Evaluation of nanocellulose carriers produced by four different bacterial strains for laccase immobilization

Accepted Manuscript Title: Evaluation of Nanocellulose Carriers Produced by Four Different Bacterial Strains for Laccase Immobilization Authors: Haibin Yuan, Lin Chen, Feng F. Hong, Meifang Zhu PII: DOI: Reference:

S0144-8617(18)30597-6 https://doi.org/10.1016/j.carbpol.2018.05.055 CARP 13637

To appear in: Received date: Revised date: Accepted date:

6-2-2018 16-5-2018 16-5-2018

Please cite this article as: Yuan, Haibin., Chen, Lin., Hong, Feng F., & Zhu, Meifang., Evaluation of Nanocellulose Carriers Produced by Four Different Bacterial Strains for Laccase Immobilization.Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.05.055 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.

Evaluation of Nanocellulose Carriers Produced by Four Different Bacterial Strains for Laccase Immobilization

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Haibin Yuana,b, Lin Chena*, and Feng F. Honga,b*, Meifang Zhua

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State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, P. R. China. E-mail: [email protected] b Group of Microbiological Engineering and Industrial Biotechnology, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, North Ren Min Road 2999, Shanghai 201620, P. R. China. E-mail: [email protected]

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Corresponding Author：*E-mail: [email protected]; [email protected].

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Table of Contents (TOC)：

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Highlights 

Properties of BNC produced by four different strains were compared for laccase immobilization.

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Four types of BNC had significant structural differences in fiber density, diameter and distribution.

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BNC materials had various specific surface area, total pore volume, and average 1

pore size. 

Structural diversity of BNC may directly result in different efficiency in enzyme immobilization.

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A looser fiber network in BNC with larger porosity is helpful for enzyme

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immobilization.

ABSTRACT:

Properties of bacterial nanocellulose (BNC) produced by four different strains were studied

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and compared for laccase immobilization. Scanning electron microscope inspection indicated the four types of BNC had obvious differences in fiber density, diameter and distribution. BNC hydrogel demonstrated the highest fracture stress of 2.44 Mpa and the highest Young's

modulus of 12.76 Mpa. Brunauer-Emmett-Teller analysis suggested the four BNC had

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significant difference in specific surface area, total pore volume and average pore size. Laccase was immobilized on BNC carriers via adsorption. Kinetic studies showed that the

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four types of BNC-immobilized laccase had different affinity with substrate, and all types of immobilized laccase showed high operational stability after ten consecutive biocatalytic

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cycles of reaction. The results suggest that the structure diversity of BNC from various strains may directly result in different efficiency in laccase immobilization, and a looser fiber

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network in BNC with larger porosity is helpful for enzyme immobilization.

KEYWORDS: bacterial nanocellulose; laccase; immobilization; strain effect; structural

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difference

1. Introduction

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Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) belongs to multicopper oxidase

family and can be roughly divided into two major groups based on sources: laccase from higher plants and fungal laccase (Mayer, 2006). The range of substrates which laccase can attack is very wide due to its strong oxidation ability. Basically, laccase can not only oxidize substrates with characteristics similar to phenolic compounds, but also oxidize other substrates including syringaldazine, aromatic

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amines and non-phenolic compounds to form free radicals (Hong, Jönsson, Lundquist, & Wei, 2006; Strong & Claus, 2011). Depending on the strong oxidation ability, laccase has been studied in different industrial applications, such as dye decolorization (Jaiswal, Pandey, & Dwivedi, 2016; Soares, de Amorim, & Costa-Ferreira, 2001), pulp delignification and bleaching (Gamelas, Tavares, Evtuguin, & Xavier, 2005), soil bioremediation (Couto & Herrera, 2006), food industry (Osma, Toca-Herrera & Rodriguez-Couto, 2010) and detection of specific analytes (Gomes & Rebelo, 2003). Although laccase has a lot of advantages, there are some obstacles limiting its applications, for instance low stability and high production costs. In order to overcome these drawbacks, immobilization 2

of laccase has been proposed and studied for several years, for instance entrapment with alginate–chitosan (Lu, Zhao, & Wang, 2006), adsorption on bimodal mesoporous Zr-metal organic framework (Zr-MOF) (Pang et al., 2016) and covalent coupling on Poly(maleic anhydride-alt-methyl vinyl

ether)–g-Poly

(L-lactic

acid)

