Eco-friendly polylactic acid/rice husk ash mixed matrix membrane for efficient purification of lysozyme from chicken egg white

Highlights

• An eco-friendly and inexpensive membrane was developed for protein purification.

• The IEC and lysozyme adsorption capacity of PLA/RHA MMM were 844 and 4.49 μmol/g.

• In dynamic operation at 122 L/h/m², the breakthrough volume was ca. 50 MMM volume.

• Lysozyme purification fold was 35 after an adsorption/washing/elution cycle.

• The performance was deteriorated at a minor percentage during successive cycles.

Abstract

An eco-friendly and inexpensive ion-exchange membrane was developed for efficient purification of lysozyme from chicken egg white. Agricultural waste particles (rice husk ash, RHA) were incorporated into biodegradable polymeric matrix (polylatic acid, PLA) to fabricate porous PLA/RHA mixed matrix membrane (MMM) via water-vapour-induced phase inversion. The resulted membrane had an ion-exchange capacity (IEC) of 844 μmol/g membrane. In batch operation at pH 8, the protein adsorption equilibrium was attained within 2 h of contact time and the maximum lysozyme adsorption capacity was 4.49 μmol/g membrane. The adsorptivity of PLA/RHA MMM was essentially contributed from the filled RHA particles. In dynamic operation using one piece of PLA/RHA MMM at 122 L/h/m² for a feed lysozyme concentration identical to that in mucin-free egg white (0.5 mg/mL), the breakthrough volume was about 50 MMM volume. The lysozyme recovery was 68–70% with a near 100% purity and a purification fold of 35 after an adsorption/washing/elution cycle treating mucin-free egg white mixture. The MMM performance was only deteriorated at a minor percentage during three successive cycles. These results demonstrate that the PLA/RHA MMM process is a green and efficient approach for isolating lysozyme from chicken egg white.

Introduction

Since discovered by A. Fleming in 1921, lysozyme has long been a valuable protein for its enzymatic and antibacterial applications in food industry as well as its potential usages for the treatment of ulcers and infections [1], [2], [3]. Among a couple of natural substances containing lysozyme, chicken egg white is an important source for lysozyme production owing to its high lysozyme content (2500–3500 μg/mL) [1,4] and good availability. However, chicken egg white consists of many other functional proteins [5]: 54 wt% ovalbumin, 12–13 wt% ovotransferrin, 11 wt% ovomucoid, 1.5–3.5 wt% ovomucin, etc. (lysozyme composition: 3.4–3.5 wt%). In order to produce high-purity lysozyme from chicken egg white, an efficient and economical purification process is very important.

Protein purification method is generally based on the distinction in property of protein molecule such as: molecular size (e.g. size exclusion chromatography), molecular charge (e.g. ion-exchange chromatography), molecular polarity (e.g. hydrophobic interaction chromatography), or specific affinity (e.g. affinity chromatography). Because of the big difference in isoelectric point (pI) between lysozyme and other proteins, ion-exchange chromatography could achieve excellent performance for the isolation of lysozyme from egg white [3,[6], [7], [8]]. Nevertheless, process time and cost issue are still the major concerns for a practical chromatographic process applying in protein purification. There have been a couple of crucial problems for the utilization of traditional column chromatography: large pressure drop, slow mass transfer, lengthy time duration, clogging, bed compression, etc. usually arise for long and compact column; conversely, non-uniform packing (e.g. channelling and dead volume) may occur for short and wide bed [3,[9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]]. These problems impede the efficacy of packed-bed process. To avoid the above limitations, recommended is a more proper design by making the adsorbent in the form of thin film, i.e. membrane chromatography (or membrane adsorber, or adsorptive membrane) [3,[9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]]. Uneven packing and intraparticle diffusion do not exist in inherent membrane matrix. Membrane structure provides shorter bed height, higher porosity, and larger pores for convective flow, resulting in the operation at lower pressure drop, greater flow velocity, and smaller process time [3,9–19]. Additionally, membrane chromatography can offer a benefit of simpler scale-up by either stacking more pieces of flat membranes together, or winding a large flat membrane spirally, or gathering a bundle of hollow fibers [3,9–19]. In recent two decades, adsorptive membrane studies have particularly focused on porous mixed matrix membranes (MMMs) via filling the particulate adsorbents in polymeric matrix and successfully applied in food, biomedical, and environmental processes [9–13,[15], [16], [17], [18], [19], [20], [21], [22], [23], [24]]. The adopted fillers include ion-exchange resin, clay, plant waste, functionalized silica, functionalized graphene oxide, etc. Especially, ion-exchange resins are the most popular fillers for MMMs in protein purification.

In view of membrane technologies for protein purification in bio-industry, the common strategies include: filtration, dialysis, ion exchange, affinity membrane, membrane reactor [25], etc. The membrane shapes could be categorized as flat sheet, hollow fibre (or tubular, or capillary), spiral wound, and monolith. The aim of this study is to develop a green and cost-effective ion-exchange membrane process for lysozyme purification from chicken egg white. In order to fabricate eco-friendly membrane product, a biodegradable polymer, polylactic acid (PLA), was selected as the polymer matrix of flat-sheet MMM. Moreover, natural substance was considered as the raw material for MMM fillers. Since rice is a major agricultural crop in many countries, its main wastes, rice husks, account for a big portion of the annual gross rice production of the world [[26], [27], [28], [29]]. The reuse of rice husks, as an abundant agricultural waste, is of vital importance so as to reduce the possible pollution from incineration or landfilling. They were accordingly adopted as the resource of the MMM fillers in our study. Rice husks can be easily obtained from our local rice mills without any cost, which is another benefit. By leaching with acid solution and then treating thermally from 500 to 1400 °C, the metallic impurities of rice husks could be effectually removed to produce white ashes with high specific surface area and plentiful silica composition [27], [28], [29], [30], [31], [32]. As reported in the literature [[26], [27], [28],33], white rice husk ash (RHA) have been successfully applied in the removal of heavy metal ions and cationic dye from aqueous solutions, implying that RHA is an effective cation-exchanger. Via blending RHA with PLA solution together, the porous PLA/RHA MMM was prepared by non-solvent-induced phase-inversion process in this work. The resultant membrane is a biodegradable product and will not cause secondary pollution, making the protein purification process more environmentally-friendly. Furthermore, the direct material cost of the PLA/RHA MMM is low since the base materials came from inexpensive polymer and free agricultural wastes. The prepared PLA/RHA MMM was hence utilized in the isolation of lysozyme from chicken egg white, and its performance in dynamic operation with the influence of operational parameters was investigated.