Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles ...

Biotechnol Lett (2012) 34:1307–1313 DOI 10.1007/s10529-012-0902-y

ORIGINAL RESEARCH PAPER

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides Saurabh Sudha Dhiman • Sujit Sadashiv Jagtap Marimuthu Jeya • Jung-Rim Haw • Yun Chan Kang • Jung-Kul Lee

•

Received: 2 December 2011 / Accepted: 4 March 2012 / Published online: 16 March 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The kcat value (4,261 s-1) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-012-0902-y) contains supplementary material, which is available to authorized users. S. S. Dhiman S. S. Jagtap M. Jeya Y. C. Kang (&) J.-K. Lee (&) Department of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, Republic of Korea e-mail: [email protected] J.-K. Lee e-mail: [email protected] S. S. Dhiman M. Jeya J.-R. Haw J.-K. Lee Institute of SK-KU Biomaterials, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, Republic of Korea

Keywords Immobilization Response surface methodology SiO2 Xylanase Xylooligosaccharide

Introduction Endoxylanases (b-1,4-d-xylanohydrolase, EC 3.2.1.8) hydrolyze polysaccharides, such as, xylan and have versatile applications (Belanic et al. 1995). Complete hydrolysis of xylan requires the synergistic action of different hydrolytic enzymes to convert xylan to xylose (Li et al. 1993). Endo-b-1,4-d-xylanases attack the xylan backbone by cleaving the 1,4-b-d-xylosidic linkages and catalyze the release of different xylool igosaccharides (XOSs), which in turn can be converted to xylose by b-xylosidase (Ximenes et al. 1999). On the basis of sequence homology and hydrophobic cluster analysis, xylanases have been classified mainly into two families of glycosyl hydrolases (GHs): GH10 and GH11 (Tenkanen et al. 1992) in different microorganisms. Among microbial sources, filamentous fungi are especially interesting, as their xylanase activities are much higher than those found in yeasts and bacteria (Raj and Chandra 1996). Xylanases have occupied the central stage in the scientific community due to their enormous applications in various industrial processes but production of XOSs using xylanase is a relatively untouched area (Dhiman et al. 2008). Moreover, most of the reports focus on the application of free rather than

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immobilized xylanase for XOS production (Kapoor and Kuhad 2007). These XOSs have potential application as prebiotics for humans and animals to improve their health (Gawande and Kamat 1998). The emerging trend of nanobiocatalysis has opened the scope of utilization of biomolecules in association with nanostructured materials (Zhang et al. 2011). Different modification methods, i.e., physical adsorption, electrostatic binding etc. are available for stable attachment of proteins onto support matrices. Of these, covalent bond formation provides the most stable attachment, fixing the enzyme to the carrier (Singh et al. 2011). Most commonly, the amino group of the enzyme are employed for covalent immobilization (Singh et al. 2011) and thus, eliminates the possibility of enzyme leaching and hence no protein contamination of the product (Zhang et al. 2011). The current paper focuses on the production of fungal xylanase which has exceptionally high catalytic activity when produced in a 7-liter jar fermenter. Furthermore, the enzyme was purified and characterized. Upon immobilization of the crude xylanase on SiO2 nanoparticles, it showed enhanced stability. The efficiency of immobilized xylanase was evaluated for the synthesis of different XOSs and compared with the efficiency of free crude xylanase.

Materials and methods Organism and culture conditions By employing the capillary tube method, the soil samples collected from Sorak Mountain, Republic of Korea, were diluted in sterile 0.9 % saline. For screening of fungal cultures, aliquots were spread on agar plates containing 0.5 % birchwood xylan, and then incubated for 3 days. The isolated strain was identified on the basis of the internal transcribed spacer of ribosomal DNA (ITS rDNA) sequence analysis. For the sequence analysis, the ITS1-5.8 S-ITS2 rDNA region of the fungus was amplified by polymerase chain reaction (PCR) using the primer set pITS1 (50 -TCCGTAGGTGAACCTGCCG-30 ) and pITS4 (50 -TCCTCCGCTTATTGAT-ATGC-30 ) (White et al. 1990). A 592 bp amplicon was obtained, cloned, and sequenced. Pairwise evolutionary distances and a phylogenetic tree were constructed with the MegAlign software.

