In-silico evaluation of Oroxylum indicum vent compounds in the plausible treatment and prevention of nasopharyngeal cancer.

BACKGROUND: Shyonaka (Oroxylum indicum Vent) is widely used in Ayurveda and in ethnomedical practice for the treatment of inflammation, pain, diarrhea, non-healing ulcers, and cancer. Owing to the high prevalence of Epstein-Barr virus (EBV) infection in Nasopharyngeal carcinoma (NPC) patients, simultaneous targeting of proteins involved in both EBV replication and NPC proliferation might help to manage the disease effectively. OBJECTIVES: This study is designed to identify potential dual targeting inhibitors from Oroxylum indicum having the potential to inhibit both EBV and NPC. This study also attempted quantitative analysis of Shyonaka Bark Decoction (SBD) to confirm the presence of Baicalein and Chrysin which are predominant marker compounds of Shyonaka. METHODOLOGY: The HPLC analysis of stem bark and root bark of Oroxylum indicum was done to estimate the presence of marker compounds Baicalein and Chrysalin. The in-silico analysis included ADMET analysis followed by molecular docking of known compounds from Oroxylum indicum (retrieved from IMPPAT database) onto the target proteins of EBV (BHRF1, NEC1, dUTPase, Uracil DNA glycosylase) and NPC (COX-2, EGFR, and MDM2) using DOCK6 tool. Further validations were done using the molecular dynamics simulations of top screened molecules onto the selected target proteins using AMBER20 package and their corresponding MMGBSA binding free-energy values were calculated. RESULTS: The molecular docking revealed that the key molecules from the plant, scutellarein 7-rutinoside (S7R), scutellarin (SCU) and 6-hydroxyluteolin, Baicalein and 5,7-Dihydroxy-2-phenyl-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one (57D) are effectively intervening with the target proteins of EBV, one of the key causative factors of NPC and the NPC specific targets which have the potential to reduce tumor size and other consequences of NPC. The molecular dynamics simulations of S7R, Baicalein and 57D, Baicalein with MDM-2 protein and dUTPase protein, respectively, showed stable interactions between them which were further assessed by the binding energy calculations. CONCLUSION: Overall, the in-silico evaluation of these phytochemicals with target proteins indicates their potential to inhibit both EBV and NPC which needs further in-vitro and in-vivo validations.

Functional Outcomes in Anterior Cruciate Ligament (ACL) Reconstruction: A Nine-Month Follow-up Study Using Lysholm Score in a Rural Tertiary Care Center in India.

Introduction The knee joint, an extraordinary feat of biomechanics, is prone to injuries, with the anterior cruciate ligament (ACL) often being a common victim. The intricate coordination of joint movements relies heavily on the ACL's screw-home mechanism, a crucial element for synchronizing knee movement with neighboring joints. Despite its indispensable role, the ACL is susceptible to injury, necessitating surgical intervention. While many patients experience positive outcomes following ACL reconstruction surgeries, a significant proportion face the challenge of procedure failure. The key to success lies in the healing process within the tibial and femoral bone tunnels. The post-ACL reconstruction phase introduces its own set of challenges, particularly in the context of returning to sports (RTS), underscoring the importance of reinstating neuromuscular and motor function. The trajectory of rehabilitation is influenced by factors such as graft healing, patient age, gender, pain levels, and concurrent injuries. Materials and methods This prospective observational study spanned 2.5 years, enrolling 71 patients with diagnosed ACL injuries. Arthroscopic reconstruction utilized hamstring autografts and peroneus longus autografts. A nine-month post-surgery follow-up employed the Lysholm scoring system for comprehensive evaluations. Results Over 2.5 years, 87.3% of male and 12.6% of female participants underwent arthroscopic reconstruction. Lysholm scores revealed 28.1% excellent, 45.0% good, and 26.7% fair outcomes, with no participants in the unsatisfactory range. Lysholm scores demonstrated positive outcomes, indicating the efficacy of arthroscopic reconstruction in enhancing knee function. Findings align with existing literature, emphasizing positive results from ACL reconstruction techniques and specific implants. Comparisons with related studies highlight challenges in standardized return-to-sport guidelines and underscore the need for outcome measure standardization. Conclusion The study contributes nuanced insights into ACL reconstruction outcomes, emphasizing positive functional recovery trends at the nine-month follow-up. Lysholm scores indicate favorable outcomes, supporting the procedure's effectiveness. Consideration of specific implants adds practical value. Despite limitations, this study enriches ACL reconstruction research, promoting advancements in patient care and outcomes. Ongoing research with extended follow-ups and larger cohorts will enhance understanding and refine ACL reconstruction strategies.

