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Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose
Accepted Manuscript Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose Yi Wei Chan, Caleb Acquah, Eugene M. Obeng, Elvina C. Dullah, Jaison Jeevanandam, Clarence M. Ongkudon PII:
S0300-9084(18)30344-4
DOI:
https://doi.org/10.1016/j.biochi.2018.11.019
Reference:
BIOCHI 5557
To appear in:
Biochimie
Received Date: 19 September 2018 Accepted Date: 30 November 2018
Please cite this article as: Y.W. Chan, C. Acquah, E.M. Obeng, E.C. Dullah, J. Jeevanandam, C.M. Ongkudon, Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose, Biochimie, https://doi.org/10.1016/j.biochi.2018.11.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose
Jaison Jeevanandam3 and Clarence M. Ongkudon1* 1
RI PT
Yi Wei Chan1, Caleb Acquah2,3, Eugene M. Obeng1,4, Elvina C. Dullah1,
Bioprocess Engineering Research Group, Biotechnology Research Institute, Universiti
SC
Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
School of Nutrition Sciences, University of Ottawa, K1N 6N5, Ontario, Canada.
3
Department of Chemical Engineering, Curtin University, 98009 Miri, Sarawak,
Malaysia. 4
M AN U
2
Bioengineering Laboratory, Department of Chemical Engineering, Monash University,
TE D
Victoria 3800, Australia.
*Correspondence: Clarence M. Ongkudon
AC C
EP
[email protected]; +6088-320 000 Ext: 8536
ACCEPTED MANUSCRIPT
ABSTRACT Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be
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less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate
SC
particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay.
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Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 oC. The maximum CMCase
TE D
activity was achieved at pH 5 and 50 oC. Residual activity of ~50 % was retained after three iterations and dipped to ~18 % on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ±1.11 %
EP
relative to similar amount of free cellulase.
AC C
Keywords: Polymethacrylate porous particle, cellulase, immobilization, reusability, carboxymethyl cellulose
AC C
EP
TE D
M AN U
SC
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose
Jaison Jeevanandam3 and Clarence M. Ongkudon1* 1
RI PT
Yi Wei Chan1, Caleb Acquah2,3, Eugene M. Obeng1,4, Elvina C. Dullah1,
Bioprocess Engineering Research Group, Biotechnology Research Institute, Universiti
SC
Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
School of Nutrition Sciences, University of Ottawa, K1N 6N5, Ontario, Canada.
3
Department of Chemical Engineering, Curtin University, 98009 Miri, Sarawak,
Malaysia. 4
M AN U
2
Bioengineering Laboratory, Department of Chemical Engineering, Monash University,
TE D
Victoria 3800, Australia.
*Correspondence: Clarence M. Ongkudon
AC C
EP
[email protected]; +6088-320 000 Ext: 8536
1
ACCEPTED MANUSCRIPT
ABSTRACT Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be
RI PT
less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate
SC
particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay.
M AN U
Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 oC. The maximum CMCase
TE D
activity was achieved at pH 5 and 50 oC. Residual activity of ~50 % was retained after three iterations and dipped to ~18 % on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ±1.11 %
EP
relative to similar amount of free cellulase.
AC C
Keywords: Polymethacrylate porous particle, cellulase, immobilization, reusability, carboxymethyl cellulose
2
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ABBREVIATIONS
immobilized cellulase-polymethacrylate particle
EDA-P
EDA derivatized polymethacrylate particles
GA-P
Aldehyde terminated surface of polymethacrylate particles
WICP
Dry weight of ICP powder
WGA-P
Dry weight of GA-P powder
ASample
Absorbance reading of sugar yield
AStandard
Absorbance reading of standard (Blank)
U
Hydrolytic activity of cellulase
U/mg
Specific activity of cellulase
M AN U
SC
RI PT
ICP
Temperature of cellulolytic reaction of free and immobilized cellulase
AC C
EP
TReaction
TE D
TImmobilization Temperature setpoint of cellulase immobilization
3
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1.
INTRODUCTION The development of cellulases for biomass saccharification continues to be a
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dominant research in the quest for green chemicals. These enzymes ensure the synergistic conversion of cellulose to simple sugars which are subsequently converted to platform chemicals such as alcohols and organic acids [1,2]. Advancements in this area of research
SC
has witnessed enzymes in free, immobilized and microbial surface displayed forms [3,4]. The progression is as a result of the search for high enzyme efficiency, enzyme
M AN U
robustness and favorable process economics. Cellulases in the free forms are the most popularly used paradigm but are difficult to recycle; the immobilized forms are recyclable but often experience reduced enzyme efficiency and are mostly expensive [3]. The cell surface displayed forms are also recyclable; however, the cells most often
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experience physiological burdens that somehow influence their overall functionality [5]. Focusing on enzyme immobilization, the practice involves the anchorage of the biocatalyst onto stationary phases to enhance the enzyme’s technical performance (e.g.,
EP
stability, reactivity, substrate specificity, and enantioselectivity) and reusability potentials [3,6]. This further promotes continuous economic operation and recovery of product with
AC C
a high degree of purity [7–9]. In this practice, the choice of the biocarrier and the immobilization procedure remain critical since they can alter the innate structure and conformation of the enzymes and, thus, leading to a suppression of their catalytic efficiencies. Physical adsorption and covalent binding have been noted to be the most applied chemistries in immobilizing enzymes [10–12]. Other chemistries applicable for enzyme immobilization are entrapment, copolymerization, and encapsulation. Physical
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adsorption offers a facile immobilization strategy which retains the native conformation of the enzyme, and hence, limit the loss of its activity [13,14]. The mode of interaction between the enzyme and the biocarrier in physical adsorption usually occur through
RI PT
hydrogen, hydrophobic and electrical binding. Nevertheless, the weak interaction between the biocatalyst and biocarrier results in excessive leaching/desorption and minimum reusability [10,13,15,16]. Contrary, covalent bonding results in high reusability
SC
of immobilized enzyme and, therefore, renders the application of the enzymatic process in industrial operations commercially viable [10,11,17,18]. That notwithstanding,
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application of covalent chemistries may result in major loss of enzymatic activity and liable changes in conformation of the multimeric enzyme, in exchange for more stable enzyme-carrier linkage of covalent nature.
Screening of biocarriers is imperative to the development of an innovative
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biocatalytic system with targeted industrial application (food, pharmaceutical and chemical industries). Fundamental criteria for the solid support selection comprises of chemical stability, physical/mechanical strength and cost effectiveness [19]. A plethora of
EP
biocarriers such as silica gel [7], copolymers [20], multiwall carbon nanotubes [21], magnetic nanoparticles [22] and clay mineral [23] with different physical forms has been
AC C
experimented with cellulolytic enzyme. For instances, in the work of Lima et al., 2017, Fe3O4 magnetic particle as the support permitted easy separation of enzymes from the solution via modulation of magnetic field, but it was susceptible to agglomeration which affected the mass transfer rate. Copolymeric particles, in the context of poly(glycidyl methacrylate-co-ethylene dimethacrylate) resins, are well known for its robustness and biocompatibility with several ligands. The epoxy-containing copolymers are modifiable
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and fine-tunable for varied immobilization chemistries and for tethering bioligands such as biocatalysts [25]. To the best of our knowledge, polymethacrylate particles have yet to be exploited as biocarriers for cellulase in the extant literature.
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Herein, the prospects of polymethacrylate particles as a biocarrier for cellulases have been assessed and reported. The cellulases were anchored covalently to an aldehyde-activated moiety to form immobilized cellulase-polymethacrylate particle (ICP).
SC
The scope of this paper entails the characterization of the parameters (pH and temperatures) that enhance immobilization yield and enzymatic activity of the ICP. Also,
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the reusability of the ICP was included as a yardstick for the selection of optimal parameters. Furthermore, the performance of the ICP was compared to free cellulolytic enzyme so that a more comprehensive assessment can be obtained thereof.
EXPERIMENTAL
2.1
Materials
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2.
FTIR Cary 630 (Agilent, United States), vacufuge (Eppendorf, Germany), thermomixer R
EP
(Eppendorf, Germany), sieve shaker (UTS, Malaysia), thermostated water bath (TW20, Julabo, Germany), incubator shaker (New Brunswick Scientific Innova 40, Eppendorf,
AC C
Selangor, Malaysia), centrifuge (TGL-40, Sichuan Shuke Instrument, Chengdu, China), spectrophotometer (Genesys 20, Thermo Scientific, Düsseldorf, Germany), microplate reader (Infinite m200, Tecan, Männedorf, Switzerland), microplates (Greiner 96 flat transparent, Sigma-Aldrich, Darmstadt, Germany), thermocycler (PTC-200, MJResearch Inc., Quebec, Canada), Celluclast (Sigma-Aldrich, Germany), carboxymethyl cellulose (CMC, low viscosity, Sigma-Aldrich, Germany), 3,5-dinitrosalicyclic acid (DNS, Acros
6
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Organics, Düsseldorf, Germany), sodium sulfite (Sigma-Aldrich, Germany), Phenol ACS (VWR, Gul, Singapore), potassium sodium tartrate tetrahydrate (Na-K-tartrate, Carl Roth, ᴅ-glu-cose (Roth), glutaraldehyde (Sigma-Aldrich, Germany),
Karlsruhe, Germany),
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ethylenediamine (Acros Organics, Belgium), glycidyl methacrylate (Sigma-Aldrich, Germany), methanol (J.T.Baker, USA), ethylene glycol dimethacrylate (Sigma-Aldrich,
Aldrich, Germany).
