Exploring the Antarctic aminopeptidase P from Pseudomonas sp. strain AMS3 through structural analysis and molecular dynamics simulation.

Aminopeptidase P (APPro) is a crucial metalloaminopeptidase involved in amino acid cleavage from peptide N-termini, playing essential roles as versatile biocatalysts with applications ranging from pharmaceuticals to industrial processes. Despite acknowledging its potential for catalysis in lower temperatures, detailed molecular basis and biotechnological implications in cold environments are yet to be explored. Therefore, this research aims to investigate the molecular mechanisms underlying the cold-adapted characteristics of APPro from Pseudomonas sp. strain AMS3 (AMS3-APPro) through a detailed analysis of its structure and dynamics. In this study, structure analysis and molecular dynamics (MD) simulation of a predicted model of AMS3-APPro has been performed at different temperatures to assess structural flexibility and thermostability across a temperature range of 0-60 °C over 100 ns. The MD simulation results revealed that the structure were able to remain stable at low temperatures. Increased temperatures present a potential threat to the overall stability of AMS3-APPro by disrupting the intricate hydrogen bond networks crucial for maintaining structural integrity, thereby increasing the likelihood of protein unfolding. While the metal binding site at the catalytic core exhibits resilience at higher temperatures, highlighting its local structural integrity, the overall enzyme structure undergoes fluctuations and potential denaturation. This extensive structural instability surpasses the localized stability observed at the metal binding site. Consequently, these assessments offer in-depth understanding of the cold-adapted characteristics of AMS3-APPro, highlighting its capability to uphold its native conformation and stability in low-temperature environments. In summary, this research provides valuable insights into the cold-adapted features of AMS3-APPro, suggesting its efficient operation in low thermal conditions, particularly relevant for potential biotechnological applications in cold environments.Communicated by Ramaswamy H. Sarma.

Enhancement in T1 lipase purification recovery using the novel construct pGEX4T1/His-T1.

The Geobacillus zalihae strain T1 produces a thermostable T1 lipase that could be used for industrial purposes. Previously, the GST-T1 lipase was purified through two chromatographic steps: affinity and ion exchange (IEX) but the recovery yield was only 33%. To improve the recovery yield to over 80%, the GST tag from the pGEX system was replaced with a poly-histidine at the N-terminal of the T1 lipase sequence. The novel construct of pGEX/His-T1 lipase was developed by site-directed mutagenesis, where the XbaI restriction site was introduced upstream of the GST tag, allowing the removal of tag via double digestion using XbaI and EcoRI (existing cutting site in the pGEX system). Fragment of 6 × His-T1 lipase fusion was synthesized, cloned into the pGEX4T1 system, and expressed in Escherichia coli BL21 (DE3) pLysS, resulting in lipase-specific activity at 236 U/mg. The single purification step of His-T1 lipase was successfully achieved using nickel Sepharose 6FF with an optimized concentration of 5 mM imidazole for binding, yielding the recovery of 98%, 1,353 U/mg lipase activity, and a 5.7-fold increase in purification fold. His-T1 lipase was characterized and was found to be stable at pH 5-9, active at 70 °C, and optimal at pH 9.

Worldwide trend discovery of structural and functional relationship of metallo-ß-lactamase for structure-based drug design: A bibliometric evaluation and patent analysis.

Metallo-ß-lactamase (MBL) is an enzyme produced by clinically important bacteria that can inactivate many commonly used antibiotics, making them a significant concern in treating bacterial infections and the risk of having high antibiotic resistance issues among the community. This review presents a bibliometric and patent analysis of MBL worldwide research trend based on the Scopus and World Intellectual Property Organization databases in 2013-2022. Based on the keywords related to MBL in the article title, abstract, and keywords, 592 research articles were retrieved for further analysis using various tools such as Microsoft Excel to determine the frequency analysis, VOSviewer for bibliometric networks visualization, and Harzing's Publish or Perish for citation metrics analysis. Standard bibliometric parameters were analysed to evaluate the field's research trend, such as the growth of publications, topographical distribution, top subject area, most relevant journal, top cited documents, most relevant authors, and keyword trend analysis. Within 10 years, MBL discovery has shown a steady and continuous growth of interest among the community of researchers. United States of America, China, and the United Kingdom are the top 3 countries contribute high productivity to the field. The patent analysis also shows several impactful filed patents, indicating the significance of development research on the structural and functional relationship of MBL for an effective structure-based drug design (SBDD). Developing new MBL inhibitors using SBDD could help address the research gap and provide new successful therapeutic options for treating MBL-producing bacterial infections.

