Physicochemical and antioxidant properties of Apis cerana honey from Lombok and Bali Islands.

Limited honey production worldwide leads to higher market prices, thus making it prone to adulteration. Therefore, regular physicochemical analysis is imperative for ensuring authenticity and safety. This study describes the physicochemical and antioxidant properties of Apis cerana honey sourced from the islands of Lombok and Bali, showing their unique regional traits. A comparative analysis was conducted on honey samples from Lombok and Bali as well as honey variety from Malaysia. Moisture content was found slightly above 20% in raw honey samples from Lombok and Bali, adhering to the national standard (SNI 8664:2018) of not exceeding 22%. Both honey types displayed pH values within the acceptable range (3.40-6.10), ensuring favorable conditions for long-term storage. However, Lombok honey exhibited higher free acidity (78.5±2.14 meq/kg) than Bali honey (76.0±1.14 meq/kg), surpassing Codex Alimentarius recommendations (≤50 meq/kg). The ash content, reflective of inorganic mineral composition, was notably lower in Lombok (0.21±0.02 g/100) and Bali honey (0.14±0.01 g/100) compared to Tualang honey (1.3±0.02 g/100). Electric conductivity, indicative of mineral content, revealed Lombok and Bali honey with lower but comparable values than Tualang honey. Hydroxymethylfurfural (HMF) concentrations in Lombok (14.4±0.11 mg/kg) and Bali (17.6±0.25 mg/kg) were slightly elevated compared to Tualang honey (6.4±0.11 mg/kg), suggesting potential processing-related changes. Sugar analysis revealed Lombok honey with the highest sucrose content (2.39±0.01g/100g) and Bali honey with the highest total sugar content (75.21±0.11 g/100g). Both honeys exhibited lower glucose than fructose content, aligning with Codex Alimentarius guidelines. The phenolic content, flavonoids, and antioxidant activity were significantly higher in Lombok and Bali honey compared to Tualang honey, suggesting potential health benefits. Further analysis by LC-MS/MS-QTOF targeted analysis identified various flavonoids/flavanols and polyphenolic/phenolic acid compounds in Lombok and Bali honey. The study marks the importance of characterizing the unique composition of honey from different regions, ensuring quality and authenticity in the honey industry.

Baseline amplicon sequencing data for the ITS2 region in the green honey of Banggi Island, Sabah.

Green honey, was discovered on Banggi Island, Sabah, showing high in essential amino acids and chlorophyll derivatives. Despite its lucrative market potential owing to its distinctive color, uncertainties persist regarding its nature. This study leverages amplicon sequencing by targeting micro- and macro-organisms present in honey environmental DNA (eDNA) using Internal Transcribed Spacer 2 (ITS2) region, enabling the identification of floral and microorganism sources that represent the honey's composition. The investigation into green honey from Banggi Island concerns the prevalence of honey adulteration and authenticity for economic gain. Adulteration methods, such as the addition of sugar syrups, compromise honey purity. Using a sequencing approach would help in determining the geographic origin and verifying the authenticity of the honey. The study aims to identify plant species or microorganisms in honey's eDNA. To authenticate honey, we utilized ITS2 with Illumina sequencing, exploring the diversity of green honey samples. Raw sequence reads obtained for the green honey sample revealed 1,438,627 raw reads, with a GC average of 49.22 %. A total of 44 amplicon sequence variances (ASVs) were identified, including three genera: Zygosaccharomyces with two species, Fraxinus with three species, and the genus Ficaria with only one species. Their respective relative abundances were 98.55%, 0.94%, and 0.51%. Zygosaccharomyces rouxii and Zygosaccharomyces mellis were identified as the pre-dominant yeast species in honey, while the Fraxinus and Ficaria genus represent common plant species in Sabah, particularly in Banggi Island. The dominance of Zygosaccharomyces species aligns with their known prevalence in honey, affirming the reliability of our findings. The presence of Fraxinus and Ficaria in the honey sample correlates with its abundance in the local environment. This amplicon sequencing approach not only contributes to our understanding of green honey composition but also serves as a valuable resource for authenticating honey origin in Malaysia, particularly for green honey from Banggi Island, Sabah. Our study pioneers the application of ITS2 amplicon sequencing for green honey amplicon sequencing, providing valuable insights into its composition and origin. This methodology, with a focus on eDNA, contributes to the authentication and quality determination of honey in Malaysia, addressing the pressing concerns of adulteration and variability in production practices.