Preparation of polymethacrylate particles
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2.2
SC
Germany), glycidyl methacrylate (Sigma-Aldrich, Germany), cyclohexanol (Sigma-
The porous biocarrier was synthesized via thermally induced free-radical polymerization of the functional monomer (GMA) and cross-linker monomer (EDMA) blended at a ratio of 3:2 v/v% (GMA/EDMA) in the presence of 60% porogenic solvent, cyclohexanol. In
TE D
addition, free radical polymerization process was initiated by addition of 1% w/v AIBN. The resulting mixture was sonicated and sparged with N2 gas for 15 min respectively to acquire homogeneous blend freed from dissolved O2. 50 mL of the homogenized
EP
polymerization mixture was gently transferred into a mold and sealed with a parafilm sheet on top. The polymeric feedstocks containing mold was immersed in a thermostated
AC C
water bath preset to 60 oC for 5 h to commence the polymerization reaction. Solidified polymer resin was extracted and immersed in methanol-filled beaker kept in the incubator shaker overnight. Methanol-drenched polymethacrylate resin was immersed in deionized water for 4 h. The porogen-free porous material was oven-dried overnight [26]. The parched resin was fragmented into smaller parts, that were later ground and crushed to
7
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powder in a mortar with a pestle. Refined porous polymethacrylate particles (≤60 µm) was obtained via sieving with 60 µm mesh. 2.3
Functionalization of polymethacrylate particles
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The functional group, epoxy moieties, of polymethacrylate particles were activated via a Schiff-base covalent reaction. About 25 g of the copolymers powder was mixed with 50 mL of ethylenediamine (EDA) solution (50% v/v prepared with deionized water) and
SC
incubated in the thermomixer at 60 °C, 300 rpm shaking speed and for 12 h. EDA derivatized polymethacrylate particles (EDA-P) solution was subjected to centrifugation
M AN U
at 15,000 x g for 15 mins, followed by discarding the supernatant and rinsing the pellet with deionized water to remove the residues. EDA-P pellet was dissolved in 50 mL of 10% glutaraldehyde solution prepared with deionized water and incubated in the thermomixer at room temperature, 300 rpm shaking speed for 4 h. Aldehyde terminated surface of
TE D
polymethacrylate particles (GA-P) mixture was centrifuged at 15,000 x g for 15 mins and then washed with deionized water [27]. GA-P powder was reconcentrated from the
mode.
Immobilization of cellulase
AC C
2.4
EP
residue-free aqueous solution via an overnight incubation in the vacufuge preset to V-AQ
50 mg of GA-P powder was dissolved in 500 µL of 3 % Celluclast solution (prepared with 0.1 M pH 6 sodium citrate buffer). The microcentrifuge tube containing the mixture was vortexed and incubated in the thermomixer at 20 oC, 300 rpm shaking speed and for 12 h. ICP solution was then centrifuged (15,000 x g, 15 mins) and washed 3 times with sodium citrate buffer (0.1 M pH) to remove the unbound cellulase. The residue-free
8
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solution formed the ICP powder after a 3 h centrifugal drying using the vacufuge (V-AQ
2.5
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mode).
Immobilization yield estimation
SC
Given the sensitivity of spectroscopic protein assay methods, physical quantification method was opted to assay the loading of cellulase onto polymethacrylate particles. The
M AN U
dry weights of GA-P and ICP were measured with analytical balance and recorded. The dry weight differential between ICP and GA-P was quantified (1) and determined as the immobilization yield of cellulase (mg) [23].
(1)
(
)
CMCase activity assay
EP
2.6
=
TE D
−
About 500 µL of 1 % CMC solution dissolved in sodium citrate buffer (0.1 M pH 6) was
AC C
added into microcentrifuge tube containing 50 mg ICP, vortexed and incubated in the thermomixer (50 oC, 300 rpm, 30 mins). The supernatant was collected from the reaction mixture via centrifugation(15,000 x g for 15 mins) and the associated reducing sugar content was quantified via DNS colorimetric method [28]. Figure 1 demonstrates a simplified activity assay. The DNS reagent was formulated via blending DNS (1 g), phenol (0.2 g), sulfite (0.05 g), Na-K-tartrate (20 g) in 150 ml of 0.168 M NaOH. 1 g/L of
9
ACCEPTED MANUSCRIPT
glucose solution (MW = 180 g/mol) was selected as analytical standard. The absorbance (A540nm) readings of samples and standard were implemented in Equation (2) – (4) to
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=
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%$−
-!./!0/
(2)
2-343-5 67'8
78.7:;
=
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1
M
N 7O
(4)
2.7
PQ*+:R:+S
""BT3$3U!-3B. 53%$/ BV 2%$$W$!X%
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=
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(3)
KL
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compute the specific activity of cellulase [29].
Effect of pH on the immobilization yield of cellulase
EP
3% Celluclast solutions were prepared with sodium citrate buffer of pH 4, 5, 6, 7 and 8. The immobilization of cellulase onto GA-P was carried out in accordance with section
AC C
2.4. The amount of cellulase anchored onto activated polymethacrylate particles was quantified with the method outlined in section 2.5.
2.8
Effect of temperature on the immobilization yield of cellulase
The immobilization of the cellulase onto GA-P was carried out in accordance with section 2.4 albeit the cloned mixtures were incubated across a spectrum of temperature
10
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setpoints 20, 30 40 50 and 60 oC, respectively. The immobilization yield of the cellulase was quantified via the method described in section 2.5.
Effect of temperature on the CMCase activity of the ICP
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2.9
The ICP with good immobilization characteristics from the above experiments was used hereof. Microcentrifuge tubes containing 50 mg of the ICP were mixed with 500 µL of 1 %
SC
CMC solutions prepared with sodium citrate buffer (0.1 M, pH 6). The hydrolysis of CMC and its activity was carried out as described in section 2.6 with slight modification.
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Herein, the blends were incubated in the vacufuge preset to the respective discrete temperature setpoint of 20, 30, 40, 50 and 60 oC. The activity trends of CMCase incubated at different temperature settings were studied over time by taking the absorbance measurement of sample per hour for 4 h. The reusability behavior of the ICP
TE D
was also evaluated by recording its activity conducted in different temperature setpoints for four consecutive days (1 run per day).
Effect of pH on CMCase activity of the ICP
EP
2.10
This section also made use of the best performing ICP that resulted from sections 2.7 and
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2.8. Microcentrifuge tubes comprising 50 mg of ICP were mixed with 500 µL of 1 % CMC solutions prepared with sodium citrate buffer of varied pH of 4, 5, 6, 7 and 8. The hydrolysis of CMC and the associated activity measurements were carried out as described in section 2.6. The activity of the ICP conducted in the range of pH mentioned was measured for 4 days (1 run per day) to characterize the reusability of the ICP.
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2.11
Imaging and spectra analysis
The porous particles of pre- and post-immobilization states were coated with gold and observed under a Scanning Electron Microscope (Hitachi S3400, 15 kV). Fourier transform infrared
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(FTIR) spectroscopic analysis was conducted to assess the chemical composition of the porous copolymers particles and the ICP using Agilent Cary 630 FTIR (USA) with diamond attenuated total reflectance. Standard operating procedures were used. Both ICP
SC
and polymethacrylate particles were oven dried overnight prior to the commencement of
M AN U
analysis.
RESULTS AND DISCUSSION
3.1
Characterization of the ICP
TE D
3.
Methacrylate-based polymers have proven to be effective as biocarriers in multi-omics application [25,30]. A considerable amount of literature studies pertaining to the
EP
characterization of copolymers (GMA-co-EDMA) in various formats have been well documented [26,27,31]. To date, there are no reported works on exploring porous
AC C
poly(GMA-co-EDMA) particles as biocarrier for cellulolytic enzymes. The present work explored the molecular binding of cellulase on polymethacrylate particles (≤ 60 µm particle size) for compatibility assessment. Figure 2 shows the SEM images of the nascent polymethacrylate particles and ICP under x1.00 and x10.00 magnification. The surface morphology from the two different stages showed that there was no significant alteration in surface areas and pore sizes after functionalization and immobilization of cellulase to form the ICP. The epoxy bearing porous particles were activated by adopting 12
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Schiff-based chemistry, allowing cellulase to be covalently anchored to the biocarrier as illustrated in Figure 3. Covalent immobilization chemistries minimize/prevent hydrolysis and subsequent leaching of immobilized ligands [32,33].