Immobilization of Hyperthermostable Carboxylesterase EstD9 from Anoxybacillus geothermalis D9 onto Polymer Material and Its Physicochemical Properties.

Carboxylesterase has much to offer in the context of environmentally friendly and sustainable alternatives. However, due to the unstable properties of the enzyme in its free state, its application is severely limited. The present study aimed to immobilize hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9 with improved stability and reusability. In this study, Seplite LX120 was chosen as the matrix for immobilizing EstD9 by adsorption. Fourier-transform infrared (FT-IR) spectroscopy verified the binding of EstD9 to the support. According to SEM imaging, the support surface was densely covered with the enzyme, indicating successful enzyme immobilization. BET analysis of the adsorption isotherm revealed reduction of the total surface area and pore volume of the Seplite LX120 after immobilization. The immobilized EstD9 showed broad thermal stability (10-100 °C) and pH tolerance (pH 6-9), with optimal temperature and pH of 80 °C and pH 7, respectively. Additionally, the immobilized EstD9 demonstrated improved stability towards a variety of 25% (v/v) organic solvents, with acetonitrile exhibiting the highest relative activity (281.04%). The bound enzyme exhibited better storage stability than the free enzyme, with more than 70% of residual activity being maintained over 11 weeks. Through immobilization, EstD9 can be reused for up to seven cycles. This study demonstrates the improvement of the operational stability and properties of the immobilized enzyme for better practical applications.

Bacteria consortia enhanced hydrocarbon degradation of waxy crude oil.

Waxy crude oil is a problem to the oil and gas industry because wax deposition in pipelines reduces the quality of the crude oil. Currently, the industry uses chemicals to solve the problem but it is not environmentally friendly. As an alternative, the biodegradation approach is one of the options. Previously eleven thermophilic bacteria were isolated and exhibited high ability to degrade hydrocarbon up to 70% of waxy crude oil. However, despite the successful study on these single bacteria strains, it is believed that biodegradation of paraffin wax requires more than a single species. Five consortia were developed based on the biodegradation efficiency of 11 bacterial strains. Consortium 3 showed the highest biodegradation (77.77%) with more long-chain alkane degraded throughout the incubation compared to other consortia. Enhancement of hydrocarbon degradation was observed for all consortia especially in long chain alkane (C18-C40). Consortium 3 exhibited higher alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase activities. Moreover, the dominant bacteria in the consortia were determined by denaturing gradient gel electrophoresis (DGGE), which showed the domination of genera Geobacillus, Parageobacillus, and Anoxybacillus. It can be concluded that the bacterial consortia showed higher biodegradation and improved degrading more long-chain hydrocarbon compared to a single isolate.

A new hyper-thermostable carboxylesterase from Anoxybacillus geothermalis D9.

