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Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides
Bioresource Technology 115 (2012) 152–157
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Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides Sujata Sinha a,⇑, Sanjay R. Dhakate b, Pankaj Kumar b, R.B. Mathur b, Pushplata Tripathi c, Subhash Chand a,⇑ a
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Physics and Engineering of Carbon, Division of Materials Physics and Engineering, National Physical Laboratory (CSIR), Dr. K.S. Krishnan Marg, New Delhi 110012, India c School of Sciences, Indira Gandhi National Open University, Maidan Garhi, New Delhi 110068, India b
a r t i c l e
i n f o
Article history: Available online 2 December 2011 Keywords: Nanofibers Immobilization Chitosan Chitooligosaccharide
a b s t r a c t Polyacrylonitrile nanofibrous membranes (PANNFM) were prepared by electrospinning from 10 wt.% of PAN solution and its surface was modified by amidination reaction. A new chitosan degrading enzyme from Aspergillus sp. was covalently immobilized on PANNFM. Immobilization efficiency of 80% was achieved by activating PANNFM surface for 30 min followed by 2 h treatment with enzyme solution. The optimum temperature and pH for immobilized enzyme were 50 °C and 5.8, respectively. The immobilized chitosanase retained >70% activity after ten repeated batch reaction and could be stored up to 60 days at 4 °C with minor loss in activity. Chitosan hydrolysis using different substrates were studied using immobilized chitosanase in batch conditions. Continuous selective production of chitooligosaccharides (dimer to hexamer) by changing the temperature was achieved by PANNFM-chitosanase. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Chitosanases are group of hydrolytic enzymes which act on chitosan and can be of great industrial application due to enormous availability of chitin and chitosan in nature (Somashekar and Joseph, 1996). Chitosan is deacetylated chitin; most abundant polymer after cellulose and due to its biological properties and biocompatibility (Kim, 2010) has great potential to be applied in different areas like agricultural, industrial, biomedical, etc. Poor solubility of chitosan makes it difficult to be used at large scale. Interestingly, low molecular weight chitosan (LMWC) and chitooligosaccharides (COS) are readily soluble in water due to free amino group in D-glucosamine (Jeon et al., 2000) and show excellent biological activities (Kim and Rajapakse, 2005). Monomers of chitosan (D-glucosamine and N-acetyl D-glucosamine) has biomedical applications like arthritis treatment, dentistry, wound healing, etc. and has been studied as food supplement (Kajimoto et al., 1998). Chitosan can be depolymerized either by chemically or enzymatically. However, chemical method is avoided because of low yield, toxicity, pollution, high cost and non fitness for human consumption. Use of chitosan degrading enzyme is limited at industrial scale due to its high cost and limited availability (Kim and Rajapakse, 2005). Chitosanase enzymes have been found in large number of microbes including bacteria and fungi (Somashekar and ⇑ Corresponding authors. Tel.: +91 11 2659 1004; fax: +91 11 2658 2282. E-mail addresses: [email protected] (S. Sinha), [email protected], [email protected] (S. Chand). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.101
Joseph, 1996) which can be used in production of COS, but most of these enzymes have low substrate specificity and enzyme is inducible in nature (Shimosaka et al., 1995). In order to find a novel microbial chitosanase, we screened microbes from soil sample rich in fish waste and isolated partially purified enzyme was immobilized on electrospun polyacrylonitrile nanofibrous membranes (PANNFM). Enzyme immobilization improves reusability and has other advantages like scale up, ease in recycling, continuous operation and product purification. Performance of immobilized enzyme generally depends on choice of matrix and method of immobilization. One dimensional nanofibers have extremely high surface area to volume ratio and excellently interconnected pore structure. The interconnectivity of electrospun supports circumvent the mass transfer limitations and have been used as immobilization matrix for a number of enzymes (Wang et al., 2009). NFMs from natural polymers are generally less stable chemically and mechanically than those from synthetic polymers. PAN is a polymer with good stability and mechanical properties (Kim et al., 2005). Derivatives of PAN have also been used for enzyme immobilization with an aim to introduce functional groups into the polymer backbone due to the inertness and hydrophobicity of acrylonitrile monomer (Ye et al., 2006). In this study, PANNFM was used for immobilization of chitosanase after activation of surface by amidination reaction and were studied for residual activity, reusability, optimum pH and temperature. PANNFMchitosanase was used for batch hydrolysis of different chitosan substrates and selective production of glucosamine and COS was achieved.
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2. Methods
2.5. Immobilization of chitosanase enzyme
2.1. Materials
All the immobilization steps were carried out by cutting the PANNFM into standard pieces and weighed. Adsorption of enzyme was done by adding the enzyme solution (10 ml of 1 mg/ml) to NFM kept in 100 ml flask for 2 h with mild shaking. NFM surface was activated by placing it in absolute ethanol and bubbling with 1 N solution of hydrogen chloride (6–7 ml) to produce the corresponding imidoester derivatives, a method followed by Li et al. (2007) for lipase immobilization on PAN. NFMs were washed with acetate buffer and were treated with chitosanase enzyme solution in 0.1 M acetate buffer solution (pH 5.5). Flask was put for shaking (100 rpm) at room temperature for 2 h. After the immobilization reaction, the membrane was removed from the solution and washed with acetate buffer several times to remove any unbound enzyme. NFM bound enzyme was lyophilized and was stored till further use.
Commercial chitosan from crab shell, average molecular weight 290 kDa, 93% N-deacetylated (DAC); were kind gift from Marine Chemicals, Chennai, India. Chitosan from shrimp shell (>75% deacetylated) and PAN powder were procured from Sigma–Aldrich, Germany and N,N-Dimethylformamide (DMF) was purchased from Fisher Scientific, India. Glucosamine hydrochloride and Chitin were purchased from Hi-media. All other chemicals were procured from SRL, Mumbai, India and were of analytical grade. 2.2. Preparation of PANNFM by electrospinning The PAN solution (10 wt.%) was prepared in DMF by mixing it in multi frequency ultrasonic bath (Life Care Equipment Pvt. Ltd., Mumbai, India) for one hour at 50 °C. Continuous stirring for 15–20 h was done to get uniformly mixed solution and was spun using electrospinning unit (ESPIN-NANO). Conditions for electrospinning were as follows: needle orifice: 0.55 mm, rotating cylindrical collector diameter: 30 mm, voltage applied: 5–20 kV, rotational speed of the collector: 2000 rpm, distance between tip to collector: 20 cm and flow rate of solution 0.2 ml/h which was maintained using computer control program. 2.3. Screening and isolation of chitosanase producing microbial strain from soil
2.6. Analytical methods Surface morphology of NFM before and after immobilization was studied by scanning electron microscope (SEM, Zeiss-EVO, MA-10, variable UK). The electrospun PANNFM, amidinated or surface modified PANNFM, PANNFM-chitosanase were characterized by Fourier transform infra red spectrometer (FTIR Nicolet 5700). Small amounts of samples were separately mixed with KBr and prepared pellet was used for FTIR spectra.
