Catalytic, kinetic and thermodynamic properties of free and immobilized caseinase on mica glass-ceramics

Ahmed, Samia A.; Saleh, Shireen A.A.; Abdel-Hameed, Salwa A.M.; Fayad, A. M.

doi:10.1016/j.heliyon.2019.e01674

Cited by 55 publications

(16 citation statements)

References 42 publications

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“…The decrease in activation energy upon entrapment was expected, as it is well documented that various types of immobilization cause such a decrease, usually attributed to conformational changes upon interaction with the support. 61 A large decrease is also observed for the Arrhenius exponential pre-factor, A, from 149 mM s À1 of the free enzyme in solution to 10.2 mM s À1 when entrapped. We recall that A is related to the number of collisions (fruitful or not) per second occurring with the proper orientation to react: the caging and the diffusional restrictions in the entrapped case, reduce this number.…”

Section: The Enzymatic Activitiesmentioning

confidence: 82%

Enzymes in a golden cage

Baruch-Shpigler

Avnir

2020

Chem. Sci.

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We describe a general method for the entrapment of enzymes within bulk metallic gold. This is a new approach for the immobilization of enzymes on metals, which is commonly carried out by 2D adsorption or covalent biding, that is, the enzyme is in contact with the metal at a specific contact zone of the enzyme, while most of the rest of it remains exposed to the environment. The 3D metallic encaging of the enzymes is quite different: the enzyme is in contact with the metallic cage walls all around it and is well protected inside. The porous nature of the metallic matrix enables substrate molecules to diffuse inside, reach the active site, and let product molecules diffuse out. The generality of the approach was proven by the successful entrapment of five enzymes representing different classes and different bioand medical applications: L-asparaginase (Asp), collagenase, horseradish peroxidase (HRP), laccase and glucose oxidase (GOx). GOx-gold conjugates have been of particular interest in the literature. The main challenge we had to solve was how to keep the enzyme active in the process of gold-synthesis from its cationthis required careful tailoring of reaction conditions, which are detailed in the paper. The gold entrapped enzymes gain thermal stability and protectability against harsh conditions. For instance, we could keep Asp alive at the extreme pH of 13, which normally kills the enzyme instantly. The entrapped enzymes obey the Michaelis-Menten kinetics, and activation energies were determined. Good recyclability for eight cycles was found. Multi-enzymatic reactions by combinations of the off-the-shelf bioactive enzyme@gold powders are possible, as demonstrated for the classical detection of GOx activity with HRP. Detailed material characterization and proposed mechanisms for the 3D protectability of the enzymes are provided. The new enzyme immobilization method is of wide potential uses in medicine, biotechnology, bio-fuel cells and enzymatic (electro)sensing applications.Scheme 1 (Top) Entrapped enzyme (glucose oxidase, GOx) within a golden cage. In redthe surface amino acids of GOx which contain gold binding moieties (thiols, amines, imines and carboxylate anions, taken from PDB file 1CF3). (Bottom) The entrapment processsee also Scheme 2. Scheme 2The mechanism of entrapmentsee text for details. Bottom: 2D adsorption, a completely different architecture compared with the 3D entrapment.This journal is

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Section: The Enzymatic Activitiesmentioning

confidence: 82%

Enzymes in a golden cage

Baruch-Shpigler

Avnir

2020

Chem. Sci.

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“…The half-life (t 1/2 ) is an economically important factor since it allows to establish (according to conditions) expiration and storage dates for industrial or environmental applications, forasmuch as the higher the half-life (t 1/2 ) the higher the thermostability [ 82 ].…”

Section: Discussionmentioning

confidence: 99%

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study

et al. 2021

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Background Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others. Methods A real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K. Results In the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t1/2 values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an Ed value of 41.40 KJ mol− 1, a low variation of KM and Vmax, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K. Conclusions Laccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t1/2 of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.

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“…α-Amylase enzyme was produced under submerged fermentation from isolated strain B. subtilis strain-MK1 as reported in the previous work by Ahmed et al 2019aAhmed et al , 2019b. The enzyme was partially purified using 60% (v/ v) ethanol precipitation; the precipitate was collected by centrifugation, dried, weighed, and used for α-amylase immobilization.…”

Section: Enzyme Productionmentioning

confidence: 99%

“…High stability of enzyme under industrial conditions is considered an economic advantage due to low enzyme loss. Enzyme immobilization is the simplest way to solve the stability problem of protein and reduce the expensive cost of applying them on an industrial scale (Ahmed et al 2019a(Ahmed et al , 2019b. Also, immobilization improves enzyme properties as activity, reduces the inhibition, increases stability, specificity to substrates, and avoids contamination of product by enzyme (Ahmed et al 2019a(Ahmed et al , 2019b.…”

Section: Introductionmentioning

confidence: 99%

Statistical optimization of chemical modification of chitosan-magnetic nano-particles beads to promote Bacillus subtilis MK1 α-amylase immobilization and its application

et al. 2020

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Background: α-Amylase randomly hydrolyzes starch molecule to produce oligosaccharides of different chain length. It is among the most significant hydrolytic enzymes used in industrial applications. Enzyme immobilization is the simplest way to solve the stability problem of protein under industrial harsh conditions. Magnetic nano-particles considered suitable for immobilization due to their unique characteristics. The polymer nanocarriers still the feature of modifiable surfaces of carriers for further conjugation with biomolecule. This study aims to promote the immobilization of Bacillus subtilis MK1 α-amylase using the statistical optimization of the chemical modification of the chitosan-magnetic nano-magnetic particle beads and their ability to apply. Results: B. subtilis MK1 α-amylase was successfully immobilized on chitosan-magnetic nano-particles using a method combining the advantages of both physical adsorption and covalent binding. The beads were chemically modified using polyethyleneimine (PEI) followed by glutaraldehyde (GA). Aminated beads by (PEI), activated beads by (GA), and immobilized enzyme on activated beads were characterized using FTIR. Morphological examinations of the beads surface before and after conjugation with the α-amylase enzyme were carried out using scanning electron microscope (SEM). Chemical modification parameters of the beads were optimized using response surface methodology based on central composite design. Statistical approach enhanced the immobilization yield (IY%) by 1.5-fold. The application of immobilized enzyme in the baking process enhanced dough-raising about 2.3-fold and can be reused for 5 cycles with 100% activity. Conclusions: Statistical methods are an important way to improve immobilization yield and efficiency. The ANOVA data confirmed the fitness of the model which possessed R 2 value (0.975) and the adjusted R 2 value (0.940). The results confirm the ability to reuse the immobilized enzyme in industrial processes.

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Catalytic, kinetic and thermodynamic properties of free and immobilized caseinase on mica glass-ceramics

Cited by 55 publications

References 42 publications

Enzymes in a golden cage

Enzymes in a golden cage

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study

Statistical optimization of chemical modification of chitosan-magnetic nano-particles beads to promote Bacillus subtilis MK1 α-amylase immobilization and its application

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