PEGylation increases antitumoral activity of arginine deiminase of Streptococcus pyogenes

Arginine auxotrophy is a metabolic defect that renders tumor cells vulnerable towards arginine-depleting substances, such as arginine deiminase (ADI) from Streptococcus pyogenes (SpyADI). Previously, we confirmed SpyADI susceptibility on patient-derived glioblastoma multiforme (GBM) models in vitro and in vivo. For application in patients, serum half-life of the enzyme has to be increased and immunogenicity needs to be reduced. For this purpose, we conjugated the S. pyogenes-derived SpyADI with 20 kDa polyethylene glycol (PEG20) moieties, achieving a PEGylation of seven to eight of the 26 accessible primary amines of the SpyADI. The PEGylation reduced the overall activity of the enzyme by about 50% without affecting the Michaelis constant for arginine. PEGylation did not increase serum stability of SpyADI in vitro, but led to a longer-lasting reduction of plasma arginine levels in mice. Furthermore, SpyADI-PEG20 showed a higher antitumoral capacity towards GBM cells in vitro than the native enzyme. Key points • PEGylation has no effect on the affinity of SpyADI for arginine • PEGylation increases the antitumoral effects of SpyADI on GBM in vitro • PEGylation prolongs plasma arginine depletion by SpyADI in mice

Approximations for the Concentration and Effectiveness Factor in Porous Catalysts of Arbitrary Shape: Taylor Series and Akbari-Ganji’s Methods

A mathematical model of reaction-diffusion problem with Michaelis-Menten kinetics in catalyst particles of arbitrary shape is investigated. Analytical expressions of the concentration of substrates are derived as functions of the Thiele modulus, the modified Sherwood number, and the Michaelis constant. A Taylor series approach and the Akbari-Ganji's method are utilized to determine the substrate concentration and the effectiveness factor. The effects of the shape factor on the concentration profiles and the effectiveness factor are discussed. In addition to their simple implementations, the proposed analytical approaches are reliable and highly accurate, as it will be shown when compared with numerical simulations.

A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analysed. Thus, it is possible that the correlations observed were an artefact of the presence of phylogenetic signal in rubisco kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically-resolved analysis of rubisco kinetics and show that there is a significant phylogenetic signal in rubisco kinetic traits. We re-evaluated the extent of catalytic trade-offs accounting for this phylogenetic signal and found that all were attenuated. Following phylogenetic correction, the largest catalytic trade-offs were observed between the Michaelis constant for CO2 and carboxylase turnover (∼21-37%), and between the Michaelis constants for CO2 and O2 (∼9-19%), respectively. All other catalytic trade-offs were substantially attenuated such that they were marginal (<9%) or non-significant. This phylogenetically resolved analysis of rubisco kinetic evolution also identified kinetic changes that occur concomitant with the evolution of C4 photosynthesis. Finally, we show that phylogenetic constraints have played a larger role than catalytic trade-offs in limiting the evolution of rubisco kinetics. Thus, although there is strong evidence for some catalytic trade-offs, rubisco adaptation has been more limited by phylogenetic constraint than by the combined action of all such trade-offs.

In-silico development of a method for the selection of optimal enzymes using L-asparaginase II against Acute Lymphoblastic Leukemia as an example