Carboxylesterases are attractive biocatalysts for various industrial applications, especially hyperthermophilic carboxylesterases, due to their high tolerance toward extreme environments. Such ability confers many advantages, including cost-effectiveness and an increased manufacturing rate. In the current work, we first described the characterization of EstD9, a new carboxylesterase from thermophilic Anoxybacillus geothermalis D9. Sequence analysis of EstD9 revealed a significant identity (80 %) with thermophilic Est30 and a catalytic triad, composed of Ser93-His22-Asp193. As the protein sequence contained a conserved pentapeptide (GLSLG), EstD9 could be proposed as a new member of family XIII. The putative carboxylesterase was recombinantly expressed in E. coli BL21 (DE3) with a molecular mass of 28 kDa and successfully purified via affinity chromatography with recovery of 88.36 %. Using p-nitrophenyl butyrate, EstD9 presented excellent stability at high temperature range (70 °C-100 °C) and a broad pH tolerance (pH 6-9), with optimal activity at 80 °C and pH 7. Notably, EstD9 activity was stimulated in the presence of 1-propanol and DMSO with 107.8 % and 108.9 % relative activities, respectively. The purified EstD9 maintained 60 % residual activity after 30 min exposure to various surfactants and metal ions. Additionally, the inhibition studies demonstrated strong deactivation by phenylmethylsulfonyl fluoride, dithiothreitol, and ß-mercaptoethanol. The estimated Tm value was 72.12 °C. Unlike typical carboxylesterases, in silico 3D model of EstD9 disclosed a topological α/ß hydrolase fold with a small α-helix cap. The enzymatic properties of EstD9 suggest this enzyme to be a highly suitable catalyst for industrial bioprocesses under harsh conditions.

Characterization of Carboxylic Acid Reductase from Mycobacterium phlei Immobilized onto Seplite LX120.

A multi-domain oxidoreductase, carboxylic acid reductase (CAR), can catalyze the one-step reduction of carboxylic acid to aldehyde. This study aimed to immobilize bacterial CAR from a moderate thermophile Mycobacterium phlei (MpCAR). It was the first work reported on immobilizing bacterial CAR onto a polymeric support, Seplite LX120, via simple adsorption. Immobilization time and protein load were optimized for MpCAR immobilization. The immobilized MpCAR showed optimal activity at 60 °C and pH 9. It was stable over a wide range of temperatures (10 to 100 °C) and pHs (4-11), retaining more than 50% of its activity. The immobilized MpCAR also showed stability in polar solvents. The adsorption of MpCAR onto the support was confirmed by Scanning Electron Microscopy (SEM), Fourier-Transform Infrared (FTIR) spectroscopy, and Brunauer-Emmett-Teller (BET) analysis. The immobilized MpCAR could be stored for up to 6 weeks at 4 °C and 3 weeks at 25 °C. Immobilized MpCAR showed great operational stability, as 59.68% of its activity was preserved after 10 assay cycles. The immobilized MpCAR could also convert approximately 2.6 mM of benzoic acid to benzaldehyde at 60 °C. The successfully immobilized MpCAR on Seplite LX120 exhibited improved properties that benefit green industrial processes.

Structure Prediction and Characterization of Thermostable Aldehyde Dehydrogenase from Newly Isolated Anoxybacillus geothermalis Strain D9.

In nature, aldehyde dehydrogenase (ALDH) is widely distributed and mainly involved in the oxidation of aldehydes. Thermostability is one of the key features for industrial enzymes. The ability of enzymes to withstand a high operating temperature offers many advantages, including enhancing productivity in industries. This study was conducted to understand the structural and biochemical features of ALDH from thermophilic bacterium, Anoxybacillus geothermalis strain D9. The 3D structure of A. geothermalis ALDH was predicted by YASARA software and composed of 24.3% ß-sheet located at the center core region. The gene, which encodes 504 amino acids with a molecular weight of ~56 kDa, was cloned into pET51b(+) and expressed in E.coli Transetta (DE3). The purified A. geothermalis ALDH showed remarkable thermostability with optimum temperature at 60 °C and stable at 70 °C for 1 h. The melting point of the A. geothermalis ALDH is at 65.9 °C. Metal ions such as Fe3+ ions inhibited the enzyme activity, while Li+ and Mg2+ enhanced by 38.83% and 105.83%, respectively. Additionally, this enzyme showed tolerance to most non-polar organic solvents tested (xylene, n-dedocane, n-tetradecane, n-hexadecane) in a concentration of 25% v/v. These findings have generally improved the understanding of thermostable A. geothermalis ALDH so it can be widely used in the industry.

Unravelling protein -organic solvent interaction of organic solvent tolerant elastase from Pseudomonas aeruginosa strain K crystal structure.

The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- ß-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.

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.

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.