Multidomain chimeric enzymes as a promising alternative for biocatalysts improvement: a minireview.

Searching for new and better biocatalysts is an area of study in constant development. In nature, mechanisms generally occurring in evolution, such as genetic duplication, recombination, and natural selection processes, produce various enzymes with different architectures and properties. The recombination of genes that code proteins produces multidomain chimeric enzymes that contain two or more domains that sometimes enhance their catalytic properties. Protein engineering has mimicked this process to enhance catalytic activity and the global stability of enzymes, searching for new and better biocatalysts. Here, we present and discuss examples from both natural and synthetic multidomain chimeric enzymes and how additional domains heighten their stability and catalytic activity. Moreover, we also describe progress in developing new biocatalysts using synthetic fusion enzymes and revise some methodological strategies to improve their biological fitness.

Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.

The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism, and protein-protein interactions. WT1 is involved in the development of several organs, including the kidneys and gonads, heart, spleen, adrenal glands, liver, diaphragm, and neuronal system. We previously provided evidence of transient WT1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of Wt1 in the cardiac troponin T lineage caused abnormal cardiac development. A low expression of WT1 has also been reported in adult cardiomyocytes. Therefore, we aimed to explore its function in cardiac homeostasis and in the response to pharmacologically induced damage. Silencing of Wt1 in cultured neonatal murine cardiomyocytes provoked alterations in mitochondrial membrane potential and changes in the expression of genes related to calcium homeostasis. Ablation of WT1 in adult cardiomyocytes by crossing αMHCMerCreMer mice with homozygous WT1-floxed mice induced hypertrophy, interstitial fibrosis, altered metabolism, and mitochondrial dysfunction. In addition, conditional deletion of WT1 in adult cardiomyocytes increased doxorubicin-induced damage. These findings suggest a novel role of WT1 in myocardial physiology and protection against damage.

AmyJ33, a truncated amylase with improved catalytic properties.

Biochemical and kinetic properties are of special interest for the specific applications of α-amylases in industrial sectors such as textile industries, detergents, biofuels and food among others. Therefore, protein engineering is currently directed towards a continuous demand to improve the properties of amylases and thus meet the specific characteristics for various industrial sectors. In the present work, modular protein engineering was performed to improve the biochemical and kinetic properties of AmyJ33r an α-amylase isolated from Bacillus siamensis JJC33M consisting of five domains, A, B, C, D and E (SBD) (Montor-Antonio et al. in 3 Biotech 7:336, 2017. https://doi.org/10.1007/s13205-017-0954-8 ). AmyJ33r is not active on native starch, only showing activity on gelatinized starch. At the C-terminal, AmyJ33r has a starch binding domain (SBD, domain E) belonging to the CBM26 family. In this study, four truncated versions were constructed and expressed in E. coli (AmyJ33-AB, AmyJ33-ABC, AmyJ33-ABCD, and SBD) to determine the role of the A, B, C, D, and E domains in the biochemical behavior of AmyJ33r on starch. Biochemical and kinetic characterization of the truncated versions showed that domain C is essential for catalysis; domain D improved enzyme activity at alkaline pH values, is also involved negatively in thermostability at 40, 50, and 60 °C and its presence favored the production of maltooligosaccharides with a higher degree of polymerization (DP4). E domain have interaction with raw starch, also the deletion of E domain (SBD) favors the affinity for the substrate while the deletion of D domain increased enzyme kcat at the time of product release. In conclusion, AmyJ33-ABC has better kinetic parameters than AmyJ33-ABCD and AmyJ33r, but is less stable than these two enzymes.

Enzymatic hydrolysis of lignocellulosic biomass using native cellulase produced by Aspergillus niger ITV02 under liquid state fermentation.

