Mg Of Enzyme Research Articles

The Use of Crude Alpha-Galactosidase in the Removal of Semi-Complex Raffinose Sugars from some Types of Legumes

The study measured the proportions of the semi-complex raffinose group in selected types of legumes using active yeast Saccharomyces as a source to obtain the enzyme alpha-galactosidase in its raw form and adding this enzyme to different levels of legumes soaked under different soaking conditions of temperature, pH, and soaking period. The rates of removal of semi-complex raffinose sugars were estimated. The study aims to remove the group of semi-complex raffinose sugars (which is one of the most important anti-nutritional compounds in legumes) to obtain some types of legumes that are largely free of these compounds in order to be ready for consumption by humans, especially children. The results of the table show that the amount of semi-complex sugars of the family of RFOs, represented by raffinose. It was 0.03 gm in chickpeas, and 0.03 gm in beans, and it was concluded that the best result was obtained when using the pure enzyme α-galactosidase at a temperature of 30 ° C and a concentration of 20 mg of enzyme during 8 hours of treatment with the enzyme pH = 4 in chickpeas. As for white beans, the best result was reached when using the pure enzyme α-galactosidase at a temperature of 40°C and at a concentration of 20 mg of enzyme during 8 hours of treatment with enzyme pH = 4.

Enzymatic Degradation of Polylactic Acid Fibers Supported on a Hydrogel for Sustained Release of Lactate.

The incorporation of exogenous lactate into cardiac tissues is a regenerative strategy that is rapidly gaining attention. In this work, two polymeric platforms were designed to achieve a sustained release of lactate, combining immediate and prolonged release profiles. Both platforms contained electrospun poly(lactic acid) (PLA) fibers and an alginate (Alg) hydrogel. In the first platform, named L/K(x)/Alg-PLA, lactate and proteinase K (x mg of enzyme per 1 g of PLA) were directly loaded into the Alg hydrogel, into which PLA fibers were assembled. In the second platform, L/Alg-K(x)/PLA, fibers were produced by electrospinning a proteinase K:PLA solution and, subsequently, assembled within the lactate-loaded hydrogel. After characterizing the chemical, morphological, and mechanical properties of the systems, as well as their cytotoxicity, the release profiles of the two platforms were determined considering different amounts of proteinase K (x = 5.2, 26, and 52 mg of proteinase K per 1 g of PLA), which is known to exhibit a broad cleavage activity. The profiles obtained using L/Alg-K(x)/PLA platforms with x = 26 and 52 were the closest to the criteria that must be met for cardiac tissue regeneration. Finally, the amount of lactate directly loaded in the Alg hydrogel for immediate release and the amount of protein in the electrospinning solution were adapted to achieve a constant lactate release of around 6 mM per day over 1 or 2 weeks. In the optimized bioplatform, in which 6 mM lactate was loaded in the hydrogel, the amount of fibers was increased by a factor of ×3, the amount of enzyme was adjusted to 40 mg per 1 g of PLA, and a daily lactate release of 5.9 ± 2.7 mM over a period of 11 days was achieved. Accordingly, the engineered device fully satisfied the characteristics and requirements for heart tissue regeneration.

Synthesis and Characterization of PVA-Enzyme/GA/PPy/ PVC-KTpClPB-o-NPOE Indicator Electrodes, XRD Analysis, FTIR and Variable Signal Analysis

This study aims to synthesize and characterize PVA-Enzyme/GA/PPy/PVC-KTpClPB-o-NPOE indicator indicator electrodes, XRD and FTIR analysis and variable signal analysis for urea sensors. The method used is biosensor potentiometry with urease enzyme immobilization technique which analytes urea. Modify 6 mg of enzyme from the amount of 1 drop and 3 drops in 0.5 mL (50% water: 50% ethanol) in PVA solution and o-NPOE plasticizer in PVC-KTpClPB. The indicator electrode is designed in multi-membrane, namely PVA-Enzyme/GA/PPy/PVC-KTpClPB-o-NPOE. PPy was dissolved in H2SO4 at a concentration of 8 M. Modification of PVA-Enzyme/(GA 2.9%)/PVC-KTpClPB-(o-NPOE 61%) was carried out in one layer each. The number of enzyme drops to see the difference in the intensity of the XRD diffraction spectrum and the difference in the transmittance of the FTIR spectrum from the indicator electrode with multi membrane modified PPy with H2SO4 denoted H2SO4-1 and H2SO4-3, respectively. The best results were obtained on the indicator electrode with the notation H2SO4-1

Catalytically self-sufficient CYP116B5: Domain switch for improved peroxygenase activity.

Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions toward a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2 O2 without the addition of NAD(P)H. In this work, protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2 O2 . The full-length enzyme (CYP116B5-fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2 O2 (CYP116B5-hd), and sarcosine (CYP116B5-SOX) as source of electrons. CYP116B5-SOX performs better than CYP116B5-fl and CYP116B5-hd showing 10- and 3-folds higher activity, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.

Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC-cutinase highlights the key role of residue 243.

Enzymatic degradation of poly(ethylene terephthalate) (PET) is becoming a reality because of the identification of novel PET-hydrolysing enzymes (PHEs) and the engineering of evolved enzyme variants. Here, improved variants of leaf-branch compost cutinase (LCC), a thermostable enzyme isolated by a metagenomic approach, were generated by a semi-rational protein engineering approach. Starting from a deleted LCC form lacking the secretion signal (ΔLCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T ΔLCC variant partially (~ 67%) depolymerized amorphous PET film producing ~ 21.9 mm of products after 27 h of reaction at 72 °C. The S101N/F243T ΔLCC double variant reached a further increase in activity on PET. Notably, for both single and double variants the highest conversion yield was obtained at 55 °C. Kinetics studies and molecular dynamics simulations support that a slight decreased affinity for PET is responsible for the superior degradation performance of the S101N/F243T variant and that this stems from a slightly higher flexibility of the active site region close to position 243. Furthermore, our findings question the need for a high reaction temperature for PET degradation, at least for LCC: at ≥ 70 °C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T ΔLCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in ≤ 3 days at 55 °C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process.

Unprecedented continuous elastic foam-bed reactor for CO2 capture

Open-cell polymer foams are memory shape materials with remarkable transport properties. We have designed an elastic foam-bed reactor based on these features to intensify gas–liquid mass transfer of CO2 through in situ foam compression/decompression cycles. The cycles are ensured by a crankshaft and only a low additional electric power (<50 Watts) is required under upflow co-current conditions in order to improve the mass transfer by one order to magnitude compared to fixed foam-bed reactor (i.e. without cycles). CO2 capture is improved thanks to carbonic anhydrase-supported foam (1.3 mg of enzyme per cm3 of foam) using a supramolecular hydrogel for continuous applications.

Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase.

Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme‐based hydrolysis of post‐consumer plastic is an emerging strategy for closed‐loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Vertical scanning interferometry shows degradation rates of the PET film of 6.8 μm h−1. Structural analysis indicates the importance of leucine at position 210 for the extraordinarily high PET‐hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET −1 completely degrades post‐consumer thermoform PET packaging in an aqueous buffer at 70 °C without any energy‐intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.

Optimization of an organic solvent-tolerant lipase production by Staphylococcus capitis SH6. Immobilization for biodiesel production and biodegradation of waste greases

Using the statistical approach, this work seeks to optimize the process parameters to boost the generation of an organic solvent-tolerant lipase by Staphylococcus capitis SH6. The main parameters influencing the enzyme production were identified by using Plackett–Burman’s screening design. Among the test variables, only tryptone (25 g/L), malt extract (2.5 g/L), NaCl (10 g/L) and pH (7.0) contributed positively to enzyme production. Then, the crude lipase was immobilized by adsorption on CaCO3 at pH 10. A maximum immobilization efficiency of 82% was obtained by incubating 280 mg of enzyme with CaCO3 (1 g) during 30 min. The immobilized lipase was more stable toward organic solvents than the free enzyme. It retained about 90% of its original activity in the presence of ethanol and methanol. After that, the immobilized enzyme was used for biodiesel production by transesterification process between waste oil and methanol or ethanol during 24 h at 30 °C. Our results show that the lipase can be utilized efficiently in biodiesel industry. Likewise, we have demonstrated that the immobilized enzyme may be implicated in the biodegradability of waste grease; the maximum conversion yield into fatty acids obtained after 12 h at 30 °C, was 57%.

Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 50 °C

Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 50 °C. Cellulose obtained by the Kürschner-Hoffer method from the wood of 3-year-old poplar (Populus trichocarpa) was subjected to enzymatic hydrolysis. Cellic® CTec2 enzymes (Novozymes, Denmark) were used. The enzymatic hydrolysis was tested within the conditions recommended by the manufacturer and the literature. The process was carried out at 50 °C at various pH – 4.8, 5.0, 5.5 and enzymes doses - 25, 50 and 100 mg per 100 mg of the dry mass of cellulose. The process was ended after 24 h. The hydrolysates were analysed by high-performance liquid chromatography (HPLC) to determine the glucose content, and then the highest glucose yield. The highest glucose yield was obtained for pH 4.8 and 100 mg of enzymes per 100 mg of the dry mass of cellulose – 72 %.

Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 45 °C

Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 45 °C. The enzymatic hydrolysis with the use of industrial enzymes Cellic® CTec2 (Novozymes, Denmark) was carried out within the conditions recommended by the manufacturer and literature. Cellulose obtained by the Kürschner-Hoffer method from a wood of 3-year-old poplar (Populus trichocarpa) was used for the study. Three pH values of 4.8, 5.0 and 5.5 were applied. Also, three amounts of enzymes were used: 25, 50 and 100 mg per 100 mg of the dry mass of cellulose for each pH used. The temperature was 45 °C. Samples were taken after 24 h and subjected to chromatographic analysis to determine the glucose content in the hydrolysates, and then the process parameters allowing for the highest glucose yield after the enzymatic hydrolysis process. The highest glucose yield was obtained for pH 5.0 and 100 mg of enzymes per 100 mg of the dry mass of cellulose – 79 %.

Insights in the biocatalyzed hydrolysis, esterification and transesterification of waste cooking oil with a vegetable lipase

The present contribution investigates the biocatalytic performance of the lipase obtained from the latex of the native plant known as Araujia sericifera (ASL) in the hydrolysis followed by the esterification of released free fatty acids and transesterification of glycerides of sunflower waste cooking oil WCO. A specific enzymatic activity of 719.05 μmol mg−1 h−1 (60 % conversion of triglycerides towards free fatty acids) was obtained in the hydrolysis of WCO with 0.05 % of biocatalyst (typically, 10.0 g WCO and 5.0 mg of enzyme) and 50 % w/w of water added in 30 min of reaction at 25 °C in homogeneous type of reaction system. The lipase has the capacity to catalyze the hydrolysis of the triglycerides mainly towards monoglycerides and diglycerides in a lesser extent. The released FFAs and the remaining glycerides reacted in tandem by addition of short chain alcohols immediately after the hydrolysis (without addition of more enzyme to the reaction media). Above 90 % conversion of the FFAs was obtained with methanol, ethanol, n-propanol and n-butanol at contents as low as 1:0.2 oil: alcohol molar ratio at 25 °C. The transesterification of the remaining monoglycerides (about 20 %) was also observed although no further reaction of the triglycerides was detected even under a great excess of alcohol.

Evaluación da la actividad de tres enzimas proteolíticas como biocatalizadores lácteos

El estudio de las enzimas proteolíticas a nivel industrial tiene una gran importancia ya que su utilidad catalítica podrá ser utilizada especialmente en la industria láctea, por lo que el objetivo del estudio fue la obtención de tres enzimas proteolíticas a partir de la piña (Annanas-cosmosus L), papaya (Carica-papaya) y el higo (Ficus-carica L), para lo cual se utilizaron técnicas diferentes de extracción del látex, aplicando un diseño completamente al azar y Tukey como prueba estadística, contando con 5 repeticiones y un tamaño de unidad experimental de 5kg, dando como resultado diferencias altamente significativas entre las tres enzimas estudiadas, en tanto que, para la obtención del látex se utilizaron los métodos de la ruptura mecánica para la piña, cortes longitudinales para la papaya e higo. Se obtiene valoraciones físico-químicas de 7,62 Brix y 5,48 de pH para la papaya, 12,12 Brix y 2,20 de pH de para la piña y 7,24 Brix con 6,48 pH para el higo, mientras que, en lo que corresponde al rendimiento se puede mencionar que con 18 g/Kg la ficina es la enzima con mejor resultado. Para la actividad enzimática se empleó un diseño completamente al azar con un tamaño de unidad de 0,0375mg de enzima utilizando leche como sustrato para el proceso de catálisis reportándose valores de, 15,74 U/mg proteína con un tiempo de 73,92seg para la bromelina, 15,09 U/mg proteína con un tiempo de coagulación de 61,44seg para la papaína y 14,87 U/mg proteína con un tiempo de 58 segundos para la ficina indicando que los resultados presentan diferencias altamente significativas y demostrando que la ficina es la enzima que presenta una velocidad enzimática y tiempo de coagulación eficaces y eficientes.

