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  1. Fulltext Stabilization of Eversa® Transform 2.0 lipase with sorbitol to enhance the efficiency of ultrasound-assisted biodiesel production
    Liow MY, Chan ES, Ng WZ, Song CP
    Int J Biol Macromol, 2024 Sep;276(Pt 1):133817.
    PMID: 39002902 DOI: 10.1016/j.ijbiomac.2024.133817
    Ultrasound technology has emerged as a promising tool for enhancing enzymatic biodiesel production, yet the cavitation effect induced can compromise enzyme stability. This study explored the efficiency of polyols in enhancing lipase stability under ultrasound conditions to further improve biodiesel yield. The incorporation of sorbitol resulted in the highest fatty acid methyl ester (FAME) content in the ultrasound-assisted biodiesel production catalyzed by Eversa® Transform 2.0 among the investigated polyols. Furthermore, sorbitol enhanced the stability of the lipase, allowing it to tolerate up to 100 % ultrasound amplitude, compared to 60 % amplitude in its absence. Enzyme activity assays revealed that sorbitol preserved 99 % of the lipase activity, in contrast to 84 % retention observed without sorbitol under an 80 % ultrasound amplitude. Circular dichroism (CD) and fluorescence spectroscopy analyses confirmed that sorbitol enhanced lipase rigidity and preserved its conformational structure under ultrasound exposure. Furthermore, employing a stepwise methanol addition strategy in ultrasound-assisted reactions with sorbitol achieved an 81.2 wt% FAME content in 8 h with only 0.2 wt% enzyme concentration. This promising result highlights the potential of sorbitol as a stabilizing agent in ultrasound-assisted enzymatic biodiesel production, offering a viable approach for enhancing biodiesel yield and enzyme stability in industrial applications.
  2. Fulltext Exploring combi-lipase catalysis of Eversa® Transform and Candida rugosa lipases for enzymatic hydrolysis of crude palm oil: Effects on fatty acid yield and retention of carotene and tocols
    Adiiba SH, Chan ES, Ng WZ, Song CP
    Int J Biol Macromol, 2025 Mar 06;306(Pt 4):141650.
    PMID: 40044000 DOI: 10.1016/j.ijbiomac.2025.141650
    Several lipases have been used to catalyze the hydrolysis of crude palm oil (CPO) to concentrate palm phytonutrients. Previously, the retention of phytonutrients during hydrolysis has been improved with a small addition of ethylenediaminetetraacetic acid. However, the production of free fatty acids (FFA) via enzymatic hydrolysis has yet to be optimised. Thus, for the first time, this study explored the combined use of AY 30SD (from Candida rugosa) and Eversa® Transform 2.0, ET 2.0 (from Aspergillus oryzae) to investigate the effect of reaction time and temperature on the lipases' performance towards the production of FFA during the alcohol-free process of producing phytonutrients concentrates. The results revealed that the application of combi-lipases at 45 °C yielded the highest final FFA content due to ET 2.0's affinity towards longer fatty acid chain lengths and AY 30SD's effectiveness in hydrolysing TAG molecules. With total enzyme concentration of 0.2 wt%, incorporating combi-lipase AY 30SD and ET 2.0 produced >95 wt% of FFA. Furthermore, a basic economic analysis validated that the combination of 0.15 wt% AY 30SD and 0.05 wt% ET 2.0 reduced the reaction time by seven times (completed within 24 h), potentially leading to a substantial increase in profit without sacrificing the phytonutrients retention.
  3. Fulltext Purification of the Recombinant Green Fluorescent Protein Using Aqueous Two-Phase System Composed of Recyclable CO2-Based Alkyl Carbamate Ionic Liquid
    Song CP, Liew PE, Teh Z, Lim SP, Show PL, Ooi CW
    Front Chem, 2018;6:529.
    PMID: 30430106 DOI: 10.3389/fchem.2018.00529
    The formation of aqueous two-phase system (ATPS) with the environmentally friendly and recyclable ionic liquid has been gaining popularity in the field of protein separation. In this study, the ATPSs comprising N,N-dimethylammonium N',N'-dimethylcarbamate (DIMCARB) and thermo-responsive poly(propylene) glycol (PPG) were applied for the recovery of recombinant green fluorescent protein (GFP) derived from Escherichia coli. The partition behavior of GFP in the PPG + DIMCARB + water system was investigated systematically by varying the molecular weight of PPG and the total composition of ATPS. Overall, GFP was found to be preferentially partitioned to the hydrophilic DIMCARB-rich phase. An ATPS composed of 42% (w/w) PPG 1000 and 4.4% (w/w) DIMCARB gave the optimum performance in terms of GFP selectivity (1,237) and yield (98.8%). The optimal system was also successfully scaled up by 50 times without compromising the purification performance. The bottom phase containing GFP was subjected to rotary evaporation of DIMCARB. The stability of GFP was not affected by the distillation of DIMCARB, and the DIMCARB was successfully recycled in three successive rounds of GFP purification. The potential of PPG + DIMCARB + water system as a sustainable protein purification tool is promising.
