GC-MS Analysis, Antioxidant and Antifungal Studies of Different Extracts of Chaetomium globosum Isolated from Urginea indica.

To discover new natural resources with biological effects, the chemical investigation of antioxidant and antimicrobial activities of extract's Chaetomium globosum isolated from roots of Urginea indica. Gas chromatography-mass spectrometry (GC-MS) analysis demonstrated the presence of the major chemical constituents present in the methanol extract (1,3-oxathiolane, 1,3-cyclopentadiene, 5-(1-methylethylidene), 5,9-hexadecadienoic acid, methyl ester, decane), chloroform extract (acetic acid, diethoxy-, ethyl ester, 2,2-bis(ethylsulfonyl)propane, 3-methyl-2-(2-oxopropyl) furan), and hexane extract (3-hexanone, 4,4-dimethyl, decane,2,6-dimethyldecane, decane, 2,4,6-trimethyl, decane, 2,4,6-trimethyl, 1-butanesulfinamide, 1,1,2,2,3,3,4,4,4-nonafluoro-N-methyl, decane). The total compound identified (56.2%) in chloroform extract, (54.72%) in hexane extract, and (65%) in methanol extract. The antioxidant effects were performed using diphenylpicrylhydrazyl radical (DPPH). The results showed that the methanol extract showed significantly the highest anti-DPPH with an IC50 value of 37.61 ± 1.37 µg/mL, followed by chloroform and hexane extracts with IC50 values of 40.82 ± 3.60 and 45.20 ± 2.54 µg/mL, respectively. The antifungal activity of extracts was evaluated against pathogens fungi including Fusarium oxysporum, Rosellinia necatrix, Cladosporium xanthochromaticum, and Sclerotinia sclerotiorum. Methanolic and chloroform extracts showed maximum inhibition against all test pathogens, while hexane extract showed minimum inhibition.

Nanocarriers-based immobilization of enzymes for industrial application.

Nanocarriers-based immobilization strategies are a novel concept in the enhancement of enzyme stability, shelf life and efficiency. A wide range of natural and artificial supports have been assessed for their efficacy in enzyme immobilization. Nanomaterials epitomize unique and fascinating matrices for enzyme immobilization. These structures include carbon nanotubes, superparamagnetic nanoparticles and nanofibers. These nano-based supports offer stable attachment of enzymes, thus ensuring their reusability in diverse industrial applications. This review attempts to encompass recent developments in the critical role played by nanotechnology towards the improvement of the practical applicability of microbial enzymes. Nanoparticles are increasingly being used in combination with various polymers to facilitate enzyme immobilization. These endeavors are proving to be conducive for enzyme-catalyzed industrial operations. In recent years the diversity of nanomaterials has grown tremendously, thus offering endless opportunities in the form of novel combinations for various biotransformation experimentations. These nanocarriers are advantageous for both free enzymes and whole-cell immobilization, thus demonstrating to be relatively effective in several fermentation procedures.

Nitrile hydratase mediated green synthesis of lactamide by immobilizing Rhodococcus pyridinivorans NIT-36 cells on N, N'-Methylene bis-acrylamide activated chitosan.

In this paper green synthesis of an important commodity chemical lactamide has been undertaken using chitosan immobilized Rhodococcus pyridinivorans NIT-36 harbouring nitrile hydratase (NHase) enzyme. The cells immobilization (300 mg/g) is based on the partial entrapment of cells by suspension cross-linking technique facilitated by N, N'-Methylene bis-acrylamide. In the repeated-use experiments, the immobilized cells retained 80% of its initial activity when stored at 4 °C for 30 days. NHase activity of free and immobilized cells was studied over temperature ranging from 25 °C to 60 °C. The activity for free cells showed a sharp decline of 70% when the reaction temperature was elevated from 45 °C to 50 °C whereas chitosan immobilized cells retained their activity in the same temperature range. A fed-batch reaction was designed and the immobilized cells showed 100% similar enzymatic pattern for five consecutive rounds which gradually decreased in following cycles. A volumetric productivity of 20 g/L and catalytic productivity of 8.33 g/g dcw/h for lactamide were achieved.

Random Mutagenesis of Thermophilic Xylanase for Enhanced Stability and Efficiency Validated through Molecular Docking.