2.7. Chitosanase assay Chitosan minimal salt medium was used for screening and isolation. Soil samples, taken from marine waste dumping area in local market (Chittaranjan Park, New Delhi, India) was weighed, suspended in sterile water and serial dilutions were prepared. Plating was done on chitosan minimal salt agar medium. Composition of medium was as follows: pH 6.8, colloidal chitosan 1% (w/v), KH2PO4 (0.15 g/l), K2HPO4 (0.35 g/l), MgSO45H2O (0.25 g/l), FeSO47H2O (0.005 g/l), ZnSO4 (0.001 g/l), MnCl2 (0.001 g/l) and agar (2%) was added for making plates. Microbial colonies grown on the surface of agar plates were transferred to chitosan broth. Composition of chitosan broth medium was as follows (g/l): KH2PO4, 2.0; K2HPO4, 1.0; MgSO47H2O, 0.5; NaCl, 0.5; CaCl2, 0.1; yeast extract 0.5 and chitosan 15; the medium was adjusted to pH 6.0 with the help of acetic acid (1 N). Inoculated chitosan broth medium, after incubation for 6–7 days was centrifuged to remove microbial growth and supernatant was used for enzyme assay. Microbes showing good chitosanase (endo and exo) activities were selected and used for further enzyme production. 2.4. Preparation of partially purified chitosanase Microbial cultures, stored on agar slants at 4 °C were scrapped off and washed out with sterile distilled water. One milliliter of microbial suspension was inoculated into a 250 ml flask containing 50 ml of the medium and incubated on a rotary shaker at 200 rpm for 3–4 days at 32 °C. The composition of the medium (pH 5.6) was same as that of chitosan broth. Finally, the cells of the culture broth were removed from the medium by centrifugation at 7500 rpm for 15 min at 4 °C and the supernatant was collected. The chitosanase was precipitated out by chilled acetone (80–90%) at 4 °C; precipitates were collected by centrifugation at 10,000 rpm for 20 min, washed repeatedly with sodium acetate buffer and dissolved in an appropriate volume of same buffer (100 mM, 5.5). This partially purified chitosanase enzyme was stored at 4 °C was used for immobilization.
The activity of free and immobilized chitosanase was determined by 3,5-dinitrosalicylic acid (Miller, 1959) by measuring the rate of release of reducing sugar. Immobilized enzyme was weighed, added to 3 ml 1% chitosan solution and incubated at 37 °C for 30 min. The reaction was stopped by adding 0.5 ml 1 N NaOH solution in case of free enzyme while nanofibers were withdrawn to stop the reaction for immobilized enzyme. The withdrawn reaction mixture was centrifuged at 7000 rpm for 15 min to remove the chitosan and the concentration of reducing sugar was determined in supernatant. One unit of enzyme activity was defined as the amount of enzyme that could produce l lmol reducing sugar per min. The amount of protein was measured using the Bradford method (1976).
2.8. Properties of immobilized chitosanase Immobilization efficiency was calculated by following formula:
Specific activity of immobilized chitosanase enzyme ðU=mgÞ Specific activity of soluble chitosanase enzyme ðU=mgÞ 100 Activity and stability of free and immobilized enzyme was measured over the temperature range of 20–90 °C. Thermal stability of enzyme was checked by incubating PANNFM-chitosanase in acetate buffer at different temperatures for 30 min. pH optimum was determined by using buffer of different range for assay, glycine HCl (2.2–3.6); sodium acetate (3.6–5.6); sodium phosphate (5.8– 8.5); and glycine NaOH (8.6–10.6). To evaluate the reusability, PANNFM-chitosanase, after each reaction was washed with acetate buffer (100 mM, 5.5) and introduced into substrate solution to start the next batch of reaction. This process was repeated up to 10 cycles. The storage stability of immobilized enzyme was determined by incubating the PANNFM-chitosanase in acetate buffer at
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4 °C and assay was done for residual activity at predetermined times. Soluble enzyme was simply stored at 4 °C and assay was done at frequent time interval. 2.9. Chitosan hydrolysis Qualitative and quantitative estimation of COS was done by high performance thin layer chromatography (HPTLC). Chitosan hydrolysis study by immobilized chitosanase was done by adding 5 g PANNFM-chitosanase to 50 ml of chitosan (1% w/v) in 0.1 M of acetate buffer (pH 5.5) at different temperatures (20–80 °C) for 0–24 h. At appropriate time interval, reaction mixtures were withdrawn, absolute ethanol was added to the concentration of 70% and insoluble chitosan was discarded by centrifugation. The supernatant was concentrated by a rotary vacuum evaporator and then subjected to HPTLC on a silica gel plate (Merck 60, GF-254). Solvent system composed of n-propanol:water:concentrated ammonia water (7:2:1) was used. The TLC plate was stained by spraying 0.1% ninhydrin dissolved in ethanol and the products were visualized by keeping the plate in an oven at 100 °C for 10 min. To know the concentration of COS, different dilutions of chitooligomers standard were prepared and loaded on TLC plates. TLC plates were scanned at 366 nm using win CATS software and peak area values were plotted against concentration of different oligomers. Wherever, chitosan hydrolysis using soluble enzymes were performed, conditions were kept same as that of PANNFM-chitosanase except, soluble partially purified enzyme (6 U/ml) was used in place of PANNFM-chitosanase. Chitosan substrates studied for hydrolysis were: chitosan of different degree of deacetylation (40–90%), shrimp shell chitosan (>70% deacetylation), chitin, colloidal chitin/chitosan and glycol chitosan. Colloidal chitin and chitosan was prepared, respectively by method of Lee et al. (2008) and Yabuki et al. (1988), respectively. All the substrates were studied for hydrolysis at 50–60 °C, except >90% DAC chitosan which was studied from 30 °C to 80 °C. Acetylation of chitosan was done by acetic anhydride (Kiang et al., 2004) and degree of acetylation was determined by UV spectrophotometer (Optizen UV3220) using dual standard (Dasheng et al., 2006). 3. Results and discussion 3.1. Synthesis and characterization of PANNFM and PANNFMchitosanase Nanofibres were characterized using scanning electron microscopy (figure not shown). Variation in diameter was observed, which can be explained by instability of jets from the needle. Uniform morphology of fibers was checked and average diameter was found to be 300 nm. Diameter increased with polymer concentration, applied voltage and decreased with increasing tip to collector distance and speed. Upon increasing voltage, more charged solutions having higher solid contents are ejected, which in turn leads to increase in diameter. A detail of this PANNFM preparation has been reported by Dhakate et al. (2011). Adsorption of protein/enzyme on PANNFM was tried before covalent attachment of enzyme, but showed poor reusability. Enzyme molecules, attached non covalently on the surface and/or entrapped between the porous networks of the PANNFM tends to be washed out easily in aqueous solution. Lipase from Pseudomonas cepacia has been immobilized on PANNFM by adsorption and has been used for biodiesel production (Sakai et al., 2010). However, for covalent immobilization, PANNFM surface was activated by ethanol in hydrochloric acid solution (1 N) which led to formation of imidoester derivative. Activated surface was then treated with enzyme containing solution for binding with amino group of
enzyme. Surface morphology of fibers changed from smooth to rough which could be explained due to the formation of covalent bond between (–C–N–) nitrile group of nanofibers and amino (–NH2) group of enzyme which was further confirmed by FTIR (figure not shown). Prominent peaks of nitrile groups (C–N) were observed at 2250–2235 cm1 and peaks at 2930–2850 cm1, 2670–2650 cm1and 1350–1380 cm1 were explained due to aliphatic –CH vibration of different mode in –CH and –CH2. The peak at 1570 cm1 corresponded to the N–H bending vibration of primary amine. The peaks between 1000 and 1300 cm1 was due to C–O stretching vibration. However, upon enzyme adsorption, intensity and sharpness of these peaks increased and two additional peaks appeared at 1460–1450 cm1 (–CH2 group), and 1732 cm1 due to the chitosanase backbone network and stretching vibration of –C'O from saturated aldehyde groups, respectively. After amidination reaction, two more peaks appeared at 1644 cm1 and 1720 cm1 due to amide bond and carboxylic stretch vibration. It was assumed that covalent bonding between amidised nanofibres surface and protein of chitosanase enzyme took place which led to increased peak intensity at 2244 cm1, 1734 and 1622 cm1. 3.2. Screening and isolation Soil plating on chitosan minimal salt agar plates led to growth of around 32 types of isolates i.e. bacteria, actinomycetes and fungi which were differentiated on basis of colony morphology. Majority of these groups were bacteria but their colony sizes were very small and only small number of them showed good growth (data not shown). Enzyme production from these organisms was studied when transferred to chitosan minimal salt broth medium. Five isolates, having relatively high (2–6 U/ml) chitosanase activity were selected. Among which, three were fungal sp. and two were bacterial sp. These isolates were further screened for their exo and endo activity and finally, two fungal sp. (A-4 and A-24) were isolated as potential chitosanase producer which were showing 4 U/ml and 6 U/ml of enzyme activity, respectively. Immobilization study of partially purified chitosanase enzyme from A-4 strain on PANNFM has been previously reported by us (Sinha et al., 2011) and was showing only exo hydrolyzing activity towards chitosan. Chitosanase from other strain (A-24), showing both exo and endo hydrolyzing activity was used for this immobilization study. Chitosanase activity after 4 days of growth was found to be 6 U/ml at 37 °C. Nucleotide sequence data have been deposited in GenBank accession No. JF708947. Strain showed 99.5% sequence homology with Aspergillus fumigatus sp. Chitosanases occur widely in soil microorganism which may be due to common soil microbes like zygomycetes fungi which contain chitosan in their cell walls (Davis and Eveleigh, 1984); another reason could be availability of chitosan through deacetylation of chitin abundantly present in marine waste which was present in soil taken for screening. Chitosan degradation occurs by combined action of chitosanases, exo-b-D-glucosaminidase (exo chitosanases) and N-acetyl-b-Dhexosaminidases (Somashekar and Joseph, 1996), thus these soil microbes prevent deposition of large amount of polysaccharides from marine waste, fungi and dead animal. Screening of fungal strains for enzymatic production of N-acetyl glucosamine has been done previously by Binod et al., 2007. Fungal strain producing chitobiase and endochitinase were isolated after screening. Maximum yield of 1.8% N-acetyl glucosamine was achieved by hydrolysis of colloidal chitin using these two crude enzymes in combination. 3.3. Chitosanase immobilization Results of protein loading were comparable (80%) for both adsorption and covalent attachment of enzyme, but reusability
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Fig. 1. (a) Operational stability: retention of enzymatic activity when operated in batch condition at room temperature and (b) storage stability: retention of enzymatic activity when stored at 4 °C.
