Enzymatic Pretreatment Research Articles

Laccase driven biocatalytic oxidation to reduce polymeric surface hydrophobicity: An effective pre-treatment strategy to enhance biofilm mediated degradation of polyethylene and polycarbonate plastics

Plastic pollution is a major global environmental issue due to its structural complexity and poor biodegradability. Biological approaches are appropriate due to cost effectiveness and environmental friendliness, however effective polymer degradation is still in its infancy. As biological treatments are slower than physical and chemical approaches, they could be applied in conjunction with pre-treatment techniques such as photo-oxidation, heat treatment, and chemical treatments. But these processes lead to high energy consumption and hazardous secondary pollution. To address these concerns, an enzymatic pre-treatment strategy has been proposed in this study, with an aim of promoting surface oxidation on the plastics leading to improved hydrophilicity. This in turn, facilitates the surface attachment of microbes, ultimately, accelerating biodegradation. Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopy analyses confirmed the surface oxidation of the polyethylene (PE) and polycarbonate (PC) plastics mediated by the action of laccase enzyme. Contact angle measurement witnessed the increased hydrophilicity of the treated plastics. Following, a potential biofilm forming microbial consortium has been employed for the biodegradation of enzyme treated plastics. SEM analysis indicated the increased formation of corrosive pits and surface aberrations on the enzymatically pre-treated plastics and Confocal Laser Scanning microscopy (CLSM) analysis exhibited the enhanced biofilm formation and exopolysaccharide deposition on the pre-treated PE and PC. In addition, X-ray photoelectron spectroscopy (XPS) revealed the reduction in the elemental composition of carbon with an increment in the oxygen composition of plastics. Gel permeation chromatography (GPC) further confirmed the greater reduction in the molecular weights of the plastics subjected to integrated enzymatic and biofilm treatment than only biofilm treated plastics. This is the first report on the integration of enzymatic pre-treatment with the biofilm mediated microbial degradation to achieve enhanced treatment of plastics which demonstrated to be a promising technology for the effective mitigation of plastic pollution.

Comparative evaluation of different enzyme pretreatment on the oxidative stability and volatile compounds of sunflower oil

In order to determine the influence of enzyme pretreatment to modify the aromas of sunflower seed oil (SSO). In this study, different enzyme combinations prepared SSO as raw material, using reducing sugar, amino acids, oxidation stability, and basic aroma characteristics to compare the whole. The results show that reducing sugar increased approximately 1.5-fold after enzymatic hydrolysis. Arginine, lysine, and histidine decreased most after roasting. The combination of cellulase and protease showed the greatest increase in heterocyclic substances (from 1681.81 to 13865.84 μg/kg), resulting in a strong caramel and nutty flavor. Interestingly, it was identified by aroma extract dilution analysis (AEDA) combined with relative odor activity value (ROAV) that 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) was a common key aroma compound in enzyme-treated SSO, and phenylacetaldehyde, furfuryl alcohol and acetic acid were considered to be important contributors to the aroma after cellulase, hemicellulose and pectinase treatment, respectively. All of these were newly identified as odorants in enzyme-treated sunflower oil. The enzyme-treated oil also had advantages in terms of oxidative stability, including the reduction of acid value and peroxide value. This work provides a feasible method to enhance the aroma flavor and extend the shelf-life of sunflower oil in future production.

Polysaccharide extracted from cultivated Sanghuangporous vaninii spores using three-phase partitioning with enzyme/ultrasound pretreatment: Physicochemical characteristics and its biological activity in vitro