Impact of Meniscus Repair in Conjunction With Anterior Cruciate Ligament Reconstruction on Functional Outcomes at Six Months.

Background and aim Anterior cruciate ligament (ACL) injuries often occur along with menisci tears. ACL reconstruction with meniscectomy has long been the preferred technique for such injuries; however, it has been postulated to increase the chances of osteoarthritis (OA). Therefore, recent techniques have involved preserving menisci while reconstructing ACL to prevent OA and improve overall functional outcomes. This study aimed to evaluate the functional outcomes of arthroscopic meniscal repair performed concurrently with ACL reconstruction at six months post-surgery. Methodology We conducted a cross-sectional study at a tertiary care center after getting appropriate ethics committee approval. A total of 67 participants who met the inclusion and exclusion criteria were enrolled in the study after obtaining informed consent. Their demographics were recorded retrospectively from hospital records, while their Lysholm Knee Score (LKS) responses were collected prospectively during their sixth-month follow-up visit to our department. Analysis was done using Microsoft Excel. Appropriate statistical tests including chi-square, analysis of variance (ANOVA), and independent t-tests were applied to keep an alpha of 0.05. Results We found that the mean age of participants was 35 years. The mean LKS of patients who underwent isolated ACL reconstruction (ACLR) was 86.02 ± 9.38. For those who underwent ACLR plus meniscus repair (MR), the mean LKS was marginally higher at 87.4 ± 7.41 during their sixth-month follow-up, with a P-value of 0.27. Furthermore, the mean LKS of patients who underwent ACLR plus meniscectomy was 86 ± 10.48. Comparing the means of all three groups revealed no statistical difference among any surgical approach with a P-value of 0.69. A total of 33 (49.25%) participants achieved an LKS falling within the Good category (84-94). Comparing between three surgical groups and their LKS categories also revealed no statistical difference with a P-value of 0.7. Conclusions Short-term functional outcomes in patients undergoing ACLR or ACLR plus MR using patient-reported knee scores like LKS demonstrate favorable outcomes but fail to demonstrate statistical significance. On a longer follow-up period, a reduction in the prevalence of OA is a possibility with the preservation of menisci; however, conflicting evidence in the literature about the approach to ACL injuries with menisci involvement warrants large-scale randomized controlled trials to decide upon the standard of care.

Chemoprofiling and medicinal potential of underutilized leaves of Cyperus scariosus.