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Enzyme production and assay The fungal strain was sub-cultured every 3 weeks on potato/dextrose (PD) agar plates and 50 ml PD broth was used for seeding the culture. After 5 days, 5 ml pre-culture was inoculated into 50 ml production medium for optimizing the different nutritional components. The concentration of the nitrogen source was adjusted to the same content of nitrogen by using the Kjeldahl method (Elegir et al. 1994). The advanced central composite design of response surface methodology (CCD-RSM) was applied to three independent physical parameters (temperature [A]; pH [B]; agitation speed [C]) that affect the xylanase production significantly. For optimizing the physical parameters, 7-liter jar fermenter containing 3 l optimized production medium was used (Supplementary Table 4). Xylanase activity of the crude extract was assayed with birchwood xylan through the modified Bailey’s method using the dinitrosalicylic acid reagent. Similarly, endoglucanase (EG), b-glucosidase (BGL), and cellobiohydrolase (CBH) activity of the crude extract was determined using standard methods. Purification of Pholiota adiposa xylanase Liquid cultures were harvested and pooled after 8 days, and cells were removed by centrifugation at 10,0009g for 20 min. The supernatant was concentrated to 5 ml by ultrafiltration (10 kDa cutoff). The sample was applied to a DEAE-Sepharose fast flow column (1.6 9 10 cm) equilibrated with 50 mM sodium acetate buffer (pH 5.0). The column was washed with the same buffer, and absorbed proteins were eluted by a linear concentration gradient of NaCl (0–1 M) at 1 ml/min. The fractions containing xylanase activity were pooled, dialyzed, concentrated to *2 ml by ultrafiltration with (30 kDa cutoff), and applied to a Hiload 16/60 Superdex 200 pg column (Amersham Pharmacia Biotech, UK) equilibrated with 25 mM sodium acetate buffer containing 15 mM NaCl at pH 5.0. Proteins were eluted with the same buffer at 0.5 ml/min. Xylanase-rich fractions were pooled, concentrated by ultrafiltration as described above, and stored at 4 °C for further use. The chromatographic separation was performed using a BioLogic FPLC system. SDS-PAGE was performed a 5 % stacking gel and a 10 % resolving gel through electrophoresis system (Tan et al. 1985).


Internal amino acid sequence and characterization of PaXyl Protein bands of interest were cut from polyacrylamide gels and digested with trypsin using a standard protocol. The cleaved peptides were eluted and analyzed by nanoLC–MS/MS for internal amino acid sequencing. To identify protein sequence homology, a search method was employed using the MS data analysis program SEQUEST (Thermo Finnigan, San Jose, CA) against a fungal protein database obtained from the National Center for Biotechnology Information (NCBI) protein sequence database. Protein concentration was determined by the method of Bradford with bovine serum albumin as the standard. Km and kcat values of the pure xylanase were determined by measurement of the enzyme activity with various concentrations (0.01– 0.1 mM) of birchwood xylan and other oligosaccharides as substrate at the optimum pH in each case. Immobilization of PaXyl P. adiposa-derived crude xylanase was immobilized onto functionalized SiO2 nanoparticles 4830HT with an average particle size of 80 nm (Nanostructured & Amorphous Material Inc., Houston, USA) through covalent linkage. The activation of SiO2 nanoparticles was achieved by treating the nanoparticles with glutaraldehyde. SiO2 nanoparticles were washed twice with deionized water and then recovered by centrifugation. The crude enzyme preparation (325 mg protein per g activated support matrix) was mixed with the activated SiO2 in 100 mM sodium acetate buffer, pH 5.0 (Singh et al. 2011). Functionalized SiO2 was considered as a reference immobilization matrix (IM). As SiO2 was considered as reference IM, it was subjected to statistical modeling using CCD-RSM, including different physical parameters (temperature [D]; pH [E]; agitation speed [F]; incubation time [G]), and the immobilization process was performed under different combinations of independent variables. Non-covalently adsorbed protein was removed thereafter by thorough washing of the nanoparticles with deionized water and sodium acetate buffer (Kapoor and Kuhad 2007). The supernatant was used for xylanase protein analysis and residual enzyme activity. The washed carrier was directly used for the determination of activity and stability. Immobilization efficiency (IE) and immobilization