Cross-linked enzyme aggregates of polyethylene terephthalate hydrolyse (PETase) from Ideonella sakaiensis for the improvement of plastic degradation.

Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower Km of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation.

Green honey of Banggi Island: A preliminary anti-diabetic study on zebrafish model.

Zebrafish is a developing vertebrate model with several advantages, including its small size, and high experimental efficiency. Malaysia exhibit one of the highest diabetes rates in the Western Pacific and incurring an annual cost of 600 million US dollars. The objective of the study is to determine the antidiabetic properties of green honey (GH) using a zebrafish model. Adult zebrafish, aged 3-4 months, were subjected to overfeeding and treated with streptozotocin (STZ) through intraperitoneal injection (IP) on days 7 and 9. The study assessed the oral sucrose tolerance test (OSTT) and the anti-diabetic effects of green honey. The evaluation was conducted at three time points: 30, 60, and 120 min after treatment and sucrose administration. The study utilised a model with a sample size of 5. The study was performed in six groups. These groups are (1) Normal control (non-diabetic, no intervention), (2) Normal control + GH (non-diabetic, supplemented with GH 3 µl), (3) DM control (diabetic, no intervention), (4) DM Gp1 (diabetic, 3 µL GH), (5) DM Gp2 (diabetic, 6 µ L GH), (6) DM Acarbose (diabetic, treated with acarbose). Fasting blood glucose levels for non-diabetic (non-DM) and diabetic (DM) groups were evaluated before and after the 10 days of diabetic induction. DM groups (excess of food and two injections of STZ) have caused a significant increment in the fasting blood glucose to 11.55 mmol/l (p < 0.0001). Both GH treatments effectively decreased postprandial blood glucose levels and the area under the curve in the oral glucose tolerance test (OSTT). Based on these results, it is concluded that green honey could play a role in hyperglycemia management and show potential as a natural alternative to conventional diabetes therapy. The underlying mechanisms need to be clarified, and their potential use in human diabetes therapy needs to be investigated.

An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications.

Enzymes are commonly used as biocatalysts for various biological and chemical processes in industrial applications. However, their limited operational stability, catalytic efficiency, poor reusability, and high-cost hamper further industrial usage. Thus, crosslinked enzyme aggregates (CLEAs) are developed as a better enzyme immobilization tool to extend the enzymes' operational stability. This immobilization method is appealing because it is simpler due to the absence of ballast and permits the collective use of crude enzyme cocktails. CLEAs, so far, have been successfully developed using a variety of enzymes, viz., hydrolases, proteases, amidases, lipases, esterases, and oxidoreductase. Recent years have seen the emergence of novel strategies for preparing better CLEAs, which include the combi- and multi-CLEAs, magnetics CLEAs, and porous CLEAs for various industrial applications, viz., laundry detergents, organic synthesis, food industries, pharmaceutical applications, oils, and biodiesel production. To better understand the different strategies for CLEAs' development, this review explores these strategies and highlights the relevant concerns in designing innovative CLEAs. This article also details the challenges faced during CLEAs preparation and solutions for overcoming them. Finally, the trending strategies to improve the preparation of CLEAs alongside their industrial application trends are also discussed.

Comparative modeling and enzymatic affinity of novel haloacid dehalogenase from Bacillus megaterium strain BHS1 isolated from alkaline Blue Lake in Turkey.

