Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding different oxygen species, and (iv) glycosidic bond cleavage. However, complete is poorly understood may vary across families even within same family. Here, we have investigated protonation state a secondary co-ordination sphere histidine, conserved AA9 family LPMOs that has previously been proposed to be...

Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear site. This site is defined by an N-terminal histidine second internal side chain in configuration called the brace. To understand better determinants of reactivity, biochemical structural properties well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) compared with that Pseudomonas fluorescens (PfCopC) LPMO-like Bim1 Cryptococcus neoformans. PfCopC not reduced...

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that act in synergy with glycoside hydrolases to saccharify the most abundant polysaccharides nature. Both O2 and H2O2 can be cosubstrates for LPMOs. The Lentinus similis LPMO (LsAA9A) has previously been shown oxidatively cleave oligosaccharides when supplied ascorbate as a reductant. This study demonstrates LsAA9A is unable complete catalytic cycle cellulose without H2O2. Instead, cellooligomers efficiently prevent...

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading substrates oxidatively. generally accepted first step in the LPMO reaction is reduction active-site metal ion from Cu 2+ to + . Here we have used a systematic diffraction data collection method monitor structural changes two AA9 LPMOs, one Lentinus similis ( Ls AA9_A) and Thermoascus aurantiacus Ta AA9_A), as photoreduced X-ray beam. For AA9_A, protein produced different...

Abstract Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus , enable one-pot exponential amplification known polymerase chain reaction (PCR). However, properties other than thermostability - fidelity, processivity, and compatibility with modified nucleotides are important in contemporary molecular biology applications. Here, we describe engineering characterization of a fusion between identified marine archaea Nanoarchaeum equitans binding...

Environmentally friendly sources of energy and chemicals are essential constituents a sustainable society. An important step toward this goal is the utilization biomass to supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) enzymes that play critical role in breaking chemical bonds most abundant polymers found recalcitrant biomass, such as cellulose chitin. To use them industrial processes they need be produced high titers cell factories. Predicting...

The histidine brace (His‐brace) is a copper‐binding motif that associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical structural methods to characterize mutants of His‐brace‐containing chaperone from Pseudomonas fluorescens (PfCopC). A total 15 amino acid variants in primary second‐sphere residues were produced characterized terms their binding redox properties. PfCopC has very high affinity for Cu(II) also binds Cu(I). reorganization barrier...

Abstract Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation filamentous fungi to protect from auto-oxidative inactivation, however, the responsible methyltransferase enzyme unknown. Using mass-spectrometry-based quantitative proteomics...

The market for recombinant proteins is on the rise, and Gram-positive strains are widely exploited this purpose. Bacillus subtilis a profitable host protein production thanks to its ability secrete large amounts of proteins, Lactococcus lactis an attractive organism with long history in food fermentation. We have developed synbio approach increasing gene expression two bacteria. First all, interest was coupled antibiotic resistance create growth-based selection system. then randomised...

Abstract This study presents a comparative analysis of three LysC homologues from Achromobacter lyticus, Pseudomonas aeruginosa, and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing protein aggregation capture (PAC) workflow with HeLa cell lysate, we assessed the enzymes’ cleavage specificity, digestion efficiency, performance across various experimental conditions. Results showed that while all exhbihited high specificity at lysine residues, A. lyticus outperformed...

Abstract Environmentally friendly sources of energy and chemicals are essential constituents a sustainable society. An important step towards this goal is the utilization non-edible biomass as supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) enzymes that play critical role in breaking chemical bonds most abundant polymers found recalcitrant biomass, such cellulose chitin. Predicting optimal strategies producing LPMOs often non-trivial, methods...

E. coli is a gram-negative bacteria used mainly in academia and some industrial scenarios, as protein production workhorse. This due to its ease of manipulation the range genetic tools available. protocol describes how express proteins periplasm with strain BL21 (DE3) using T7 expression system. Specifically, it series steps tips "hard-to-express" coli, for instance, LPMOs. The adapted from Hemsworth, G. R., Henrissat, B., Davies, J., Walton, P. H. (2014) Discovery characterization new...

The methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) is the most commonly used species in production of recombinant proteins. This likely due to its ability grow high cell density, express genes a tightly controlled manner and efficiently secrete Despite biotechnological importance wide use industry, relatively few genetic tools are readily available for academic research. Here, we present protocol proteins K. phaffii. found here modified from provided Expression Kit...

This protocol describes the expression and extraction of heterologously cytoplasmically expressed proteins in bacterium E.coli, for T7 based systems. Specifically, it how to produce "hard-to-produce", as example, LPMOs. It is on a modified version of: "IPTG Induction Extraction Proteins from Bacteria" by Swathi Arur Sudhir Nayak, Schedl Lab. Washington University Genetics, St. Louis. Hemsworth, G. R., Henrissat, B., Davies, J., Walton, P. H. (2014) Discovery characterization new family lytic...

Abstract Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus , enable one-pot exponential amplification known polymerase chain reaction (PCR). However, other properties than thermostability - fidelity, processivity, and compatibility with modified nucleotides are important in contemporary molecular biology applications. Here, we describe engineering characterization of a fusion between identified marine archaea Nanoarchaeum equitans binding...

ABSTRACT Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in biotechnological purposes. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation filamentous fungi to protect from auto-oxidative inactivation, however, the responsible methyltransferase enzyme unknown. Using mass-spectrometry-based quantitative proteomics...

B. subtilis is a gram-positive bacteria used by both academia and industry as protein production workhorse. This due to its' excellent fermentation properties, high titers, capacity secrete proteins into the extracellular medium. protocol describes transformation of natural competence. The method utilizes stress-induced competence take up heterologous DNA. requires cells be grown for specific amount time in starvation media (SM). adapted from Vojcic, L., Despotovic, D., Martinez, R., Maurer,...

B. subtilis is a gram-positive bacteria used by both academia and industry as protein production workhorse. This due to its' their excellent fermentation properties, high titers, capacity secrete proteins into the extracellular medium. protocol describes how express in subtilis. The developed using KO7-S, although it might also work for other strains well. method adapted from Rasmussen, M. D.; Bjoernvad, E.; Diers, I. Pectate Lyase Fusion Expression Secretion of Polypeptides. WO 00/75344,...

Display of proteins on the bacterial cell surface has always been an attractive technique for production functional cell-anchored proteins, thereby reducing cost, time and effort related to enzyme purification. Thus, display can enable fast easy screening protein libraries, e.g. activity variants, or different substrates, while maintaining a connection between phenotype genotype. Additionally, cells displaying enzymes potentially be used as whole-cell catalysts. This protocol describes how...

Lytic polysaccharide monooxygenses (LPMOs) are enzymes that play a critical role in breaking the chemical bonds of most abundant polymers found recalcitrant biomass, such as cellulose and chitin. LyGo cloning (Lytic Polysaccharide Monooxygenase Golden Gate cloning) is versatile heterologous expression platform for LPMOs, which compatible with both PCR products synthetic gene fragments simple 15-minute assembly step. The method allows parallel construction multiple vectors, enabling...

Abstract Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in biotechnological purposes. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation filamentous fungi to protect from auto-oxidative inactivation, however, the responsible methyltransferase enzyme unknown. Using mass-spectrometry-based quantitative proteomics...

E. coli is a gram-negative bacteria used mainly in academia and some industrial scenarios, as protein production workhorse. This due to its ease of manipulation the range genetic tools available. protocol describes how express proteins periplasm with strain BL21 (DE3) using T7 expression system. Specifically, it series steps tips "hard-to-express" coli, for instance, LPMOs. The adapted from Hemsworth, G. R., Henrissat, B., Davies, J., Walton, P. H. (2014) Discovery characterization new...