scholarly journals Fenton-type chemistry by a copper enzyme: molecular mechanism of polysaccharide oxidative cleavage

2016 ◽  
Author(s):  
Bastien Bissaro ◽  
Asmund K Rohr ◽  
Morten Skaugen ◽  
Zarah Forsberg ◽  
Svein J Horn ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Reaction Kinetics ◽  
Mode Of Action ◽  
Process Conditions ◽  
Enzyme Assays ◽  
Enzymatic Conversion ◽  
Redox Enzymes ◽  
Anaerobic Conditions ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has been instrumental for the development of economically sustainable lignocellulose biorefineries. Despite the obvious importance of these exceptionally powerful redox enzymes, their mode of action remains enigmatic and their activity and stability under process conditions are hard to control. By using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show that H2O2, and not O2 as previously thought, is the co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics and high enzymatic rates are achieved, the LPMOs work under anaerobic conditions, and the need for adding stoichiometric amounts of reductants is alleviated. These results offer completely new perspectives regarding the mode of action of these unique mono-copper enzymes, the enzymatic conversion of biomass in Nature, and industrial biorefining.

scholarly journals Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1098
Author(s):  
Federica Calderaro ◽  
Loes E. Bevers ◽  
Marco A. van den Berg
Keyword(s):  
Experimental Design ◽  
Mode Of Action ◽  
Data Interpretation ◽  
Electron Donors ◽  
Cellulolytic Enzymes ◽  
Inhibiting Factors ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Power Tools ◽  
Reliable Methods

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

scholarly journals LPMO-oxidized cellulose oligosaccharides evoke immunity in Arabidopsis conferring resistance towards necrotrophic fungus B. cinerea

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Marco Zarattini ◽  
Massimiliano Corso ◽  
Marco Antonio Kadowaki ◽  
Antonielle Monclaro ◽  
Silvia Magri ◽  
...  
Keyword(s):  
Cell Wall ◽  
Crucial Role ◽  
Pairwise Comparison ◽  
Leucine Rich Repeat ◽  
Redox Enzymes ◽  
Transcriptional Reprogramming ◽  
Specific Effects ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Plant Pathogen Interactions

AbstractLytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.

scholarly journals Exploitation of Enzymes for the Production of Biofuels: Electrochemical Determination of Kinetic Parameters of LPMOs

2021 ◽  
Vol 11 (11) ◽  
pp. 4715
Author(s):  
Dimitrios Zouraris ◽  
Anthi Karnaouri ◽  
Raphaela Xydou ◽  
Evangelos Topakas ◽  
Antonis Karantonis
Keyword(s):  
Kinetic Parameters ◽  
Filamentous Fungus ◽  
Electrode Surface ◽  
Plant Biomass ◽  
Electrochemical Determination ◽  
Redox Enzymes ◽  
Electrode Surfaces ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) consist of a class of enzymes that boost the release of oxidised products from plant biomass, in an approach that is more eco-friendly than the traditional ones, employing harsh chemicals. Since LPMOs are redox enzymes, they could possibly be exploited by immobilisation on electrode surfaces. Such an approach requires knowledge of kinetic and thermodynamic information for the interaction of the enzyme with the electrode surface. In this work, a novel methodology is applied for the determination of such parameters for an LPMO from the filamentous fungus Thermothelomyces thermophila, MtLPMO9H.

scholarly journals Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lukas Rieder ◽  
Katharina Ebner ◽  
Anton Glieder ◽  
Morten Sørlie
Keyword(s):  
Pichia Pastoris ◽  
Biomass Conversion ◽  
Single Step ◽  
Additional Advantage ◽  
Lytic Polysaccharide Monooxygenases ◽  
Expression Host ◽  
Shake Flask Cultivation ◽  
Polysaccharide Monooxygenases ◽  
Efficient Expression ◽  
High Yields

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

Abstract 657: Increased Urinary Angiotensin Converting Enzyme 2 (ACE2) In Type 2 Diabetic Patients

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Sridevi Gutta ◽  
Nadja Grobe ◽  
Hassan Osman ◽  
Mohammad Saklayen ◽  
Khalid M Elased
Keyword(s):  
Mass Spectrometry ◽  
Medical Center ◽  
Diabetic Patients ◽  
Enzyme Assays ◽  
Renal Tubules ◽  
Type 2 Diabetic Patients ◽  
Major Health ◽  
Angiotensin Converting Enzyme 2 ◽  
Noninvasive Biomarker

Diabetes and its associated chronic kidney disease (CKD) is a major health burden and there is an urgent need for new sensitive biomarkers to detect and monitor the progression of CKD. Albuminuria is still the gold standard for the evaluation of kidney function. However, its sensitivity and reliability have recently been questioned. ACE2 is highly expressed in renal tubules and has been shown to be shed in the urine of diabetic patients with CKD. The aim of the study was to investigate whether urinary ACE2 is increased in diabetic patients with CKD before the onset of microalbuminuria. Participants were recruited from Dayton VA Medical Center (Dayton, OH, USA). Baseline urinary albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) were determined three months before initiation of the study in non-diabetic patients (UACR <30 mg/g, eGFR=97.40±16 ml/min/1.73 m 2 ), and in diabetic patients with normoalbuminuria (UACR <30 mg/g, eGFR=83.08±17 ml/min/1.73 m 2 ), microalbuminuria (UACR = 30-300 mg/g, eGFR=47.13±23 ml/min/1.73 m 2 ), and macroalbuminuria (UACR >300 mg/g, eGFR=39.68±20 ml/min/1.73 m 2 ). Using fluorogenic and mass spectrometry-based enzyme assays, we measured urinary and plasma ACE2 activity in patients. Urinary ACE2 activity was significantly increased in diabetic patients with normoalbuminuria (0.58±0.2 nmol/hr/mg creatinine), microalbuminuria (1.19 ±0.5 nmol/hr/mg creatinine), and macroalbuminuria (2.265±0.4 nmol/hr/mg creatinine) compared with non-diabetic controls (0.06 ± 0.02 nmols/hr/mg creatinine) (p<0.0001). These results were confirmed by detecting the ACE2 product Ang-(1-7) ( m/z 899) in incubations of urine samples with the natural substrate Ang II ( m/z 1046) using mass spectrometry-based enzyme assays. In addition, urinary ACE2 expression was significantly increased in diabetic patients as determined by western blot analysis (p<0.05). Plasma ACE2 activity was not detectable in control and diabetic patients. In conclusion, urinary ACE2 is increased in diabetic patients with CKD which suggests that urinary ACE2 could be used as an early, noninvasive biomarker for diabetic nephropathy before the onset of microalbuminuria.

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