Subject: Chemistry, Analytical Chemistry Keywords: Cow milk; Enzymatic hydrolysis; Allergic protein; Antigenicity
Online: 19 October 2020 (11:04:35 CEST)
In this paper, the effects of different food-grade enzymes on the antigenicity of casein (CN), β- lactoglobulin (β-LG) and ɑ-lactalbumin (ɑ-LA) in natural cow milk were studied. The degree of hydrolysis (DH), SDS-PAGE and molecular mas (MW) distribution of cow milk (CM) hydrolysates was assessed. Additionally, the residual antigenicity of CM hydrolysates was evaluated by using ELISA and western blotting with anti-CN, anti-β-LG and anti-ɑ-LA rabbit polyclonal antibody. The results showed that Alcalase and Protamex hydrolysis could efficiently reduce the antigenicity of CN, β-LG, ɑ-LA, showed a higher DH and the loss of density of CM proteins, as indicated by SDS-PAGE. The increasing of the low MW (<3 kDa) in CM hydrolysates was also presented. It was also found that Protamex, Alcalase could be more efficiently hydrolyzed major allergenic of CM than other enzymes for the development of hypoallergenic cow milk. Our research will lay a theoretical foundation for the study of hypoallergenic cow milk.
ARTICLE | doi:10.20944/preprints201902.0037.v1
Subject: Engineering, Energy & Fuel Technology Keywords: Biomass, valorisation, ionic liquid, crystallinity, enzymatic hydrolysis, pre-treatment
Online: 4 February 2019 (13:57:18 CET)
Ionic liquids have been recognised as interesting solvents applicable in the efficient lignocellulosic biomass valorisation, especially in the biomass fractionation into individual polymeric components or direct hydrolysis some of biomass fractions. Considering the chemical character of ionic liquids, two different approaches, paved the way for a fractionation of biomass. The first strategy integrated a pre-treatment, hydrolysis and conversion of biomass through the employment of hydrogen-bond acidic 1-ethyl-3-methyimidazolim hydrogen sulfate ionic liquid. The second one relied on the use of a three-step fractionation process with hydrogen-bond basic 1-ethyl-3-methylimidazolium acetate to produce high purity cellulose, hemicellulose and lignin fractions. The proposed approaches were scrutinised for wheat straw and eucalyptus residues. Those different biomasses allowed understanding that enzymatic hydrolysis yields are dependent on the crystallinity of pre-treated biomass. The use of acetate based ionic liquid allowed to change crystalline cellulose I to cellulose II and consequently enhanced glucan to glucose yield to 93.14.1 mol% and 82.91.2 mol% for wheat straw and eucalyptus, respectively. Whereas for hydrogen sulfate ionic liquid, the same enzymatic hydrolysis yields were 61.6 0.2 mol% for wheat straw and only 7.90.3 mol% for eucalyptus residues. These results demonstrate the importance of either ionic liquid character or biomass type on the efficient biomass processing.
ARTICLE | doi:10.20944/preprints202208.0436.v1
Online: 25 August 2022 (13:21:39 CEST)
Indole and indole-3-lactate are known dominant microbial tryptophan catabolites (MICT). In obesity, the fecal indole concentration corresponds to the normal one, and that of indole-3-lactate significantly decreases along with other MICT, while it increases in blood plasma. During the analysis of the «enzymatic landscape» of the intestinal microbiota we find an almost twofold increase in the correlation between the concentrations of fecal MICT and the «enzymatic landscape», with indole-3-lactate having the closest relationships with the “enzymatic landscape” of all MICT. Here, we report statistically significant correlations of indole-3-lactate and the gut microbial enzymes for fructose, amino sugars, nucleotides, polyamines metabolism, and sulfoglycolysis. We also demonstrate that indole-3-lactate producing microbiota representatives increase three-fold in obesity. The phenotype of the microbiotic population is thus represented by completely different genera and species of microorganisms in obese individuals compared to healthy donors.
Subject: Materials Science, Biomaterials Keywords: enzyme; bast fibers; enzymatic retting; characterizations
Online: 26 November 2019 (10:27:02 CET)
Natural fibers are a gift from nature that we yet fully utilized until now. It can be classified into several groups and bast fibers are the group having the most promising performance when reinforced in polymer composites. However, numerous factors have been reported that influences mechanical properties of fiber reinforcements in the composite. In this review, bast fiber retting process and the effect of enzymatic retting on fiber and fibers reinforced polymer composites have been discussed and reviewed for the latest researches. Retting precedes mechanical processing (i.e. scutching) of the fiber from the stem and is essential for reduction of fiber breakage. All retting methods except chemical retting process are involving secretes of enzymes by bacteria or fungi under controlled (enzymatic retting) or random conditions (water and dew retting). Besides, enzymatic retting is claimed to have more environmentally friendly wastewater products, shorter retting period and controllable fiber biochemical components under mild incubation conditions. This review comprehensively assesses the enzymatic retting process for producing high-quality bast fiber and will become a reference for future development on bast fiber reinforced polymer composites.
Subject: Chemistry, Analytical Chemistry Keywords: nanocellulose; ionic liquids; Trichoderma reesei; enzymatic hydrolysis
Online: 11 May 2021 (10:42:38 CEST)
Nanocellulose has gained increasing attention during the past decade, which is related to its unique properties and wide application. In this paper, nanocellulose was produced by hydrolysis with ionic liquids (1-ethyl-3-methylimidazole acetate (EmimOAc) and 1-allyl-3-methylimidazolium chloride (AmimCl)) from microcrystalline cellulose (Avicel and Whatman) subjected to enzymatic pretreatment. The obtained material was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscope (SEM) and thermogravimetric analysis (TG). The results showed that the nanocellulose had a regular and spherical structure with a diameter of 30-40 nm and exhibited lower crystallinity and thermal stability than the material after hydrolysis with Trichoderma reesei enzymes. However, the enzyme-pretreated Avicel had a particle size of about 200 nm and a cellulose II structure. A two-step process involving enzyme-pretreatment and hydrolysis with ionic liquids resulted in the production of nanocellulose. Moreover, the particle size of nanocellulose and its structure depend on the ionic liquid used.
