance

Puji syukur saya panjatkan kehadirat Tuhan Yang Maha Esa karena dengan rahmat serta karunia-Nya saya hendaknya dapat menyelesaikan makalah berjudul "Mekanisme kerja obat antibiotik β-lactam dan resistensinya" ini dengan cukup baik meskipun mungkin banyak kekurangan didalamnya. Dan terimakasih kepada semua pihak terutama dosen pengampu Enzimologi yang mana menugaskan pembuatan makalah dengan tujuan memperluas pengetahuan tentang aplikasi enzim dalam industri. Saya sangat berharap makalah ini dapat berguna dalam rangka menambah wawasan serta pengetahuan mengenai mekanisme kerja obat terhadap enzim serta resistensinya sehingga bisa kita ketahui langkah yang harus diambil ketika menciptakan obat penarget enzim. Saya juga menyadari bahwa di dalam makalah ini pastinya terdapat kekurangan dan jauh dari kata sempurna. Oleh sebab itu, saya berharap adanya kritik, saran dan usulan demi adanya perbaikan. Semoga makalah sederhana ini dapat dipahami bagi siapapun yang membacanya. Sebelumnya mohon maaf apabila banyak kesalahan kata-kata yang kurang berkenan. Terimakasih Surakarta, 11 Desember2015 Penulis DAFTAR ISI BAB I: PENDAHULUAN 1

. Enzim amilase merupakan enzim yang mampu mengkatalis proses hidrolisa pati untuk menghasilkan molekul lebih sederhana seperti glukosa, maltosa, dan dekstrin. Proses hidrolisa pati tersebut dilakukan melalui tiga tahapan yaitu gelatinisasi, likuifikasi, dan sakarifikasi. Ketiga tahapan tersebut memerlukan energi yang relatif tinggi sehingga meningkatkan biaya produksi pada produk berbasis pati. Saat ini, sebagai salah satu upaya dalam penghematan energi, berbagai penelitian telah difokuskan mengenai enzim amilase pemecah pati mentah (APPM) yaitu enzim yang dapat bekerja langsung pada granula pati mentah tanpa melalui proses gelatinisasi. APPM dapat diproduksi dari berbagai sumber seperti tanaman, hewan, atau mikroba. Paper ini akan mengulas tentang perkembangan dari eksplorasi enzim APPM tersebut khususnya dari sumber mikroba. Ulasan diarahkan pada enzim APPM dari mikroba sebab amilase dari mikroba lebih diminati untuk nantinya diproduksi dalam skala industri.

A detailed description of the microbial glycoside hydrolases able to degrade lignocellulosic biomass is very important for a better understanding of the new processes involving biorefinery  the conversion of biomass into biofuels. Cellulose and hemicellulose, the major carbohydrates of plant biomass, together with lignin, constitute the most abundant organic compounds present in nature. Cellulose and hemicellulose are converted enzymatically into glucose, xylose or other sugars, which may be fermented by yeasts into second-generation bioethanol or other chemicals. In order to process the lignocellulosic biomass in biorefinery, the use of efficient degrading enzymes is essential for the bioconversion. In this chapter we describe recent advances in the characterization of glycoside hydrolases, auxiliary activities and synergism between cellulases and accessory proteins involved in cellulose hydrolysis. Furthermore, considering that pretreatments are necessary for efficient biomass degradation and exposition of the lignocellulosic components, a detailed description of several physical, chemical, physicochemical, biological and integrated pretreatments is also presented. Modern and classical methods, including spectroscopy, coupled chromatography, electron microscopy, MALDI-imaging MS, and others, are also discussed as strategies for improving both fiber characterization and understanding of the saccharification of lignocellulose and subsequent biofuel production.

Phytic acid is an anti-nutritional constituent of plant derived feeds. As a reactive anion, it forms a wide variety of insoluble salts with minerals including phosphorus, calcium, zinc, magnesium and copper. Phytic acid is also known to form complexes with protein and proteolytic enzymes (pepsin and trypsin). Because of the lack of endogenous phytase enzymes that hydrolyze phytic acid; phosphorus, calcium, protein and other phytic acid bound nutrients are less available to poultry.
Commercially-available microbial phytase has great nutritional significance and ecological importance. Poultry diets supplemented with microbial phytase result in improved growth performance and increased digestibility and availability of
phytate bound phosphorous, calcium, zinc and copper. Microbial phytase supplementation has also been shown to increase ileal digestibility of crude protein and amino acids. The efficacy of supplemental microbial phytase depends on its rate of inclusion, dietary calcium and phosphorus ratio, vitamin D3, nature of
diet, age and genotype of birds. Thus, microbial phytase can be used to exploit the inherent nutritional potential of feedstuffs for more economic and environmentally friendly poultry production.

