Downstream purification of surfactin produced by Bacillus subtilis ATCC 21332

Dlamini, Bagcinele (2017-12)

Thesis (MEng)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANS OPSOMMING: Onder siektes wat deur 'n aansteeklike agense veroorsaak word, is tuberkulose (TB), wat deur Mycobacterium tuberculosis veroorsaak word, die dodelikste. In 2014 het 9.6 miljoen mense siek geword van TB en 1.5 miljoen het van die siekte gesterf. Surfactin blyk 'n belowende alternatiewe antimikrobiese agens te wees teen organismes wat TB veroorsaak, soos dit die vermoë het om selmembrane te vernietig, asook om membrane se deurlaatbaarheid te verander; dus is dit in staat om M. tuberculosis te denatureer. Die gebruik van surfactin as mediese middel is beperk deur sy hemolitiese aktiwiteit. Dit kan dus gebruik word in ander toepassings in die stryd teen TB, soos handverzorgers of in toerusting en oppervlaksterilisators. Die antimikrobiese werking van surfactin is beperk in omgewings met hoë proteïen- en lipoïedonsuiwerhede, en dus is dit nodig om surfactin te suiwer van die medium waarin dit vervaardig word. Die doel van hierdie studie is dus om 'n gepaste strategie vir die suiwering van surfactin voor te stel, met bedryfstoestande. Aangesien die studie van surfactinsuiwering is afhanklik van die analise van surfactinkonsentrasie; is 'n verdere doel van hierdie studie om 'n tegniek te ontwikkel en te valideer vir die analise van surfactinkonsentrasie. Die voorgestelde stappe vir surfactinsuiwering was suurpresipitasie, oplosmiddelonttrekking en adsorpsie. Voor optimalisering van hierdie stappe, is surfactin bondelgewys uit Bacillus subtilis ATCC 21332, met 'n maksiumum surfactinkonsentrasie van 1109 mg/L en selektiwiteit (verhouding surfactin tot antifungale middel) van 5.5 gs/ga, vervaardig .Dunlaag-chromatografie (DLC) is ontwikkel vir surfactin konsentrasie analise, was verder gevalideer deur na lineariteit en werksomvang, perke van opsporing, perke van kwantifisering, instrumentperke van opsporing, akkuraatheid, presisie, spesifisiteit, en selektiwiteit en robuustheid onder verskillende oplosmiddel- en monsternemingstoestande te verwys. Na die surfactin produksie en ontwikkeling van die DLC analitiese tegniek, is surfactin deur suurpresipitasie tot 'n presipitaat geïsoleer van selvrye supernatante van B. subtilis. Suurpresipitasie is uitgevoer deur chloorwaterstof by selvrye supernatante te voeg om die pH, en verhaal surfactin in die presipitaat deur sentrifugasie. Suurpresipitasie is geoptimaliseer deur die effek van die werkende pH op suiwerheid, herstel, en selektiwiteit van surfactin, en relatiewe konsentrasies van proteïen- en lipoïedonsuiwerhede te ondersoek. Die aanbevole werkende pH vir suurpresipitasie was pH 4, en die suiwerheid, herstel, en selektiwiteit van surfactin was onderskeidelik 97%, 43% en 5.7 gs/ga. Die presipitaat van die suurpresipitasie is verder gesuiwer deur oplosmiddelekstraksie. Oplosmiddelekstraksie is uitgevoer deur surfactin selektief in die presipitaat op te los deur verskeie organiese oplosmiddels met verskillende polariteite te gebruik. Hierdie oplosmiddels, in volgorde van afnemende polariteit, was metanol, i-propanol, chloroform:metanol (1:1 v/v), asetonitriel, chloroform:metanol (2:1 v/v), asetoon, chloroform, etielasetaat, metiel tert-butieleter(MTBE), petroleum-eter en n-heksaan. Oplosmiddelekstraksie is geoptimaliseer deur ondersoek in te stel na die uitwerking van die polariteit van die oplosmiddel op suiwerheid, herstel, en selektiwiteit van surfactin, en relatiewe konsentrasies van proteïen- en lipoïedonsuiwerhede in oplosmiddels na ekstraksie. Daar is bevind dat polêre oplosmiddels beter surfactinherstel en surfactinsuiwerheid toon in vergelyking met nie-polêre oplosmiddels. MTBE het die hoogste surfactinsuiwerheid (80%) en surfactinherstel (100%) gelewer, en was dus die beste oplosmiddel vir surfactinsuiwering deur ekstraksie van oplosmiddels. Hierdie herstel en suiwerheid was ook hoër as die herwinnings en suiwerhede wat deur adsorpsie en suur neerslag verkry is, dus was oplosmiddelwinning die beste suiweringstegniek in hierdie studie. Die presipitaat van suurpresipitasie is ook deur adsorpsie gesuiwer. Adsorpsie is uitgevoer deur die gebruik van HP-20 nie-polêre hars op adsorpsievloeistowwe wat gevorm is deur die presipitate in alkaliese water meer oplosbaar te maak. Die meer oplosbare presipitaat is, verder, met water of metanol gemeng om die adsorpsievloeistof te vorm. Adsorpsie is geoptimaliseer deur ondersoek in te stel na die effek van aanvanklike pH, bedryfstemperatuur, die verhouding van harskonsentrasie tot surfactinkonsentrasie (HK/SK) op die persentasie surfactin in die adsorpsievloeistof wat deur die hars geadsorbeer is (%SA), en die faktor waarteen die surfactinselektiwiteit verbeter het na die adsorpsie (VS) deur aanwending van oppervlakontwerpe. Die bestudering van surfactinadsorbsie deur oppervlakontwerpe is 'n nuwe eksperimentele benadering, nie voorheen geraporteer nie. % SA verbeter met aanvanklike pH, bedryfstemperatuur, HK/SK-verhouding en metanol konsentrasie.. VS was onafhanklik van bedryfstemperatuur, het afgeneem met toenames in HK/SK-verhouding en verhoog met pH en metanol konsentrasie. Die aanbevole aanvanklike pH, bedryfstemperatuur, HK/SK-verhouding en metanol konsentrasie was onderskeidelik 11.5, 45°C, 31 gr/gs en 30% (v/v). Die surfactin herstel en suiwerheid na suiwering deur adsorpsie by die geselekteerde bedryfsomstandighede was onderskeidelik 91% en 58%. Die waarneming was dat die teenwoordigheid van metanol in die adsorpsievloeistof die koers waarteen surfactin geabsorbeer is, verbeter het. Ekwilibrium is vyf keer vinniger bereik in adsorpsievloeistowwe met metanol as in dié sonder metanol. Adsorpsie van surfactin kan gedefinieer word as multilaagadsorpsie met die feit dat dit geskik was vir die Freundlich-model. Ekstraksie en adsorpsie was suksesvol in die versywering van surfactin B.subtillis cultures. Dit is van groot relevansie siendat surfactin gebruik kan word in die geveg teen TB, wat die grootste oorsaak van sterftes van ‘n enkele agent global is.

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