Purified Enzyme Preparation Research Articles

An oxidant and organic solvent tolerant alkaline lipase by P. aeruginosa mutant: downstream processing and biochemical characterization.

An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45 °C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca(2+), Mg(2+) and Na(+) tend to increase the enzyme activity, whereas, Fe(2+) and Mn(2+) ions resulted in discreet decrease in the activity. Divalent cations Ca(+2) and Mg(+2) seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca(+2) (5 mM) the optimum temperature shifted from 45 °C to 55 °C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of K(m) and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL(-1) and 5000 μg.mL(-1).min(-1), respectively.

Purification and Characterization of 1,3-β-D-Glucan Phosphorylase fromOchromonas danica

1,3-β-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.

Partial Purification and Characterization of Peroxidase from Pearl Millet (Pennisetum Glaucum [L.] R. Br.) Grains

Peroxidase from pearl millet hybrid HHB 94 was purified stepwise by (NH4)2SO4 fractionation, gel filtration and ion exchange chromatography using Sephadex G-100 and DEAE-cellulose to near homogeneity. The protocol yielded 41% of peroxidase with 33.2-fold purification. The purified enzyme preparation has molecular masses of 31 kDa, as determined by gel filtration through Sephadex G-100, and 30.4 kDa, as determined by SDS-PAGE, suggesting that the enzyme is a monomer. The enzyme exhibited maximum activity at pH 5.6 and 30C. It was significantly more stable in the pH range of 6.0–6.8 and at temperature below 35C. Peroxidase showed Km values of 0.10 and 11 mM for o-dianisidine and H2O2, respectively. The activity of peroxidase was highly inhibited by DTT, β-mercaptoethanol and hydrazine, and moderately by sodium azide, sodium borohydride, oxalic acid, EDTA, DTNB, Mn2+, Na+ and K+. The enzyme activity was enhanced by Ca2+ and Fe3+. Practical Applications Peroxidase is considered to have an empirical relationship to off-flavors and odors in foods. Earlier we have shown that peroxidase activity varied in different genotypes, i.e., HHB 94 (high) and 94222A × 78/711 (low), which appeared to be responsible for odor generation in stored pearl millet meal. In 94222A × 78/711, less off-odor was observed. The enzyme purified from mature pearl millet grains is strongly inhibited by DTT. This new information has been used to assay the enzyme in situ. We have found that in comparison to extractable activity, in situ activity of the enzyme is more strongly correlated with the development of off-odor in stored flour. Thus, in situ assay is being used to screen germplasm for selecting contrasting genotypes (with respect to off-odor) that will be used to develop pearl millet hybrids with improved shelf life of flour.

Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011

An extracellular α-l-rhamnosidase has been purified to electrophoretic homogeneity from the culture filtrate of Penicillium corylopholum MTCC-2011 using a simple procedure consisting of concentration by ultrafiltration and cation exchange column chromatography on carboxymethyl cellulose. The sodium dodesyl sulphate polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass of 67.0kDa. The native – polyacrylamide gel electrophoresis analysis also gave a single protein band confirming the purity of the enzyme and also showing that the enzyme is a monomer in the native state. The Km and kcat values of the enzyme were 0.42mM and 35.7s−1, respectively, using p-nitrophenyl α-l-rhamnopyranoside as the substrate. The pH and temperature optima of the enzyme were 6.5 and 57.0°C, respectively. The purified enzyme preparation successfully hydrolyzed naringin and rutin to prunin and quercetin glucoside, respectively. Thus it can be used for the preparation of these pharmaceutically important compounds.

Reporter Enzyme Inhibitor Study To Aid Assembly of Orthogonal Reporter Gene Assays

Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.

