Initial Enzyme Concentration Research Articles

Nanobiocatalyst based on covalent immobilization of proteases onto functionalized nanocellulose for efficient production of sunflower meal protein hydrolysates

This study aimed to engineer bio-based and environmentally friendly nanobiocatalyst systems using commercially available protease preparations, Alcalase® 2.4L (Alcalase) and Flavourzyme® (Flavourzyme), along with functionalized cellulose nanocrystals (CNC). The study highlights the advantageous properties of the obtained glutaraldehyde-activated amino-modified CNC support (GA-amino-CNC), characterized as nanorods with dimensions ranging between 100-800 nm in length and 40-60 nm in width, particularly for protease immobilization. Maximum specific activity of immobilized Alcalase (15 IU/mg of proteins) was achieved at an initial enzyme concentration of 52 mg/g of support after 3 hours, with 80% of covalently attached enzyme molecules. In the case of Flavourzyme, maximum exo-hydrolytic activity of 21 L-pNAU/g of support was observed after 20 min, while a higher yield of covalently bound enzyme molecules (53%) detected after 1 hour. The covalently bound proteases on glutaraldehyde-activated amino-modified cellulose nanocrystals were evaluated for their efficacy in producing high-quality sunflower-based protein hydrolysates suitable for food and feed applications. Sequential hydrolysis of sunflower meal protein isolate (SMPI) using the developed nanobiocatalysts yielded a maximum hydrolysis degree of 20%. In addition, more than 80% of the initial activity of nanobiocatalysts were retained after 3 reuses, suggesting that these protease nanobiocatalysts have potential application in production of SMP hydrolysates.

Fructooligosaccharides: Production by recombinant fructosyltransferase from Festuca arundinacea in a continuous reactor and kinetic modeling profile

In the quest for maximum conversion of sucrose into 1-kestose using fructosyltransferases in a continuous reactor, consideration of the reversible reactions at competing concentrations of sucrose and 1-kestose is crucial. In this study, a kinetic model based on a combined approach of rapid equilibrium and steady-state kinetics was developed to predict the profile of FOS production using recombinant sucrose: sucrose 1-fructosyltransferase from Festuca arundinacea produced in Pichia pastoris. The purified enzyme selectively converted sucrose into 1-kestose with a final yield of 0.66 g/g sucrose consumed. The reaction rate laws adequately described the kinetic profile of the substrate and the products and accurately predicted the composition of the reaction mixture. The model was fitted to experimental data obtained at three enzyme dosages and three initial sucrose concentrations. The model could predict the effect of substrate concentration on initial rate kinetics, half-saturation constant, and transferase to hydrolase ratio. For the known values of initial sucrose and enzyme concentration, the optimum reaction time and the required dilution rate were predicted for the development of a continuous process to produce 1-kestose-enriched short-chain FOS. A volumetric productivity of ∼110 g/L/h of 1-kestose with 40 % carbohydrate composition in the permeate flux was demonstrated.

The Michaelis–Menten Reaction at Low Substrate Concentrations: Pseudo-First-Order Kinetics and Conditions for Timescale Separation

We demonstrate that the Michaelis–Menten reaction mechanism can be accurately approximated by a linear system when the initial substrate concentration is low. This leads to pseudo-first-order kinetics, simplifying mathematical calculations and experimental analysis. Our proof utilizes a monotonicity property of the system and Kamke’s comparison theorem. This linear approximation yields a closed-form solution, enabling accurate modeling and estimation of reaction rate constants even without timescale separation. Building on prior work, we establish that the sufficient condition for the validity of this approximation is s0≪K\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$s_0 \\ll K$$\\end{document}, where K=k2/k1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$K=k_2/k_1$$\\end{document} is the Van Slyke–Cullen constant. This condition is independent of the initial enzyme concentration. Further, we investigate timescale separation within the linear system, identifying necessary and sufficient conditions and deriving the corresponding reduced one-dimensional equations.

An experimental and modeling approach to describe the deactivation of cellulases at the air-liquid interface.