and storage stability improved remarkably by covalent attachment. Protein loading was checked at different activation and immobilization and it reached the highest value at 30 min of activation and 120 min of enzyme immobilization time. Lipase immobilization (Li et al., 2007) by same method required only 5 min activation, which may be explained due to the different processing of fibers and may vary with batches of NFM fabrication. Higher immobilization time may also be explained due to the partially purified enzyme which contains more than one protein. The Km value of immobilized enzyme was found to be 8.11 g/l by Line weaver–Burk plots, while for free enzyme it was 3.66 g/l, which showed low affinity of immobilized enzyme towards substrate. Active site of enzyme is distorted due to covalent binding and access of enzyme active site to substrate is restricted hence required more substrate for showing equal enzyme activity as that of free enzyme. 3.4. Temperature and pH Optimum temperature for immobilized enzyme and free enzyme was 50 °C and 40 °C, respectively (data not shown). This result could be ascribed to the restriction of conformational mobility of the immobilized enzyme resulting due to covalent bond formation between the enzyme and the support matrix, thus requires higher activation energy. Lower activity at higher temperature for soluble enzyme was also observed for lipase enzyme (Li et al., 2007) and explained by restriction in the diffusion of the substrates and denaturation of enzyme at high temperature in soluble form. Thermal stability studies showed that soluble enzyme was stable up to 50 °C while PANNFM chitosanase was having >50% residual activity even at 80 °C. Thermal stability study for immobilized chitosanase has been reported only in case of chitin based chitosanase, where immobilized enzyme was stable up to 60 °C (Zeng and Zheng, 2002). The optimal pH for the immobilized enzyme was 5.8, and for the free enzyme 5.5. The charges on the support materials could influence the pH optimum. The binding of enzymes to polycationic supports, such as chitosan would result in an acidic shift of the pH optimum; however, PAN is a non-ionic polymer and neutral amidine bonds are formed after reacting amino groups of enzyme with
imidoester derivative. The optimum pH did not shift much as compared to that of free enzyme as was expected.
3.5. Reusability and stability PANNFM-chitosanase retained about >70% of its original specific activity even after ten batches of use (Fig. 1a) in aqueous solution, which is better than previous study of chitosanase immobilization on nanoparticles (Kuroiwa et al., 2008). Storage stability of PANNFM-chitosanase improved over free enzyme due to covalent binding by amidination reaction. Sharp decrease in activity was observed for soluble enzyme (>60%) within five day and further 20% decrease in next four days. Activity decreases rapidly if enzyme is stored in crude form and loss is attributed to denaturation of protein. More than 60% residual activity of PANNFM-chitosanase was observed even after storing for 30 days (Fig. 1b). Thus, amidination reaction played role in improving chitosanase stability. The number of covalent bonds between enzyme molecule and nanofibers support system can be controlled by the degree of surface activation of NFs. PANNFM-chitosanase could be stored for more than 60 days with minor loss (10–20%) in activity.
Fig. 2. Thin layer chromatography of glucosamine produced at different time interval (10–70 min) at 50 °C.
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Fig. 3. (a) Time course of concentration of chitooligosaccharides (COS) produced by PANNFM-chitosanase at 60 °C and (b) thin layer chromatography of Chitooligosaccharides produced.
3.6. Chitosan hydrolysis Product profile changed with temperature and time when batch hydrolysis of chitosan was carried out with PANNFM-chitosanase. Chitosan (DAC > 90%) hydrolysis at 50 °C led to production of reducing sugar equivalent to D-glucosamine, concentration of which increased linearly up to 20 min, nonlinear afterwards and became almost constant after 60 min, TLC of glucosamine produced at different time interval has been shown in Fig 2. Final concentration of glucosamine at 24 h was found to be 0.3 mg/ml and yield of hydrolysis was 73% as compared to free enzyme. 100% yield was assumed, if same substrate was used, with which the soluble enzyme showed highest activity. This could be due to the mass transfer limitations associated with PANNFM-chitosanase and/or improper mixing of reaction mixture. Vigorous mixing of reaction mixture with PANNFM was avoided due to fragile nature of membrane. Yield of hydrolysis decreased with increased degree of acetylation (40–90%) of chitosan and it ranged from 20% to 73% at 50 °C. COS were produced after 30–40 min reaction time when incubated at 60 °C and above temperature. Changing product profile along with temperature can be explained by the fact that crude enzymes could be mixtures of more than one enzyme having different optimum temperatures. Two types of chitosanases from same microorganism having different optimum temperatures have been reported by Wang et al. (2011) and Chen et al. (2005). At 60 °C, pentamers and hexamers were produced and their concentrations increased with time (Fig. 3a and b). Dimers and trimers concentration increased after 4–6 h along with decrease in larger oligomers. Maximum concentration of tetramers (0.8 mg/ml) was produced at 2 h. However, after 10 h of reaction only monomers, small amount of dimers and trimers were present in the reaction mixtures. It suggested that controlling the reaction immediately at particular point is an important factor in order to get physiologically important COS. PANNFM-chitosanase enzyme could be of
great use for selective production of desired oligomers as shown by this study. Comparison of yield of monomers and COS from same batch of soluble and PANNFM-chitosanase enzyme after using three times were done. It was observed that 400% more glucosamine production can be achieved using same amount of enzyme when used in immobilized form, this system was more advantageous for small oligomers (dimer) as compared to large olimers (trimer to hexamer). Further use (10 times) of PANNFM bound 30 U of chitosanase gave chitosan hydrolytic products equivalent to approximately 250–300 U of crude soluble enzymes. COS profile changed with different batches of soluble enzyme (data not shown) while, it was not the case with PANNFM-chitosanase. Ratio of two enzymes in crude mixture cannot be controlled if, taken in soluble form and this problem can be removed by PANNFM-chitosanase.COS production at 60 °C were studied on chitosan having varied degree of deacetylation (40–90%). It was observed that product profile was same with all the chitosan (>50–90% DAC), though yield was low with chitosan having lower DAC and no COS were observed at chitosan having <50% DAC. PANNFM-chitosanase hydrolyzed shrimp shell chitosan to produce monomer, but COS were not found even after 24 h incubation at any of the temperature. Colloidal chitosan hydrolysis showed lower activity (0.8 U/ml) but was able to selectively produce COS (pentamers only) at 60 °C. Chitin couldnot be hydrolyzed, which may be due to their inability to cleave bond between two N-acetylated glucosamine units of chitin. Yield of glucosamine from colloidal chitin was very low and oligomers production was not studied using PANNFM-chitosanase. According to cleavage site, chitosanase can be divided into two major categories: endo and exo chitosanases (GlcN-ase). Exo chitosanases cleaves glucosamine continuously from the non reducing end of the substrate while endo types cut chitosan at random positions and produce COS. Crude chitosanase exhibits wide range of substrate specificity, since they contains mixture of enzymes (Fenton and Evelegih,
