January 2016, Vol.82:127–133, doi:10.1016/j.ijbiomac.2015.10.012

Immobilization of pectin depolymerising polygalacturonase using different polymers

• Haneef Ur Rehman ^a,,
• Afsheen Aman ^b
• Muhammad Asif Nawaz ^c
• Asad Karim ^b
• Maria Ghani ^b
• Abdul Hameed Baloch ^d
• Shah Ali Ul Qader ^b

• aDepartment of Chemistry, University of Turbat, Kech, Balochistan, Pakistan
• bThe Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi 75270, Pakistan
• cDepartment of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, Dir (Upper), KPK, Pakistan
• dFaculty of Agriculture, Lasbela University of Agriculture, Water and Marine Sciences (LUAWMS), Uthal 90150, Balochistan, Pakistan

Received 20 April 2015. Revised 1 October 2015. Accepted 5 October 2015. Available online 25 October 2015.

Highlights

• •
  Calcium alginate beads, agar-agar and polyacrylamide gel were used as carriers for the immobilization of polygalacturonase.
• •
  Polyacrylamide showed most promising results in term of immobilization yield.
• •
  The thermal stability of polygalacturonase was increased after entrapment with polymers.
• •
  Polymers entrapped polygalacturonases showed good reusability and retained more than 80% during 2nd cycle.

Abstract

Polygalacturonase catalyses the hydrolysis of pectin substances and widely has been used in food and textile industries. In current study, different polymers such as calcium alginate beads, polyacrylamide gel and agar-agar matrix were screened for the immobilization of polygalacturonase through entrapment technique. Polyacrylamide gel was found to be most promising one and gave maximum (89%) immobilization yield as compared to agar-agar (80%) and calcium alginate beads (46%). The polymers increased the reaction time of polygalacturonase and polymers entrapped polygalacturonases showed maximum pectinolytic activity after 10 min of reaction as compared to free polygalacturonase which performed maximum activity after 5.0 min of reaction time. The temperature of polygalacturonase for maximum enzymatic activity was increased from 45 °C to 50 °C and 55 °C when it was immobilized within agar-agar and calcium alginate beads, respectively. The optimum pH (pH 10) of polygalacturonase was remained same when it was immobilized within polyacrylamide gel and calcium alginate beads, but changed from pH 10 to pH 9.0 after entrapment within agar-agar. Thermal stability of polygalacturonase was improved after immobilization and immobilized polygalacturonases showed higher tolerance against different temperatures as compared to free enzyme. Polymers entrapped polygalacturonases showed good reusability and retained more than 80% of their initial activity during 2nd cycles.

Keywords

• Polygalacturonase
• Immobilization
• Entrapment
• Calcium alginate beads
• Agar-agar matrix
• Polyacrylamide gel

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1 Introduction

Advancement of biotechnology over last three decades, enzyme have drawn considerable interest of many biotechnologist because of having their excellent characteristics (high activity, selectivity and specificity under mild reaction conditions). Enzymes have been used in different industries including food, textiles, detergents and pharmaceutical industry. Enzymes are environmentally and economically more attractive as compared to organic catalyst because utilization of enzymes avoids the need of functional groups protections/activation, and provide a shorter synthetic routs for formation fine chemicals. Pectinase is a generic term used for a group of enzymes which catalyze the degradation of pectin substances by hydrolysis, trans-elimination and de-esterification reactions [1]. Among them, polygalacturonase is the most promising that completely hydrolysed pectin polymers into galacturonic acids monomers. Polygalacturonase has significant role in various industries with wide range of applications in different industrial processes such as fruit juices extraction, textile processing, paper making, pectin containing waste water treatment, degumming of plants bast fibers, wine clarification, oil extraction, coffee and tea fermentation [2], [3] and [4]. But, the industrial applications of enzymes are often hampered due to low operational stability in harsh industrial conditions [5]. So there are many techniques such as protein engineering, chemical modification, adding additives and immobilization have utilized to engineer the enzymes from their native form to designed industrial bioreactor [6], [7], [8] and [9]. Immobilization not only increases the operational stability of enzymes but also makes them reusable for continuous industrial process. Immobilization is a process to confine or localize the enzyme within/onto the carrier and retained its activity for continuous uses. Different methods have been used for the immobilization of enzyme which can be categorized into three types such as binding of enzyme to a carrier, enzymes crosslinking and entrapment or encapsulation of enzymes within polymers. Entrapment is a simple and easy method for the immobilization of enzymes which induces no conformational modification on to the biological activities of enzymes. Up to date various supports have been used for the immobilization of polygalacturonase by different methods [10], [11], [12], [13], [14] and [15]. In this study, calcium alginate beads, polyacrylamide gel and agar-agar matrix were screened for the immobilization of polygalacturonase through entrapment technique. The kinetic properties of polymers entrapped polygalacturonases were evaluated with the comparison of free enzyme. The thermal and operational stabilities of entrapped polygalacturonases were investigated to evaluate the industrial feasibilities of polygalacturonase.