CHARACTERISTICS OF ELECTRODE MEMBRANE PVA-ENZIME COATING PVC-PLASTICIZER KTpClPB FOR UREA BIOSENSOR

This study aims to determine the effect of the composition comparison of the PVA-Enzyme urease coating PVC-Plastisizer KTpClPB membrane with memmbran characterization using FTIR and analyze membrane electrodes with XRD. The method used is the potentiometric method using the Electrode Selective Ion (ISE), where the polymer used is PVA and PVC as a coating with a composition ratio of PVA: PVC is 1: 1. The same PVA and PVC were used, namely 0.0350 g, with 2 mg of enzyme and 0.0500 g and 0.0120 g of KTpClPB plasticizer. The best composition result is the PVA-Enzyme PVC-Plastisizer coating enzyme KTpClPB 0.0500 g membrane electrode characterization using XRD shows the highest intensity value is at an angle of 2θ = 44.020 which is 1,626 count second (cts) and membrane characterization using FTIR shows that there is a functional group OH, CH, C≡C, NH, CO and CH. The addition of PVA-Enzyme coating will reduce or even eliminate the amount of Wolframm and can reduce the amorphous properties of the membrane electrodes.

CHARACTERIZATION OF MEMBRANE PVA-ENZYME COATED PVC-KTpClPB AS UREA SENSOR WITH POTENTIOMETRIC METHOD

Membranes with PVA, enzymes, and PVC-KTpClPB contribute as pores on the indicator electrode of potentiometric cells to produce voltage. This study aimed to characterize membranes modified with PVA, enzymes, and PVC-KTpClPB as urea sensors. Membrane modification was done in three variations: membrane A (0.504 g of PVA, 0.504 g of PVC, 1 mg of enzymes, and 0.012 g of KTpCIPB), membrane B (0.035 g of PVA, 0.035 g of PVC, 1 mg of enzymes, and 0.012 g of KTpCIPB), and membrane C (0.035 g of PVA, 0.035 g of PVC, 1 mg of enzymes, and 0.050 g of KTpCIPB). The analysis was performed using UV-Visible to obtain the best membranes, namely membrane A and C. The preparation of electrodes was carried out using membrane A and C with various PVA coatings, such as one coating for membrane electrode A1 and C1, two coatings for membrane electrode A2 and C2, and three coatings for membrane electrode A3 and C3. XRD analysis was performed to obtain the best membrane electrode which was the membrane electrode C2. Based on the measurement analysis of electrode membrane in the potentiometric cells with electrochemical techniques, the sensitivity obtained was 19.069 mV/decade with a confidence level of R2 = 0.9434 in the range of 1.10-5-5.10-4 M and detection limit of 1.10-5M.

Biocatalytic Ethanolysis of Waste Chicken Fat for Biodiesel Production

The use of residues from vegetable oils and animal fats is an attractive alternative for the biodiesel production with lower costs, besides providing benefits to the environment. In this study, were performed enzymatic transesterification reactions of residual chicken fat with ethanol and without the presence of co-solvents. Ten lipases were tested, and the lipase of Candida antarctica (CALB) provided the best yields. Several reaction conditions were evaluated, such as: amount of enzyme, reaction time, rotation (rpm), temperature and reuse of the biocatalyst. CALB provided a 96% yield of ethyl biodiesel with the use of 20 mg of enzyme. After 10 h of reaction the yield was higher than 90% and the rotation did not significantly influence the reaction. Temperatures up to 50 °C have been tolerated by the enzyme, and it can be reused without significative loss of yield for up to ten reaction cycles.