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1981); however, PANNFM-chitosanase showed specificity towards chitosan. Production performance of chitosanase from different organism varies in their catalytic action. Differentially deacetylated chitosan have four different types of randomly distributed glycosidic bonds in their structure and enzymatic activity mainly depends on degree of deacetylation (Aiba, 1994; Kurita et al., 1977; Sakai et al., 1991). In colloidal chitosan preparation, acetyl group is removed and degree of deacetylation increases, hence substrate specificity changes and shows different hydrolytic activity. 4. Conclusions Nanofibers diameter can be changed by factors like needle diameter, rpm of collector drum, applied voltage, viscosity of solution, etc. PANNFM could be efficiently used as support for immobilization of chitosanase and chitosan hydrolysis. Selective production of desired product by can be achieved by changing parameters like time, temperature, etc. Improvement in storage stability and reusability of chitosanase enzyme was seen using PANNFM as matrix. Recovery of chitosanase is desirable by using PAN matrix and can be used for large scale production of glucosamine and COS to be used in food, pharmaceutical, cosmetics industry, etc. Acknowledgements Authors are grateful to Director, NPL (CSIR), for his kind permission to publish the results. DST for providing financial support in the form of project Grant (SR/S2/CMP-10/2010) and one of the authors under WOS-A scheme is highly acknowledged and would like to thank Dr. Sukhavir Singh and K.N. Sood for providing SEM facility. References Aiba, S., 1994. Preparation of N-acetylchitooligosaccharides by lysozymic hydrolysates of partially N-acetylated chitosans. Carbohydr. Res. 261, 297–306. Binod, P., Sandhya, C., Suma, P., Szakacs, G., Pandey, Ashok., 2007. Fungal biosynthesis of endo chitinase and chitobiase in solid state fermentation and their application for the production of N-acetyl-D-glucosamine from colloidal chitin. Bioresour. Technol. 98, 2742–2748. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chen, X., Xia, W., Yu, X., 2005. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22–326. Food Res. Int. 38, 315–322. Dasheng, L., Yuanan, W., Pingija, Y., Linbin, J., 2006. Determination of degree of acetylation of chitosan by UV spectrophotometry using dual standards. Carbohydr. Res. 341, 782–785. Davis, B., Eveleigh, D.E., 1984. Chitosanases: occurence, production and immobilization. In: Zikakis, P. (Ed.), Chitin, Chitosan and Related Enzymes. Academic Press, FL, pp. 161–179.
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Dhakate, S.R., Gupta, A., Chahudhari, A., Tawale, J., Mathur, R.B., 2011. Morphology and thermal properties of PAN copolymer based electrospun nanofibers. Synth. Met. 161 (5–6), 411–419. Fenton, D.M., Evelegih, D.E., 1981. Purification and mode of action of chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126, 151–165. Jeon, Y.J., Shahidi, F., Kim, S.K., 2000. Preparation of chitin and chitosan oligomer and their application in physiological functional foods. Food Rev. Int. 61, 59–76. Kajimoto, O., Sakamoto, K., Takamori, Y., Kajitani, N., Imanishi, T., Matsuo, R., Kajitani, Y., 1998. Therapeutic activity of oral glucosamine hydrochloride in osteoarthritis of the knee: a placebo-controlled, double-blind, cross-over study. Nippon Rinsho Eiyo Gakkaishi 20, 41–47. Kiang, T., Wen, J., Lim, H.W., Leong, K.W.K.W., 2004. The effect of the degree of chitosan deacetylation on the efficiency of gene transfection. Biomaterials 22, 5293–5301. Kim, S.K., 2010. Chitin, Chitosan and Oligosaccharides and Their Derivatives: Biological Activities and Their Applications, vol. 15. CRC Press, New York, pp. 195–215. Kim, S.K., Rajapakse, N., 2005. Enzymatic production and biological activities of chitosan oligosaccharides (COS): a review. Carbohydr. Polym. 62, 357–368. Kim, J., Grate, W., Wang, P., 2005. Nanostructures for enzyme stabilization. Chem. Eng. Sci. 61, 1017–1026. Kurita, K., Sannan, T., Iwakura, Y., 1977. Studies on chitin. 4. Evidence for formation of block and random copolymers of N-acetyl-D-glucosamine and D-glucosamine by hetero and homogeneous hydrolyses. Makromol. Chem. 178, 3197–3202. Kuroiwa, T., Noguchi, Y., Nakajima, M., Sato, S., Sukekuni, M., Ichikawa, S., 2008. Production of chitosan oligosaccharides using chitosanase immobilized on amylose-coated magnetic nanoparticles. Process Biochem. 43, 62–69. Lee, D.X., Xia, W.X., Zhang, J.L., 2008. Enzymatic preparation of chitooligosaccharides by commercial lipase. Food Chem. 111, 291-195. Li, S.F., Chen, J.P., Wu, W.T., 2007. Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization. J. Mol. Catal. B: Enzym. 47, 117–124. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Sakai, K., Katsumi, R., Isobe, A., Nanjo, F., 1991. Purification and hydrolytic action of a chitosanase from Nocardia orientalis. Biochim. Biophys. Acta. 1097, 65–72. Sakai, S., Liu, Y., Yamaguchi, T., Watanabe, R., Kawabe, M., Kawakami, K., 2010. Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun polyacrylonitrile fibers. Bioresour. Technol. 101, 7344–7349. Shimosaka, M., Nogawa, M., Wang, X.Y., Kumehara, M., Okazaki, M., 1995. Production of two chitosanases from chitosan assimilating bacterium Acinetobacter sp. strain CHB101. Appl. Environ. Microbiol. 61 (2), 438–442. Sinha, S., Kumar, R., Dhakate, S., Chand, S., 2011. Chitosanase linked PAN nanofibres for enzymatic production of glucosamine. IJBBB 1 (2), 153–158. Somashekar, D., Joseph, R., 1996. Chitosanase – properties and application: a review. Bioresour. Technol. 55, 35–45. Wang, Z.J., Wan, L.S., Liu, Z.M., Huang, X.J., Xu, Z.K., 2009. Enzyme immobilization on electrospun polymer nanofibers: an overview. J. Mol. Catal. B: Enzym. 56, 189– 195. Wang, S.-L., Tseng, W.N., Liang, T.W., 2011. Biodegradation of shellfish waste and production of chitosanase by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU02. Biodegradation. doi:10.1007/s10532-011-9453-5. Yabuki, M., Uchiyama, A., Suzuki, K., Ando, A., Fujii, T., 1988. Purification and properties of chitosanase from Bacillus circulans MH-K1. J. Gen. Appl. Microbiol. 34, 255–270. Ye, P., Xu, Z.K., Wu, J., Innocent, C., Seta, P., 2006. Nanofibrous poly(acrylonitrile-comaleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization. Biomaterials 27, 4169–4176. Zeng, J., Zheng, L.Y., 2002. Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction. Process Biochem. 38, 531–535.