Sanghuangporous vaninii, as a valuable dietary supplement and medicinal ingredient, contains abundant bioactive polysaccharides that have health-promoting effects. In the present study, four polysaccharides (SVSPs-C, SVSPs-E, SVSPs-U, and SVSPs-E/U) were extracted for the first time from S. vaninii spores by three-phase partitioning (TPP), enzyme pretreatment before TPP (E-TPP), ultrasonic pretreatment before TPP (U-TPP), and enzyme pretreatment followed by ultrasonic before TPP (E/U-TPP) methods, respectively. Their physicochemical characteristics and in vitro pharmacological functions were determined and compared. Results showed that four TPP-based extraction methods had remarkable impacts on the extraction yield, chemical properties, monosaccharide compositions, and molecular weights (Mw) of SVSPs. Specifically, SVSPs-E/U obtained by E/U-TPP showed the highest extraction yield (25.40 %), carbohydrate content (88.50 %), and the lowest protein content (0.86 %). The four SVSPs had high-Mw (183.8–329.1 kDa) and low-Mw (23.0–156.4 kDa) fractions and mainly consisted of galactose, glucose, and mannose with different contents. In vitro bioactivities assays indicated that SVSPs-E/U possessed stronger antioxidant, hypoglycemic, hypouricemic, immunostimulatory, and antitumor activities than those of SVSPs-C, SVSPs-E, and SVSPs-U. Therefore, our results provide an efficient and promising extraction technique for bioactive polysaccharides from S. vaninii spores, as well as SVSPs had the potential to be applied in functional food, pharmaceutical, and cosmetics fields.

A novel approach to enhance methane production during anaerobic digestion of waste activated sludge by combined addition of trypsin, nano-zero-valent iron and activated carbon

A novel approach with a combination of trypsin, nano-zero-valent iron (NZVI) and activated carbon (AC) was conducted to promote the methane production of waste activated sludge (WAS) during the anaerobic digestion (AD) processes. Results showed that the combined addition of trypsin-NZVI-AC exhibited the synergistic effect during different AD stages. Trypsin mainly facilitated the hydrolysis process and the acetic acid conversion, while NZVI-AC enhanced the substrate metabolism and the electronic transfer to subsequently produce methane. A dose of 1000 mg/L trypsin was optimal to maximize this synergistic effect. Metagenomic analysis showed that trypsin-NZVI-AC addition effectively improved the relative abundance of acetyl-CoA carboxylase, and then strengthened both acetoclastic methanogenesis (M00357) and hydrogenotrophic methanogenesis (M00567). Hydrogenotrophic methanogens such as Methanobacterium, Methanoculleus, and Methanosarcina were greatly enriched with trypsin-NZVI-AC compared with trypsin or NZVI-AC addition. Moreover, electroactive bacteria G. sulfurreducens and G. metallireducens were also enriched by this method to conduct direct interspecies electron transfer among methanogens, leading to the better improvement of methane production. These findings supply a promising way to optimize the enzyme pretreatment technology and elevate the methanogenic efficiency of WAS.

A qPCR technology for direct quantification of methylation in untreated DNA

DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.

Development of bio-mechanical rice straw pulp by high consistency kneader with thermal and Alkaline

The purpose of this study was to develop a bio-mechanical pulp from rice straw with thermal and low chemical dosage. The process is carried out by thermal alkali kneading pretreatment (0–5% NaOH) with a high concentration kneader, enzyme pretreatment (0.2–4%) and Hollander beater. The results showed that the addition of NaOH was the main factor affecting the properties of pulp and paper, and the best properties of straw pulp could be obtained when NaOH and enzyme dosage were 5% and 0.2%, respectively. Although the optimum rice straw pulp is inferior to straw soda pulp in terms of tensile, burst and ring crush index, it is superior to old corrugated cardboard pulp and partially meets the CNS standards of corrugated medium paper and linerboard without the addition of chemicals. Therefore, the bio-mechanical rice straw pulp in this study has the potential to be produced as industrial paper.

Methods for enhancing the properties of products from food waste via hydrothermal carbonation (HTC): Gradient-enzymatic-pretreatment-nitrogen-migration-strategy

Gradient enzymatic pretreatment is an effective method for improving the quality of hydrothermal carbonation products from food waste. Food waste mainly contains polysaccharides, proteins, fats, and indigestible fibers. In this study, food waste model compounds formulated according to the survey were subjected to enzyme-assisted hydrothermal carbonation. Results showed that the high heating value of the hydrochar reached 31.62 MJ/kg at 240 °C for 25 min when the meat content of the raw material was high. Increases in reaction temperature and residence time remarkably changed the species of compounds in the aqueous phase. The increase in the amount of the key nitrogen-containing components doping in the aqueous phase after enzyme-assisted hydrothermal carbonation was the major factor that enhanced its luminescence intensity. The concentration of total nitrogen in the aqueous phase reached 1403.31 mg/L at 220 °C for 25 min. The gradient enzymatic pretreatment method combined with hydrothermal carbonation process facilitates the nitrogen migration of food waste in the aqueous phase, thus ensuring sustainable production and enhancing the product properties of food waste for high-valued applications.

