Enzyme Solution Research Articles

Effect of ultrasound on the activity and structure of polyphenol oxidase purified from mango (Mangifera indica cv. "Xiaotainong")

This study separated, purified, and identified mango polyphenol oxidase (PPO) in Xiaotainong mango, and the inactivation effect and possible inactivation mechanism of mango PPO by ultrasound (US) was investigated. The purified mango PPO, identified as Alfonso mango chloroplast PPO (NCBI number: XP:044462061.1), exhibited specific enzyme activity of 178,192 U/mg, with a yield of 1.2%, which was 9.85-fold higher than crude enzyme solution. Bioinformatics analysis of XP_044462061.1 indicated that the PPO consisted of a stable hydrophilic protein with a molecular weight of 66.77 kDa, an amino acid number of 593, and a structural domain similar to the TYROSINASE_1 (PS00497, tyrosinase) CuA and TYROSINASE_2 (PS00498; tyrosinase) CuB binding regions. The result showed that increased US treatment intensity and time better facilitated PPO inactivation, with a higher US intensity performing better than extended treatment time. US changed and disrupted the secondary and endogenous hydrophobic structures of the mango PPO, altered the exogenous hydrophobicity, and caused PPO molecule aggregation, ultimately leading to PPO inactivation. This article provided a theoretical basis for the application of US technology for processing mango products.

Effect of enzymatic processing on the physicochemical properties of wheat gluten protein enzymatic products

AbstractBackground and ObjectivesWheat gluten is a high‐quality, low‐priced, and nutritious plant protein. Applications of wheat gluten protein could be expanded by improving its solubility through enzymatic modifications. Herein, we investigated the effect of proteolytic processing on the physicochemical properties of wheat gluten protein.FindingsThe degree of hydrolysis, trichloroacetic acid‐soluble nitrogen (TCA‐SN), and peptidyl nitrogen content of the enzymatic digestion products increased, and the TCA‐SN content exceeded 60% in all cases. The ζ‐potentials of all products after enzymatic digestion were significantly decreased and were negative. Three enzymatic solutions increased the stability of the solution system and the surface hydrophobicity of the enzymatic products to different degrees. The products after enzymatic digestion by alkaline protease had better antioxidant capacity.ConclusionsThis study provides theoretical support for expanding the applications for wheat protein to achieve the added value of wheat products and enhance the utilization rate of these readily available raw materials.Significance and NoveltyThis study investigates the dual enzyme stepwise enzymatic hydrolysis of wheat gluten protein by alkaline protease and neutral protease, which increases the water‐soluble protein content of wheat gluten protein. The resulting hydrolysate has a high peptide nitrogen content.

Pressurized microwave-assisted hydrothermal treatment with various salts for efficient production of monosaccharides from rice straw

This study proposed a two-stage pressurized microwave hydrothermal treatment with a catalyst, followed by enzymatic saccharification, as a pretreatment method for efficiently converting cellulose and hemicellulose from rice straw into glucose and xylose. The use of various inorganic salts and dilute sulfuric acid as catalysts enhances sugar production. Using 1 wt% sulfuric acid as a catalyst at 150 °C for 5 min for the first-stage and then 180 °C for 5 min for the second-stage yielded the highest sugar production from rice straw compared with other inorganic salts tested. The filtrate and enzymatic saccharification solution contained a total sugar of 0.434 g/g-untreated rice straw (i.e. 0.302 g-glucose/g-untreated rice straw and 0.132 g-xylose/g-untreated rice straw). When inorganic salts such as NaCl, MgCl2, CaCl2, and FeCl3 were used as catalysts, the highest sugar yield of 0.414 g/g-untreated rice straw (i.e. 0.310 g-glucose/g-untreated rice straw and 0.104 g-xylose/g-untreated rice straw) was obtained when using 1 wt% FeCl3 at 170 °C for 5 min in the first-stage and 190 °C for 5 min in the second-stage, with a value close to that of 1 wt% sulfuric acid. These findings suggest that two-stage treatment with a catalyst is a suitable pretreatment method for the production of glucose and xylose from rice straw owing to the different hydrolysis temperatures of cellulose and hemicellulose.