This study presents the initial structural model of L-haloacid dehalogenase (DehLBHS1) from Bacillus megaterium BHS1, an alkalotolerant bacterium known for its ability to degrade halogenated environmental pollutants. The model provides insights into the structural features of DehLBHS1 and expands our understanding of the enzymatic mechanisms involved in the degradation of these hazardous pollutants. Key amino acid residues (Arg40, Phe59, Asn118, Asn176, and Trp178) in DehLBHS1 were identified to play critical roles in catalysis and molecular recognition of haloalkanoic acid, essential for efficient binding and transformation of haloalkanoic acid molecules. DehLBHS1 was modeled using I-TASSER, yielding a best TM-score of 0.986 and an RMSD of 0.53 Å. Validation of the model using PROCHECK revealed that 89.2% of the residues were located in the most favored region, providing confidence in its structural accuracy. Molecular docking simulations showed that the non-simulated DehLBHS1 preferred 2,2DCP over other substrates, forming one hydrogen bond with Arg40 and exhibiting a minimum energy of -2.5 kJ/mol. The simulated DehLBHS1 exhibited a minimum energy of -4.3 kJ/mol and formed four hydrogen bonds with Arg40, Asn176, Asp9, and Tyr11, further confirming the preference for 2,2DCP. Molecular dynamics simulations supported this preference, based on various metrics, including RMSD, RMSF, gyration, hydrogen bonding, and molecular distance. MM-PBSA calculations showed that the DehLBHS1-2,2-DCP complex had a markedly lower binding energy (-21.363 ± 1.26 kcal/mol) than the DehLBHS1-3CP complex (-14.327 ± 1.738 kcal/mol). This finding has important implications for the substrate specificity and catalytic function of DehLBHS1, particularly in the bioremediation of 2,2-DCP in contaminated alkaline environments. These results provide a detailed view of the molecular interactions between the enzyme and its substrate and may aid in the development of more efficient biocatalytic strategies for the degradation of halogenated compounds.Communicated by Ramaswamy H. Sarma.

The first ITS2 sequence data set of eDNA from honey of Malaysian giant honeybees (Apis dorsata) and stingless bees (Heterotrigona itama) reveals plant species diversity.

OBJECTIVES: Pollen is a useful tool for identifying the provenance and complex ecosystems surrounding honey production in Malaysian forests. As native key pollinators in Malaysia, Apis dorsata and Heterotrigona itama forage on various plant/pollen species to collect honey. This study aims to generate a dataset that uncovers the presence of these plant/pollen species and their relative abundance in the honey of A. dorsata and H. itama. The information gathered from this study can be used to determine the geographical and botanical origin and authenticity of the honey produced by these two species. RESULTS: Sequence data were obtained for both A. dorsata and H. itama. The raw sequence data for A. dorsata was 5 Mb, which was assembled into 5 contigs with a size of 6,098,728 bp, an N50 of 15,534, and a GC average of 57.42. Similarly, the raw sequence data for H. itama was 6.3 Mb, which was assembled into 11 contigs with a size of 7,642,048 bp, an N50 of 17,180, and a GC average of 55.38. In the honey sample of A. dorsata, we identified five different plant/pollen species, with only one of the five species exhibiting a relative abundance of less than 1%. For H. itama, we identified seven different plant/pollen species, with only three of the species exhibiting a relative abundance of less than 1%. All of the identified plant species were native to Peninsular Malaysia, especially the East Coast area of Terengganu. DATA DESCRIPTION: Our data offers valuable insights into honey's geographical and botanical origin and authenticity. Metagenomic studies could help identify the plant species that honeybees forage and provide preliminary data for researchers studying the biological development of A. dorsata and H. itama. The identification of various flowers from the eDNA of honey that are known for their medicinal properties could aid in regional honey with accurate product origin labeling, which is crucial for guaranteeing product authenticity to consumers.

Biological and molecular approaches of the degradation or decolorization potential of the hypersaline Lake Tuz Bacillus megaterium H2 isolate.

