Biodegradation of polyvinyl alcohol using cross-linked

International Journal of Biological Macromolecules 124 (2019) 10–16

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Biodegradation of polyvinyl alcohol using cross-linked enzyme aggregates of degrading enzymes from Bacillus niacini Hongjie Bian a, Mengfei Cao a, Huan Wen a,b, Zhilei Tan b, Shiru Jia b,⁎, Jiandong Cui a,b,⁎⁎ a

Research Center for Fermentation Engineering of Hebei, College of Bioscience and Bioengineering, Hebei University of Science and Technology, 26 Yuxiang Street, Shijiazhang 050000, PR China Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China

b

a r t i c l e

i n f o

Article history: Received 8 September 2018 Received in revised form 3 November 2018 Accepted 20 November 2018 Available online 22 November 2018 Keywords: Biodegradation Cross-linked enzyme aggregates Immobilization enzyme Bacillus niacini strain Polyvinyl alcohol

a b s t r a c t In this study, polyvinyl alcohol (PVA)-degrading bacteria were screened from sludge samples using PVA as a sole source of carbon. A novel strain was obtained and identified as Bacillus niacini based on the analysis of a partial 16S rDNA nucleotide sequence and morphological characteristics. PVA-degrading enzyme (PVAase) from Bacillus niacini was immobilized as cross-linked enzyme aggregates (CLEAs) via precipitation with ammonium sulfate followed by glutaraldehyde cross-linking. The effects of precipitation and cross-linking on PVAase-CLEAs activity were investigated and characterized. 70% ammonium sulfate and 1.5% glutaraldehyde were used for precipitation and 1-h cross-linking reaction. The activity recovery of PVAase-CLEAs was approximately 90% starting from free PVAase, suggesting non-purification steps are required for extended use. No significant differences in optimum pH and temperature values of the PVAase were recorded after immobilization. The PVAase-CLEAs showed a ball-like morphology and enhanced PVA degradation efficiency in comparison with the free PVAase in solution. Furthermore, the PVAase-CLEAs exhibited excellent thermal stability, pH stability and storage stability compared to free PVAase. The PVAase-CLEAs retained about 75% of initial PVAase activity after 4 cycles of use. These results suggest that this CLEA is potentially usable for PVA degradation in industrial applications. © 2018 Published by Elsevier B.V.

1. Introduction Poly(vinyl alcohol) (PVA) is a water soluble synthetic polymer, and used widely for pharmaceutical products, paper coating, and adhesives and textile industries [1,2]. Moreover, PVA has been used as an immobilization carrier for enzymes and biomedical application [3,4]. However, PVA is difficult to degrade, and tends to accumulate and eventually cause environmental problems [5,6]. Thus, there is a significant pollution problem caused by PVA. In the past few years, biodegradation is viewed as a promising alternative for efficient PVA removal. Various studies were carried out to investigate the biodegradability of PVA. Suzuki et al. first found that PVA could be removed by a bacterial strain (Pseudomonas O-3) when PVA was used as the sole source of carbon and energy [7]. The biodegradation behaviour of PVA by Flammulina velutipes was also observed [8]. Furthermore, the white-rot fungus (Fomitopsis pinicola) was reported to degrade PVA [1]. In addition, the

⁎ Corresponding author. ⁎⁎ Correspondence to: J. Cui, Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China. E-mail addresses: [email protected] (S. Jia), [email protected] (J. Cui).

https://doi.org/10.1016/j.ijbiomac.2018.11.204 0141-8130/© 2018 Published by Elsevier B.V.