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Fourier Transform Infrared (FTIR) spectra analysis was performed to screen for the available functional groups in the polymethacrylate particles and enzyme loaded configuration of the polymer network. The FTIR spectra of porous polymethacrylate
SC
particles and ICP are depicted in Figure 4. The peaks of ~756.87 cm-1, ~906.01 cm-1 and ~3000 cm-1 are characteristic of epoxy group moiety in the polymethacrylate particles. A
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reduction in the peaks, as observed in Figure 4, is indicative of a successful activation of the polymethacrylate particles with EDA and GA [27,34,35]. The characteristic band of enzyme after immobilization was observed in the ICP spectrum at 1578.88 cm-1 emanated by C—O stretching vibration. Additional band at 1653.56 cm-1 suggests the
TE D
presence of imine bond (C=N) linking EA-P to GA and GA-P to cellulase [9,36]. The peak at ~1724.40 cm-1 emerged from copolymers particles spectrum can be attributed to the presence of C=O bond. Lower intensity of the C=O band as shown by the ICP
EP
spectrum could be attributed to the amine reductive reactions [27]. The broad peak across the range of 3200 - 3500 cm-1 was due to N—H and O—H stretching vibrations of the
AC C
amino and OH groups in cellulase [9,34]. The overall FTIR analysis validates successful cellulase coupling on polymethacrylate particles
13
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3.2
Screening for the optimal configuration of enzyme immobilization
The optimal parameters setting amid enzyme immobilization is crucial to maximize the activity of CMCase. Inappropriate pH or reaction temperature setpoint may lead to
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enzyme inactivation or lower the amount of enzyme anchored onto the biocarrier. Hence, immobilization of cellulolytic enzymes onto the porous copolymers particles were
3.21
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immobilization parameters for subsequent experiments.
SC
conducted across a spectrum of pH and temperature setting to determine the optimum
Effect of pH on immobilization yield
The cellulase solutions prepared with sodium citrate buffer of varied pH (4-8) were
TE D
respectively loaded on aldehyde-activated polymethacrylate particles (GA-P) at 25 oC. Figure 5A displays the amount of ICP formed under the influence of distinct pH. The GA-P-cellulase conjugate prepared at pH 6 resulted as the highest loaded ICP with 10 mg
EP
of anchored cellulase, followed by pH 5 (9.5 mg), pH 4 (8.5 mg), pH 8 (7.8 mg) and pH 7 (7.3 mg). To ensure that quantity commensurate with quality, activity assay in terms of
AC C
the sugar yield per mg of cellulase was conducted at 50 oC. By referring to Figure 5B, the specific activities of respective ICPs in descending
order were pH 6 (12.26 U/mg) > pH 5 (11.64 U/mg) > pH 7 (10.4 U/mg) > pH 8 (8.16 U/mg) > pH 4 (7.03 U/mg). Considering the highest immobilization yield and specific activity of ICP, pH 6 emerged as the most optimal hydrogen ion concentration for the immobilization of active cellulase onto the porous polymethacrylate particles. Herein, the
14
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pH with highest immobilization (i.e., pH 6) yield gave the highest specific enzyme activity. However, no apparent correlation could be derived for pH 4, pH 7 and pH 6 in terms of their immobilization yields and activities. For instance, pH 4 with an enzyme
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loading of 8.5 mg gave the lowest activity (i.e., 7.03 U/mg) in comparison with pH 7 with 7.3 mg enzyme loading but a better specific enzyme activity of 10.4 U/mg. Since Celluclast
is
a
concoction
of
hydrolytic
enzymes
(i.e.,
endoglucanases,
SC
cellobiohydrolases, cellobiases and other accessory proteins), it is possible that the pH 4 adsorbed other proteins which are not specific to the degradation of CMC. It is worth
M AN U
knowing that CMC is specific for CMC degrading enzymes such as endoglucanases (thus, the name CMCase). CMC is an amorphous substrate [37] and therefore would hardly be hydrolyzed by non-CMCase.
It is perceivable that the enzymes could undergo conformational changes (severity
TE D
order: pH 4 > pH 8 > pH 7) amid immobilization process, thus switching binding functional group from the non-catalytic site to restricted region, and consequently limiting the access of substrates into the anchored cellulases.
Effect of temperature on immobilization yield
AC C
3.22
EP
The pH was maintained at pH 6 for this section. In this section, the immobilization of cellulase on GA-P was carried out across a broad range of temperature settings (TImmobilization : 20, 30, 40, 50 and 60 oC) to elucidate the influence of temperature on ICP preparation. Figure 6A depicts the immobilization yield of the ICP from the respective incubation temperatures. The outcome of the test revealed TImmobilization of 20 oC as the
15
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temperature with the lowest enzyme loading (10 mg) followed by 30 oC (13.06 mg), 50 o
C (25.7 mg), 40 oC (26.18 mg) and 60 oC (29.89 mg). The result indicates an apparent
relationship between the increase in TImmobilization and enzyme loading for a single run for
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each different sample.
The ICPs prepared at varied TImmobilization were further assessed for their relative cellulose hydrolyzing performance as represented in Figure 6B. The figure illustrates an
SC
inverse relationship between the specific enzyme activity and TImmobilization, which happens to be a complete opposite of that of ICP yield verses thermal input. The highest
M AN U
specific activity of the ICP was recorded at TImmobilization of 20 oC, followed by an exponential decrease at 30 oC and 40 oC and eventually comes to a complete halt beyond 50 oC. Despite recording the lowest ICP yield, 20 oC emerged as the optimal TImmobilization which putatively facilitated the retention of the native conformation of the anchored
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cellulase thus resulting in the high enzyme activity in comparison to the others. The low CMC conversion of the ICP prepared at high TImmobilization could be attributed to progressive thermal denaturation of the cellulase over the course of the 12-hour
EP
immobilization phase. In this case, the structure/conformation of the enzymes might have transitioned from partial unfolding to permanent change [35] upon covalently linked to
AC C
GA-P. Amid that span, the enzyme could have been irreversibly altered, joint covalently to GA-P via functional group at restriction site that leads to steric hindrance or at partially folded state which wipes off any chance of renaturation. In general, high TImmobilization increased the ICP yield but negatively impacted the catalytic activity thereof. The subsequent section hereafter employed the ICP prepare at pH 6 and 20 oC because of its performance as discussed above.
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3.3
Effect of reaction temperature and associated glucose yields of the ICP
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The temperature of cellulose hydrolysis, TReaction, plays a pivotal role in maximizing sugar yield. Therefore, the range of temperature settings, viz 20 oC, 30 oC, 40 oC, 50 oC and 60 o
C, were assessed for their respective impact on the activity of the ICP. Figure 7A depicts
SC
the glucose yield by the ICP prepared at pH 6 and 20 oC. The ICP was subjected to varied TReaction for a total time interval of 2 h. In this experiment, comparing the trend of glucose
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yield for the reaction temperatures from 20 oC to 50 oC showed an increasing sugar profile for each reaction temperature. In this range, the sugar yield increased with respect to time in the order of 20 oC < 30 oC< 40 oC < 50 oC. In other words, the higher the reaction temperature, the better the sugar yield for that temperature over time. However,
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there was a deviation from the above-stated trend for TReaction of 60 oC, which happened to exhibit an increasing glucose profile until the hour mark after which a decline in sugar yield was observed. This could be a consequence of periodic thermal denaturation, which
EP
causes the enzymes to lose their functionality over time. Maximal sugar yield was recorded at 50 oC. Similar finding was also reported by Acharya and Chaudhary (2012)
AC C
and Chang et al. (2009). Furthermore, as per the study conducted by Andreaus et al. (1999), the catalytic activity of cellulase diminished when TReaction was above the temperature optima (> 50 oC), during which thermal denaturation likely took place. The assessment of the reusability of the ICP was executed with the same range of
TReation (20 – 60 oC). The common issue of immobilized enzyme activity loss due to leaching after repeated run was eminent during this trial. Figure 7B shows a sharp decline
17
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in activity of 54.2 % and 43.8 % for 40 oC and 50 oC, respectively, after three iterations. The reaction temperature of 20 oC and 30 oC only managed to retain a residual activity of 25.3 % and 26.4 %, respectively, after three cycles. The reusability potential at 60 oC was
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poor since it could only retain 3.9% of enzyme activity after the third batch. Despite recording the highest value in reusability, the cumulative specific activity of 40 oC throughout the 4 recycles (26.2 U/mg) did not exceed that conducted at 50 oC (29.1 u/mg).
SC
On grounds of all the data collected, 50 oC, which also happens to be the customary
maximize the hydrolysis of CMC.