The presence of synthetic dyes in water bodies and soil is one of the major issues affecting the global ecology, possibly impacting societal well-being adversely due to the colorants' recalcitrance and toxicity. Herein, the study spectrophotometrically monitored the ability of the Bacillus megaterium H2 azoreductase (AzrBmH2) to degrade four synthetic dyes, reactive blue 4, remazol brilliant red, thymol blue, and methyl red, followed by in-silico assessment using GROMACS. We found that the bacterium degraded as much as 60% of all four synthetic dyes at various tested concentrations. The genome analysis revealed five different azoreductase genes, which were then modeled into the AzrBmH21, AzrBmH22/3, and AzrBmH24/5 templates. The AzrBmH2-substrate complexes showed binding energies with all the dyes of between -10.6 to -6.9 kcal/mol and formed 4-6 hydrogen bonds with the predicted catalytic binding residues (His10, Glu 14, Ser 58, Met 99, Val 107, His 183, Asn184 and Gln 191). In contrast, the lowest binding energies were observed for the AzrBmH21-substrates (-10.6 to -7.9). Molecular dynamic simulations revealed that the AzrBmH21-substrate complexes were more stable (RMSD 0.2-0.25 nm, RMSF 0.05 - 0.3 nm) and implied strong bonding with the dyes. The Molecular Mechanics Poisson-Boltzmann Surface Area results also mirrored this outcome, showing the lowest azoreductase-dye binding energy in the order of AzrBmH21-RB4 (-78.18 ± 8.92 kcal/mol), AzrBmH21-RBR (-67.51 ± 7.74 kcal/mol), AzrBmH21-TB (-46.62 ± 5.23 kcal/mol) and AzrBmH21-MR (-40.78 ± 7.87 kcal/mol). In short, the study demonstrated the ability of the B. megaterium H2 to efficiently decolorize the above-said synthetic dyes, conveying the bacterium's promising use for large-scale dye remediation.Communicated by Ramaswamy H. Sarma.

In silico analysis of a putative dehalogenase from the genome of halophilic bacterium Halomonas smyrnensis AAD6T.

Microbial-assisted removal of natural or synthetic pollutants is the prevailing green, low-cost technology to treat polluted environments. However, the challenge with enzyme-assisted bioremediation is the laborious nature of dehalogenase-producing microorganisms' bioprospecting. This bottleneck could be circumvented by in-silico analysis of certain microorganisms' whole-genome sequences to predict their protein functions and enzyme versatility for improved biotechnological applications. Herein, this study performed structural analysis on a dehalogenase (DehHsAAD6) from the genome of Halomonas smyrnensis AAD6 by molecular docking and molecular dynamic (MD) simulations. Other bioinformatics tools were also employed to identify substrate preference (haloacids and haloacetates) of the DehHsAAD6. The DehHsAAD6 preferentially degraded haloacids and haloacetates (-3.2-4.8 kcal/mol) and which formed three hydrogen bonds with Tyr12, Lys46, and Asp182. MD simulations data revealed the higher stability of DehHsAAD6-haloacid- (RMSD 0.22-0.3 nm) and DehHsAAD6-haloacetates (RMSF 0.05-0.14 nm) complexes, with the DehHsAAD6-L-2CP complex being the most stable. The detail of molecular docking calculations ranked complexes with the lowest binding free energies as: DehHsAAD6-L-2CP complex (-4.8 kcal/mol) = DehHsAAD6-MCA (-4.8 kcal/mol) < DehHsAAD6-TCA (-4.5 kcal/mol) < DehHsAAD6-2,3-DCP (-4.1 kcal/mol) < DehHsAAD6-D-2CP (-3.9 kcal/mol) < DehHsAAD6-2,2-DCP (-3.5 kcal/mol) < DehHsAAD6-3CP (-3.2 kcal/mol). In a nutshell, the study findings offer valuable perceptions into the elucidation of possible reaction mechanisms of dehalogenases for extended substrate specificity and higher catalytic activity.Communicated by Ramaswamy H. Sarma.

Molecular interactions of trichoderma ß-1,4-glucosidase (ThBglT12) with mycelial cell wall components of phytopathogenic Macrophomina phaseolina.

Efficacy of a ß-1,4-glucosidase from Trichoderma harzianum T12 (ThBglT12) in disrupting the cell wall of the phytopathogenic fungus M. phaseolina (Macrophomina phaseolina) was studied, as the underlying molecular mechanisms of cell wall recognition remains elusive. In this study, the binding location identified by a consensus of residues predicted by COACH tool, blind docking, and multiple sequence alignment revealed that molecular recognition by ThBglT12 occurred through interactions between the α-1,3-glucan, ß-1,3-glucan, ß-1,3/1,4-glucan, and chitin components of M. phaseolina, with corresponding binding energies of -7.4, -7.6, -7.5 and -7.8 kcal/mol. The residue consensus verified the participation of Glu172, Tyr304, Trp345, Glu373, Glu430, and Trp431 in the active site pocket of ThBglT12 to bind the ligands, of which Trp345 was the common interacting residue. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), total energy, and minimum distance calculation from molecular dynamics (MD) simulation further confirmed the stability and the closeness of the binding ligands into the ThBglT12 active site pocket. The h-bond occupancy by Glu373 and Trp431 instated the role of the nucleophile for substrate recognition and specificity, crucial for cleaving the ß-1,4 linkage. Further investigation showed that the proximity of Glu373 to the anomeric carbon of ß-1,3/1,4-glucan (3.5 Å) and chitin (5.5 Å) indicates the nucleophiles' readiness to form enzyme-substrate intermediates. Plus, the neighboring water molecule appeared to be correctly positioned and oriented towards the anomeric carbon to hydrolyze the ß-1,3/1,4-glucan and chitin, in less than 4.0 Å. In a nutshell, the study verified that the ThBglT12 is a good alternative fungicide to inhibit the growth of M. phaseolina.Communicated by Ramaswamy H. Sarma.