Association of enzymatic and optimized ultrasound-assisted aqueous extraction of flavonoid glycosides from dried Hippophae rhamnoides L. (Sea Buckthorn) berries

The main purpose of the present study was to determine the effect of associating an optimized ultrasound-assisted extraction (UAE) protocol with enzyme-assisted extraction (EAE) in aqueous media, using the dried berries of Hippophae rhamnoides L. (sea buckthorn) as plant material. A specialized software was used for the determination of potential optimal extraction parameters, leading to the development of four optimized extracts with different characteristics (UAE ± EAE). For these extracts, buffered or non-buffered solutions have been used, with the aim to determine the influence of adjustable pH on extractability. As enzymatic solution, a pectinase, cellulase, and hemicellulase mix (2:1:1) has been applied, acting as pre-treatment for the optimized protocol. The highest extractive yields have been identified for non-buffered extracts, and the E-UAE combination obtained extracts with the highest overall in vitro antioxidant activity. The HPLC-MSn analysis demonstrated a rich composition in different types of isorhamnetin-O-glycosides, as well as some quercetin-O-glycosides, showing a high recovery of specific flavonol-type polyphenolic species. Moreover, we have tentatively identified two flavanols (i.e., catechin and epigallocatechin) and one flavone derivative (i.e., luteolin).

Optimisation of some technological factors of pretreatment by pullulanase enzymatic hydrolysis combined with hydrothermal process to produce resistant starch (RS3) from rice flour ingredients

The purpose of the study was to conduct a multi-factor experiment and optimize some technological factors of pretreatment by pullulanase enzymatic hydrolysis combined with hydrothermal process to produce resistant starch (RS3) from rice flour of IR50404 paddy variety. The multi-factor experimental plan used the Box-Behnken planning model with 3 independent variables corresponding to the variation range: Pullulanase enzyme concentration (1.5-2.5%); Hydrolysis temperature (52-58oC); Hydrolysis time (8-12 hours). The objective functions include: RS3 resistant starch content (%), reducing sugar content (%), and solubility (%). The experiments were conducted under the same conditions with the parameters determined from single-factor experiments: Prepare enzymatic hydrolysis solution with rice starch/water concentration of 15% at pH 5.5; Hydrothermal mode includes 3 heat treatment/cooling cycles: Autoclaving (130oC, 60minutes) →Cooling and storage time(4oC, 18 hours). Experimental data processing and optimization used Design -Expert 7.1 software with expected function algorithm with the desire that the RS3 resistant starch content, reducing sugar content and solubility reach their maximum values, corresponding to the important coefficients of 5/5, 4/5, and 4/5. The results were the determination of the optimal enzymatic hydrolysis pretreatment regime with pullulanase enzyme concentration of 2.2%, hydrolysis temperature of 54.4oC for 10.4 hours, corresponding to RS3 resistant starch content reached 47.58±0.22%, reducing sugar content reached 14.12±0.08%, and solubility reached 68.52 ± 0.18% after hydrothermal process.

Ultrasonic/enzymatic extraction, characteristics and comparison of leechee peel polysaccharide

In this study, the process conditions, physicochemical properties, structural composition and activity of polysaccharides isolated from leechee peel (LPP) by ultrasound-assisted extraction (UAE) with enzyme and alkali solution extraction (ASE) were compared. The results showed that the total sugar content of LPP extracted by UAE accounted for 75.65 %, which was significantly higher than that extracted by alkali solution. The optimum conditions were as follows: extraction temperature of 68.78 ℃, ultrasonic enzymolysis time of 39.68 min, pectinase dosage of 4.03 %, solid–liquid ratio of 1:30 g/mL, and ultrasonic power of 360 W. The antioxidant activities and structure of leechee peel polysaccharide (LPP) prepared under different conditions were compared. It was found that UAE-LPP was an α-type polysaccharide containing 15.83 % uronic acid. Moreover, LPP extracted by UAE showed strong activity in anti-lipid peroxidation and reducing ability. Ultrasound-assisted enzymatic method is an effective means to improve the content and activity of natural plant polysaccharides, and this method has the advantages of short time-consuming, simple process and easy operation, which can greatly improve the utilization rate of polysaccharides and lay a theoretical and scientific basis for the development and utilization of LPP.

Developing a new ethylene glycol/H2O pretreatment system to achieve efficient enzymatic hydrolysis of sugarcane bagasse cellulose and recover highly active lignin: Countercurrent extraction