ARTICLE | doi:10.20944/preprints202212.0059.v1
Subject: Life Sciences, Biotechnology Keywords: nanocellulose; enzymatic synergism; optimal mixture; CCRD; Colby factor
Online: 5 December 2022 (06:03:10 CET)
A study to produce cellulose nanofibrils (CNF) from Kraft cellulose pulp, an optimal enzyme mixture, was defined using a centroid simplex mixture design. The enzyme blend contains 69% endoglucanase and 31% exoglucanase. The central composite rotational design (CCRD) optimized the CNF production process by achieving a higher crystallinity index. It thus corresponded to a solid loading of 15 g/L and an enzyme loading of 0.974. Using the Segal formula, the crystallinity index (CrI) of CNF was determined by X-ray diffraction to be 80.87%. The average diameter of nanocellulose fibers measured by scanning electron microscopy between 550 - 600 nm for the CNF prepared by enzymatic hydrolysis and between 250 - 300 nm for the CNF produced by enzymatic hydrolysis with the optimal enzyme mixture followed by ultrasonic dispersion. Finally, synergistic interactions between the enzymes involved in nanocellulose production were demonstrated, with Colby factor values greater than one.
ARTICLE | doi:10.20944/preprints202007.0726.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: Carboxymethylcellulose; Pectin; Plum; Qualitative attributes; Enzymatic activity; Postharvest
Online: 31 July 2020 (03:04:33 CEST)
Polysaccharide-based edible coatings are served as an attractive preservation method for postharvest maintenance of most fruits. The current study examined the effect of carboxymethylcellulose (CMC)- and pectin (Pec)- based edible coatings on weight loss, firmness, total soluble solids (TSS), pH¬, titratable acidity (TA), vitamin C (vit C), total phenolics, anthocyanin and flavonoid contents, total antioxidant capacity (based on DPPH) and the activities of peroxidase (POD), polyphenol oxidase (PPO) and polygalacturonase (PG) enzymes during cold storage. The results showed that each coating and their combinations caused positive effects in all measured parameters except weight loss. The applied coatings preserved firmness and improved total phenols, anthocyanin and flavonoid contents, antioxidant capacity and POD activity. In addition, the coatings retarded TSS and pH enhancement and TA and vit C loss and decreased PPO and PG activities. It could be stated that CMC at 1 % and Pec at 1.5 % separately demonstrated the best results at most measured parameters; and among the combinations 0.5 % Pec + 1.5 % CMC acted better than the other treatments. Henceforth, application of CMC and/or Pec and/or their combinations would be considered as favorable approaches to improve postharvest quality characteristics of plum fruit.
ARTICLE | doi:10.20944/preprints201903.0227.v2
Subject: Biology, Horticulture Keywords: oxidative stress; enzymatic antioxidants; malondialdehyde; membrane permeability; chlorophyll
Online: 28 March 2019 (11:15:50 CET)
Scarcity of water is one of the most serious concerns in plant biology with diverse implications at all the levels of molecular, biochemical, and physiological phenomena of plant growth, development, and consequently the productivity. Most of the strategies to induce or enhance drought tolerance in plants are unreasonably expensive and/or time-consuming. Some studies conducted in the recent past have shown that plant growth regulators (PGRs) may induce/improve physiological tolerance in plants to cope with adverse environmental conditions including drought. The present study was aimed at investigating the effects of foliar spray of GABA (0, 1, 2, and 4 mM) applied 20 days following the germination of seeds, on vegetative growth, morphological characteristics, integrity of cell-membrane, and the levels of photosynthetic pigments and enzymatic antioxidants in carrot cvs. Supertaj and Bharat, grown under 100% and 50% field capacity of soil moisture. The treated and untreated (control) carrot plants were harvested and analyzed 2 weeks following the GABA application. The results revealed that foliar application of GABA improved the vegetative growth and significantly increased the levels of free amino acids, plastid pigments, enzymatic antioxidants, and the relative water content in the root crop grown under 50% field capacity of soil moisture, compared to control. Additionally, the GABA application decreased the electrolyte leakage of ions and melondialdehyde (MDA) content in carrot leaves. The carrots harvested from GABA-treated or untreated (control) plants were not significantly different for their protein contents. In conclusion, the incorporation of GABA in the production management of carrots may help plants to mitigate the adverse effects of water deficit stress.
ARTICLE | doi:10.20944/preprints201810.0597.v1
Subject: Chemistry, Analytical Chemistry Keywords: NiFe alloy; graphene oxide; glucose; non-enzymatic sensor
Online: 25 October 2018 (06:20:16 CEST)
NiFe alloy nanoparticles/graphene oxide hybrid (NiFe/GO) was prepared for electrochemical glucose sensing. The as-prepared NiFe/GO hybrid was characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The results indicated that NiFe alloy nanoparticles can be successfully deposited on GO. The electrochemical glucose sensing performance of the as-prepared NiFe/GO was studied by cyclic voltammetry and amperometric measurement. Results showed that NiFe/GO modified glassy carbon electrode had sensitivity of 173 μA mM−1cm−2 for glucose sensing with a linear range up to 5 mM, which was superior to commonly used Ni nanoparticles. Furthermore, high selectivity for glucose detection can be achieved by NiFe/GO. All the results demonstrated that NiFe/GO hybrid was promising for using in electrochemical glucose sensing.
