A review on the immobilization of pepsin: A Lys-poor enzyme that is unstable at alkaline pH values

International Journal of Biological Macromolecules, Год журнала: 2022, Номер 210, С. 682 - 702

Опубликована: Май 1, 2022

Pepsin is a protease used in many different applications, and instances, it utilized an immobilized form to prevent contamination of the reaction product. This enzyme has two peculiarities that make its immobilization complex. The first one related poor presence primary amino groups on surface (just Lys terminal group). second stability at alkaline pH values. Both features this be considered complicated goal, as most protocols utilize for immobilization. review presents some attempts get pepsin biocatalyst their applications. high density anionic (Asp Glu) anion exchange simpler, but makes strategies immobilize (e.g., amino-glutaraldehyde supports) more mixed ion exchange/hydrophobic adsorption than real covalent Finally, we propose possibilities can permit not only enzyme, also stabilization via multipoint attachment.

Язык: Английский

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Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features

International Journal of Biological Macromolecules, Год журнала: 2023, Номер 248, С. 125853 - 125853

Опубликована: Июль 17, 2023

Lipase B from Candida antarctica (CALB) and lipase Thermomyces lanuginosus (TLL) have been immobilized on octyl agarose at low loading a exceeding the maximum support capacity. Then, enzymes treated with glutaraldehyde inactivated pH 7 in Tris-HCl, sodium phosphate HEPES, giving different stabilities. Stabilization (depending buffer) of highly loaded biocatalysts was found, very likely as consequence detected intermolecular crosslinkings. This did not occur for lowly biocatalysts. Next, were chemically aminated then glutaraldehyde. In case TLL, intramolecular crosslinkings (visible by apparent reduction protein size) increased enzyme stability biocatalysts, an effect that further due to Using CALB, less intense, stabilization lower, even though quite intense biocatalyst. The depended inactivation buffer. interactions between inactivating buffer effects chemical modifications suggest modification studies must be performed under target conditions.

Язык: Английский

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Robust Prediction of Enzyme Variant Kinetics with RealKcat

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Фев. 15, 2025

Accurate prediction of kinetic parameters is crucial for understanding known and tailoring novel enzymes biocatalysis. Current models fail to capture mutation effects on catalytically essential residues, limiting their utility in enzyme design. We grid-searched through ten model architectures (25,671 hyperparameter combinations) identify a gradient-based additive framework called RealKcat, trained 27,176 experimental entries curated manually (KinHub-27k) by screening 2,158 articles. Clustering catalytic turnover (k cat ) substrate affinity (K M rational orders magnitude, RealKcat achieves >85% test accuracy, demonstrating highest sensitivity mutation-induced variability thus far, the first-of-its-kind-model demonstrate complete loss activity upon deletion apparatus. Finally, state-of-the-art k validation accuracy (96%) alkaline phosphatase (PafA) mutant industrial dataset confirms RealKcat's generalizability learning per-residue relevance.

Язык: Английский

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One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

Catalysts, Год журнала: 2020, Номер 10(6), С. 605 - 605

Опубликована: Май 29, 2020

Lipases are among the most utilized enzymes in biocatalysis. In many instances, main reason for their use is high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate pursued, enzyme selectivity become problem. This case hydrolysis oils fats to produce free fatty acids alcoholysis biodiesel, which can be considered cascade reactions. these cases, original heterogeneity substrate, presence intermediate products, such as diglycerides monoglycerides, an additional drawback. Using substrates, promote that some substrates (initial products) may not recognized (in worst scenario they acting inhibitors) by enzyme, causing yields reaction rates drop. To solve this situation, mixture lipases with different specificity, differently affected conditions offer much better results than single lipase exhibiting very initial activity even best global course. from sources has been called “combilipases” becoming increasingly popular. They include liquid formulations immobilized lipases. have coimmobilized. Some discussion offered regarding problems coimmobilization give rise to, strategies proposed. The combilipases future extended other processes enzymes.

Язык: Английский

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Rhizopus oryzae Lipase, a Promising Industrial Enzyme: Biochemical Characteristics, Production and Biocatalytic Applications

Catalysts, Год журнала: 2020, Номер 10(11), С. 1277 - 1277

Опубликована: Ноя. 3, 2020

Lipases are biocatalysts with a significant potential to enable shift from current pollutant manufacturing processes environmentally sustainable approaches. The main reason of this prospect is their catalytic versatility as they carry out several industrially relevant reactions hydrolysis fats in water/lipid interface and synthesis solvent-free or non-aqueous media such transesterification, interesterification esterification. Because the outstanding traits Rhizopus oryzae lipase (ROL), 1,3-specificity, high enantioselectivity stability organic media, its application energy, food pharmaceutical industrial sector has been widely studied. Significant advances have made biochemical characterisation ROL particularly how activity affected by presence prosequence. In addition, native heterologous production ROL, latter cell factories like Escherichia coli, Saccharomyces cerevisiae Komagataella phaffii (Pichia pastoris), thoroughly described. Therefore, review, we summarise knowledge about R. (i) characteristics, (ii) strategies (iii) applications.

Язык: Английский

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