capacities of many PVA-degrading strains for PVA utilisation are often reliant upon cooperation with other bacteria supplying them with various growth factors. For example, the Pseudomonas sp. strain VM15A could produce the key growth factor pyrroloquinoline quinone (PQQ) which was necessary for PVA biodegradation with the strain Pseudomonas sp. VM15C [9]. Similarly, Sphingomonas sp. was able to degrade PVA only in the presence of PQQ and another growth factor produced by Rhodococcus erythropolis strain [10]. In fact, the PVA-biodegradation in such microorganism relied on PVA-degrading enzyme (PVAase), and the PVAase systems involved in the combination of PVA dehydrogenase (PVADH) and oxidized PVA hydrolase [2,9]. Generally, the enzymatic degradation of PVA is considered an enabling technology because it is a valuable, cost-effective and environmentally friendly method [11,12]. Until now, many studies on the enzymatic degradation of PVA have been focused on new microbial isolates and PVAase purification and characterization [13,14]. Furthermore, purification process can also lead to enzyme inactivation and increase production costs. Especially, the industrial application of PVAase is still hampered due to a lack of long-term operational stability and the difficulty in recycling PVAase [14,15]. Therefore, an efficient enzymatic degradation process of PVA is highly desirable. Enzyme immobilization techniques are well recognized as a common way to improve stability and get a simpler recovery of the enzyme

H. Bian et al. / International Journal of Biological Macromolecules 124 (2019) 10–16

[15,16]. Sometimes, the immobilization protocol (including support, enzyme modification and immobilization conditions) can be designed to couple the immobilization of the enzyme and its purification in just one process preferably [16,17]. Among various immobilization methods, the cross-linked enzyme aggregates (CLEAs) methodology have been recently proposed as a rapid, gentle and low-cost enzyme immobilization method. The preparation of CLEAs essentially combines a two-step process (primary purification and immobilization) into a single operation [18]. Moreover, CLEAs can even take the crude enzyme extract from fermentation broth and produce the immobilized enzyme in one simple operation. For example, cross linked invertase aggregates (invertase-CLEAs) from crude enzyme soluble solution was prepared. 100% activity recovery was achieved in invertase-CLEAs with enhanced thermal and acidic condition stabilities [19]. Cross-linked enzyme aggregates of cutinase (cut-CLEA) directly from the solid state fermentation (SSF) crude broth were prepared. The resultant cut-CLEA showed better thermal, solvent, detergent and storage stability, making it more elegant and efficient for industrial biocatalytic process [20]. In addition, the crude extract of Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was directly subjected to enzyme immobilization using CLEAs method. The obtained HheC-CLEAs retained N90% activity of the free enzyme, and maintained N70% activity after 10 reusability cycles [21]. These advances of CLEAs make it possible to prepare efficient biocatalyst by coupling the primary purification and the immobilization of industrial enzymes in a single step. However, to the best of our knowledge, no report of PVA biodegradation by CLEAs has been found in the literature. In this study, we screened Bacillus niacini capable of producing intracellular PVAase from activated sludge samples in a waste water treatment plant, extracted crude PVAase from Bacillus niacini and optimized preparation conditions of cross-linked PVAase aggregates (PVAase-CLEAs) by using crude PVAase. Furthermore, the properties of the prepared PVAase-CLEAs such as optimum temperature and pH, thermal stabilities, storage stability and reusability were investigated. We show that PVAase-CLEAs have better application potential than free crude PVAase in the biodegradation of PVA. 2. Materials and methods 2.1. Isolation and culture of PVA-degrading strains Activated sludge sample was harvested from the watercourse of waste water treatment (Shijiazhuang, China), and used as a source of the microorganisms. One gram of an air-dried sludge sample was added to 10 mL of 0.9% (w/v) sterile saline. Then, the samples were incubated at 30 °C for 24 h with 150 rpm agitation on a shaking incubator. After centrifugation at 10000 ×g for 10 min, 0.1 mL of the supernatant was added to 0.9 mL of sterile saline, and a serial dilution (10−1– 10−6) was prepared. About 0.1 mL of each dilution was spread on screening agar plates using PVA as the sole carbon source and incubated at 30 °C for 48 h. 8 mL I2-KI was added to the plates and reacted for 20 min in the dark. The promising strains capable of degrading PVA were selected on the basis of the formation of a clear and large discolored zone. The seed inocula of isolated strains were cultured in Luria-Bertani medium broth (0.5% yeast extract, 1% tryptone, 1% NaCl) at 30 °C with 140 rpm for 20 h. Subsequently, 5 mL of inoculum culture was added to 45 mL of the screening medium in a 250 mL Erlenmeyer flask. The screening medium contained the following per liter: 1.0 g PVA, 1 g yeast extract, 1.0 g (NH4)2SO4, 0.5 gKH2PO4·3H2O, 0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.1 g NaCl, with the pH adjusted to 7.0. The culture was incubated at 30 °C in a rotary set at 200 rpm for 3 days.

