Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting ...considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale ; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from Curcuma amada , identified as Talaromyces pinophilus , was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. Talaromyces pinophilus initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family.
The partitioning of Lactoferrin (LF) into the reverse micellar phase formed by a cationic surfactant, cetyltrimethylammonium bromide (CTAB) in
n
-heptanol from the synthetic solution of LF was ...studied. The solubilization behaviour of LF into the reverse micellar phase and back extraction using a fresh stripping phase were improved by studying the effect of processing parameters, including surfactant concentration, solution pH, electrolyte salt concentration and addition of alcohol as co-solvent. Forward extraction of 100% was achieved at CTAB concentration of 50 mM in
n
-heptanol solvent, pH of 10 and 1 M NaCl. The electrostatic force and hydrophobic interaction have major influence on LF extraction during forward and back extraction respectively. The size of the reverse micelles and their corresponding water content were measured at different operating conditions to assess their role on the LF extraction. The present reverse micellar system has potential to solubilise almost all the LF into the reverse micelles during forward extraction and could able to allow back extraction from the reverse micellar phase with addition of small amount of co-solvent.
Fibrinolytic enzymes are agents administered for the treatment of myocardial infarctions, strokes, cardiac and respiratory failure. Although several microorganisms are known to produce these ...fibrinolytic enzymes, only a few of such enzymes, along with the age-old oral anticoagulants, have been employed in the clinical and therapeutic applications in humans. The use of these agents is associated with drawbacks such as allergic reactions and bleeding complications; therefore, it necessitates frequent monitoring of drug levels in the blood. Due to this, there is an impetus on the current effort to identify newer potential candidates from the novel microbial sources which show longer half-life, higher fibrin specificity, higher therapeutic index and lesser allergic reactions. Various methods are available for the preliminary evaluation of a potential drug candidate for the therapeutic use. Choosing the right combination of in vitro and in vivo methods would give crucial insight on the therapeutic potential of the chosen test compound. This article discusses various assay techniques, in vitro trails and in vivo models available, to help researchers in choosing right biological methods and its combinations to evaluate efficacy of potential drug candidate.
Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in ...clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U mg
−1
protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4-80°C for 6 h. The enzymatic activity was not influenced by metal ions and chemical agents (K
+
, Na
+
, Zn
2+
, Fe
3+
, Mn
2+
, Mg
2+
, glucose, urea, lactate) commonly found in serum and urine, with Cu
2+
being the exception. The enzyme appears to be a metalloprotein stimulated by Ca
2+
and Fe
2+
. Its K
m
and K
cat
for oxalate were found to be 0.45 mM and 85 s
−1
, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50 mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.
Enzymatic synthesis of propyl gallate in organic solvent was studied using cell-associated tannase (EC 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as the ...biocatalyst. The effects of solvent, surfactant treatment, and bioimprinting on the propyl gallate synthesis were studied and subsequently optimized. Among various solvents, benzene followed by hexane was found to be the most favorable. Treatment of the biocatalyst with Triton X-100 at a lower concentration (0.2% w/v), before lyophilization, increased the propyl gallate yield by 24.5% compared to the untreated biocatalyst. The biocatalyst was imprinted with various concentrations of gallic acid and tannic acid. Biocatalyst imprinted with tannic acid showed 50% enhancement in the propyl gallate yield compared to the non-imprinted biocatalyst.
Heavy metal removal efficiency of indigenously present metal tolerant fungal isolates obtained from a scrap dumpsite was assessed in this study. Four fungal isolates were selected based on their ...ability to grow in multi-metal supplemented media. Minimum inhibition concentrations of these four isolates were determined against individual metals; lead (II) (50–400 mgL−1), cadmium (II) (50–400 mgL−1), arsenic (III) (10–100 mgL−1) and mercury (II) (10–100 mgL−1). Their ability to remove metals from synthetic aqueous medium was tested and the heavy metal–fungi combination which showed the highest removal efficiency was selected. Live biomass of the selected isolate dispensed in lead solution with concentrations of 50 mgL−1, 100 mgL−1 and 150 mgL−1 showed a removal of 92.27%, 92.73% and 89.36% respectively at the end of the 40th h. Scanning Electron Microscopy with Electron Dispersive X-ray (SEM-EDX) of the treated biomass confirmed the biosorptive ability of the isolate for lead when compared with the control biomass. Fourier Transforms Infra-red (FTIR) Spectroscopy showed the probable involvement of amide, carboxylic acid, hydroxyl and isocyanate groups in the adsorption of lead from the synthetic metal solution.