An Overview into Polyethylene Terephthalate (PET) Hydrolases and Efforts in Tailoring Enzymes for Improved Plastic Degradation.

Plastic or microplastic pollution is a global threat affecting ecosystems, with the current generation reaching as much as 400 metric tons per/year. Soil ecosystems comprising agricultural lands act as microplastics sinks, though the impact could be unexpectedly more far-reaching. This is troubling as most plastic forms, such as polyethylene terephthalate (PET), formed from polymerized terephthalic acid (TPA) and ethylene glycol (EG) monomers, are non-biodegradable environmental pollutants. The current approach to use mechanical, thermal, and chemical-based treatments to reduce PET waste remains cost-prohibitive and could potentially produce toxic secondary pollutants. Thus, better remediation methods must be developed to deal with plastic pollutants in marine and terrestrial environments. Enzymatic treatments could be a plausible avenue to overcome plastic pollutants, given the near-ambient conditions under which enzymes function without the need for chemicals. The discovery of several PET hydrolases, along with further modification of the enzymes, has considerably aided efforts to improve their ability to degrade the ester bond of PET. Hence, this review emphasizes PET-degrading microbial hydrolases and their contribution to alleviating environmental microplastics. Information on the molecular and degradation mechanisms of PET is also highlighted in this review, which might be useful in the future rational engineering of PET-hydrolyzing enzymes.

Prominent bactericidal characteristics of silver-copper nanocomposites produced via pulse laser ablation.

This paper reports the characterization and antibacterial performance evaluation of some spherical and stable crystalline silver (Ag)/copper (Cu) nanocomposites (Ag-CuNCs) prepared in deionized water (DIW) using pulse laser ablation in liquid (PLAL) method. The influence of various laser fluences (LFs) on the structural, morphological, optical and antibacterial properties of these NCs were determined. The UV-Vis absorbance of these NCs at 403 nm and 595 nm was gradually increased accompanied by a blue shift. XRD patterns disclosed the nucleation of highly crystalline Ag-CuNCs with their face centered cubic lattice structure. TEM images showed the existence of spherical NCs with size range of 3-20 nm and lattice fringe spacing of approximately 0.145 nm. EDX profiles of Ag-CuNCs indicated their high purity. The antibacterial effectiveness of the Ag-CuNCs was evaluated by the inhibition zone diameter (IZD) and optical density (OD600) tests against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The proposed NCs revealed the IZD values in the range of 22-26 mm and 20-25 mm when tested against E. coli and S. aureus bacteria, respectively. The Ag-CuNCs prepared at LF of 14.15 J/cm2 revealed the best bactericidal activity. It is established that by controlling the laser fluence the bactericidal effectiveness of the Ag-CuNCs can be tuned.

Physicochemical Properties of a New Green Honey from Banggi Island, Sabah.