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cells were diluted and observed by a scanning electron microscope (S4800-I; Hitachi Research and Development Corp., Japan). 2.3. 16S rDNA gene amplification and sequence analysis The 16S rRNA gene sequence from the isolated colonies was amplified using genomic DNA as a template for polymerase chain reaction (PCR) [22]. The universal primer 27f (5′-AGAGTTTGATCCTGGCTCAG3′) and 1492r (5′-CGGTTACCTTGTTACGACTT-3′) were used for PCR in a thermal cycler. The amplified PCR product was checked by electrophoresis in a 1.5% (w/v) agarose gel, and then confirmed by commercial DNA sequencing. The 16S rRNA gene sequence identification was estimated by using the BLASTN facility of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/). 2.4. Extraction and preliminary purification of PVAase The PVA-degrading strain cells were incubated at 30 °C in a rotary set at 200 rpm for 72 h. The cultured cells were harvested by centrifugation (6000 ×g, 10 min) and resuspended in a potassium phosphate buffer (10 mM, pH 7.5). The cells were disrupted by sonication and the supernatant was obtained by centrifugation (10,000 ×g 15 min). And then, solid ammonium sulfate was added to the supernatant to 80% saturation. The precipitate was collected and dissolved in phosphate buffer (10 mM, pH 7.5). This cell-free extract was utilized as a crude PVAase for the preparation of CLEA. Furthermore, to determine whether PVAase is an intracellular enzyme or an extracellular enzyme, we examined the activity of PVAase enzyme in fermentation broth and cell-free extract. 2.5. Preparation of PVAase-CLEAs PVAase-CLEA was prepared using the crude PVAase alone. The different precipitants (ammonium sulfate, methanol, ethanol, isopropanol or acetone) were added dropwise to 1 mL the crude PVAase solution (about 4 mg/mL), under gentle stirring at 30 °C for 1 h. The different concentrations of glutaraldehyde (0.5%, 1%, 1.5%, 2%, 2.5%) were added in the mixture for CLEAs. Then, mixture was kept at 4 °C for 1 h with constant shaking at 200 rpm and then centrifuged at 10000 ×g for 10 min. Afterwards, The precipitates were recovered and washed with the relevant solvents until no more activity was determined in the supernatant. The resulted CLEA was washed three times with phosphate buffer (10 mM, pH 7.5). Finally, CLEA preparation was kept in the same buffer at 4 °C prior to use. 2.6. PVAase activity assay and PVA degradation efficiency PVA-degrading enzyme activity was monitored by the absorbance at 645 nm by the modification of the procedure [11]. A reaction mixture contained 1 mL of crude enzyme solution and 100 μL of 0.1% PVA dissolved in 0.1 M phosphate buffer (pH 8.0), and was incubated at 30 °C for 3 h. One unit PVAase activity was defined as an absorbance decrease of 0.001 per minute at 645 nm. For PVA degradation efficiency, 1 g/L of a standard PVA solution was diluted in a solution containing 0.5 mL of distilled water, 1 mL PVAase example, 0.75 mL of 4% boric acid and 0.15 mL of I2-KI (12.7 g/L I2 and 25 g/L KI), and was incubated at 30 °C for 3 h. The PVA concentration was finally analyzed on a UV spectrophotometer at 645 nm, and calculated on the basis of the calibration curve. A calibration curve was linear from 0 to 100 μg/mL. PVA degradation efficiency is percentage of degraded PVA number (concentration, g/L) and initial PVA number (1 g/L), and was calculated by using Eq. (1). The PVAase activity recovery in CLEAs was calculated as given in Eq. (2) [25].

2.2. Morphological characteristics The features of colony morphologies on the plates, that is, color and texture, were observed by Gram's method. The PVA-degrading strain

PVA degradation efficiency Total number of PVA‐residual number of PVA ¼ 100% Total number of PVA

ð1Þ

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cycle, respectively. The reusability was defined as the ratio of the activity for the PVAase-CLEAs after recycling to its initial activity.

