hydrolytic activity
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2021 ◽  
Vol 23 (1) ◽  
pp. 264
Author(s):  
Valentina Pirillo ◽  
Marco Orlando ◽  
Davide Tessaro ◽  
Loredano Pollegioni ◽  
Gianluca Molla

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL−1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL−1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL−1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.


Crystals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 18
Author(s):  
Sergio Martinez-Rodríguez ◽  
Rafael Contreras-Montoya ◽  
Jesús M. Torres ◽  
Luis Álvarez de Cienfuegos ◽  
Jose Antonio Gavira

L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 °C, with an apparent thermal melting temperature of 46 °C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity.


Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1891
Author(s):  
Nikita K. Lapshin ◽  
Michail S. Piotrovskii ◽  
Marina S. Trofimova

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.


2021 ◽  
Vol 22 (23) ◽  
pp. 13123
Author(s):  
Anastasia D. Teplova ◽  
Marina V. Serebryakova ◽  
Raisa A. Galiullina ◽  
Nina V. Chichkova ◽  
Andrey B. Vartapetian

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.


2021 ◽  
pp. 1-2
Author(s):  
Vincent H. Tam ◽  
Cole S. Hudson ◽  
Paul R. Merlau ◽  
Ryan K. Shields

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259181
Author(s):  
Andrea Olga Papadopoulos ◽  
Christopher Ealand ◽  
Bhavna Gowan Gordhan ◽  
Michael VanNieuwenhze ◽  
Bavesh Davandra Kana

Mycobacterium tuberculosis, the causative agent of tuberculosis remains a global health concern, further compounded by the high rates of HIV-TB co-infection and emergence of multi- and extensive drug resistant TB, all of which have hampered efforts to eradicate this disease. As a result, novel anti-tubercular interventions are urgently required, with the peptidoglycan component of the M. tuberculosis cell wall emerging as an attractive drug target. Peptidoglycan M23 endopeptidases can function as active cell wall hydrolases or degenerate activators of hydrolases in a variety of bacteria, contributing to important processes such as bacterial growth, division and virulence. Herein, we investigate the function of the Rv0950-encoded putative M23 endopeptidase in M. tuberculosis. In silico analysis revealed that this protein is conserved in mycobacteria, with a zinc-binding catalytic site predictive of hydrolytic activity. Transcript analysis indicated that expression of Rv0950c was elevated during lag and log phases of growth and reduced in stationary phase. Deletion of Rv0950c yielded no defects in growth, colony morphology, antibiotic susceptibility or intracellular survival but caused a reduction in cell length. Staining with a monopeptide-derived fluorescent D-amino acid, which spatially reports on sites of active PG biosynthesis or repair, revealed an overall reduction in uptake of the probe in ΔRv0950c. When stained with a dipeptide probe in the presence of cell wall damaging agents, the ΔRv0950c mutant displayed reduced sidewall labelling. As bacterial peptidoglycan metabolism is important for survival and pathogenesis, the role of Rv0950c and other putative M23 endopeptidases in M. tuberculosis should be explored further.


Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1657
Author(s):  
Luisa Ugolini ◽  
Giovanni Cilia ◽  
Eleonora Pagnotta ◽  
Lorena Malaguti ◽  
Vittorio Capano ◽  
...  

The microsporidian fungus Nosema ceranae represents one of the primary bee infection threats worldwide and the antibiotic fumagillin is the only registered product for nosemosis disease control, while few alternatives are, at present, available. Natural bioactive compounds deriving from the glucosinolate–myrosinase system (GSL–MYR) in Brassicaceae plants, mainly isothiocyanates (ITCs), are known for their antimicrobial activity against numerous pathogens and for their health-protective effects in humans. This work explored the use of Brassica nigra and Eruca sativa defatted seed meal (DSM) GSL-containing diets against natural Nosema infection in Apis mellifera colonies. DSM patties from each plant species were obtained by adding DSMs to sugar candy at the concentration of 4% (w/w). The feeding was administered in May to mildly N. ceranae-infected honey bee colonies for four weeks at the dose of 250 g/week. In the treated groups, no significant effects on colony development and bee mortality were observed compared to the negative controls. The N. ceranae abundance showed a slight but significant decrease. Furthermore, the GSL metabolism in bees was investigated, and MYR hydrolytic activity was qualitatively searched in isolated bee midgut and hindgut. Interestingly, MYR activity was detected both in the bees fed DSMs and in the control group where the bees did not receive DSMs. In parallel, ITCs were found in gut tissues from the bees treated with DSMs, corroborating the presence of a MYR-like enzyme capable of hydrolyzing ingested GSLs. On the other hand, GSLs and other GSL hydrolysis products other than ITCs, such as nitriles, were found in honey produced by the treated bees, potentially increasing the health value of the final product for human consumption. The results are indicative of a specific effect on the N. ceranae infection in managed honey bee colonies depending on the GSL activation within the target organ.


2021 ◽  
Author(s):  
Bhargava Nemmaru ◽  
Jenna Douglass ◽  
John M Yarbrough ◽  
Antonio De Chellis ◽  
Srivatsan Shankar ◽  
...  

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ~330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increase hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.


Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2427
Author(s):  
Boyu Tian ◽  
Chenxia Zhou ◽  
Dongxiao Li ◽  
Jiawei Pei ◽  
Ailiang Guo ◽  
...  

This study investigated the effects of a hemicellulase dosage (20, 40, and 60 mg kg−1 of flour) on the bread quality and rheological properties of wheat aleurone-rich flour. The results showed that hemicellulase could soften dough and improve extensibility. At the optimum hemicellulase dosage (40 mg kg−1 of flour), the bread specific volume increased by 40.91% and firmness of breadcrumb decreased by 104.57% compared to those of the control. Intermolecular forces indicated that the gluten network during the proofing was mainly strengthened via disulfide bonds, hydrophobic interactions, and hydrogen bonds but not through ionic bonds after hemicellulase addition. Fourier infrared spectroscopy indicated that the hydrolytic activity of hemicellulase catalyzed the transition from α-helix to β-sheet, which verified that viscoelasticity of gluten was enhanced at a dosage of 40 mg kg−1 of flour. These results suggested that hydrolyzation of hemicellulase contributed to the structural of gluten changes, thereby improving the quality of wheat aleurone-rich bread.


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