Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi

L-asparaginase is used for the treatment of acute lymphoblastic leukaemia (ALL); however, its formulation presents drawbacks such as a lack of stability and formation of aggregates. Osmolytes are small molecules accumulated by cells in response to environmental stresses and present a protective behaviour, favouring the equilibrium of macromolecules towards the native conformation. Therefore, osmolytes are employed as excipients in pharmaceutical protein formulations. Herein, recombinant L-ASNase II from Erwinia chrysanthemi (ErA II) was analysed with respect to the effect of osmolytes on kinetic and stability of this biopharmaceutical. The aggregation profiles were analysed trough nanotracking particle analysis and dynamic light scattering. The majority of the tested osmolytes increased ErA II specific activity and stability, being more pronounced for sucrose and sorbitol, which increased almost 70% of ErA II activity. The polyol preserved total enzyme activity for 30 days while sucrose preserved 81.1 ± 5.3% total enzyme activity over this period. Each osmolyte resulted in a specific aggregation profile and the presence of sucrose or sorbitol resulted in a lower quantity of aggregates in the range of 100–300 nm. The present findings may contribute to the improvement of adjuvants in L-ASNase formulations and the optimization of other biopharmaceutical formulations.publishe

"Antarctic yeasts as a source of L-asparaginase: Characterization of a glutaminase-activity free L-asparaginase from psychrotolerant yeast Leucosporidium scottii L115"

"Microorganisms from extreme environments, such as the Antarctic ecosystems, have a great potential to produce enzymes with novel characteristics. Within this context, L-asparaginase (ASNase) obtained from yeast species has been poorly studied. In this study, yeasts isolated from samples collected at Admiralty Bay (King George Island, Antarctica) were tested to produce ASNase. From an initial screening of 40 strains, belonging to 13 different species, Leucosporidium scottii L115 produced an ASNase activity (LsASNase activity: 6.24 U g-1 of dry cell weight) with the lowest glutaminase activity. The LsASNase was purified 227-fold, with a specific activity of 137.01 U mg-1 at 37 ◦C, without glutaminase activity. Moreover, the maximum enzyme activity was observed at pH 7.5 and at a temperature of 55 ◦C. The enzyme is a multimer of 462 kDa, presenting a single band of 53 kDa molecular mass in reduced conditions; after PGNase F treatment, a single band of 45 kDa was observed. The enzymatic kinetic evaluation revealed an allosteric regulation of the enzyme and the kinetic parameters were determined at 37 ◦C, pH 7.0 as substrate affinity constant, K0.5 = 233 μM, kcat = 54.7 s − 1 and Hill coefficient, nH = 1.52, demonstrating positive cooperativity by the enzyme and the substrate. This is the first study to report L. scottii as a source of glutaminase-activity free L-asparaginase, an acute lymphoblastic leukemia drug feature suitable for the treatment of asparagine synthetase negative cancer cells.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, b- cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5 degrees C and 40% at 25 degrees C after 8 months. The lipase dried with 10% of beta-cyclodextrin retained 72% of activity at 5 degrees C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[04/07935-6]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[08/52732-7]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[Proc. 06/50955-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[Proc. 08/52732-7

Normal sonographic anatomy of the abdomen of coatis (Nasua nasua Linnaeus 1766)

Submitted by Franciele Moreira ([email protected]) on 2017-11-28T16:51:46Z No. of bitstreams: 2 Artigo - Rejane Guerra Ribeiro - 2013.pdf: 2052707 bytes, checksum: e3b34e0079f028003a4b96373c00c3d1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2017-11-29T11:19:51Z (GMT) No. of bitstreams: 2 Artigo - Rejane Guerra Ribeiro - 2013.pdf: 2052707 bytes, checksum: e3b34e0079f028003a4b96373c00c3d1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2017-11-29T11:19:51Z (GMT). No. of bitstreams: 2 Artigo - Rejane Guerra Ribeiro - 2013.pdf: 2052707 bytes, checksum: e3b34e0079f028003a4b96373c00c3d1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013Background: The use of ultrasound in veterinary medicine is widespread as a diagnostic supplement in the clinical routine of small animals, but there are few reports in wild animals. The objective of this study was to describe the anatomy, topography and abdominal sonographic features of coatis. Results: The urinary bladder wall measured 0.11 ± 0.03 cm. The symmetrical kidneys were in the left and right cranial quadrant of the abdomen and the cortical, medullary and renal pelvis regions were recognized and in all sections. The medullary rim sign was visualized in the left kidney of two coatis. The liver had homogeneous texture and was in the cranial abdomen under the rib cage. The gallbladder, rounded and filled with anechoic content was visualized in all coatis, to the right of the midline. The spleen was identified in the left cranial abdomen following the greater curvature of the stomach. The parenchyma was homogeneous and hyperechogenic compared to the liver and kidney cortex. The stomach was in the cranial abdomen, limited cranially by the liver and caudo-laterally by the spleen. The left adrenal glands of five coatis were seen in the cranial pole of the left kidney showing hypoechogenic parenchyma without distinction of cortex and medulla. The pancreas was visualized in only two coatis. The left ovary (0.92 cm x 0.56 cm) was visualized on a single coati in the caudal pole of the kidney. The uterus, right adrenal, right ovary and intestines were not visualized. Conclusions: Ultrasound examination of the abdomen of coatis may be accomplished by following the recommendations for dogs and cats. It is possible to evaluate the anatomical and topographical relationships of the abdominal organs together with the knowledge of the peculiarities of parenchymal echogenicity and echotexture of the viscera