ARTICLE | doi:10.20944/preprints201709.0143.v1
Subject: Medicine & Pharmacology, Other Keywords: fibrinolytic acticity; FGFC1; plasminogen activator; plasminogen; enzymatic kinetic
Online: 28 September 2017 (09:17:34 CEST)
A marine fibrinolytic compound FGFC1 enhancing fibrinolysis was obtained involving in enzymatic kinetic parameters of reciprocal activation system with single chain urokinase type plasminogen activator and plasminogen. FGFC1, a kind of bisindole alkaloid from a metabolite of rare marine fungi Starchbotrys longispora FG216, modulated enzymatic kinetic parameters including fibrinolytic reaction rate and fibrin degradation characteristics. The enzymatic kinetics of fibrinolysis was described based on enzymatic reaction of chromogenic-substrate associated with p-nitroaniline (p-NA). While single chain urokinase-type plasminogen activator (pro-uPA) actived plasminogen, Kcat and kcat/km increased significantly with increase of FGFC1 concentration. Moreover, Kcat and kcat/km exhibited 26.5-fold and 22.8-fold enhanced activity at the concentration of 40 μg•mL−1 of FGFC1, respectively. The results suggested that FGFC1 improved significantly the maximum catalytic efficiency and the total catalytic activity of fibrinolysis base on the reciprocal activation of pro-uPA and plasminogen. Km increased with increasing FGFC1 concentration, which indicated that FGFC1 decreased slightly the affinity activity of pro-uPA and plasminogen versus enzyme substrate. The marine bisindole alkaloid FGFC1 enhanced fibrinolysis which was taken on enzymatic kinetic characteristics.
ARTICLE | doi:10.20944/preprints202212.0266.v1
Subject: Materials Science, Biomaterials Keywords: enzymatic hydrolysis; biomodified kraft pulp; cellulose; xylan; cryogel; crystallinity
Online: 15 December 2022 (06:23:58 CET)
The global development of bioeconomy is impossible without technologies for comprehensive processing of plant renewable resources. The use of proven pretreatment technologies raises the possibility of industrial implementation of enzymatic conversion of polysaccharides from lignocellulose considering the process complexity. For instance, a well-tuned kraft pulping produces a substrate easily degraded by cellulases and hemicelulases. Enzymatic hydrolysis of bleached hardwood kraft pulp was carried out using an enzyme complex of endoglucanases, cellobiohydrolases, b-glucosidases and xylanases produced by recombinant strains of Penicillium verruculosum at a 10 FPU/g mixture rate and a 10 % substrate concentration. As a result of biocatalysis the following products are obtained: sugar solution, mainly glucose, xylobiose, xylose, as well as other minor reducing sugars; modified complex based on cellulose and xylan. The composition of biomodified kraft pulp was determined involving the use of HPLC. The method for determining the degree of crystallinity on an X-ray diffractometer was used to characterize the properties. The article shows the possibility of producing modified cellulose cryogels by amorphization with concentrated 85 % H3PO4 followed by precipitation with water and supercritical drying. Analysis of the enzymatic hydrolysate composition reveals the predominance of glucose (55...67 %) in reducing sugars with a maximum content of up to 6 % in the solution after 72 hours. The properties and structure of modified kraft pulp were shown to change during biocatalysis, in particular, the degree of crystallinity increased by 5 % after 3 hours of enzymatic hydrolysis. We obtained cryogels based on the initial and biomodified kraft pulp with conversion rates of 35, 50 and 70 %. The properties of these cryogels are not inferior to those based on industrial microcrystalline cellulose, as confirmed by the specific surface area, degree of swelling, porosity and SEM images. Thus, kraft pulp enzymatic hydrolysis offers prospects not only for producing sugar-rich hydrolysates for microbiological synthesis, but also for producing cellulose powders and cryogels with specified properties.
ARTICLE | doi:10.20944/preprints202111.0130.v1
Subject: Life Sciences, Biotechnology Keywords: biorefining; bran; enzymatic hydrolysis; green-extraction; hydroxycinnamates; sustainability; valorization
Online: 8 November 2021 (12:04:08 CET)
Biorefining by enzymatic hydrolysis (EH) of lignocellulosic waste material due to low costs and affordability has received enormous interest amongst scientists as a potential strategy suitable for the production of bioactive ingredients and chemicals. In the present study, a sustainable and eco-friendly approach to the extraction of bound ferulic acid (FA) has been demonstrated using a single-step EH by a mixture of lignocellulose-degrading enzymes. For comparative purposes of the efficiency of EH, an online SFE-SFC-MS extraction and analysis approach was applied. The experimental results demonstrated up to 369.3 mg 100 g−1 FA released from rye bran after 48 h EH with Viscozyme L. The EH of wheat and oat bran with Viscoferm for 48 h resulted in 255.1 and 33.5 mg 100 g−1 of FA, respectively. The extraction of FA from bran matrix using the SFE-CO2-EtOH delivered up to 464.3 mg 100 g−1 of FA, though the extractability varied depending on the parameters used. The 10-fold and 30-fold scale-up experiments confirmed the applicability of EH as a bioprocessing method valid for industrial-scale. The highest yield of FA in both scale-up experiments was obtained from rye bran after 48 h of EH with Viscozyme L. In purified extracts, the absence of xylose, arabinose, and glucose as final degradation products of lignocellulose was proven by a HPLC-RID system. Up to 94.0% purity of FA was achieved by SPE using the polymeric reversed-phase Strata X column and 50% EtOH as eluent.
REVIEW | doi:10.20944/preprints202101.0194.v1
Subject: Engineering, Automotive Engineering Keywords: ultrasound; ultrasonic reactors; enzyme kinetic; enzymatic reaction; sonochemical reaction
Online: 11 January 2021 (12:49:39 CET)
The use of low-intensity ultrasound has gotten surprising consideration over the last decade as a method for enhancing the catalytic activity of the enzyme. Ultrasounds have the potential to significantly influence the activity of the enzymatic processes, provided that the energy input is not too high to inactivate the enzyme. By providing the variation in parameters, various physical and chemical effects can be attained that can enhance the enzymatic reaction. Ultrasonic reactors are known for their application in bioprocesses. However, the potential of their applications is still limited broadly due to the lack of proper information about their operational and performance parameters. In this review, the detailed information about ultrasonic reactors is provided by defining the different types of reactors, number and position of ultrasonic transducers. Also, it includes the mechanism of intensification and influence of ultrasonic parameters (intensity, duty cycle and frequency) and enzymatic factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment.