Understanding the reaction mechanisms involved in the enzymatic hydrolysis of cellulose is important because it is kinetically the most limiting step of the bioethanol production process. The present work focuses on the enzymatic deactivation at the air-liquid interface, which is one of the aspects contributing to this global deactivation. This phenomenon has already been experimentally proven, but this is the first time that a model has been proposed to describe it. Experiments were performed by incubating Celluclast cocktail solutions on an orbital stirring system at different enzyme concentrations and different surface-to-volume ratios. A 5-day follow-up was carried out by measuring the global FPase activity of cellulases for each condition tested. The activity loss was proven to depend on both the air-liquid surface area and the enzyme concentration. Both observations suggest that the loss of activity takes place at the air-liquid surface, the total amount of enzymes varying with volume or enzyme concentration. Furthermore, tests performed using five individual enzymes purified from aTrichoderma reesei cocktail showed that the only cellulase that is deactivated at the air-liquid interface is cellobiohydrolaseII. From the experimental data collected by varying the initial enzyme concentration and the ratio surface to volume, it was possible to develop, for the first time, a model that describes the loss of activity at the air-liquid interface for this configuration.

Micro‐kinetic model of fructo‐oligosaccharide synthesis for prebiotic products

AbstractA new micro‐kinetic model of the enzyme‐catalyzed synthesis of fructo‐oligosaccharides (FOS) was developed. A commercial enzyme mixture Pectinex® Ultra SP‐L derived from Aspergillus aculeatus was used. A variety of initial enzyme concentrations (1–5 vol%) and sucrose concentrations (400–600 g/L) were experimentally investigated and included in kinetic modeling. Several variations of kinetic mechanisms and corresponding models have been examined. A hybrid genetic algorithm was used to predict the kinetic parameters simultaneously for all experimental data. The best fitting model has been adopted, and with an average error of 13.34%, it describes the experimental data very well. The influence of initial concentrations on the conversion of sucrose and production of FOS is being carefully investigated. It was shown that the initial sucrose concentration significantly affects the highest level of FOS concentration, but the enzyme concentration controls the time at which maximum is reached as well as the rate of FOS decomposition.

Stability and Esterification Activity of Candida rugosa Lipase Immobilized on Celite with Acetone Solvent

Candida rugosa lipase (CRL) is an enzyme that is widely used in biopharmaceutical and biopesticide industries due to its high catalytic ability and reusability. However, conditions from industrial processes that are often outside CRL stability range may cause denaturation or destabilization of the enzyme, causing it to be not reusable, hence lowering its economic value. To increase its catalytic activity, stability, and reusability, CRL enzyme can be immobilized in a matrix. In this study, the CRL was physically adsorbed onto Celite-545. The immobilization process was done using buffer solution with the addition of acetone. The influence of initial enzyme concentration and immobilization condition (time, temperature, and pH) were optimized using OFAT method. The data were taken in terms of esterification activity, thermal stability, and protein content. The result showed good thermal stability of the immobilized biocatalysts and an increase in esterification activity at optimum conditions that supported the efficiency of Celite-545 as lipase immobilization supports.

Kinetic Model for Galacto-Oligosaccharide Synthesis

A new micro-kinetic model is proposed for galacto-oligosaccharide (GOS) synthesis with β-galactosidase from Aspergillus oryzae. Several kinetic models with similar reaction mechanisms, i.e., steps and kinetic constants, have been investigated covering a range of initial lactose (20–40 wt %) and enzyme concentrations (0.5–2 mg/mL). The kinetic parameters were estimated simultaneously for all experimental results using a hybrid genetic algorithm. A detailed analysis of the effect of process parameters on the concentrations of lactose and GOS is performed. The proposed model has a very good quantitative fit of the experimental data, with an average error of 9.34%. More importantly, qualitative trends for the change in the concentration of lactose and GOS are predicted excellently, especially if one takes into account the variation of both concentrations of enzyme and lactose in a range wider than reported in the literature to date. Also, the analysis between the two comparative reactions of transglycosylation and lactose hydrolysis confirmed the positive effect of increasing lactose concentration on the production of GOS. On the other hand, a higher enzyme concentration resulted in a faster production of GOS but with more intensive decomposition at longer reaction times.