Improving pretreatment efficiency is a critical premise in achieving efficient biomass conversion, and obtaining high-performance natural polymers is the guarantee of high-value conversion of biomass. In this study, a new pilot-scale continuous countercurrent pretreatment reaction unit about ethylene glycol-alkali solution was designed for pretreating sugarcane bagasse in order to achieve efficient separation of the three major components of lignocellulose when expanding the scale of pretreatment, reduce lignin deposition on the fiber surface, and obtain highly active lignin and excellent enzymatic hydrolysis efficiency of cellulose. X-ray diffractometer (XRD), X-ray photoelectron spectrometer (XPS), brunauer-emmett-teller (BET) and scanning electron microscope (SEM) methods are used to analyze the structural properties of sugarcane bagasse before and after pretreatment, and high-performance liquid chromatography (HPLC) is used to analyze the monosaccharide components in the enzymatic solution. In addition, the structural properties of the recovered lignin are analyzed by gel permeation chromatography (GPC), 31P NMR and 2D-HSQC-NMR methods. The results indicate that the system can gain a high cellulose recovery of 92.99% along with a lignin removal of 95.33%, and recovered lignin has low lignin carbohydrate complexes, low condensation, and rich in phenolic hydroxyl groups for 1.95 mmol/g. Meanwhile, the countercurrent pretreatment system can effectively reduce the deposition of lignin on the cellulose surface, which is evidently superior to the non-countercurrent pretreatment and facilitates the efficiency of enzymatic saccharification of substrate, achieving a high glucose yield of 99% as well as a total sugar yield of 91.11%. The method efficiently separates biomass in a green manner, and solid residues are easily hydrolyzed, showing potential for industrial-scale production.

Low frequency magnetic field assisted production of acidic protease by Aspergillus niger.

Acid protease is widely used in industries such as food processing and feed additives. In the study, low frequency magnetic field (LF-MF) as an aid enhances acid protease production by Aspergillus niger (A. niger). The study assessed mycelial biomass, the enzymic activity of the acidic protease and underlying mechanism. At low intensities, alternating magnetic field (AMF) is more effective than static magnetic fields (SMF). Under optimal magnetic field conditions, acid protease activity and biomass increased by 91.44% and 16.31%, as compared with the control, respectively. Maximum 19.87% increase in enzyme activity after magnetic field treatment of crude enzyme solution in control group. Transcriptomics analyses showed that low frequency alternating magnetic field (LF-AMF) treatment significantly upregulated genes related to hydrolases and cell growth. Our results showed that low-frequency magnetic fields can enhance the acid protease production ability of A. niger, and the effect of AMF is better at low intensities. The results revealed that the effect of magnetic field on the metabolic mechanism of A. niger and provided a reference for magnetic field-assisted fermentation of A. niger.

Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Molecular characterization revealed the role of thaumatin-like proteins of Rhizoctonia solani AG4-JY in inducing maize disease resistance.

The gene family of thaumatin-like proteins (TLPs) plays a crucial role in the adaptation of organisms to environmental stresses. In recent years, fungal secreted proteins (SP) with inducing disease resistance activity in plants have emerged as important elicitors in the control of fungal diseases. Identifying SPs with inducing disease resistance activity and studying their mechanisms are crucial for controlling sheath blight. In the present study, 10 proteins containing the thaumatin-like domain were identified in strain AG4-JY of Rhizoctonia solani and eight of the 10 proteins had signal peptides. Analysis of the TLP genes of the 10 different anastomosis groups (AGs) showed that the evolutionary relationship of the TLP gene was consistent with that between different AGs of R. solani. Furthermore, it was found that RsTLP3, RsTLP9 and RsTLP10 were regarded as secreted proteins for their signaling peptides exhibited secretory activity. Prokaryotic expression and enzyme activity analysis revealed that the three secreted proteins possess glycoside hydrolase activity, suggesting they belong to the TLP family. Additionally, spraying the crude enzyme solution of the three TLP proteins could enhance maize resistance to sheath blight. Further analysis showed that genes associated with the salicylic acid and ethylene pathways were up-regulated following RsTLP3 application. The results indicated that RsTLP3 had a good application prospect in biological control.

Processing and performance of 3D‐printed gelatin‐based edible composite hydrogels

Abstract3D‐printed food exhibits unique advantages in precision nutrition food and personalized customization, achieving disease prevention and treatment through personalized nutrition plans and scientific addition. In this study, 3D‐printing and stable structure of gelatin‐based foods were achieved by heating extrusion. On this basis, we compared the relationship between gelatin viscosity and formability, quantified the water diffusion of composite hydrogels, and verified the edibility of each stage and component by gel strength test and texture profile analysis. The optimal content of the composite hydrogel was 35% gelatin and 5% glycerol, and the concentration of TG enzyme solution was 5%. On this basis, the content of brown sugar increased to 15% did not affect the 3D‐printing formability. Therefore, the 3D‐printed gelatin‐based composite hydrogels in this study provided a processing scheme for high adding nutrients to food 3D‐printed products.