Agro-waste is the outcome of the under-utilization of bioresources and a lack of knowledge to re-use this waste in proper ways or a circular economy approach. In the Indian medicinal system, the root of Cyperus scariosus (CS) is used at a large scale due to their vital medicinal properties. Unfortunately, the aerial part of CS is treated as agro-waste and is an under-utilized bioresource. Due to a lack of knowledge, CS is treated as a weed. This present study is the first ever attempt to explore CS leaves as medicinally and a nutrient rich source. To determine the food and nutritional values of the neglected part of Cyperus scariosus R.Br. (CS), i.e. CS leaves, phytochemicals and metal ions of CS were quantified by newly developed HPLC and ICPOES-based methods. The content of the phytochemicals observed in HPLC analysis for caffeic acid, catechin, epicatechin, trans-p-coumaric acid, and trans-ferulic acid was 10.51, 276.15, 279.09, 70.53, and 36.83 µg/g, respectively. In GC-MS/MS analysis, fatty acids including linolenic acid, phytol, palmitic acid, etc. were identified. In ICPOES analysis, the significant content of Na, K, Ca, Cu, Fe, Mg, Mn, and Zn was observed. The TPC and TFC of the CS leaves was 17.933 mg GAE eq./g and 130.767 mg QCE eq./g along with an IC50 value of 2.78 mg/mL in the DPPH assay and better antacid activity was measured than the standard (CaCO3). The methanolic extract of CS leaves showed anti-microbial activity against Staphylococcus aureus (15 ± 2 mm), Pseudomonas aeruginosa (12 ± 2 mm) and Escherichia coli (10 ± 2 mm). In silico studies confirmed the in vitro results obtained from the antioxidant, antiacid, and anti-microbial studies. In addition, in silico studies revealed the anti-cancerous and anti-inflammatory potential of the CS leaves. This study, thus, demonstrated the medicinal significance of the under-utilized part of CS and the conversion of agro-waste into mankind activity as a pharmaceutical potent material. Consequently, the present study highlighted that CS leaves have medicinal importance with good nutritional utility and have a large potential in the pharmaceutical industry along with improving bio-valorization and the environment.

Establishment of the mechanism of purification and levigation of green chemistry-assisted biocomposites of red ochre (Gairika): synthesis, characterization, and antibacterial, prebiotic, antioxidant, and antacid activities of the traditional Ayurvedic medicine Laghu Sutashekhara Rasa.

Gairika (red ochre) has a long history of influencing human civilization. Gairika is a rich source of nutrients used for reproductive and brain health. Gairika is mentioned as an antacid drug in Indian Ayurvedic medicine under Laghu Sutashekhara Rasa (LSR). However, a detailed study on LSR has not been reported to date. In the present study, LSR was prepared, and a pharmaceutical SOP (standardization procedure) was reported to obtain batch-to-batch reproducibility. LSR was characterized using FTIR, XRD, SEM-EDX, and TGA analyses. LSR was tested in vitro for its antacid activity. Advanced instrumentation revealed that LSR formation produced symmetrical particles (5-8 µm) with kaolin, kaolinite, quartz, goethite, and hematite, along with the phytoconstituents of Goghrita (clarified cow's butter), Shunthi, and Nagawalli, as confirmed by GC-MS/MS analysis. The FTIR study revealed the formation of a chelating complex of goethite and hematite along with their phytoconstituents. XRD analysis confirmed the presence of kaolin, kaolinite, quartz, goethite, and hematite. Using in vitro antacid experiments, LSR and Shunthi demonstrated significant antacid activity as compared to antacid drugs and standards in the market, such as CaCO3. The DPPH assay revealed IC50 values of 12.16 ± 1.23 mg/mL, which is 0.0029 of Trolox-equivalent antioxidant activity. The inhibition (18 ± 4 mm) against pathogens (S. aureus, E. coli, P. aeruginosa, and B. subtilis) and the prominent growth of gut microbiota-supported strains (S. boulardii, L. paracasei, and L. plantarum) observed on LSR formulation were indicative of LSR application as a prebiotic. Here, the mechanism of purification and levigation mentioned in the classical literature of LSR was established. Overall, purification of Gairika with cow ghee and levigation with Nagawalli may enhance the solubility, bioavailability, and shelf-life of LSR through hydration and co-crystallization mechanisms. This is the first comprehensive report on the pharmaceutical validation of LSR and its characterization. The results of the present study could contribute to the development and reliable reproduction of LSR and the utility of environmental red ochre as a medicine in combination with Shunthi (Zingiber officinale Roxb.), as prescribed under Indian Ayurvedic medicine.

A Comparative Analysis of Phytochemicals, Metal Ions, Volatile Metabolites in Heart Wood, Stem Bark and Leaves of Salix alba L. along with in Vitro Antioxidant, Antacid, Antimicrobial Activities for Sake of Environment Conservation by Substitution of Stem Bark With Leaf.