Green honey is exclusively available on the island of Banggi in Sabah, and its uniqueness sees the commodity being sold at a high market price. Therefore, green honey is prone to adulteration by unscrupulous individuals, possibly compromising the health of those consuming this food commodity for its curative properties. Moreover, an established standard for reducing sugar in green honey is unavailable. Ipso facto, the study aimed to profile green honey's physical and chemical properties, such as its pH, moisture content, free acidity, ash content, electroconductivity, hydroxymethylfurfural (HMF), total phenolic content, total flavonoid content, DPPH, colour, total sugar content, total protein content, and heavy metals as well as volatile organic compounds, the data of which are profoundly valuable in safeguarding consumers' safety while providing information for its quality certification for local consumption and export. The results revealed that the honey's physicochemical profile is comparable to other reported kinds of honey. The honey's naturally green colour is because of the chlorophyll from the nectar from various flowers on the island. The raw honey showed free acidity between 28 and 33 Meq/100 g, lower than the standard's 50 Meq/100 g. The hydroxymethylfurfural content is the lowest compared to other reported honey samples, with the total phenolic content between 16 and 19 mg GAE/100 g. The honey's reducing sugar content is lower (~37.9%) than processed ones (56.3%) because of water removal. The protein content ranged from 1 to 2 gm/kg, 4- to 6-fold and 2-fold higher than local and manuka honey, respectively. The exceptionally high content of trans-4-hydroxyproline in raw honey is its source of collagen and other healing agents. Interestingly, low levels of arsenic, lead, nickel, cadmium, copper, and cobalt were detected in the honey samples, presumably due to their subterranean hives. Nevertheless, the honey is fit for general consumption as the concentrations were below the maxima in the Codex Alimentarius Commission of 2001.

Lactic acid bacteria and their bacteriocins: new potential weapons in the fight against methicillin-resistant Staphylococcus aureus.

Alternative solutions are eminently needed to combat the escalating number of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Bacteriocins produced by lactic acid bacteria are promising candidates for next-generation antibiotics. Studies have found that these stable and nontoxic ribosomally synthesized antimicrobial peptides exhibit significant potency against other bacteria, including antibiotic-resistant strains. Here the authors review previous studies on bacteriocins that have been effectively employed to manage MRSA infections. The authors' review focuses on the beneficial traits of bacteriocins for further application as templates for the design of novel drugs. Treatments that combine bacteriocins with other antimicrobials to combat pervasive MRSA infections are also highlighted. In short, future studies should focus on the pharmacodynamics and pharmacokinetics of bacteriocins-antimicrobials to understand their interactions, as this aspect would likely determine their efficacy in MRSA inhibition.

Exploring the genome of Lactobacillaceae spp. Sy-1 isolated from Heterotrigona itama honey.

Background: Honey produced by Heterotrigona itama is highly preferred among consumers due to its high-value as a functional food and beneficial lactic acid bacteria (LAB) reservoir. Fructophilic lactic acid bacteria (FLAB) are a group of LAB with unique growth characteristics and are regarded as promising producers of bioactive compounds. Hence, it is not surprising that LAB, especially FLAB, may be involved with the excellent bioactivity of H. itama honey. With the trending consumer preference for H. itama honey coupled with increasing awareness for healthy food, the genomic background of FLAB isolated from this honey must, therefore, be clearly understood. In this study, one FLAB strain designated as Sy-1 was isolated from freshly collected H. itama honey. Its FLAB behavior and genomic features were investigated to uncover functional genes that could add value to functional food. Methods: The fructophilic characteristics of strain Sy-1 were determined, and the genome was sequenced using Illumina iSeq100 and Oxford Nanopore. The average nucleotide identity and phylogenetic analyses based on 16S rRNA, 92 core genes, and whole-genome sequence were performed to unravel the phylogenetic position of strain Sy-1. NCBI Prokaryotic Genome Annotation Pipeline annotated the genome, while the EggNOG-mapper, BLASTKoala, and GHOSTKoala were used to add functional genes and pathways information. Results: Strain Sy-1 prefers D-fructose over D-glucose and actively metabolizes D-glucose in the presence of electron acceptors. Genomic annotation of strain Sy-1 revealed few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, in line with the characteristic of FLAB. The 16S rRNA gene sequence of strain Sy-1 showed the highest similarity to unknown LAB species isolated from the gut of honeybees. The phylogenetic analyses discovered that strain Sy-1 belonged to the Lactobacillaceae family and formed a separate branch closer to type strain from the genera of Acetilactobacillus and Apilactobacillus. The ANI analysis showed the similarity of the closest relative, Apilactobacillus micheneri Hlig3T. The assembled genome of Sy-1 contains 3 contigs with 2.03 Mbp and a 41% GC content. A total of 1,785 genes were identified, including 1,685 protein-coding genes, 68 tRNA, and 15 rRNA. Interestingly, strain Sy-1 encoded complete genes for the biosynthesis of folate and riboflavin. High-performance liquid chromatography analysis further confirmed the high production of folic acid (1.346 mg/L) by Sy-1. Discussion: Based on phylogenetic and biochemical characteristics, strain Sy-1 should be classified as a novel genus in the family of Lactobacillaceae and a new member of FLAB. The genome information coupled with experimental studies supported the ability of strain Sy-1 to produce high folic acid. Our collective findings support the suitable application of FLAB strain Sy-1 in the functional food and pharmaceutical industries.