Activity recovery Total activity of CLEAsðUÞ ¼ Total free enzyme activity used for CLEA productionðUÞ 100%

ð2Þ

3. Results and discussion 3.1. Isolation and identification of PVA-degrading strains

2.7. Optimal conditions for PVAase activity The optimal temperature of the PVAase-CLEAs and free PVAase were determined by adding the enzyme samples into the substrate solution (pH 7.0) at different temperatures (30–70 °C) for 3 h, and the optimal pH of the PVAase-CLEAs and free PVAase was determined by adding the enzyme samples into the substrate solution of different pH 5, 6, 7, 8 and 9 at 30 °C for 3 h. The results for optimum temperature and pH were given in relative form with the highest value being 100% activity. 2.8. Stability of PVAase-CLEAs and free PVAase Thermal stability of PVAase-CLEAs and free PVAase was examined by incubating enzyme samples at 0.1 M phosphate buffer (pH 7.0) without substrate at 50 °C for 1–5 h before measuring activity. The pH stability of PVAase-CLEAs and free PVAase was tested in the system over a pH range between 3 and 11 for 2 h at 30 °C, respectively. The storage stability was determined as follows: PVAase samples were stored at 25 °C in 0.1 mM phosphate buffer (pH 7.0). The activities of PVAase samples were determined in a certain storage time. Herein, the residual activities of PVAase samples were defined as the ratio of the activities for the PVAase samples after treatment to its initial activity, and calculated by taking the initial activity of the enzyme as 100%. The reusability of PVAase-CLEAs was assessed by performing several consecutive operating cycles using 0.1% PVA solution as the substrate. The PVAase-CLEAs were collected by centrifugation, and washed with 0.1 M phosphate buffer (pH 7.0) solution after each batch and then added to the next

Several bacterial strains that are capable of PVA-degrading were isolated to single colonies from sludge samples by using PVA agar plates containing a solution of iodine and boric acid. To obtain stable strains, the strains were subcultured nearly three times, and bacterial colonies surrounded with clear and large discolored zone were selected based on cell growth and their PVA biodegradation capacity (Fig. 1A). Then, the ability of these strains to degrade PVA was examined by shake flask experiments. A novel strain (PVA-6) that could degrade 80% of PVA was obtained. The colonies appeared moist, smooth, white, and round (Fig. 1B). The isolate PVA-6 was Gram-positive and rod-shaped (Fig. 1C and D). Besides, genomic DNA of strain PVA-6 was extracted (Fig. 2A). Then, an approximately 1300 bp sized fragment of the 16S rDNA gene sequence of strain PVA-6 was amplified and sequenced. The fragment band was confirmed by electrophoresis after performance of PCR (Fig. 2B). The alignments of the sequences were carried out by the ClustalX software to determine the sequence consensus. The obtained sequences were subjected via Internet using the BLAST software for comparison with the homologous sequences contained in the data bank (GenBank). The 16S rDNA gene of the strain PVA-6 showed the closest match to Bacillus niacini with a homology of 99%. Therefore, it could be identified as a Bacillus niacini. 3.2. Optimization of preparation conditions of the PVAase-CLEAs Generally, the precipitant has an important effect on the activity recovery in CLEAs as it causes physical aggregation of enzyme molecules

Fig. 1. Strain morphology of the isolated colonies (A) Screening agar plates, (B) Luria broth agar plates, (C) Gram stain image, (D) SEM image.


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Fig. 2. Agarose gel electrophoresis images of (A) genomic DNA, (B) PCR product.

into supramolecular structures [23,24]. Consequently, it is necessary to screen a number of precipitants for CLEAs preparation. Fig. 3A showed effects of different precipitants on PVAase activity recovery. Compared

to methanol, ethanol, isopropanol and acetone, the PVAase activity recoveries were relatively high when ammonium sulfate was used as precipitants. The results might be due to hydrophobic interactions between

Fig. 3. Effect of precipitants on PVAase activity recovery. (A) different precipitants, (B) ammonium sulfate with different concentrations. (C) effect of glutaraldehyde concentration, (D) effect of cross-link time.