Enzyme technology in bioethanol production from lignocellulosic biomass: Recent trends with a focus on immobilized enzymes

Enzyme immobilization is a useful tool to produce biocatalysts with improved performance, such as high activity, high stability at operational conditions, and easy recovery and reuse. In this context, the production of second-generation ethanol using immobilized enzymes was reviewed. Emphasis was placed on the pre-treatment and/or hydrolysis steps to increase efficiency and reduce the cost of the process. In addition, the process design of bioethanol using immobilized enzymes was critically reviewed. In the enzymatic pre-treatment, laccases, manganese peroxidases, lignin peroxidases, and lytic polysaccharide monooxygenases are the main enzymes involved. When considering processes with heterogeneous biocatalysts, laccases are the most explored enzyme. In the hydrolysis step, cellulases and hemicellulases or a mixture of them are the main enzymes explored in the literature frequently using magnetic nanoparticles as support for enzyme immobilization. Although enzyme immobilization is a mature technology, the use of these biocatalysts is frequently limited to only one step of the bioethanol production process, such as pre-treatment or hydrolysis performed using Benchmark Technology. However, some recent papers have explored innovative design processes using a mixture of immobilized enzymes or co-immobilized enzymes to join some process steps, thereby decreasing the cost of enzymes and equipment.

Enhancement of Soybean Meal Alters Gut Microbiome and Influences Behavior of Farmed Atlantic Salmon (Salmo salar).

Atlantic salmon (Salmo salar) is one of the worlds most domesticated fish. As production volumes increase, access to high quality and sustainable protein sources for formulated feeds of this carnivorous fish is required. Soybean meal (SBM) and soy-derived proteins are the dominant protein sources in commercial aquafeeds due to their low-cost, availability and favorable amino acid profile. However, for Atlantic salmon, the inclusion of soybean meal (SBM), and soy protein concentrate (SPC) in certain combinations can impact gut health, which has consequences for immunity and welfare, limiting the use of soy products in salmonid feeds. This study sought to address this challenge by evaluating two gut health-targeted enhancements of SBM for inclusion in freshwater phase salmon diets: enzyme pre-treatment (ETS), and addition of fructose oligosaccharide (USP). These were compared with untreated soybean meal (US) and fish meal (FM). This study took a multi-disciplinary approach, investigating the effect on growth performance, gut microbiome, and behaviors relevant to welfare in aquaculture. This study suggests that both enhancements of SBM provide benefits for growth performance compared with conventional SBM. Both SBM treatments altered fish gut microbiomes and in the case of ETS, increased the presence of the lactic acid bacteria Enterococcus. For the first time, the effects of marine protein sources and plant protein sources on the coping style of salmon were demonstrated. Fish fed SBM showed a tendency for more reactive behavior compared with those fed the FM-based control. All fish had a similar low response to elicited stress, although ETS-fed fish responded more actively than US-fed fish for a single swimming measure. Furthermore, SBM-fed fish displayed lower repeatability of behavior, which may indicate diminished welfare for intensively farmed fish. The implications of these findings for commercial salmonid aquaculture are discussed.

(Pre)analytical considerations concerning the analysis of synovial calprotectin.

