Kinetic and computational analysis of the reversible inhibition of porcine pancreatic elastase: a structural and mechanistic approach

Structural and mechanistic insights were revealed for the reversible inhibition of Porcine Pancreatic Elastase (PPE); the kinetics of uninhibited and inhibited hydrolysis of substrate Suc-AAA-pNA was analyzed thoroughly. Additionally, the interactions between PPE and its inhibitor were studied by computational techniques. The uninhibited hydrolysis of Suc-AAA-pNA by PPE proceeds through a virtual transition state, involving an inferior physical and another dominating chemical step, where two stabilized reactant states precede the predominant acyl-enzyme. Different kinds of bonding with the PPE-backbone residues, including those of the catalytic triad, were found during the MD simulation of 5 ns, as key interactions favoring a higher stabilization of the best ranked complex PPE-CF3C(O)-KA-NHPh-p-CF3. The proton inventories of the inhibited hydrolysis of Suc-AAA-pNA by PPE, were ruled out the existence of any virtual transition state and thus they argue for a different mode of catalysis involving a structurally disturbed PPE molecule. Thereafter, a novel inhibition mechanism was suggested.</p

Kinetic Analysis of the Lipase-Catalyzed Hydrolysis of Erythritol and Pentaerythritol Fatty Acid Esters: A Biotechnological Application for Making Low-Calorie Healthy Food Alternatives

Contemporary consumers demand healthier and more nourishing food, and thus, alternative foods that are low-calorie in fats and/or sugars are preferred. These desired properties may be attained by substituting the fatty acid esters of erythritol and pentaerythritol due to their antioxidant action and low toxicity for humans. In this work, the catalyzed hydrolysis of five fatty acid tetraesters of erythritol and/or pentaerythritol by both porcine pancreas type VI-s lipase (PPL) and Candida antarctica lipase-B (CALB) were studied kinetically. In all cases, except the hydrolysis of pentaerythritol tetrastearate by CALB, Michaelis–Menten kinetics were observed. In addition, the pKa values of the fatty acids released due to the catalyzed hydrolysis of the studied tetraesters by CALB were estimated. In the course of the aforementioned procedures, it was found that the CALB-catalyzed hydrolysis was incomplete to various degrees among four of the five studied tetraesters (excluding erythritol tetraoleate), and one or more estimated apparent pKa values were obtained. These results are novel, and by means of applied methodology, they reveal that erythritol and/or pentaerythritol tetraesters of medium- and long-chain fatty acids are suitable candidates for use as beneficial alternatives to butter and/or sweeteners.</jats:p

The mechanism of lipase-catalyzed synthesis of food flavoring ethyl butyrate in a solvent-free system

Immobilized Lipase-B from Candida antarctica catalyzed the esterification of butyric acid and ethanol under anhydrous solvent-free reaction conditions toward the synthesis of ethyl butyrate, a compound significant as food and perfume flavoring, as well as biofuel. The proton inventory technique was efficiently applied in mixtures of the anhydrous polar solvents ethanol and deuterated ethanol (CH3CH2OD). Subsequently, and by suitable analysis of the experimental data, the aforementioned synthetic procedure seems likely to follow the kinetic mechanism ordered bi-bi involving single substrate dead-end inhibition by ethanol, whereas were estimated values of important parameters. So far is the first experimental evidence that the synthesis of ethyl butyrate, which is catalyzed by immobilized lipase, it follows an entirely different mechanism when it is performed in anhydrous solvent-free system vs. that in anhydrous n-hexane; then it may be constructive for the industrial production of fixed quality of ethyl butyrate.</jats:p

Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation

1045-1051The kinetics of immobilized enzymes can not be analyzed by means of the simple Michaelis-Menten concept, which generally fails to describe the immobilized state due to both its probable barriers, and because the active concentration of the enzyme approaches, or even exceeds this of its substrate(s). In such cases, the various experimental data are usually treated by complex rate equations comprising too many parameters acquiring different natures and meanings, depending on both the properties of the immobilization state and the experimental conditions; thus, more likely, only apparent values of the Michaelis-Menten kinetic parameters can be estimated experimentally. Likewise, immobilization is often a key method in optimizing the operational performance of enzymes, in both laboratory and industrial scale, and affects considerably the kinetics in non-aqueous and non-conventional media due to several issues as the structural changes of the enzyme molecule, the heterogeneity of the system, and the partial or total absence of water. In this work a theoretical approach is described on the formulation of simplified rate equations, reflecting also the actual mass balances of the reactants, in the case where esterification synthetic reactions are catalyzed by immobilized lipases, in either a non-aqueous organic solvent or in a non-solvent system

Correction to: Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi‑1, the first member of a new bacterial lipase family (XIX)

We have recently (8th February 2018) published our article entitled “Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi-1, the first member of a new bacterial lipase family (XVIII)” [1]. While our manuscript was going through the final stages of publication, an article by Samoylova et al. [2] was published (12th January 2018) in the journal Extremophiles, entitled “Cloning, expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII”. Since we could not have known of the work of Samoylova et al. [2] when we submitted our manuscript, and in order to avoid confusion in the scientific community, we propose to reclassify LipSm as the first characterized member of the new bacterial lipase family XIX. Therefore throughout our article [1] “lipase family XVIII” should read “lipase family XIX” (title included)

Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi-1, the first member of a new bacterial lipase family (XVIII)

Abstract Background Microbial lipases catalyze a broad spectrum of reactions and are enzymes of considerable biotechnological interest. The focus of this study was the isolation of new lipase genes, intending to discover novel lipases whose products bear interesting biochemical and structural features and may have a potential to act as valuable biocatalysts in industrial applications. Results A novel lipase gene (lipSm), from a new environmental Stenotrophomonas maltophilia strain, Psi-1, originating from a sludge sample from Psittaleia (Greece), was cloned and sequenced. lipSm was further overexpressed in E. coli BL21(DE3) and the overproduced enzyme LipSm was purified and analyzed in respect to its biochemical and kinetic properties. In silico analysis of LipSm revealed that it is taxonomically related to several uncharacterized lipases from different genera, which constitute a unique clade, markedly different from all other previously described bacterial lipase families. All members of this clade displayed identical, conserved consensus sequence motifs, i.e. the catalytic triad (S, D, H), and an unusual, amongst bacterial lipases, Y-type oxyanion hole. 3D-modeling revealed the presence of a lid domain structure, which allows LipSm to act on small ester substrates without interfacial activation. In addition, the high percentage of alanine residues along with the occurrence of the AXXXA motif nine times in LipSm suggest that it is a thermostable lipase, a feature verified experimentally, since LipSm was still active after heating at 70 °C for 30 min. Conclusions The phylogenetic analysis of LipSm suggests the establishment of a new bacterial lipase family (XVIII) with LipSm being its first characterized member. Furthermore, LipSm is alkaliphilic, thermostable and lacks the requirement for interfacial activation, when small substrates are used. These properties make LipSm a potential advantageous biocatalyst in industry and biotechnology