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the organic solvent and the nonpolar groups of the enzyme, which causing denaturation of enzymes [25]. Moreover, the best results (recovery of about 90% of the initial PVAase activity is 2 U/mg) were obtained using 70% saturated ammonium sulfate as precipitant (Fig. 3B). In contrast, a small amount of saturated ammonium sulfate solution is not enough to precipitate enzymes. However, the excessive adding of saturated ammonium sulfate solution can form bigger particle, which influences mass transfer of the substrates to contact with the inner enzymes of the particles [26]. Several reports suggested that ammonium sulfate has been most successfully applied for precipitation during CLEA preparation. Several reports on CLEAs such as CLEAs of Candida rugosa lipase [27], penicillin G acylase [28], alpha amylase [29], laccase [30], etc. showed ammonium sulfate acting as the best precipitating agent. Therefore, 70% ammonium sulfate was used as the optimized precipitating agent for CLEA preparation. Besides, the concentration of cross linker is also key parameter in determining the morphology and hence catalytic properties of CLEAs. The most commonly used cross linker is glutaraldehyde. The different concentrations of glutaraldehyde (0.5%, 1%, 1.5%, 2%, and 2.5%) were used to prepare with the same concentration of free PVAase (4 mg/mL) at the same preparation conditions. Effects of glutaraldehyde concentration on activity recovery of PVAase-CLEAs were shown in Fig. 3C. It is observed that the activity recovery increased with an increase in glutaraldehyde concentration to a maximum value and then decreased with further increase in glutaraldehyde concentration. The suitable concentration of glutaraldehyde for crosslinking was found to be 1.5%. Generally, lower glutaraldehyde concentration causes sufficient cross linking, affording very little insoluble aggregates that would result in operationally unstable CLEAs releasing free enzyme into the reaction medium [24]. In contrast, when glutaraldehyde concentration is high, CLEA with a strong diffusion resistance and rigidification of the enzyme molecule occurs due to excessive cross-linking [31,32]. Similarly, a short cross linking time resulted in inadequate cross linking, leading to poor activity recovery of PVAase-CLEAs (Fig. 3D). On the contrary, prolonged cross linking time could restrict the enzyme flexibility, abolishing enzyme activity due to more intensive cross linking [33–35]. The highest activity recovery could be obtained when 1 h cross-linked time were adopted.

Fig. 4. SEM images of PVAase-CLEAs. (A) 30,000× magnification, (B) 100,000× magnification.

compared with free PVAase in the determination of pH range. Moreover, the PVAase-CLEAs retained its most activity under acidic conditions (pH b 6) while free PVAase almost lost activity. Besides, we further determined PVA degradation efficiency of fermentation broth, crude PVAase, and PVAase-CLEAs, respectively. The results showed that the PVA degradation efficiency of PVAase-CLEAs (68.5%) was 1.3 times higher than that of crude PVAase (51.2%) which were prepared with the same concentration of free PVAase (Fig. 6). Moreover, we compared the activity of PVAase enzyme in fermentation broth, cell-free extract, and PVAase-CLEAs. The results showed that low PVA degradation efficiency was observed in the fermentation broth, indicating that PVAase exists in cells. 3.5. Stability of PVAase-CLEAs In order to evaluate the stabilities of PVAase-CLEAs under different conditions, the activities of PVAase-CLEAs at high temperature and a series of pH were detected. The results showed that the PVAase-CLEAs exhibited the higher stability against high temperature than free PVAase after incubating at 50 °C for 5 h. Free PVAase almost lost activity, whereas the activity of PVAase-CLEAs still remained 70% of initial

3.3. Scanning electron microscopy (SEM) Several reports showed that the shape and size of CLEAs have a significant effect on the CLEAs activity [36,37]. There are usually two types of CLEAs' structure that can be observed under SEM [38]. One is spherical structure (Type 1), the other is a less-structured form (Type 2). In this study, the shape and surface morphology of the CLEA were examined by SEM. Fig. 4 showed the SEM micrographs of PVAase- CLEA at a magnification of 30,000× and 100,000×. It can be clearly seen that spherical CLEAs formed (Type 1), and were relative uniform morphologies with ball- like shape (Fig. 4A, B). The spherical morphology of CLEAs could minimize the diffusion effect within the catalyst, and maintain the structural stabilization, which is beneficial to improving activity recovery of PVAase-CLEAs. 3.4. Properties of PVAase-CLEAs The effect of temperature on the activity of free PVAase and PVAaseCLEAs preparations are given in Fig. 5A. The results showed that the optimum temperature both PVAase and PVAase-CLEAs were at 40 °C. The free PVAase rapidly began to lose its activity after 60 °C, and did not show any activity at 70 °C which was completely inactive. However, the PVAase-CLEAs retained its activity up to 70 °C. It seems that immobilization elevates the tolerance of the enzyme against high temperatures. The results are consistent with the previous reports [39,40]. In addition, the activities of free PVAase and PVAase-CLEAs were measured at various pH values and both showed maximum activity around pH 7.0 (Fig. 5B). However, PVAase-CLEAs revealed broader enzyme activity

