Catalytically active inclusion bodies as a potential tool for biotechnology.

The initial assumption that viewed inclusion bodies as a hindrance to the efficient production of protein is no longer held due to the emergence of catalytically active inclusion bodies (CatIBs). Recent studies revealed their potential to be used in free form or immobilized as biocatalysts. The curiosity to acquire suitable catalysts has remained the measure of concern for researchers and industrialists. Numerous processes and production in various sectors of food industries, petroleum, pharmaceutical, cosmetics, and many others are still searching for a robust catalyst with outstanding features such as recyclability, resistance to pH, as well as temperature. CatIBs are forms of inclusion bodies that possess catalytic activity, which can improve catalysis efficiency, stability, and recyclability. One of the advantages of CatIBs is their potential to be used as catalysts for numerous bioprocesses when generated by an enzyme. These aggregates can efficiently be used as a replacement for traditional enzyme immobilization. This review tends to focus on the possibility of its application in various processes. The novelty of this review is that it considered the production of CatIBs both from artificial and natural perspectives, as well as how to improve it. Inclusion bodies' immobilization may provide an efficient alternative in the area of biocatalysis, and hence it will improve industrial sectors and substantially provide a means of achieving excellent performance in the near future.

Effect of cysteine mutation at Ca2+ coordinating residues to the autolysis, folding and hydrophobicity of full length and mature Rand protease: molecular dynamics simulation and essential dynamics.

Rand protease is a serine protease that shared common characteristics with members of the MEROPS S8 subtilisin family. It is thermostable, highly stable in organic solvent and broad in specificity. Many structures of homologous protein solved by X-ray crystallography and NMR have been deposited to Protein Data Bank (PDB) which allowed this study to rely on structure prediction by deep learning to build three-dimensional (3D) structure of full length and mature Rand protease (flRP and mRP). In silico cysteine mutation to 7 predicted high affinity Ca2+ coordinating residues were introduced, and the mutants were subjected to molecular dynamics simulation to study its effect on flRP and mRP. MD simulation showed a marked increase in flexibility of the pro-peptide segment indicating the impact of single cysteine substitution at high affinity Ca2+ coordinating residues to autolysis of flRP. MD simulation for mRP reaffirmed the role of Ca2+ coordinating sites in providing stability to Rand protease. In addition, these residues also affect the autolysis, folding and hydrophobicity of RP. Essential dynamics observed large contribution of the first few eigenvectors of flRP, mRP and their high affinity Ca2+ coordinating residues mutants to the TMSF values which indicates that these values account for a large portion of the overall atomic fluctuations. These results have given a more comprehensive understanding on the role of cysteine substituted Ca2+ coordinating surface loop to the structure of flRP and mRP which are important in contributing to the structural stability of subtilisin.Communicated by Ramaswamy H. Sarma.

In silico identification and characterization of potential druggable targets among hypothetical proteins of Leptospira interrogans serovar Copenhageni: a comprehensive bioinformatics approach.

Leptospirosis is one of the neglected zoonosis, affecting human and animal populations worldwide. Reliable effective therapeutics and concerns to look for more research into the molecular analysis of its genome is therefore needed. In the genomic pool of the Leptospira interrogans many hypothetical proteins are still uncharacterized. In the current research, we performed extensive in silico analysis to prioritize the potential hypothetical proteins of L. interrogans serovar Copenhageni via stepwise reducing the available hypothetical proteins (Total 3606) of the assembly to only 15, based on non-homologous to homosapien, essential, functional, virulent, cellular localization. Out of them, only two proteins WP_000898918.1 (Hypothetical Protein 1) & WP_001014594.1 (Hypothetical Protein 2) were found druggable and involved in protein-protein interaction network. The 3 D structures of these two target proteins were predicted via ab initio homology modeling followed by structures refinement and validation, as no structures were available till date. The analysis also revealed that the functional domains, families and protein-protein interacting partners identified in both proteins are crucial for the survival of the bacteria. The binding cavities were predicted for both the proteins through blind and specific protein-ligand docking with their respective ligands and inhibitors and were found to be in accordance with the druggable sites predicted by DoGSiteScorer. The docking interactions were found within the active functional domains for both the proteins while for Hypothetical Protein 2, the same residues were involved in interactions with Cytidine-5'-triphosphate in blind and specific docking. Furthermore, the simulations of molecular dynamics and free binding energy revealed the stable substrate binding and efficient binding energies, and were in accordance to our docking results. The work predicted two unique hypothetical proteins of L. interrogans as a potential druggable targets for designing of inhibitors for them.Communicated by Ramaswamy H. Sarma.