BACKGROUND: Xylanases of thermophilic origin are more robust and stable and hence more suitable for industrial applications. The aim of the research was to develop a patent using a robust mutant exhibiting enhanced xylanase activity. The strain (Bacillus aestuarii SC-2014) subjected to mutagenesis is thermophilic in origin and hence it is envisioned that the enhancement of its catalytic potential will enhance its industrial applicability. OBJECTIVE: The main aim was to develop a stable and vigorous mutant having higher xylanase activity and improved thermostability. METHODS: The bacterial strain isolated from the Tattapani hot springs of Himachal Pradesh (India) was mutagenized by single separate exposure of Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG). RESULTS: A mutant library was generated and extensive screening led to the identification of the most potent mutant strain selected and designated as Bacillus sp. SC-2014 EMS200 (MTCC number 25046) which displayed not only enhanced xylanase activity and thermo stability but also appreciable genetic stability. This strain displayed a 3-fold increase in enzyme activity and simultaneously, a significant reduction in fermentation time from 72 h to 48 h was also observed. The xylanase gene from wild and mutant strain was cloned, sequenced and subjected to molecular docking. Two mutations H121D and S123T were present inside the binding pocket. CONCLUSION: Mutation H121D made the binding pocket more acidic and charged, thus enhancing the xylanase activity for mutant protein. Mutations also resulted in charged amino acids (Y99K and H121D) which were identified as a probable cause for enhancing the thermostability of mutant protein.

Purification and characterization of thermostable superoxide dismutase from Anoxybacillus gonensis KA 55 MTCC 12684.

A thermostable superoxide dismutase from thermophilic bacterium Anoxybacillus gonensis KA 55 MTCC 12684 which was isolated from Manikaran hotspring of Himachal Pradesh was purified to apparent homogeneity by fractional ammonium sulphate precipitation and anion exchange chromatography. A purification factor of 33.1-fold was achieved, with the purified enzyme exhibiting specific activity of 5758.4 U/mg protein. The purified superoxide dismutase was optimally active at pH 9.0 and displayed stability over a broad pH range of 7.0-10.0 and was stable up to 70 °C. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide ion. The molecular weight of superoxide dismutase was calculated as 31 kDa by SDS-PAGE. The Km and Vmax values of purified enzyme were found to be 1.002 mM and 14,285.71 U/mg of protein respectively. Tests of inhibitors indicated that the enzyme activity was inhibited by hydrogen peroxide and potassium cyanide but not by sodium azide showing that purified SOD was Cu/ZnSod.

Restriction enzyme-mediated insertional mutagenesis: an efficient method of Rosellinia necatrix transformation.

Rosellinia necatrix: causing root rot disease is a very destructive pathogen of woody plants and is responsible for yield losses to a large number of fruit trees. The genetic analysis of this pathogen has not been picked up because of difficulty in generating mutations in Rosellinia necatrix for many reasons. A number of methods have been proposed for inducing mutations in Rosellinia necatrix but none of them proved worth because of very low transformation efficiencies. Here, we propose an efficient method for Rosellinia necatrix protoplast production, where protoplasts in the tune of 107 per ml can be easily generated. We also propose a restriction enzyme-mediated integration (REMI)-based methods for efficient transformation of Rosellinia necatrix. In the present study, an approximate of 800 transformants was obtained from 5 µg of linearized plasmid. Out of 47 single spored transformants analyzed, only 33 showed hygromycin gene amplification using PCR and only 19 transformants showed single gene integration in southern hybridization, which accounted for single gene integration percentage of 42%, highest amongst all the previous reports on Rosellinia necatrix transformations. Some of the transformants studied for pathogenicity phenotype also showed a marked reduction in pathogenicity. Thus, in the present investigation, 42% single gene integrations among the transformed colonies can be considered as excellent transformation efficiency.

Improving stability and reusability of Rhodococcus pyridinivorans NIT-36 nitrilase by whole cell immobilization using chitosan.

Immobilized enzymes have great significance for industrial processes. In this study whole cell immobilization of Rhodococcus pyridinivorans NIT-36 has been undertaken using chitosan microspheres as an immobilized matrix. R. pyridinivorans NIT-36 harbors a significant intracellular enzyme nitrilase. Chitosan microspheres were generated by supplementing chitosan with glutaraldehyde and results, supported the porous microsphere structure via SEM. The resultant microspheres exhibited cell immobilization capacity of 450mg/g. The immobilized cells exhibited a considerable increase in temperature tolerance at 60°C as compared to free cells. The immobilized microspheres also demonstrated higher substrate tolerance. The immobilized nitrilase retained 80% activity when stored at 4°C for 10 days and retained 50% activity after 7 reuse cycles. It may be concluded that chitosan microspheres are a novel immobilization agent for whole cell immobilization which enhances the stability and reusability of nitrilase enzyme.