The genus of Salix is used in food, medicine and nutraceuticals, and standardized by using the single marker compound Salicin only. Stem bark is the official part used for the preparation of various drugs, nutraceuticals and food products, which may lead to overexploitation and damage of tree. There is need to search substitution of the stem bark with leaf of Salix alba L. (SA), which is yet not reported. Comparative phytochemicals viz. Salicin, Procyanidin B1 and Catechin were quantified in the various parts of SA viz. heart wood (SA-HW), stem bark (SA-SB) and leaves (SA-L) of Salix alba L.by using newly developed HPLC method. It was observed that SA-HW and SA-L contained far better amount of Salicin, Procyanidin B and Catechin as compared to SA-SB (SA-HW~SA-L≫SA-SB). Essential and toxic metal ions of all three parts were analysed using newly developed ICP-OES method, where SA-L were founded as a rich source of micronutrients and essential metal ions as compared to SA-SB and SA-HW. GC-MS analysis has shown the presence of fatty acids and volatile compounds. The observed TPC and TFC values for all three parts were ranged from 2.69 to 32.30 mg GAE/g of wt. and 37.57 to 220.76 mg QCE/g of wt. respectively. In DPPH assay the IC50 values of SA-SB, SA-HW, and SA-L were 1.09 (±0.02), 5.42 (±0.08), and 8.82 (±0.10) mg/mL, respectively. The order of antibacterial activities against E. coli, S. aureus, P. aeruginosa, and B. subtilis strains was SA-L>SA-HW>SA-SB with strong antibacterial activities against S. aureus, and B. subtilis strains. The antacid activities order was SA-L>SA-SB>SA-HW. The leaves of SA have shown significant source of nutrients, phytochemicals and medicinal properties than SA-HW and SA-SB. The leaves of SA may be considered as substitute of stem bark to save the environment or to avoid over exploitation, but after the complete pharmacological and toxicological studies.

Quantification of Phytochemicals and Metal Ions as well as the Determination of Volatile Compounds, Antioxidant, Antimicrobial and Antacid Activities of the Mimosa pudica L. Leaf: Exploration of Neglected and Under-Utilized Part.

Mimosa pudica L. (MP) is well-known plant in traditional medicinal system, especially in India. Unfortunately, leaves of MP are less explored. To determine the food and nutritional value of the neglected part of Mimosa pudica L. (MP), that is MP leaves, phytochemicals and metal ions of MP were quantified by newly developed HPLC and ICPOES-based methods. The content of phytochemicals observed using HPLC analysis for chlorogenic acid, catechin, and epicatechin was 141.823 (±8.171), 666.621 (±11.432), and 293.175 (±12.743) µg/g, respectively. Using GC/MS/MS analysis, fatty acid like oleic acid were identified. In ICP-OES analysis, a significant content of Na, K, Ca, Cu, Fe, Mg, Mn, and Zn was observed. The observed TPC and TFC for MP leaf extracts was 44.327 (±1.041) mg GAE/ g of wt. and 214.217 (±4.372) mg QCE/ g of wt., respectively. The DPPH assay depicted a strong antioxidant activity of MP leaf extracts with IC50 values of 0.796 (±0.081) mg/mL and a TEAC value of 0.0356 (±0.0003). A significant antacid activity (666 mg MP+400 mg CaCO3 >400 mg CaCO3 ≫666 mg Gelusil) of MP leaves was noticed. The methanolic extract of MP leaves demonstrated anti-microbial activity against Staphylococcus aureus (15±2mm), Pseudomonas aeruginosa (12±2mm) and Escherichia coli (10±2mm). In silico studies confirmed the in vitro results obtained for antioxidant, antiacid, and anti-microbial activities. In addition, in silico studies revealed the anti-cancerous and anti-inflammatory potential of the MP leaves. In summary, this study demonstrated the medicinal significance of MP leaves and the conversion of agro-waste or the under-utilized part of MP into pharmaceutical potent materials. Consequently, the present study highlighted that MP leaves alone have medicinal importance with good nutritional utility and possess large promise in the pharma industry along with improving bio-valorization and the environment.

Identification of potential inhibitors of HER2 targeting breast cancer-a structure-based drug design approach.

