Enhanced activity of immobilized transglutaminase for cleaner production technologies

Gajšek, Marjetka; Jančič, Urška; Vasić, Katja; Knez, Željko; Leitgeb, Maja

doi:10.1016/j.jclepro.2019.118218

Cited by 21 publications

(15 citation statements)

References 28 publications

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“…The combi-CLEA with pectinase, xylanase, and cellulase retained 86%, 90%, and 88% activity, respectively [ 58 ]. The addition of proteic feeder BSA (at concentrations 1.25 mg/mL, 1.88 mg/mL, and 2.50 mg/mL) was used as it can potentially improve CLEAs and mCLEAs activity and more over its stability, as confirmed by our previous studies [ 75 ]. BSA can be used as protein matrix for improving enzyme stability and is usually used while working with low protein concentrations in enzyme preparation.…”

Section: Resultsmentioning

confidence: 98%

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst

et al. 2023

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Cross-linked enzyme aggregates (CLEAs) represent an effective tool for carrier-free immobilization of enzymes. The present study promotes a successful application of functionalized magnetic nanoparticles (MNPs) for stabilization of cellulase CLEAs. Catalytically active CLEAs and magnetic cross-linked enzyme aggregates (mCLEAs) of cellulase from Trichoderma reesei were prepared using glutaraldehyde (GA) as a cross-linking agent and the catalytic activity and stability of the CLEAs/mCLEAs were investigated. The influence of precipitation agents, cross-linker concentration, concentration of enzyme, addition of bovine serum albumin (BSA), and addition of sodium cyanoborohydride (NaBH3CN) on expressed activity and immobilization yield of CLEAs/mCLEAs was studied. Particularly, reducing the unsaturated Schiff’s base to form irreversible linkages is important and improved the activity of CLEAs (86%) and mCLEAs (91%). For increased applicability of CLEAs/mCLEAs, we enhanced the activity and stability at mild biochemical process conditions. The reusability after 10 cycles of both CLEAs and mCLEAs was investigated, which retained 72% and 65% of the initial activity, respectively. The thermal stability of CLEAs and mCLEAs in comparison with the non-immobilized enzyme was obtained at 30 °C (145.65% and 188.7%, respectively) and 50 °C (185.1% and 141.4%, respectively). Kinetic parameters were determined for CLEAs and mCLEAs, and the KM constant was found at 0.055 ± 0.0102 mM and 0.037 ± 0.0012 mM, respectively. The maximum velocity rate (Vmax) was calculated as 1.12 ± 0.0012 µmol/min for CLEA and 1.17 ± 0.0023 µmol/min for mCLEA. Structural characterization was studied using XRD, SEM, and FT-IR. Catalytical properties of immobilized enzyme were improved with the addition of reducent NaBH3CN by enhancing the activity of CLEAs and with addition of functionalized aminosilane MNPs by enhancing the activity of mCLEAs.

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Section: Resultsmentioning

confidence: 98%

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst

et al. 2023

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“…The protocol was previously described by Primožič et al [ 73 ]: one unit is expressed as Tu cas mL −1 , which represents amount of casein hydrolyzed per mL of sample per minute. Transglutaminase activity was determined according to the protocol described by Gajšek et al [ 74 ], using CBZ-Glutaminylglycine as substrate; one unit form 1 μmole of hydroxamate per minute at pH 6.0 and 37 °C.…”

Section: Methodsmentioning

confidence: 99%

A Comprehensive Study of the Antibacterial Activity of Bioactive Juice and Extracts from Pomegranate (Punica granatum L.) Peels and Seeds

