Identification and characterization of a promiscuous metallohydrolase in metallo-ß-lactamase superfamily from a locally isolated organophosphate-degrading Bacillus sp. strain S3wahi.

In this present study, characteristics and structure-function relationship of an organophosphate-degrading enzyme from Bacillus sp. S3wahi were described. S3wahi metallohydrolase, designated as S3wahi-MH (probable metallohydrolase YqjP), featured the conserved αß/ßα metallo-ß-lactamase-fold (MBL-fold) domain and a zinc bimetal at its catalytic site. The metal binding site of S3wahi-MH also preserves the H-X-H-X-D-H motif, consisting of specific amino acids at Zn1 (Asp69, His70, Asp182, and His230) and Zn2 (His65, His67, and His137). The multifunctionality of S3wahi-MH was demonstrated through a steady-state kinetic study, revealing its highest binding affinity (KM) and catalytic efficiency (kcat/KM) for OP compound, paraoxon, with values of 8.09 × 10-6 M and 4.94 × 105 M-1 s-1, respectively. Using OP compound, paraoxon, as S3wahi-MH native substrate, S3wahi-MH exhibited remarkable stability over a broad temperature range, 20 °C - 60 °C and a broad pH tolerance, pH 6-10. Corresponded to S3wahi-MH thermal stability characterization, the estimated melting temperature (Tm) was found to be 72.12 °C. S3wahi-MH was also characterized with optimum catalytic activity at 30 °C and pH 8. Additionally, the activity of purified S3wahi-MH was greatly enhanced in the presence of 1 mM and 5 mM of manganese (Mn2+), showing relative activities of 1323.68 % and 2073.68 %, respectively. The activity of S3wahi-MH was also enhanced in the presence of DMSO and DMF, showing relative activities of 270.37 % and 307.41 %, respectively. The purified S3wahi-MH retained >60 % residual activity after exposure to non-ionic Tween series surfactants. Nevertheless, the catalytic activity of S3wahi-MH was severely impacted by the treatment of SDS, even at low concentrations. Considering its enzymatic properties and promiscuity, S3wahi-MH emerges as a promising candidate as a bioremediation tool in wide industrial applications, including agriculture industry.

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.

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.

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues.

Cold environments characterised by diverse temperatures close to or below the water freezing point dominate about 80% of the Earth's biosphere. One of the survival strategies adopted by microorganisms living in cold environments is their expression of cold-active enzymes that enable them to perform an efficient metabolic flux at low temperatures necessary to thrive and reproduce under those constraints. Cold-active enzymes are ideal biocatalysts that can reduce the need for heating procedures and improve industrial processes' quality, sustainability, and cost-effectiveness. Despite their wide applications, their industrial usage is still limited, and the major contributing factor is the lack of complete understanding of their structure and cold adaptation mechanisms. The current review looked at the recombinant overexpression, purification, and recent mechanism of cold adaptation, various approaches for purification, and three-dimensional (3D) crystal structure elucidation of cold-active lipases and esterase.

Membrane fatty acid desaturase: biosynthesis, mechanism, and architecture.

Fatty acid desaturase catalyzes the desaturation reactions by inserting double bonds into the fatty acyl chain, producing unsaturated fatty acids, which play a vital part in the synthesis of polyunsaturated fatty acids. Though soluble fatty acid desaturases have been described extensively in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to their difficulties in producing a sufficient amount of recombinant desaturases. However, the advancement of technology has shown substantial progress towards the development of elucidating crystal structures of membrane fatty acid desaturase, thus, allowing modification of structure to be manipulated. Understanding the structure, mechanism, and biosynthesis of fatty acid desaturase lay a foundation for the potential production of various strategies associated with alteration and modifications of polyunsaturated fatty acids. This manuscript presents the current state of knowledge and understanding about the structure, mechanisms, and biosynthesis of fatty acid desaturase. In addition, the role of unsaturated fatty acid desaturases in health and diseases is also encompassed. This will be useful in understanding the molecular basis and structural protein of fatty acid desaturase that are significant for the advancement of therapeutic strategies associated with the improvement of health status. KEY POINTS: • Current state of knowledge and understanding about the biosynthesis, mechanisms, and structure of fatty acid desaturase. • The role of unsaturated fatty acid desaturase. • The molecular basis and structural protein elucidated the crystal structure of fatty acid desaturase.

Clinical and Preclinical Studies of Fermented Foods and Their Effects on Alzheimer's Disease.

The focus on managing Alzheimer's disease (AD) is shifting towards prevention through lifestyle modification instead of treatments since the currently available treatment options are only capable of providing symptomatic relief marginally and result in various side effects. Numerous studies have reported that the intake of fermented foods resulted in the successful management of AD. Food fermentation is a biochemical process where the microorganisms metabolize the constituents of raw food materials, giving vastly different organoleptic properties and additional nutritional value, and improved biosafety effects in the final products. The consumption of fermented foods is associated with a wide array of nutraceutical benefits, including anti-oxidative, anti-inflammatory, neuroprotective, anti-apoptotic, anti-cancer, anti-fungal, anti-bacterial, immunomodulatory, and hypocholesterolemic properties. Due to their promising health benefits, fermented food products have a great prospect for commercialization in the food industry. This paper reviews the memory and cognitive enhancement and neuroprotective potential of fermented food products on AD, the recently commercialized fermented food products in the health and food industries, and their limitations. The literature reviewed here demonstrates a growing demand for fermented food products as alternative therapeutic options for the prevention and management of AD.