Breast cancer is one of the most prevalent and malignant cancers in women. Most breast cancer patients show overexpression of the HER2 protein. The current study focused on identifying potent inhibitors of HER2 using a structure-based drug design approach. Prefiltered compounds from the Drugbank and the ZINC database were docked on HER2 protein using the FlexX docking tool of LeadIT. The docking study identified the 12 best molecules that interacted strongly with the active site of HER2 and also fulfilled the ADMET parameters. The complexes of these compounds with HER2 were further subjected to molecular dynamics simulation using GROMACS 2021.4, followed by the end-state MMGBSA binding energy calculations. The RMSD analysis was conducted to study the conformational changes, which revealed stability throughout the 100 ns simulation period. The local flexibility and dynamics of the simulated ligand-protein complexes were studied using RMSF analysis. The values of the radius of gyration were computed to analyze the compactness of HER2. The MMGBSA analysis provided insights into the energetic aspects of the system. The compound DB15187 emerged as the most potent candidate, showing MMGBSA-computed binding energy of -63.60 ± 3.39 kcal/mol. The study could help develop targeted therapies for HER2-positive breast cancer.Communicated by Ramaswamy H. Sarma.

Identification of potential neuroprotective compound from Ganoderma lucidum extract targeting microtubule affinity regulation kinase 4 involved in Alzheimer's disease through molecular dynamics simulation and MMGBSA.

Objective: Alzheimer's disease (AD) is one of the most prevalent neurological ailments, affecting around 50 million individuals globally. The condition is characterized by nerve cell damage due to the formation of amyloid-beta plaques and neurofibrillary tangles. Only a few US Food and Drug Administration (FDA)-approved medications are available in the market which are devoid of side effects, thus, making it imperative to investigate new alternatives for countering this disease. According to a recent study, microtubule affinity regulation kinase 4 (MARK4) is attributed as one of the most promising drug targets for AD, thus, being selected for this study. Compounds from Ganoderma lucidum (Reishi mushroom) extracts were selected to be used as ligands for this study. Methods: In this study, the five most potent compounds from Ganoderma lucidum were selected and their absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was performed, followed by molecular docking, and molecular dynamics simulation of each compound with MARK4 and supported by molecular mechanics generalized born surface area (MMGBSA) binding free energy calculations. Results: The promising compounds were selected based on their ADMET profile and interactions with the active site residues of MARK4. Based on docking scores of -9.1 and -10.3 kcal/ mol, respectively, stability assessment by molecular dynamics simulation, and MMGBSA calculations, ganoderic acid A and ganoderenic acid B were found to be the most promising compounds against MARK4 which will require further in vitro and in vivo validations. Conclusion: Through this study, it is suggested that ganoderic acid A and ganoderenic acid B might be a class of promising compounds against AD, based on computational research, and can be further studied for preclinical and clinical studies.

Dioxinodehydroeckol: A Potential Neuroprotective Marine Compound Identified by In Silico Screening for the Treatment and Management of Multiple Brain Disorders.

Neurodegenerative disorders such as Alzheimer's disease (AD), Glioblastoma multiforme (GBM), Amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD) are some of the most prevalent neurodegenerative disorders in humans. Even after a variety of advanced therapies, prognosis of all these disorders is not favorable, with survival rates of 14-20 months only. To further improve the prognosis of these disorders, it is imperative to discover new compounds which will target effector proteins involved in these disorders. In this study, we have focused on in silico screening of marine compounds against multiple target proteins involved in AD, GBM, ALS, and PD. Fifty marine-origin compounds were selected from literature, out of which, thirty compounds passed ADMET parameters. Ligand docking was performed after ADMET analysis for AD, GBM, ALS, and PD-associated proteins in which four protein targets Keap1, Ephrin A2, JAK3 Kinase domain, and METTL3-METTL14 N6-methyladenosine methyltransferase (MTA70) were found to be binding strongly with the screened compound Dioxinodehydroeckol (DHE). Molecular dynamics simulations were performed at 100 ns with triplicate runs to validate the docking score and assess the dynamics of DHE interactions with each target protein. The results indicated Dioxinodehydroeckol, a novel marine compound, to be a putative inhibitor among all the screened molecules, which might be effective against multiple target proteins involved in neurological disorders, requiring further in vitro and in vivo validations.