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

Hydrothermal enzyme pretreatment on structure and properties of apple branches

Abstract Pretreatment serves as an important step in maximizing the efficient utilization of woody biomass feedstock. This study focused on enhancing the component and structural properties of apple wood to optimize the utilization of apple wood resources effectively. This study presents the optimization of, the apple wood pretreatment system, combining bioenzyme and hydrothermal treatments. This study aimed to investigate the effect of different bioenzyme pretreatment conditions on the components and structural properties of apple wood extract and to analyze the feasibility of enzyme solution reuse. Fourier transform infrared spectroscopy analysis revealed an increase in cellulose content in the wood flour following enzyme treatment. Concurrently, changes in characteristic peaks indicated an increased hemicellulose content and a reduction in lignin content after the pretreatment, correlating with fraction analysis results. Scanning electron microscopy illustrated that the dense structure of the fiber surface was disrupted after treatment. Notably, the results indicated that the recovered biocomposite enzymes exhibited excellent activity, suggesting their potential for reuse.

Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 UmL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 UmL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.

Lead and copper removal from sterile dumps by phytoremediation with Robinia pseudoacacia

In Romania, huge quantities of gangue material from the mining activity practiced in the past were improperly stored and led to the pollution of the environment. Thus, this work is framed to manage the sterile dump of the “Radeș” mine (Alba, Romania) through a 12-week phytoremediation process. The efficient use of Robinia pseudoacacia was studied through the implementation, at the laboratory level, of a phytoremediation experiment based on various variants prepared by mixtures of gangue material, uncontaminated soil, and dehydrated sludge. The prepared variants, all planted with R. pseudoacacia, were watered with tap water, potassium monobasic phosphate, and enzyme solution. The bioconcentration and translocation factors for lead showed values ˂ 1, which indicates a potential presence of an exclusion system for Pb or a reduced Pb bioavailability since the R. pseudoacacia accumulates high concentrations of metals absorbed on and inside the roots. For copper, both factors had values > 1 indicating the suitability of R. pseudoacacia to readily translocate copper into the epigean organs. In the investigated experimental conditions, the highest efficiency in the removal of copper (93.0%) and lead (66.4%) by plants was obtained when gangue material was not mixed with other materials and wetted with enzymatic solution.

Inhibition kinetics and mechanism of genistein against α‐glucosidase

AbstractSeveral studies have documented the effective inhibition of genistein, a component in soybeans against α‐glucosidase. However, the details of the inhibition mechanism and kinetics remain unfulfilled. Here, the authors aim to investigate the anti‐diabetic potential of genistein through IC50 value, fluorescence quenching and inhibition kinetics. Genistein was found to exhibit an inhibition activity on α‐glucosidase with an IC50 value of 7.79 ± 0.36 µm. Analysis of fluorescence spectra indicated that genistein quenched enzyme emission through a static mechanism with a bimolecular quenching constant, kq = 4.04 × 1013 m−1 s−1. In addition, it was found that genistein was bound to α‐glucosidase with a high affinity of binding constant, Ka = 3.38 × 105 m−1 and a 1:1 stoichiometric ratio (n = 1.06). In order to suggest a possible contact involved, 8‐anilino‐1‐naphthalenesulfonic acid (ANS) was added as an extrinsic fluorescence probe to enzyme solution and analyzed fluorescence spectra of the α‐glucosidase‐ANS complexes. When treated with genistein, fluorescence intensities of the complexes were reduced remarkably, indicating that genistein interacts with the enzyme via a hydrophobic domain. Finally, the inhibition constant and inhibition mode were studied by inhibition kinetics. The results revealed that genistein bound easily to α‐glucosidase with a Ki value of 25.61 × 10−6 m through a non‐competitive type.

Inactivation kinetics of polyphenol oxidase and peroxidase enzymes in buffer solutions by ultraviolet light sources at multiple UV-C wavelengths

This study aimed to evaluate the effect of multiple wavelengths of UV-C light sources on the inactivation of polyphenol oxidase (PPO) and peroxidase (POD) in buffer solutions. Enzyme solutions were treated with a UV-C excimer lamp (EL) emitting at 222 nm, low-pressure mercury (LPM) lamp at 253.7 nm, four UV-LEDs sources emitting at 255, 265, 275 and 285 nm, and a novel pulsed UV-C lamp (PL) emitting at 232, 242, 261 and 272 nm. Three kinetic models were used to compare UV-C wavelengths enzymatic inactivation efficacy considering the volume average fluence for 90% reduction (HD). Validation of kinetic parameters was performed under continuous-flow treatment in a UV-C LED and EL system. EL treatment resulted in the highest inactivation efficacy of PPO and POD enzymes with HD of 52.8 and 74.8 mJ/cm2, respectively. PL inactivation efficacy for both enzymes was higher than with LPM lamp or UV-LEDs. The two-component first-order model best described the inactivation of PPO and POD in a buffer solution. The adjusted kinetic parameters for this model were validated under UV-C light continuous flow processing. This study contributes to the development of liquid food treatment by ultraviolet technology.

