Hui Suan Ng

Primary recovery of a bacteriocin-like inhibitory substance derived from Pediococcus acidilactici Kp10 by an aqueous two-phase system

A polymer-salt aqueous two-phase system (ATPS) consisting of polyethylene-glycol (PEG) with sodium citrate was developed for direct recovery of a bacteriocin-like inhibitory substance (BLIS) from a culture of Pediococcus acidilactici Kp10. The influences of phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and sodium chloride (NaCl) on the partition behaviour of BLIS was investigated. Under optimum conditions of ATPS, the purification of BLIS was achieved at 26.5% PEG (8000)/11% sodium citrate with a TLL of 46.38% (w/w), VR of 1.8, and 1.8% crude load at pH 7 without the presence of NaCl. BLIS from P. acidilactici Kp10 was successfully purified by the ATPS up to 8.43-fold with a yield of 81.18%. Given that the operation of ATPS is simple, environmentally friendly and cost-effective, as it requires only salts and PEG, it may have potential for industrial applications in the recovery of BLIS from fermentation broth.

Extractive bioconversion of cyclodextrins by Bacillus cereus cyclodextrin glycosyltransferase in aqueous two-phase system

An extractive bioconversion with Bacillus cereus cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) in aqueous two-phase system (ATPS) was investigated for the synthesis and recovery of cyclodextrins (CDs). Optimum condition for the extractive bioconversion of CDs was achieved in ATPS consisted of 7.7% (w/w) polyethylene glycol (PEG) 20,000 and 10.3% (w/w) dextran T500 with volume ratio (VR) of 4.0. Enzymatic conversion of starch occurred mainly in dextran-rich bottom phase whereas the product, CDs was transferred to top phase and a higher partition coefficient of CDs was achieved. Repetitive batch of CDs synthesis was employed by replenishment of the top phase components and addition of starch every 8h. An average total CDs concentration of 13.7 mg/mL, (4.77 mg/mLα-CD, 5.02 mg/mLβ-CD and 3.91 mg/mLγ-CD) was recovered in the top phase of PEG 20,000/dextran T500 ATPS. This study showed the effectiveness of ATPS application in extractive bioconversion of CDs synthesis with B. cereus CGTase.

Purification of β-mannanase derived from Bacillus subtilis ATCC 11774 using ionic liquid as adjuvant in aqueous two-phase system

The partitioning of β-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of β-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of β-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of β-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the β-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of β-mannanase from fermentation broth in one single-step strategy.

Partitioning behavior of recombinant lipase in Escherichia coli by ionic liquid-based aqueous two-phase systems

There has been significant interest in ionic liquid aqueous two-phase systems (ILATPSs) with properties such as rapid phase segregation which can lead to a reduction in time taken for protein recovery. Evaluations of ILATPSs were performed with various types of ionic liquid ((Emim)BF4, (Emim)Br, (Bmim)BF4, and (Bmim)Br) and salts (potassium-, sodium-, and magnesium-based) as phase components to figure out their competencies in the recovery of lipase from a fermentation broth of E. coli using banana waste as a substrate. The results of this study revealed that an ILATPS comprising (Emim)Br/potassium phosphate significantly enhanced lipase recovery upon partitioning lipase. Optimization of the composition of the ILATPS using response surface methodology (RSM) significantly improved the purification factor (PF) and partition coefficient (Ke). The influences of crude loading (CL), pH changes, and the presence of NaCl on the recovery performance were also studied. Recovery of E. coli BL21 lipase was accomplished in an (Emim)Br/potassium phosphate ILATPS using 26.5% (w/w) (Emim)Br, 19% (w/w) potassium phosphate at pH 7.6, 3% NaCl and a crude loading of 7% (w/w). Using this ILATPS, lipase was successfully recovered in a single purification step, which gave a yield, PF and Ke of 93.75%, 3.394 and 1.352, respectively. A high PF value indicates that (Emim)Br/potassium phosphate is capable of attaining an excellent degree of lipase purity, suggesting that the proposed ILATPS is suitable for implementation in a large scale process for lipase purification.

Optimization of recovery of esterase from Serratia marcescens using combination of the solvent impregnated resin and aqueous two-phase extraction techniques

ABSTRACT The performance of tunable aqueous polymer phase impregnated resins (TAPPIR) which is the combination of the solvent impregnated resin principle and an aqueous two-phase system for the separation of esterase from Serratia marcescens was evaluated in this study. Different molecular weight of polyethylene glycol (PEG) (2000, 4000 and 6000) at concentration ranging from 5% to 20% (w/w) and potassium citrate were used to construct the aqueous phase in TAPPIR technology. Optimum composition of PEG and salt for esterase partitioning was determined using response surface methodology. The optimum condition for the purification of esterase was impregnation of 25% (w/w) of PEG 2000 into 4 mm porous glass beads and extraction of esterase using 15% (w/w) potassium citrate at pH 8 containing 12% (w/w) crude loading with the addition of 4% (w/w) NaCl. Esterase from S. marcescens was successfully purified by the TAPPIR technology up to 5.32 of purification factor with a yield of 75.98%.