Chew, Pey Ling (2015) Extractive bioconversion of polycaprolactone by lipase in aqueous two-phase system / Chew Pey Ling. Masters thesis, University of Malaya.
Abstract
Aqueous two-phase system (ATPS) is a technique used for recovery and purification of biomolecules. Extractive bioconversion is a system which integrates biotransformation into ATPS to allow the in situ recovery of bioproducts immediately after they are formed. The extractive bioconversion of poly-ε-caprolactone (PCL) in an ATPS had not been established. In addition, the properties of the products obtained following the extractive bioconversion process are not well understood. Therefore, this study aimed to determine the ideal conditions for Burkholderia cepacia lipase-mediated extractive bioconversion of PCL in a polyethylene glycol (PEG)-based ATPS, as well as to examine the properties of the products obtained. A sequential optimization strategy, which comprised of full factorial design (FFD) followed by one-factor-at-a-time testing, was employed for the optimization. FFD was employed to screen for significant variables, namely pH, bioconversion temperature, molecular weight of PEG, tie-line length (TLL), volume ratio (VR) and concentration of NaCl additive in the recovery of the hydrolyzed products and enzyme. Qualitative analysis, which included gas chromatography-tandem mass spectrometry (GC-MS/MS), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) were subsequently used to characterize the products recovered. Results from the FFD screening indicated that temperature, VR and concentration of NaCl additive were the significant factors which influenced the partitioning of the hydrolyzed products into the upper phase. On the other hand, factors which could significantly influence the partitioning of lipase into the lower phase were temperature, TLL and VR. With the exception of NaCl additive, all other significant factors were subsequently subjected to the one-factor-at-a-time optimization. It was found that the balanced tradeoff between yields of hydrolyzed PCL in the upper phase and lipase in the lower phase was observed iv at a temperature of 40 °C, TLL of 28 % (w/w), and VR of 80:20. When these conditions were applied in a system composed of 19 % (w/w) PEG 3000 and 8.1 % (w/w) potassium phosphate at pH 7.0 without the presence of NaCl additive, 79.8 % hydrolyzed PCL was recovered from the upper phase and 42.0 % lipase was partitioned into the lower phase. GC-MS/MS analysis revealed and confirmed that the products of the extractive bioconversion were monomers and oligomers of PCL. Results of TGA analysis demonstrated that the higher the temperature of hydrolysis, the lower the onset temperature at which decomposition of PCL occurred. In addition, DSC data demonstrated a negative relationship between temperature of PCL hydrolysis and the melting as well as crystallization temperatures of the excess reactants. Besides, GPC analysis illustrated that the molecular weight of the PCL samples showed a decreasing trend with the temperature of hydrolysis, and that the higher the temperature of hydrolysis, the higher the polydispersity index. In conclusion, an optimized system for extractive bioconversion of PCL using B. cepacia lipase was described, which preferentially partitioned the hydrolyzed products into the upper phase and lipase into the lower phase.
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