Application of foam separation in production of β-glucanase in Pichia

Abstract β-glucanase is widely used in many fields and has great economic value and development space, but it faces the difficulties of separation and nutrient destruction in the process of industrial production. Foam separation is a simple, mild and efficient adsorption separation technique that enables efficient separation and extraction of β-glucanase. In this study, five single factors (loading volume, pH, separation gas velocity, fermentation loading concentration, surfactant concentration) of foam separation and harvest of β-glucanase produced by Pichia pastoris were studied. The best univariate condition was: 600 mL/min separation gas velocity, loading volume of 200 mL, initial enzyme concentration of 100 g/mL, surfactant concentration of 0.3 mg/mL and pH of 5. Based on the best univariate condition, the optimal separation conditions of β-glucanase were further explored, and the five-factor four-level orthogonal test was designed. From the experimental results, the best separation condition was: 600 mL/min, loading volume of 200 mL, initial enzyme concentration of 100 μg/mL, surfactant concentration of 0.5 mg/mL and pH of 5. Under this separation condition, the enrichment ratio (E) was 0.56 and the recovery rate (R) was 96.01%.

Conversion of golden oyster mushroom, Pleurotus citrinopileatus to sugar syrup using enzymatic hydrolysis as a substrate for novel bacterial cellulose (Nata) fermentation.

Enzymatic hydrolysis of the golden oyster mushroom (Pleurotus citrinopileatus) generated a new bacterial cellulose (BC). The sugar syrup obtained from the hydrolysis of mushroom powder by commercial enzymes gave maximum total soluble solids (TSS) content at 8.83 ± 0.29°Brix, while 8.82 ± 0.06mg GAE/g substrate of total phenolic content(TPC) was obtained when using initial substrate and enzyme concentrations at 125g/L and 5.0%, respectively. Glutamic acid, aspartic acid, alanine and valine were determined as the main amino acids found in P. citrinopileatus hydrolysis at 524.74 ± 0.03, 247.09 ± 0.04, 176.82 ± 0.07 and 174.57 ± 0.01mg/100g sample, respectively. Thin-layer chromatography revealed that the obtained sugar syrup was glucose. The hydrolyzed mushroom fermented with Komagataeibacter xylinus AGR 60 at 30 ± 2°C for 9days produced optimal conditions at 4.0°Brix of the initial mushroom syrup and 12.0% (v/v) of the starter culture. Maximum BC thickness was 0.88 ± 0.03cm with 7.90 ± 0.07g dry weight, equivalent to 39.50 ± 0.35g/L and 4.39 ± 0.04g/L/day for BC production (P) and BC production rate (R p), respectively. The obtained BC was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, small-angle X-ray scattering and wide-angle X-ray diffraction. These showed the structure and functional properties as a natural source of fiber from the fermentation of a novel substrate.

Effective immobilization of lactate dehydrogenase onto mesoporous silica.

This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica. To develop mesoporous silica as support material for use in l-LDH immobilization, the particle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. The effect of some parameters such as glutaraldehyde concentration, immobilization pH, initial enzyme concentration, and immobilization time was investigated and the optimum conditions for these parameters were determined as 1% (w/v), pH 8.0, 1mg/ml, and 120min, respectively. The maximum working pH and temperature for the oxidation of lactate to pyruvate reaction were determined as 10.0 and 35°C for free and 9.0 and 40°C for immobilized l-LDH, respectively. The kinetic parameters (Km and Vmax ) of l-LDH for the oxidation of lactate to pyruvate reaction were examined as 1.02 mM and 7.58 U/mg protein for free and 0.635mM and 1.7 U/mg protein for immobilized l-LDH, respectively. Moreover, the immobilized l-LDH was 1.3-fold more stable than free l-LDH at 25°C according to calculated t1/2 values. The immobilized l-LDH retained 80% of its initial activity in a batch reactor after 14 reuses.