2 Materials and methods

2.1 Production of polygalacturonase

The production of polygalacturonase was carried out from Bacillus licheniformisKIBGE-IB21 in pectin containing production medium using submerged fermentation technology at 37 °C for 48 h [16]. After 48 h of fermentation crude polygalacturonase was separated from bacterial cell by centrifugation (10,000 rpm for 15 min) and precipitated through 50% ammonium sulphate saturation. The obtained precipitates were then dissolved in minimum amount of glycine–NaOH buffer (50 mM and pH 10) and dialyzed against the same buffer to remove the salts. The dialyzed enzyme solutions were used for the immobilization within different polymers.

2.2 Immobilization of polygalacturonase

The polygalacturonase was immobilized through entrapment technique using different natural and synthetic polymers. The immobilization yield was calculated through following equation;

2.2.1 Entrapment within calcium alginate beads

The immobilization of polygalacturonase was carried out by mixing sodium alginate solution (3.0%) with partially purified enzyme solution in 1:1 ratio [17]. The calcium alginate beads with 2.0 mm diameter of size were formed by adding mixture of sodium alginate and enzyme solution in 0.2 M CaCl₂ solution drop-wise at 4.0 °C through needle. Substitution of calcium with the sodium due to having strong affinity to alginate leads to gelatinization of alginate to form calcium alginate beads. Beads were hardened by keeping in CaCl₂ solution for 30.0 min at 4.0 °C. The calcium alginate beads were then washed dried, weighted and stored in buffer (50 mM glycine–NaOH buffer, pH 10).

2.2.2 Entrapment within agar-agar matrix

Agar-agar solution (3.0%) was prepared in glycine–NaOH buffer (50 mM and pH 10) by gently heating. Equal volume of enzyme and agar-agar solution (1:1 ratio) was mixed and immediately casted in pre-assembled glass plate. After solidification at the room temperature, the gel was cut into small pieces (2.0 mm in diameter) and washed several times before use. These pieces were stored in glycine–NaOH buffer (50 mM, pH 10.0) at 4 °C for further studies [18].

2.2.3 Entrapment within polyacrylamide gel

The entrapment of polygalacturonase within polyacrylamide gel was done by polymerization of acrylamide (9.5%) and N,N′-methylenebisacrylamide (0.5%) in the presence of 4.0 ml enzyme solution by the addition of 0.375 ml ammonium persulphate (20%) and 0.003 ml commercially prepared 14.4 M tetramethylethylenediamine (TEMED). Gel was cut into pieces of equal size and washed with glycine–NaOH buffer (50 mM, pH 10) and deionized water before use.

2.3 Enzyme assay

The enzymatic activity of free and polymers entrapped polygalacturonase were estimated through DNS method using citrus pectin as a substrate (1.0%) and galacturonic acid monohydrate as standard [19]. The enzyme assay was performed by addition of 0.05 ml of partially purified enzyme solution and 0.5 g of immobilized enzyme matrixes separately into 1.0 ml citrus pectin solution of pH 10 and incubated at 45 °C for 5 min. After 5 min, 0.5 ml reaction mixtures were withdraw from each tubes and added separately into 1.0 ml DNS reagent for the estimation of reducing sugar.