  4. Purification of lysozyme from chicken egg white using nanofiber membrane immobilized with Reactive Orange 4 dye
    Ng IS, Song CP, Ooi CW, Tey BT, Lee YH, Chang YK
    Int J Biol Macromol, 2019 Aug 01;134:458-468.
    PMID: 31078593 DOI: 10.1016/j.ijbiomac.2019.05.054
    Nanofiber membrane chromatography integrates liquid membrane chromatography and nanofiber filtration into a single-step purification process. Nanofiber membrane can be functionalised with affinity ligands for promoting binding specificity of membrane. Dye molecules are a good affinity ligand for nanofiber membrane due to their low cost and high binding affinity. In this study, a dye-affinity nanofiber membrane (P-Chitosan-Dye membrane) was prepared by using polyacrylonitrile nanofiber membrane modified with chitosan molecules and immobilized with dye molecules. Reactive Orange 4, commercially known as Procion Orange MX2R, was found to be the best dye ligand for membrane chromatography. The binding capacity of P-Chitosan-Dye membrane for lysozyme was investigated under different operating conditions in batch mode. Furthermore, desorption of lysozyme using the P-Chitosan-Dye membrane was evaluated systematically. The recovery percentage of lysozyme was found to be ~100%. The optimal conditions obtained from batch-mode study were adopted to develop a purification process to separate lysozyme from chicken egg white. The process was operated continuously using the membrane chromatography and the characteristic of the breakthrough curve was evaluated. At a lower flow rate (i.e., 0.1 mL/min), the total recovery of lysozyme and purification factor of lysozyme were 98.59% and 56.89 folds, respectively.
  5. Direct recovery of enhanced green fluorescent protein from unclarified Escherichia coli homogenate using ion exchange chromatography in stirred fluidized bed
    Song CP, Ooi CW, Tey BT, Lu CX, Liu BL, Chang YK
    Int J Biol Macromol, 2020 Dec 01;164:4455-4465.
    PMID: 32937154 DOI: 10.1016/j.ijbiomac.2020.09.051
    A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
  6. Purification of lysozyme from chicken egg white by high-density cation exchange adsorbents in stirred fluidized bed adsorption system
    Show PL, Ooi CW, Song CP, Chai WS, Lin GT, Liu BL, et al.
    Food Chem, 2021 May 01;343:128543.
    PMID: 33187742 DOI: 10.1016/j.foodchem.2020.128543
    Lysozyme from crude chicken egg white (CEW) feedstock was successfully purified using a stirred fluidized bed adsorption system ion exchange chromatography where STREAMLINE SP and SP-XL high density adsorbents were selected as the adsorption carrier. The thermodynamic and kinetic studies were carried out to understand the characteristics of lysozyme adsorption by adsorbents under various conditions, including adsorption pH, temperature, lysozyme concentration and salt concentrations. Results showed that SP and SP-XL adsorbents achieved optimum lysozyme adsorption at pH 9 with capacity of ~139.77 and ~251.26 mg/mL, respectively. The optimal conditions obtained from batch studies were directly employed to operate in SFBA process. For SP-XL adsorbent, the recovery yield and purification factor of lysozyme were 93.78% and ~40 folds, respectively. For SP adsorbent, lysozyme can be eluted ~100% with purification factor of ~26 folds. These two adsorbents are highly suitable for use in direct recovery of lysozyme from crude CEW.
  7. Antibacterial efficacy of poly(hexamethylene biguanide) immobilized on chitosan/dye-modified nanofiber membranes
    Xu FX, Ooi CW, Liu BL, Song CP, Chiu CY, Wang CY, et al.
    Int J Biol Macromol, 2021 Jun 30;181:508-520.