•Indigenous fungal isolates were studied for efficient heavy metal removal.•Live biomass of the selected isolates was used for the study.•Selected isolate exhibited 90% efficiency in lead removal in synthetic solution.•SEM analysis of the fungal biomass shows bio-adsorption as the primary mechanism.•FTIR confirms the participation of several organic groups associated with cell wall.
Enzymatic synthesis of propyl gallate in an organic solvent was studied using cell‐associated tannase (E.C. 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used ...as a biocatalyst. The influence of buffer pH and strength, water activity, temperature, biocatalyst loading, gallic acid concentration, and 1‐propanol concentration was studied by the one‐factor‐at‐a‐time method. Subsequently, response surface methodology was applied based on a central composite design to determine the effects of three independent variables (biocatalyst loading, gallic acid concentration, and 1‐propanol concentration) and their mutual interactions. A total of 20 experiments were conducted, and a statistical model was developed, which predicted the maximum propyl gallate yield of 20.28 μg/mL in the reaction mixture comprising 40.4 mg biocatalyst, 0.4 mM gallic acid, and 6.52 % (v/v) 1‐propanol in 9.5 mL benzene at 30°C. The subsequent verification experiments established the validity of the model. Under optimal conditions, 25% conversion of gallic acid to propyl gallate was achieved on a molar basis. The absence of the need for enzyme purification and subsequent immobilization steps and good conversion efficiency makes this enzyme system an interesting one. Reports on the applications of bacterial whole cell systems for synthetic reactions in organic solvents are scarce, and perhaps this is the first report on bacterial cell‐associated tannase‐mediated esterification in a nonaqueous medium.
Taro (Colocasia esculenta) starch is known to possess unique physical and functional properties such as low amylose content, A-crystalline form, small granules, higher swelling power, etc. Due to the ...presence of significant amount of calcium oxalate crystals, the food industry is reluctant to explore this unique and cheap starch source for various food applications. Traditional processes utilizing various physical and chemical methods to remove oxalate content of starch inevitably change its physical and functional properties. However, using oxalate oxidase can effectively remove oxalates without altering the unique properties of starch. Hence, an attempt was made to optimize oxalate oxidase assisted starch extraction process from taro flour using response surface methodology. A central composite design comprising 20 experimental trials with 10 cube points augmented with six axial points and four replicates at the center point was applied. A mathematical model was developed to show the effect of taro flour concentration, enzyme load and incubation time on the oxalate removal. Validity of the model was experimentally verified and found that 98.3% of total oxalates can be removed under optimal conditions. This is the first report of optimization of the production of starch from taro flour using microbial oxalate oxidase.
Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation ...conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.
Five strains of naringin-degrading bacteria were isolated and found to be positive for extracellular naringinase activity. The one that showed highest activity in the selective medium was identified ...by 16S rRNA analysis as Bacillus methylotrophicus . The best combination of carbon–nitrogen source was determined by employing two-level full factorial analyses, comprising 24 experiments in shake flasks. Sucrose–yeast extract showed significant increase in naringinase activity (7.46 U/L) compared to the basal medium. Naringinase production was found to be inducible and naringin was found to be the best inducer among naringin, naringenin, hesperidin, and L-rhamnose. Inoculum size of 2% (v/v) and age of 48 hr favored naringinase and biomass production. Highest naringinase activity of 8 U/L was observed at the initial medium pH of 6. Response surface modeling was applied based on central composite design to determine the effects of three independent variables (sucrose, yeast extract, and naringin) and their mutual interactions. In total, 20 experiments were conducted and a statistical model was developed, which predicted naringinase production of 10.61 U/L. Subsequently, verification experiments were conducted and validity of the model was verified. Bioreactor studies conducted with the optimized medium showed an enzyme production of 12.05 U/L within 34 hr of fermentation.
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