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Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction ( E *) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol -1 respectively). Half-life decreased progressively with increasing temperature, and longer half-life was observed for PEG-crisantaspase (87.74 min) at 50 °C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol -1 ) was higher than for crisantaspase (218.1 kJ mol -1 ) and Glyco-crisantaspase (120.0 kJ mol -1 ), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋 𝑚𝑎𝑥 ) of 2.0 g L -1 was reached after 5 h of cultivation, corresponding to 𝑃 X  = 0.4 g L -1 h. The µ 𝑚𝑎𝑥 corresponded to 1.0 h -1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L -1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL -1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase k M of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products., (© 2024 Wiley-VCH GmbH.)

Phage display links the phenotype of displayed polypeptides with the DNA sequence in the phage genome and offers a universal method for the discovery of proteins with novel properties. However, the display of large multisubunit proteins on phages remains a challenge. A majority of protein display systems are based on monovalent phagemid constructs, but methods for the robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼ 200 kDa tetrameric l-asparaginase protein on M13 and fd phages produced by ligation of SpyCatcher-Asparaginase fusion (ScA) and PEGylated-ScA (PEG-ScA) to barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 or p8 coat proteins yielded constructs with five copies of ScA displayed on p3 (ScA-p3), ∼100 copies of ScA on p8 protein (ScA-p8) and ∼300 copies of PEG-ScA on p8 protein (PEG-ScA-p8). Display constructs of different valencies and chemical modifications on protein (e.g., PEGylation) can be injected into mice and analyzed by deep sequencing of the DNA barcodes associated with phage clones. In these multiplexed studies, we observed a density and protein-dependent clearance rate in vivo . Our observations link the absence of PEGylation and increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo . In conclusion, we demonstrate that a multivalent display of l-asparaginase on phages could be used to study the circulation life of this protein in vivo , and such an approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multisubunit proteins in vivo .

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L -1  H -1 , respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg -1 , 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a K M value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL., (© 2018 International Union of Biochemistry and Molecular Biology, Inc.)

L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUT s under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 g dcw .L -1 during glycerol batch phase, corresponding to a biomass yield of 0.77 g dcw. g glycerol - 1 and a specific growth rate of 0.21 h -1 . After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 g dcw. L -1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 g dcw. L -1 , periplasmic specific enzyme activity 37.1 U.g dcw - 1 , volumetric enzyme activity 3,315 U.L -1 , overall enzyme volumetric productivity 31 U.L -1 .h -1 , while the specific growth rate fell to 0.039 h -1 . Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.