T. A. Costa-Silva

Bio: T. A. Costa-Silva is an academic researcher from University of São Paulo. The author has contributed to research in topics: Lipase & Cercospora kikuchii. The author has an hindex of 11, co-authored 25 publications receiving 299 citations.

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Abstract: L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

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

Abstract: 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, β-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°C and 40% at 25°C after 8 months. The lipase dried with 10% of β-cyclodextrin retained 72% of activity at 5°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.

Characterization and spray drying of lipase produced by the endophytic fungus Cercospora kikuchii

Abstract: A lipase from the endophytic fungus Cercospora kikuchii was purified, biochemically characterized and the effects of spray drying on stabilization of the purified enzyme were studied. The lipase was purified 9.31-fold with recovery of 26.6% and specific activity of 223.6 U/mg. The optimum pH and temperature were 4.6 and 35 oC, respectively, while the Vmax was 10.28 µmol/min.mg-1 protein and Km 0.0324 mM. All the metal ions tested enhanced the enzyme activity. The lipase retained almost 100% activity in the presence of strong oxidants and was also resistant to Triton X, Tween 80 and 20 and SDS, as well as to proteases. The purified lipase was spray dried and kept until 85.2% of enzymatic activity. At least 70% of the enzymatic activity was maintained for spray dried purified lipase during the storage period. The lipase produced by Cercospora kikuchii has properties useful for industrial application and showed adequate stabilization and retention of its enzymatic activity after spray drying.

Penicillium and Talaromyces endophytes from Tillandsia catimbauensis , a bromeliad endemic in the Brazilian tropical dry forest, and their potential for l -asparaginase production

Abstract: This study was conducted to report the richness of endophytic Penicillium and Talaromyces species isolated from Tillandsia catimbauensis, a bromeliad endemic in the Brazilian tropical dry forest (Caatinga), to verify their ability to produce the enzyme l-asparaginase and to partially optimise the production of biomass and l-asparaginase of the best enzyme producer. A total of 184 endophytes were isolated, of which 52 (29%) were identified through morphological and phylogenetic analysis using β-tubulin sequences into nine putative species, four in Penicillium and five in Talaromyces. Talaromyces diversus and T. cf. cecidicola were the most frequent taxa. Among the 20 endophytic isolates selected for l-asparaginase production, 10 had the potential to produce the enzyme (0.50–2.30 U/g), especially T. cf. cecidicola URM 7826 (2.30 U/g) and Penicillium sp. 4 URM 7827 (1.28 U/g). As T. cf. cecidicola URM 7826 exhibited significant ability to produce the enzyme, it was selected for the partial optimisation of biomass and l-asparaginase production. Results of the 23 factorial experimental design showed that the highest dry biomass (0.66 g) was obtained under pH 6.0, inoculum concentration of 1 × 108 and 1% l-proline. However, the inoculum concentration was found to be statistically significant, the pH was marginally significant and the concentration of l-proline was not statistically significant. l-Asparaginase production varied between 0.58 and 1.02 U/g and did not reach the optimal point for enzyme production. This study demonstrates that T. catimbauensis is colonised by different Penicillium and Talaromyces species, which are indicated for enzyme production studies.

On the taught new tricks of enzymes immobilization: An all-inclusive overview

Abstract: The primary means of immobilizing enzymes are to boost the enzyme productivity and operational stability, alongside facilitating the reuse of enzymes. Notwithstanding the aforementioned benefits, enzyme immobilization promotes high catalytic activity and stability, convenient handling of enzymes, in addition to their facile separation from reaction mixtures without contaminating the products. This review describes the choices of support materials and cross-linkers together with several mechanisms that influence the performance, stabilization and hyperactivation of immobilized enzymes. Altering enzyme properties often changes the enzyme structure due to random modifications in the behavior, which in some cases can be positive or negative. Future strategy to develop new generations of immobilized enzymes should capitalize on the rapid advances of genetic manipulation, organic chemistry, computational chemistry and bioinformatics, reactor and reaction design. Upcoming efforts to improve enzymes as industrial biocatalysts must consider their development for increased selective promiscuity suitable for multiple biotransformations, either independently or as catalytic cascade processes thereby enhance the cost-effectiveness of the processes.

Single droplet drying for optimal spray drying of enzymes and probiotics

Abstract: Spray drying is a mild and cost-effective convective drying method. It can be applied to stabilise heat sensitive ingredients, such as enzymes and probiotic bacteria, albeit in industrial practice for example freeze drying or freezing are often preferred. The reason is that optimum drying conditions and tailored matrix formulations are required to avoid severe heat damage leading to loss in enzyme activity or reduced survival of bacteria. An overview is provided on the use of protective carbohydrate-rich formulations in the spray drying of enzymes and probiotics. Subsequently, single droplet drying experimentation methods are reviewed for mapping drying trajectories of individual droplets. The advantage of these is to provide insight in inactivation kinetics of enzymes and probiotics and thus contribute to unravelling of stabilisation mechanisms. Finally, it is shown that detailed modelling of single droplet drying and insight in micro-structural changes during drying can be complementary to the experimental single droplet approaches.

Endophytic Fungi: Biodiversity, Ecological Significance, and Potential Industrial Applications

Abstract: Endophytic fungi are abundant and have been reported from all tissues such as roots, stems, leaves, flowers, and fruits. In recent years, research into the beneficial use of endophytic fungi has increased worldwide. In this chapter, we critically review the production of a wide range of secondary metabolites, bioactive compounds from fungal endophytes that are a potential alternative source of secondary plant metabolites and natural producers of high-demand drugs. One of the major areas in endophytic research that holds both economic and environmental potential is bioremediation. During their life span, microbes adapt fast to environmental pollutants and remediate their surrounding microenvironment. In the last two decades, bioremediation has arisen as a suitable alternative for remediating large polluted sites. Endophytic fungi producing ligninolytic enzymes have possible biotechnological applications in lignocellulosic biorefineries. This chapter highlights the recent progress that has been made in screening endophytic fungi for the production and commercialization of certain biologically active compounds of fungal endophytic origin.