Several studies have demonstrated that synovial calprotectin is a highly accurate biomarker in diagnosing periprosthetic joint infections (PJI). Assuring reliability is of great importance and coincides with adequate preanalytical handling. This study focuses on potentially interfering factors. To assess the stability of synovial calprotectin, the effect of time, storage temperature, EDTA, freeze-thaw cycles, viscosity, and blood and lipid contamination was investigated. In the blood and lipid contamination experiments, hemolyzed and non-hemolyzed blood, homogenized adipose tissue, intralipid and chylomicrons were added. The effect of viscosity was investigated using freeze-thaw cycles, enzymatic pretreatment and sonification. No effect on synovial calprotectin levels was observed in synovial samples kept at room temperature compared to samples kept at 4 °C for up to seven days of storage. Freeze-thaw cycles did not result in significantly different calprotectin levels, although samples without EDTA resulted in higher recoveries after 1 and 2 freeze-thaw cycles. Blood and lipid contamination did not interfere with accurate synovial calprotectin analysis. Sample pretreatment to reduce sample viscosity by pretreating samples with DNAse and/or hyaluronidase did not influence calprotectin analysis. Sonification, however, resulted in increased calprotectin values. Synovial calprotectin is a stable biomarker and its analysis is not easily influenced by potential interfering factors.

Multidisciplinary treatment in the long-term management of Fabry disease

Fabry disease is a rare X-linked hereditary condition caused by mutations in the α-galactosidase A (GLA) gene, resulting in decreased α-GAL A enzyme activity. The clinical manifestations of Fabry disease are diverse, which leads to delays in diagnosis and treatment, thereby increasing the disease burden for patients and their families. Given its characteristics, multidisciplinary treatment (MDT) is critical for the long-term management of Fabry disease, and should include nephrology departments, cardiovascular departments, neurology departments, and pediatric department, among others. This study focuses on early screening for Fabry disease, the indication for initiating enzyme replacement therapy, pre-treatment evaluation, and monitoring to provide practical guidance for Chinese clinicians.

Optimization of solid-state fermentation for enhanced production of pectinolytic complex by Aspergillus tubingensis FAT43 and its application in fruit juice processing.

The main goal of this study was to examine the efficiency of a newly isolated fungus from quince, Aspergillus tubingensis FAT43, to produce the pectinolytic complex using agricultural and industrial waste as the substrate for solid state fermentation. Sugar beet pulp was the most effective substrate inducer of pectinolytic complex synthesis out of all the waste residues examined. For endo-pectinolytic and total pectinolytic activity, respectively, statistical optimization using Placked-Burman Design and Optimal (Custom) Design increased production by 2.22 and 2.15-fold, respectively. Liquification, clarification, and an increase in the amount of reducing sugar in fruit juices (apple, banana, apricot, orange, and quince) processed with pectinolytic complex were identified. Enzymatic pre-treatment considerably increases yield (14%-22%) and clarification (90%). After enzymatic treatment, the best liquefaction was observed in orange juice, whereas the best clarification was obtained in apricot juice. Additionally, the pectinolytic treatment of apricot juice resulted in the highest increase in reducing sugar concentration (11%) compared to all other enzymatically treated juices. Optimizing the production of a highly active pectinolytic complex and its efficient utilization in the processing of fruit juices, including the generation of an increasing amount of waste, are the significant outcomes of this research.

Efficient environmentally friendly enzymatic and ultrasonic pretreatment of lignocellulosic wastes for enhanced methane production

Efficient environmentally friendly enzymatic and ultrasonic pretreatment of lignocellulosic wastes for enhanced methane production

Effect of size press coating of cationic starch/ nanofibrillated cellulose on physical and mechanical properties of recycled papersheets

This study aimed to evaluate the effect of size press coating on two types of recycled papers using different types of nanofibrillated cellulose (NFC) and cationic starch (CS) on physical and mechanical properties. Wheat straw was used as a cellulose source, and NFC was obtained through oxidative and enzymatic pretreatments. Recycled fluting and coreboard papers were coated with cationic starch/NFC blends at various concentrations of NFC (0.5%, 1%, 2%, 3%, and 4%) using a three-time repetitive size press application, followed by one-time drying section, and compared to uncoated papers. The application of a coating suspension containing 4% periodate-oxidized NFC on both paper surfaces resulted in a significant improvement in the tensile index, burst index, and internal bond strength of the papersheets, with increases of up to 60.6%, 96.3%, and 119.9%, respectively. Furthermore, significant decreases in air permeability were also observed with reductions of 75% and 81.6% for coreboard and fluting papers, respectively. Compared to other pretreatment methods, periodate oxidation resulted in higher viscosity values in NFC samples. Therefore, the application of periodate-oxidized NFC with a size press resulted in a significant improvement in the mechanical and barrier properties of papers made from recycled pulps.