Parametrically optimized feather degradation by Bacillus velezensis NCIM 5802 and delineation of keratin hydrolysis by multi-scale analysis for poultry waste management.

Enormous amounts of keratinaceous waste make a significant and unexploited protein reserve that can be utilized through bioconversion into high-value products using microbial keratinases. This study was intended to assess the keratinase production from a newly isolated B. velezensis NCIM 5802 that can proficiently hydrolyze chicken feathers. Incubation parameters used to produce keratinase enzyme were optimized through the Response Surface Methodology (RSM) with chicken feathers as substrate. Optimization elevated the keratinase production and feather degradation by 4.92-folds (109.7 U/mL) and 2.5 folds (95.8%), respectively. Time-course profile revealed a direct correlation among bacterial growth, feather degradation, keratinase production and amino acid generation. Biochemical properties of the keratinase were evaluated, where it showed optimal activity at 60 °C and pH 10.0. The keratinase was inhibited by EDTA and PMSF, indicating it to be a serine-metalloprotease. Zymography revealed the presence of four distinct keratinases (Mr ~ 100, 62.5, 36.5 and 25 kDa) indicating its multiple forms. NMR and mass spectroscopic studies confirmed the presence of 18 free amino acids in the feather hydrolysates. Changes in feather keratin brought about by the keratinase action were studied by X-ray diffraction (XRD) and spectroscopic (FTIR, Raman) analyses, which showed a decrease in the total crystallinity index (TCI) (1.00-0.63) and confirmed the degradation of its crystalline domain. Scanning electron microscopy (SEM) revealed the sequential structural changes occurring in the feather keratin during degradation. Present study explored the use of keratinolytic potential of the newly isolated B. velezensis NCIM 5802 in chicken feather degradation and also, unraveled the underlying keratin hydrolysis mechanism through various analyses.

Molecular insight into Aspergillus oryzae ß-mannanase interacting with mannotriose revealed by molecular dynamic simulation study.

Fungal ß-mannanases hydrolyze ß-1, 4-glycosidic bonds of mannans and find application in the generation of mannose and prebiotic mannooligosaccharides (MOS). Previously, a MOS generating ß-mannanase from Aspergillus oryzae MTCC 1846 (ßManAo) was characterized and its structural and functional properties were unraveled through homology modeling and molecular dynamics in this study. The ßManAo model was validated with 92.9% and 6.5% of the residues found to be distributed in the most favorable and allowed regions of the Ramachandran plot. Glu244 was found to play a key role in the interaction with mannotriose, indicating conserved amino acids for the catalytic reaction. A detailed metadynamic analysis of the principal components revealed the presence of an α8-helix in the C-terminus which was very flexible in nature and energy landscapes suggested high conformation sub-states and the complex dynamic behavior of the protein. The binding of the M3 substrate stabilized the ß-mannanase and resulted in a reduction in the intermediate conformational sub-states evident from the free energy landscapes. The active site of the ß-mannanase is mostly hydrophilic in nature which is accordance with our results, where the major contribution in the binding energy of the substrate with the active site is from electrostatic interactions. Define Secondary Structure of Proteins (DSSP) analysis revealed a major transition of the protein from helix to ß-turn for binding with the mannotriose. The molecular dynamics of the ßManAo-mannotriose model, and the role and interactions of catalytic residues with ligand were also described. The substrate binding pocket of ßManAo was found to be highly dynamic and showed large, concerted movements. The outcomes of the present study can be exploited in further understanding the structural properties and functional dynamics of ßManAo.

Hierarchically Porous Metal-Organic Gel Hosting Catholyte for Limiting Iodine Diffusion and Self-Discharge Control in Sustainable Aqueous Zinc-I2 Batteries.

