VOLUME: 10, Special Issue 02, (IC-IMAF-2023) Paper id-IJIERM-X-II, January 2023 26
A COMPARATIVE STUDY BASED ON SELECTION OF THE CELLULASE ENZYME FOR DOCKING STUDY
Aafreen Shaikh
Department of Biotechnology, Shri Umiya Kanya Mahavidhyalaya, Rangwasa Rau, Indore (M.P.)
Abstract- An enzyme that degrade the linear polysaccharide like cellulose into glucose with the aid of breaking the β-1,4- glycosidic bonds is cellulose . Those enzymes are the 0.33 largest enzymes with a tremendous capacity closer to the ethanol manufacturing and play a important function in degrading the biomass. The production of ethanol is based totally upon the capability of the cellulose to make use of the massive sort of substrates. In this take a look at, the shape of cellulase from bacillus have emerge as from PDB (PROTIEN DATBANK). Proper right here, SUGARS are used as (cellobiose, cellotetraose, cellotetriose and laminaribiose) ligand, while our enzymes (endo-β-1, 4-glucanases, exo- β-1,4glucanases (or cellobiohydrolase) and β-glucosidases) used as proteins. The docking of cellobiose, cellotetraose, cellotetriose and laminaribiose with cellulase exhibited the maximum favorable binding energy. Those docking studies determine that cellulase has more capacity closer to the cellotetraose as a substrate for the immoderate yield of ethanol.
Keywords: Protien databank (PDB), Cellobiohydrolase, Cellulase enzyme, Ethanol production, Docking studies.
1 INTRODUCTION 1.1 About Cellulase:
Cellulases are induced enzymes that can be produced by microbes when they are growing on cellulose materials. Due to their usage in the hydrolysis of cellulose, the largest biopolymer and potential source of utilisable sugars, which is used as a raw material in the production of chemicals and fuel, they are extensively investigated [1]. A D-glucose unit and a 1, 4-D glucoside bond bind together to form the unbranched glucose polymer known as cellulose. It is broken down by enzymes made with the help of fungus and bacteria. Since cellulose is a totally stable polymer, numerous enzymes, including, must work in concert to hydrolyze it effectively. Exo-glucanases, Endo-glucanases, and -glucosidases are three types of glucanases.
1.2 Based on the kind of reaction they catalyse, there are five basic categories of cellulases:
Endocellulases (EC 3.2.1.4)
Exocellulases or cellobiohydrolases (EC 3.2.1.91)
Cellobiose dehydrogenase).
Cellulose
2 ACTION OF CELLULASE ENZYME ON CELLULOSE
Fig.1 Phylogeny of the genus Bacillus according to [8].
https://en.wikipedia.org/wiki/Cellulase
VOLUME: 10, Special Issue 02, (IC-IMAF-2023) Paper id-IJIERM-X-II, January 2023 27
Cellulases are a group of enzymes that can hydrolyze the structure of cellulose to produce cellobiose, which can then be broken down into glucose monomers. Cellulase systems contain three main enzymes that cooperate to hydrolyze cellulose into glucose monomer:
B-glucosidase,
Exocellulase, and
Endo-Cellulase.
Beginning with endo-cellulase, which restricts randomly at 1, 4-, the biodegradation of cellulose begins. Glucosidic connections in the cellulose through the production of various oligosaccharide lengths with the fresh chain links. Eventually, this action causes the crystalline structure of the cellulose.
Exocellulase then cleaves the reducing and non-reducing ends of this new oligosaccharides chain, resulting in the main products being either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase).
The leftover cellobiose or cellotetraose is then hydrolyzed into glucose by glucosidase to finish this procedure. The intermediate product cellobiose, which is a potent inhibitor of endoglucanase and exoglucanase, becomes one of the main obstacles to enzymatic hydrolysis.
The catalytic interactions between -glucosidase and cellobiose are not well understood, yet. Therefore, in this investigation, molecular docking studies have been used to try to understand the binding effectiveness of the cellulase enzyme of the BACILLUS species with the four polysaccharides subunits cellobiose, cellotetraose, cellotetriose, and laminaribiose [12–14].
https://en.wikipedia.org/wiki/Bacillus (16) 2.1 Tools & Database Used:
1. PDB (Protein Databank):
2. Pymol Structure Viewer:
3. Pubchem:
4. Autodock Tool [20]
3 METHODOLOGY
The bacillus sp. cellulase 3D structures are available in Protein Data Bank (PDB), the homologous sequences for building the 3D structure was searched against PDB using NCBI-BLAST (Basic Local Alignment Search Tool). Thus, here we got the 100% sequence similarity. To determine the binding affinities between two polysaccharide sub units, cellobiose, cellotetraose, the structure of cellulase from bacillus sp. are predicted by using auto dock.