REVIEW | doi:10.20944/preprints202009.0449.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: oleaginous microorganisms; microbial lipids; lignocellulose pretreatment; enzymatic hydrolysis; inhibitors
Online: 19 September 2020 (05:16:42 CEST)
The microorganisms able of accumulating lipids in high percentages, known as oleaginous microorganisms, have been widely studied as an alternative for producing oleochemicals and biofuels. Microbial lipid, so called Single Cell Oil (SCO), production depends on several growth parameters, including the nature of the carbon substrate, which must be efficiently taken up and converted into storage lipid. Οn the other hand, substrates considered for large scale applications must be abundant and of low acquisition cost. Among others, lignocellulosic biomass is a promising renewable substrate containing high percentages of assimilable sugars (hexoses and pentoses). However, it is also highly recalcitrant and therefore it requires specific pretreatments in order to release its assimilable components. The main drawback of lignocellulose pretreatment is the generation of several by-products that can inhibit the microbial metabolism. In this review, we discuss the main aspects related to the cultivation of oleaginous microorganisms using lignocellulosic biomass as substrate, hoping to contribute to the development of a sustainable process for SCO production in the near future.
ARTICLE | doi:10.20944/preprints201703.0032.v1
Subject: Chemistry, Applied Chemistry Keywords: organic pollutants; enzymatic remediation; sulforhodamine B; soybean peroxidase; chloroperoxidase
Online: 6 March 2017 (07:45:32 CET)
Enzymatic degradation of organic pollutants is a new and promising remediation approach. Peroxidases are one of the most commonly used classes of enzymes to degrade organic pollutants. However, it is generally assumed that all peroxidases behave similarly and produce similar degradation products. In this study, we conducted detailed studies of the degradation of a model aromatic pollutant, Sulforhodamine B dye (SRB dye), using two peroxidases—soybean peroxidase (SBP) and chloroperoxidase (CPO). Our results show that these two related enzymes had different optimum conditions (pH, temperature, H2O2 concentration...etc.) for efficiently degrading SRB dye. High-performance liquid chromatography and LC-mass spectrometry analyses confirmed that both SBP and CPO transformed the SRB dye into low molecular weight intermediates. While most of the intermediates produced by the two enzymes were the same, the CPO treatment produced at least one different intermediate. Furthermore, toxicological evaluation using lettuce (Lactuca sativa) seeds demonstrated that the SBP-based treatment was able to eliminate the phytotoxicity of SRB dye, but the CPO-based treatment did not. Our results show, for the first time, that while both of these related enzymes can be used to efficiently degrade organic pollutants, they have different optimum reaction conditions and may not be equally efficient in detoxification of organic pollutants.
ARTICLE | doi:10.20944/preprints202208.0271.v1
Subject: Chemistry, Inorganic & Nuclear Chemistry Keywords: non-enzymatic sensor; glucose; cobalt metal-organic framework; electrochemical detection
Online: 16 August 2022 (03:26:09 CEST)
The integration of metal nanoparticles and solid carriers can achieve ideal stability, high load and good conductivity. In this work, copper nanoparticles (Cu NPs) were sequentially deposited on a cobalt metal-organic framework (Co-MOF) by bonding with exposed imino groups, followed by a reduction reaction to prepare a new Cu@Co-MOF composite. Cu@Co-MOF acts as a non-enzymatic electrochemical sensor to detect glucose (Glu) in an alkaline medium. The composite working electrode of Cu@Co-MOF/GCE (GCE = glassy carbon electrode) improves the electrocatalytic activity for Glu oxidation. Cu@Co-MOF/GCE shows excellent electrocatalytic performances in Glu concentration ranging 0.005~1.8 mmol∙L−1 (mM): the sensitivities are 282.89 μA∙mM−1∙cm−2 in 0.005-0.4 mM Glu and 113.15 μA∙mM−1∙cm−2 in 0.4-1.8 mM Glu respectively with low detection limit of 1.6 μM (S/N = 3) and high selectivity and stability.
ARTICLE | doi:10.20944/preprints201810.0590.v1
Subject: Life Sciences, Other Keywords: biofortification; antioxidants; soilless culture; nutraceutical quality; enzymatic activity; plant resistance
Online: 25 October 2018 (05:22:51 CEST)
Selenium (Se) is an essential element of the human diet. Therefore, it is necessary to implement Se in agricultural fertilization, although it is not considered as an essential element for plants, Se provides benefits at the level of redox metabolism, increasing the resistance of plants to various stress factors. The increase of the availability of selenium with the use of biopolymer complexes was sought in Great Lakes lettuce grown in substrate pots treated with SeO2 (5 mg L Se), Cs-PAA + Se (5 mg L Se), and Cs-PAA. The redox metabolism was modified by increasing the enzymatic activity of glutathione peroxidase. The use of Cs-PAA + Se biopolymer complexes increase selenium up to 24 mg/Kg dry weight (DW) in plant tissues.
ARTICLE | doi:10.20944/preprints202212.0566.v1
Subject: Life Sciences, Microbiology Keywords: Alternaria alternata; pear pathogens; multi-infections; enzymatic activities; mycotoxins; plant necrosis.
Online: 30 December 2022 (01:57:03 CET)
This lack of information is due to the primary role of HSTs in fungal pathogenesis, which often masks the functions of NSTs and CWDEs. So, the toxic effects of A. alternata metabolites due to NSTs and CWDEs have received minor attention than those reported for HSTs mycotoxins . A wider study of the activity of isolated fungal metabolites can allow the identification of compounds directly related to the pathogenic activity of the fungus, making it possible to create chemo libraries that facilitate the linking of the structure of the compounds with the species that produce it and its effect on host and non-host crops, as well as with biosynthetic features . In this context, our work reports a study focused on NSTs and CWDEs used by an A. alternata strain isolated from infected pears in Italy. To this aim, the characterization of hydrolytic enzyme activities of A. alternata and the identification of the metabolites produced in vitro were performed. Furthermore, the phytotoxic activity of the isolated compounds was evaluated on pear (host and non-host varieties) and lemon fruits . Finally, the competition of A. alternata with other pathogens was evaluated to investigate the role of NSTs on co-infections.