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yield (IY) were calculated as follows: %IE = (ai/af) 9 100; %IY = [{Pi - (Pw ? Ps)}/Pi] 9 100. Where ai is the total activity of the immobilized enzyme and af is the total activity of the free enzyme. Pi is the total protein content of the crude enzyme preparation; Pw and Ps are the protein concentrations of the wash solution and supernatant after immobilization, respectively. After evaluating the interaction among different independent variables for the reference IM, its %IE and %IY were also compared with other IMs, e.g., resins. Characterization of immobilized xylanase Optimal activity and half-life of immobilized crude xylanase were determined from 35–65 °C and pH 2–7 and compared with crude unimmobilized (free) xylanase activity. The kinetic parameters, i.e., kcat and Km, were determined from non-linear regression fitting of the Michaelis–Menten equation using Prism 5 (Graphpad Software). The reusability of the immobilized preparation was assessed at 50 °C by carrying out hydrolysis of birchwood xylan under the standardized assay condition. The activity of the immobilized enzyme after the first cycle was considered 100 %. Each cycle is defined here as the complete hydrolysis of the substrate present in the reaction mixture. Production of XOSs Free and immobilized crude enzyme preparations (200 U/mg g birchwood xylan) were mixed with 10 ml of 100 mM sodium phosphate buffer (pH 5.0), containing 1 % (w/v) birchwood xylan. The mixture was incubated at 55 °C for 10 h with mild agitation (100 rpm) and centrifuged (1,0009g; 4 °C; 20 min) to obtain the clear supernatant. The supernatant was subjected to HPLC using a Shodex sugar column (SP0810, 8 9 300 mm) at 70 °C with water/acetonitrile (25:75 v/v) as mobile phase at 0.5 ml/min. The retention times of the hydrolytic products (XOSs) were compared with known standards with an evaporative light scattering detector. Results and discussion Identification of the isolated strain From 340 strains screened for xylanase activity, one microorganism was selected on the basis of the

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fluorescence observed when agar plates containing 10 mM of birchwood xylan were exposed under UV light. The ITS rDNA region of the isolated fungus was sequenced, and the ITS sequence was submitted to GenBank with the accession no. JF719544. A BLAST search revealed that the strain showed the highest identity (99 %) with P. adiposa. The approximate phylogenetic position of the strain is shown in Supplementary Fig. 1. The identified strain P. adiposa SKU714 was deposited at the Korean Culture Center of Microorganisms (KCCM) and was given the KCCM accession number 11187P. Xylanase production Various nutritional parameters were tested and optimized through the conventional ‘‘one-variable-at-atime’’ approach using Erlenmeyer flasks (250 ml) (Supplementary Table 1). Single and cumulative effects of different independent variables were evaluated at five different levels (Supplementary Table 2) to analyze the response (Y1). Regression equation analysis (Table S3), determination coefficient (R2 = 0.9872), adequate precision (16.54), and adjacent R2 (0.9768) indicate linearity of the model and hence, confirmed its significance. Second-order polynomial equation was used to evaluate the final response (Y1), i.e., xylanase yield (U/ml): Y1 = 5.24-(0.34 9 A)-(0.22 9 B)-(0.23 9 C) ? (0.09 9 A 9 B)-(0.16 9 A 9 C)-(0.041 9 B 9 C) ? (0.10 9 A2)-(0.028 9 B2) ? (0.013 9 C2). Statistical modeling (Run 10; 30 °C, pH 5.5, rpm 350) resulted in 1,680 ± 148 U/ml xylanase under optimized conditions (Supplementary Table 4). The culture broth also contained EG (26 U/ml), CBH (32 U/ml), and BGL (39 U/ml) activities. Purification and characterization of PaXyl PaXyl was purified using ultrafiltration, ion-exchange, gel filtration, and Mono-Q column chromatography. The results are summarized in Table 1. At the end of Table 1 Different steps involved in purification of xylanase from P. adiposa

Immobilization of PaXyl onto SiO2 nanoparticles CCD-RSM was also applied for the optimization of different physical parameters for immobilization of crude PaXyl using glutaraldehyde-activated SiO2 nanoparticles. Individual and cumulative effects of different independent variables (i.e., temperature [D], pH [E], agitation speed [F], incubation time [G]) were analyzed at five different levels (Table S3) for %IE and %IY (Supplementary Table 6). Regression equation analysis (Supplementary Table 7), determination coefficient (R2 = 0.9722), adequate precision (12.34), and adjacent R2 (0.8532) clearly indicated the significance of the model. Furthermore, second-order

Stage

Protein (mg)

Activity (U)