    PMID: 33775766 DOI: 10.1016/j.ijbiomac.2021.03.151
    This study aimed to develop a novel electrospun polyacrylonitrile (PAN) nanofiber membrane with the enhanced antibacterial property. The PAN nanofiber membrane was first subjected to alkaline hydrolysis treatment, and the treated membrane was subsequently grafted with chitosan (CS) to obtain a CS-modified nanofiber membrane (P-COOH-CS). The modified membrane was then coupled with different dye molecules to form P-COOH-CS-Dye membranes. Lastly, poly(hexamethylene biguanide) hydrochloride (PHMB) was immobilized on the modified membrane to produce P-COOH-CS-Dye-PHMB. Physical characterization studies were conducted on all the synthesized nanofiber membranes. The antibacterial efficacies of nanofiber membranes prepared under different synthesis conditions were evaluated systematically. Under the optimum synthesis conditions, P-COOH-CS-Dye-PHMB was highly effective in disinfecting a high concentration of Escherichia coli, with an antibacterial efficacy of approximately 100%. Additionally, the P-COOH-CS-Dye-PHMB exhibited an outstanding wash durability as its antibacterial efficacy was only reduced in the range of 5%-7% even after 5 repeated cycles of treatment. Overall, the experimental results of this study suggested that the P-COOH-CS-Dye-PHMB is a promising antibacterial nanofiber membrane that can be adopted in the food, pharmaceutical, and textile industries.
  8. Adsorption and purification performance of lysozyme from chicken egg white using ion exchange nanofiber membrane modified by ethylene diamine and bromoacetic acid
    Chang YK, Cheng HI, Ooi CW, Song CP, Liu BL
    Food Chem, 2021 Oct 01;358:129914.
    PMID: 34000689 DOI: 10.1016/j.foodchem.2021.129914
    A high-performance polyacid ion exchange (IEX) nanofiber membrane was used in membrane chromatography for the recovery of lysozyme from chicken egg white (CEW). The polyacid IEX nanofiber membrane (P-BrA) was prepared by the functionalization of polyacrylonitrile (PAN) nanofiber membrane with ethylene diamine (EDA) and bromoacetic acid (BrA). The adsorption performance of P-BrA was evaluated under various operating conditions using Pall filter holder. The results showed that optimal conditions of IEX membrane chromatography for lysozyme adsorption were 10% (w/v) of CEW, pH 9 and 0.1 mL/min. The purification factor and yield of lysozyme were 402 and 91%, respectively. The adsorption process was further scaled up to a larger loading volume, and the purification performance was found to be consistent. Furthermore, the regeneration of IEX nanofiber membrane was achieved under mild conditions. The adsorption process was repeated for five times and the adsorption capacity of adsorber was found to be unaffected.
  9. Fulltext Towards an alcohol-free process for the production of palm phytonutrients via enzymatic hydrolysis of crude palm oil using liquid lipases
    Adiiba SH, Chan ES, Lee YY, Amelia, Chang MY, Song CP
    J Sci Food Agric, 2022 Dec;102(15):6921-6929.
    PMID: 35662022 DOI: 10.1002/jsfa.12053
    BACKGROUND: Crude palm oil (CPO) is rich with phytonutrients such as carotenoids and tocols which possesses many health benefits. The aim of this research was to develop a methanol-free process to produce palm phytonutrients via enzymatic hydrolysis. In this work, triacylglycerol was hydrolyzed into free fatty acids (FFAs) using three different types of liquid lipases derived from Aspergillus oryzae (ET 2.0), Aspergillus niger (Habio) and Candida antartica (CALB).

    RESULTS: ET 2.0 was found to be the best enzyme for hydrolysis. Under the optimum condition, the FFA content achievable was 790 g kg-1 after 24 h of reaction with 1:1 water-to-oil mass ratio at 50 °C and stirring speed of 9 × g. Furthermore, with the addition of 2 g kg-1 ascorbic acid, it was found that 98% of carotenoids and 96% of tocols could be retained after hydrolysis.

    CONCLUSION: This work shows that enzymatic hydrolysis, which is inherently safer, cleaner and sustainable is feasible to replace the conventional methanolysis for the production of palm phytonutrients. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

  10. Fatty acid esters of 3-monochloropropane-1,2-diol and glycidol in palm oil: A review on current industrial-scale mitigation strategies, challenges and perspectives
    Ong YH, Song CP, Choo WS, Lee YY, Qua KS, Quek WP, et al.
    Food Res Int, 2025 Feb;202:115697.