Effect of xylanase addition and ultrasonic pretreatment on the yield and volatile profile of bay laurel and sage essential oil

In order to increase the yield of essential oils in hydrodistillation of bay laurel and sage, the effect of pretreatment with ultrasound and enzymes (under different conditions of xylanase pretreatment) was studied. The effects of the optimized conditions (concentration of xylanase 0.20 mg/mL in buffer pH=6.5 at 40°C for 4 h) during the enzymatic pretreatment showed that despite the optimization the activity of xylanase did not significantly affect the quantity and quality of essential oils of bay laurel and sage. Ultrasonic pretreatment prior to enzymatic pretreatment with the addition of optimized xylanase resulted in increased yields of dominant compounds (α-thujone 27.42%, β-thujone 12.15%, and camphor 20.94%) in sage essential oil, while in bay laurel the amount of dominant compounds (1,8-cineole 24.65% and α-terpenyl acetate 11.05%) was not significantly increased. Despite minor changes, ultrasonic and enzymatic pretreatment of the plant materials did not significantly affect the chemical composition of the essential oils of bay laurel and sage compared with the control without pretreatment, as confirmed by the Spearman rank test (p < 0.05). The main advantage of ultrasonic extraction is the reduction in pretreatment time. Ultrasonic pretreatment for only 10 min before enzymatic pretreatment resulted in a 50% increase in sage essential oil yield and a 40% increase in bay laurel essential oil yield.

Structural characterization of lignin-carbohydrate complexes from Chinese quince fruits extracted after enzymatic hydrolysis pretreatment

Chinese quince fruit (CQF) contains abundant pectin; however, the pectin cannot be efficiently separated by conventional approaches because of strong lignin-carbohydrate complexes (LCC). In this study, to elucidate the structural characteristics of the original LCC formed by lignin and pectin in CQF, single and multiple enzymatic hydrolysis pretreatments were innovatively performed, and the resulting LCC preparations were comprehensively characterized using a series of techniques. The enzymatic hydrolysis pretreatments significantly increase the LCC yield, releasing LCC fractions with low molecular weights (Mw = 4660–8288 Da). LCC-4, isolated by pretreatment with cellulase plus xylanase, had the highest galacturonic acid content (15.5 %), followed by LCC-2 (isolated by xylanase pretreatment) of 14.0 %. In CQF, lignin develops lignin-carbohydrate (LC) bonds with pectin to form LCC, with phenyl-glycoside bond being the dominant linkage. Although the pectinase pretreatment reduced the pectin content, signals of the LC linkages in the 2D-HSQC spectra were enhanced. LCC-4 could be considered as the most representative of the original LCC in CQF due to its high pectin content and multiple LCC signals in the 2D-HSQC spectrum. The structural understanding of the original LCC in CQF will lay a foundation for designing appropriate methods for extracting pectin from CQF.

Ferulic acid production from wheat bran by integration of enzymatic pretreatment and a cold-adapted carboxylesterase catalysis

High-value chemical production from natural lignocellulose transformation is a reliable waste utilization approach. A gene encoding cold-adapted carboxylesterase in Arthrobacter soli Em07 was identified. The gene was cloned and expressed in Escherichia coli to obtain a carboxylesterase enzyme with a molecular weight of 37.2 KDa. The activity of the enzyme was determined using α-naphthyl acetate as substrate. Results showed that the optimum enzyme activity of carboxylesterase was at 10 °C and pH 7.0. It was also found that the enzyme could degrade 20 mg enzymatic pretreated de-starched wheat bran (DSWB) to produce 235.8 μg of ferulic acid under the same conditions, which was 5.6 times more than the control. Compared to the chemical strategy, enzymatic pretreatment is advantageous because it is environmentally friendly, and the by-products can be easily treated. Therefore, this strategy provides an effective method for high-value utilization of biomass waste in agriculture and industry.