/

octadecyl

amine-montmorillonite

[Poly(MA-alt-MVE)-g-PLA/ODA-MMT] (İLk, DemİRcan, SaĞLam, SaĞLam, & Rzayev, 2016). Although these materials have a good performance on laccase immobilization, each has its own limitation. For example, some of the materials are very difficult to produce, which would increase immobilization cost; some of the materials have toxicity, which would limit the range of industrial applications; and some of the materials belong to small particles, which would increase the difficulty in

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recycling the immobilized enzymes (Skoronski et al., 2014).

Bacterial nanocellulose (BNC) is the polysaccharide that is mainly excreted by bacteria, such as Komagataeibacter xylinus (Gama, Gatenholm & Klemm, 2012; Yamada et al., 2013). Compared with

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plant cellulose, BNC displays unique structural and mechanical properties including three-dimensional

nanostructure, high porosity, high purity, high water absorption capacity, and excellent wet mechanical strength (Bielecki, Krystynowicz, Turkiewicz, & Kalinowska, 2005; Gama, Gatenholm & Klemm, 2012). Based on these properties, BNC has attracted much more attentions as a new basic material for advanced applications, including artificial skin (Barud et al., 2016), blood vessels (Tang, Bao, Li,

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Chen, & Hong, 2015), wound dressings (Chang & Chen, 2016; Zhang, Chen, Zhang, & Hong, 2016), scaffold for tissue engineering of cartilage (Svensson et al., 2005) and proton-conducting membranes

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of fuel cells (Jiang, Qiao, & Hong, 2012; Jiang et al., 2015). Due to the unique three-dimensional (3D) network consisting of 1D nanofiber in diameter, BNC has been used for immobilization of enzymes

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including glucoamylase (Wu & Lia, 2008), glutamate decarboxylase (Yao, Wu, Zhu, Sun, & Miller,

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2013), laccase (Chen, Zou, & Hong, 2015; Frazão et al., 2014; Sampaio et al., 2016), urease (Akduman et al., 2013; Pesaran, Amoabediny, & Yazdian, 2015), and lipase (Wu, Wu, & Su, 2017). However, influence of BNC structure and the structural difference originated from strain type on enzyme

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immobilization have not been investigated. Since the size of BNC fibrils and their spatial arrangement depends on the type of cellulose-synthesizing microorganism (Sampaio et al., 2016), BNC from different bacterial strains may lead to different results. To our knowledge, no comparison among the

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BNC carriers produced by different strains has been made for enzyme immobilization. In this work, BNC produced by four different strains (K. xylinus ATCC 23770, DHU-ZCY-1, DHU-ZGD-1, and DHU-ATCC-1) were evaluated for laccase immobilization. Performance and

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stability of the immobilized laccase were evaluated for ten consecutive biocatalytic cycles by using a reaction with 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate.

2. Material and Methods

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2.1. Materials

Coriolus versicolor laccase was purchased from Shandong Sukahan Bio-Technology Co., Ltd.

(Shandong, China) and used without further purification. ABTS was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). K. xylinus ATCC 23770 was purchased from the American Type Culture Collection and other three K. xylinus strains (DHU-ATCC-1, DHU-ZGD-1, and DHU-ZCY-1) were stored in our laboratory. DHU-ZCY-1 and DHU-ZGD-1 were obtained from Hainan Yeguo Foods Co., Ltd, and DHU-ATCC-1 was the mutant of ATCC 23770, which was obtained through random mutagenesis using chemical and physical standard methods (nitrite 3

impregnation combined with UV radiation). All other chemicals were analytical grade unless otherwise stated.

