Analysis of the Propionate Metabolism in Bacillus subtilis during 3-Indolacetic Production.

The genera Bacillus belongs to the group of microorganisms that are known as plant growth-promoting bacteria, their metabolism has evolved to produce molecules that benefit the growth of the plant, and the production of 3-indole acetic acid (IAA) is part of its secondary metabolism. In this work, Bacillus subtilis was cultivated in a bioreactor to produce IAA using propionate and glucose as carbon sources in an M9-modified media; in both cases, tryptophan was added as a co-substrate. The yield of IAA using propionate is 17% higher compared to glucose. After 48 h of cultivation, the final concentration was 310 mg IAA/L using propionate and 230 mg IAA/L using glucose, with a concentration of 500 mg Trp/L. To gain more insight into propionate metabolism and its advantages, the genome-scale metabolic model of B. subtilis (iBSU 1147) and computational analysis were used to calculate flux distribution and evaluate the metabolic capabilities to produce IAA using propionate. The metabolic fluxes demonstrate that propionate uptake favors the production of precursors needed for the synthesis of the hormone, and the sensitivity analysis shows that the control of a specific growth rate has a positive impact on the production of IAA.

Propionate as the preferred carbon source to produce 3-indoleacetic acid in B. subtilis: comparative flux analysis using five carbon sources.

3-Indoleacetic acid (IAA) is a phytohormone that promotes plant root growth, improving the use of nutrients and crop yield and it is been reported that bacteria of the genus Bacillus are capable of producing this phytohormone under various growth conditions. Considering this metabolic capability, in this work, Bacillus subtilis was cultivated in five different carbon sources: glucose, acetate, propionate, citrate and glycerol; and l-tryptophan (Trp) was used as an inducer for the IAA production. Based on the experimental results it was observed that the highest growth rate was achieved using glucose as a carbon source (µ = 0.12 h-1) and the lowest value was for citrate (µ = 0.08 h-1). On the other hand, the highest IAA production was obtained using propionate Yp/s = 0.975 (gIAA gTrp-1) and the lowest was when glucose was the substrate Yp/s = 0.803 (gIAA gTrp-1). In order to explore the metabolism and understand these differences, the experimental data was used to calculate the flux distribution using the genomic-scale metabolic model of Bacillus subtilis. Performing a comparative analysis it is observed that the fluxes towards precursors increase when propionate is the carbon source.

Modeling and Experimental Validation of Algorithms for Maximum Quantity of Protein to be Immobilized on Solid Supports by Electrostatic Adsorption in the Strategy of Rational Design of Immobilized Derivatives.

Protein immobilization by electrostatic adsorption to a support could represent a good option. On the other hand, lysozyme (EC 3.2.1.17) is a little and basic protein. The objective of this work was to test the functionality of the strategy of Rational Design of Immobilized Derivatives for the immobilization by electrostatic adsorption of egg white lysozyme on SP-Sepharose FastFlow support. The RDID1.0 software was used to predict the superficial lysozyme clusters, the electrostatic configuration probability for each cluster, and the theoretical and estimated maximum quantity of protein to be immobilized. In addition, immobilization was performed and the experimental parameter practical maximum quantity of protein to be immobilized and the enzymatic activity of the immobilized derivative were assessed. The estimated maximum quantity of protein to be immobilized (9.49 protein mg/support g) was close to the experimental practical maximum quantity of protein to be immobilized (14.73 ± 0.09 protein mg/support g). The enzymatic activity assay with the chitosan substrate showed the catalytic functionality of the lysozyme-SP-Sepharose immobilized derivative (35.85 ± 3.07 U/support g), which preserved 78% functional activity. The used algorithm to calculate the estimated maximum quantity of protein to be immobilized works for other proteins, porous solid supports and immobilization methods, and this parameter has a high predictive value, useful for obtaining optimum immobilized derivatives. The applied methodology is valid to predict the most probable protein-support configurations and their catalytic competences, which concur with the experimental results. The produced biocatalyst had a high retention of functional activity. This indicates its functionality in enzymatic bioconversion processes.

Optimization of theoretical maximal quantity of cells to immobilize on solid supports in the rational design of immobilized derivatives strategy.

Current worldwide challenges are to increase the food production and decrease the environmental contamination by industrial emissions. For this, bacteria can produce plant growth promoter phytohormones and mediate the bioremediation of sewage by heavy metals removal. We developed a Rational Design of Immobilized Derivatives (RDID) strategy, applicable for protein, spore and cell immobilization and implemented in the RDID1.0 software. In this work, we propose new algorithms to optimize the theoretical maximal quantity of cells to immobilize (tMQCell) on solid supports, implemented in the RDIDCell software. The main modifications to the preexisting algorithms are related to the sphere packing theory and exclusive immobilization on the support surface. We experimentally validated the new tMQCell parameter by electrostatic immobilization of ten microbial strains on AMBERJET® 4200 Cl- porous solid support. All predicted tMQCell match the practical maximal quantity of cells to immobilize with a 10% confidence. The values predicted by the RDIDCell software are more accurate than the values predicted by the RDID1.0 software. 3-indolacetic acid (IAA) production by one bacterial immobilized derivative was higher (~ 2.6 µg IAA-like indoles/108 cells) than that of the cell suspension (1.5 µg IAA-like indoles/108 cells), and higher than the tryptophan amount added as indole precursor. Another bacterial immobilized derivative was more active (22 µg Cr(III)/108 cells) than the resuspended cells (14.5 µg Cr(III)/108 cells) in bioconversion of Cr(VI) to Cr(III). Optimized RDID strategy can be used to synthesize bacterial immobilized derivatives with useful biotechnological applications.