Heterologous Expression and Structural Elucidation of a Highly Thermostable Alkaline Serine Protease from Haloalkaliphilic Actinobacterium, Nocardiopsis sp. Mit-7.

A highly thermostable alkaline serine protease gene (SPSPro, MN429015) obtained from haloalkaliphilic actinobacteria, Nocardiopsis sp. Mit-7 (NCIM-5746), was successfully cloned and overexpressed in Escherichia coli BL21 under the control of the T7 promoter in the pET Blue1 vector leading to a 20-kDa gene product. The molecular weight of the recombinant alkaline protease, as determined by SDS-PAGE and the Mass Spectrometer (MALDI-TOF), was 34 kDa. The structural and functional attributes of the recombinant thermostable alkaline serine protease were analyzed by Bioinformatic tools. 3D Monomeric Model and Molecular Docking established the role of the amino acid residues, aspartate, serine, and tryptophan, in the active site of thealkaline protease.The activity of the recombinant alkaline protease was optimal at 65 °C, 5 °C higher than its native protease. The recombinant protease was also active over a wide range of pH 7.0-13.0, with a maximal activity of 6050.47 U/mg at pH 9. Furthermore, the thermodynamic parameters of the immobilized recombinant alkaline protease suggested its reduced vulnerability against adverse conditions under which the enzyme has to undergo varied applications.

Solvent tolerant enzymes in extremophiles: Adaptations and applications.

Non-aqueous enzymology has always drawn attention due to the wide range of unique possibilities in biocatalysis. In general, the enzymes do not or insignificantly catalyze substrate in the presence of solvents. This is due to the interfering interactions of the solvents between enzyme and water molecules at the interface. Therefore, information about solvent-stable enzymes is scarce. Yet, solvent-stable enzymes prove quite valuable in the present day biotechnology. The enzymatic hydrolysis of the substrates in solvents synthesizes commercially valuable products, such as peptides, esters, and other transesterification products. Extremophiles, the most valuable yet not extensively explored candidates, can be an excellent source to investigate this avenue. Due to inherent structural attributes, many extremozymes can catalyze and maintain stability in organic solvents. In the present review, we aim to consolidate information about the solvent-stable enzymes from various extremophilic microorganisms. Further, it would be interesting to learn about the mechanism adapted by these microorganisms to sustain solvent stress. Various approaches to protein engineering are used to enhance catalytic flexibility and stability and broaden biocatalysis's prospects under non-aqueous conditions. It also describes strategies to achieve optimal immobilization with minimum inhibition of the catalysis. The proposed review would significantly aid our understanding of non-aqueous enzymology.

Comparative analysis of the catalysis and stability of the native, recombinant and metagenomic alkaline proteases in organic solvents.

The effect of organic solvents on alkaline proteases was assessed for native, recombinant, and metagenomically derived alkaline proteases. Their stability and the effects of physicochemical parameters were studied in the presence of hexane. The native enzyme was comparatively more resistant against the organic solvents than the recombinant counterparts. On the other hand, the metagenomically derived alkaline protease was minimally resistant against solvents. A similar trend was apparent for the stability of enzyme in organic solvents. The novelty of this study lies in the fact that the majority of the studies on the solvent tolerance have focused on the mesophilic enzymes, while those from the haloalkaliphilic bacteria have received little attention. The comparative tolerance of the native, recombinant, and metagenomic alkaline proteases against the organic solvent has practical importance. The phylogenetic relatedness among the various protease sequences will be described.