2.2. Preparation of BNC carriers BNC membranes were produced at 30 oC in static cultures inoculated with four different types of K. xylinus under the same conditions, respectively. The basal composition of culture medium contained: 25 g/L glucose, 5 g/L yeast extract, and 3 g/L tryptone. The pH was adjusted to 5.0 with 80% (v/v) sulfuric acid. The BNC membranes were allowed to grow to a thickness of 2.5 mm after which they were washed with distilled water to remove residual medium components, and then were purified by

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boiling in a 0.5 M NaOH aqueous solution for 2 h, in order to eliminate the bacterial cells (Chen et al.,

2015). Afterwards, BNC membranes were washed with distilled water until the pH became neutral. Finally, the BNC membranes were cut into many wafers with the diameter of 1 cm and were

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lyophilized as supports for enzyme immobilization. In order to avoid errors, five independent

fermentation batches were performed and at least 3 wafers from the BNC membranes of each strain were randomly selected for laccase immobilization.

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2.3. Characterization of BNC 2.3.1. Morphological inspection

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Field emission scanning electron microscopy (FE-SEM, HITACHI, S-4800, Japan) was used to inspect structural diversities of BNC membranes produced with different strains. Lyophilized BNC

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samples with surface and cross-section were coated with gold and the center areas of the samples were

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viewed with different magnification. In order to determine the mean diameter (MD) of the fibers in the four BNC membranes, more than 100 fibers were randomly selected from the SEM images and the diameter was measured by using an image analysis software Image-J (NIH, USA) (Tang, Bao, Li,

2.3.2. Tensile test

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Chen & Hong, 2015) .

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Membranes thickness was measured using a vernier caliper. Each sample was sliced into rectangle shaped gels (length: 50 mm, width: 10 mm) and were tested in wet state with an electronic universal testing machine (Hengyu, HY-940FS, China) at a fixed tensile velocity of 50 mm/min. For

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each essay, at least 5 replicates from each group of BNC were used for the test. The evaluation parameters of BNC consisted of fracture stress, breaking elongation and Young's modulus. The fracture stress was defined as the stress at the breaking point. Breaking elongation was calculated from the following equation: W = (B - S) / S, where B is the length of breaking point, S is the initial length of samples. Young's modulus was calculated from the slope of the linear region of the stress-strain curve

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(Backdahl et al., 2006).

2.3.3. Brunauer–Emmett–Teller (BET) analysis The structural property (surface area, pore diameter and pore volume) of freeze-dried BNC membranes was analyzed by using BET nitrogen adsorption-desorption isotherm measurements at liquid nitrogen temperature (-196 oC) on a Micromeritics instrument (Tristar Ⅱ 3020M, Atlanta, USA). The sample was out gassed in vacuum at 150 oC for 4-5 h prior to the measurement. All samples were investigated for at least 3 replicates and data are presented as mean±standard deviation (mean± 4

SD) of three independent measurements.

2.4. Immobilization of laccase Laccase was dissolved in 0.2 M phosphate-citrate buffer at pH 3.0. In all tests, 10 mg of lyophilized BNC wafers were incubated in 10 mL of the buffered laccase solution under stirring at 60 rpm. Laccase immobilization was carried out under different conditions including free enzyme concentration (0.5-11 U/mL) and immersing time (0-240 min) at room temperature. For the study on effects of concentration of free laccase, the immersing time was 24 h. After immobilization, the membrane supports were washed three times (each time for 15 min) with the same buffer solution and

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the membranes were immediately used.

2.5. Measurements of activity of free and immobilized laccase

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The activity of free laccase was determined spectrophotometrically (PerkinElmer, Lambda 35,

USA) by measuring the slope of the initial linear portion of the catalytic kinetic curve of ABTS at 420 nm (ε= 3.6×104 M-1cm-1) (Hong, Meinander, & Jönsson, 2002). The assay mixture in a quartz cuvette contained 1 mL of the phosphate-citrate buffered laccase solution, 1 mL phosphate-citrate buffer (pH 3.0) and 1 mL of 1 mM ABTS. Immobilized enzyme activity was assayed by mixing 100