Kinetic Studies of Enzymatic Hydrolysis Pre-Treatment of Waste Bread Under Preliminary Conditions

This study investigate the process conditions of enzyme concentration (2.0, 4.0, 6.0% w/v), temperature (30, 40, 50 °C), and reaction time (0, 60, 90, 120, 180, 240 min) of enzymatic hydrolysis for sugar recovery from waste bread. The kinetics of the enzyme (α-amylase) was studied to analyse the chemical reaction and the activity of the α-amylase towards the starch present in the waste bread. Enzymatic hydrolysis with different substrate concentration (5, 10, 15, 20 g) was conducted to analyse the kinetics of the enzyme with increasing amount of substrate concentration. The highest reducing sugar yield from the waste bread was 119.73 mg/mL at 15g of substrate concentration, 6.0% w/v enzyme concentration, temperature 50°C and 180 mins reaction time. From this value, the percentage recovery of the reducing sugar was 71.84%. The Michaelis-Menten equation was applied to study the kinetics of enzymatic hydrolysis of waste bread. The Vmax value was 3443.234 µmol/L/min and the Km value was 6.0g.

Saccharification of starchy food waste through thermochemical and enzymatic pretreatment, towards enhanced bioethanol production via newly isolated non-conventional yeast strains

A starchy food waste containing mainly cooked wasted rice (WR) was exploited for bioethanol production using novel yeast strains was investigated. Different pretreatment schemes of the waste at solids loading 10%–30% TS WR (w/v) i.e. enzymatic, thermochemical and combined thermochemical/enzymatic pretreatment, were evaluated aiming to the maximum liberation of fermentable carbohydrates and their subsequent bioconversion to ethanol. Fermentation tests of the whole pretreated slurries were initially performed with the yeasts strains that were identified as Kluyveromyces marxianus isolate V3-19,Pichia kudriavzevii strain YF1702 and K. marxianus strain TTG-428, and their fermentation efficiencies (FE) were comparatively assessed. It was shown that the combined pretreatment led to the maximum saccharification, whereas FEs were higher for K. marxianus, V3-19, exceeding 90% of the theoretical maximum. In the case of the highest organic loading of WR, though, up to 25% of soluble carbohydrates remained unexploitable after 72 h of fermentation, indicating that kinetic restrictions occurred in the process. Further experiments with the hydrolysates that were recovered after combined pretreatment, revealed that the removal of solids enhances the consumption of sugars and leads to complete uptake for the loading 20% TS WR (w/v).

Proteinase K Pretreatment for the Quantitative Recovery and Sensitive Detection of the Tuberculosis Biomarker Mannose-Capped Lipoarabinomannan Spiked into Human Serum.

This paper reports on an investigation of an enzymatic pretreatment protocol using proteinase K (ProK) for the analysis of human serum samples spiked with mannose-capped lipoarabinomannan (ManLAM). ManLAM is an antigenic biomarker found in the serum, urine, and other body fluids of individuals infected with tuberculosis (TB). Immunometric measurements of ManLAM are compromised by steric effects due to its complexation with high-molecular-weight components in these matrices that interfere with its capture and/or labeling. Recent work has shown that deproteinization of these types of samples by perchloric acid acidification or ProK digestion releases ManLAM from complexation. Releasing ManLAM greatly improves its detectability and, as a result, its utility as a TB biomarker. The work detailed herein examined how different ProK reaction conditions (e.g., enzyme concentration and digestion time and temperature) affect the recovery and detectability of ManLAM in human serum. As measured by enzyme-linked immunosorbent assay (ELISA), we show that using the optimal set of digestion conditions to free ManLAM, which also yield a small, quantitatively reproducible level of sample concentration, it is possible to achieve a spiked ManLAM recovery of 98 ± 13% and a limit of detection of 10 pg/mL (0.6 pM). Experiments also demonstrated that the ELISA responses measured for a given ManLAM concentration in serum after pretreatment were statistically indistinguishable from those directly determined for the same amounts of ManLAM added to an innocuous buffered solution. Possible adaptations of the digestion protocol for use in point-of-care TB testing are also briefly discussed.