One unit of polygalacturonase is defined as “the amount of enzyme required to release 1 μmole of galacturonic acid per minute under standard assay conditions”.

2.4 Scanning electron microscopy

Scanning electron microscopy was used to study the surface topologies of calcium alginate beads, agar-agar and polyacrylamide gel before and after entrapment of polygalacturonase. The dried samples were sputter-coated with gold using Auto coater (Model JFC-1500 Jeol, Japan) and micrographs were taken using scanning electron microscope (JSM 6380A Jeol, Japan).

2.5 Characterization of polymers entrapped polygalacturonases

2.5.1 Effect of reaction time on the activities of polymers immobilized polygalacturonases

The effect of reaction time on the activity of calcium alginate beads, agar-agar matrix and polyacrylamide gel entrapped polygalacturonases were separately investigated by performing the enzyme assay for different time interval (5.0–60.0 min) with the comparison of free enzyme.

2.5.2 Effect of temperature on the activities of polymers immobilized polygalacturonases

The influence of temperature on the activity of entrapped polygalacturonases were determined by measuring the enzyme reaction in different incubation temperatures ranging from 30 °C to 60 °C, while keeping the pH and reaction time constant.

2.5.3 Effect of pH on the activities of polymers immobilized polygalacturonases

Effect of pH on the activities of calcium alginate, agar-agar matrix and polyacrylamide gel entrapped polygalacturonases were determined by performing the enzyme substrate reaction in various pH levels ranging from 5.0 to 12.0 at constant conditions. Different buffers such as sodium acetate buffer (pH 5.0 and pH 6.0), potassium phosphate buffer (pH 7.0 and pH 8.0) and glycine–NaOH buffer (pH 9.0–12) were used in the experiment.

2.5.4 Kinetic parameters of polymers immobilized polygalacturonases

The kinetic parameters such as maximum reaction rate (V_max) and Michaelis–Menten constant values (K_m) of entrapped polygalacturonases were determined by measuring the rate of reaction in different substrate concentration ranging from 1.0 to 20 mg ml⁻¹ in glycine–NaOH buffer (pH 10).

2.5.5 Thermal stability of polymers immobilized polygalacturonases

The thermal de-activation of entrapped polygalacturonases was determined by keeping the immobilized enzymes in glycine–NaOH buffer (50 mM, pH 10.0) at different temperatures ranging from 20.0 to 60.0 °C for different time intervals (24–120 h). After every 24 h, enzyme assay of different entrapped polygalacturonases were performed separately under standard assay conditions for the estimation of residual activity of each entrapped polygalacturonases.

2.5.6 Reusability of polymers immobilized polygalacturonases

The operational stabilities of immobilized polygalacturonases were determined by repeatedly reusing the same calcium alginate beads, agar-agar matrix and polyacrylamide gel entrapped enzymes for pectin hydrolysis. After each reaction, the entrapped enzymes were washed with glycine–NaOH buffer (pH 10) and deionized water to remove residual substrate onto the immobilized polygalacturonases. Fresh substrates were added for next reaction. The initial activities of polygalacturonase entrapped polymers were considered as 100%.

3 Results and discussion

3.1 Immobilization of polygalacturonase

For enhancing the operational stability and continuous reusability of enzyme, the partially purified polygalacturonase from B. licheniformis KIBGE-IB21 was immobilized within different polymers including calcium alginate beads, polyacrylamide gel and agar-agar matrix. The concentration of each polymer was individually optimized for the immobilization of maximum polygalacturonase and maximum immobilization yield (%) were obtained using 3.0% sodium alginate, 9.5% acrylamide and 3.0% agar-agar (Fig. 1). Among these polymers, polyacrylamide gel was found to be most promising and gave higher immobilization yield (89%), followed by agar-agar that retained 80% activity after immobilization. While less immobilization yield was observed in case of calcium alginate beads that only retained 46% activity. The immobilization yield of enzyme not only depends on the concentration of polymers used but also on the molecular size of the enzyme and polyacrylamide might be tightly wrapped the enzyme as compared to others.