ARTICLE | doi:10.20944/preprints201809.0575.v1
Subject: Chemistry, Chemical Engineering Keywords: melanin like nanoparticles; sodium periodate; enzymatic activity; layer-by-layer films
Online: 29 September 2018 (04:16:12 CEST)
Polydopamine (PDA) deposition, obtained from the oxidation of dopamine and other catecholamines is an universal way to coat all known materials with a conformal coating which can subsequently be functionalyzed at will. The structural analogies between polydopamine and eumelanin, the black-brown pigment of the skin, incited to produce stable polydopamine nanoparticles in solution instead of amorphous precipitates obtained from the oxidation of dopamine. Herein, we demonstrate that size controlled and colloidally stable PDA based nanoparticles can be obtained in acidic conditions, where spontaneous auto-oxidation of dopamine is suppressed, using sodium periodate as the oxidant and a protein like alkaline phosphatase as a templating agent. The size of the PDA@Alp nanoparticles depends on the dopamine/enzyme ratio and the obtained particles display the enzymatic activity of alkaline phosphatase with an activity extending up to two weeks after particle synthesis. The PDA@ alkaline phosphatase (Alp) nanoparticles can be engineered in polyelectrolyte multilayered films to potentially design model biosensors.
ARTICLE | doi:10.20944/preprints201711.0074.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: cuprous oxide; non-enzymatic glucose sensor; alkaline solution; differential pulse voltammetry
Online: 13 November 2017 (03:15:30 CET)
A cuprous oxide (Cu2O) thin layer served as the base for a non-enzymatic glucose sensor in an alkaline medium, 0.1 NaOH solution, with a linear range of 50-200 mg/dL using differential pulse voltammetry (DPV) measurement. An X-ray photoelectron spectroscopy (XPS) study confirmed the formation of the cuprous oxide layer on the thin gold film sensor prototype. Quantitative detection of glucose in both phosphate-buffered saline (PBS) and undiluted human serum were carried out. Neither ascorbic acid nor uric acid even at a relatively high concentration level of 100mg/dL in serum interfered with the glucose detection, demonstrating the excellent selectivity of this non-enzymatic cuprous oxide thin layer based glucose sensor. Chronoamperometry (CA) and single potential amperometric voltammetry were used to verify the measurements obtained by differential pulse voltammetry (DPV), and the positive results validated that the detection of glucose in a 0.1 M NaOH alkaline medium by DPV measurement was effective. Nickel, platinum and copper are commonly used metals for non-enzymatic glucose detection. The performance of these metal-based sensors for glucose detection using DPV were also evaluated. Cuprous oxide (Cu2O) thin layer based sensor showed the best sensitivity for glucose detection among the sensors evaluated.
ARTICLE | doi:10.20944/preprints202207.0085.v1
Subject: Biology, Other Keywords: Crown of thorns starfish (CoTS); actinomycetes; venom extract; anti-enzymatic; antibacterial activity
Online: 6 July 2022 (04:03:02 CEST)
Crown of thorns starfish (Acanthaster planci) are coral predators with advantages of having toxicity in their venom and tissue regeneration capabilities. With all these characteristics, only a handful of studies have highlighted the association of microorganisms with this organism. Crown of thorns starfish are common in Fiji and their analyses of microbial diversity for secondary metabolites could be of great interest to the scientific community. This study is an attempt to investigate Fijian-based A. planci for their venom and associated actinomycetes antibacterial activity and further identify the type of enzymes present in the crude venom extract. The CoTS venom extract (0.192 g) harbor enzymes such as gelatinase, caesinase, and amylase. An abundant and potent actinomycete strain, represented as FJA1 showed antibacterial activity against Enterococcus faecium with an inhibition zone of 10 mm. Moreover, all pathogenic test microorganisms were resistant against concentrations of 500 µg and 1 mg of A. planci venom extract.
ARTICLE | doi:10.20944/preprints202204.0236.v1
Subject: Life Sciences, Biochemistry Keywords: biorefining; by-products; enzymatic hydrolysis; essential fatty acids; green-extraction; lipids; sustainability
Online: 26 April 2022 (10:37:03 CEST)
The main intention of the present work was to investigate the ability of cellulose-degrading enzymes (C-DE) to release fatty acids (FAs) from complex matrices of cereal by-products during enzymatic hydrolysis (EH). For this purpose, three types of cereal bran (CB), i.e., wheat, rye, and oat were used as a lignocellulose substrate for three commercially available hydrolytic enzymes, i.e., Viscozyme L, Viscoferm, and Celluclast 1.5 L. The yield and composition of FAs after EH was assessed and confronted with the yield obtained after either conventional Soxhlet extraction or alkaline-assisted hydrolysis (A-AH) with 10% KOH in 80% MeOH and subsequent liquid-liquid extraction. The experimental results demonstrated that up to 6.3% and 43.7% higher total FAs yield can be achieved within EH of rye bran using Celluclast 1.5 L than by A-AH and Soxhlet extraction, respectively. However, the application of Viscoferm for EH of wheat bran ensured up to 7.7% and 13.4% higher total FAs yield than A-AH and Soxhlet extraction, respectively. The concentration of essential linolenic acid (C18:3) in lipids extracted after EH of rye bran with Celluclast 1.5 L was up to 24.4% and 57.0% higher than in lipids recovered by A-AH and Soxhlet extraction, respectively. In turn, the highest content of linolenic in wheat bran lipids was observed after EH with Viscoferm and Viscozyme L, ensuring 17.0 and 13.6% higher yield than after A-AH, respectively. SEM analysis confirmed substantial degradation of CB matrix promoted by the ability of C-DE to act specifically on 1,4-β-D-glycosidic bonds in cellulose and on 1,2-α-,1,3-α-, and 1,5-α-L- arabinofuranoside and 1,4-β-D-xylosidic bonds in arabinoxylans, arabinans, and other arabinose-containing hemicelluloses. Structural alteration in cells integrity greatly contributed to the release of bound FAs and their better transfer into the extraction solvent. It has been shown that the proposed process of EH can be used for the efficient release of FAs from the CB matrix more sustainably and with a safer profile, thereby representing the further sustainable production of FAs for certain purposes.
