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Biocatalytic Membrane Based on Polydopamine Coating: A Platform for Studying Immobilization Mechanisms Huiru Zhang, Jianquan Luo, Sushuang Li, Yuping Wei, and Yinhua Wan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02860 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018
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Biocatalytic Membrane Based on Polydopamine Coating: A Platform for Studying Immobilization Mechanisms Huiru Zhang, Jianquan Luo*, Sushuang Li, Yuping Wei, Yinhua Wan* a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China b
University of Chinese Academy of Sciences, Beijing 100049, PR China
ABSTRACT Application of biocatalytic membrane is promising in food, pharmaceutical and water treatment industries, while 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, while 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 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. KEYWORDS: Enzyme immobilization, biocatalytic membrane, enzymatic membrane reactor, polydopamine coating, membrane modification
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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 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 membrane. Adsorption, entrapment, crosslinking, 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 used for the reaction involving substrate and product with small molecular weight due to the diffusion barrier. Cross-linking consumes a large amount of toxic crosslinking agent and requires functional groups on the membrane. Affinity method also needs the presence of specific groups on enzyme (e.g. histidine, 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. Since 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 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 ACS Paragon Plus Environment
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Raaijmakers et al. prepared a ultrathin active skin layer by interfacial polymerization of pepsin and trimesoyl chloride on a polyacrylonitrile (PAN) membrane 9. Cooper et al. immobilized enzyme on a polyvinylidene 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 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 while 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 PDA surface are easy to
react with thiol- and amino- containing compounds via Micheal 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 co-deposition 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,
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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 Fig. 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 effect of immobilization mechanisms on enzyme performance, and to screen a suitable strategy for constructing an biocatalytic membrane as well as enzymatic membrane reactor (EMR) for a specific enzyme and reaction.
Figure 1 Schematic of biocatalytic membrane preparation based on dopamine coating via different immobilization mechanisms
2. EXPERIMENTAL 2.1 Materials Five enzymes were tested in this work, and their main properties are shown in Table 1 and Table S1 according to the literature
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and manufacturers’ information. As listed in
Table 2, NF90 membrane (polyamide, molecular weight cut-off (MWCO ∼100-200 Da) for laccase and lipase immobilization, NT101 membrane (polyamide, MWCO∼500 Da) for glucose oxidase immobilization, PAN membrane (polyacrylonitrile, MWCO∼20 kDa) for ACS Paragon Plus Environment
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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 ∼10000) and 1-dodecanethiol (DDM) were supplied by Aladdin. Glutaraldehyde (GA) was supplied by Tianjin Fuchen Chemical Reagents Factory. 2, 6-Dimethoxy phenol (DMP, 154.16 Da) as the assay substrate for laccase activity measurement, horseradish peroxidase (HRP) and 4-aminoantipyrine (4-AAP, 203.25 Da) for glucose oxidase activity determination were supplied by Aladdin. D-glucose (198.17 Da) as the assay substrate for glucose oxidase was supplied by Xilong Science Co., Ltd. Dextran (40 kDa) as the assay substrate for dextranase activity measurement was supplied by Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA, 66 kDa) supplied by Beijing Solarbio Science& Technology Co., Ltd was used as the assay substrate for pepsin.
para-Nitrophenyl palmitate (p-NPP, 377.53 Da) as the assay substrate for lipase was supplied by Alfa Asesar, and para-Nitrophenol (p-NP, 139.11 Da) for lipase activity determination was supplied by Aladdin. All chemicals used were of analytical grade without any further purification. Table 1 Main properties of the tested enzymes Enzymes
Laccase
Glucose oxidase
Lipase
Pepsin
Dextranase
Origin
Trametes versicolor
Aspergillus niger
Aspergillus oryzae
Porcine stomach
Penicillium sp.
E.C. number
1.10.3.2
1.1.3.4
3.1.1.3
3.4.23.1
3.2.1.11
35
67 e
Molecular weight(kDa)
63
Isoelectric point Specific activity (U/mg) d
a
b
160
41
3.5-4.0
4.2
5.0 c
2.8 d
3.9 e
11.7
74.0
258
113
43.0
a
Data from Collins et al.24
d
Own measurement
b
Data from Toida et al. 25 e
c
Data from Ying et al. 26
Data from Larsson et al. 27
Table 2 Membrane substrates for enzymes immobilization Enzymes
Membrane substrates
MWCO
Potential applications
Laccase
NF90
100-200 Da
Micropollutant removal
Glucose oxidase
NT101