Molecular docking and molecular dynamics simulation of Bacillus thuringiensis dehalogenase against haloacids, haloacetates and chlorpyrifos.

The high dependency and surplus use of agrochemical products have liberated enormous quantities of toxic halogenated pollutants into the environment and threaten the well-being of humankind. Herein, this study performed molecular docking, molecular dynamic (MD) simulations, molecular mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis, to identify the order of which the enzyme degrades different substrates, haloacids, haloacetate and chlorpyrifos. The study discovered that the DehH2 favored the degradation of haloacids and haloacetates (-3.3 - 4.6 kcal/mol) and formed three hydrogen bonds with Asp125, Arg201 and Lys202. Despite the inconclusive molecular docking result, chlorpyrifos was consistently shown to be the least favored substrate of the DehH2 in MD simulations and MM-PBSA calculations. Results of MD simulations revealed the DehH2-haloacid- (RMSD 0.15 - 0.25 nm) and DehH2-haloacetates (RMSF 0.05 - 0.25 nm) were more stable, with the DehH2-L-2CP complex being the most stable while the least was the DehH2-chlorpyrifos (RMSD 0.295 nm; RMSF 0.05 - 0.59 nm). The Molecular Mechanics Poisson-Boltzmann Surface Area calculations showed the DehH2-L-2CP complex (-24.27 kcal/mol) having the lowest binding energy followed by DehH2-MCA (-22.78 kcal/mol), DehH2-D-2CP (-21.82 kcal/mol), DehH2-3CP (-21.11 kcal/mol), DehH2-2,2-DCP (-18.34 kcal/mol), DehH2-2,3-DCP (-8.34 kcal/mol), DehH2-TCA (-7.62 kcal/mol), while chlorpyrifos was unable to spontaneously bind to DehH2 (+127.16 kcal/mol). In a nutshell, the findings of this study offer valuable insights into the rational tailoring of the DehH2 for expanding its substrate specificity and catalytic activity in the near future.Communicated by Ramaswamy H. Sarma.

In silico assessment of dehalogenase from Bacillus thuringiensis H2 in relation to its salinity-stability and pollutants degradation.

Increased scientific interest has led to the rise in biotechnological uses of halophilic and halotolerant microbes for hypersaline wastewater bioremediation. Hence, this study performed molecular docking, molecular dynamic (MD) simulations, and validation by Molecular Mechanic Poisson-Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis H2. We aimed to identify the interactions of DehH2 with substrates haloacids, haloacetates, and chlorpyrifos under extreme salinity (35% NaCl). MD simulations revealed that DehH2 preferentially degraded haloacids and haloacetates (-6.3 to -4.7 kcal/mol) by forming three or four hydrogen bonds to the catalytic triad, Asp125, Arg201, and Lys202. Conversely, chlorpyrifos was the least preferred substrate in both MD simulations and MM-PBSA calculations. MD simulation results ranked the DehH2-L-2CP complex (RMSD □0.125-0.23 nm) as the most stable while the least was the DehH2-chlorpyrifos complex (RMSD 0.32 nm; RMSF 0.0 - 0.29). The order of stability was as follows: DehH2-L-2CP > DehH2-MCA > DehH2-D-2CP > DehH2-3CP > DehH2-2,2-DCP > DehH2-2,3-DCP > DehH2-TCA > DehH2-chlorpyrifos. The MM-PBSA calculations further affirmed the DehH2-L-2CP complex's highest stability with the lowest binding energy of -45.14 kcal/mol, followed closely by DehH2-MCA (-41.21 kcal/mol), DehH2-D-2CP (-31.59 kcal/mol), DehH2-3CP (-30.75 kcal/mol), DehH2-2,2- DCP (-29.72 kcal/mol), DehH2-2,3-DCP (-22.20 kcal/mol) and DehH2-TCA (-18.46 kcal/mol). The positive binding energy of the DehH2-chlorpyrifos complex (+180.57 kcal/mol) proved the enzyme's non-preference for the substrate. The results ultimately illustrated the unique specificity of the DehH2 to degrade the above-said pollutants under a hypersaline condition.Communicated by Ramaswamy H. Sarma.