Effect of pH on ICP
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3.4
M AN U
TReaction for commercial cellulases [35], appears to be the optimal TReaction for the ICP to
Also, the pH-activity characteristics of the ICP prepared at pH 6 and 20 oC were assessed within a range of pH spectrum to elucidate the dynamics thereof. In reference to Figure
EP
8A, both pH 5 and pH 6 recorded comparable enzymatic activities for the 1st and 2nd batches. However, there was a subsequent dip in residual activity (29 %) for pH 5 while
AC C
the latter hits a plateau (50 %) after 3rd batch. The higher degrees of activity loss of 21.7 %, 18.1 % and 14.2 % were observed for pH 4, 7 and 8, respectively, at the 3rd cycle. The activity recorded across the tested range of pH spectrum (descending order: pH 5 > pH 6 > pH 4 > pH 7 > pH 8) suggests the ineffectiveness of the ICP under alkaline operating conditions. According to Ben Hmad and Gargouri (2017), the local charge balance may be disrupted under alkaline conditions, consequently affecting the
18
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hydrolysis performance of cellulase. On account of achieving maximal activity and reusability, pH 5 was selected as the optimal operating pH of the ICP. Additionally, Dragomirescu et al. (2010), Manasa et al. (2017) and Hosseini et al., (2018) reported
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maximum activity of immobilized cellulase achieved at pH 5, which coincides with our findings.
Chemical adaptability of the ICP and free cellulase was examined and compared
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as shown in Figure 8B. A relatively consistent trend of activity was observed for the ICP as compared to free cellulase, which indicates an improvement of pH stability during
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hydrolysis of CMC for cellulase anchored onto the polymethacrylate carrier. Apart from that, there was a decreased shift in pH optima from pH 6 (free cellulase) to pH 5 (ICP). Interestingly, Hosseini et al., (2018) obtained an optimum activity at pH 5 for both free and immobilized enzymes whereas Zhang et al. (2010) reported an increasing pH shift
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whereby the immobilized cellulase had an optimum activity at pH 5.25 in comparison to pH 4.75 for free enzymes. Bohara et al. (2016) attributed the altered pH stability of the anchored cellulase to the restriction in stretching of enzyme molecules as a function of
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the covalent interaction of enzyme-carrier composite. The ICP maintained about 65.14 ±1.11 % of enzyme activity relative to a similar amount of the free cellulase. Despite
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achieving promising maximal activity, a low residual activity (~18 %) after 4 cycles was observed; lowest relative to previously reported biocarriers as stipulated in Table 1. This could be due to the occurrence of multipoint covalent anchorage of the enzyme unto the aldehyde-activated biocarrier resulting in steric hindrance, restricted conformational transitions and reduction in activity. This drawback can be potentially mitigated in future
19
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developments of ICP by decreasing the porogen content of the porous particles to further increase its surface area.
4.
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CONCLUSION
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This study investigated the effect of various parameters affecting cellulase coupling to glutaraldehyde activated polyGMA-co-EDMA particles of ≤ 60 µm dimension in a
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column and their corresponding enzymatic activity. The ICP displayed optimum immobilization yield (10 mg) of cellulase in active state at pH 6 and 20 oC. Also, the pH and reaction temperature optima of the ICP that enhance its activity and reusability were determined to be pH 5 and 50 oC. Also, the ICP featured better pH adaptability relative to
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free cellulase. Additionally, the specific activity of the ICP was 65.14 ± 1.11 % relative to similar amount of free cellulase. Despite showing promising maximal sugar yield, residual activity of ~50 % was retained by ICP after three recycles, followed by a
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significant dip to ~18 % on the 4th cycle. The severe enzymatic leaching can be potentially improved in future works by reducing the pore size of biocarrier, and
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therefore increasing its surface area. The polymethacrylate based particles employed herein demonstrated to be highly potent biocarrier for cellulase immobilization. Results acquired serves as a good baseline for the advancement in research and development of ICP.
Acknowledgements
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The authors would like to thank Universiti Malaysia Sabah, Malaysia for the financial support to conduct this project. There is no conflict of interest to declare.
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Table
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Table 1. Comparison of similar researches with current work in terms of carriers, strategy and reusability. Adapted from Zhang et al. (2016)
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Figure Legends
Figure 1. Schematic description of CMCase activity assay. ICP was blended with carboxymethyl cellulose and incubated for a given time interval. Product yield was
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quantified from the supernatant via colorimetric means.
Figure 2. SEM images of pre- and post-immobilization of cellulase onto GA-P.
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Figure 3. Graphical representation of the molecular interactions [(A) Amino group derivatization, (B) Incorporation of aldehyde spacer group and (C) Immobilization of cellulase] that culminated in the formation of ICP.
Figure 4. FTIR spectra of nascent polymethacrylate particles (blue) and ICP (red).
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Figure 5. (A) Immobilization yield as a function of pH. Increasing pH resulted in an increased immobilization yield until pH 6, where a sudden decline in immobilization yield occurred. (B) The specific activity of ICPs buffered with different pH amid
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immobilization phase. The ICP immobilized at pH 6 gave the highest specific activity in agreement with its high immobilization yield.
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Figure 6. (A) Immobilization yield as a function of immobilization temperature. Increasing temperature facilitated the cohesion between the cellulase-polymethacrylate particles and, thus, leading to an improved immobilization yield. (B) The specific activity of ICPs prepared at different temperatures (TImmobilization). The ICPs prepared at lower temperature yielded the highest specific enzyme activity. Figure 7. (A) The effect of varied TReaction on sugar yield over the span of 2 h. (B) Reusability analysis of the ICP subjected to various TReaction. All the variants in (B) 27
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retained a certain amount of their residual activity in the order of 50 oC > 40 oC > 30 oC > 20 oC > 60 oC. Figure 8. (A) The effect of varied pH on activity and reusability of the ICP. (B)
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Chemical stability assessment of the ICP and free cellulase across different pH.
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Table 1. Comparison of similar researches with current work in terms of carriers, strategy and reusability. Adapted from Zhang et al. (2016) Reusability
Sol-gel matrix
Sol-gel entrapment
Sodium alginate gel
Sol-gel entrapment and
beads
crosslinking
Functionalized Physical adsorption
nanotubes
*Poly (glycidyl
*Current work
7
N/A
8
30 min
20
58.37
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6
30 min
48.2
Covalent Binding
4
1h
55
Covalent Binding
6
30 min
40
Covalent Binding
10
3h
60
Covalent Binding
4
30 min
18
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dimethacrylate)
24 h
Covalent Binding
methacrylate-coethylene
6
50
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Modified Silica gel
activity (%)
10 min
terpolymers
nanoparticles
Interval
8
Magnetic porous
Magnetic
Repeat
Physical adsorption
microparticles
nanoplatelets
Residual
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Ultrafine Eri silk
Magnetic graphene
Reaction
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multiwall carbon
No. of
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Immobilization Strategy
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Biocarrier
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Figure 1. Schematic description of CMCase activity assay. ICP was blended with carboxymethyl cellulose and incubated for a given time interval. Product yield was
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quantified from the supernatant via colorimetric means.
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Figure 2. SEM images of pre- and post-immobilization of cellulase onto GA-P.
Post-Immobilization
x 1.00
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x 1.00
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Pre-Immobilization
x 10.00
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x 10.00
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Figure 3. Graphical representation of the molecular interactions [(A) Amino group derivatization, (B) Incorporation of aldehyde spacer group and (C) Immobilization of
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cellulase] that culminated in the formation of ICP.
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Figure 4. FTIR spectra of nascent polymethacrylate particles (blue) and ICP (red).
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~ 3200-3500 cm-1
~ 3000 cm-1 Poly(GMA)-co-(EDMA) particle
~ 1578.88 cm-1
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ICP
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~ 1653.56 cm-1
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~ 1724.40 cm -1
~906.01 cm -1 ~ 756.87 cm-1
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Figure 5. (A) Immobilization yield as a function of pH. Increasing pH resulted in an increased immobilization yield until pH 6, where a sudden decline in immobilization yield occurred. (B) The specific activity of ICPs buffered with different pH amid immobilization
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phase. The ICP immobilized at pH 6 gave the highest specific activity in agreement with
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its high immobilization yield.
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Figure 6. (A) Immobilization yield as a function of immobilization temperature. Increasing temperature facilitated the cohesion between the cellulase-polymethacrylate particles and, thus, leading to an improved immobilization yield. (B) The specific activity of ICPs
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yielded the highest specific enzyme activity.
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prepared at different temperatures (TImmobilization). The ICPs prepared at lower temperature
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Figure 7. (A) The effect of varied TReaction on sugar yield over the span of 2 h. (B) Reusability analysis of the ICP subjected to various TReaction. All the variants in (B) retained a certain amount of their residual activity in the order of 50 oC > 40 oC > 30 oC > 20 oC >
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60 oC.
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(B)
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(A)
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Figure 8. (A) The effect of varied pH on activity and reusability of the ICP. (B) Chemical
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stability assessment of the ICP and free cellulase across different pH.
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(A)
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(B)
120
80
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Relative activity [%]
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100
60
ICP-Cellulase Free cellulase
40 20 0
2
4
6
pH
8
10
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Highlights: Poly(GMA-co-EDMA) particles were applied as biocarrier for cellulase.