Antimicrobial activities of Bacillus velezensis strains isolated from stingless bee products against methicillin-resistant Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have reached epidemic proportions globally. Therefore, there is an urgent need for a continuous supply of antibiotics to combat the problem. In this study, bacteria initially identified as species belonging to the Bacillus amyloliquefaciens operational group were re-identified based on the housekeeping gene, gyrB. Cell-free supernatants (CFS) from the strains were used for antimicrobial tests using the agar well diffusion assay against MRSA and various types of pathogenic bacteria. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and physicochemical characteristics of the CFS were determined. Based on gyrB sequence analysis, five strains (PD9, B7, PU1, BP1 and L9) were identified as Bacillus velezensis. The CFS of all B. velezensis strains showed broad inhibitory activities against Gram-negative and -positive as well as MRSA strains. Strain PD9 against MRSA ATCC 33742 was chosen for further analysis as it showed the biggest zone of inhibition (21.0 ± 0.4 mm). The MIC and MBC values obtained were 125 µl/ml. The crude antimicrobial extract showed bactericidal activity and was stable at various temperatures (40-80°C), pH (4-12), surfactants (Tween 20, Tween 80, SDS and Triton X-100) and metal ions (MgCI2, NaCI2, ZnNO3 and CuSO4) when tested. However, the crude extract was not stable when treated with proteinase K. All these properties resembled the characteristics of peptides. The antimicrobial compound from the selected strain was purified by using solvent extraction method and silica gel column chromatography. The purified compound was subjected to High Performance Liquid Chromatography which resulted in a single peak of the anti-MRSA compound being detected. The molecular weight of the anti-MRSA compound was determined by using SDS-PAGE and zymogram. The size of the purified antimicrobial peptide was approximately ~ 5 kDa. The antimicrobial peptide produced from B. velezensis strain PD9 is a promising alternative to combat the spread of MRSA infections in the future.

Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase.

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

Effect of Cultural Conditions on Protease Production by a Thermophilic Geobacillus thermoglucosidasius SKF4 Isolated from Sungai Klah Hot Spring Park, Malaysia.

Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.

New Recombinant Cold-Adapted and Organic Solvent Tolerant Lipase from Psychrophilic Pseudomonas sp. LSK25, Isolated from Signy Island Antarctica.

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.

Isolation, Characterisation, and Lipase Production of a Cold-Adapted Bacterial Strain Pseudomonas sp. LSK25 Isolated from Signy Island, Antarctica.

In recent years, studies on psychrophilic lipases have been an emerging area of research in the field of enzymology. This study focuses on bacterial strains isolated from anthropogenically-influenced soil samples collected around Signy Island Research Station (South Orkney Islands, maritime Antarctic). Limited information on lipase activities from bacteria isolated from Signy station is currently available. The presence of lipase genes was determined using real time quantification PCR (qPCR) in samples obtained from three different locations on Signy Island. Twenty strains from the location with highest lipase gene detection were screened for lipolytic activities at a temperature of 4 °C, and from this one strain was selected for further examination based on the highest enzymatic activities obtained. Analysis of 16S rRNA sequence data of this strain showed the highest level of sequence similarity (98%) to a Pseudomonas sp. strain also isolated from Antarctica. In order to increase lipase production of this psychrophilic strain, optimisation of different parameters of physical and nutritional factors were investigated. Optimal production was obtained at 10 °C and pH 7.0, at 150 rev/min shaking rate over 36 h incubation.

