Monomeric Enzyme Research Articles

CK2 expression in cancer and associated clinical characteristics.

e15156 Background: The protein casein kinase 2 (CK2) is a ubiquitously expressed, highly pleiotropic kinase. CK2 may have incredibly varied substrate specificity and catalytic activity, as it functions as both a tetrameric and monomeric enzyme. CK2 participates in many cellular processes such as cellular proliferation, growth, and survival, and has significant potential to assist in oncogenic signaling. We and others have demonstrated CK2 gene and protein overexpression, and sometimes underexpression, in a variety of cancers. Here we investigate how the expression of CK2 subunits in cancer is associated with clinical characteristics in several distinct cancer types. Methods: Data analysis was performed on 32 non-overlapping cancer datasets obtained from The Cancer Genome Atlas (TCGA) and Broad GDAC Firehose Legacy projects, one dataset from Cancer Cell Line Encyclopedia (CCLE), and normal tissue datasets from Gene Expression Omnibus. Statistical analysis was performed using RStudio to explore the relationship between the expression levels of the three CK2 alpha transcripts (CSNK2A1, CSNK2A2, and CSNK2A3) and beta transcripts (CSNK2B) with sample clinical characteristics provided by TCGA/Broad. Relationships between CK2 expression and cancer-type specific characteristics were also explored, such as KRAS mutations in colorectal adenocarcinoma and serum AFP at sample procurement in hepatocellular carcinoma. CCLE data was used to explore correlations between cancer-type dependent CK2 gene expression and half maximal inhibitory concentration (IC50) of over 250 anticancer drugs. Tumor Immune Estimation Resource (TIMER) 2.0 was used to assess the relationship between CK2 subunit expression and tumor immune infiltration. Results: As seen in our previous analysis, CK2 transcripts were upregulated in the majority of cancer types analyzed. We also found that CK2 transcript upregulation correlated with worse survival outcomes in several previously unstudied cancers. CK2 expression differed significantly between cancer subtypes such as in ductal carcinoma and lobular carcinoma in breast cancer. CK2 expression significantly correlated with tumor grade, TNM staging, clinical staging in certain cancer types. CK2 expression significantly correlated with altered response to various anticancer drugs in certain cancers. CK2 expression was associated with both increased and decreased immune infiltration in a variety of cancers. Conclusions: Overall, this study demonstrates that CK2 transcript expression correlates with a variety of cancer-type dependent characteristics and clinical outcomes. As a result, CK2 may be useful as a prognostic biomarker and therapeutic target.

Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy.

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/β-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p-nitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅1). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅2). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.

Stabilization of galactose oxidase by high hydrostatic pressure: Insights on the role of cavities size.

High hydrostatic pressure stabilized galactose oxidase (GaOx) at 70.0-80.0°C against thermal inactivation. The pseudo-first-order rate constant of inactivation kinact decreased by a factor of 8 at 80°C and by a factor of 44 at 72.5°C. The most pronounced effect of pressure was at the lowest studied temperature of 70.0°C with an activation volume of inactivation ΔV‡ of 78.8 cm3 mol-1. The optimal pressure against thermal inactivation was between 200 and 300 MPa. Unlike other enzymes, as temperature increased the ΔV‡ of inactivation decreased, and as pressure increased the activation energy of inactivation Eai increased. Combining the results for GaOx with earlier research on the pressure-induced stabilization of other enzymes suggests that ΔV‡ of inactivation correlates with the total molar volume of cavities larger than ~100 Å3 in enzyme monomers for enzymes near the optimal pH and whose thermal unfolding is not accompanied by oligomer dissociation.

Abatement of the binding of human hexokinase II enzyme monomers by in-silico method with the design of inhibitory peptides.

The online version contains supplementary material available at 10.1007/s40203-024-00201-8.

A novel endo-1,4-β-xylanase from Alicyclobacillus mali FL18: Biochemical characterization and its synergistic action with β-xylosidase in hemicellulose deconstruction

A novel endo-1,4-β-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (kcat/KM = 91.89 s−1mLmg−1). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized β-xylosidase AmβXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmβXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmβXyl among the most promising biocatalysts for the saccharification of agricultural waste.