•
Parameters for cellulase linkage and hydrolytic activity of ICP were optimized.
•
ICP displayed better chemical stability than free cellulase.
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~50 % and ~18 % of residual activity are retained after 3rd and 4th cycles.
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ICP recorded specific activity of 65.14 ± 1.11 % of free cellulase.
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S0300-9084(18)30344-4
DOI:
https://doi.org/10.1016/j.biochi.2018.11.019
Reference:
BIOCHI 5557
To appear in:
Biochimie
Received Date: 19 September 2018 Accepted Date: 30 November 2018
Please cite this article as: Y.W. Chan, C. Acquah, E.M. Obeng, E.C. Dullah, J. Jeevanandam, C.M. Ongkudon, Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose, Biochimie, https://doi.org/10.1016/j.biochi.2018.11.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose
Jaison Jeevanandam3 and Clarence M. Ongkudon1* 1
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Yi Wei Chan1, Caleb Acquah2,3, Eugene M. Obeng1,4, Elvina C. Dullah1,
Bioprocess Engineering Research Group, Biotechnology Research Institute, Universiti
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Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
School of Nutrition Sciences, University of Ottawa, K1N 6N5, Ontario, Canada.
3
Department of Chemical Engineering, Curtin University, 98009 Miri, Sarawak,
Malaysia. 4
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Bioengineering Laboratory, Department of Chemical Engineering, Monash University,
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Victoria 3800, Australia.
*Correspondence: Clarence M. Ongkudon
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[email protected]; +6088-320 000 Ext: 8536
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ABSTRACT Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be
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less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate
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particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay.
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Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 oC. The maximum CMCase
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activity was achieved at pH 5 and 50 oC. Residual activity of ~50 % was retained after three iterations and dipped to ~18 % on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ±1.11 %
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relative to similar amount of free cellulase.
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Keywords: Polymethacrylate porous particle, cellulase, immobilization, reusability, carboxymethyl cellulose
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Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose
Jaison Jeevanandam3 and Clarence M. Ongkudon1* 1
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Yi Wei Chan1, Caleb Acquah2,3, Eugene M. Obeng1,4, Elvina C. Dullah1,
Bioprocess Engineering Research Group, Biotechnology Research Institute, Universiti
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Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
School of Nutrition Sciences, University of Ottawa, K1N 6N5, Ontario, Canada.
3
Department of Chemical Engineering, Curtin University, 98009 Miri, Sarawak,
Malaysia. 4
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2
Bioengineering Laboratory, Department of Chemical Engineering, Monash University,
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Victoria 3800, Australia.
*Correspondence: Clarence M. Ongkudon
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[email protected]; +6088-320 000 Ext: 8536
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ABSTRACT Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be
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less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate
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particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay.
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Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 oC. The maximum CMCase
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activity was achieved at pH 5 and 50 oC. Residual activity of ~50 % was retained after three iterations and dipped to ~18 % on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ±1.11 %
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relative to similar amount of free cellulase.
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Keywords: Polymethacrylate porous particle, cellulase, immobilization, reusability, carboxymethyl cellulose
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ABBREVIATIONS
immobilized cellulase-polymethacrylate particle
EDA-P
EDA derivatized polymethacrylate particles
GA-P
Aldehyde terminated surface of polymethacrylate particles
WICP
Dry weight of ICP powder
WGA-P
Dry weight of GA-P powder
ASample
Absorbance reading of sugar yield
AStandard
Absorbance reading of standard (Blank)
U
Hydrolytic activity of cellulase
U/mg
Specific activity of cellulase
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ICP
Temperature of cellulolytic reaction of free and immobilized cellulase
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TReaction
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TImmobilization Temperature setpoint of cellulase immobilization
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1.
INTRODUCTION The development of cellulases for biomass saccharification continues to be a
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dominant research in the quest for green chemicals. These enzymes ensure the synergistic conversion of cellulose to simple sugars which are subsequently converted to platform chemicals such as alcohols and organic acids [1,2]. Advancements in this area of research
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has witnessed enzymes in free, immobilized and microbial surface displayed forms [3,4]. The progression is as a result of the search for high enzyme efficiency, enzyme
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robustness and favorable process economics. Cellulases in the free forms are the most popularly used paradigm but are difficult to recycle; the immobilized forms are recyclable but often experience reduced enzyme efficiency and are mostly expensive [3]. The cell surface displayed forms are also recyclable; however, the cells most often
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experience physiological burdens that somehow influence their overall functionality [5]. Focusing on enzyme immobilization, the practice involves the anchorage of the biocatalyst onto stationary phases to enhance the enzyme’s technical performance (e.g.,
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stability, reactivity, substrate specificity, and enantioselectivity) and reusability potentials [3,6]. This further promotes continuous economic operation and recovery of product with
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a high degree of purity [7–9]. In this practice, the choice of the biocarrier and the immobilization procedure remain critical since they can alter the innate structure and conformation of the enzymes and, thus, leading to a suppression of their catalytic efficiencies. Physical adsorption and covalent binding have been noted to be the most applied chemistries in immobilizing enzymes [10–12]. Other chemistries applicable for enzyme immobilization are entrapment, copolymerization, and encapsulation. Physical
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adsorption offers a facile immobilization strategy which retains the native conformation of the enzyme, and hence, limit the loss of its activity [13,14]. The mode of interaction between the enzyme and the biocarrier in physical adsorption usually occur through
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hydrogen, hydrophobic and electrical binding. Nevertheless, the weak interaction between the biocatalyst and biocarrier results in excessive leaching/desorption and minimum reusability [10,13,15,16]. Contrary, covalent bonding results in high reusability
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of immobilized enzyme and, therefore, renders the application of the enzymatic process in industrial operations commercially viable [10,11,17,18]. That notwithstanding,
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application of covalent chemistries may result in major loss of enzymatic activity and liable changes in conformation of the multimeric enzyme, in exchange for more stable enzyme-carrier linkage of covalent nature.
Screening of biocarriers is imperative to the development of an innovative
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biocatalytic system with targeted industrial application (food, pharmaceutical and chemical industries). Fundamental criteria for the solid support selection comprises of chemical stability, physical/mechanical strength and cost effectiveness [19]. A plethora of
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biocarriers such as silica gel [7], copolymers [20], multiwall carbon nanotubes [21], magnetic nanoparticles [22] and clay mineral [23] with different physical forms has been
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experimented with cellulolytic enzyme. For instances, in the work of Lima et al., 2017, Fe3O4 magnetic particle as the support permitted easy separation of enzymes from the solution via modulation of magnetic field, but it was susceptible to agglomeration which affected the mass transfer rate. Copolymeric particles, in the context of poly(glycidyl methacrylate-co-ethylene dimethacrylate) resins, are well known for its robustness and biocompatibility with several ligands. The epoxy-containing copolymers are modifiable
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and fine-tunable for varied immobilization chemistries and for tethering bioligands such as biocatalysts [25]. To the best of our knowledge, polymethacrylate particles have yet to be exploited as biocarriers for cellulase in the extant literature.
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Herein, the prospects of polymethacrylate particles as a biocarrier for cellulases have been assessed and reported. The cellulases were anchored covalently to an aldehyde-activated moiety to form immobilized cellulase-polymethacrylate particle (ICP).
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The scope of this paper entails the characterization of the parameters (pH and temperatures) that enhance immobilization yield and enzymatic activity of the ICP. Also,
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the reusability of the ICP was included as a yardstick for the selection of optimal parameters. Furthermore, the performance of the ICP was compared to free cellulolytic enzyme so that a more comprehensive assessment can be obtained thereof.
EXPERIMENTAL
2.1
Materials
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2.
FTIR Cary 630 (Agilent, United States), vacufuge (Eppendorf, Germany), thermomixer R
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(Eppendorf, Germany), sieve shaker (UTS, Malaysia), thermostated water bath (TW20, Julabo, Germany), incubator shaker (New Brunswick Scientific Innova 40, Eppendorf,
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Selangor, Malaysia), centrifuge (TGL-40, Sichuan Shuke Instrument, Chengdu, China), spectrophotometer (Genesys 20, Thermo Scientific, Düsseldorf, Germany), microplate reader (Infinite m200, Tecan, Männedorf, Switzerland), microplates (Greiner 96 flat transparent, Sigma-Aldrich, Darmstadt, Germany), thermocycler (PTC-200, MJResearch Inc., Quebec, Canada), Celluclast (Sigma-Aldrich, Germany), carboxymethyl cellulose (CMC, low viscosity, Sigma-Aldrich, Germany), 3,5-dinitrosalicyclic acid (DNS, Acros
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Organics, Düsseldorf, Germany), sodium sulfite (Sigma-Aldrich, Germany), Phenol ACS (VWR, Gul, Singapore), potassium sodium tartrate tetrahydrate (Na-K-tartrate, Carl Roth, ᴅ-glu-cose (Roth), glutaraldehyde (Sigma-Aldrich, Germany),
Karlsruhe, Germany),
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ethylenediamine (Acros Organics, Belgium), glycidyl methacrylate (Sigma-Aldrich, Germany), methanol (J.T.Baker, USA), ethylene glycol dimethacrylate (Sigma-Aldrich,
Aldrich, Germany).