The potential bioproduction of the pharmaceutical agent sakuranetin, a flavonoid phytoalexin in rice

Sakuranetin, the major flavonoid phytoalexin in rice, can be induced by ultraviolet (UV) irradiation, treatment with CuCl2 or jasmonic acid (JA), or phytopathogenic infection. In addition to sakuranetin’s biological significance on disease resistance in rice, its broad bioactivities have recently been described. Results from these studies have shown that sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin, a precursor of sakuranetin, by naringenin 7-O-methyltransferase (NOMT), but the relevant gene has not yet been identified in rice. Recently, we identified the OsNOMT gene, which is involved in the final step of sakuranetin biosynthesis in rice. In previous studies, OsNOMT was purified to apparent homogeneity from UV-treated wild-type rice leaves; however, the purified protein, termed OsCOMT1, exhibited caffeic acid 3-O-methyltransferase (COMT) activity, but not NOMT activity. Based on the analysis of an oscomt1 T-DNA tagged mutant, we determined that OsCOMT1 did not contribute to sakuranetin production in rice in vivo. Therefore, we took advantage of the oscomt1 mutant to purify OsNOMT. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps resulting in a 400-fold purification from the crude enzyme preparation with a minor band at an apparent molecular mass of 40 kDa in the purest enzyme preparation. Matrix-assisted laser desorption/ionization time of flight/time of flight analysis showed that the 40 kDa protein band included two O-methyltransferase-like proteins, but one of the proteins encoded by Os12g0240900 exhibited clear NOMT activity; thus, this gene was designated OsNOMT. Gene expression was induced by treatment with jasmonic acid in rice leaves prior to sakuranetin accumulation, and the recombinant protein showed reasonable kinetic properties to NOMT. Identification of the OsNOMT gene enables the production of large amounts of sakuranetin through transgenic rice and microorganisms. This finding also allows for the generation of disease-resistant and sakuranetin biofortified rice in the future.

Desymmetrization of cbz-serinol catalyzed by crude pig pancreatic lipase reveals action of lipases with opposite enantioselectivity

Abstract Lipase preparations from pig pancreas are widely used in industrial production of fine chemicals and pharmaceuticals. In our attempts to synthesize enantiopure building blocks for production of iminocyclitols, we used a crude preparation of pig pancreatic lipase in acetylation of carboxybenzyl-2-amino-1,3-propanediol (cbz-serinol) with vinyl acetate in tetrahydrofuran. The enantiomeric excess of the product monoester changed from (2S)-monoacetate to (2R)-monoacetate after prolonged reaction. By using vinyl acetate both as solvent and acyl donor only the (2R)-monoacetate was obtained. Purified enzyme preparation gave only the (2S)-monoacetate. It is likely that the crude enzyme preparation contains lipases with opposite stereoselectivity, acting at different rates in the desymmetrization reaction. The same enzyme preparation was used in hydrolysis of the diacetylated propanediol in phosphate buffer and acetonitrile as co-solvent. After 24 h the (2S)-monoacetate was obtained in high enantiomeric excess. Our results show that monitoring the progress of biocatalyzed reactions are of utmost importance, in particular when crude enzyme preparations are used as catalysts.

Extraction, purification and characterization of L-asparaginase from Penicillium sp. by submerged fermentation.

A fungal strain identified as Penicillium sp. was evaluated for its L–asparaginase enzyme production. The L-asparaginase enzyme was purified to homogeneity from Penicillium sp. that was grown on submerged fermentation. Different purification steps including salt precipitation, followed by separation on sephadex G-100-120 gel filtration and DEAE were applied to obtain pure enzyme preparation. The purified enzyme showed 13.97 IU/mg specific activity and 36.204% yield. The polyacrylmide gel electrophoresis of the pure enzyme exhibited one protein of 66 kDa. The enzyme showed maximum activity at 7.0 pH and 37°C and Km value 4.00 × 10-3 M. Key words: L – Asparaginase, fungi, Penicillium.

The cofactor preference of glucose‐6‐phosphate dehydrogenase from Escherichia coli– modeling the physiological production of reduced cofactors

In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.

