Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
In silico design and virtual screening of inulinase immobilization ligands with highest affinity
Inulinase (EC 22.214.171.124, inulase, endoinulinase, endo-inulinase, exoinulinase, 2,1-beta-D-fructan fructanohydrolase) is an enzyme with system name 1-beta-D-fructan fructanohydrolase. This enzyme catalyses the hydrolysis of inulin and other fructose polymers to fructose, which is a source of carbon and energy for the microorganisms and plants.
Enzymes used in modern food production, are immobilized on inert, insoluble material (carrier) by chemical or physical interactions. They have a number of significant technical advantages compared with their soluble precursors, therefore, special attention in the scientific community is given to problems of selection and modification of carriers. It is known that immobilization can lead to significant loss of enzyme activity, but selecting the appropriate carrier and method for forming a bond between the interacting components, can significantly reduce the adverse effect of matrix on the structural and functional properties of the protein, and thereby increase the specific activity of the immobilized biocatalyst.
The aim of the work was to carry out in silico design and virtual screening of inulinase immobilization ligands with highest affinity - using modern methods of computer simulation. The structure of inulinase from Aspergillus ficuum (code molecule: 3SC7) was taken as a model of the native enzyme, and used as a target for docking from a database of spatial structures of proteins (Protein Data Bank, PDB). It is important to note that inulinase was crystallized together with some organic ligands, including D-mannose, which according to the literature, is the activator of the enzyme. Known position of the sugar on the surface of the enzyme gave us the opportunity to calibrate the selected package for docking (Autodock VINA) on a quite specific example of a complex formed by mannose and inulinase.
The ligand set used in our study included relatively simple sugars (activators, inhibitors, products of enzymatic catalysis), and macromolecular compounds, glycoproteins, Pro-Phe peptide, polylactate, caffeine, and polymers. We propose a matrix for enzyme immobilization. Each of the ligands based docking had a maximum conformational freedom allowing rotation of functional groups around all single bonds. Charges on the ligand molecule were placed automatically - in the MGLTools 1.5.6.rc3 software package. Based on comparative analysis of energy and ligand binding sites, as well as on published data about the structure of the catalytic center of inulinase, assumptions were made about the localization and interaction mechanisms already in use and matrices for immobilization of the enzyme molecule were offered.
During the implementation of the research on the model of the enzyme inulinase, as well as models of its ligands and fragments, matrices for immobilization were estimated for their binding affinities. Based on the conclusions about the prospects from the pilot testing some of the compounds were proposed as agents for inulinase immobilization. The main candidates for such agents are glycoproteins, which assumed the inclusion of an additional Cys aminoacid residue - to create a possibility to form a disulfide Â«anchorÂ» with the substrate.
Maxim S. Kondratyev1
1 Institute of Cell Biophysics