Fig. 5. The optimal temperature (A) and pH (B) curves of PVAase-CLEAs.


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initial activity (Fig. 7B). These results strongly suggest that the CLEA methods significantly improved the thermal and pH stabilities of the PVAase. The increased pH stability could be due to the interactions between basic residues of enzyme and glutaraldehyde, which caused the change in acidic and basic amino acid side chain ionization in the microenvironment around the enzyme active site [42].

3.6. The reusability and storage of the PVAase-CLEAs

Fig. 6. Comparison of PVA degradation efficiency of different PVAase.

activity (Fig. 7A). This result may be explained that the conformational flexibility/stretchability of PVAase was decreased due to cross-linking of the aggregated PVAase molecules, and thus, increasing the thermal tolerance of the PVAase. Therefore, enzyme required higher activation energy to attain the proper functional conformation [41]. Similar results were obtained from the pH variance study on the free PVAase and PVAase-CLEAs. PVAase-CLEAs retained higher activity than free PVAase at extreme pH. Free PVAase almost lost activity at acidic pH (pH 3.0) or alkaline pH environment (pH 8–11), while retained about 40% of their

In addition, storage stability of free PVAase and PVAase-CLEAs was determined. As shown in Fig. 7C, free PVAase lost activity after 6 days, whereas the PVAase-CLEAs still retained 60% of their initial activity, demonstrating the PVAase-CLEAs had good resistance to room temperature storage. Furthermore, the reusability of PVAaseCLEAs was also investigated because the reused efficiency of immobilized enzymes represents one of the essential factors for their application as biocatalysts. The results of the reusability of PVAase-CLEAs are shown in Fig. 7D. The PVAase-CLEAs remained 74% of its initial activity after four cycles. However, the PVAaseCLEAs only retained about 49% of its initial activity after five recycles. Similar results were also observed in previous reports [43,44]. These results indicate that the PVAase-CLEAs can be reused without appreciable loss of activity. The lower recoveries of catalytic activity after five cycles could be due to the fact that centrifugation might result in increased clumping due to the low resistance to compression of CLEAs, which would hamper their resuspension in solution and so reduce the catalytic efficiency [45,46].

Fig. 7. Stability of PVAase-CLEAs (A) thermostability, (B) pH stability, (C) storage stability, (D) reusability.

