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Frontiers in Biology

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Front. Biol.    2015, Vol. 10 Issue (6) : 508-519    https://doi.org/10.1007/s11515-015-1379-6
RESEARCH ARTICLE
Selection of effective and highly thermostable Bacillus subtilis lipase A template as an industrial biocatalyst-A modern computational approach
B. Senthilkumar, D. Meshachpaul, Rao Sethumadhavan, R. Rajasekaran()
School of Biosciences and Technology, Bioinformatics Division, VIT University, Vellore 632014, Tamil Nadu, India
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Abstract

Biocatalysts are intrinsically reactive and hence their operational stability is of vital significance for any bioprocess. The setback in biocatalyst stability has been tackled from diverse prospects. Inherently, stable biocatalysts are markedly realized and a regular attempt is being made to seek out new organisms that harbor them. Here, we analyzed the industrial biocatalyst lipase A (Native) of Bacillus subtilis and its six thermostable mutants (2M, 3M, 4M, 6M, 9M and 12M) computationally using conformational sampling technique. Consequently, the various structural events deciphering thermostability like root mean square deviation, root mean square fluctuation, radius of gyration and polar surface area showed mutant 12M to be highly stable with statistical validation. Besides, static model analysis involving intra-molecular interactions, secondary structure, solvent accessibility, hydrogen bond pattern, simulated thermal denaturation and desolvation energy also supported 12M comparatively. Of note, the presence of high secondary structural rigidity and hydrogen bonds increased thermostability and functionality of 12M, thus selecting it as a best template for designing thermostable lipases in future. Also, this study has a significant implication toward a better understanding of conformational sampling in enzyme catalysis and enzyme engineering.
Keywords thermophilic      Bacillus subtilis      lipase A      conformational analysis      docking     
Corresponding Author(s): R. Rajasekaran   
Just Accepted Date: 24 November 2015   Online First Date: 25 December 2015    Issue Date: 26 January 2016
 Cite this article:   
B. Senthilkumar,D. Meshachpaul,Rao Sethumadhavan, et al. Selection of effective and highly thermostable Bacillus subtilis lipase A template as an industrial biocatalyst-A modern computational approach[J]. Front. Biol., 2015, 10(6): 508-519.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-015-1379-6
https://academic.hep.com.cn/fib/EN/Y2015/V10/I6/508
Fig.1  Flowchart illustration of the methodology and various bioinformatics tools used in this study.
SI No.PDB IDMutant nameNumber of mutationsTotal No. of IMMHydrophobic
(Å)		Hydrophilic
(Å)
11I6WNativeNative1525782.874558.73
21T4M2M21545428.724131.86
31T2N3M31505651.674194.25
43D2A4M41415913.044213.21
53D2B6M61545528.734359.09
63D2C9M91525683.364199.47
73QMM12M121605497.664101.97
Tab.1  Bacillus subtilis LipA structures along with their computed intra-molecular interactions (IMM), hydrophobic and hydrophilic values of SASA
Fig.2  Geometrical observables of native LipA and its mutants (A) Root Mean Square Deviation (RMSD), (B) Root Mean Square Fluctuation (RMSF), (C) Radius of Gyration (Rg) and (D) Polar surface area (PSA)
Fig.3  Two dimensional view of variable relative solvent solubility of native LipA and its mutants.
Fig.4  Scatter plot showing the distribution of secondary structures in the native LipA and its mutants.
Fig.5  Two dimensional representation of secondary structures for native LipA and its mutants.
ParametersRange and type of H-bondingNative2M3M4M6M9M12M
Number of hydrogen bonds
RangeClass I0000000
Class II189188187185186192197
Class III5134452
TypeM – M126119114112114118119
M – S 17131415131621
S – M33333639393534
S – S 18272623242825
Tab.2  Structural distribution of hydrogen bonds in native LipA and its mutants
ParametersRange and type of H-bondingNative2M3M4M6M9M12M
Number of hydrogen bonds
ClassClass I0000000
Class II23201626222330
Class III1000010
TypeM – M 114254510
M – S 1001012
S – M 119914111113
S – S 1756775
Tab.3  Structural distribution of bifurcated hydrogen bonds for native LipA and its mutants
SI No.PDB IDCluster positionsNumber of cluster residuesPercent rigidity (%)
1Native2-9, 28-39, 48-64, 68-86, 93-100, 153-1728442.9
22M4-9, 14-42, 47-103, 123-130, 133-136, 147-17913773
33M5-9, 16-28, 51-66, 71-76, 79-89, 98-102, 124-128, 164-1747234.9
44M4-11, 18-31, 36-41, 50-67, 72-77, 78-90, 98-103, 124-129, 162-1759147.3
56M4-31, 35-41, 51-67, 70-91, 96-102, 106-110, 123-130, 147-17612465.8
69M4-10, 16-41, 50-90, 96-103, 123-131, 134-141, 146-17713170.9
712M4-10, 15-42, 47-51, 51-67, 70-89, 96-103, 124-130, 146-151, 162-17511258.7
Tab.4  Percent rigidity and cluster residues that constitute Bacillus subtilis Lip A native and its mutants
Fig.6  A comparative graphical representation of hydrogen bond dilution, showing the number of hydrogen bonds broken and the number of hydrogen bonds remained to be broken, at different energy levels (A-H).
Fig.7  Diagramatic view of the docked native LipA and its mutants with triglyceride (A) mean ACE (B) superimposed structure of the docked Lip A native and mutants and (C) the orientation of triglyceride at the active site.
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