    PMID: 39967094 DOI: 10.1016/j.foodres.2025.115697
    Glycidyl ester (GE) and 3-monochloropropane-1,2-diol ester (3-MCPDE) are food contaminants formed during edible oil refining. Due to their potential human carcinogenicity, the European Commission has established regulatory limits for vegetable oil, including palm oil. While laboratory-scale studies have shown promising reductions, large-scale implementation remains challenging. This review examines current mitigation technologies for palm oil, evaluated from lab to plant scale, and explores the challenges of large-scale implementation. Effective methods, such as crude palm oil washing, bleaching with non-hydrochloric acid-activated bleaching earth, dual-temperature deodorisation, and double refining, have been successfully tested at pilot scale or larger. Particularly, crude palm oil washing is widely adopted to reduce 3-MCPDE, whereas double refining effectively targets GE. However, a combination of strategies is required to simultaneously reduce 3-MCPDE and GE. These measures often lead to increased costs, oil loss and wastewater generation, with potential impacts on product quality. Furthermore, maintaining crude palm oil quality through good harvesting and milling practices is essential for consistent mitigation results. Continued research is necessary to improve mitigation methods for greater cost efficiency and reduced environmental impact, with large-scale studies needed to validate lab-scale findings and facilitate industry adoption.
  11. Affordable CO2-derived alkyl carbamate ionic liquids boosting enzymatic production of renewable hydrocarbon fuels via Chlorella variabilis NC64A fatty acid photodecarboxylase (CVFAP)
    Ng WZ, Chan ES, Liow MY, Ooi CW, Tey BT, Song CP
    Int J Biol Macromol, 2025 Feb 17.
    PMID: 39971058 DOI: 10.1016/j.ijbiomac.2025.141187
    The development of sustainable aviation fuel (SAF) presents a promising alternative to conventional jet fuel, with biofuels offering net-zero CO2 emissions. However, the conventional SAF production process typically involves expensive metal catalysts and extreme operating conditions. In contrast, the enzymatic approach offers a milder alternative; however, it is hindered by several limitations, including prolonged reaction times, reliance on cosolvents and low productivity. This study investigates the impact of CO2-based alkyl carbamate ionic liquids (ILs) on the enzymatic photodecarboxylation reaction catalyzed by Chlorella variabilis algal fatty acid photodecarboxylase (CvFAP). Notably, the study examines four ILs: N,N-dimethylammonium N',N'-dimethylcarbamate (DIMCARB), N,N-dipropylammonium N',N'-dipropylcarbamate (DPCARB), N,N-diallylammonium N',N'-diallylcarbamate (DACARB), and bis(2-ethylhexyl)-ammonium bis(2-ethylhexyl)carbamate (DBCARB). The results demonstrated that DACARB was the most optimal IL as it enhanced hydrocarbon conversion by 40 % and was able to reduce DMSO usage up to 86.7 % as compared to the absence of DACARB. This enhancement is attributed to DACARB's optimal balance of hydrophobic and hydrophilic properties, which increased enzyme activity while serving as a viable DMSO replacement, as well as acting as an allosteric modulator, as shown by Michaelis-Menten model fitting. Overall, this study highlights the potential of DACARB to facilitate process intensification in enzymatic SAF production, contributing to sustainable aviation practices.
  12. Antibacterial efficacy of quaternized chitosan/poly (vinyl alcohol) nanofiber membrane crosslinked with blocked diisocyanate
    Wu JY, Ooi CW, Song CP, Wang CY, Liu BL, Lin GY, et al.
    Carbohydr Polym, 2021 Jun 15;262:117910.
    PMID: 33838797 DOI: 10.1016/j.carbpol.2021.117910
    N-[(2-hydroxyl-3-trimethylammonium) propyl] chitosan chloride (HTCC), which is a type of chitosan derivative with quaternary ammonium groups, possesses a higher antibacterial activity as compared to the pristine chitosan. The nanofiber membranes made of HTCC are attractive for applications demanding for antibacterial function. However, the hydrophilic nature of HTCC makes it unsuitable for electrospinning of nanofibers. Hence, biodegradable polyvinyl alcohol (PVA) was proposed as an additive to improve the electrospinnability of HTCC. In this work, PVA/HTCC nanofiber membrane was crosslinked with the blocked diisocyanate (BI) to enhance the stability of nanofiber membrane in water. Microbiological assessments showed that the PVA/HTCC/BI nanofiber membranes possessed a good antibacterial efficacy (∼100 %) against E. coli. Moreover, the biocompatibility of PVA/HTCC/BI nanofiber membrane was proven by the cytotoxicity test on mouse fibroblasts. These promising results indicated that the PVA/HTCC/BI nanofiber membrane can be a promising material for food packaging and as a potential wound dressing for skin regeneration.
  13. Fulltext Unveiling the role of mechanical process intensifications and chemical additives in boosting lipase-catalyzed hydrolysis of vegetable oil for fatty acid production: A comprehensive review
    Ng WZ, Chan ES, Gourich W, Adiiba SH, Liow MY, Ooi CW, et al.