3.1 Protein Preparation
The protein structure is taken from pdb (Protein databank). Then, protein is prepared for docking by applying the following steps:
By removing water
By adding kolmann charges
By adding hydrogen bond (for better protein ligand binding)
Save as “pdb”.
3.2 Ligand Generation Docking Parameters
The structures of cellobiose, cellotetraose, cellotetriose and laminaribiose were docked with the binding sites of cellulase (endocellulase, exocellulase and beta glucosidase) from bacillus sp. by using autodock with following parameters like:
Binding energy
Ligand efficiency
Intermol energy
Total internal
Number of hydrogen bonds
VOLUME: 10, Special Issue 02, (IC-IMAF-2023) Paper id-IJIERM-X-II, January 2023 28
The interactions between cellobiose, cellotetraose, from bacillus sp. as docked compound were analyzed by the auto dock.
3.3 Docking Studies
In this study, we have performed flexible ligand and rigid receptor docking by using four polysaccharides subunits and protein. The interactions of cellobiose, cellotetraose, cellotetriose and laminaribiose were docked with in the predicted site of cellulase from BACILLUS sp. The docking interactions between the binding site residues and the compounds with their respective binding score were given in table 2.1, 2.1, 2.3 respectively.
The binding efficiency of cellulase enzyme of BACILLUS sp. with two polysaccharide sub units, cellobiose, cellotetraose was determined by the docking interactions have shown in table 2.1, 2.1, 2.3 respectively.
This study suggest that one can enhance the efficiency of enzymatic hydrolysis activity of cellulases by modifying the residues in the catalytic site for the better yield and a significant impact on the production of biofuels like ethanol.
4 RESULTS AND DISCUSSION
In this project, the bacillus sp. cellulase 3D structures are available in Protein Data Bank (PDB), the homologous sequences for building the 3D structure was searched against PDB using NCBI-BLAST (Basic Local Alignment Search Tool).
The protein structure from pdb are:
Protein structure Accession number 1. Endocellulase
2. Exocellulase 3. Beta glucosidase
accession no: 4zyp Accession no: 5BV9 Accession no:1QOX Table1.1 protein structure accession no
After that, the sugars which are used as ligand are extracted from PubChem. Then, it is converted into pdb format by using pymol for their better compatibility.
The ligand (sugars) used:
Structure of ligand Accession number
Cellobiose 10712
cellotetroase 439626
Table 1.2 structure of ligand from pubchem
The protein we have been used then analyzed or compared in order to find out the best compatible ligand binding docking.
The best compatible can be used for the biofuel production of ethanol by the degradation sugars (cellobiose, cellotetriose) by the several classes of cellulase enzymes (endocellulases, exocellulases, beta glucosidase) which has been used as protein and sugars structure are used as ligand .
The best protein ligand compatible can be find out by comparing the following parameters:
Binding energy
Ligand efficiency
Intermol energy
Total internal energy
Number of hydrogen bond
Docking complex Binding energy Endocellulase with cellobiose -7.33 Endocellulase with cellotetrose -14.75 Exocellulase with cellobiose -7.53 Exocellulasewith cellotetrose -14.21 Beta glucosidase with cellobiose -6.06 Beta glucosidase with cellotetrose -13.68
VOLUME: 10, Special Issue 02, (IC-IMAF-2023) Paper id-IJIERM-X-II, January 2023 29 Fig. 3 Docking of exocellulase with cellotetroase
Fig.4 Docking of endocellulase with cellotetroas
Fig.5 Docking of beta glucosidase with cellotetroase And from the above data we can conclude that,
From our topic of comparison of binding efficiency of cellulase enzyme with sugars (cellobiose, cellotetroase), we came to conclude that the binding efficiency of cellulase shows best compatibility with cellotetroase. This docking studies revealed that the cellulase has greater potential towards the cellotetroase as a substrate for high yield of ethanol. The results of this study may prompt out new ideas for strategic approaches by experimentalists to design higher enzymes aiming more efficient enzymatic hydrolysis process with higher yields while also maintain the cost of production of biofuel.
VOLUME: 10, Special Issue 02, (IC-IMAF-2023) Paper id-IJIERM-X-II, January 2023 30 REFERENCE
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