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Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides Sujata Sinha a,⇑, Sanjay R. Dhakate b, Pankaj Kumar b, R.B. Mathur b, Pushplata Tripathi c, Subhash Chand a,⇑ a
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Physics and Engineering of Carbon, Division of Materials Physics and Engineering, National Physical Laboratory (CSIR), Dr. K.S. Krishnan Marg, New Delhi 110012, India c School of Sciences, Indira Gandhi National Open University, Maidan Garhi, New Delhi 110068, India b
a r t i c l e
i n f o
Article history: Available online 2 December 2011 Keywords: Nanofibers Immobilization Chitosan Chitooligosaccharide
a b s t r a c t Polyacrylonitrile nanofibrous membranes (PANNFM) were prepared by electrospinning from 10 wt.% of PAN solution and its surface was modified by amidination reaction. A new chitosan degrading enzyme from Aspergillus sp. was covalently immobilized on PANNFM. Immobilization efficiency of 80% was achieved by activating PANNFM surface for 30 min followed by 2 h treatment with enzyme solution. The optimum temperature and pH for immobilized enzyme were 50 °C and 5.8, respectively. The immobilized chitosanase retained >70% activity after ten repeated batch reaction and could be stored up to 60 days at 4 °C with minor loss in activity. Chitosan hydrolysis using different substrates were studied using immobilized chitosanase in batch conditions. Continuous selective production of chitooligosaccharides (dimer to hexamer) by changing the temperature was achieved by PANNFM-chitosanase. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Chitosanases are group of hydrolytic enzymes which act on chitosan and can be of great industrial application due to enormous availability of chitin and chitosan in nature (Somashekar and Joseph, 1996). Chitosan is deacetylated chitin; most abundant polymer after cellulose and due to its biological properties and biocompatibility (Kim, 2010) has great potential to be applied in different areas like agricultural, industrial, biomedical, etc. Poor solubility of chitosan makes it difficult to be used at large scale. Interestingly, low molecular weight chitosan (LMWC) and chitooligosaccharides (COS) are readily soluble in water due to free amino group in D-glucosamine (Jeon et al., 2000) and show excellent biological activities (Kim and Rajapakse, 2005). Monomers of chitosan (D-glucosamine and N-acetyl D-glucosamine) has biomedical applications like arthritis treatment, dentistry, wound healing, etc. and has been studied as food supplement (Kajimoto et al., 1998). Chitosan can be depolymerized either by chemically or enzymatically. However, chemical method is avoided because of low yield, toxicity, pollution, high cost and non fitness for human consumption. Use of chitosan degrading enzyme is limited at industrial scale due to its high cost and limited availability (Kim and Rajapakse, 2005). Chitosanase enzymes have been found in large number of microbes including bacteria and fungi (Somashekar and ⇑ Corresponding authors. Tel.: +91 11 2659 1004; fax: +91 11 2658 2282. E-mail addresses: [email protected] (S. Sinha), [email protected], [email protected] (S. Chand). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.101
Joseph, 1996) which can be used in production of COS, but most of these enzymes have low substrate specificity and enzyme is inducible in nature (Shimosaka et al., 1995). In order to find a novel microbial chitosanase, we screened microbes from soil sample rich in fish waste and isolated partially purified enzyme was immobilized on electrospun polyacrylonitrile nanofibrous membranes (PANNFM). Enzyme immobilization improves reusability and has other advantages like scale up, ease in recycling, continuous operation and product purification. Performance of immobilized enzyme generally depends on choice of matrix and method of immobilization. One dimensional nanofibers have extremely high surface area to volume ratio and excellently interconnected pore structure. The interconnectivity of electrospun supports circumvent the mass transfer limitations and have been used as immobilization matrix for a number of enzymes (Wang et al., 2009). NFMs from natural polymers are generally less stable chemically and mechanically than those from synthetic polymers. PAN is a polymer with good stability and mechanical properties (Kim et al., 2005). Derivatives of PAN have also been used for enzyme immobilization with an aim to introduce functional groups into the polymer backbone due to the inertness and hydrophobicity of acrylonitrile monomer (Ye et al., 2006). In this study, PANNFM was used for immobilization of chitosanase after activation of surface by amidination reaction and were studied for residual activity, reusability, optimum pH and temperature. PANNFMchitosanase was used for batch hydrolysis of different chitosan substrates and selective production of glucosamine and COS was achieved.
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2. Methods
2.5. Immobilization of chitosanase enzyme
2.1. Materials
All the immobilization steps were carried out by cutting the PANNFM into standard pieces and weighed. Adsorption of enzyme was done by adding the enzyme solution (10 ml of 1 mg/ml) to NFM kept in 100 ml flask for 2 h with mild shaking. NFM surface was activated by placing it in absolute ethanol and bubbling with 1 N solution of hydrogen chloride (6–7 ml) to produce the corresponding imidoester derivatives, a method followed by Li et al. (2007) for lipase immobilization on PAN. NFMs were washed with acetate buffer and were treated with chitosanase enzyme solution in 0.1 M acetate buffer solution (pH 5.5). Flask was put for shaking (100 rpm) at room temperature for 2 h. After the immobilization reaction, the membrane was removed from the solution and washed with acetate buffer several times to remove any unbound enzyme. NFM bound enzyme was lyophilized and was stored till further use.
Commercial chitosan from crab shell, average molecular weight 290 kDa, 93% N-deacetylated (DAC); were kind gift from Marine Chemicals, Chennai, India. Chitosan from shrimp shell (>75% deacetylated) and PAN powder were procured from Sigma–Aldrich, Germany and N,N-Dimethylformamide (DMF) was purchased from Fisher Scientific, India. Glucosamine hydrochloride and Chitin were purchased from Hi-media. All other chemicals were procured from SRL, Mumbai, India and were of analytical grade. 2.2. Preparation of PANNFM by electrospinning The PAN solution (10 wt.%) was prepared in DMF by mixing it in multi frequency ultrasonic bath (Life Care Equipment Pvt. Ltd., Mumbai, India) for one hour at 50 °C. Continuous stirring for 15–20 h was done to get uniformly mixed solution and was spun using electrospinning unit (ESPIN-NANO). Conditions for electrospinning were as follows: needle orifice: 0.55 mm, rotating cylindrical collector diameter: 30 mm, voltage applied: 5–20 kV, rotational speed of the collector: 2000 rpm, distance between tip to collector: 20 cm and flow rate of solution 0.2 ml/h which was maintained using computer control program. 2.3. Screening and isolation of chitosanase producing microbial strain from soil
2.6. Analytical methods Surface morphology of NFM before and after immobilization was studied by scanning electron microscope (SEM, Zeiss-EVO, MA-10, variable UK). The electrospun PANNFM, amidinated or surface modified PANNFM, PANNFM-chitosanase were characterized by Fourier transform infra red spectrometer (FTIR Nicolet 5700). Small amounts of samples were separately mixed with KBr and prepared pellet was used for FTIR spectra.