ALDOSE REDUCTASE ENZYME'S CLINICAL AND THERMODYNAMIC CHARACTERISTICS IN THE SERUM OF IRAQI DIABETIC PATIENTS

A superfamily of aldo-keto reductases includes the monomeric NADPH-dependent cytosolic enzyme aldose reductase (AR). When hyperglycemia, increased glucose levels stimulate the AR, which activates the polyol pathway and results in glucose metabolism, the dangerous aldehydes created by reactive oxygen species (ROS) into innocuous alcohols. NADPH is a crucial cofactor in the formation of glutathione (GSH), a crucial antioxidant, and its consumption lowers GSH levels. The main causes problems of diabetes are osmotic stress brought on by the buildup of excess AR and oxidative stress brought on by a decline in the NADPH/NADP+ ratio and decreased NAD+. The aim of this work is to measure the AR enzyme's activity in healthy and diabetes patients. A spectrophotometric was used to measure the serum (AR) activity in 120 type II diabetes patients and 60 healthy individuals. Increase in serum (AR) is related to the body's internalized oxygen free radicals, which cause oxidative damage to human tissues. The thermodynamic constants of activation, ΔH, Ea, and ΔS, were calculated using the Arrhenius plot in order to reconcile our findings with the thermodynamic behavior of (AR) enzyme's. These values were found to be (10.55, 19.93, and 2.85) KJ/mol, respectively. Due to its capacity to change NADPH into NADH, the active polyol pathway in diabetes mellitus significantly contributes to the NADH/NAD+ redox imbalance. Lack of NADPH can cause oxidative stress via limiting glutathione metabolism, the excess NADH's ability to cause it generating ROS through the mitochondrial electron transport chain.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide‐Activated Sirtuin 7**

AbstractThe sirtuins are NAD+‐dependent lysine deacylases, comprising seven isoforms (SIRT1–7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ‐N‐acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small‐angle X‐ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism‐based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA‐display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Production of enzymes for the removal of odorous substances in plant biomass

Residual plant biomass collected from agricultural, technical or biopharmaceutical processes contains odorous substances. The latter are often unacceptable for customers if the biomass is used in sustainable products such as building materials, paints, glues or flame-resistant foils. The objective of this study was to identify enzymes that can prevent the formation or facilitate the degradation of odorous substances such as butanol, eugenol or ethyl acetate and their derivatives in residual biomass. We used plant cell packs (PCPs) as a small-scale screening platform to assess the expression of enzymes that break down odorous substances in tobacco biomass. First, we compiled a list of volatile compounds in residual plant biomass that may give rise to undesirable odors, refining the list to 10 diverse compounds representing a range of odors. We then selected five monomeric enzymes (a eugenol oxidase, laccase, oxidase, alkane mono-oxidase and ethyl acetate hydrolase) with the potential to degrade these substances. We transiently expressed the proteins in PCPs, targeting different subcellular compartments to identify optimal production conditions. The maximum yield we achieved was ∼20 mg kg−1 for Trametes hirsute laccase targeted to the chloroplast. Our results confirm that enzymes for the removal of odorous substances can be produced in plant systems, facilitating the upcycling of residual biomass as an ingredient for sustainable products.

Role and structure of the small subunit forming heterodimers with laccase-like enzymes.

Unlike laccases sensu stricto, which are usually monomeric enzymes, laccase-like enzymes recently re-classified as Novel Laccases (NLACs) are characterized by the formation of heterodimers with small proteins (subunits) of unknown function. Here the NLAC from Pleurotus eryngii (PeNL) and a small protein selected from the fungal genome, that is homologous to reported POXA3 from Pleurotus ostreatus, were produced in Aspergillus oryzae separately or together. The two proteins interacted regardless of whether the small subunit was co-expressed or exogenously added to the enzyme. The stability and catalytic activity of PeNL was significantly enhanced in the presence of the small subunit. SEC-MALS analysis confirmed that the complex PeNL-ss is a heterodimer of 77.4 kDa. The crystallographic structure of the small protein expressed in Escherichia coli was solved at 1.6 Å resolution. This is the first structure elucidated of a small subunit of a NLAC. The helix bundle structure of the small subunit accommodates well with the enzyme model structure, including interactions with specific regions of NLACs and some amino acid residues of the substrate-binding loops. This article is protected by copyright. All rights reserved.

