Downstream purification of surfactin produced by Bacillus subtilis ATCC 21332

[摘要] ENGLISH SUMMARY: (TB), caused by Mycobacterium tuberculosis, is the most fatal disease from a single infectious agent. In 2014, 9.6 million people fell ill with TB and 1.5 million died from the disease. Surfactin offers a promise as an alternative antimicrobial agent against the organisms causing TB, as it possesses the ability to lyse cell membranes as well as the ability to alter membrane permeability. The use of surfactin as a medical drug is limited by its haemolytic activity, thus it can be used in other applications in the fight against TB such as hand sanitizers or in equipment and surface sterilisers. The antimicrobial activity of surfactin is limited in environments with high protein and lipid impurities thus it is necessary to purify surfactin from the medium in which it is produced. The aim of this study is therefore to propose a suitable strategy with operating conditions for surfactin purification. Since the study of surfactin purification is dependent on the analysis of surfactin concentration, an additional aim of this study is to develop and validate an analytical technique for surfactin concentration.The proposed unit operations for surfactin purification were acid precipitation, solvent extraction and adsorption. Prior to optimisation of these unit operations, surfactin was produced batch-wise from Bacillus subtilis ATCC 21332 with a maximum surfactin concentration of 1109 mg/L and selectivity (surfactin to antifungal ratio) of 5.5 gs/ga. Thin layer chromatography (TLC) was developed for quantifying surfactin concentration, and . was further validated using the linearity and working range, limit of detection, limit of quantification, instrument limit of detection, accuracy, precision, specificity, selectivity and robustness under diverse solvent and sampling conditions.After surfactin production and development of the TLC analytical technique, surfactin was isolated from cell-free supernatants of B. subtilis into a precipitate by acid precipitation. Acid precipitation was carried out by adding hydrochloric acid to cell-free supernatants to lower the pH, and recovering surfactin with the precipitate by centrifugation. Acid precipitation was optimised by studying the effect of the operating pH on surfactin purity, surfactin recovery, surfactin selectivity and relative concentrations of protein and lipid impurities. The recommended operating pH for acid precipitation was pH 4, where the surfactin purity, recovery and selectivity were 97%, 43% and 5.7 gs/ga respectively.The precipitate from acid precipitation was further purified by solvent extraction. Solvent extraction was carried out by selectively dissolving surfactin in dry acid precipitates using various organic solvents of differing polarity. These solvents, in order of decreasing polarity, were: methanol, i-propanol, chloroform:methanol (1:1 v/v), acetonitrile, chloroform:methanol (2:1 v/v), acetone, chloroform, ethyl acetate, methyl tert-butyl ether (MTBE), petroleum ether and n-hexane. Solvent extraction was optimised by studying the effect of solvent polarity on surfactin purity, surfactin recovery and relative concentration of protein and lipid impurities in solvents after extraction. Polar solvents had better surfactin recoveries and purities in comparison to non-polar solvents. MTBE gave both the highest recovery (100%) and highest purity (80%), thus was the best solvent for extraction. This recovery and purity was also higher than the recoveries and purities achieved by adsorption and acid precipitation, thus solvent extraction was the best purification technique in this study.The precipitate from acid precipitation was also purified by adsorption. Adsorption was carried out using HP-20 non-polar resins on adsorption liquids formed by solubilising the precipitates in alkaline water. The solubilised precipitates were further mixed with water or methanol to form the adsorption liquid. Adsorption was optimised by studying the effect of initial pH, operating temperature, resin concentration to surfactin concentration (RC/SC) ratio and methanol concentration on the percentage of surfactin in the adsorption liquid adsorbed onto resins (% SA) and the factor by which surfactin selectivity improved after the adsorption (IS) using surface designs. The study of surfactin adsorption using surface designs or using methanol based adsorption liquid were new experimental approaches, not having being previously reported.% SA improved with initial pH, operating temperature, RC/SC ratio and methanol concentration. IS independent of operating temperature, decreased with increase in RC/SC ratio and increased with pH and methanol concentration. The recommended initial pH, operating temperature, RC/SC ratio and methanol concentration was 11.5, 45 °C, 5 gr/gs and 30 % (v/v) respectively. The surfactin recovery and purity after purification by adsorption at the selected operating conditions were found to be 91% and 58% respectively. The presence of methanol in adsorption liquid was seen to improve surfactin adsorption rates. Equilibrium was reached 5 times faster in adsorption liquids with methanol compared to adsorption liquids without methanol. Surfactin adsorption can be defined as multilayer adsorption as it fitted the Freundlich model.Acid precipitation, solvent extraction and adsorption were successful in purifying surfactin from B. subtilis cultures. This is of major significance as surfactin can be used in the fight against TB, which is the major cause of death from a single infectious agent globally.

[发布机构] Stellenbosch University