Rechargeable aqueous zinc-iodine batteries (AZIBs) represent excellent zinc-iodine redox chemistry and emerged as a promising aspirant due to their high safety, low cost, ease of fabrication, and high energy density. Nevertheless, the high-dissolution-induced iodide diffusion toward the zinc anode brings the self-discharge, which governs the capacity fading and poor cycling life of the battery. Herein, a multipurpose sponge-like porous matrix of a metal-organic gel to host a substantial amount of an iodine-based catholyte and uniform distribution of iodine with controlled iodide diffusion is introduced. Limiting the iodine diffusion due to increased viscosity provides superior electrochemical performance of this promising cathode for solid-state AZIBs. As a result, AZIBs delivering high performance and long-term stability are fabricated with a capacity of 184.9 mA h g-1 with a superior capacity retention of 95.8% even after 1500 cycles at 1 C rate. The unique concept of self-discharge protection is successfully evaluated. Prototype flexible band-aid-type AZIBs were fabricated, which delivered 166.4 mA h g-1 capacity in the bending state, and applied to real-scale wearable applications.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

Production optimization and characterization of mannooligosaccharide generating ß-mannanase from Aspergillus oryzae.

A multi-tolerant ß-mannanase (ManAo) was produced by Aspergillus oryzae on copra meal, a low-cost agro waste. Under statistically optimized conditions, 4.3-fold increase in ß-mannanase production (434 U/gds) was obtained. Purified ManAo had MW ∼34 kDa and specific activity of 335.85 U/mg with optimum activity at 60 °C and at pH 5.0. Activity of ManAo was enhanced by most metal ions and modulators while maximum enhancement was noticed with Ag+ and Triton X-100. Km and Vmax were 2.7 mg/mL and 1388.8 µmol/min/mg for locust bean gum while the enzyme showed lower affinity towards konjac gum (8.8 mg/mL, 555.5 µmol/min/mg). Evaluation of various thermodynamic parameters indicated high-efficiency of the ManAo with activation energy 12.42 KJ/mol and 23.31 KJ/mol towards LBG and konjac gum, respectively. End product analysis of ß-mannanase action by fluorescence assisted carbohydrate electrophoresis (FACE) revealed the generation of sugars from DP 1-4 with some higher DP MOS from different mannans.

Enzymatic monoesterification of symmetric diols: restriction of molecular conformations influences selectivity.

We have experimentally demonstrated that by 'locking' the molecular conformation through the introduction of a double or triple bond in the center of a symmetric diol, enzymatic monoesterification can be achieved selectively. The enzyme Candida antarctica lipase B, generally used for the transesterification of diols, can be effectively used for the monoesterification of symmetrical diols in an unbuffered system also. By varying the chain length of a carboxylic acid moiety, we have established that optimum selectivity and efficiency can be achieved in the range of 4.8 to 5.0 pKa values. Selectivity can be improved up to 98.75% for a monoester in an overall 73% yield (mixture of a monoester and a diester) when but-2-yne-1,4-diol reacted with hexanoic acid. Water, a by-product, provides an interfacial environment for the enzyme to work in the organic reaction medium. The uniqueness of the reported monoesterification protocol is that it involves only the mechanical stirring of the reaction mixture at room temperature in the presence of the enzyme for 24 h. High percentage yield with selectivity for a monoester, easier product isolation and overall, environmental sustainability are added advantages. The synthesized monoesters are characterized by using HNMR and high resolution mass spectrometry (HRMS).

Biotechnological potential of microbial inulinases: Recent perspective.

Among microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Screening, statistical optimized production, and application of ß-mannanase from some newly isolated fungi.

