Predicting the partition coefficients of a recombinant cutinase in polyethylene glycol/phosphate aqueous two-phase systems

A model for the prediction of protein partition coefficients in aqueous two-phase systems has been developed. This model accounts for both charge-independent and electrostatic effects. The determination of nonelectrostatic effects was based on the model of Eiteman and Gainer for uncharged solutes while the electrostatic contribution was computed using TITRA, a program that uses a continuum electrostatic model to treat charge interactions in proteins and considers the effect of pH and ionic strength. The partition coefficients of Fusarium solani pisi recombinant cutinase have been satisfactorily predicted in polyethylene glycol (PEG) 1000 and phosphate aqueous two-phase systems at a pH range of 6.0-9.0. The model failed to predict the enzyme partitioning behavior at pH 4.5. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 248-257, 1997.     

A bibliometric overview of Brazilian LCA research

Purpose

We bibliometrically evaluated the scientific literature outlined around Brazilian life cycle assessment (LCA). Our aim is twofold: (1) Analyze the Brazilian scientific literature on LCA, forming a current view of how the LCA methodology is applied in the country; (2) within this view, trace the evolution of themes, characterize institution collaboration, and indicate major influences in Brazilian LCA community.

Methods

Data were outlined around academic production and publications, from 1993 to 2015, indexed by the Institute for Scientific Information (ISI- SCIE and SSCI) through a specific group of keywords. Initially, a temporal evolution and projection of papers, PhD and Master Theses, were performed. In sequence, indexed papers were analyzed through performance indicators (i.e., number of authors, impact factor, among others) and content evaluation (for instance, major addressed themes). Finally, a mapping of science was performed, with the aid of Cite Space software application, where coword (and evolution), cocollaboration (and evolution), and cocitation maps were created.

Results and discussion

The survey identified 429 documents divided among international and national papers, PhD and Master Theses. From those documents, 165 were indexed. In terms of production and performance, the results indicate an undeniable evolution of the Brazilian LCA research, as affirmed by relations solidified through time. The main research field is “LCA application” with 84 % of papers, whereas “LCA methodology” completes the framework. In LCA applications, 25 % of publications are related to Biofuels—divided into bioethanol and biodiesel—which makes it the current dominant LCA research area in Brazil. The collaboration network demonstrates three main institution groups, whereas evolution through the years indicates that this situation may further improve. Influential authors are linked to LCA of biofuels, general LCA guidelines, and methodological LCA developments.

Conclusions

Brazilian LCA research has been growing and more complex relations between themes and institutions denote that further developments can be expected. Cocollaboration indicates three main clusters, led by USP, Unicamp, and UFRJ. “Biofuels” is the main research area where sugarcane ethanol and biodiesel from different sources are the domain product systems. Cocitation analysis solidifies this statement, pointing to Isaias Macedo (and other biofuel researchers) as the main author in Brazilian LCA after ISO and Mark Goedkoop.

     

Quantification of protopectinase SE, an endopolygalacturonase with pectin-releasing activity from Geotrichum klebahnii

Pectin releasing activity of protopectinase SE (PPase-SE) from Geotrichum klebahnii (= G. penicillatum = Trichosporon penicillatum) ATCC 42397 was determined using different batches of lemon protopectin as substrate. Results obtained showed a high degree of variability depending on the batch of protopectin used. As PPase-SE also shows polygalacturonase (PGase) activity, a method for the assay of this activity was optimised. The best assay conditions were: substrate (polygalacturonic acid) concentration of 2.0 g 1^–1, reaction time of 10 min and up to 0.17 PGase units per test tube.     

Spectral attenuation of brain and retina tissues in the near‐infrared range measured using a fiber‐based supercontinuum device

A novel setup for the efficient constant optical measurements of biological tissues in the near infrared is presented. The system combines the use of a fiber‐based supercontinuum source with a simple optics fiber collimator. This configuration allows a wide spectral range of measurement and, at the same time, can efficiently filter the straightforward transmitted light while avoiding scattered light. As a performance example, the optical characterization of rat brain and retina tissues are shown. The attenuation coefficient for both tissues in the near infrared region is also obtained. This technique could be applied in clinical research as a noninvasive method with several potential practical applications.     