Preparation of immobilized lipase on Co2+-chelated carboxymethyl cellulose based MnFe2O4 magnetic nanocomposite particles

Lipase immobilization was performed via physical adsorption on a novel metal-chelating nanostructure consisted of (S)-N-(5-Amino-1-carboxypentyl) iminodiacetic acid (NTA), as a tetra-dentate chelating agent, grafted onto MnFe2O4 nanoparticles coated with carboxymethyl cellulose (CMC) for Co2+-chelated affinity lipase adsorption. The influential immobilization parameters for enzyme adsorption were investigated. The maximum amount of immobilized lipase (8.5 mg/g adsorbent) was obtained at modified conditions with initial enzyme concentration of 2 mg/mL and adsorbent dosage of 1 mg/mL after 90 min incubation at 50 mM buffer citrate at pH 5. The Fourier transform infrared spectroscopy (FTIR), energy dispersive spectroscopy (EDS), and elemental mapping provided evidence of successful immobilization of lipase. The results of kinetic study revealed that the adsorption of lipase followed pseudo second order kinetic model. Investigation on the enzymatic properties demonstrated that the immobilized lipase possesses remarkable thermal stability at 60 °C for 120 min, as the catalytic activity of immobilized lipase was 75% higher than that of free lipase at the same temperature. Beside, the immobilized lipase still retained 70% of its initial activity after 7 successive recycles, suggesting the new designed Co2+-chelated magnetic nanostructure as a great matrix for the non-covalent enzyme immobilization.

Evolution of enzyme levels in metabolic pathways: A theoretical approach. Part 1

The central role of metabolism in cell functioning and adaptation has given rise to countless studies on the evolution of enzyme-coding genes and network topology. However, very few studies have addressed the question of how enzyme concentrations change in response to positive selective pressure on the flux, considered a proxy of fitness. In particular, the way cellular constraints, such as resource limitations and co-regulation, affect the adaptive landscape of a pathway under selection has never been analyzed theoretically. To fill this gap, we developed a model of the evolution of enzyme concentrations that combines metabolic control theory and an adaptive dynamics approach, and integrates possible dependencies between enzyme concentrations. We determined the evolutionary equilibria of enzyme concentrations and their range of neutral variation, and showed that they differ with the properties of the enzymes, the constraints applied to the system and the initial enzyme concentrations. Simulations of long-term evolution confirmed all analytical and numerical predictions, even though we relaxed the simplifying assumptions used in the analytical treatment.

Study of silanized nanostructures with immobilized fumarase for production of L-malate.

The conversion of fumaric acid into L-malate by fumarase immobilized on silanized nanostructures was analyzed experimentally. The enzyme was bound to the silanized nanostructures. We carried out scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) analysis, zeta size analysis and surface area calculation for the characterization of the nanostructures. The effect of initial enzyme concentration and pH on immobilization procedure were investigated and the change of Michaelis-Menten constants (Km and Vmax) with immobilization was examined. The change in the storage stability of the enzyme by immobilization was also investigated. The stability of the immobilized enzyme was very good. We observed that the fumarase was bound to silanized nanostructures [p(HEMA)-3-MTES] in much greater amounts. We have compared the activities of free fumarase and immobilized fumarase and we have observed a significant increase in the activity of the fumarase after immobilization for L-malate production. Moreover, we came to the conclusion that this activity can be better preserved for 30 days compared to free fumarase.