Fig. 1. Concentration effect of different polymers on the immobilization yield of polygalacturonase. Immobilization yield of immobilized polygalacturonase after entrapment within calcium alginate (A), polyacrylamide (B) and agar-agar (C) (means ± S.E., n = 6).

3.2 Scanning electron microscopy

The surface topologies of polymers before and after entrapment of polygalacturonase were determined through scanning electron microscopic technology. The surface topologies of alginate beads, agar-agar matrix and polyacrylamide gel were significantly changed after entrapment of polygalacturonase (Fig. 2). The SEM images showed that the polymers before entrapment of polygalacturonase appeared smooth but after immobilization of polygalacturonase the surface of polymers showed a considerable extent of irregularity. These changes were due to the entrapment of different molecules of enzyme on to the porous surface of polymers. The structural topology of calcium alginate beads was maintained after immobilization and various black particles appeared in pores of calcium alginates beads of entrapped polygalacturonase (Fig. 2A). Similar observation was also reported on immobilization of penicillin acylase on APTES functionalized SBA-15 [20]. Similar image was observed in the micrographs of agar-agar matrix and polyacrylamide gel, the surface topologies of agar-agar matrix and polyacrylamide gel were not disturbed by immobilization of polygalacturonases but different particles were found to be entrapped on the surface of these polymers (Fig. 2A and B). Zhao et al. also reported similar findings according to the surface topologies of polymer with and without immobilized enzyme using SEM [21].

Fig. 2. Scanning electron micrographs (SEM) of polymers before and after entrapment of polygalacturonase. (A) Micrographs of calcium alginate beads without (a) and with (b) entrapped polygalacturonase at magnification scales of 10,000×. (B) Micrographs of polyacrylamide gel without (a) and with (b) entrapped polygalacturonase at magnification scales of 10,000×. (C) Micrographs of Agar-agar matrix without (a) and with (b) entrapped polygalacturonase at magnification scales of 10,000×.

3.3 Influence of reaction time on the activities of polymer entrapped polygalacturonases

The influence of reaction time on the activities of polymers entrapped polygalacturonases were determined by measuring the rate of enzymatic reaction with different incubation periods (5.0–60 min) with the evaluation of free polygalacturonase. Entrapment increased the incubation period of polygalacturonase from 5.0 to 10 min. Calcium alginate beads, agar-agar matrix and polyacrylamide gel entrapped enzyme showed maximum relative activities after 10 min of reaction as compared to free enzyme which showed maximum activity after 5.0 min of reaction time (Fig. 3). The increased of reaction time could be due to the diffusion limitation of polymers for the substrate molecules and substrate take time to diffuse into porous structure of polymers in order to react with the entrapped enzyme. Similar finding was reported earlier that the immobilized dextransucrase showed maximum enzyme activity after 60 min of reaction as compared to free enzyme that achieved it within 15 min [22].

Fig. 3. Influence of different range of reaction times on the activities of polymers entrapped polygalacturonases with comparison of free polygalacturonase at constant temperature (45 °C), pH (pH 10) and substrate concentration (1.0%) (means ± S.E., n = 6).