High yield process for the production of active human α‐galactosidase a in CHO‐K1 cells through lentivirus transgenesis

Fabry disease is an X‐linked recessive disorder caused by a deficiency in lysosomal α‐Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high‐expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α‐Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO‐K1) cells. Herein, we describe for the first time the application of a strategy based on third‐generation lentiviral particles (LP) transduction of suspension CHO‐K1 cells to obtain high‐producing rhαGAL clones (3.5 to 59.4 pg cell^?1 d^?1). After two purification steps, the active enzyme was recovered (2.4 × 10⁶ U mg^?1) with 98% purity and 60% overall yield. Michaelis‐Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU‐α‐Gal at a comparable rate to Fabrazyme®, the current CHO‐derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose‐6‐phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334–1345, 2017     

Patterns of <Emphasis Type="Italic">Azteca</Emphasis> ants’ defence of <Emphasis Type="Italic">Cecropia</Emphasis> trees in a tropical rainforest: support for optimal defence theory

Optimal defence theory (ODT) predicts that, whereas high risk of herbivory should select for high constitutive levels of defence, induced defences should be more advantageous in environments with a low probability of herbivory. In the present field study, conducted on the Azteca–Cecropia ant–plant system in a Neotropical rainforest, we evaluated whether the constitutive and induced ant defence of leaves are directly and inversely related to an estimate of herbivory risk, respectively. To assess the constitutive level of Azteca defence in Cecropia obtusifolia trees, we recorded the number of ants patrolling undamaged leaves. To evaluate the induced level of Azteca defence, the same leaves were subjected to simulated herbivory by punching circular holes in them. We recorded the maximum number of ants patrolling the damaged leaves from 2 to 15 min after damage. Past herbivory (% defoliation of old leaves) was assumed to indicate a risk of herbivory. Regression analyses showed that, whereas the constitutive level of ant patrolling was positively associated with the magnitude of herbivory on old leaves, there was a negative association between the magnitude of induced ant defence and past herbivory. These preliminary results lend support to ODT.     

Characterization and biotechnological application of an acid alpha-galactosidase from Tachigali multijuga Benth. seeds

Tachigali multijuga Benth. seeds were found to contain protein (364 mg g(-1)dwt), lipids (24 mg g(-1)dwt), ash (35 mg g(-1)dwt), and carbohydrates (577 mg g(-1)dwt). Sucrose, raffinose, and stachyose concentrations were 8.3, 3.0, and 11.6 mg g(-1)dwt, respectively. alpha-Galactosidase activity increased during seed germination and reached a maximum level at 108 h after seed imbibition. The alpha-galactosidase purified from germinating seeds had an M(r) of 38,000 and maximal activity at pH 5.0-5.5 and 50 degrees C. The enzyme was stable at 35 degrees C and 40 degrees C, but lost 79% of its activity after 30 min at 50 degrees C. The activation energy (E(a)) values for p-nitrophenyl-alpha-d-galactopyranoside (pNPGal) and raffinose were 13.86 and 4.75 kcal mol(-1), respectively. The K(m) values for pNPGal, melibiose, raffinose, and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was sensitive to inhibition by HgCl(2), SDS, AgNO(3), CuSO(4), and melibiose. d-Galactose was a competitive inhibitor (K(i)=2.74 mM). In addition to its ability to hydrolyze raffinose and stachyose, the enzyme also hydrolyzed galactomannan.     

Partitioning of functional diversity reveals the scale and extent of trait convergence and divergence

Questions: Trait differentiation among species occurs at different spatial scales within a region. How does the partitioning of functional diversity help to identify different community assembly mechanisms? Location: Northeastern Spain. Methods: Functional diversity can be partitioned into within‐community (α) and among‐communities (β) components, in analogy to Whittaker's classical α and β species diversity concept. In light of ecological null models, we test and discuss two algorithms as a framework to measure α and β functional diversity (the Rao quadratic entropy index and the variance of trait values). Species and trait (specific leaf area) data from pastures under different climatic conditions in NE Spain are used as a case study. Results: The proposed indices show different mathematical properties but similarly account for the spatial components of functional diversity. For all vegetation types along the climatic gradient, the observed α functional diversity was lower than expected at random, an observation consistent with the hypothesis of trait convergence resulting from habitat filtering. On the other hand, our data exhibited a remarkably higher functional diversity within communities compared to among communities (α?β). In contrast to the high species turnover, there was a limited functional diversity turnover among communities, and a large part of the trait divergence occurred among coexisting species. Conclusions: Partitioning functional diversity within and among communities revealed that both trait convergence and divergence occur in the formation of assemblages from the local species pool. A considerable trait convergence exists at the regional scale in spite of changes in species composition, suggesting the existence of ecological redundancy among communities.