Development of a continuous aqueous two-phase flotation process for the downstream processing of biotechnological products

• Process intensification by ATPF as downstream for biotechnological products. • High initial product concentration leads to high final product concentration. • Main mass transfer during first 30 min of batch ATPF. • 2.5 times the volume purified by continuous ATPF compared to batch ATPF. Aqueous two-phase flotation (ATPF) is an alternative downstream process for direct recovery of biotechnological products from crude biosuspensions. ATPF is a combination of two phase extraction and flotation, resulting in high recoveries and product concentrations. In addition to selecting a suitable two-phase system, the process design must be optimized to realize an efficient ATPF process. In this study, the focus was on the process strategy. As an example of a biotechnological product, the technical enzyme phospholipase was purified. The influence of different initial enzyme concentrations on the separation process was investigated and limitations of the separation efficiency were identified. Based on these findings, batch ATPF was transferred first to semicontinuous ATPF and later to continuous ATPF. Thus, this work describes for the first time a continuous ATPF, which showed 2.5 times the purification capacity of batch ATPF in the same flotation time.

Ethanol Production from Olive Stones through Liquid Hot Water Pre-Treatment, Enzymatic Hydrolysis and Fermentation. Influence of Enzyme Loading, and Pre-Treatment Temperature and Time

Olive table industry, olive mills and olive pomace oil extraction industries annually generate huge amounts of olive stones. One of their potential applications is the production of bioethanol by fractionation of their lignocellulose constituents and subsequent fermentation of the released sugars using yeasts. In this work, we studied the influence of temperature (175–225 °C) and residence time (0–5 min) in the liquid hot-water pre-treatment of olive stones as well as the initial enzyme loading (different mixtures of cellulases, hemicellulases and β–glucosidases) in the later enzymatic hydrolysis on the release of fermentable sugars. The Chrastil’s model was applied to the d-glucose data to relate the severity of pre-treatment to enzyme diffusion through the pre-treated cellulose. Finally, the hydrolysate obtained under the most suitable conditions (225 °C and 0 min for pre-treatment; 24 CE initial enzyme concentration) was fermented into ethanol using the yeast Pachysolen tannophilus ATCC 32691. Considering the overall process, 6.4 dm3 ethanol per 100 kg olive stones were produced.

Kinetic model for the enzymolysis of Paphia undulate by marine low-temperature alkaline proteinase

The kinetics of enzymatic hydrolysis of paphia sinensis was studied. The molecular weight distribution of the small peptide mixture was determined by liquid and mass spectrometry. The effects of initial substrate concentration on enzymatic hydrolysis reaction, the effects of initial enzyme concentration on enzymatic hydrolysis reaction, the effects of enzymatic hydrolysis time on enzymatic hydrolysis reaction and the mechanism of enzymatic hydrolysis reaction were investigated. In this experiment, the basic kinetic model of enzymatic hydrolysis of protein was derived, and the kinetic parameters and enzyme inactivation constant were determined.

Hydrolysis of lactose using β-D-galactosidase immobilized in pectin-based hydrogels: Modeling and optimization by factorial design

In this work, we studied the hydrolysis of lactose using β-d-galactosidase immobilized in pectin-based hydrogels and optimized the process using a central composite rotatable design and response surface methodology. The experimental conditions were established by optimizing the enzyme immobilization capacity of the hydrogels and glucose concentrations formed during the hydrolysis of lactose using the immobilized enzyme (dependent variables). Optimal temperature, initial enzyme concentration, and pH (independent variables) for immobilization in the hydrogel without pine fiber were 44.0 ± 1.0 °C, 7.0%, and 6.4, respectively. These values were approximately 35.0 ± 1.0 °C, 6.3%, and 5.6 for both composite hydrogels containing 5 and 10% pine fiber. The immobilization of β-d-galactosidase at optimum pH led to the absence of hydrolytic activity owing to enzyme overload, whereas higher glucose concentrations were found during the hydrolysis of lactose when β-d-galactosidase was immobilized at pH 4.0. Thus, the immobilization protocols led to β-d-galactosidase immobilized in the hydrogels with high catalytic capacity. In conclusion, the hydrolysis of lactose can be performed using β-d-galactosidase immobilized in pectin-based hydrogels for the production of low-lactose foods.