2.7. Chitosanase assay Chitosan minimal salt medium was used for screening and isolation. Soil samples, taken from marine waste dumping area in local market (Chittaranjan Park, New Delhi, India) was weighed, suspended in sterile water and serial dilutions were prepared. Plating was done on chitosan minimal salt agar medium. Composition of medium was as follows: pH 6.8, colloidal chitosan 1% (w/v), KH2PO4 (0.15 g/l), K2HPO4 (0.35 g/l), MgSO45H2O (0.25 g/l), FeSO47H2O (0.005 g/l), ZnSO4 (0.001 g/l), MnCl2 (0.001 g/l) and agar (2%) was added for making plates. Microbial colonies grown on the surface of agar plates were transferred to chitosan broth. Composition of chitosan broth medium was as follows (g/l): KH2PO4, 2.0; K2HPO4, 1.0; MgSO47H2O, 0.5; NaCl, 0.5; CaCl2, 0.1; yeast extract 0.5 and chitosan 15; the medium was adjusted to pH 6.0 with the help of acetic acid (1 N). Inoculated chitosan broth medium, after incubation for 6–7 days was centrifuged to remove microbial growth and supernatant was used for enzyme assay. Microbes showing good chitosanase (endo and exo) activities were selected and used for further enzyme production. 2.4. Preparation of partially purified chitosanase Microbial cultures, stored on agar slants at 4 °C were scrapped off and washed out with sterile distilled water. One milliliter of microbial suspension was inoculated into a 250 ml flask containing 50 ml of the medium and incubated on a rotary shaker at 200 rpm for 3–4 days at 32 °C. The composition of the medium (pH 5.6) was same as that of chitosan broth. Finally, the cells of the culture broth were removed from the medium by centrifugation at 7500 rpm for 15 min at 4 °C and the supernatant was collected. The chitosanase was precipitated out by chilled acetone (80–90%) at 4 °C; precipitates were collected by centrifugation at 10,000 rpm for 20 min, washed repeatedly with sodium acetate buffer and dissolved in an appropriate volume of same buffer (100 mM, 5.5). This partially purified chitosanase enzyme was stored at 4 °C was used for immobilization.
The activity of free and immobilized chitosanase was determined by 3,5-dinitrosalicylic acid (Miller, 1959) by measuring the rate of release of reducing sugar. Immobilized enzyme was weighed, added to 3 ml 1% chitosan solution and incubated at 37 °C for 30 min. The reaction was stopped by adding 0.5 ml 1 N NaOH solution in case of free enzyme while nanofibers were withdrawn to stop the reaction for immobilized enzyme. The withdrawn reaction mixture was centrifuged at 7000 rpm for 15 min to remove the chitosan and the concentration of reducing sugar was determined in supernatant. One unit of enzyme activity was defined as the amount of enzyme that could produce l lmol reducing sugar per min. The amount of protein was measured using the Bradford method (1976).
2.8. Properties of immobilized chitosanase Immobilization efficiency was calculated by following formula:
Specific activity of immobilized chitosanase enzyme ðU=mgÞ Specific activity of soluble chitosanase enzyme ðU=mgÞ 100 Activity and stability of free and immobilized enzyme was measured over the temperature range of 20–90 °C. Thermal stability of enzyme was checked by incubating PANNFM-chitosanase in acetate buffer at different temperatures for 30 min. pH optimum was determined by using buffer of different range for assay, glycine HCl (2.2–3.6); sodium acetate (3.6–5.6); sodium phosphate (5.8– 8.5); and glycine NaOH (8.6–10.6). To evaluate the reusability, PANNFM-chitosanase, after each reaction was washed with acetate buffer (100 mM, 5.5) and introduced into substrate solution to start the next batch of reaction. This process was repeated up to 10 cycles. The storage stability of immobilized enzyme was determined by incubating the PANNFM-chitosanase in acetate buffer at
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4 °C and assay was done for residual activity at predetermined times. Soluble enzyme was simply stored at 4 °C and assay was done at frequent time interval. 2.9. Chitosan hydrolysis Qualitative and quantitative estimation of COS was done by high performance thin layer chromatography (HPTLC). Chitosan hydrolysis study by immobilized chitosanase was done by adding 5 g PANNFM-chitosanase to 50 ml of chitosan (1% w/v) in 0.1 M of acetate buffer (pH 5.5) at different temperatures (20–80 °C) for 0–24 h. At appropriate time interval, reaction mixtures were withdrawn, absolute ethanol was added to the concentration of 70% and insoluble chitosan was discarded by centrifugation. The supernatant was concentrated by a rotary vacuum evaporator and then subjected to HPTLC on a silica gel plate (Merck 60, GF-254). Solvent system composed of n-propanol:water:concentrated ammonia water (7:2:1) was used. The TLC plate was stained by spraying 0.1% ninhydrin dissolved in ethanol and the products were visualized by keeping the plate in an oven at 100 °C for 10 min. To know the concentration of COS, different dilutions of chitooligomers standard were prepared and loaded on TLC plates. TLC plates were scanned at 366 nm using win CATS software and peak area values were plotted against concentration of different oligomers. Wherever, chitosan hydrolysis using soluble enzymes were performed, conditions were kept same as that of PANNFM-chitosanase except, soluble partially purified enzyme (6 U/ml) was used in place of PANNFM-chitosanase. Chitosan substrates studied for hydrolysis were: chitosan of different degree of deacetylation (40–90%), shrimp shell chitosan (>70% deacetylation), chitin, colloidal chitin/chitosan and glycol chitosan. Colloidal chitin and chitosan was prepared, respectively by method of Lee et al. (2008) and Yabuki et al. (1988), respectively. All the substrates were studied for hydrolysis at 50–60 °C, except >90% DAC chitosan which was studied from 30 °C to 80 °C. Acetylation of chitosan was done by acetic anhydride (Kiang et al., 2004) and degree of acetylation was determined by UV spectrophotometer (Optizen UV3220) using dual standard (Dasheng et al., 2006). 3. Results and discussion 3.1. Synthesis and characterization of PANNFM and PANNFMchitosanase Nanofibres were characterized using scanning electron microscopy (figure not shown). Variation in diameter was observed, which can be explained by instability of jets from the needle. Uniform morphology of fibers was checked and average diameter was found to be 300 nm. Diameter increased with polymer concentration, applied voltage and decreased with increasing tip to collector distance and speed. Upon increasing voltage, more charged solutions having higher solid contents are ejected, which in turn leads to increase in diameter. A detail of this PANNFM preparation has been reported by Dhakate et al. (2011). Adsorption of protein/enzyme on PANNFM was tried before covalent attachment of enzyme, but showed poor reusability. Enzyme molecules, attached non covalently on the surface and/or entrapped between the porous networks of the PANNFM tends to be washed out easily in aqueous solution. Lipase from Pseudomonas cepacia has been immobilized on PANNFM by adsorption and has been used for biodiesel production (Sakai et al., 2010). However, for covalent immobilization, PANNFM surface was activated by ethanol in hydrochloric acid solution (1 N) which led to formation of imidoester derivative. Activated surface was then treated with enzyme containing solution for binding with amino group of
enzyme. Surface morphology of fibers changed from smooth to rough which could be explained due to the formation of covalent bond between (–C–N–) nitrile group of nanofibers and amino (–NH2) group of enzyme which was further confirmed by FTIR (figure not shown). Prominent peaks of nitrile groups (C–N) were observed at 2250–2235 cm1 and peaks at 2930–2850 cm1, 2670–2650 cm1and 1350–1380 cm1 were explained due to aliphatic –CH vibration of different mode in –CH and –CH2. The peak at 1570 cm1 corresponded to the N–H bending vibration of primary amine. The peaks between 1000 and 1300 cm1 was due to C–O stretching vibration. However, upon enzyme adsorption, intensity and sharpness of these peaks increased and two additional peaks appeared at 1460–1450 cm1 (–CH2 group), and 1732 cm1 due to the chitosanase backbone network and stretching vibration of –C'O from saturated aldehyde groups, respectively. After amidination reaction, two more peaks appeared at 1644 cm1 and 1720 cm1 due to amide bond and carboxylic stretch vibration. It was assumed that covalent bonding between amidised nanofibres surface and protein of chitosanase enzyme took place which led to increased peak intensity at 2244 cm1, 1734 and 1622 cm1. 3.2. Screening and isolation Soil plating on chitosan minimal salt agar plates led to growth of around 32 types of isolates i.e. bacteria, actinomycetes and fungi which were differentiated on basis of colony morphology. Majority of these groups were bacteria but their colony sizes were very small and only small number of them showed good growth (data not shown). Enzyme production from these organisms was studied when transferred to chitosan minimal salt broth medium. Five isolates, having relatively high (2–6 U/ml) chitosanase activity were selected. Among which, three were fungal sp. and two were bacterial sp. These isolates were further screened for their exo and endo activity and finally, two fungal sp. (A-4 and A-24) were isolated as potential chitosanase producer which were showing 4 U/ml and 6 U/ml of enzyme activity, respectively. Immobilization study of partially purified chitosanase enzyme from A-4 strain on PANNFM has been previously reported by us (Sinha et al., 2011) and was showing only exo hydrolyzing activity towards chitosan. Chitosanase from other strain (A-24), showing both exo and endo hydrolyzing activity was used for this immobilization study. Chitosanase activity after 4 days of growth was found to be 6 U/ml at 37 °C. Nucleotide sequence data have been deposited in GenBank accession No. JF708947. Strain showed 99.5% sequence homology with Aspergillus fumigatus sp. Chitosanases occur widely in soil microorganism which may be due to common soil microbes like zygomycetes fungi which contain chitosan in their cell walls (Davis and Eveleigh, 1984); another reason could be availability of chitosan through deacetylation of chitin abundantly present in marine waste which was present in soil taken for screening. Chitosan degradation occurs by combined action of chitosanases, exo-b-D-glucosaminidase (exo chitosanases) and N-acetyl-b-Dhexosaminidases (Somashekar and Joseph, 1996), thus these soil microbes prevent deposition of large amount of polysaccharides from marine waste, fungi and dead animal. Screening of fungal strains for enzymatic production of N-acetyl glucosamine has been done previously by Binod et al., 2007. Fungal strain producing chitobiase and endochitinase were isolated after screening. Maximum yield of 1.8% N-acetyl glucosamine was achieved by hydrolysis of colloidal chitin using these two crude enzymes in combination. 3.3. Chitosanase immobilization Results of protein loading were comparable (80%) for both adsorption and covalent attachment of enzyme, but reusability
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Fig. 1. (a) Operational stability: retention of enzymatic activity when operated in batch condition at room temperature and (b) storage stability: retention of enzymatic activity when stored at 4 °C.