A novel endoribonuclease from the marine sponge Tethya aurantium specific to 2′,5′-phosphodiester bonds

In the marine sponge Tethya aurantium a novel endoribonuclease was found which specifically catalyzed the degradation of 2',5'-phosphodiester linkages and was therefore named endo-2',5'-ribonuclease. This enzymatic reaction yielded 2',3'-cyclic phosphate and 5'-OH products similarly to the 3'-5' bond cleavage in RNA, catalyzed by metal-independent ribonucleases. The partially purified enzyme preparation was used for its biochemical characterization. The enzyme did not require the presence of metal ions for its activity. The novel nuclease exhibited a preference for 5'-phosphorylated 2',5'-oligoadenylates, but 2'-5' linkage in 5'-triphosphorylated hetero-oligomers or homo-dimers comprising guanylate or uridylate residues instead of adenylate was cleaved as well. The enzyme was also able to catalyze the degradation of 5'-unphosphorylated 2',5'-oligoadenylates, except for 2',5'-diadenylate, which were weaker substrates for the enzyme than the respective 5'-triphosphorylated forms. The observed substrate specificity may refer to the specific role of the enzyme in the degradation of natural 2',5'-oligoadenylates (2-5A) that function in the interferon-induced mammalian 2-5A system as allosteric regulators of ribonuclease L. They are produced by 2-5A synthetases (OAS) that are also present in sponges, the most ancient phylum of Metazoa. We suggest that the newly discovered endoribonuclease found in the marine sponge T.aurantium could be a representative of the group of 2',5'-specific ribonucleases that primarily control the cellular levels of 2',5'-oligoadenylates.

Molecular characterization of the C-methyltransferase NovO of Streptomyces spheroides, a valuable enzyme for performing Friedel–Crafts alkylation

Abstract The methyltransferase NovO cloned from Streptomyces spheroides could be heterologously produced as soluble and active enzyme in Escherichia coli . Sequencing of the cloned novO gene revealed differences to the GenBank entry AAF67508.1 resulting in a different amino acid at position 223 (Cys instead of Ser). A generated variant containing a Ser residue at this position, however, resulted in poor ability to express soluble and enzymatically active protein. Characterization of NovO revealed a type I methyltransferase that performs its action as a dimer in solution. Functional elements include the conserved S-adenosyl- l -methionine (SAM) binding site (consensus: E/DXXXGXG) as DLCCGSG (residues 45–51). Mutation analyses of the respective amino acids verified their importance for cofactor binding and enzyme activity. In soluble protein fractions of mutants D45N and G49A the calculated k cat values decreased from 2.5 × 10 −2 s −1 of the wild-type protein to 9.7 × 10 −4 s −1 and 1.2 × 10 −3 s −1 , respectively. A histidine at position 15 was identified as the catalytic base in the methyl transfer reaction. The analysis of purified enzyme preparations showed that the transfer of allyl groups via the SAM analog allyl-SAH occurs with a fourfold increased k cat of 11 × 10 −3 s −1 compared to 3.2 × 10 −3 s −1 for methyl transfer. However, the evolutionary design toward SAM is obvious from the K m value of 0.06 mM compared to 0.22 mM for allyl-SAH.

Enhancement of protease production by Pseudomonas aeruginosa isolated from dairy effluent sludge and determination of its fibrinolytic potential

ObjectiveThe present study aimed at isolating proteolytic bacteria from dairy effluent sludge, designing the process parameters for the enhanced production of protease and determination of its fibrinolytic potential. MethodsThe dairy sludge was processed according to the microbiological criteria for the isolation of proteolytic bacteria. All the isolates were screened for their protease production ability and the isolate showing highest proteolysis was selected for further studies. Effects of various media components and process parameters like carbon and nitrogen supplementation, temperature, pH and incubation period were investigated. Partial purification of the protease was done using ammonium sulphate fractionation, following which its molecular weight and fibrinolytic activity were determined. ResultsBased on the biochemical studies, the selected isolate was identified as Pseudomonas aeruginosa. The highest protease yield was obtained with maltose and yeast extract as supplements. The optimum pH, temperature and incubation period for protease production by the isolate was found to be 7.0, 37°C and 48 h respectively. The partially purified enzyme preparation showed a single protein band in sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealing the apparent molecular weight of the enzyme to be 35 kDa. The efficient removal of the blood stain emphasized its fibrinolytic potential. ConclusionsFrom the present study it is envisaged that cultural parameters significantly affect the protease production. Based upon the fibrinolytic activity, this protease may find broad applications in detergent and pharmaceutical industries.