Abstract P1060: Correlation Of Plasma Glutathione Peroxidase 4 Concentration With Coronary Heart Disease And The Severity Of Coronary Artery Stenosis

Introduction: Selenium-dependent glutathione peroxidase (GPX4) is an antioxidative, monomeric enzyme that can protect cells from membrane lipid oxidation-linked damage. Given the potential influence of GPX4 concentration on cardiovascular risk, we investigated how GPX4 levels change in patients with coronary heart disease (CHD), and whether GPX4 concentration correlates with the severity of coronary artery stenosis (CAS). Methods: Patients admitted at our institution between August 2022 and January 2023 for suspected CHD and completed coronary angiography were included. Patients were stratified according to their coronary angiography results: CHD group (positive) and control group (negative). The severity of CAS in patients with CHD was evaluated by dividing patients into low (<43 points) or high (≥43 points) Gensini score groups. Plasma GPX4 concentration was measured by enzyme-linked immunosorbent assay (ELISA). Results: Eighty-one patients were included based on our eligibility criteria, 41 patients in the CHD group and 40 patients in the control group. Patients with CHD were more commonly male and hypertensive, and presented with significantly greater levels of triglycerides and LDL-cholesterol at baseline. ELISA analysis indicates that patients with CHD have significantly lower plasma GPX4 concentrations as compared to patients in the control group. Furthermore, Spearman’s correlation analysis shows an inverse association between plasma GPX4 concentration and Gensini score in patients with CHD. Conclusion: Our results suggest that plasma GPX4 concentration was significantly lower in patients with CHD and is negatively correlated with the severity of CAS.

In silico study of novel alpha tocopheroids as effective inhibitors of aldo-keto reductase 1c3 (AKR1C3) enzyme

Aldo-keto reductase 1C3 (AKR1C3) is a monomeric enzyme expressed in steroidogenic tissues such as the testis, prostate, uterus, and breast. Overexpression of this AKR1C3 is associated with vast cancers such as breast, colon, colorectal, endometrial, prostate, and acute myeloid leukaemia. Regarding the treatment of castration-resistant prostate cancer, breast cancer, and acute myeloid leukaemia, AKR1C3 inhibitors may offer clear advantages over currently available therapies. Thus, discovering novel and specific AKR1C3 inhibitors is a promising way to obstruct drug resistance in cancer. Derivatives of alpha-tocopherol and alpha-tocopheroids were selected as possible therapeutics to act as AKR1C3 inhibitors. The precise targets of several ligands were determined using computational screening methods. The molecular structure of AKR1C3 and its ligands were used as the foundation for in silico predictions, modelling, and dynamic simulations. Compounds were selected based on their biological properties and filtered according to their ADMET and drug-likeness properties. Additionally, simulations of all-atom molecular dynamics on AKR1C3 with the cleared compounds revealed stability over the simulated trajectories of 100 ns. When seen collectively, alpha-tocospiro A may be considered prospective AKR1C3 inhibitors for creating anticancer therapies. Communicated by Ramaswamy H. Sarma

Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD+/NADH redox method

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors.

Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase.

Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme's conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme's function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK's conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme's conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme's active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

Production and application of purified mutanase from novel Cellulosimicrobium funkei SNG1 in the in vitro biofilm degradation.

Mutanase (α-1,3-glucanase) is an inducible extracellular enzyme with potential medical applications in dentistry. A novel Cellulosimicrobium funkei strain SNG1 producing mutanase enzyme using α-1,3-glucans was isolated, and the enzyme was optimized for increased productivity using the one-factor-at-a-time approach. Maximum growth and enzyme-specific activity (2.12 ± 0.4U/mg) were attained in a production medium with pH 7.0 and 1% α-1,3-glucans as carbon source, incubated at 37°C for 30h. The result showed a five-fold increase in activity compared to unoptimized conditions (0.40U/mg). The enzyme was purified by gel-filtration chromatography, and recovered with a yield of 29.03% and a specific activity increase of 10.9-fold. The molecular mass of the monomeric enzyme is 137kDa. The pH and temperature optima are 6.0 and 45°C with Km of 1.28 ± 0.11mg for α-1,3-glucans. The enzyme activity was stimulated by adding Co2+ , Ca2+ , Cu2+ , and was entirely inhibited by Hg2+ . On 2-h incubation, the purified enzyme effectively degraded in vitro film with an 82.68% degradation rate and a saccharification yield of 30%.