Ultrafiltration

734.6

150800

205.3

100

1.0

24.7

47050

1903

31.2

9.3

6.7 1.5

16610 7776

2472 5184

11 5.2

12 25.2

DEAE GFC Mono-Q

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the process, xylanase was purified 25-fold. The optimum temperature and pH of PaXyl was 60 °C and 5.5, respectively (Supplementary Fig. 2). Purified PaXyl appeared as a single band on SDS-PAGE as visualized by Coomassie brilliant blue staining, with a mobility corresponding to a molecular mass (Mr) of 37 kDa (Fig. 1). Size exclusion chromatography on a Sephacryl S-300 high resolution column resulted in the elution of the enzyme as a symmetrical peak corresponding to a Mr of approx. 37 kDa and hence, confirmed that PaXyl is a monomer. The monomeric purified PaXyl showed high activity toward birchwood xylan (Supplementary Table 5). The kcat and Km value of the enzyme for birchwood xylan was 4,261 s-1 and 2.4 mg/ml, respectively (Supplementary Fig. 3). Purified PaXyl has exceptional kcat and kcat/Km values which are the highest ever reported for any fungal xylanase and hence, provide significance to the current study (Table 2). PaXyl had a half life of more than 22 h at 50 °C (Supplementary Fig. 4) and did not show any increase in activity at different concentrations of various metal ions, salts, or other chemical agents. The internal peptide sequences (PEKAQWGGGASAGQKL, PEKAEWNAAVNRG, and PERDPGDDVMRHTQG) of purified PaXyl confirmed that it belongs to the GH11 family.

Specific activity (U/mg)

Yield (%)

Purification fold


1311

functionalized silicon oxide nanoparticles, 66 % (IY) of the loaded enzyme (216 mg-protein/g-activated SiO2) was retained on the SiO2 particle. In comparison with the IY (66 %) and IE (144 %) obtained with SiO2 as IM, none of the other tested IMs gave significant values, supporting the use of SiO2 as suitable candidate for immobilization (Supplementary Table 8). Characterization of immobilized PaXyl

Fig. 1 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified xylanase and leaching out of the enzyme during reusability cycles L1 Marker lane; L2 crude protein; L3 purified PaXyl (37 kDa); L4 leached out enzyme after the 17th reusability cycle of immobilized enzyme; L5 leached out enzyme after the 16th reusability cycle during XOS synthesis

polynomial equation was used to evaluate the statistical equation to describe the interaction among the different independent variables and final response, i.e. IE (Y2): Y2 = 122.5 ? (7.65 9 D) ? (9.03 9 E) ? (0.80 9 F) ? (13.1 9 G)-(1.72 9 D 9 E) ? (2.31 9 D 9 F)-(0.22 9 D 9 G)-(1.06 9 E 9 F) ? (3.12 9 E 9 G) ? (4.81 9 F 9 G)-(4.17 9 D2)-(6.32 9 E2) ? (3.27 9 F2)-(11.82 9 G2). On covalent immobilization of the crude xylanase preparation (325 mg protein/g activated SiO2) onto

Although no change in the optimal temperature was observed for immobilized crude PaXyl, an increase (?0.5 U) in pH optima for the immobilized enzyme was observed in comparison to crude free PaXyl (Supplementary Fig. 5). Unoptimized protein loading or low enzyme dose (50 mg/g of activated SiO2) did not give any significant change in the optimal temperature and pH for the immobilized enzyme. However, the relative activity with less enzyme on the support was low in comparison to the relative activity observed with optimized enzyme dose (high protein loading, 200 mg/g activated SiO2) (Supplementary Fig. 6). Furthermore, immobilization provides extra stability to the enzyme (Gawande and Kamat 1998) which can be seen from the thermal stability profile (Supplementary Fig. 7) and reusability profile (Supplementary Fig. 8). Covalently immobilized enzyme retained 97 % of its original activity even after the 17th cycle (Supplementary Fig. 8). Only after the 17th cycle, did immobilized PaXyl begin to leach out in the washing buffer (Fig. 1). This increase in stability of immobilized PaXyl might be due to the fact that

Table 2 Comparison of different purified fungal xylanases Strain

Mw (kDa)

Optimum pH

Temp (°C)

Km (mg ml-1)

kcat (s-1)

kcat/Km (mg ml-1 s-1 )

References

Ximenes et al. (1999)

Acrophialophora nainiana

17

6.0

50

0.7

–

–

Aspergillus fischeri

31

6.0

60

4.9

3.03

0.62

Raj and Chandra (1996)

Aspergillus nidulans

34

6.0

56

1

622

641

Belanic et al. (1995)

Thermomyces lanuginosus

23.6

6.5

70

3.3

2480

760

Lin et al. (1999)

Aureobasidium pullulans

25

4.4

54

7.6

1100

145

Li et al. (1993)

Trichoderma harzianum

20

5.0

50

0.6

0.03

0.06

Tan et al. (1985)