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mg immobilized laccase (containing about 0.1 mL water) with 1.9 mL phosphate-citrate buffer and 1 mL of 1 mM ABTS at room temperature, as similar as the free laccase. Changes in absorbance of the

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mixture were determined every 1 min and lasted for 5 min. After linear regression over the obtained

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U / g = (Aabs/min×fdil×Vr×106) / (ɛ×mBNC)

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data, enzyme activity was determined by using the following equation:

where U/g is the enzyme amount per mass unit of carrier that is capable to oxidize 1 µmol of ABTS per minute, Aabs/min is the absorbance variation during a certain period of reaction time, fdil is the

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dilution factor of the sample, Vr is the volume of reaction, ε is the ABTS molar absorption coefficient (3.6×104 M-1cm-1 at 420 nm), mBNC is the mass of immobilized laccase added to the

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solution (g).

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2.6. Thermal stability of free and immobilized laccase Thermal stability was investigated by incubating free and immobilized laccase in 0.2 M phosphate-citrate buffer at pH 3.0 and 70 oC for 1 h. After incubation, the samples were taken out and then residual activity was measured. 2.7. Catalytic kinetics of free and immobilized laccase Michaelis–Menten kinetic parameters (Km and Vmax) of free and four different types of

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immobilized laccase were determined by measuring enzymatic activity with ABTS solutions ranging from 0.01 to 4 mM, according to the method as mentioned above. Kinetic parameters were calculated according to a simple Michaelis–Menten kinetics model by a nonlinear regression fitting the experimental kinetics data using ORIGIN software (ver. 8.0).

2.8. Application stability of immobilized laccase In order to investigate the operational stability of BNC-immobilized laccase, ten consecutive operating cycles of reaction with substrate ABTS at pH 3.0 and room temperature were performed 5

under magnetic stirring at 100 rpm. At the end of each cycle, the reaction was stopped by filtration to remove substrate from immobilized-laccase quickly, and the immobilized laccases were washed three times (each time 15 min) with 0.2 M phosphate-citrate buffer at pH 3. Enzyme activity of first circle was set as 100% enzyme activity. For each assay, triplicate runs were made.

2.9. Statistical analysis All data were presented as mean±SD, and were analyzed statistically by the paired Student's t-test method and comparisons among more than two groups were obtained by Analysis of Variance

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(ANOVA). A value of p＜0.05 was considered statistically significant.

3. Results and Discussion

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3.1. Characterization of bacterial nanocellulose 3.1.1. Morphological inspection

As shown in Fig. 1, four BNC membranes produced by different strains exhibited different transparency. SEM images (Fig. 2) of the BNC surface indicated that the nanofibrous network of BNC produced by ATCC 23770 was much looser than other three kinds of BNC. In addition, statistic

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analysis of fiber diameter showed the mean diameter (MD) of BNC fibers produced by ATCC 23770 was the thinnest (35 nm) as compared to other three BNC (DHU-ZCY-1: 53 nm, DHU-ZGD-1: 59 nm,

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and DHU-ATCC-1: 52 nm). The section images of the four BNC exhibit different internal structures. The fibers of BNC produced by ATCC 23770 and DHU-ZCY-1 were distributed evenly while BNC

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from DHU-ATCC-1 and DHU-ZGD-1 showed an orientation structure. The difference in

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microstructure such as fiber density, fiber distribution, fiber diameter and internal spaces may result in different physical property. For instance, the different transparency of the four BNC membranes may be closely related to the fiber density, since a looser fiber density would improve the transparency of

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the membrane. In general, different BNC microstructure may be ascribed to different metabolic processes of bacterial strains (Fujiwara et al., 2013) and the type of cellulose-synthesizing microorganisms since the size of BNC fibrils and their spatial arrangement correlate with strains

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(Mohite & Patil, 2014).