The development of alternative energy sources by applying lignocellulose-based biofuel technology is critically important because of the depletion of fossil fuel resources, rising fossil fuel prices, security issues regarding the fossil fuel supply, and environmental issues. White-rot fungi have received much attention in recent years for their valuable enzyme systems that effectively degrade lignocellulosic biomasses. These fungi have powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers, respectively. Lignocellulosic biomasses from either agricultural or forestry wastes are abundant, low-cost feedstock alternatives in nature but require hydrolysis into simple sugars for biofuel production. This review provides a complete overview of the different lignocellulose biomasses and their chemical compositions. In addition, a complete list of the white-rot fungi-derived lignocellulolytic enzymes that have been identified and their molecular structures, mechanism of action in lignocellulose hydrolysis, and biochemical properties is summarized in detail. These enzymes include ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase) and cellulolytic enzymes (endo-glucanase, cellobiohydrolase, and beta-glucosidase). The use of these fungi for low-cost lignocellulolytic enzyme production might be attractive for biofuel production.

Cladodes of the cactus plant Opuntia ficus-indica (L.) Mill, known as nopal, are used in ethnic gastronomy, such as Mexican cuisine, and for medicinal purposes.
This study aimed at obtaining a method to extract bioactive phenolic compounds from nopal. Five nopal aqueous extracts were prepared using different methodologies and  their composition was determined by HPLC-DAD and LC-MS/MS. The anti-acetylcholinesterase (AChE) and antioxidant activities were assayed as models of biological effects. The main components identified were piscidic acid, eucomic acid and isorhamnetin derivatives. The highest biological activities were found for aqueous extracts after precipitating the ethanol-insoluble material. The best IC 50 values obtained were 780±20 g/ml and 330±40 g/ml for AChE and DPPH, respectively. In silico  molecular docking studies indicated that both piscidic acid and isorhamnetin glycosides are able to interact with the AChE active site, but with significantly different binding constants. These results indicate that nopal is a promising plant for the development of  polyphenol-based new functional foods.

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged

Proteases are enzymes which catalyze the hydrolysis of peptide bonds. Microorganisms are capable of producing these enzymes intracellularly and extracellularly. The isolation of proteases especially the extracelluar proteases of microbial origin is easy and economical. Seasonal fluctuations in the availability of raw material usually do not affect the enzyme production by microbes. There are possibilities for genetic and environmental manipulations to improve yield and properties of the enzymes. Alkaline proteases are referring to proteolytic enzymes which work optimally in alkaline pH (Barett, 1994; Gupta et al., 2002). The vast diversity of proteases, in contrast to the specificity of their action has attracted worldwide attention in attempts to exploit their physiological and biotechnological applications

MH (2015) Array-comparative genomic hybridization analysis of a cohort of saudi patients with epilepsy. CNS Neurol Disord Drug Targets. 14(4):468-75. 3. M.A. Kamal (2015) Biological aspect of various global health issues. Saudi J. Biol. Sci.; 22(1): 1-2.

Peroxidase (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) is an oxidoreductase enzyme found in many fruits and vegetables. This enzyme was purified from sweet gourd (Cucurbita moschata Lam. Poiret) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. Furthermore, optimum pH, optimum temperature, optimum ionic strength, stable pH, and stable temperature conditions were determined as 7.2, 50°C, 0.4 M, 8.0, and 40°C, respectively. The molecular weight (MW) of the enzyme was estimated to be 85 kDa by SDS-PAGE method. The values of Km and Vmax were calculated from the Lineweaver-Burk graph for guaiacol/H2O2 substrate patterns.

IntEnz is the name for the Integrated relational Enzyme database and is the official version of the Enzyme Nomenclature. The Enzyme Nomenclature comprises recommendations of the Nomenclature Committee of the International Union of Bio chemistry and Molecular Biology (NC‐IUBMB) on the nomenclature and classification of enzyme‐catalysed reactions. IntEnz is supported by NC‐IUBMB and contains enzyme data curated and approved by this committee. The database IntEnz is available at http://www.ebi.ac.uk/intenz.