The Effect of N-Terminal Domain Removal towards the Biochemical and Structural Features of a Thermotolerant Lipase from an Antarctic Pseudomonas sp. Strain AMS3.

Lipase plays an important role in industrial and biotechnological applications. Lipases have been subject to modification at the N and C terminals, allowing better understanding of lipase stability and the discovery of novel properties. A thermotolerant lipase has been isolated from Antarctic Pseudomonas sp. The purified Antarctic AMS3 lipase (native) was found to be stable across a broad range of temperatures and pH levels. The lipase has a partial Glutathione-S-transferase type C (GST-C) domain at the N-terminal not found in other lipases. To understand the influence of N-terminal GST-C domain on the biochemical and structural features of the native lipase, the deletion of the GST-C domain was carried out. The truncated protein was successfully expressed in E. coli BL21(DE3). The molecular weight of truncated AMS3 lipase was approximately ~45 kDa. The number of truncated AMS3 lipase purification folds was higher than native lipase. Various mono and divalent metal ions increased the activity of the AMS3 lipase. The truncated AMS3 lipase demonstrated a similarly broad temperature range, with the pH profile exhibiting higher activity under alkaline conditions. The purified lipase showed a substrate preference for a long carbon chain substrate. In addition, the enzyme activity in organic solvents was enhanced, especially for toluene, Dimethylsulfoxide (DMSO), chloroform and xylene. Molecular simulation revealed that the truncated lipase had increased structural compactness and rigidity as compared to native lipase. Removal of the N terminal GST-C generally improved the lipase biochemical characteristics. This enzyme may be utilized for industrial purposes.

Lid opening and conformational stability of T1 Lipase is mediated by increasing chain length polar solvents.

The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the solvent molecules influenced its gradual opening. Formation of additional hydrogen bonds and hydrophobic interactions indicates that the contribution of the cooperative network of interactions could retain the stability of the protein in some solvent systems. Time-correlated atomic motions were used to characterize the correlations between the motions of the atoms from atomic coordinates. The resulting cross-correlation map revealed that the organic solvent mixtures performed functional, concerted, correlated motions in regions of residues of the lid domain to other residues. These observations suggest that varying lengths of polar organic solvents play a significant role in introducing dynamic conformational diversity in proteins in a decreasing order of polarity.

Expression and characterization of thermotolerant lipase with broad pH profiles isolated from an Antarctic Pseudomonas sp strain AMS3.

A gene encoding a thermotolerant lipase with broad pH was isolated from an Antarctic Pseudomonas strain AMS3. The recombinant lipase AMS3 was purified by single-step purification using affinity chromatography, yielding a purification fold of approximately 1.52 and a recovery of 50%. The molecular weight was approximately ∼60 kDa including the strep and affinity tags. Interestingly, the purified Antarctic AMS3 lipase exhibited broad temperature profile from 10-70 °C and stable over a broad pH range from 5.0 to pH 10.0. Various mono and divalent metal ions increased the activity of the AMS3 lipase, but Ni2+ decreased its activity. The purified lipase exhibited the highest activity in the presence of sunflower oil. In addition, the enzyme activity in 25% v/v solvents at 50 °C particularly to n-hexane, DMSO and methanol could be useful for catalysis reaction in organic solvent and at broad temperature.

3D structure elucidation of thermostable L2 lipase from Thermophilic Bacillus sp. L2.

The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 × 0.1 × 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 Å and the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 Å, α = ß = γ = 90°. There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew's coefficient of 2.85 Å Da(-1). The 3D structure of L2 lipase revealed topological organization of α/ß-hydrolase fold consisting of 11 ß-strands and 13 α-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca(2+) and one Zn(2+) were found in the L2 lipase molecule.