3.1.2. Mechanical testing

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The different microstructure of BNC membrane may lead to different mechanical properties. Superior mechanical properties are important for immobilization carriers because this can broaden the application of immobilized enzyme, especially for applications in membrane reactors (Rajeswari, Vismaiya, & Pius, 2017). Fig. 3 shows the stress-strain curves of the four BNC samples under quasi-static conditions. It can be seen obviously that the four BNC membranes had different

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mechanical properties. The fracture stress, Young's modulus and breaking elongation of BNC samples are given in Table 1. BNC from DHU-ATCC-1 had the highest fracture stress (2.44 Mpa) and BNC from DHU-ZCY-1 had the highest Young's modulus (12.76 Mpa). By comparing these values it can be seen that the BNC produced by ATCC 23770 had obviously lower mechanical property than others. The different BNC mechanical property may be ascribed to different structure properties, including fiber density, fiber distribution, and even could be due to polymerization degree and crystallinity since the mechanical strength of the materials composed of plant cellulose has been found to be related to polymerization degree (Vizárová, et al. 2012) and crystallinity (Abe & Yano, 2012). 6

3.1.3. Brunauer–Emmett–Teller (BET) surface area analysis The surface area was measured by the BET method and the pore size and pore volume were determined by the Barrett-Joyner-Halenda (BJH) model according to N2 adsorption-desorption isotherms and calculated by corresponding formula (Leofanti, Padovan, Tozzola & Venturelli, 1998). As shown in Fig. 4 the isotherms of all samples look like IUPAC type IV with H3 hysteresis loop. This is because when relative pressure is low, formation of a monolayer of adsorbed nitrogen molecules is the prevailing process, while at high relative pressure, a multilayer adsorption takes place: the adsorbate thickness progressively increases until condensation pressure has been reached (Bera et al.,

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2014; Leofanti, Padovan, Tozzola, & Venturelli, 1998). This phenomenon shows that lots of slit-like

holes have condensed and the range of pore size is very large (Bera, Khan, Biswas, & Jana, 2016). The results obtained demonstrated that the BNC membrane from ATCC 23770 had the highest surface area

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and greatest total pore volume as well as pore size among the four BNC membranes (Table 2). As a result of the increased total pore volume (DHU-ZCY-1 < DHU-ATCC-1 < DHU-ZGD-1 < ATCC 23770), an increase in the specific surface area (DHU-ZCY-1 < DHU-ATCC-1 < DHU-ZGD-1 < ATCC 23770) was verified within the four kinds of BNC membranes, which indicates that the total pore volume correlates directly to the specific surface area, as reported previously (Stumpf, Pertile,

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Rambo, & Porto, 2013).

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Table 2 shows average pore size of the BNC from the four strains is in the range of 9.7-16.5 nm, however Fig. 2 shows that the space between the BNC fibers is in the range of micron. The pore size

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determined via the nitrogen adsorption is just a group mean value, but not for the pore size of a specific

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visible pore in the SEM images. The values were calculated ones by using the software accompanied with the BET analytical equipment. What was determined for calculation of group mean values of pore sizes included the space between fibers, cavity formed by fiber intertwine as well as invisible pores on

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fiber surface. The pore size of BNC from K. xylinus ATCC 53582 has already been reported to be around 13 nm (Li, Qing, Zhou, & Yang, 2014), which is similar to the current results.

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3.2. Immobilization of laccase

Bacterial nanocellulose has been found to have a cage-like structure, which could efficiently entrap enzymes without covalent binding, while still allowing substrate to diffuse easily with only a

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minor loss in activity due to the different diffusion rate and microenvironment variations (Iguchi, Yamanaka, & Budhiono, 2000). Depending on this theory, the method of physical adsorption was adopted to immobilize laccase.

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3.2.1. Effects of concentration of free laccase The effects of free laccase concentration on the immobilization are shown in Fig. 5A. After

immerging for 24 h, all types of immobilized laccase attained the maximal activity when the initial concentration of free laccase reached 4 U/mL. It can be observed that the adsorption amount and activity of immobilized laccase increased with increasing initial concentration of laccase if the concentration was less than 4 U/mL, while the concentration was above 4 U/mL, the activity recovery showed a downward trend (for most BNC p<0.01 except for DHU-ZCY-1 0.01
surface area and space to load laccase molecules when immerged in low laccase concentration, and thus, the activity was improved; (ii) too much of laccase on the surface of BNC impeded the external substrate and oxygen diffuse into the interior, and subsequently decreased the enzyme activity. Therefore, too high concentration of free laccase would not be helpful in improving the efficiency of laccase immobilization.