Kupnik

Primožič

Vasić

et al. 2021

Plants

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Due to the growing awareness of Punica granatum fruit’s health-promoting properties, the pomegranate is increasingly used for food purposes. This results in the formation of biological waste products such as peels. A biowaste circular bioeconomy strategy holds great prospective for a sustainable economy. Therefore, a sustainable and environmentally friendly way of disposing of waste (e.g., use of biowaste to obtain high-value components (e.g., punicalagins, enzymes)) is crucial for the protection of the environment and human health. In the presented study, the content of total phenols and proanthocyanidins in ten samples of Punica granatum fruit (juice, aqueous (H2O) and ethanolic (EtOH) extracts of peels and seeds) was determined. Peel extracts were found to be the richest in the content of secondary metabolites and showed extremely high antioxidant potential (approximately 90% inhibition: DPPH radical scavenging activity). To the best of our knowledge, this is the first comparative study to determine the enzymatic activity of α-amylase, lipase, peroxidase, protease, and transglutaminase in different P. granatum samples. Furthermore, the antibacterial efficacy of all P. granatum samples was qualitatively determined against three strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens) and three strains of Gram-positive (Bacillus cereus, Staphylococcus aureus, and Streptococcus pyogenes) bacteria, susceptible to gaining antibiotic resistance. Moreover, the most promising peel extracts were quantified for antibacterial efficacy against tested bacteria at five different concentrations. All samples slowed down and inhibited the growth of all tested bacteria. MIC90 values (2.7 or 0.3 mg/mL) were determined in 18 out of 24 experiments (four samples, six bacteria tested). There is no research in the reviewed literature that is current with such detailed and comprehensive determination of P. granatum peel extracts antibacterial activity. The results of the research showed great potential for the use of P. granatum in the field of antibacterial activity in biomedicine applications and in the cosmetic, food, and pharmaceutical industries.

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“…In the presence of TGM, hydroxylamine was incorporated into Z-Gln-Gly to form Z-glutamyl-hydroxamate-glycine, which developed a colored complex with iron (III) detectable at 525 nm on UV-spectrophotometer. One unit of TGM activity was defined as the amount of enzyme that causes the formation of 1.0 μmol of hydroxamate per minute by catalyzing the reaction between Z-Gln-Gly and hydroxylamine at 37°C and pH 6.0. l -glutamic acid γ-monohydroxamate was used as a standard probe ( Gajšek et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

“…One unit of TGM activity was defined as the amount of enzyme that causes the formation of 1.0 μmol of hydroxamate per minute by catalyzing the reaction between Z-Gln-Gly and hydroxylamine at 37 °C and pH 6.0. L-glutamic acid γ-monohydroxamate was used as a standard probe (Gajšek et al, 2019). Activity (U/ml) of the TGM was calculated according to Eq.…”

Section: Tgm Activitymentioning

confidence: 99%

The Synthesis of (Magnetic) Crosslinked Enzyme Aggregates With Laccase, Cellulase, β-Galactosidase and Transglutaminase

Podrepšek

Knez

Leitgeb

2022

Front. Bioeng. Biotechnol.

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Immobilized enzymes have important aspects due to the fact that they possess higher stability, have the possibility to be easily removed from the reaction mixture, and are much easier to use when compared to free enzymes. In this research, the enzymes laccase, cellulase, β-galactosidase (β-gal), and transglutaminase (TGM) were immobilized by two different methods: crosslinked enzyme aggregates (CLEAs) and magnetic crosslinked enzyme aggregates (mCLEAs). The processes for CLEAs and mCLEAs preparation with different enzymes have been optimized, where the aim was to achieve the highest possible relative activity of the immobilized enzyme. The optimal conditions of the synthesis of CLEAs in mCLEAs are described, thus emphasizing the difference between the two types of immobilization based on different enzymes. This comparative study, which represents the synthesis of crosslinked enzyme aggregates using different enzymes, has not been performed so far. Moreover, the obtained activity of CLEAs and mCLEAs is presented, which is important for further use in different biocatalytic processes. Specifically, of a higher importance is the selection of enzymes involved in immobilization, as they belong to the three different most applicable enzymes (oxidoreductases, hydrolases, and transferases). The study confirmed that the resulting activity of the immobilized enzyme and the optimization of enzyme immobilization depended on the type of the enzyme. Moreover, the prepared CLEAs and mCLEAs were exposed to the supercritical carbon dioxide (scCO2) at different pressures to determine the effect of scCO2 on enzyme activity in immobilized form. Additionally, to demonstrate the reuse and stability of the immobilized enzyme, the stability and reusability tests of CLEAs and mCLEAs were performed. The catalytic performance of immobilized enzyme was tested, where the catalytic efficiency and long-term operational stability of mCLEAs were obviously superior to those of CLEAs. However, the higher activity observed for CLEAs compared to mCLEAs suggests a significant effect of magnetic nanoparticles in the stabilization of an enzyme crosslinked aggregate structure.

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Enhanced activity of immobilized transglutaminase for cleaner production technologies

Cited by 21 publications

References 28 publications

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst

A Comprehensive Study of the Antibacterial Activity of Bioactive Juice and Extracts from Pomegranate (Punica granatum L.) Peels and Seeds

The Synthesis of (Magnetic) Crosslinked Enzyme Aggregates With Laccase, Cellulase, β-Galactosidase and Transglutaminase

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