Exploring laccase: a sustainable enzymatic solution for the paper recycling domain.

The global advocacy of resource conservation and waste management emphasizes the significance of sustainable practices, particularly in sectors such as paper manufacturing and recycling. Currently, conventional chemical methods are predominant for paper production, necessitating the use of substantial amount of toxic chemicals. This chemical-intensive approach compromises the recycled fiber quality, generates hazardous effluent causing serious ecological threats which triggers regulatory complexities for the mills. To address these challenges modern research suggests adopting sustainable eco-friendly practices such as employing enzymes. This review aims to explore the applicability of 'laccase' enzyme for paper recycling, investigating its properties and contribution to improved recycling practices. By delving into the potential application of laccase integration into the papermaking process, this article sheds light on the limitations inherent in traditional methods surmounted within both research and translational landscapes. Culture and process optimization studies, supporting the technological improvements and the future prospects have been documented.

Quality of edible bird’s nest treated by keratinolytic enzymes-based cleaning solution

Edible bird's nest (EBN) is one of the luxury foods that is widely used as a healthy food due to its nutritional value and therapeutic benefits. The traditional EBN washing process causes a reduction in weight and nutrient content and an increase in pollutants due to the use of hydrogen peroxide. An exploratory observation method was used to examine the quality of EBN from Collocalia fuciphaga before and after washing using a washing solution based on keratinolytic enzymes. There are four stages of EBN cleaning i.e. cleaning by tap water, ethanol solution, enzyme solution at room temperature and 50oC and drying at 40oC for 42 hrs. A total of 60 EBN (unclean, n = 30) and clean, n = 30) were analyzed using the AOAC method. The mineral content of Ca, Fe, K and Mg using atomic absorption spectrophotometry (AAS) except that P was measured by spectrophotometer. A carbohydrate estimation kit was used to measure glycoprotein content and the amino acid content was determined using the HPLC method. Evaluation of the cleanliness was carried out using a scoring system by semi-trained panelists. The results obtained showed that the clean EBNs were slightly yellowish-white in color, and the cleanliness of EBN before and after cleaning increased from 2.35 to 3.84. Clean EBN protein content decreased by 7.2% while total amino acids decreased from 38.51% to 32.71%. Clean EBN contains eight essential amino acids of 17.93% with high levels of leucine, valine, arginine, and threonine (2.42-2.96%). EBN's clean ash content increased from 3.7% to 7.8%. Carbohydrate content and iron in clean EBN were 39.19±0.76% and 14.35 mg/100 g dry matter, respectively. The high levels of carbohydrates and iron appear to be good sources of glycoprotein to support health and have the potential as an alternative source of iron for people with anemia. The stepwise EBN washing method using tapwater, ethanol and a washing solution based on keratinolytic enzymes can be applied to reduce weight loss and improve the quality of EBN.

Abstract 6085: Leveraging targeted epigenetic and genetic detection for cost-effective cancer classification

Abstract The combinatorial power of genetics and epigenetics is vital to understanding biology in healthy and cancerous states. Utilizing a recent novel approach, we enable the simultaneous identification of modified cytosine and the canonical bases A, C, T & G in an enzymatic single-workflow solution. Generating this information across the whole genome provides a much-improved understanding of genetic and epigenetic changes, but can require a large amount of sequencing, particularly when exploring at depth. Enrichment protocols such as target enrichment enable deep interrogation of target areas whilst maintaining cost effectiveness. Target enrichment panels enable the selective isolation of sequences of interest through target hybridization. Using commercially available panels combined with the novel enzymatic single-workflow solution we show uniform performance across a range of input amounts with reduced sequencing requirements. Samples used were cell free DNA (cfDNA) from patient samples with either Stage I or IV Colorectal Cancer and reportedly healthy controls and were targeted using pan-cancer and methylome panels. Widespread differences such as hypermethylation are clearly observed in late-stage cancer vs early-stage or healthy controls demonstrating utility of the approach for applications such as liquid biopsy. Refining genetic and epigenetic investigations to a narrower region of interest can improve detection of low prevalence disease associated biomarkers at significant sequencing depth and lower sequencing costs. Citation Format: Robert Crawford, Ben Krajacich, Ermira Lleshi, Aurel Negrea, Lisabet Andreasen, Jens Fullgrabe, Jack Monahan, David Morley, Fabio Puddu, Donna McDade Walker, Semyon Kruglyak, Páidí Creed. Leveraging targeted epigenetic and genetic detection for cost-effective cancer classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6085.