Preparation of polymethacrylate particles
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2.2
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Germany), glycidyl methacrylate (Sigma-Aldrich, Germany), cyclohexanol (Sigma-
The porous biocarrier was synthesized via thermally induced free-radical polymerization of the functional monomer (GMA) and cross-linker monomer (EDMA) blended at a ratio of 3:2 v/v% (GMA/EDMA) in the presence of 60% porogenic solvent, cyclohexanol. In
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addition, free radical polymerization process was initiated by addition of 1% w/v AIBN. The resulting mixture was sonicated and sparged with N2 gas for 15 min respectively to acquire homogeneous blend freed from dissolved O2. 50 mL of the homogenized
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polymerization mixture was gently transferred into a mold and sealed with a parafilm sheet on top. The polymeric feedstocks containing mold was immersed in a thermostated
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water bath preset to 60 oC for 5 h to commence the polymerization reaction. Solidified polymer resin was extracted and immersed in methanol-filled beaker kept in the incubator shaker overnight. Methanol-drenched polymethacrylate resin was immersed in deionized water for 4 h. The porogen-free porous material was oven-dried overnight [26]. The parched resin was fragmented into smaller parts, that were later ground and crushed to
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powder in a mortar with a pestle. Refined porous polymethacrylate particles (≤60 µm) was obtained via sieving with 60 µm mesh. 2.3
Functionalization of polymethacrylate particles
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The functional group, epoxy moieties, of polymethacrylate particles were activated via a Schiff-base covalent reaction. About 25 g of the copolymers powder was mixed with 50 mL of ethylenediamine (EDA) solution (50% v/v prepared with deionized water) and
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incubated in the thermomixer at 60 °C, 300 rpm shaking speed and for 12 h. EDA derivatized polymethacrylate particles (EDA-P) solution was subjected to centrifugation
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at 15,000 x g for 15 mins, followed by discarding the supernatant and rinsing the pellet with deionized water to remove the residues. EDA-P pellet was dissolved in 50 mL of 10% glutaraldehyde solution prepared with deionized water and incubated in the thermomixer at room temperature, 300 rpm shaking speed for 4 h. Aldehyde terminated surface of
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polymethacrylate particles (GA-P) mixture was centrifuged at 15,000 x g for 15 mins and then washed with deionized water [27]. GA-P powder was reconcentrated from the
mode.
Immobilization of cellulase
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2.4
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residue-free aqueous solution via an overnight incubation in the vacufuge preset to V-AQ
50 mg of GA-P powder was dissolved in 500 µL of 3 % Celluclast solution (prepared with 0.1 M pH 6 sodium citrate buffer). The microcentrifuge tube containing the mixture was vortexed and incubated in the thermomixer at 20 oC, 300 rpm shaking speed and for 12 h. ICP solution was then centrifuged (15,000 x g, 15 mins) and washed 3 times with sodium citrate buffer (0.1 M pH) to remove the unbound cellulase. The residue-free
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solution formed the ICP powder after a 3 h centrifugal drying using the vacufuge (V-AQ
2.5
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mode).
Immobilization yield estimation
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Given the sensitivity of spectroscopic protein assay methods, physical quantification method was opted to assay the loading of cellulase onto polymethacrylate particles. The
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dry weights of GA-P and ICP were measured with analytical balance and recorded. The dry weight differential between ICP and GA-P was quantified (1) and determined as the immobilization yield of cellulase (mg) [23].
(1)
(
)
CMCase activity assay
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2.6
=
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−
About 500 µL of 1 % CMC solution dissolved in sodium citrate buffer (0.1 M pH 6) was
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added into microcentrifuge tube containing 50 mg ICP, vortexed and incubated in the thermomixer (50 oC, 300 rpm, 30 mins). The supernatant was collected from the reaction mixture via centrifugation(15,000 x g for 15 mins) and the associated reducing sugar content was quantified via DNS colorimetric method [28]. Figure 1 demonstrates a simplified activity assay. The DNS reagent was formulated via blending DNS (1 g), phenol (0.2 g), sulfite (0.05 g), Na-K-tartrate (20 g) in 150 ml of 0.168 M NaOH. 1 g/L of
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glucose solution (MW = 180 g/mol) was selected as analytical standard. The absorbance (A540nm) readings of samples and standard were implemented in Equation (2) – (4) to
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(2)
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(4)
2.7
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""BT3$3U!-3B. 53%$/ BV 2%$$W$!X%
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=
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(3)
KL
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compute the specific activity of cellulase [29].
Effect of pH on the immobilization yield of cellulase
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3% Celluclast solutions were prepared with sodium citrate buffer of pH 4, 5, 6, 7 and 8. The immobilization of cellulase onto GA-P was carried out in accordance with section
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2.4. The amount of cellulase anchored onto activated polymethacrylate particles was quantified with the method outlined in section 2.5.
2.8
Effect of temperature on the immobilization yield of cellulase
The immobilization of the cellulase onto GA-P was carried out in accordance with section 2.4 albeit the cloned mixtures were incubated across a spectrum of temperature
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setpoints 20, 30 40 50 and 60 oC, respectively. The immobilization yield of the cellulase was quantified via the method described in section 2.5.
Effect of temperature on the CMCase activity of the ICP
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2.9
The ICP with good immobilization characteristics from the above experiments was used hereof. Microcentrifuge tubes containing 50 mg of the ICP were mixed with 500 µL of 1 %
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CMC solutions prepared with sodium citrate buffer (0.1 M, pH 6). The hydrolysis of CMC and its activity was carried out as described in section 2.6 with slight modification.
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Herein, the blends were incubated in the vacufuge preset to the respective discrete temperature setpoint of 20, 30, 40, 50 and 60 oC. The activity trends of CMCase incubated at different temperature settings were studied over time by taking the absorbance measurement of sample per hour for 4 h. The reusability behavior of the ICP
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was also evaluated by recording its activity conducted in different temperature setpoints for four consecutive days (1 run per day).
Effect of pH on CMCase activity of the ICP
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2.10
This section also made use of the best performing ICP that resulted from sections 2.7 and
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2.8. Microcentrifuge tubes comprising 50 mg of ICP were mixed with 500 µL of 1 % CMC solutions prepared with sodium citrate buffer of varied pH of 4, 5, 6, 7 and 8. The hydrolysis of CMC and the associated activity measurements were carried out as described in section 2.6. The activity of the ICP conducted in the range of pH mentioned was measured for 4 days (1 run per day) to characterize the reusability of the ICP.
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2.11
Imaging and spectra analysis
The porous particles of pre- and post-immobilization states were coated with gold and observed under a Scanning Electron Microscope (Hitachi S3400, 15 kV). Fourier transform infrared
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(FTIR) spectroscopic analysis was conducted to assess the chemical composition of the porous copolymers particles and the ICP using Agilent Cary 630 FTIR (USA) with diamond attenuated total reflectance. Standard operating procedures were used. Both ICP
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and polymethacrylate particles were oven dried overnight prior to the commencement of
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analysis.
RESULTS AND DISCUSSION
3.1
Characterization of the ICP
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3.
Methacrylate-based polymers have proven to be effective as biocarriers in multi-omics application [25,30]. A considerable amount of literature studies pertaining to the
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characterization of copolymers (GMA-co-EDMA) in various formats have been well documented [26,27,31]. To date, there are no reported works on exploring porous
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poly(GMA-co-EDMA) particles as biocarrier for cellulolytic enzymes. The present work explored the molecular binding of cellulase on polymethacrylate particles (≤ 60 µm particle size) for compatibility assessment. Figure 2 shows the SEM images of the nascent polymethacrylate particles and ICP under x1.00 and x10.00 magnification. The surface morphology from the two different stages showed that there was no significant alteration in surface areas and pore sizes after functionalization and immobilization of cellulase to form the ICP. The epoxy bearing porous particles were activated by adopting 12
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Schiff-based chemistry, allowing cellulase to be covalently anchored to the biocarrier as illustrated in Figure 3. Covalent immobilization chemistries minimize/prevent hydrolysis and subsequent leaching of immobilized ligands [32,33].