Enhancing the activity of a monomeric alcohol dehydrogenase for site-specific applications by site-directed mutagenesis.

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications. In this study, we engineered one of the rare examples of monomeric alcohol dehydrogenases for improved catalytic characteristics by site-directed mutagenesis. The enzyme from the hyperthermophilic archaeon Thermococcus kodakarensis naturally exhibits high thermostability and a broad substrate spectrum, but only low activity at moderate temperatures. The best enzyme variants showed an ~5-fold (2-heptanol) and 9-fold (3-heptanol) higher activity while preserving enantioselectivity and good thermodynamic stability. These variants also exhibited modified kinetic characteristics regarding regioselectivity, pH dependence and activation by NaCl.

Mycobacterial helicase Lhr abets resistance to DNA crosslinking agents mitomycin C and cisplatin.

Mycobacterium smegmatis Lhr exemplifies a novel clade of helicases composed of an N-terminal ATPase/helicase domain (Lhr-Core) and a large C-terminal domain (Lhr-CTD) that nucleates a unique homo-tetrameric quaternary structure. Expression of Lhr, and its operonic neighbor Nei2, is induced in mycobacteria exposed to mitomycin C (MMC). Here we report that lhr deletion sensitizes M. smegmatis to killing by DNA crosslinkers MMC and cisplatin but not to killing by monoadduct-forming alkylating agent methyl methanesulfonate or UV irradiation. Testing complementation of MMC and cisplatin sensitivity by expression of Lhr mutants in Δlhr cells established that: (i) Lhr-CTD is essential for DNA repair activity, such that Lhr-Core does not suffice; (ii) ATPase-defective mutant D170A/E171A fails to complement; (iii) ATPase-active, helicase-defective mutant W597A fails to complementand (iv) alanine mutations at the CTD-CTD interface that interdict homo-tetramer formation result in failure to complement. Our results instate Lhr's ATP-driven motor as an agent of inter-strand crosslink repair in vivo, contingent on Lhr's tetrameric quaternary structure. We characterize M. smegmatis Nei2 as a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Counter to previous reports, we find Nei2 is inactive as a lyase at a THF abasic site and has feeble uracil glycosylase activity.

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Detection of macroenzymes: establishing upper reference limits for eight enzymes after polyethylene glycol precipitation.

The presence of macroenzymes in blood can cause diagnostic confusion. Therefore, confirming the presence of macroenzymes is important to reduce unnecessary (non-)invasive investigations. Polyethylene glycol (PEG) precipitation is a simple and fast first-line method for the detection of macroenzymes. However, there is no consensus on the upper reference limit for the PEG-precipitable activity (%PPA) of monomeric enzymes. The aim of this study was to verify a PEG precipitation protocol for the detection of macroenzymes in our laboratory by establishing upper reference limits (URLs) and determining imprecision for eight enzymes after PEG precipitation. In addition, we aimed to clinically verify the URLs using samples containing macroenzymes as identified by electrophoresis. Per enzyme, at least 40 leftover blood samples from adult patients with either normal or increased enzyme activities were diluted 1:1 with 25% PEG 6000 and 1:1 with 0.9% NaCl. Mixtures were incubated for 10 min at 37°C and centrifuged. Supernatant enzyme activity was measured on Cobas c702 and the %PPA was calculated. The following URLs were obtained: 26% PPA for amylase, 29% PPA for alkaline phosphatase (ALP), 61% PPA for alanine aminotransferase, 48% PPA for aspartate aminotransferase, 24% PPA for creatine kinase (CK), 55% PPA for gamma-glutamyltransferase, 65% PPA for lactate dehydrogenase, and 56% PPA for lipase. The within-lab imprecision was < 15%. Regarding the clinical verification, the two historical samples with proven macroCK showed a %PPA of 69% and 43%, respectively, and a sample with proven macroALP had a %PPA of 52%. In this study, URLs for monomeric enzyme activities after PEG precipitation for eight different enzymes were established. The URLs are suitable for clinical use, but are only partially in line with other studies. Therefore, our data highlight the importance of establishing laboratory-specific upper reference limits for %PPA to allow a correct interpretation.