ABSTRACTL-Asparaginase II (asnB), a periplasmic protein, commercially extracted from E. coli and Erwinia, is often used to treat Acute Lymphoblastic Leukemia. L-Asparaginase is an enzyme that converts L-asparagine to aspartic acid and ammonia. Cancer cells are dependent on asparagine from other sources for growth and when these cells are deprived of asparagine by the action of the enzyme the cancer cells selectively die. Questions remain as to whether asnB from E. coli and Erwinia is the best asparaginase as they have many side-effects. asnB with the lowest Michaelis constant (Km) (most potent), and with the lowest immunogenicity is considered the most optimal enzyme. In this paper asnB sequence of E. coli was used to search for homologous proteins in different bacterial and archaeal phyla and a maximum likelihood phylogenetic tree was constructed. The sequences that are most distant from E. coli and Erwinia were considered best candidates in terms of immunogenicity and were chosen for further processing. The structures of these proteins were built by homology modeling and asparagine was docked with these proteins to calculate the binding energy. asnBs from Streptomyces griseus, Streptomyces venezuelae and Streptomyces collinus were found to have the highest binding energy i.e. −5.3 kcal/mol, −5.2 kcal/mol, and −5.3 kcal/mol respectively (Higher than the E.coli and Erwinia asnBs) and were predicted to have the lowest Kms as we found that there is an inverse relationship between binding energy and Km. Besides predicting the most optimal asparaginase, this technique can also be used to predict the most optimal enzymes where the substrate is known and the structure of one of the homologs is solved.

RuBisCO adaptation is more limited by phylogenetic constraint than by catalytic trade-off in plants

RuBisCO assimilates CO2 to form the sugars that fuel life on earth. Correlations between RuBisCO kinetic traits across species have led to the proposition that RuBisCO adaptation is constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analysed. Thus, it is possible that the observed correlations between RuBisCO kinetic traits are an artefact of the presence of phylogenetic signal in RuBisCO kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically resolved analysis of RuBisCO kinetics and show that there is significant phylogenetic signal in all carboxylase kinetic traits, and significant phylogenetic signal in the Michaelis constant for O2 in species that conduct C3 photosynthesis. When accounting for this phylogenetic non-independence between enzymes, we show that the catalytic trade-off between carboxylase turnover and the Michaelis constant for CO2 is weak (~30 % dependency) and that the correlations between all other RuBisCO kinetic traits are either not-significant or marginal (<9 % dependency). Finally, we demonstrate that phylogenetic constraints have limited RuBisCO evolution to a greater extent than catalytic trade-offs. Thus, RuBisCO adaptation in angiosperms is predominantly limited by phylogenetic constraint (most likely caused by a slow rate of molecular evolution) and a partial trade-off between carboxylase turnover and the Michaelis constant for CO2.

Potential Degradation and Kinetics of Melanoidin by Using Laccase from White Rot Fungus

This study was attempted to use laccase extracted from white rot fungus to remove melanoidin in the ethanol production wastewater. The isolated fungus producing the highest laccase was identified as Megaspororia sp. The highest degradation efficiencies of the purified and crude laccases were 48.00% and 44.60%, respectively. Both degradation kinetics well fit Michaelis-Menten model. The Michaelis constant (Km) and maximum rate of reaction (Vmax) were 0.82% melanoidin and 0.0045% melanoidin h-1 for the degradation by the purified laccase and 0.71% melanoidin and 0.0037% melanoidin h-1 for the degradation by the crude laccase. Turnover number (Kcat) of purified and crude laccases were 0.00023 and 0.00019% melanoidin U-1 h-1, respectively. Catalytic efficiency (Kcat/Km) of purified and crude laccases were 0.00028 and 0.00027 U-1 h-1, respectively. The affinity of the crude laccase was slightly higher because of its non-specificity. Kcat and Kcat/Km of the purified laccase were higher than the crude laccase. Proposed potential degradation result showed that laccase could oxidize CH3, carbonyl groups, haloalkanes (C–H), C–O and C–N bondings which probably caused decolorization of melanoidin in wastewater. Thus, the purified and crude laccases can be used to decolorize melanoidin-containing wastewater from ethanol industries. As the attempt to use purified laccase consumed times and costs especially in purification steps, the crude laccase can be used to degrade color of melanoidin in wastewater with only 3.4% lower than the purified laccase.

Xylanase SMXL1 from Stenocarpella maydis: Purification and biochemical characterization

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5  0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (Km) value was 2.61 mg/mL and the Vmax was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn2+ and by Fe3+ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.