A protease from the medicinal mushroom Pleurotus sajor-caju; production, purification and partial characterization

ObjectiveTo produce protease using solid state fermentation from Pleurotus sajor-caju. MethodsOptimization of saccharification conditions like time, pH, temperature, inoculum size, different carbon sources and its concentration, various nitrogen sources and its concentrations were studied. ResultsThe results showed that the optimum conditions for maximum protease production under SSF (Solid State Fermentation) by Pleurotus sajor-caju was found to be on the fourth day of incubation, pH, 7; temperature, 30°C, 3%; corn as substituted carbon source; 0.8% ammonium nitrate as nitrogen source concentration. Protease was partially purified by ammonium sulphate precipitation (70% saturation) to 2.72 fold with recovery of 53% characterized for few properties. Homogeneous preparation of protease from the culture filtrate of the fungus has been achieved using ammonium sulphate precipitation, anion exchange chromatography on DEAE cellulose. The purified enzyme preparation gave a single protein band in SDS-PAGE analysis indicating a molecular weight of 48 kDa. The purified enzyme exhibited its optimal activity at 60 °C and at pH 8. Metal ions (Ca2+ and Na 2+) caused moderate reduction in enzyme activity. ConclusionsConsidering the importance and application of the proteases, this study provides better understanding of condition for the activity of protease by fungal culture.

Ethylmalonyl-CoA Decarboxylase, a New Enzyme Involved in Metabolite Proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.

Abstract 18385: Serum Amyloid A is an Evolutionary Conserved Apolipoprotein That Functions to “Fine Tune” Reverse Cholesterol Transport During the Acute-Phase Response

In response to severe injury, the acute-phase protein, serum amyloid A (SAA) can replace apoA-I as the major apo-protein of HDL. The inclusion of SAA in HDL changes a number of HDL's properties. Our data indicates that SAA's presence reduces HDL's capacity to accept free cholesterol. SAA also increases HDL's binding affinity for macrophages and is dependent on the cell surface glycosaminoglycan, heparin sulfate (HS). SAA contains two HS binding sites and HS binding to SAA can cause it to dissociate from HDL, leaving behind lipid poor apoA-I rich oligomers, with enhanced cholesterol accepting potential. Under "amyloidogenic" condition this dissociated SAA can be readily converted into amyloid fibrils by macrophages and our data suggests this occurs largely on the cell surface. SAA can also be taken up by macrophages and once in the cytoplasm we have shown that it can stimulate neutral cholesteryl ester hydrolase (nCEH) and inhibit acetyl-coenzyme A acetyltransferase (ACAT) activities, respectively, with the net effect of mobilizing stored cholesteryl ester and making it available for export via the ABCA1/ABCG1 transporters. Under experimental conditions SAA can promote the export of over 70% of the cholesterol loaded into macrophages within 24 h. SAA's activities complement those of apoA-I/HDL by increasing the pool of exportable (de-esterified) cholesterol for efflux to an extracellular cholesterol acceptor like apoA-I/HDL and entry into the reverse cholesterol transport (RCT) pathway. The nCEH and ACAT modulating domains of SAA have been identified and as synthetic peptides have been also demonstrated to be potent promoters and inhibitors of nCEH and ACAT activities, respectively, with purified enzyme preparations, intact cells in culture and in vivo in mice, consistent with an allosteric mechanisms of action. When delivered in liposomes to a mouse apoE knockout model of atherosclerosis, these SAA peptides were shown to be highly effective in the prevention and regression of atherosclerotic lesions. We believe SAA has been evolutionary conserved to aid in the recycling of cellular cholesterol.

Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.

The CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold.

English

&nbsp; Investigation of the production of cellulase complex enzymes by the fungus&nbsp;Aspergillus oryzae&nbsp;on soluble bio- cellulosic waste like cheese whey was the goal of this work. The effects of incubation period, substrate concentration, initial pH and temperature on the production of endoglucanase, exoglucanase and cellulase (filter paper activity FPA) activities, were investigated. Seven days incubation period with an initial pH value of 6 and temperature of 40&deg;C, and cheese whey concentration of 10% yielded the highest specific activities with 1.5 fold increases for endoglucanase and 1.4 fold increases for exoglucanase. Cellulase specific activity represented as FPA showed no fold increase at all. The optimum temperature of endoglucanase was observed at 55&deg;C, and the enzyme activity has abroad pH range between 3.8 to 8.0 and 24.91% of the original activity was retained after heat treatment at 90&deg;C for 30 min. Analysis of the partial purified enzyme preparation by SDS &ndash; PAGE revealed one protein band showing cellulolytic activity, The molecular weight of this band was estimated to be around 45.000 Daltons. &nbsp; Key words:&nbsp;Cellulolytic enzymes,&nbsp;Aspergillus oryzae,&nbsp;cheese whey,&nbsp;dinitrosalicylic acid(DNS), fermentation variables.

Nucleoside triphosphate diphosphohydrolases (NTPDase) inhibitory activity of some medicinal plants.

Nucleoside triphosphate diphosphohydrolasess (NTPDases, EC 3.6.1.5) are well characterized ectoenzymes which are involved in the hydrolysis of ATP and ADP resulting in the formation of nucleoside monophosphates and orthophosphate. By hydrolyzing circulating ATP to ADP to AMP, NTPDases play a crucial role in preventing the activation of platelets and their aggregation. NTPDase inhibitors therefore are currently target for various cardiovascular diseases and some types of cancer. In search for new enzyme inhibitors of therapeutic importance, the present study was conducted to find out the inhibitors of NTPDase from methanolic extracts of 50 medicinal plants used in folk medicine. Data shows that only 6 plants exhibited anti-NTPDase activity above 50% with crude enzyme preparation of chicken liver at 125 μg plant extract. These plants included, Cymbopogan jawarancusa, (51.16±4.81%), Asparagus officinalis (58.24±0.93%), Tribulus terrestris (50.84±6.19%), Rubia cordizolla (53.11±3.49%), Echimops echimatus (51.48±6.29%) Portulaca oleracea (52.79±0.10%). These studies are priliminary findings and further work is required to explore these plants using purified enzyme preparations. Keywords: Nucleoside triphosphate diphosphohydrolases, NTPDases, medicinal plants.

Enhancing Jatropha oil extraction yield from the kernels assisted by a xylan-degrading bacterium to preserve protein structure.

We investigated the use of bacterial cells isolated from paddy crab for the extraction of oil from Jatropha seed kernels in aqueous media while simultaneously preserving the protein structures of this protein-rich endosperm. A bacterial strain—which was marked as MB4 and identified by means of 16S rDNA sequencing and physiological characterization as either Bacillus pumilus or Bacillus altitudinis—enhanced the extraction yield of Jatropha oil. The incubation of an MB4 starter culture with preheated kernel slurry in aqueous media with the initial pH of 5.5 at 37 °C for 6 h liberated 73% w/w of the Jatropha oil. Since MB4 produces xylanases, it is suggested that strain MB4 facilitates oil liberation via degradation of hemicelluloses which form the oil-containing cell wall structure of the kernel. After MB4 assisted oil extraction, SDS-PAGE analysis showed that the majority of Jatropha proteins were preserved in the solid phase of the extraction residues. The advantages offered by this process are: protein in the residue can be further processed for other applications, no purified enzyme preparation is needed, and the resulting oil can be used for biodiesel production.