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In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.

ABSTRACT: In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.

SUBMITTER: Fang H

PROVIDER: S-EPMC4790935 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The aim of this work was to demonstrate an optimization methodology to reliably obtain stable macrodispersions (i.e., for ≥24 h) of carbon nanotubes in water using sonication. Response surface methodology (RSM) was utilized to assess and optimize the sonication parameters for the process. The studied input parameters were (i) sonication time (duration), (ii) amplitude (of vibration), and (iii) pulse-on/off (duration) of the sonicator. The analyzed responses were mean diameter and size distribution of multiwalled carbon nanotube (MWNT) aggregates in water, which were measured by the dynamic light scattering technique. A semiempirical model was developed and statistically tested to estimate the magnitude of sonicator parameters required to obtain specified MWNT macrodispersions (i.e., aggregates' mean diameter and distribution) in water. The results showed that MWNT aggregates of 2 ± 0.5 μm can be obtained by optimizing sonicator parameters to a sonication time of 89 s, amplitude of 144 μm, and pulse-on/off cycle of 44/30 s. These process settings for 100 mg/L MWNTs in a 30 mL aliquot of deionized water would consume 863 J/mL of sonication energy. Contrary to the popular belief, "sonication time" and/or "sonication energy input" were not found to be proportional to the degree of dispersion of MWNTs in water. This might be the reason for the frequent disparity and nonreproducibility of sonication results reported in scientific literature, especially for dispersing nanomaterials in a number of different systems. The amplitude of vibration was noted to be the most sensitive parameter affecting MWNT aggregates' diameter and distribution in water. The characterization of MWNTs was performed using electron microscopy, surface area analyzer, thermogravimetric analyzer, and zeta potential analyzer. This study can be helpful in evaluating sonication dispersion of particulate matter in other incompressible fluids such as graphene dispersion in organic solvents.

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In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.

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