Eighty-eight fungi isolated from soil and decaying organic matter were screened for mannanolytic activity. Twenty-eight fungi produced extracellular mannanase on locust bean gum as evidenced by zone of hydrolysis produced on mannan agar gel. Six prominent producers, including four Fusarium species namely Fusarium fusarioides NFCCI 3282, Fusarium solani NFCCI 3283, Fusarium equiseti NFCCI 3284, Fusarium moniliforme NFCCI 3287 with Cladosporium cladosporioides NFCCI 3285 and Acrophialophora levis NFCCI 3286 produced the ß-mannanase in the range of 84-140 nkat/mL. All these grew well on particulate substrates in solid-state fermentation (SSF), producing relatively higher titers on mannan-rich palm kernel cake (PKC) and copra meal. Two high yielding strains, F. equiseti (1747 nkat/gds) and A. levis (897 nkat/gds) were selected for statistical optimization of mannanase on PKC. Interaction of two critical solid state fermentation parameters, pH and moisture on mannanase production by these two molds was studied by response surface method. Optimized production on PKC resulted in three- to fourfold enhancement in enzyme yield was observed in case of F. equiseti (5945 nkat/gds) and A. levis (4726 nkat/gds). HPLC analysis of mannan hydrolysate indicated that F. equiseti and A. levis mannanase performed efficient hydrolysis of konjac gum (up to 99%) with exclusive mannooligosaccahride (DP of 4) production. A seminative SDS-PAGE revealed that A. levis and F. solani produced three isoforms, F. moniliforme produced two isoforms while F. fusarioides, F. equiseti, and C. cladosporioides produced a single enzyme.

Experimental design of response surface methodology used for utilisation of palm kernel cake as solid substrate for optimised production of fungal mannanase.

The results obtained from this work strongly indicate that the solid state fermentation (SSF) system using the palm kernel cake (PKC) as a substrate is an economical method for the production of ß-mannanase at extremely low operational cost based on the fact that PKC is one of the cheap and abundant agro-waste by-products of the palm oil industry. Under initial conditions, i.e. 2 mm particle size of PKC, the moisture ratio of 1:1 of PKC:moistening agent and pH 7, Malbranchea cinnamomea NFCCI 3724 produced 109 U/gram distribution of the substrate (gds). The production of ß-mannanase was optimised by the statistical approach response surface methodology (RSM) using independent variables, namely initial moisture (12.5), pH (9.0) and solka floc (100 mg). Noticeably, six fold enhancement of ß-mannanase production (599 U/gds) was obtained under statistically optimised conditions. HPLC results revealed that ß-mannanase is an endo-active enzyme that generated manno-oligosaccharides with a degree of polymerisation (DP) of 3 and 4. Semi-native PAGE analysis revealed that M. cinnamomea produced three isoforms of mannanase. Selective production of oligosaccharide makes M. cinnamomea ß-mannanase an attractive enzyme for use in food and nutraceutical industries.

Purification and characterization of ß-mannanase from Aspergillus terreus and its applicability in depolymerization of mannans and saccharification of lignocellulosic biomass.

Aspergillus terreus FBCC 1369 was grown in solid-state culture under statistically optimized conditions. ß-Mannanase was purified to apparent homogeneity by ultrafiltration, anion exchange and gel filtration chromatography. A purification factor of 10.3-fold was achieved, with the purified enzyme exhibiting specific activity of 53 U/mg protein. The purified ß-mannanase was optimally active at pH 7.0 and 70 °C and displayed stability over a broad pH range of 4.0-8.0 and a 30 min half-life at 80 °C. The molecular weight of ß-mannanase was calculated as ~49 kDa by SDS-PAGE. The enzyme exhibited K m and V max values of 5.9 mg/ml and 39.42 µmol/ml/min, respectively. ß-Mannanase activity was stimulated by ß-mercaptoethanol and strongly inhibited by Hg2+. The ß-Mannanase did not hydrolyze mannobiose and mannotriose, but only mannotetraose liberating mannose and mannotriose. This indicated that at least four mannose residues were required for catalytic activity. Oligosaccharide with a degree of polymerization (DP) three was the predominant product in the case of locust bean gum (16.5 %) and guar gum (15.8 %) hydrolysis. However, the enzyme liberated DP4 oligosaccharide (24 %) exclusively from konjac gum. This property can be exploited in oligosaccharides production with DP 3-4. ß-Mannanase hydrolyzed pretreated lignocelluloses and liberated reducing sugars (% theoretical yield) from copra meal (30 %). This property is an important factor for the bioconversion of the biomass.