and storage stability improved remarkably by covalent attachment. Protein loading was checked at different activation and immobilization and it reached the highest value at 30 min of activation and 120 min of enzyme immobilization time. Lipase immobilization (Li et al., 2007) by same method required only 5 min activation, which may be explained due to the different processing of fibers and may vary with batches of NFM fabrication. Higher immobilization time may also be explained due to the partially purified enzyme which contains more than one protein. The Km value of immobilized enzyme was found to be 8.11 g/l by Line weaver–Burk plots, while for free enzyme it was 3.66 g/l, which showed low affinity of immobilized enzyme towards substrate. Active site of enzyme is distorted due to covalent binding and access of enzyme active site to substrate is restricted hence required more substrate for showing equal enzyme activity as that of free enzyme. 3.4. Temperature and pH Optimum temperature for immobilized enzyme and free enzyme was 50 °C and 40 °C, respectively (data not shown). This result could be ascribed to the restriction of conformational mobility of the immobilized enzyme resulting due to covalent bond formation between the enzyme and the support matrix, thus requires higher activation energy. Lower activity at higher temperature for soluble enzyme was also observed for lipase enzyme (Li et al., 2007) and explained by restriction in the diffusion of the substrates and denaturation of enzyme at high temperature in soluble form. Thermal stability studies showed that soluble enzyme was stable up to 50 °C while PANNFM chitosanase was having >50% residual activity even at 80 °C. Thermal stability study for immobilized chitosanase has been reported only in case of chitin based chitosanase, where immobilized enzyme was stable up to 60 °C (Zeng and Zheng, 2002). The optimal pH for the immobilized enzyme was 5.8, and for the free enzyme 5.5. The charges on the support materials could influence the pH optimum. The binding of enzymes to polycationic supports, such as chitosan would result in an acidic shift of the pH optimum; however, PAN is a non-ionic polymer and neutral amidine bonds are formed after reacting amino groups of enzyme with
imidoester derivative. The optimum pH did not shift much as compared to that of free enzyme as was expected.
3.5. Reusability and stability PANNFM-chitosanase retained about >70% of its original specific activity even after ten batches of use (Fig. 1a) in aqueous solution, which is better than previous study of chitosanase immobilization on nanoparticles (Kuroiwa et al., 2008). Storage stability of PANNFM-chitosanase improved over free enzyme due to covalent binding by amidination reaction. Sharp decrease in activity was observed for soluble enzyme (>60%) within five day and further 20% decrease in next four days. Activity decreases rapidly if enzyme is stored in crude form and loss is attributed to denaturation of protein. More than 60% residual activity of PANNFM-chitosanase was observed even after storing for 30 days (Fig. 1b). Thus, amidination reaction played role in improving chitosanase stability. The number of covalent bonds between enzyme molecule and nanofibers support system can be controlled by the degree of surface activation of NFs. PANNFM-chitosanase could be stored for more than 60 days with minor loss (10–20%) in activity.
Fig. 2. Thin layer chromatography of glucosamine produced at different time interval (10–70 min) at 50 °C.
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Fig. 3. (a) Time course of concentration of chitooligosaccharides (COS) produced by PANNFM-chitosanase at 60 °C and (b) thin layer chromatography of Chitooligosaccharides produced.
3.6. Chitosan hydrolysis Product profile changed with temperature and time when batch hydrolysis of chitosan was carried out with PANNFM-chitosanase. Chitosan (DAC > 90%) hydrolysis at 50 °C led to production of reducing sugar equivalent to D-glucosamine, concentration of which increased linearly up to 20 min, nonlinear afterwards and became almost constant after 60 min, TLC of glucosamine produced at different time interval has been shown in Fig 2. Final concentration of glucosamine at 24 h was found to be 0.3 mg/ml and yield of hydrolysis was 73% as compared to free enzyme. 100% yield was assumed, if same substrate was used, with which the soluble enzyme showed highest activity. This could be due to the mass transfer limitations associated with PANNFM-chitosanase and/or improper mixing of reaction mixture. Vigorous mixing of reaction mixture with PANNFM was avoided due to fragile nature of membrane. Yield of hydrolysis decreased with increased degree of acetylation (40–90%) of chitosan and it ranged from 20% to 73% at 50 °C. COS were produced after 30–40 min reaction time when incubated at 60 °C and above temperature. Changing product profile along with temperature can be explained by the fact that crude enzymes could be mixtures of more than one enzyme having different optimum temperatures. Two types of chitosanases from same microorganism having different optimum temperatures have been reported by Wang et al. (2011) and Chen et al. (2005). At 60 °C, pentamers and hexamers were produced and their concentrations increased with time (Fig. 3a and b). Dimers and trimers concentration increased after 4–6 h along with decrease in larger oligomers. Maximum concentration of tetramers (0.8 mg/ml) was produced at 2 h. However, after 10 h of reaction only monomers, small amount of dimers and trimers were present in the reaction mixtures. It suggested that controlling the reaction immediately at particular point is an important factor in order to get physiologically important COS. PANNFM-chitosanase enzyme could be of
great use for selective production of desired oligomers as shown by this study. Comparison of yield of monomers and COS from same batch of soluble and PANNFM-chitosanase enzyme after using three times were done. It was observed that 400% more glucosamine production can be achieved using same amount of enzyme when used in immobilized form, this system was more advantageous for small oligomers (dimer) as compared to large olimers (trimer to hexamer). Further use (10 times) of PANNFM bound 30 U of chitosanase gave chitosan hydrolytic products equivalent to approximately 250–300 U of crude soluble enzymes. COS profile changed with different batches of soluble enzyme (data not shown) while, it was not the case with PANNFM-chitosanase. Ratio of two enzymes in crude mixture cannot be controlled if, taken in soluble form and this problem can be removed by PANNFM-chitosanase.COS production at 60 °C were studied on chitosan having varied degree of deacetylation (40–90%). It was observed that product profile was same with all the chitosan (>50–90% DAC), though yield was low with chitosan having lower DAC and no COS were observed at chitosan having <50% DAC. PANNFM-chitosanase hydrolyzed shrimp shell chitosan to produce monomer, but COS were not found even after 24 h incubation at any of the temperature. Colloidal chitosan hydrolysis showed lower activity (0.8 U/ml) but was able to selectively produce COS (pentamers only) at 60 °C. Chitin couldnot be hydrolyzed, which may be due to their inability to cleave bond between two N-acetylated glucosamine units of chitin. Yield of glucosamine from colloidal chitin was very low and oligomers production was not studied using PANNFM-chitosanase. According to cleavage site, chitosanase can be divided into two major categories: endo and exo chitosanases (GlcN-ase). Exo chitosanases cleaves glucosamine continuously from the non reducing end of the substrate while endo types cut chitosan at random positions and produce COS. Crude chitosanase exhibits wide range of substrate specificity, since they contains mixture of enzymes (Fenton and Evelegih,