The objective of this work was to evaluate the biochemical characteristics of an enzymatic extract obtained from autochthonous fungus Aspergillus niger ITV02 and its application in the enzymatic hydrolysis of wheat straw and corn stubble pretreated by steam explosion. The enzymatic extract was obtained by submerged fermentation using delignified sweet sorghum bagasse as a carbon source. The results obtained showed that the enzymatic extract had ß-glucosidase and endoglucanase activities. The effects of pH and temperature on cellulase activity were evaluated and its thermostability was determined. The optimal parameters of the ß-glucosidase and endoglucanase activities obtained were pH 5 and 70 °C. The enzymatic extract of A. niger ITV02 was used to hydrolyze wheat straw and corn stubble, and the hydrolysis yields were compared with those obtained by a commercial cellulase (Celluclast 1.5L NS 50013) and CellicCTec3. The results showed that with the use the mixture of Celluclast 1.5L-A. niger ITV02 and CellicCTec3-A. niger ITV02 in the hydrolysis, conversions of 86.36% and 67.8% were obtained, respectively. Glucose production for the mixture extract increased 2.15 times more than when the enzyme was used independently alone. The present work shows that A. niger ITV02 has a potential as an enzyme producer for lignocellulosic hydrolysis.

Deletion of the Wilms' Tumor Suppressor Gene in the Cardiac Troponin-T Lineage Reveals Novel Functions of WT1 in Heart Development.

Expression of Wilms' tumor suppressor transcription factor (WT1) in the embryonic epicardium is essential for cardiac development, but its myocardial expression is little known. We have found that WT1 is expressed at low levels in 20-25% of the embryonic cardiomyocytes. Conditional ablation of WT1 using a cardiac troponin T driver (Tnnt2 Cre ) caused abnormal sinus venosus and atrium development, lack of pectinate muscles, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects, ventricular diverticula and aneurisms. Coronary development was normal and there was not embryonic lethality, although survival of adult mutant mice was reduced probably due to perinatal mortality. Adult mutant mice showed electrocardiographic anomalies, including increased RR and QRS intervals, and decreased PR intervals. RNASeq analysis identified differential expression of 137 genes in the E13.5 mutant heart as compared to controls. GO functional enrichment analysis suggested that both calcium ion regulation and modulation of potassium channels are deeply altered in the mutant myocardium. In summary, together with its essential function in the embryonic epicardium, myocardial WT1 expression is also required for normal cardiac development.

The Insulin-like Growth Factor Signalling Pathway in Cardiac Development and Regeneration.

Insulin and Insulin-like growth factors (IGFs) perform key roles during embryonic development, regulating processes of cell proliferation and survival. The IGF signalling pathway comprises two IGFs (IGF1, IGF2), two IGF receptors (IGFR1, IGFR2), and six IGF binding proteins (IGFBPs) that regulate IGF transport and availability. The IGF signalling pathway is essential for cardiac development. IGF2 is the primary mitogen inducing ventricular cardiomyocyte proliferation and morphogenesis of the compact myocardial wall. Conditional deletion of the Igf1r and the insulin receptor (Insr) genes in the myocardium results in decreased cardiomyocyte proliferation and ventricular wall hypoplasia. The significance of the IGF signalling pathway during embryonic development has led to consider it as a candidate for adult cardiac repair and regeneration. In fact, paracrine IGF2 plays a key role in the transient regenerative ability of the newborn mouse heart. We aimed to review the current knowledge about the role played by the IGF signalling pathway during cardiac development and also the clinical potential of recapitulating this developmental axis in regeneration of the adult heart.

Embryonic circulating endothelial progenitor cells.

The development of vascular system in vertebrates has been traditionally explained by early vasculogenic assembly of angioblasts followed by angiogenic outgrowth of pre-existing vessels. The discovery of adult endothelial progenitor cells (Asahara et al. in Science 275(5302):964-967, 1997) challenged this view, since postnatal vascular growth could be accomplished by recruitment of circulating cells with the ability to differentiate into endothelial cells. However, the existence of embryonic circulating endothelial progenitor cells and their actual contribution to vascular development is far less known. We review in this paper the literature concerning the features, origin and physiological functions of embryonic and foetal circulating endothelial progenitors. Our review includes the early (E7.5) progenitors isolated from yolk sac, the hematovascular progenitors identified in the foetal liver, the yolk sac-derived erythro-myeloid progenitors, circulating hematopoietic cells from the G2-GATA4 lineage and the endothelial colony-forming cells isolated from the placenta and umbilical cord blood. We highlight the need of further characterization of these populations and the relationships between them.