    Int J Biol Macromol, 2025 Jan;284(Pt 1):138144.
    PMID: 39613062 DOI: 10.1016/j.ijbiomac.2024.138144
    The enzymatic production of fatty acids from vegetable oils is becoming a preferred method due to its mild conditions, simplicity, and scalability. This review analyzes studies on enzymatic hydrolysis, exploring various feedstocks, lipases, reaction conditions, and conversion yields. However, a key limitation is the longer reaction time compared to conventional methods. This limitation is primarily due to the immiscibility of triacylglycerols (TAGs) with water at low temperatures and pressures, as well as the lower activity of enzymes compared to chemical catalysts. To overcome these issues, chemical additives are identified as the most effective process intensification strategy. They are easy to implement, cause less damage to lipases, and are more efficient than mechanical methods. The impact of various chemical additives was thoroughly examined for potential improvements in the enzymatic hydrolysis of vegetable oils. A synergistic combination of chemical additives comprising ionic liquids (ILs) and polyols, along with ultrasound, as well as the consideration of immobilization techniques were explored. Overall, this review highlights the potential of chemical additives and their synergistic feasibility in enhancing the enzymatic performance of lipase-catalyzed hydrolysis reactions.
  14. Fulltext Tripolyphosphate-chitosan-pea protein interactions confers long-term stability to 3D printed high internal phase Pickering emulsions
    Karandagaspitiya CO, Mahendra CK, Lim HP, Chan DK, Tey YS, Kam CF, et al.
    Food Chem, 2025 Feb 28;466:142228.
    PMID: 39608119 DOI: 10.1016/j.foodchem.2024.142228
    This research explores the interactions of tripolyphosphate-chitosan-pea protein (TPP-CS-PP) in improving the stability and storage of 3D printing food inks. Chitosan (CS) and pea protein (PP) were complexed at various concentrations with 80 % palm olein to produce high internal phase Pickering emulsions (HIPPEs) 3D printing food inks. The resulting CSPP HIPPEs exhibited shear-thinning behaviour and the flexibility to switch between solid and liquid states, ideal for 3D printing. CSPP1:150 achieved the best 3D printing resolution and shape fidelity due to electrostatic attraction of CS-PP and excess PP enhancing adhesion at the oil/water interface. After spraying tripolyphosphate (TPP), crosslinking with CS and phosphorylation of PP further improved HIPPE resistance to deformation and oiling off for 2 days post-printing. This is a significant improvement over the control. Thus, further investigation on the interaction of TPP with CS and PP is warranted to further improve the storage stability of 3D printed food inks.
  15. Fulltext A dual-function Savinase-catalyzed strategy for enhanced bioactive protein hydrolysate production from Nannochloropsis oculata for personal care and cosmetic applications
    Susanto S, Ng WZ, Liow MY, Sahadevan S, Chan ES, Borhan N, et al.
    Int J Biol Macromol, 2026 Nov;381(Pt 2):154294.
    PMID: 42680025 DOI: 10.1016/j.ijbiomac.2026.154294
    Microalgae are a sustainable source of bioactive proteins for personal care and cosmetic applications. Conventional alkaline extraction can achieve high protein recovery but relies on harsh chemical conditions that often compromise protein integrity, reduce bioactivity, and raise environmental concerns. Moreover, the extracted proteins often require additional enzymatic hydrolysis to enhance their functionality, thus increasing process complexity. This study proposes a dual-function Savinase-assisted in situ extraction strategy for the simultaneous extraction and functionalization of protein from Nannochloropsis oculata (N. oculata). Process optimization using improvement in protein extraction yield (IPEY) as the performance indicator identified optimal conditions of pH 7, 50 °C, 0.5 wt% Savinase and 2 h hydrolysis, under which a maximum IPEY of 85.1% was achieved. Fluorescence spectroscopy and zeta potential measurements confirmed that Savinase remained structurally stable under the optimized conditions while promoting protein solubilization. The resulting protein hydrolysates exhibited enhanced functional properties, with ABTS•+ radical scavenging activity reaching 83.3% at 5 mg/mL, representing a 56.9% increase compared with the non-enzymatic control. In addition, UV absorption analysis revealed a critical wavelength (λc) of 383.6 nm, indicating broad-spectrum photoprotective potential. Overall, the proposed one-step Savinase-assisted process provides a mild, sustainable, and effective strategy for producing bioactive microalgal protein hydrolysates with strong potential as natural ingredients in skin-protective cosmetic formulations.
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