3.2.2. Effect of immersing time As shown in Fig. 5B, optimal time to attain the maximum immobilization of enzyme (4 U/mL)

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varied with the type of BNC. In other words, the obtained maximal activity of the immobilized laccase

depended on the optimal immersing time and the type of BNC. For example, DHU-ZCY-1 needed 60 min to get maximal activity, while ATCC 23770 needed 180 min to get that. Enzyme molecules diffuse

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into BNC network should be closely related to the pore size of the membranes. The time of four BNC

to achieve maxim activity was in the following order: ATCC 23770 > DHU-ZGD-1 > DHU-ATCC-1 > DHU-ZCY-1, which is corresponding with the order in their specific surface area, total pore volume and average pore size (Table 2). The more transparent, the higher is the equilibrium loading capacity. Obviously, a denser and intensive fiber network is difficult for laccase to enter inside of carrier. On the

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contrary, a relatively loose structure and larger surface area are helpful for laccase adsorption, but this

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3.3. Thermal stability of free and immobilized laccase

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makes enzyme molecules need more time to penetrate into the interior to get balance.

As can be seen in Fig. 6A, inactivation of both free and immobilized laccases took place after 1 h

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incubation at 70 ℃. However, all the BNC-immobilized laccases retained more activity as compared to the free laccase, which implies that the BNC-immobilized laccases were more stable than the free enzyme under high temperatures. There was no significant difference within the four

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BNC-immobilized laccases. The inactivation may be ascribed to higher vibration of the atoms of the enzyme protein caused by high temperatures, which may break some chemical bonds resulting in drastic changes of its 3D structure and loss of catalytic capacity (Tavares et al., 2015). Furthermore,

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that all the BNC-immobilized laccases retained more activity could be due to the restricted conformational mobility of the enzyme molecules after immobilization (Frazão et al., 2014), which

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enhanced the stability of enzyme and delayed the rate of inactivation. 3.4. Kinetic properties Laccase oxidation kinetics parameters, maximum reaction rate (V max), and Michaelis–Menten

constant (Km) of free and immobilized laccase were studied by determining the initial oxidation

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reaction rates of ABTS at different substrate concentrations and fitting the experimental data to the classical Michaelis–Menten model. (Li, Li, & Shen, 2009) The non-linear regression results showed the good quality of the fit, with high values of R2 for both free and immobilized laccases (Fig. 6B). The Michaelis–Menten constant (Km) shows the affinity of an enzyme for a given substrate. The lower the value of Km, the higher is the affinity of the enzyme to the substrate. The Km and Vmax values of four different types of immobilized laccase were much lower than that of the free laccase, demonstrating a lower affinity for the substrate (Table 3). A lower affinity for the substrate for an immobilized enzyme could be caused by diffusional limitations, decreased enzyme flexibility and 8

lower accessibility of the substrate to the active site (Lloret et al., 2012). Similar results were observed in previous studies of different nanofibers used as a support for enzyme immobilization (Sathishkumar et al., 2014; Tavares et al., 2015). In addition, among the four different types of immobilized laccase, a gradient change of affinity with ABTS corresponded with BNC’s surface area and pore size (Table 2). Due to the largest surface area (125.5 m2/g) and pore size (16.5 nm) of membrane, laccase immobilized on the BNC from ATCC 23770 has the highest affinity (Km = 0.37 mM) with ABTS substrate, which indicates that a larger surface area and pore size are helpful for immobilized laccase to react with substrate.