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Fourier Transform Infrared (FTIR) spectra analysis was performed to screen for the available functional groups in the polymethacrylate particles and enzyme loaded configuration of the polymer network. The FTIR spectra of porous polymethacrylate
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particles and ICP are depicted in Figure 4. The peaks of ~756.87 cm-1, ~906.01 cm-1 and ~3000 cm-1 are characteristic of epoxy group moiety in the polymethacrylate particles. A
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reduction in the peaks, as observed in Figure 4, is indicative of a successful activation of the polymethacrylate particles with EDA and GA [27,34,35]. The characteristic band of enzyme after immobilization was observed in the ICP spectrum at 1578.88 cm-1 emanated by C—O stretching vibration. Additional band at 1653.56 cm-1 suggests the
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presence of imine bond (C=N) linking EA-P to GA and GA-P to cellulase [9,36]. The peak at ~1724.40 cm-1 emerged from copolymers particles spectrum can be attributed to the presence of C=O bond. Lower intensity of the C=O band as shown by the ICP
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spectrum could be attributed to the amine reductive reactions [27]. The broad peak across the range of 3200 - 3500 cm-1 was due to N—H and O—H stretching vibrations of the
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amino and OH groups in cellulase [9,34]. The overall FTIR analysis validates successful cellulase coupling on polymethacrylate particles
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3.2
Screening for the optimal configuration of enzyme immobilization
The optimal parameters setting amid enzyme immobilization is crucial to maximize the activity of CMCase. Inappropriate pH or reaction temperature setpoint may lead to
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enzyme inactivation or lower the amount of enzyme anchored onto the biocarrier. Hence, immobilization of cellulolytic enzymes onto the porous copolymers particles were
3.21
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immobilization parameters for subsequent experiments.
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conducted across a spectrum of pH and temperature setting to determine the optimum
Effect of pH on immobilization yield
The cellulase solutions prepared with sodium citrate buffer of varied pH (4-8) were
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respectively loaded on aldehyde-activated polymethacrylate particles (GA-P) at 25 oC. Figure 5A displays the amount of ICP formed under the influence of distinct pH. The GA-P-cellulase conjugate prepared at pH 6 resulted as the highest loaded ICP with 10 mg
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of anchored cellulase, followed by pH 5 (9.5 mg), pH 4 (8.5 mg), pH 8 (7.8 mg) and pH 7 (7.3 mg). To ensure that quantity commensurate with quality, activity assay in terms of
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the sugar yield per mg of cellulase was conducted at 50 oC. By referring to Figure 5B, the specific activities of respective ICPs in descending
order were pH 6 (12.26 U/mg) > pH 5 (11.64 U/mg) > pH 7 (10.4 U/mg) > pH 8 (8.16 U/mg) > pH 4 (7.03 U/mg). Considering the highest immobilization yield and specific activity of ICP, pH 6 emerged as the most optimal hydrogen ion concentration for the immobilization of active cellulase onto the porous polymethacrylate particles. Herein, the
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pH with highest immobilization (i.e., pH 6) yield gave the highest specific enzyme activity. However, no apparent correlation could be derived for pH 4, pH 7 and pH 6 in terms of their immobilization yields and activities. For instance, pH 4 with an enzyme
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loading of 8.5 mg gave the lowest activity (i.e., 7.03 U/mg) in comparison with pH 7 with 7.3 mg enzyme loading but a better specific enzyme activity of 10.4 U/mg. Since Celluclast
is
a
concoction
of
hydrolytic
enzymes
(i.e.,
endoglucanases,
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cellobiohydrolases, cellobiases and other accessory proteins), it is possible that the pH 4 adsorbed other proteins which are not specific to the degradation of CMC. It is worth
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knowing that CMC is specific for CMC degrading enzymes such as endoglucanases (thus, the name CMCase). CMC is an amorphous substrate [37] and therefore would hardly be hydrolyzed by non-CMCase.
It is perceivable that the enzymes could undergo conformational changes (severity
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order: pH 4 > pH 8 > pH 7) amid immobilization process, thus switching binding functional group from the non-catalytic site to restricted region, and consequently limiting the access of substrates into the anchored cellulases.
Effect of temperature on immobilization yield
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3.22
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The pH was maintained at pH 6 for this section. In this section, the immobilization of cellulase on GA-P was carried out across a broad range of temperature settings (TImmobilization : 20, 30, 40, 50 and 60 oC) to elucidate the influence of temperature on ICP preparation. Figure 6A depicts the immobilization yield of the ICP from the respective incubation temperatures. The outcome of the test revealed TImmobilization of 20 oC as the
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temperature with the lowest enzyme loading (10 mg) followed by 30 oC (13.06 mg), 50 o
C (25.7 mg), 40 oC (26.18 mg) and 60 oC (29.89 mg). The result indicates an apparent
relationship between the increase in TImmobilization and enzyme loading for a single run for
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each different sample.
The ICPs prepared at varied TImmobilization were further assessed for their relative cellulose hydrolyzing performance as represented in Figure 6B. The figure illustrates an
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inverse relationship between the specific enzyme activity and TImmobilization, which happens to be a complete opposite of that of ICP yield verses thermal input. The highest
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specific activity of the ICP was recorded at TImmobilization of 20 oC, followed by an exponential decrease at 30 oC and 40 oC and eventually comes to a complete halt beyond 50 oC. Despite recording the lowest ICP yield, 20 oC emerged as the optimal TImmobilization which putatively facilitated the retention of the native conformation of the anchored
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cellulase thus resulting in the high enzyme activity in comparison to the others. The low CMC conversion of the ICP prepared at high TImmobilization could be attributed to progressive thermal denaturation of the cellulase over the course of the 12-hour
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immobilization phase. In this case, the structure/conformation of the enzymes might have transitioned from partial unfolding to permanent change [35] upon covalently linked to
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GA-P. Amid that span, the enzyme could have been irreversibly altered, joint covalently to GA-P via functional group at restriction site that leads to steric hindrance or at partially folded state which wipes off any chance of renaturation. In general, high TImmobilization increased the ICP yield but negatively impacted the catalytic activity thereof. The subsequent section hereafter employed the ICP prepare at pH 6 and 20 oC because of its performance as discussed above.
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3.3
Effect of reaction temperature and associated glucose yields of the ICP
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The temperature of cellulose hydrolysis, TReaction, plays a pivotal role in maximizing sugar yield. Therefore, the range of temperature settings, viz 20 oC, 30 oC, 40 oC, 50 oC and 60 o
C, were assessed for their respective impact on the activity of the ICP. Figure 7A depicts
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the glucose yield by the ICP prepared at pH 6 and 20 oC. The ICP was subjected to varied TReaction for a total time interval of 2 h. In this experiment, comparing the trend of glucose
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yield for the reaction temperatures from 20 oC to 50 oC showed an increasing sugar profile for each reaction temperature. In this range, the sugar yield increased with respect to time in the order of 20 oC < 30 oC< 40 oC < 50 oC. In other words, the higher the reaction temperature, the better the sugar yield for that temperature over time. However,
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there was a deviation from the above-stated trend for TReaction of 60 oC, which happened to exhibit an increasing glucose profile until the hour mark after which a decline in sugar yield was observed. This could be a consequence of periodic thermal denaturation, which
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causes the enzymes to lose their functionality over time. Maximal sugar yield was recorded at 50 oC. Similar finding was also reported by Acharya and Chaudhary (2012)
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and Chang et al. (2009). Furthermore, as per the study conducted by Andreaus et al. (1999), the catalytic activity of cellulase diminished when TReaction was above the temperature optima (> 50 oC), during which thermal denaturation likely took place. The assessment of the reusability of the ICP was executed with the same range of
TReation (20 – 60 oC). The common issue of immobilized enzyme activity loss due to leaching after repeated run was eminent during this trial. Figure 7B shows a sharp decline
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in activity of 54.2 % and 43.8 % for 40 oC and 50 oC, respectively, after three iterations. The reaction temperature of 20 oC and 30 oC only managed to retain a residual activity of 25.3 % and 26.4 %, respectively, after three cycles. The reusability potential at 60 oC was
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poor since it could only retain 3.9% of enzyme activity after the third batch. Despite recording the highest value in reusability, the cumulative specific activity of 40 oC throughout the 4 recycles (26.2 U/mg) did not exceed that conducted at 50 oC (29.1 u/mg).
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On grounds of all the data collected, 50 oC, which also happens to be the customary
maximize the hydrolysis of CMC.
Effect of pH on ICP
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3.4
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TReaction for commercial cellulases [35], appears to be the optimal TReaction for the ICP to
Also, the pH-activity characteristics of the ICP prepared at pH 6 and 20 oC were assessed within a range of pH spectrum to elucidate the dynamics thereof. In reference to Figure
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8A, both pH 5 and pH 6 recorded comparable enzymatic activities for the 1st and 2nd batches. However, there was a subsequent dip in residual activity (29 %) for pH 5 while
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the latter hits a plateau (50 %) after 3rd batch. The higher degrees of activity loss of 21.7 %, 18.1 % and 14.2 % were observed for pH 4, 7 and 8, respectively, at the 3rd cycle. The activity recorded across the tested range of pH spectrum (descending order: pH 5 > pH 6 > pH 4 > pH 7 > pH 8) suggests the ineffectiveness of the ICP under alkaline operating conditions. According to Ben Hmad and Gargouri (2017), the local charge balance may be disrupted under alkaline conditions, consequently affecting the
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hydrolysis performance of cellulase. On account of achieving maximal activity and reusability, pH 5 was selected as the optimal operating pH of the ICP. Additionally, Dragomirescu et al. (2010), Manasa et al. (2017) and Hosseini et al., (2018) reported
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maximum activity of immobilized cellulase achieved at pH 5, which coincides with our findings.