Aims: To optimize laccase production by submerged fermentation using an edible
mushroom Pleurotus ostreatus ARC280.
Study Design: Laccase activity was assayed by monitoring the product formation rate of
enzymatic oxidation of syringaldazine spectrophotometrically at 525 nm.
Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering
and Biotechnology Division, National Research Centre, Dokki, Cairo, Egypt, between May
2009 and October 2010.
Methodology: Pleurotus ostreatus ARC280 was maintained on potato dextrose agar
medium. The liquid medium used for the laccase production by the fungal culture during
its growth in submerged fermentation was selected from eight liquid media for inducing
laccase production. Parameters such as incubation period, temperature, pH of the
production medium, carbon and nitrogen sources and other nutritional parameters were
studied using syringaldazine as a model substrate for laccase activity determination.
Results: In the present work, Eight media with different components were screened. The
enzyme formed by Pl. ostreatus ARC280 was localized mainly in the extra-cellular
fraction. Laccase formation reaches its maximum value with specific activity of about 140
U/mg protein at the twenty-sixth day of incubation, pH 5.0 and 28ºC. Among the various
wastes used, corn stover induces the highest laccase production with specific activity of75.48 U/mg protein. Soluble starch at 1.5% (w/v) and ammonium sulfate was found to be
the best carbon and nitrogen sources for laccase formation, respectively. The optimal
concentrations of Tween-80 and CuSO4. 5H2O, were found to be 0.1% (v/v) and 100μM
and cause enzyme induction by about 44% and 19% than control, respectively.
Conclusion: Laccase production by Pl. ostreatus ARC280 has been shown to depend
markedly on the composition of the culture medium, carbon, nitrogen content and inducer
compounds and governed by parameters such as pH of the production medium and other
nutrition parameters.

Ionic liquids (ILs) have evolved as a new type of non-aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions; on the other hand, it is important to systematically analyze methods that have been developed for stabilizing and activating enzymes in ILs. This review discusses the biocatalysis in ILs from two unique aspects (1) factors that impact the enzyme's activity and stability, (2) methods that have been adopted or developed to activate and/or stabilize enzymes in ionic media. Factors that may influence the catalytic performance of enzymes include IL polarity, hydrogen-bond basicity/anion nucleophilicity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, and surfactant effect. To improve the enzyme's activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, sol–gel, or CLEA), physical or covalent attachment to PEG, rinsing with n-propanol methods (PREP and EPRP), water-in-IL microemulsions, IL coating, and the design of enzyme-compatible ionic solvents. It is exciting to notice that new ILs are being synthesized to be more compatible with enzymes. To utilize the full potential of ILs, it is necessary to further improve these methods for better enzyme compatibility. This is what has been accomplished in the field of biocatalysis in conventional organic solvents. Copyright © 2010 Society of Chemical Industry

Aim: The aim of the present study was to evaluate qualitatively the decolorization of five dyes by
Pleurotus ostreatus (P. ostreatus) ARC280 using solid medium. The laccase produced by the
fungus was used in terms of its concentration and thermal stability for enzymatic decolorization and
also in combination with Hydroxybenzotriazole (HBT) as a redox mediator.
Study Design: Qualitative evaluation of decolorization of dyes and determining the best conditions
required for decolorization in the presence and absence of HBT.
Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and
Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2013 and
February 2014.
Methodology: P. ostreatus ARC280 fungal ability for dyes decolorization was qualitatively
evaluated on solid medium containing (g/L): dye, 0.1; glucose, 10; agar, 30; 100 mL mineral
solution and 100 mL wheat bran washing water obtained by boiling 50 g of wheat bran in 1000 mLof distilled water. The efficiency of decolorization was expressed in terms of decolorization
percentage as follows:
Decolorization (%) = 100 ×
Absorbance t0 - Absorbance tf
Absorbance t0
Where Absorbance t0 is the absorbance at the optimumwavelength of the reaction mixture before
incubation with the enzyme and Absorbance tf is the absorbance at the optimum wavelength after
incubation time.
Results: The enzyme was efficient in decolorizing Acid Blue C.I. 220 (100%), Dichlorophenol
indophenol sodium salt D 5110 (92.6%) and Brilliant Green C.I. 42040 (78.6%) after 6 h of
incubation at 30ºC. In the presence of HBT (1 mM), Lanasol Red 6G was greatly affected by HBT
as a laccase mediator system with decolorization percentage of 53.85% instead of 10.90 in case of
laccase alone, however the enzyme could not efficiently decolorize Foron Yellow Brown S 2RFLI
dye even in presence of HBT. The decolorization efficiency of all dyes was decreased by
increasing reaction temperature from 30 to 50ºC. The absorbance reduction at the maximum
wavelength was recorded with all the tested dyes.
Conclusion: The results obtained clearly confirmed the role of P. ostreatus ARC280 laccase and
its mediated system in the decolorization of structurally different dyes.