Whole genome strategies and bioremediation insight into dehalogenase-producing bacteria.

An integral approach to decoding both culturable and uncultured microorganisms' metabolic activity involves the whole genome sequencing (WGS) of individual/complex microbial communities. WGS of culturable microbes, amplicon sequencing, metagenomics, and single-cell genome analysis are selective techniques integrating genetic information and biochemical mechanisms. These approaches transform microbial biotechnology into a quick and high-throughput culture-independent evaluation and exploit pollutant-degrading microbes. They are windows into enzyme regulatory bioremediation pathways (i.e., dehalogenase) and the complete bioremediation process of organohalide pollutants. While the genome sequencing technique is gaining the scientific community's interest, it is still in its infancy in the field of pollutant bioremediation. The techniques are becoming increasingly helpful in unraveling and predicting the enzyme structure and explore metabolic and biodegradation capabilities.

Molecular docking and molecular dynamics simulations of a mutant Acinetobacter haemolyticus alkaline-stable lipase against tributyrin.

We previously reported on a mutant lipase KV1 (Mut-LipKV1) from Acinetobacter haemolyticus which optimal pH was raised from 8.0 to 11.0 after triple substitutions of surface aspartic acid (Asp) with lysine (Lys). Herein, this study further examined the Mut-LipKV1 by molecular docking, molecular dynamics (MD) simulations and molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) calculations to explore the structural requirements that participated in the effective binding of tributyrin and its catalytic triad (Ser165, Asp259 and His289) and identify detailed changes that occurred post mutation. Mut-LipKV1 bound favorably with tributyrin (-4.1 kcal/mol) and formed a single hydrogen bond with His289, at pH 9.0. Despite the incongruent docking analysis data, results of MD simulations showed configurations of both the tributyrin-Mut-LipKV1 (RMSD 0.3 nm; RMSF 0.05 - 0.3 nm) and the tributyrin-wildtype lipase KV1 (tributyrin-LipKV1) complexes (RMSD 0.35 nm; RMSF 0.05 - 0.4 nm) being comparably stable at pH 8.0. MM-PBSA analysis indicated that van der Waals interactions made the most contribution during the molecular binding process, with the Mut-LipKV1-tributyrin complex (-44.04 kcal/mol) showing relatively lower binding energy than LipKV1-tributyrin (-43.83 kcal/mol), at pH 12.0. All tributyrin-Mut-LipKV1 complexes displayed improved binding free energies over a broader pH range from 8.0 - 12.0, as compared to LipKV1-tributyrin. Future empirical works are thus, important to validate the improved alkaline-stability of Mut-LipKV1. In a nutshell, our research offered a considerable insight for further improving the alkaline tolerance of lipases.Communicated by Ramaswamy H. Sarma.

Dehalogenase-producing halophiles and their potential role in bioremediation.

This review aims to briefly describe the potential role of dehalogenase-producing halophilic bacteria in decontamination of organohalide pollutants. Hypersaline habitats pose challenges to life because of low water activity (water content) and is considered as the largest and ultimate sink for pollutants due to naturally and anthropogenic activities in which a substantial amount of ecological contaminants are organohalides. Several such environments appear to host and support substantial diversity of extremely halophilic and halotolerant bacteria as well as halophilic archaea. Biodegradation of several toxic inorganic and organic compounds in both aerobic and anaerobic conditions are carried out by halophilic microbes. Therefore, remediation of polluted marine/hypersaline environments are the main scorching issues in the field of biotechnology. Although many microbial species are reported as effective pollutants degrader, but little has been isolated from marine/hypersaline environments. Therefore, more novel microbial species with dehalogenase-producing ability are still desired.