Abstract
In the work, we mainly used molecular dynamics (MD) simulation and protein structure network (PSN) to study subtilisin Carlsberg (SC) immobilized onto carbon nanotube (CNT) in water, acetonitrile and heptane solvents, in order to explore activation mechanism of enzymes in non-aqueous media. The result indicates that the affinity of SC with CNT follows the decreasing order of water > acetonitrile > heptane. The overall structure of SC and the catalytic triad display strong robustness to the change of environments, responsible for the activity retaining. However, the distances between two β-strands of substrate-binding pocket are significantly expanded by the immobilization in the increasing order of water < acetonitrile < heptane, contributing to the highest substrate-binding energy in heptane media. PSN analysis further reveals that the immobilization enhances structural communication paths to the substrate-binding pocket, leading to its larger change than the free-enzymes. Interestingly, the increase in the number of the pathways upon immobilization is not dependent on the absorbed extent but the desorbed one, indicating significant role of shifting process of experimental operations in influencing the functional region. In addition, some conserved and important hot-residues in the paths are identified, providing molecular information for functional modification.
Highlights
Over last three decades, biotechnological potential of nonaqueous biocatalysis has attracted considerable interests owing to its advantages like higher selectivity, thermo-stability, lower side reactions in numerous synthetic and biocatalysis[1,2,3]
Similar to experimental operations[9,10,30], in which the enzymes were first immobilized on carbon nanotubes in aqueous solution and transferred to the organic solvents, the initial conformations of subtilisin Carlsberg (SC) adsorbed onto CNT in the organic solvents were extracted from the final snapshot of the first 100-ns Molecular dynamics (MD) trajectories of CNT-wat system
Some organic molecules could diffuse into the contact region between the enzyme and CNT, and compete with residues to interact with the hydrophobic CNT surface, in particular for the non-polar heptane molecules with stronger competition ability than the polar acetonitrile ones
Summary
Biotechnological potential of nonaqueous biocatalysis has attracted considerable interests owing to its advantages like higher selectivity, thermo-stability, lower side reactions in numerous synthetic and biocatalysis[1,2,3]. Subtilisin Carlsberg is a member of serine protease family and converts a large number of substrates to perform hydrolytic and transesterification reactions[10,21] It has been widely used for syntheses of peptides and amino acid esters in various organic solvents[21,22]. Some novel observations are obtained with respect to previous studies on adsorbed enzymes[15,16,27,28,29], which could provide valuable information for understanding the catalytic property, the specificity, the functional modification, solvent selection and some key experimental operations for the immobilized serine protease in organic media
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