Article Cite This: Langmuir 2018, 34, 2585−2594
pubs.acs.org/Langmuir
Biocatalytic Membrane Based on Polydopamine Coating: A Platform for Studying Immobilization Mechanisms Huiru Zhang,†,‡ Jianquan Luo,*,†,‡ Sushuang Li,†,‡ Yuping Wei,†,‡ and Yinhua Wan*,†,‡ †
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China ‡ University of Chinese Academy of Sciences, Beijing 100049, PR China S Supporting Information *
ABSTRACT: Application of biocatalytic membrane is promising in food, pharmaceutical, and water treatment industries, whereas enzyme immobilization is the key step of biocatalytic membrane preparation. Thus, how to minimize the negative effect of immobilization on enzyme performance is required to answer. In this work, we proposed a platform for biocatalytic membrane preparation and immobilization mechanism investigation based on polydopamine (PDA) coating, which was demonstrated by immobilizing five commonly used enzymes (laccase, glucose oxidase, lipase, pepsin, and dextranase) on three commercially available membranes via three immobilization mechanisms (electrostatic attraction, covalent bonding, and hydrophobic adsorption), respectively. By examining the enzyme loading, activity, and kinetics under different immobilization mechanisms, we found that except for dextranase, enzyme immobilization via electrostatic attraction retained the most activity, whereas covalent bonding and hydrophobic adsorption were detrimental to enzyme conformation. Enzyme immobilization via covalent bonding ensured a high enzyme loading, and hydrophobic adsorption was only suitable for lipase and dextranase immobilization. Moreover, the properties of functional groups around the enzyme active center should be considered for the selection of suitable immobilization strategy (i.e., avoid covering the active center by membrane carrier). This work not only established a versatile platform for biocatalytic membrane preparation but also provided a novel methodology to evaluate the effect of immobilization mechanisms on enzyme performance.
1. INTRODUCTION Biocatalytic membranes, with enzymes physically “anchored” in/on the membrane, integrate several distinct functions of biological membranes: localized biochemical reaction, immobilized enzyme, and physical separation of the reaction reactants and products. The porous membrane, acting as a selective barrier as well as a support for enzyme immobilization allowing enzyme reuse, may also help to stabilize the enzymes, alleviate product inhibition, and achieve continuous processing. Therefore, designed biocatalytic membranes with immobilized enzymes in commercially available membranes have attracted increasing attentions in a wide range of applications.1 Enzyme immobilization is the most important step for preparing biocatalytic membranes. Adsorption, entrapment, cross-linking, affinity, and covalent bonding have been considered as main mechanisms to immobilize enzyme onto the porous membrane.2 Adsorption is easy to operate and the process is mild, but the enzyme cannot be strongly combined with the carrier and is easy to leak. Enzyme immobilization by entrapment rarely changes the spatial conformation of the enzyme, and enzyme activity is well-maintained, but it can only be used for the reaction involving substrate and product with small molecular weight because of the diffusion barrier. Crosslinking consumes a large amount of toxic cross-linking agent © 2018 American Chemical Society
and requires functional groups on the membrane. Affinity method also needs the presence of specific groups on enzyme (e.g., histidine and biotin). Covalent bonding has good stability and reusability, but it results in serious enzyme activity loss, and it is difficult to regenerate the membrane carrier.3−6 Because the biochemical and physical properties (e.g., structure, hydrophilicity, size, charge, sensitivity, and stability) of various enzymes are distinct, even for the same enzyme, the catalyzed reactions can be different;7 it is difficult to extrapolate the most suitable immobilization mechanism for a specific enzyme and a given reaction based on the existing knowledge. From the literature, it was found that both membrane types and evaluation methods for the immobilization of a specific enzyme were quite different, making the comparison impossible. For example, Ning and Bruening immobilized the pepsin on a nylon membrane via electrostatic adsorption for rapid protein digestion and purification.8 Raaijmakers et al. prepared an ultrathin active skin layer by interfacial polymerization of pepsin and trimesoyl chloride on a polyacrylonitrile (PAN) membrane.9 Cooper et al. immobilized enzymes on a polyvinylidene Received: August 12, 2017 Revised: January 20, 2018 Published: January 30, 2018 2585
DOI: 10.1021/acs.langmuir.7b02860 Langmuir 2018, 34, 2585−2594
Article
Langmuir
Figure 1. Schematic of biocatalytic membrane preparation based on dopamine coating via different immobilization mechanisms.