Wrinkled Silica Nanoparticles: Efficient Matrix for β-Glucosidase Immobilization

β-Glucosidase (BG) was immobilized by adsorption on wrinkled silica nanoparticles (WSNs) giving an active and stable biocatalyst for the hydrolysis of cellobiose. WSNs exhibiting both a central-radial pore structure and a hierarchical trimodal micro-/mesoporous pore size distribution were synthesized. They were used as a matrix to immobilize BG, obtaining a biocatalyst (BG/WSNs) containing 150 mg of enzyme per gram of matrix. A complete textural and morphological characterization of BG/WSNs performed by the Brunauer–Emmett–Teller (BET) method, thermogravimetric (TG), Fourier transform infrared (FT-IR), and transmission electron microscopy (TEM) analyses showed that this matrix can generate a microenvironment particularly suitable for this enzyme. The immobilization procedure used allowed preserving most of the secondary structure of the enzyme and, consequently, its catalytic activity. The kinetic parameters of the cellobiose hydrolysis performed with the biocatalyst were determined and compared with thos...

Interlocking Enzymes in Graphene-Coated Cellulose Paper for Increased Enzymatic Efficiency.

A simple method for interlocking glucose oxidase and horseradish peroxidase in a network of cellulose fibers coated with bovine serum albumin (BSA)-exfoliated graphene (biographene) is reported here. The resulting paper reactor is inexpensive and stable. Biographene is expected to function as an electron shuttle, making the reaction between the enzyme and the substrate more efficient, and this hypothesis is examined here. The BSA used to separate the sheets of graphene provides extra carboxylic acid groups and primary amines to help interlock the enzymes and the graphene in between the fibers. The decrease in entropy associated with interlocking the enzymes on a solid support is likely responsible for the increase in enzymatic stability/activity observed. Each cellulose disk contained 5.2mg of enzyme per gram of paper and 93% of the enzyme is retained after washing for 0.5-2h. This simple methodology provides a low cost, effective approach for achieving high enzymatic activity and good loadings on a benign, versatile support.

Identification of Histidine 303 as the Catalytic Base of Lysyl Oxidase via Site-Directed Mutagenesis.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase enzyme that catalyzes the formation of crosslinkages of collagen and elastin in connective tissues by oxidative deamination of lysine. Using site-directed mutagenesis, Histidine 303 has been shown to be a key residue that acts as the necessary catalytic base for this enzyme to function properly. Histidine 303 was mutated to isoleucine to remove catalytic activity and to aspartate and glutamate, respectively, in order to provide alternate residues that could act as a general base that could maintain catalytic activity. Overexpression of the H303I mutant yielded 3.9mg of enzyme per liter of media, the H303D mutant yielded 3.3mg of enzyme per liter of media, and the H303E mutant yielded 3.0mg/L of media. Overexpression of wildtype LOX yielded 4.5mg/L of media, which is a slight improvement from previous yields. Total copper incorporation for H303I was calculated to be 68% and no copper was detected for the H303D and H303E mutants. As LOX requires the self-processed cofactor lysyl tyrosyl quinone (LTQ) for activity, total LTQ content was obtained by reacting the enzyme with phenylhydrazine and using the previously reported extinction coefficient of 15.4mM/cm. LTQ content for the wildtype enzyme was determined to be 92%, for H303I the total LTQ content was determined to be 36%, and no LTQ was detected for the H303D and H303E mutants. No catalytic activity was detected for any mutants when compared to the wildtype which has a previously reported activity of 0.11 U/mg. Comparison of excitation-emission matrices (EEM) of each of the mutants as compared to the wildtype indicate that all the mutations cause a change in the internal environment of the enzyme, albeit to varying degrees, as evidenced by the observed shifts.

Optimization of Eugenol Ester Using Statistical Approach of Response Surface Methodology

In this study, optimization of eugenol caprylate, a type of ester was successfully carried out using statistical approach of Response surface methodology (RSM). The eugenol ester was synthesized via green route of an enzymatic esterification reaction between eugenol and caprylic acid. Four important enzymology parameters were investigated such as reaction time, temperature, molar ratio of substrate and amount of enzyme. High percentage conversion of ester &gt;80 % was achieved at reaction time of 240.40 minute, 25.52 mg of enzyme’s amount, 1.13 molar ratio substrate and temperature of 56.57 °C based on the optimum conditions. The efficiency of a new dual enzymes system consisting of Novozym 435 and Lipozyme TL IM was also evaluated based on the effect of heat and its reusability on the esterification reaction.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus

β-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000U of β-galactosidase activity per liter of culture corresponding to approximately 170mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205gL−1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.