REVIEW | doi:10.20944/preprints202104.0652.v1
Subject: Materials Science, Biomaterials Keywords: Enzymatic reaction; crosslinking; Hydrogel; Biomedical application; Tissue engineering; Wound healing; Drug delivery
Online: 26 April 2021 (10:52:24 CEST)
Self-assembled structures mostly arises through enzyme-regulated phenomena in nature under persistent conditions. Enzymatic reactions are one of main biological processes in fabrication and construction of supramolecular hydrogel networks required for biomedical applications. The enzymatic processes provide a unique opportunity to integrate hydrogel formation. In most of cases, structure and substrates of hydrogels are adjusted by enzyme catalysis due to the chemo-, regio- and stereo-selectivity of enzymes. Hydrogels processed by using various enzyme schemes showed remarkable characteristics as dynamic frames for cells, bioactive molecules and drugs in biomedical applications. A novel class of enzyme-mediated crosslinking hydrogels mimics the extracellular matrices by displaying unique physicochemical properties and functionalities like water-retention capacity, drug loading ability, biodegradability, biocompatibility, biostability, bioactivity, optoelectronic properties, self-healing ability, shape memory ability. In recent years, many enzymatic systems investigated hydrogel cross-linking. Results of biocompatible hydrogel products show that these mechanisms of crosslinking can fulfill requirements for variety of biomedical applications including tissue engineering, wound healing and drug delivery.
ARTICLE | doi:10.20944/preprints202011.0195.v1
Subject: Chemistry, Analytical Chemistry Keywords: codfish; enzymatic hydrolysis; proteomics; bioinformatic prediction; emulsifying properties; antioxidative activity; bioactive peptides
Online: 4 November 2020 (12:44:08 CET)
Protein hydrolysates show great promise as bioactive food and feed ingredient and for valorization of side-streams from e.g. the fish processing industry. This study characterizes bulk emulsifying, foaming, and in vitro antioxidative properties of hydrolysates derived from cod frame by application of Alcalase and Neutrase, individually and sequentially as well as the influence of heat-treatment prior to hydrolysis. We present a novel approach that utilizes proteomics data for calculation of weighted mean peptide properties (length, molecular weight, and charge) and peptide-level abundance estimation. Using subsequent bioinformatic prediction of biofunctional properties to describe observed bulk properties, we are able to provide an in-depth hydrolysate characterization not previously seen. All hydrolysates displayed comparable or higher emulsifying activity and stability than sodium caseinate. Heat-treatment significantly increased stability but showed a negative effect on the activity and degree of hydrolysis. Combining peptide abundance with predicted emulsifying activity, we were able to identify several peptides that are likely linked to the observed differences in bulk emulsifying properties. In general, decreased hydrolysis resulted in significantly higher chelating activity, while the opposite was observed for radical scavenging activity. The study highlights the prospects of applying proteomics and bioinformatics for hydrolysate characterization and in food protein science.
ARTICLE | doi:10.20944/preprints202005.0427.v2
Subject: Medicine & Pharmacology, Allergology Keywords: Isolation; Neural Crest-derived Stem Cells; Mechanical Dissociation; Regenerative Periodontology, Enzymatic Digestion
Online: 2 November 2020 (11:35:16 CET)
Periodontitis is microbial infection affecting periodontium, the tooth supporting structure and affects >743 million people worldwide. Neural crest-derived stem cells (NCSCs) hold the promise to regenerate the damaged periodontium. These cells have been identified within adult adipose tissue, periodontal ligament, and palatal tissue. Typical enzymatic isolation protocols are expensive, time consuming and often not clinically compliant. Enzyme-free, mechanical dissociation has been suggested as an alternative method of generating cell suspensions required for cell separation and subsequent expansion ex vivo. In our study, samples of rat skeletal muscle tissue were used to appraise the suitability of a novel mincing method of mechanical dissociation against enzymatic digestion with collagenase and dispase. Skeletal muscle is readily available and has been shown to contain NCSC populations. We used a Rigenera-Human Brain Wave® prototype mincer to produce a suspension of skeletal muscle-derived cells modeling NCSCs. We have compared the resulting cell cultures produced via mechanical dissociation and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Cell Sorting (MACs) and Fluorescence-activated cell sorting (FACS). Despite the Countess Automated Cytometry data demonstrating that cell suspensions produced by mechanical dissociation (n=24) contain on average 26.8 times as many viable cells as enzymatic cell suspensions (n=18), enzymatic suspensions produced more successful cell cultures. Spheroids and subsequently adherent cells formed from 4 enzymatic cell suspensions (44.4%) vs. 1 mechanical cell suspension (8.3%). Enzymatic digestion protocols formed spheroids faster and more plentifully than mechanical cell suspensions. Adherent cells and spheroids isolated via both methods appear morphologically similarly to NCSCs from our previous studies.
ARTICLE | doi:10.20944/preprints201911.0378.v1
Subject: Materials Science, Nanotechnology Keywords: graphene; electrochemical; biosensor; heterostructure; non-enzymatic; schottky diode; glucose; glucometers; ald; pto
Online: 29 November 2019 (11:16:08 CET)
Non-enzymatic glucose sensing is a crucial field of study because of the current market demand. This study proposes a novel design of glucose sensor with enhanced selectivity and sensitivity by using graphene Schottky diodes, which is composed of Graphene/Platinum Oxide/n-Silicon heterostructure. The sensor was tested with different glucose concentrations and interfering solutions to investigate its sensitivity and selectivity. Different structures of the device were studied by adjusting the platinum oxide film thickness to investigate its catalytic activity. It was found that the film thickness plays a significant role in the efficiency of glucose oxidation and hence in overall device sensitivity. Moreover, theoretical investigations were conducted using Density Function Theory (DFT) to better understand the detection method and the origins of selectivity. The working principle of the sensors puts it in a competitive position with other non-enzymatic glucose sensors. DFT calculations provided a qualitative explanation of the charges distributed across the graphene sheet within a system of a platinum substrate with D-glucose molecules above. The proposed graphene/PtO/n-Si heterostructure has proven to satisfy these factors, which opens the door for further developments of more reliable non-enzymatic glucometers for continuous glucose monitoring systems.
