Introduction and literature review
1.13 Thesis organization
The present thesis entitled “Exploring Molecular adaptations of extremophilic proteins: A platform for protein engineering” is an effort in developing consummate understanding of protein extreme-stability. It has been divided into eight chapters.
Chapter I: Introduction and literature review
In this chapter a thorough literature review that provides an insight into research pertaining to the multifactorial level of extreme-stability of proteins has been comprehensively reported. Protein stability can be affected at three hierarchies of protein organization – DNA level, RNA level and protein level (including primary sequence features, various intra-protein interactions, secondary and tertiary structural features).
This chapter put forth the question whether these factors can be exploited for protein engineering to fill the caveat of attaining pre-determined mutations. The chapter lays foundation for the entire work which has been reported in this thesis.
Chapter 2: Extreme-stable Protein Database (ExProtDB)
Extremophilic adaptations though a burgeoning topic, a curated database for extremophiles and their proteins is still lacking. Thus, this chapter is a compilation of all the collected genomic and proteomic data of extremophiles into a manually curated database named as “Extreme-stable Protein Database (ExPDb)”. Architecture of the database has been built using a standard platform based on the Linux-Apache-MySQL- PHP (LAMP). It is a knowledgebase about all the information available in the World Wide Web about extremophiles and their extreme-stable proteins. The novelty of the database lies in the fact that it gives consolidated and comprehensive information of all types of extremophilic proteins and organisms.
Chapter 3: Understanding the specific codon usage patterns in extremophiles
The collected genomic and proteomic data were then utilized for comparative analyses of extremophiles and non-extremophiles in the proceeding chapters. This chapter throws light on primary level i.e. genomic adaptations of extremophilic proteins. It deciphers the rationale behind the specific codon usage patterns in extremophiles. Codon usage patterns exhibited harmony among different extremophiles and has been detailed. However, the codon attribute preferences and their selectivity of extremophiles varied in comparison to non-extremophiles. Although codon study is being partially deciphered in thermophiles, this is probably the first report of codon preferences pertaining to all extremophile classes.
Chapter 4: Analysis of attributes contributing to extreme-stability of proteins
After the codon contribution further work in chapter 4 encompassed on prediction and ranking models for analyzing protein sequence and structure attributes contributing towards extreme-stability through machine learning and multi-criteria decision making (also known as Analytic Hierarchy Process, AHP) approaches. The positively contributing attributes have been further exploited to design mutations in a mesophilic (non-extremophilic) protein in such a way as to increase the content of these attributes to endorse the prediction of generated models. This chapter reports additional contributing factors for extreme-protein stabilization, which were not reported earlier.
Chapter 5:In silico prediction of mutations to attain extreme-stability of proteins and their validation
This chapter gives a pertinent account of in silico prediction of plausible mutations and their validation in Bacillus subtilis lipase (a chosen mesophilic enzyme) by substituting the preferred protein attributes to attain extreme-stability of proteins by designing mutants. In silico validation of mutant was done by AHP generated ranking models, various mutation prediction servers (HotSpot Wizard, I-Mutant2, Cupsat, iPTREE- STAB, WET-STAB and ERIS webservers), Substrate Docking (Autodock 4.2 version), Ramachandran analysis (PROCHECK) and Contact Map analysis (CMView 1.1.1).
Interestingly, results showed that the designed mutations did not affect the catalytic activity of the lipase and thus, mutation did not disturb the catalytic properties.
Chapter 6: In vitro characterization and validation of predicted mutations
This chapter deals with experimentation employed for in vitro cloning, mutagenesis, expression, purification and characterization of mutated lipases. Results revealed that the optimum activity and stability increased from 35 °C to 55 °C in bsl_the3, pH 8 to pH 10 in bsl_alk2 but reduced to pH 6.5 in bsl_acd2 w.r.t. wild type lipase (bsl_wt). Further, the bsl_wt was halo-sensitive (0.5M NaCl) but in bsl_hal2 halostability was increased to 1.5M NaCl. The content of secondary structure enhanced extreme-stability (with increased Tm value) which was confirmed through FT-IR (ATR mode) and Differential Scanning Fluorimetry (Thermal shift assay).
Chapter 7: Employing predicted extreme-stable mutants for applications
This chapter employs generated extreme-stable mutants for applications. Detergent formulations was the chosen application here. To further increase the enzyme stability, bsl_wt and engineered lipases (bsl_the3, bsl_psy2, bsl_acd2, bsl_alk2 and bsl_hal2 have better extreme-stability) were immobilized on synthesized ZnO nanoparticles as it is cheap, biocompatible and environmental friendly. The order of performance in oil and grease stain removal from the stained cotton fabric pieces by immobilized lipases on ZnO nanoparticle- bsl_alk2 > bsl_the3 > bsl_hal2 > bsl_psy2 > bsl_acd2 > bsl_wt. Results also showed that the maximium removal of oil and grease stains were up to 90 % and 82
%, respectively.
Chapter 8: Conclusion and future perspectives
It summarizes the findings of the present investigations and states the future scopes and upcoming challenges. It can be concluded that thesis work has been successful to develop a single design strategy to mutate amino acid residues which can render proteins stable at different extremophilic range. Conclusively, this work reveals a promising perspective of using low-cost nanoparticle conjugated biocatalysts which was produced through rationalized protein engineering approach and was instrumented for detergent formulation.