Relationship between enzyme concentration and Michaelis constant in enzyme assays

The upper bound of enzyme concentration for accurately estimating the parameters in Michaelis-Menten (MM) equation is not completely determined and still under discussion, even though many researchers have investigated the equation’s validity for a long time. In the paper, we broadly investigated the correlation between the system of ordinary differential equations for monosubstrate irreversible enzyme reaction (HMM-system) and its derivative MM equation focusing on the relationship between initial enzyme concentration [E]0 and Michaelis constant Km by numerical simulation. According to the results, the initial reaction velocity v0 is still a function of initial substrate concentration [S]0 at [E]0<Km. The function is identical to the MM equation at [E]0≦0.01Km, while it is described as a new type of equation at 0.01Km≦[E]0<Km. This function is of great significance in enzyme assays as a comprehensive approximation for the HMM-system.

Applicability of recombinant Aspergillus sojae crude mannanase enzyme in carrot juice production

The mannanases can be used for enzymatic maceration in carrot juice in order to increase the extract yield that soluble compounds such as sugars and β-carotene can easily release. In this study, the effects of microbial β-mannanase and its combination with the most commonly used commercial type enzymes in the carrot juice production on the carrot juice yield, the amount of β-carotene, and turbidity were investigated. Enzyme concentrations on yield were optimized by the Response Surface Method. As a result of the maceration process, fruit juice yields were increased. While the highest yield (58.51%) was obtained from the combination of 0.15 g β-mannanase/kg carrot and 0.3 g Viscozyme/kg carrot, the highest β-carotene concentration was found to be 2.75 mg/100 g using an individual β-mannanase enzyme. Last, the quality of the carrot juice in turbidity was improved by individual β-mannanase enzyme or β-mannanase with combining industrial enzymes. Practical applications The yield and quality of carrot juice (total β-carotene content) can be improved by crude or industrial enzyme liquefaction treatment. The concentration of the initial enzyme concentration and combination strategy is the most important parameter to obtain well-produced carrot juice. Consequently, enzyme liquefaction (maceration) process with individual β-mannanase enzyme or β-mannanase with combining industrial enzymes can be used as an important production step which increases quality in carrot juice production.

Enzymatic synthesis of prebiotic carbohydrates from lactose: Kinetics and optimization of transgalactosylation activity of β‐galactosidase from Aspergillus oryzae

AbstractThe aim of the study was to analyze galactooligosaccharide (GOS) synthesis catalyzed by β‐galactosidase from Aspergillus oryzae by applying experimental data to optimization and kinetic parameter estimation methods. The apparent kinetic parameters were determined in varying initial lactose concentrations by using six‐step, 11‐parameter GOS synthesis mechanism. The results revealed that GOS yield was mainly dependent on initial lactose concentration; on the other hand, all of the reaction parameters affected the required time to achieve maximum GOS yield (TMGY) significantly. The values of 32 °Brix, 36.8°C, and 8.32 U/g lactose solution were determined as the optimum reaction experimental set which offers 26.73% GOS yield and 369.82 min TMGY. The increased enzyme‐galactose complex dissociation ratio (k3/k−3) with decreasing initial lactose concentration resulted to low GOS productivity although GOS production/GOS dissociation rate ratio (k4/k−4) increased with decreasing initial lactose concentration. This result signified that hydrolysis characteristics dominated the reaction medium at low initial lactose concentration. The role of monosaccharide inhibition became important on GOS synthesis for increasing initial lactose concentration.Practical ApplicationsThe proposed study deals with investigation of the effects of initial lactose concentration, enzyme concentration, and temperature on enzymatic synthesis of galactoligosaccharide (GOS) from lactose by β‐galactosidase from Aspergillus oryzae (βgAO) and estimation of apparent kinetic rate constants of the reaction which is important to design of futuristic strategies improving the GOS yield. Low GOS yield is the main concern of the prebiotic food industry due to thermodynamic and kinetic realities of the reaction. Deeper evaluation of the reaction mechanism will contribute valuable data to the partners in the scope of designing more productive engineering methods.