ARTICLE | doi:10.20944/preprints201704.0018.v1
Subject: Chemistry, Electrochemistry Keywords: nanomaterials; non-enzymatic glucose biosensors; nanocomposites; electrodeposition; titanium dioxide nanocomposite; XPS; EIS.
Online: 4 April 2017 (09:27:36 CEST)
The performance of modified electrode of nanocomposite film consisting of polypyrrole-chitosan-titanium dioxide (Ppy-CS-TiO2) has been explored as non-enzymatic glucose biosensors. The synergy effect of TiO2 nanoparticles and conducting polymer on the current response of electrode resulted in higher sensitivity for nanocomposite modified electrode. The incorporation of TiO2 nanoparticles in the nanocomposite films were confirmed by XPS spectra. The FESEM and HR-TEM provided more evidences for the presence of TiO2 in Ppy-CS structure. Glucose biosensing properties were determined by amperommetry and cyclic voltammetry (CV) methods. The interfacial properties of nanocomposite electrodes were studied by electrochemical impedance spectroscopy (EIS). The developed biosensors showed a good sensitivity over the liner range of 1-14 mM with a detection limit of 614 μM for glucose. It also exhibited good selectivity and long term stability with no interference effect. The Ppy-CS-TiO2 nanocomposites film presented high electron transfer kinetics.
ARTICLE | doi:10.20944/preprints201608.0072.v1
Subject: Life Sciences, Biotechnology Keywords: forest residue; pretreatment; liquefaction; enzymatic hydrolysis/saccharification; fermentation; high titer bioethanol; detoxification
Online: 8 August 2016 (10:39:56 CEST)
This study evaluated batch fermentation modes, namely, separate hydrolysis and fermentation (SHF), Quasi-simultaneous saccharification and fermentation (Q-SSF), and simultaneous saccharification and fermentation (SSF), and fermentation conditions, i.e., enzyme and yeast loadings, nutrient supplementation and sterilization, on high titer bioethanol production from SPORL-pretreated Douglas-fir forest residue without detoxification. The result indicated Q-SSF and SSF were obviously superior to SHF operation in terms of ethanol yield. The enzyme loading showed a strong positive correlation between enzyme loading and the ethanol yield. The nutrient supplementation and sterility was not necessary for ethanol production from SPORL-pretreated Douglas-fir. The yeast loading showed no significant influence on the ethanol yield for typical SSF conditions. The terminal ethanol titer of 43.2 g/L, or 75.1% theoretical based on glucose, mannose, and xylose theoretical was achieved when SSF was conducted at the condition of following: whole slurry solids loading of 15%, enzyme loading of 20 FPU/g glucan, 1.8 g/kg (wet) yeast loading, without nutrition supplementation and sterilization, at 38°C, on shake flask at 150 rpm for 96h. It is believed that with mechanical mixing, enzyme loading can be substantially reduced with affect ethanol yield by using a long fermentation time.
ARTICLE | doi:10.20944/preprints201701.0029.v2
Subject: Chemistry, General & Theoretical Chemistry Keywords: electron transfer; Marcus equation; enzymatic fuel cell; hydrogen oxidation; electrode adsorption; DFT; bioelectrochemistry
Online: 9 January 2017 (02:49:00 CET)
Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron-sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from DFT calculations. The distal cubane FeS-cluster has a three cysteine and one histidine coordination [Fe4S4](His)(Cys)3 first ligation sphere. The reorganization energy (inner- and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and the all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe4S4](Cys)4 case. The fast and efficient electron transfer from the distal iron-sulfur cluster is realized by a fine-tuned protein environment which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering.
COMMUNICATION | doi:10.20944/preprints201911.0051.v1
Subject: Chemistry, Organic Chemistry Keywords: lactones; lactames; isoindolinones; non-enzymatic biosynthesis; benzophenones; polyketides; reactive natural products; SAHA; aniline, hydroxamic acids
Online: 6 November 2019 (02:53:57 CET)
The structures of recently discovered primarolides A and B suggest their non-enzymatic formation from a common 2-formylbenzophenone precursor. This hypothesis is based on the experimentally proven facile conversion of pestalone (also a 2-formyl-benzophenone) either into the isomeric lactone pestalalactone or the structurally related isoindolinone pestalachloride A. In a related fashion, the racemic isoindolinone natural product mariline A is supposed to biosynthetically originate from the corresponding keto-aldehyde and an aniline, as experimentally supported by model studies. Due to the close structural relationship with known systems, it appears highly probable that primarolides A and B were generated under the fermentation conditions from a massarinin-related 2-formylbenzophenone (proprimarolide) by reaction either with aniline or a nucleophilic catalyst, respectively. Suberoylanilide hydroxamic acid (SAHA), used as an additive during the fermentation, is supposed to act both as a source of aniline and as a nucleophilic catalyst.
REVIEW | doi:10.20944/preprints201810.0251.v1
Subject: Engineering, Electrical & Electronic Engineering Keywords: EGFET; ISFET; electrochemical cell; enzymatic biosensor; DNA–DNA biosensor; immunosensor; antigen–antibody biosensor; ionic sensor
Online: 12 October 2018 (04:38:42 CEST)
Since 1970s, a great deal of attention has been paid to the development of semiconductor–based biosensors because of the numerous advantages they offer, including high sensitivity, faster response time, miniaturization, and low–cost manufacturing for quick biospecific analysis with reusable features. Commercial biosensors have become highly desirable in the fields of medicine, food, environmental monitoring as well as military applications (e.g., Hoffmann–La Roche, Abbott Point of Care, Orion High technologies, etc.), whereas increasing concerns on the food safety and health issues have resulted in the introduction of novel legislative standards for these sensors. Numerous devices have been developed for monitoring of biological–processes such as nucleic–acid hybridization, protein–protein interaction, antigen–antibody bonds and substrate–enzyme reactions, just to name a few. Since 1980s scientific interest moved to the development of semiconductor–based devices which also include integrated front–end electronics, such as the extended–gate–field–effect–transistor biosensor which is one of the first miniaturized chemical sensors. This work is intended to be a review of the state of the art focused on the development of biosensors based extended–gate–field–effect–transistor within the field of bioanalytical applications, which will highlight the most recent research works reported in the literature. Moreover, a comparison among the diverse EGFET devices will be presented giving particular attention to the materials and technologies.
