Plants produce flavonol compounds for vital functions regarding plant growth, fruit and flower colouring as well as fruit ripening processes. Several of these biosynthesis steps are stereo-and regioselective and are being carried out by nonheme iron enzymes. Using density functional theory calculations on a large active site model complex of flavanone-3hydroxylase (FHT), we established the mechanism for conversion of naringenin to its dihydroflavonol, which is a key step in the mechanism of flavonol biosynthesis. The reaction starts with dioxygen binding to the iron(II) centre and a reaction with -ketoglutarate cosubstrate gives succinate, an iron(IV)-oxo species and CO 2 with large exothermicity and small reaction barriers. The rate-determining reaction step in the mechanism; however, is hydrogen atom abstraction of an aliphatic C-H bond by the iron(IV)-oxo species. We identify a large kinetic isotope effect for the replacement of the transferring hydrogen atom by deuterium. In a final step the OH and substrate radicals combine to form the alcohol product with a barrier of several kcal mol-1. We show that the latter is the result of geometric constraints in the active site pocket. Furthermore, the calculations show that a weak tertiary C-H bond is shielded from the iron(IV)-oxo species in the substrate binding position and therefore the enzyme is able to activate a stronger C-H bond. As such, the flavanone-3hydroxylase enzyme reacts regioselectively with one specific C-H bond of naringenin by avoiding activation of weaker bonds through tight substrate and oxidant positioning.
BackgroundAlpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto-oligosaccharides. It is an important enzyme generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought after properties for industrial use. The Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and safe for use in food industry. Hence, Koji strain’s development for the screening of potent mutants, hyper producer of thermostable α-amylases, with desired attributes is the need of the time.ResultsA process has been developed to improve super Koji (A. oryzae cmc1) strain through γ-rays treatment. The doses i.e. 0.60, 0.80, 1.00, 1.20 & 1.40 KGy gave more than 3.0 log kill. Initially, 52 Koji mutants resistant to 1% (w/v) Triton X-100 were selected. 2nd screening was based on α-amylases hyper production and 23 mutants were sorted out by measuring clearing zones index (CI). Afterwards nine potent mutants, resistant to 2-deoxy D-glucose, were screened based on CI. These were further analyzed for thermal stability and productivity of α-amylase under submerged conditions. The mutants’ M-80(10), M-100(6) & M-120(5) gave about four fold increases in α-amylases productivity. The half life of M-100(6) α-amylase at 55 °C was 52 min and was highest among the mutants. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia depicted that exposure to gamma rays increased rigidity of the mycelium. The potent Koji mutant M-100(6) was grown on soluble starch in 10L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1 protein. Growth kinetic parameters were: μ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1.ConclusionIt was concluded that the developed five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ-rays mediated physical mutagenesis. The developed thermostable α-amylases of super Koji mutantM-100(6) has immense potential for application in saccharification process for maltose syrup production. Moreover, the developed five step strain’s development process may be used for the simultaneous improvement in productivity and thermal stability of other microbial enzymes.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1345-y) contains supplementary material, which is available to authorized users.
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