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3.5. Reuse stability of immobilized laccase

Reuse stability of the BNC-immobilized laccase was investigated by using oxidation reaction of

ABTS for ten consecutive cycles. As shown in Fig. 7, the relative activity of the immobilized laccase

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decreased slowly along the reusing cycles. However, each of the immobilized laccases retained most of its original activity after ten cycles, which indicates a notable operational stability and reusability of the immobilized enzyme. Compared with other immobilization support, for example green coconut fiber (Cristovao et al., 2011), BNC appears to have more excellent efficiency to retain enzyme activity

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during the repeated use. Good reusability of enzyme can lead to significant reduction of application cost, which is of utmost relevance for the industry (Silva, Zhang, Shen, & Cavaco-Paulo, 2006). In this

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study, low binding forces formed between enzyme and support since laccase was fixed on BNC membranes via only physical adsorption. However, the method of physical adsorption had minimal

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negative impacts on enzyme activity (Lu et al., 2006). Because of low binding force, the reduction of activity with the reuse cycles could be probably ascribed to the loss of laccase in reaction solution, and

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laccase release profiles would be investigated in further study. The residual activity of the four BNC-immobilized laccase after ten consecutive cycles was DHU-ZCY-1 (91.1%) > DHU-ATCC-1 (86.7%) > DHU-ZGD-1 (86.5%) > ATCC 23770 (75.6%), while their average pore size was

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DHU-ZCY-1 (9.7 nm) < DHU-ATCC-1 (11.7 nm) < DHU-ZGD-1 (12.3 nm) < ATCC 23770 (16.5 nm) (Table 2). Usually, a sparser fiber structure would accelerate the loss of enzyme proteins, and the results are consistent with the BET analyses.

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Adsorption/desorption strength of laccase on BNC carriers should depend on the surface properties of BNC and surface charge of the protein. However unlike a single fiber, BNC matrix has a nanofibrous network, therefore enzyme molecules are not only immobilized on the surface of BNC

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fibers, but also entrapped in the water in the cavities formed by BNC fibers, which have been described in our previous study (Chen, Zou, & Hong, 2015). For the enzyme molecules entrapped in BNC cavity or desorbed from BNC fibers, the loss of the enzyme proteins may be closely related to the fiber density because diffusivity of the proteins depends on the fiber density. More fibers in a BNC carrier

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would certainly hinder the diffusion of the proteins to outside of BNC. This is similar to the mechanism of cellulose adsorption chromatography.

Conclusions Bacterial nanocellulose synthesized from four different K. xylinus strains exhibited obvious difference in fiber density, internal spaces structure and remarkably different mechanical properties. All types of BNC membranes could be served as excellent absorbent carrier for enzyme immobilization 9

due to their wide specific surface area. However, different physical property especially the pore size of network caused different effects on laccase immobilization. The enzyme kinetics study of the BNC-immobilized laccases suggests that a properly sparse and larger pore size of BNC membrane could endow a better substrate affinity for the immobilized enzyme. All immobilized enzyme presented a very successful reusability with large residual activity after ten consecutive application cycles. This study shows that the BNC produced by different strains have different effects on laccase immobilization, and an appropriate sparse fiber network of BNC could be served as superior immobilization carriers. Since the change in pH may significantly affect the activity and stability of laccase, therefore, the effect of pH on enzyme activity and reuse stability would be investigated in

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further study.

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Notes：The authors declare no competing financial interest.

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Author Contributions：All authors have contributed to the manuscript preparation, and given approval to the final version of the manuscript.

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Acknowledgements

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We would like to thank Prof. Wankei Wan (University of Western Ontario) for helpful suggestions on polishing the manuscript. Financial supports provided by the Science and Technology Commission of Shanghai Municipality (15520720800), the Fundamental Research Funds for the Central University (2232017A-02), the National Nature Science Foundation of China (51373031), and the Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-03-E00055) are gratefully acknowledged.

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wound dressings. Frontiers in Microbiology, 7, 260.

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A B C

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Fig. 1. Appearance of four different types of BNC from (A) ATCC 23770, (B) DHU-ZCY-1, (C) DHU-ZGD-1 and (D)

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DHU-ATCC-1.