Chemical adaptability of the ICP and free cellulase was examined and compared
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as shown in Figure 8B. A relatively consistent trend of activity was observed for the ICP as compared to free cellulase, which indicates an improvement of pH stability during
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hydrolysis of CMC for cellulase anchored onto the polymethacrylate carrier. Apart from that, there was a decreased shift in pH optima from pH 6 (free cellulase) to pH 5 (ICP). Interestingly, Hosseini et al., (2018) obtained an optimum activity at pH 5 for both free and immobilized enzymes whereas Zhang et al. (2010) reported an increasing pH shift
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whereby the immobilized cellulase had an optimum activity at pH 5.25 in comparison to pH 4.75 for free enzymes. Bohara et al. (2016) attributed the altered pH stability of the anchored cellulase to the restriction in stretching of enzyme molecules as a function of
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the covalent interaction of enzyme-carrier composite. The ICP maintained about 65.14 ±1.11 % of enzyme activity relative to a similar amount of the free cellulase. Despite
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achieving promising maximal activity, a low residual activity (~18 %) after 4 cycles was observed; lowest relative to previously reported biocarriers as stipulated in Table 1. This could be due to the occurrence of multipoint covalent anchorage of the enzyme unto the aldehyde-activated biocarrier resulting in steric hindrance, restricted conformational transitions and reduction in activity. This drawback can be potentially mitigated in future
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developments of ICP by decreasing the porogen content of the porous particles to further increase its surface area.
4.
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CONCLUSION
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This study investigated the effect of various parameters affecting cellulase coupling to glutaraldehyde activated polyGMA-co-EDMA particles of ≤ 60 µm dimension in a
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column and their corresponding enzymatic activity. The ICP displayed optimum immobilization yield (10 mg) of cellulase in active state at pH 6 and 20 oC. Also, the pH and reaction temperature optima of the ICP that enhance its activity and reusability were determined to be pH 5 and 50 oC. Also, the ICP featured better pH adaptability relative to
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free cellulase. Additionally, the specific activity of the ICP was 65.14 ± 1.11 % relative to similar amount of free cellulase. Despite showing promising maximal sugar yield, residual activity of ~50 % was retained by ICP after three recycles, followed by a
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significant dip to ~18 % on the 4th cycle. The severe enzymatic leaching can be potentially improved in future works by reducing the pore size of biocarrier, and
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therefore increasing its surface area. The polymethacrylate based particles employed herein demonstrated to be highly potent biocarrier for cellulase immobilization. Results acquired serves as a good baseline for the advancement in research and development of ICP.
Acknowledgements
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The authors would like to thank Universiti Malaysia Sabah, Malaysia for the financial support to conduct this project. There is no conflict of interest to declare.
[1]
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Applications, in: Bioprocess. Technol. Biorefinery Sustain. Prod. Fuels, Chem. Polym.,
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John Wiley & Sons, Inc., Hoboken, NJ, USA, 2013: pp. 131–146. doi:10.1002/9781118642047.ch8.
V. Juturu, J.C. Wu, Microbial cellulases: Engineering, production and applications, Renew.
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Table
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Table 1. Comparison of similar researches with current work in terms of carriers, strategy and reusability. Adapted from Zhang et al. (2016)
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Figure Legends
Figure 1. Schematic description of CMCase activity assay. ICP was blended with carboxymethyl cellulose and incubated for a given time interval. Product yield was
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quantified from the supernatant via colorimetric means.
Figure 2. SEM images of pre- and post-immobilization of cellulase onto GA-P.
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Figure 3. Graphical representation of the molecular interactions [(A) Amino group derivatization, (B) Incorporation of aldehyde spacer group and (C) Immobilization of cellulase] that culminated in the formation of ICP.
Figure 4. FTIR spectra of nascent polymethacrylate particles (blue) and ICP (red).
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Figure 5. (A) Immobilization yield as a function of pH. Increasing pH resulted in an increased immobilization yield until pH 6, where a sudden decline in immobilization yield occurred. (B) The specific activity of ICPs buffered with different pH amid
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immobilization phase. The ICP immobilized at pH 6 gave the highest specific activity in agreement with its high immobilization yield.
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Figure 6. (A) Immobilization yield as a function of immobilization temperature. Increasing temperature facilitated the cohesion between the cellulase-polymethacrylate particles and, thus, leading to an improved immobilization yield. (B) The specific activity of ICPs prepared at different temperatures (TImmobilization). The ICPs prepared at lower temperature yielded the highest specific enzyme activity. Figure 7. (A) The effect of varied TReaction on sugar yield over the span of 2 h. (B) Reusability analysis of the ICP subjected to various TReaction. All the variants in (B) 27
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retained a certain amount of their residual activity in the order of 50 oC > 40 oC > 30 oC > 20 oC > 60 oC. Figure 8. (A) The effect of varied pH on activity and reusability of the ICP. (B)
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Chemical stability assessment of the ICP and free cellulase across different pH.
28
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Table 1. Comparison of similar researches with current work in terms of carriers, strategy and reusability. Adapted from Zhang et al. (2016) Reusability
Sol-gel matrix
Sol-gel entrapment
Sodium alginate gel
Sol-gel entrapment and
beads
crosslinking
Functionalized Physical adsorption
nanotubes
*Poly (glycidyl
*Current work
7
N/A
8
30 min
20
58.37
26
6
30 min
48.2
Covalent Binding
4
1h
55
Covalent Binding
6
30 min
40
Covalent Binding
10
3h
60
Covalent Binding
4
30 min
18
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dimethacrylate)
24 h
Covalent Binding
methacrylate-coethylene
6
50
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Modified Silica gel
activity (%)
10 min
terpolymers
nanoparticles
Interval
8
Magnetic porous
Magnetic
Repeat
Physical adsorption
microparticles
nanoplatelets
Residual
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Ultrafine Eri silk
Magnetic graphene
Reaction
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multiwall carbon
No. of
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Immobilization Strategy
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Biocarrier
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EP
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Figure 1. Schematic description of CMCase activity assay. ICP was blended with carboxymethyl cellulose and incubated for a given time interval. Product yield was
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quantified from the supernatant via colorimetric means.
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Figure 2. SEM images of pre- and post-immobilization of cellulase onto GA-P.
Post-Immobilization
x 1.00
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x 1.00
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Pre-Immobilization
x 10.00
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x 10.00
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Figure 3. Graphical representation of the molecular interactions [(A) Amino group derivatization, (B) Incorporation of aldehyde spacer group and (C) Immobilization of
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cellulase] that culminated in the formation of ICP.
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Figure 4. FTIR spectra of nascent polymethacrylate particles (blue) and ICP (red).
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~ 3200-3500 cm-1
~ 3000 cm-1 Poly(GMA)-co-(EDMA) particle
~ 1578.88 cm-1
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ICP
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~ 1653.56 cm-1
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~ 1724.40 cm -1
~906.01 cm -1 ~ 756.87 cm-1
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Figure 5. (A) Immobilization yield as a function of pH. Increasing pH resulted in an increased immobilization yield until pH 6, where a sudden decline in immobilization yield occurred. (B) The specific activity of ICPs buffered with different pH amid immobilization
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phase. The ICP immobilized at pH 6 gave the highest specific activity in agreement with
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its high immobilization yield.
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Figure 6. (A) Immobilization yield as a function of immobilization temperature. Increasing temperature facilitated the cohesion between the cellulase-polymethacrylate particles and, thus, leading to an improved immobilization yield. (B) The specific activity of ICPs
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yielded the highest specific enzyme activity.
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prepared at different temperatures (TImmobilization). The ICPs prepared at lower temperature
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Figure 7. (A) The effect of varied TReaction on sugar yield over the span of 2 h. (B) Reusability analysis of the ICP subjected to various TReaction. All the variants in (B) retained a certain amount of their residual activity in the order of 50 oC > 40 oC > 30 oC > 20 oC >
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60 oC.
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(B)
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(A)
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Figure 8. (A) The effect of varied pH on activity and reusability of the ICP. (B) Chemical
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stability assessment of the ICP and free cellulase across different pH.
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(A)
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(B)
120
80
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Relative activity [%]
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100
60
ICP-Cellulase Free cellulase
40 20 0
2
4
6
pH
8
10
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Highlights: Poly(GMA-co-EDMA) particles were applied as biocarrier for cellulase.
•
Parameters for cellulase linkage and hydrolytic activity of ICP were optimized.
•
ICP displayed better chemical stability than free cellulase.
•
~50 % and ~18 % of residual activity are retained after 3rd and 4th cycles.
•
ICP recorded specific activity of 65.14 ± 1.11 % of free cellulase.
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•