Production and characterization of an enzyme extract with cellulase activity produced by an indigenous strain of Fusarium verticillioides ITV03 using sweet sorghum bagasse.

OBJECTIVES: To evaluate a strain of Fusarium verticillioides ITV03 isolated from wood residues in the Veracruz region of Mexico. Endoglucanase and ß-glucosidase production by submerged fermentation was optimized using a Box-Behnken design, where the independent variables were urea, ammonium sulfate and yeast extract. RESULTS: After optimization, an endoglucanase activity of 0.27 U/mL was achieved; subsequently, three carbon sources were evaluated (carboxymethyl cellulose, sweet sorghum bagasse cellulose and delignified sweet sorghum bagasse (DSSB). The results showed that DSSB yielded the greatest endoglucanase (0.28 U/mL) and ß-glucosidase (0.12 U/mL) activities. Both enzymatic activities were characterized for the effect of pH, temperature and thermostability. The optimal parameters of ß-glucosidase and endoglucanase activity were pH 5 and 4 respectively, the optimum temperature 60 °C. These enzymes were stable at 50 °C for 150.68 h and 8.54 h, with an activation energy (Ea(day)) of 265.55 kJ/mol and 44.40 kJ/mol respectively, for ß-glucosidase and endoglucanase. CONCLUSION: The present work shows that a native strain like F. verticillioides ITV03 using DSSB supplemented with nitrogen has a great potential as a producer of cellulase for lignocellulosic residue hydrolysis.

Effect of differential processing of the native and recombinant α-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties.

AmyJ33, an α-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.

Biotechnological Strategies to Improve Plant Biomass Quality for Bioethanol Production.

The transition from an economy dependent on nonrenewable energy sources to one with higher diversity of renewables will not be a simple process. It requires an important research effort to adapt to the dynamics of the changing energy market, sort costly processes, and avoid overlapping with social interest markets such as food and livestock production. In this review, we analyze the desirable traits of raw plant materials for the bioethanol industry and the molecular biotechnology strategies employed to improve them, in either plants already under use (as maize) or proposed species (large grass families). The fundamentals of these applications can be found in the mechanisms by which plants have evolved different pathways to manage carbon resources for reproduction or survival in unexpected conditions. Here, we review the means by which this information can be used to manipulate these mechanisms for commercial uses, including saccharification improvement of starch and cellulose, decrease in cell wall recalcitrance through lignin modification, and increase in plant biomass.

Functional role of the additional domains in inulosucrase (IslA) from Leuconostoc citreum CW28.

BACKGROUND: Inulosucrase (IslA) from Leuconostoc citreum CW28 belongs to a new subfamily of multidomain fructosyltransferases (FTFs), containing additional domains from glucosyltransferases. It is not known what the function of the additional domains in this subfamily is. RESULTS: Through construction of truncated versions we demonstrate that the acquired regions are involved in anchoring IslA to the cell wall; they also confer stability to the enzyme, generating a larger structure that affects its kinetic properties and reaction specificity, particularly the hydrolysis and transglycosylase ratio. The accessibility of larger molecules such as EDTA to the catalytic domain (where a Ca2+ binding site is located) is also affected as demonstrated by the requirement of 100 times higher EDTA concentrations to inactivate IslA with respect to the smallest truncated form. CONCLUSION: The C-terminal domain may have been acquired to anchor inulosucrase to the cell surface. Furthermore, the acquired domains in IslA interact with the catalytic core resulting in a new conformation that renders the enzyme more stable and switch the specificity from a hydrolytic to a transglycosylase mechanism. Based on these results, chimeric constructions may become a strategy to stabilize and modulate biocatalysts based on FTF activity.