Table 1. Main Properties of the Tested Enzymes
a
enzymes
laccase
glucose oxidase
lipase
pepsin
dextranase
origin EC number molecular weight (kDa) isoelectric point specific activity (U/mg)d
Trametes versicolor 1.10.3.2 63a 3.5−4.0 11.7
Aspergillus niger 1.1.3.4 160 4.2 74.0
Aspergillus oryzae 3.1.1.3 41b 5.0c 258
porcine stomach 3.4.23.1 35 2.8d 113
Penicillium sp. 3.2.1.11 67e 3.9e 43.0
Data from Collins et al.24 bData from Toida et al.25 cData from Ying et al.26 dOwn measurement. eData from Larsson et al.27
fluoride (PVDF) membrane via hydrophobic adsorption to prepare a membrane-based nanoscale proteolytic reactor.10 It was also reported that enzyme could be immobilized on different membranes via covalent bonding, such as PVDF and polyamide membrane, 11,12 and their surface properties profoundly influenced the structure, orientation, and activity of the bound enzyme.13 In addition, if we use the same membrane carrier to evaluate different immobilization mechanisms, normally it is requisite to use different spacer arms. Nonetheless, the type of spacer arm has a significant influence on enzyme loading and activity. For instance, Liu et al. immobilized penicillin G acylase on metal affinity membranes (IMAM) via spacer arms with different lengths and found that the IMAM with 1,8-diaminooctane as the spacer arm had optimal enzyme adsorption capacity.14 Ozyilmaz found that inserting 1,6-diaminohexane as a hydrophobic spacer arm produced a positive effect on immobilized lipase activity, whereas a negative effect on its stability.15 Therefore, it is quite difficult to figure out the effect of immobilization mechanisms on enzyme performance when using the different membrane carriers and the spacer arms. Dopamine, as a neurotransmitter, can form a polydopamine (PDA) coating layer by self-polymerization in alkaline aqueous solutions with air, which is able to strongly adhere on a variety of solid surfaces.16 The catechol and quinone groups on the PDA surface are easy to react with thiol- and amino-containing compounds via Michael addition and/or Schiff-base reaction, providing a versatile platform for further modification and functionalization of membranes.17 For example, Yang et al. modified a polypropylene microfiltration membrane by codeposition of dopamine/polyethyleneimine (PEI) and then obtained the silica-decorated membrane via a biomineralization process.18 Fan et al. grafted PEI, dodecyl mercaptan, and histidine respectively, on the PDA-coated polyethersulfone membrane to prepare anion-exchange, hydrophobic interaction, and affinity membrane adsorbers.19 Jiang et al. immobilized heparin on the PDA-coated polyethylene membrane to increase
its anticoagulant ability.20 Moreover, dopamine could also be used for enzyme immobilization. For instance, Chao et al. modified the halloysite nanotubes surface with dopamine for laccase immobilization via covalent bonding.21 Luo et al. entrapped enzyme beneath the sublayer of ultrafiltration membranes by reverse filtration and subsequent PDA coating.22 Thanks to the hydrophilicity and biocompatibility of PDA, it is supposed to have negligible adverse effect on enzyme activity during immobilization.23 As dopamine can deposit on various membrane materials and further graft molecules with different properties, we made an attempt to fabricate biocatalytic membranes by different immobilization mechanisms based on PDA coating. As shown in Figure 1, by using commercially available polymeric membranes with different materials and pore size as carrier to immobilize five commonly used enzymes (i.e., laccase, glucose oxidase, lipase, pepsin, and dextranase) via electrostatic attraction, covalent bonding and hydrophobic adsorption, respectively, we aimed at establishing a platform to evaluate the effect of immobilization mechanisms on enzyme performance and to screen a suitable strategy for constructing a biocatalytic membrane as well as enzymatic membrane reactor (EMR) for a specific enzyme and reaction.
2. EXPERIMENTAL SECTION 2.1. Materials. Five enzymes were tested in this work, and their main properties are shown in Tables 1 and S1, according to the literature24−27 and manufacturers’ information. As listed in Table 2, NF90 membrane (polyamide, molecular weight cutoff (MWCO ≈ 100−200 Da) for laccase and lipase immobilization, NT101 membrane (polyamide, MWCO ≈ 500 Da) for glucose oxidase immobilization, PAN membrane (PAN, MWCO ≈ 20 kDa) for pepsin and dextranase immobilization were purchased from DOW FILMTEC, MICRODYN-NADIR, and Sepro, respectively. Dopamine hydrochloride and Bradford reagent used for protein assay was supplied by Sigma-Aldrich. PEI (molecular weight ≈ 10 000) and 1dodecanethiol (DDM) were supplied by Aladdin. Glutaraldehyde (GA) was supplied by Tianjin Fuchen Chemical Reagents Factory. 2,6Dimethoxy phenol (DMP, 154.16 Da) as the assay substrate for laccase activity measurement, horseradish peroxidase (HRP), and 42586
DOI: 10.1021/acs.langmuir.7b02860 Langmuir 2018, 34, 2585−2594
Article
Langmuir
membrane was immersed in 7.5 mL acetate buffer (2 M, pH = 5.4), which was reacted with 7.5 mL ninhydrin solution (1.0 g/L) at 95 °C (oil bath) for 45 min, and then the absorbance of the supernatants was detected using a spectrophotometer at the wavelength of 570 nm (the standard curve was prepared with glycine solution). Attenuated total reflection Fourier-transform infrared (ATR−FTIR) spectra was used to identify whether GA was grafted on the membrane. The FTIR spectra were acquired by a Nicolet iS50 spectrometer (Thermo Fisher Scientific Corporation, USA) with a scanning range of 400−4000 cm−1. Contact angles of the membranes before and after modification were measured by an optical contact angle goniometer (OCA20, DataPhysics Instruments Co., Germany) to verify DDM grafting. The samples were pasted on a slide, and a deionized water droplet with a volume of 2 μL was deposited on the sample surface. Each sample was tested at least three times on different locations, and the average value of contact angles was taken. 2.4. Determination of Enzyme Loading. The amount of immobilized enzymes on the membrane was calculated by mass balance, and the protein content was measured by Bradford assay. The mass balance equation is
Table 2. Membrane Substrates for Enzymes Immobilization enzymes
membrane substrates
MWCO
potential applications
laccase glucose oxidase lipase pepsin
NF90 NT101
100−200 Da