REVIEW | doi:10.20944/preprints202007.0459.v1
Subject: Chemistry, Electrochemistry Keywords: current-potential curve; multi-enzymatic cascades; multi-analyte detection; mass-transfer-controlled amperometric response; potentiometric coulometry
Online: 20 July 2020 (08:16:47 CEST)
Bioelectrocatalysis provides the intrinsic catalytic-functions of redox enzymes to non-specific electrode reactions and is the most important and basic concept for biosensors. This review starts by describing fundamental characteristics of bioelectrocatalytic reactions in mediated and direct electron transfer types from a theoretical viewpoint and summarizes amperometric biosensors based on multi-enzymatic cascades and for multi-analyte detection. The review also introduces prospective aspects of two new concepts of biosensors: mass-transfer-controlled (pseudo)steady-state amperometry at microelectrodes with enhanced enzymatic activity without calibration curves and potentiometric coulometry at enzyme/mediator-immobilized biosensors for absolute determination.
REVIEW | doi:10.20944/preprints202105.0419.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: Microgel; Janus particle; ionotropic gelation; crosslinking; cell encapsulation; enzymatic cross-linking; photopolymerization; hierarchical microgels; composite microgels; microfluidics.
Online: 18 May 2021 (11:05:01 CEST)
This article provides a systematic review of the crosslinking strategies used to produce microgel particles in microfluidic chips. Various ionic crosslinking methods for gelation of charged pol-ymers are discussed, including external gelation via crosslinkers dissolved or dispersed in the oil phase, internal gelation methods using crosslinkers added to the dispersed phase in their non-active forms, such as chelating agents, photo-acid generators, sparingly soluble or slowly hydrolyzing compounds, and methods involving competitive ligand exchange, rapid mixing of polymer and crosslinking streams, and merging polymer and crosslinker droplets. Covalent crosslinking methods using enzymatic oxidation of modified biopolymers, photo-polymerization of crosslinkable monomers or polymers, and thiol-ene “click” reactions are also discussed, as well as the methods based on sol-gel transitions of stimuli responsive polymers triggered by pH or temperature change. In addition to homogeneous microgel particles, the production of structurally heterogeneous particles such as composite hydrogel particles entrapping droplet interface bi-layers, core-shell particles, organoids, and Janus particles are also discussed. Microfluidics offers the ability to precisely tune chemical composition, size, shape, surface morphology, and internal structure of microgels by bringing in contact multiple fluid streams in a highly controlled fashion using versatile channel geometries and flow configurations and allowing controlled crosslinking.
REVIEW | doi:10.20944/preprints202111.0138.v1
Subject: Biology, Physiology Keywords: spontaneous; non-enzymatic; post translational modifications; racemization; biological clock; natural selection; allostatic load; psychological aging; psychological stress; stress response sys-tem; phase transitions.
Online: 8 November 2021 (13:08:58 CET)
In humans, age-associated degrading changes are observed in molecular and cellular processes underly the time-dependent decline in spatial navigation, time perception, cognitive and psy-chological abilities, and memory. Cross talk of biological, cognitive, and psychological clocks provides an integrative contribution to healthy and advanced aging. At the molecular level, ge-nome, proteome, and lipidome instability are widely recognized as the primary causal factors in aging. We narrow attention to the roles of protein aging linked to prevalent amino acids chirali-ty, enzymatic and spontaneous (non-enzymatic) post-translational modifications (PTMs SP), and non-equilibrium phase transitions. The homochirality of protein synthesis, resulting in the steady-state non-equilibrium condition of protein structure, makes them prone to multiple types of enzymatic and spontaneous PTMs, including racemization and isomerization. Spontaneous racemization leads to the loss of the balanced prevalent chirality. Advanced biological aging re-lated to irreversible PTMs SP has been associated with the nontrivial interplay between poor so-matic and mental health conditions. Through stress response systems (SRS), the environmental and psychological stressors contribute to the age-associated “collapse” of protein homochirality. The role of prevalent protein chirality and entropy of protein folding in biological aging is mainly overlooked. In a more generalized context, the time-dependent shift from enzymatic to the non-enzymatic transformation of biochirality might represent an important and yet un-der-appreciated hallmark of aging.
ARTICLE | doi:10.20944/preprints201812.0126.v1
Subject: Chemistry, Analytical Chemistry Keywords: advanced glycation end products (AGEs); enzymatic hydrolysis; glycation; methylglyoxal-derived hydroimidazolone 1 (MG-H1); seeds; seed ageing; seed quality; sodium dodecyl sulfate (SDS)
Online: 11 December 2018 (10:40:15 CET)
Seeds represent the major source of food protein, impacting on both human nutrition and animal feeding. Therefore, seed quality needs to be appropriately addressed in the context of viability and food safety. Indeed, long-term and inappropriate storage of seeds might result in enhancement of protein glycation, which might affect their quality and longevity. Glycation of seed proteins can be probed by exhaustive acid hydrolysis and quantification of the glycation adduct Nɛ-(carboxymethyl)lysine (CML) by liquid chromatography-mass spectrometry (LC-MS). This approach, however, does not allow analysis of thermally and chemically labile glycation adducts, like glyoxal-, methylglyoxal- and 3-deoxyglucosone-derived hydroimidazolones. Although enzymatic hydrolysis might be a good solution in this context, it requires aqueous conditions, which cannot ensure reconstitution of seed protein isolates. Because of this, the complete profiles of seed AGEs are not characterized so far. Therefore, here we propose the approach, giving access to quantitative solubilization of seed proteins in presence of sodium dodecyl sulfate (SDS) and their quantitative enzymatic hydrolysis prior to removal of SDS by reversed phase solid phase extraction (RP-SPE). Using MG-H1 as a case example, we demonstrate the applicability of this method for reliable and sensitive LC-MS-based quantification of chemically labile AGEs and its compatibility with bioassays.