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Surface

Section

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Ratio (%)

2 μm

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MD=35nm SD=16nm

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a

20 10 0

5 μm 0 10 20 30 40 50 60 70 80 90100 Fiber diameter (nm)

B

40 MD=53 nm

SD=15 nm

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20 10 0

5 μm 0 10 20 30 40 50 60 70 80 90100110 Fiber diameter (nm)

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2 μm

Ratio (%)

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25 μm

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5 μm

40

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MD=59 nm SD=15 nm

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2 μm

10

c

5 μm

0 10 20 30 40 50 60 70 80 90100110 Fiber diameter (nm)

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0

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30 20

25 μm

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5 μm

5 μm

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MD=52 nm SD=18 nm

25 μm

D

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Ratio (%)

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5 μm

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2 μm

20

25 μm

5 μm

Fig. 2. SEM images of surface (uppercase letter) and section structure (lowercase letter) of BNC from ATCC 23770 (A, a), DHU-ZCY-1 (B, b), DHU-ZGD-1 (C, c) and DHU-ATCC-1 (D, d). The embedded graphs are magnified photos and fiber diameter distribution, respectively.

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Fig. 3. Tensile stress-strain curves of four types of BNC. The right Y-axial is used for ATCC 23770.

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Fig. 4. BET nitrogen adsorption and desorption isotherms of BNC samples.

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(B)

(A)

p = 0.003

p = 0.008 p = 0.007

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p = 0.026

Fig. 5. Effects of (A) concentration of free laccase and (B) immersing time on enzyme immobilization results. For the study on effect of concentration of free laccase, the immersing time was 24 h. For the study on effect of immersing time, the

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concentration of free laccase was 4 U/mL.

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B

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Fig. 6. (A) Thermal stability of free and BNC-immobilized laccases after 1 h incubation at 70 oC; (B) Initial reaction rates for

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different concentrations of ABTS with free and BNC-immobilized laccases. *The data show significant statistical differences

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between free and BNC-immobilized laccases (p< 0.05).

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Fig. 7. Reuse stability of BNC-immobilized laccases. (A) Data show significant statistical differences than the first circle (*

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BNC-immobilized laccases after the 10th application (* p<0.05, ** p<0.01, # p>0.05).

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p<0.05, ** p<0.01, *** p<0.001, n = 3). Legends show the use cycles. (B) Statistical analyses of the residual activity of the four

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Table 1 Mechanical properties of BNC from four different strains. The data in each column marked with same superscripts means that the difference between them is not significant (p>0.05).

Strains

Fracture stress (Mpa)

Young's modulus (Mpa)

Breaking elongation (%)

ATCC 23770

0.12 ± 0.02

0.34 ± 0.04

40 ± 5*

DHU-ZCY-1

1.64 ± 0.12*

12.76 ± 2.46*

20 ± 3

DHU-ZGD-1

1.23 ±

0.21*

DHU-ATCC-1

2.44 ± 0.11

8.95 ±

1.17#

27 ± 4 38 ± 4*

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10.80 ± 2.11*,#

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Table 2 Calculated specific surface area, total pore volume and average pore size of BNC from four strains. The data in each column marked with same superscripts means that the difference between them is not significant (p>0.05).

Specific surface area (m²/g)

Total pore volume (cm3/g)

Average pore size (nm)

ATCC 23770

125.5±4.4

0.32±0.04

16.5±1.7

DHU-ZCY-1

71.8±6.1

0.12±0.02

9.7±1.6*

DHU-ZGD-1

90.6±4.2*

0.23±0.03*

12.3±2.1#

DHU-ATCC-1

87.1±6.3*

0.18±0.02*

11.7±0.8*,#

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Table 3 Kinetic parameters of free and immobilized laccase. The data in each line marked with same superscripts means that the difference between them is not significant (p>0.05).

ATCC 23770

DHU-ZCY-1

DHU-ZGD-1

DHU-ATCC-1

Km (mM)

0.29±0.06

0.37±0.03

0.53±0.06

0.42±0.03*

0.46±0.04*

Vmax (mM min-1)

9.64±0.21

4.9±0.16

2.74±0.13

3.74±0.32#

3.55±0.17#

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Free laccase

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