Ancestral sequence reconstruction of ancient lipase from family I.3 bacterial lipolytic enzymes.

Family I.3 lipase is distinguished from other families by the amino acid sequence and secretion mechanism. Little is known about the evolutionary process driving these differences. This study attempt to understand how the diverse temperature stabilities of bacterial lipases from family I.3 evolved. To achieve that, eighty-three protein sequences sharing a minimum 30% sequence identity with Antarctic Pseudomonas sp. AMS8 lipase were used to infer phylogenetic tree. Using ancestral sequence reconstruction (ASR) technique, the last universal common ancestor (LUCA) sequence of family I.3 was reconstructed. A gene encoding LUCA was synthesized, cloned and expressed as inclusion bodies in E. coli system. Insoluble form of LUCA was refolded using urea dilution method and then purified using affinity chromatography. The purified LUCA exhibited an optimum temperature and pH at 70 ℃ and 10 respectively. Various metal ions increased or retained the activity of LUCA. LUCA also demonstrated tolerance towards various organic solvents in 25% v/v concentration. The finding from this study could support the understanding on temperature and environment during ancient time. In overall, reconstructed ancestral enzymes have improved physicochemical properties that make them suitable for industrial applications and ASR technique can be employed as a general technique for enzyme engineering.

Selected Kefir Water from Malaysia Attenuates Hydrogen Peroxide-Induced Oxidative Stress by Upregulating Endogenous Antioxidant Levels in SH-SY5Y Neuroblastoma Cells.

Kefir, a fermented probiotic drink was tested for its potential anti-oxidative, anti-apoptotic, and neuroprotective effects to attenuate cellular oxidative stress on human SH-SY5Y neuroblastoma cells. Here, the antioxidant potentials of the six different kefir water samples were analysed by total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), and 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) assays, whereas the anti-apoptotic activity on hydrogen peroxide (H2O2) induced SH-SY5Y cells was examined using MTT, AO/PI double staining, and PI/Annexin V-FITC assays. The surface and internal morphological features of SH-SY5Y cells were studied using scanning and transmission electron microscopy. The results indicate that Kefir B showed the higher TPC (1.96 ± 0.54 µg GAE/µL), TFC (1.09 ± 0.02 µg CAT eq/µL), FRAP (19.68 ± 0.11 mM FRAP eq/50 µL), and DPPH (0.45 ± 0.06 mg/mL) activities compared to the other kefir samples. The MTT and PI/Annexin V-FITC assays showed that Kefir B pre-treatment at 10 mg/mL for 48 h resulted in greater cytoprotection (97.04%), and a significantly lower percentage of necrotic cells (7.79%), respectively. The Kefir B pre-treatment also resulted in greater protection to cytoplasmic and cytoskeleton inclusion, along with the conservation of the surface morphological features and the overall integrity of SH-SY5Y cells. Our findings indicate that the anti-oxidative, anti-apoptosis, and neuroprotective effects of kefir were mediated via the upregulation of SOD and catalase, as well as the modulation of apoptotic genes (Tp73, Bax, and Bcl-2).

Metagenomic and phytochemical analyses of kefir water and its subchronic toxicity study in BALB/c mice.

BACKGROUND: In recent years, researchers are interested in the discovery of active compounds from traditional remedies and natural sources, as they reveal higher therapeutic efficacies and improved toxicological profiles. Among the various traditional treatments that have been widely studied and explored for their potential therapeutic benefits, kefir, a fermented beverage, demonstrates a broad spectrum of pharmacological properties, including antioxidant, anti-inflammation, and healing activities. These health-promoting properties of kefir vary among the kefir cultures found at the different part of the world as different media and culture conditions are used for kefir maintenance and fermentation. METHODS: This study investigated the microbial composition and readily found bioactive compounds in water kefir fermented in Malaysia using 16S rRNA microbiome and UHPLC sequencing approaches. The toxicity effects of the kefir water administration in BALB/c mice were analysed based on the mice survival, body weight index, biochemistry profile, and histopathological changes. The antioxidant activities were evaluated using SOD, FRAP, and NO assays. RESULTS: The 16S rRNA amplicon sequencing revealed the most abundant species found in the water kefir was Lactobacillus hilgardii followed by Lactobacillus harbinensis, Acetobacter lovaniensis, Lactobacillus satsumensis, Acetobacter tropicalis, Lactobacillus zeae, and Oenococcus oeni. The UHPLC screening showed flavonoid and phenolic acid derivatives as the most important bioactive compounds present in kefir water which has been responsible for its antioxidant activities. Subchronic toxicity study showed no toxicological signs, behavioural changes, or adverse effects by administrating 10 mL/kg/day and 2.5 mL/kg/day kefir water to the mice. Antioxidants assays demonstrated enhanced SOD and FRAP activities and reduced NO level, especially in the brain and kidney samples. CONCLUSIONS: This study will help to intensify the knowledge on the water kefir microbial composition, available phytochemicals and its toxicological and antioxidant effects on BALB/c mice since there are very limited studies on the water kefir grain fermented in Malaysia.