Debamitra Chakravorty

Unraveling the rationale behind organic solvent stability of lipases.

Organic solvent-stable lipases have pronounced impact on industrial economy as they are involved in synthesis by esterification, interesterification, and transesterification. However, very few of such natural lipases have been isolated till date. A study of the recent past provided few pillars to rely on for this work. The three-dimensional structure, inclusive of the surface and active site, of 29 organic solvent-stable lipases was analyzed by subfamily classification and protein solvent molecular docking based on fast Fourier transform correlation approach. The observations revealed that organic solvent stability of lipases is their intrinsic property and unique with respect to each lipase. In this paper, factors like surface distribution of charged, hydrophobic, and neutral residues, interaction of solvents with catalytically immutable residues, and residues interacting with essential water molecules required for lipase activity, synergistically and by mutualism contribute to render a stable lipase organic solvent. The propensity of surface charge in relation to stability in organic solvents by establishing repulsive forces to exclude solvent molecules from interacting with the surface and prohibiting the same from gaining entry to the protein core, thus stabilizing the active conformation, is a new finding. It was also interesting to note that lipases having equivalent surface-exposed positive and negative residues were stable in a wide range of organic solvents, irrespective of their LogP values.

An insight into plant lipase research - challenges encountered.

Lipases from bacterial, fungal, and animal sources have been purified to homogeneity with very few of them being contributed from plants. Plant lipases are mostly found in energy reserve tissues, for example, oilseeds. They act as biocatalysts which are attractive due to their high substrate specificity, low production cost and easy pharmacological acceptance due to their eukaryotic origin. Hence plant lipases represent better potential for commercial applications in organic synthesis, food, detergent and pharmacological industries. However, low expression, uneconomical fold purity and the plethora of difficulties related to their recombinant expression has been limiting their commercial applicability and posing challenges to many researchers. This article focuses on comprehensive approaches that have been reported to date to address these challenges.

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.

Multifactorial level of extremostability of proteins: can they be exploited for protein engineering?

Research on extremostable proteins has seen immense growth in the past decade owing to their industrial importance. Basic research of attributes related to extreme-stability requires further exploration. Modern mechanistic approaches to engineer such proteins in vitro will have more impact in industrial biotechnology economy. Developing a priori knowledge about the mechanism behind extreme-stability will nurture better understanding of pathways leading to protein molecular evolution and folding. This review is a vivid compilation about all classes of extremostable proteins and the attributes that lead to myriad of adaptations divulged after an extensive study of 6495 articles belonging to extremostable proteins. Along with detailing on the rationale behind extreme-stability of proteins, emphasis has been put on modern approaches that have been utilized to render proteins extremostable by protein engineering. It was understood that each protein shows different approaches to extreme-stability governed by minute differences in their biophysical properties and the milieu in which they exist. Any general rule has not yet been drawn regarding adaptive mechanisms in extreme environments. This review was further instrumental to understand the drawback of the available 14 stabilizing mutation prediction algorithms. Thus, this review lays the foundation to further explore the biophysical pleiotropy of extreme-stable proteins to deduce a global prediction model for predicting the effect of mutations on protein stability.

RankProt: A multi criteria-ranking platform to attain protein thermostabilizing mutations and its in vitro applications - Attribute based prediction method on the principles of Analytical Hierarchical Process

Attaining recombinant thermostable proteins is still a challenge for protein engineering. The complexity is the length of time and enormous efforts required to achieve the desired results. Present work proposes a novel and economic strategy of attaining protein thermostability by predicting site-specific mutations at the shortest possible time. The success of the approach can be attributed to Analytical Hierarchical Process and the outcome was a rationalized thermostable mutation(s) prediction tool- RankProt. Briefly the method involved ranking of 17 biophysical protein features as class predictors, derived from 127 pairs of thermostable and mesostable proteins. Among the 17 predictors, ionic interactions and main-chain to main-chain hydrogen bonds were the highest ranked features with eigen value of 0.091. The success of the tool was judged by multi-fold in silico validation tests and it achieved the prediction accuracy of 91% with AUC 0.927. Further, in vitro validation was carried out by predicting thermostabilizing mutations for mesostable Bacillus subtilis lipase and performing the predicted mutations by multi-site directed mutagenesis. The rationalized method was successful to render the lipase thermostable with optimum temperature stability and Tm increase by 20°C and 7°C respectively. Conclusively it can be said that it was the minimum number of mutations in comparison to the number of mutations incorporated to render Bacillus subtilis lipase thermostable, by directed evolution techniques. The present work shows that protein stabilizing mutations can be rationally designed by balancing the biophysical pleiotropy of proteins, in accordance to the selection pressure.

Advance Techniques in Enzyme Research

Enzyme research encompasses the purification, characterization, identification, and applications of enzymes. Enzyme technology has entered a modern era in which bioinformatics, genetic engineering, and high-throughput screening by miniaturization of instruments and robotic handling have altogether made existing processes economical and time saving. These advances resulted in enzymes to be industrial workhorses. This chapter brings forward advances in existing techniques along with novel and innovative methodologies and instrumentations applied in high-throughput enzyme research. Highlights are the applications of advanced recombinant DNA technology like the Gateway® cloning and CODEHOP PCR for the fast and efficient cloning of enzyme genes; computational biology for in silico enzyme characterization like monitoring behavior of enzymes in real time through molecular dynamics simulations; modern analytical techniques like microfluidics, affinity ultrafiltration, automated electrophoresis, and ATP-affinity chromatography; and the use of nanoparticles, nanofibers, and mesoporous silica in enzyme stabilization.

Design of lead peptide drugs from mushroom targeting cysteine proteases

Cysteine protease inhibitors are universal regulatory proteins and have recently spawned new interest due to their ability to prevent diseases like carcinogenesis, malaria, autoimmune, and neurodegenerative diseases. However, biologically active natural products that act as protease inhibitors are rare. In the quest of a biologically active natural source of protease inhibitor and interest to find their inhibitory mechanism for drug development, we chose mushrooms as their natural source to ensure acceptability. The present research work aims at discovering motifs in cysteine protease inhibitors which can lead to future development of natural peptide drug candidates. In silico methodologies like structural modeling, protein–protein docking, and structural superimpositions were instrumental to attain the objectives. Cysteine protease inhibitors were found to dock at the active site S1 and S2 pockets of cysteine proteases with amino acid residues 111–125 in clitocypin, 69–76 and 101–115 in macrocypins having the secondary structures of β-hairpins, were the most important in recognizing target proteases. Peptides derived from the conserved regions of clitocypin and macrocypins were 25 and 18 amino acid residues in length with the primary sequences of Lys-Val-Pro-Ser-Thr-Ala-Asp-Val-Tyr-Ile-Ile-Arg-Ala-Pro-Ile-Gln-Arg-Ile-Gly-Val-Asp-Val-Glu-Val-Gly and Thr-Glu-Phe-Arg-Ile-Asp-Ser-Ile-Pro-Gly-Gln-Trp-Ala-Arg-Ser-Pro-Val-Glu, respectively. They docked with the target proteases showing similar interaction profile as their parent structures. In addition, interaction of peptides with aspartate and serine proteases at sites other than their active site pockets, ensured specificity of their binding to cysteine proteases. Thus, the knowledge derived from this analysis can be utilized for the future development of peptide drugs for cysteine proteases, aiding in modern drug discovery process.