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1981); however, PANNFM-chitosanase showed specificity towards chitosan. Production performance of chitosanase from different organism varies in their catalytic action. Differentially deacetylated chitosan have four different types of randomly distributed glycosidic bonds in their structure and enzymatic activity mainly depends on degree of deacetylation (Aiba, 1994; Kurita et al., 1977; Sakai et al., 1991). In colloidal chitosan preparation, acetyl group is removed and degree of deacetylation increases, hence substrate specificity changes and shows different hydrolytic activity. 4. Conclusions Nanofibers diameter can be changed by factors like needle diameter, rpm of collector drum, applied voltage, viscosity of solution, etc. PANNFM could be efficiently used as support for immobilization of chitosanase and chitosan hydrolysis. Selective production of desired product by can be achieved by changing parameters like time, temperature, etc. Improvement in storage stability and reusability of chitosanase enzyme was seen using PANNFM as matrix. Recovery of chitosanase is desirable by using PAN matrix and can be used for large scale production of glucosamine and COS to be used in food, pharmaceutical, cosmetics industry, etc. Acknowledgements Authors are grateful to Director, NPL (CSIR), for his kind permission to publish the results. DST for providing financial support in the form of project Grant (SR/S2/CMP-10/2010) and one of the authors under WOS-A scheme is highly acknowledged and would like to thank Dr. Sukhavir Singh and K.N. Sood for providing SEM facility. References Aiba, S., 1994. Preparation of N-acetylchitooligosaccharides by lysozymic hydrolysates of partially N-acetylated chitosans. Carbohydr. Res. 261, 297–306. Binod, P., Sandhya, C., Suma, P., Szakacs, G., Pandey, Ashok., 2007. Fungal biosynthesis of endo chitinase and chitobiase in solid state fermentation and their application for the production of N-acetyl-D-glucosamine from colloidal chitin. Bioresour. Technol. 98, 2742–2748. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chen, X., Xia, W., Yu, X., 2005. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22–326. Food Res. Int. 38, 315–322. Dasheng, L., Yuanan, W., Pingija, Y., Linbin, J., 2006. Determination of degree of acetylation of chitosan by UV spectrophotometry using dual standards. Carbohydr. Res. 341, 782–785. Davis, B., Eveleigh, D.E., 1984. Chitosanases: occurence, production and immobilization. In: Zikakis, P. (Ed.), Chitin, Chitosan and Related Enzymes. Academic Press, FL, pp. 161–179.
157
Dhakate, S.R., Gupta, A., Chahudhari, A., Tawale, J., Mathur, R.B., 2011. Morphology and thermal properties of PAN copolymer based electrospun nanofibers. Synth. Met. 161 (5–6), 411–419. Fenton, D.M., Evelegih, D.E., 1981. Purification and mode of action of chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126, 151–165. Jeon, Y.J., Shahidi, F., Kim, S.K., 2000. Preparation of chitin and chitosan oligomer and their application in physiological functional foods. Food Rev. Int. 61, 59–76. Kajimoto, O., Sakamoto, K., Takamori, Y., Kajitani, N., Imanishi, T., Matsuo, R., Kajitani, Y., 1998. Therapeutic activity of oral glucosamine hydrochloride in osteoarthritis of the knee: a placebo-controlled, double-blind, cross-over study. Nippon Rinsho Eiyo Gakkaishi 20, 41–47. Kiang, T., Wen, J., Lim, H.W., Leong, K.W.K.W., 2004. The effect of the degree of chitosan deacetylation on the efficiency of gene transfection. Biomaterials 22, 5293–5301. Kim, S.K., 2010. Chitin, Chitosan and Oligosaccharides and Their Derivatives: Biological Activities and Their Applications, vol. 15. CRC Press, New York, pp. 195–215. Kim, S.K., Rajapakse, N., 2005. Enzymatic production and biological activities of chitosan oligosaccharides (COS): a review. Carbohydr. Polym. 62, 357–368. Kim, J., Grate, W., Wang, P., 2005. Nanostructures for enzyme stabilization. Chem. Eng. Sci. 61, 1017–1026. Kurita, K., Sannan, T., Iwakura, Y., 1977. Studies on chitin. 4. Evidence for formation of block and random copolymers of N-acetyl-D-glucosamine and D-glucosamine by hetero and homogeneous hydrolyses. Makromol. Chem. 178, 3197–3202. Kuroiwa, T., Noguchi, Y., Nakajima, M., Sato, S., Sukekuni, M., Ichikawa, S., 2008. Production of chitosan oligosaccharides using chitosanase immobilized on amylose-coated magnetic nanoparticles. Process Biochem. 43, 62–69. Lee, D.X., Xia, W.X., Zhang, J.L., 2008. Enzymatic preparation of chitooligosaccharides by commercial lipase. Food Chem. 111, 291-195. Li, S.F., Chen, J.P., Wu, W.T., 2007. Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization. J. Mol. Catal. B: Enzym. 47, 117–124. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Sakai, K., Katsumi, R., Isobe, A., Nanjo, F., 1991. Purification and hydrolytic action of a chitosanase from Nocardia orientalis. Biochim. Biophys. Acta. 1097, 65–72. Sakai, S., Liu, Y., Yamaguchi, T., Watanabe, R., Kawabe, M., Kawakami, K., 2010. Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun polyacrylonitrile fibers. Bioresour. Technol. 101, 7344–7349. Shimosaka, M., Nogawa, M., Wang, X.Y., Kumehara, M., Okazaki, M., 1995. Production of two chitosanases from chitosan assimilating bacterium Acinetobacter sp. strain CHB101. Appl. Environ. Microbiol. 61 (2), 438–442. Sinha, S., Kumar, R., Dhakate, S., Chand, S., 2011. Chitosanase linked PAN nanofibres for enzymatic production of glucosamine. IJBBB 1 (2), 153–158. Somashekar, D., Joseph, R., 1996. Chitosanase – properties and application: a review. Bioresour. Technol. 55, 35–45. Wang, Z.J., Wan, L.S., Liu, Z.M., Huang, X.J., Xu, Z.K., 2009. Enzyme immobilization on electrospun polymer nanofibers: an overview. J. Mol. Catal. B: Enzym. 56, 189– 195. Wang, S.-L., Tseng, W.N., Liang, T.W., 2011. Biodegradation of shellfish waste and production of chitosanase by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU02. Biodegradation. doi:10.1007/s10532-011-9453-5. Yabuki, M., Uchiyama, A., Suzuki, K., Ando, A., Fujii, T., 1988. Purification and properties of chitosanase from Bacillus circulans MH-K1. J. Gen. Appl. Microbiol. 34, 255–270. Ye, P., Xu, Z.K., Wu, J., Innocent, C., Seta, P., 2006. Nanofibrous poly(acrylonitrile-comaleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization. Biomaterials 27, 4169–4176. Zeng, J., Zheng, L.Y., 2002. Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction. Process Biochem. 38, 531–535.