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4. Conclusions In the present study, a novel PVA-degrading bacterium was isolated from sludge samples using PVA as a sole source of carbon, and identified as Bacillus niacini on the basis of 16S rDNA gene sequencing. The PVAase-CLEAs was prepared by using crude PVAase. The optimal ammonium sulfate and glutaraldehyde concentrations, and crosslinking time were determined as 70%, 1.5% and 1 h, respectively. The newly synthesized PVAase-CLEAs presented a ball-like morphology and enhanced degradation efficiency in comparison with the free PVAase in solution. Furthermore, the thermal and storage stabilities of PVAase-CLEAs were comparably higher than those of free PVAase. Additionally, the PVAase-CLEAs had excellent reusability, which was supported by the observation that the CLEAs could still retain N50% of their original activity after successive re-use for 5 batches. High efficiency of the PVAaseCLEA in PVA degradation is an advantage and makes it a potential candidate for industrial applications. Acknowledgements This work is partially supported by the National Natural Science Foundation of China (project no. 21676069). Dr. J. D. Cui also thanks supports from the Natural Science Foundation of Hebei Province, China (project no. B2018208041), the Program for Hundreds of Outstanding Innovative Talents in Hebei province (III) under the grant number of SLRC2017036, and the Foundation (No. 2016IM001) of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (Tianjin University of Science and Technology). References [1] S. Tsujiyama, A. Okada, Biodegradation of polyvinyl alcohol by a brown-rot fungus, Fomitopsis pinicola, Biotechnol. Lett. 35 (2013) 1907–1911. [2] N.B. Halima, Poly(vinyl alcohol): review of its promising applications and insights into biodegradatiion, RSC Adv. 6 (2016) 39823–39832. [3] R. Surkatti, M.H. El-Naas, Biological treatment of wastewater contaminated with pcresol using Pseudomonas putida immobilized in polyvinyl alcohol (PVA) gel, J. Water Process Eng. (1) (2014) 84–90. [4] R.K. Balasubramanian, Antibacterial application of polyvinylalcohol-nanogold composite membranes, Colloids Surf., A 455 (2014) 174–178. [5] M.N. Kim, M.G. Yoon, Isolation of strains degrading poly(vinyl alcohol) at high temperatures and their biodegradation ability, Polym. Degrad. Stab. 95 (2010) 89–93. [6] A. Stoica-Guzun, L. Jecu, A. Gheorghe, I. Raut, M. Stroescu, M. Ghiurea, M. Danila, I. Jipa, V. Fruth, Biodegradation of poly(vinyl alcohol) and bacterial cellulose composites by Aspergillus niger, J. Polym. Environ. 19 (2011) 69–79. [7] T. Suzuki, Y. Ichihara, M. Yamada, K. Tonomura, Some characteristics of Pseudomonas O-3 which utilizes polyvinyl alcohol, Agric. Biol. Chem. 37 (1973) 747–756. [8] T. Tsujiyama, T. Maoka Nitta, Biodegradation of polyvinyl alcohol by Flammuli velutipes in an unsubmerged culture, J. Biosci. Bioeng. 112 (2011) 58–62. [9] M. Shimao, T. Tamogami, S. Kishida, S. Harayama, The gene pvaB encodes oxidized polyvinyl alcohol hydrolase of Pseudomonas sp. strain VM15C and forms an operon with the polyvinyl alcohol dehydrogenase gene pvaA, Microbiology 146 (2000) 649–657. [10] T. Vaclavkova, J. Ruzicka, M. Julinova, R. Vicha, M. Koutny, Novel aspects of symbiotic (polyvinyl alcohol) biodegradation, Appl. Microbiol. Biotechnol. 76 (2007) 911–917. [11] G. Du, L. Liu, Z. Song, Z.Z. Hua, Y. Zhu, J. Chen, Production of polyvinyl alcohol degrading enzyme with Janthinobacterium sp. and its application in cotton fabric desizing, Biotechnol. J. 2 (2007) 752–758. [12] H.Z. Zhang, Influence of pH and C/N ratio on poly (vinyl alcohol) biodegradation in mixed bacterial culture, J. Polym. Environ. 17 (2009) 286–290. [13] B. Tang, X. Liao, D. Zhang, M. Li, R. Li, K. Yan, G. Du, J. Chen, Enhanced production of poly(vinyl alcohol)-degrading enzymes by mixed microbial culture using 1,4butanediol and designed fermentation strategies, Polym. Degrad. Stab. 95 (2010) 557–563. [14] D.X. Jia, Y. Yang, Z.C. Peng, D.X. Zhang, J.H. Li, L. Liu, G.C. Du, J. Chen, High efficiency preparation and characterization of intact poly(vinyl alcohol) dehydrogenase from Sphingopyxis sp.113P3 in Escherichia coli by inclusion bodies renaturation, Appl. Biochem. Biotechnol. 172 (2014) 2540–2551. [15] D. Brady, J. Jordaan, Advances in enzyme immobilisation, Biotechnol. Lett. 31 (2009) 1639–1650. [16] C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Potential of different enzyme immobilization strategies to improve enzyme performance, Adv. Synth. Catal. 353 (2011) 2885–2904. [17] O. Barbosa, C. Ortiz, A. Berenguer-Murcia, R. Toores, R.C. Rodrigues, R. FernandezLafuente, Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts, Biotechnol. Adv. 33 (2015) 435–456.

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