Fusarium oxysporum

20.8

6.0

60

9.5

0.14

0.01

Christakopoulos et al. (1996)

Trichoderma reesei

20

5.0

45

3

–

–

Tenkanen et al. (1992)

Streptomyces sp. B-12-2

23.8

6.0

55

0.8

64.3

80.3

Elegir et al. (1994)

Pholiota adiposa

37

5.0

50

3.3

4260

1300

This study

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1312


immobilization provides extra rigidity to the enzyme support matrix without changing the conformation of the enzyme (Gawande and Kamat 1998). Kinetic parameters (kcat = 2183 s-1, kcat/Km = 921 mg ml-1 s-1) of the immobilized enzyme were similar to those of free PaXyl (kcat = 2109 s-1, kcat/Km = 909 mg ml-1 s-1). A little increase in kcat over immobilization could be explained on the basis of the increased rigidity of the enzyme due to the tight association with the supporting matrix which restricts the accessibility of the enzyme to the substrate (Kapoor and Kuhad 2007).

Conclusion

XOS synthesis

Acknowledgments This research was supported by the Converging Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-50210). This work also was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science and Technology, Republic of Korea. This research was supported by the 2011 KU Brain Pool of Konkuk University.

XOSs produced from different sources of xylans (i.e., birchwood, wheat bran, etc.) can be utilized selectively by the beneficial intestinal microflora, i.e., Bifdiobacteria, and are, thus, expected to be used as valuable food additive (Kapoor and Kuhad 2007). We investigated the potential of free crude PaXyl and immobilized crude PaXyl for XOSs synthesis. In both cases, the amount of xylopentaose (92 % for free and 94 % for immobilized PaXyl) in the product mixture was highest among different XOSs synthesized (Supplementary Fig 9). The high concentration of synthesized xylopentaose further confirmed that the secreted xylanase was an endo-(b-1,4)-xylanase (Kar et al. 2006). Xylopentaose has significant industrial applications as it is the major constituent of snack foods, bakery products, and flavorings (Gawande and Kamat 1998). In case of immobilized PaXyl, the concentration of both xylobiose (X2) and xylotriose (X3) was only 1 %, and the concentration of xylotetraose (X4) was nearly 5 % of total sugars (10.3 mg/ml). Free PaXyl produced similar concentrations of X4 (4 %) and X3 (3 %), followed by X2 (1 %) of total sugars (5.7 mg/ml). Immobilization improves the enzyme’s affinity for the respective substrate compared to the free enzyme (Singh et al. 2011) and this might be the reason for the higher yield of total sugars generated by immobilized PaXyl as compared to the free enzyme. Immobilized PaXyl gave 44.6 % higher concentrations of XOSs compared to free PaXyl. Concentration of XOSs produced is higher than previous reports (Kapoor and Kuhad 2007), which further validate the current work and also supports its potential in industrial application.

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An exceptionally active endoxylanase PaXyl has been purified, and characterized. Glutaraldehyde crosslinked SiO2 nanoparticles proved to be a suitable support matrix for PaXyl immobilization. Increased production of XOSs with immobilized PaXyl further underscores the significance of the present study. The highest ever reported turnover rate and significant stability of the covalently immobilized PaXyl broadens its scope for other applications.

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Biotechnol Lett (2012) 34:1307–1313 thermophilic fungus Thermomyces lanuginosus-SSBP. Biotechnol Applied Biochem 30:73–79 Raj KC, Chandra TS (1996) Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1. FEMS Microbiol Letter 145:457–461 Singh RK, Zhang YW, Nguyen NP, Jeya M, Lee JK (2011) Covalent immobilization of b-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles. Appl Microbiol Biotechnol 89:337–344 Tan LUL, Wong KKY, Yu EKC, Saddler JN (1985) Purification and characterization of two D-xylanases from Trichoderma harzianum. Enzyme Microbiol Technol 7:425–443

1313 Tenkanen H, Puls J, Poutanen K (1992) Two major xylanases of Trichoderma reesei. Enzyme Microbiol Technol 14:566–574 White TJ, Bruns T, Lee S, Taylor J (1990) PCR protocols: a guide to methods and applications. Academic Press, New York Ximenes FA, Sousa MV, Puls J, SilvaJr FG, Filho EXF (1999) Purification and characterization of a low-molecular weight xylanase produced by Acrophialophora nainiana. Curr Microbiol 38:18–22 Zhang YW, Tiwari MK, Jeya M, Lee JK (2011) Covalent immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase onto modified silica nanoparticles. Appl Microbiol Biotechnol 90:499–507

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