3.4 Influence of temperature on the activities of polymers entrapped polygalacturonases

The optimization of temperature is an important step for enzymatic activity for both free as well as immobilized enzyme. The temperature dependent activities of polymers entrapped polygalacturonases were determined at different temperatures ranging from 20 °C to 60 °C (Fig. 4). The optimum temperature for maximum enzymatic activity of free polygalacturonase was 45 °C but after entrapment of polygalacturonases within agar-agar matrix and calcium alginate beads the optimum temperature was shifted from 45 °C to 50 °C and 55 °C, respectively. The increased of temperature for maximum enzymatic activities could be due to physical limitation of microenvironment of polymers and higher activation energy required for the enzyme and substrate to achieved the transition state of reaction [23]. The polymers maybe increase the conformational rigidity of enzyme and protect the enzyme against denaturation at higher temperature. While in case of entrapment within polyacrylamide gel the optimum temperature of polygalacturonase did not change and both the polyacrylamide gel entrapped as well as free polygalacturonase showed maximum activities at 45 °C. Lei and Jiang also reported similar observation on the immobilization of polygalacturonase using same support [24]. The immobilized polygalacturonase showed higher relative activity at different temperatures and the range of temperature for maximum enzymatic activity was expanded after immobilization. Phadtare et al. observed similar results with greater relative activities of immobilized Pencillin G acylase at different temperatures [25].

Fig. 4. Influence of various temperatures on the activities of polymers entrapped polygalacturonases with the comparison of free polygalacturonase at constant reaction time (10 min), substrate concentration (1.0%) and pH (pH 10) (means ± S.E., n = 6).

3.5 Influence of pH on the activities of polymers entrapped polygalacturonases

The influence of pH on the activities of polymers entrapped polygalacturonases were determined by performing the assay in different pH level ranging from 5.0 to 12.0. Calcium alginate beads and polyacrylamide gel did not influence the pH of polygalacturonase and entrapped polygalacturonases showed maximum relative activities at pH 10 similar to free enzyme (Fig. 5). While, the optimum pH range of polygalacturonase was expanded to some extent after entrapment within calcium alginate beads and polyacrylamide gel. The pH profile of polymers entrapped polygalacturonases indicated that the microenvironment of calcium alginate beads and polyacrylamide might have buffered and protected the enzyme from the acidity of reaction medium; therefore, the immobilized polygalacturonases were less affected by pH as compared to free one. Similar findings were reported previously that the immobilization did not make any change on optimum pH of polygalacturonase but the range of relative activities was expanded [15] and [26]. In case of entrapment within agar-agar matrix the optimum pH of polygalacturonase was shifted to lower from pH 10 to pH 9 as compared to free enzyme. The alteration in pH value of polygalacturonase could be due to the nature as well as the structure and the composition of matrix, which might be responsible for the deviation of the optimum pH value for maximum enzyme activity [27].

Fig. 5. Influence of various pH on the activities of polymers entrapped polygalacturonase with the comparison of free polygalacturonase at constant reaction time (10 min) temperature (45 °C) and substrate concentration (1.0%) (means ± S.E., n = 6).

3.6 Thermal stability of polymers entrapped polygalacturonases

The thermal stability of polymers entrapped polygalacturonases were evaluated by pre-incubation of entrapped polygalacturonases at different temperatures ranging from 30 °C to 60 °C for 120 h and after every 24 h aliquots were taken for calculation of residual enzyme activities. The polymers entrapped polygalacturonases inactivated much slower rate as compared to free polygalacturonase at different temperatures (Fig. 6). The polymers entrapped enzymes retained more than 80% and 70% residual activity after 120 h at 30 °C and 40 °C, respectively whereas the free enzyme only showed 40% and 30% residual activity under same conditions (Fig. 6A and B). While after 72 h at 50 °C, the free polygalacturonase lost its complete activity but agar-agar, polyacrylamide gel and calcium alginate beads entrapped polygalacturonases retained 25%, 42% and 43% activity under same conditions, respectively (Fig. 6C). The stability of polyacrylamide gel entrapped polygalacturonase was found to be higher in some extent as compared to calcium alginate beads and agar-agar entrapped polygalacturonases, and retained more than 15% activity after 120 h at 50 °C. The improved thermal stability might be attributed to the stabilizing effect of supports used that restricts the conformational changes of thermal denaturation. However, at 60 °C both the free as well as polymers entrapped polygalacturonases lost their complete activities after 24 h. The reduction of enzymatic activities at certain temperatures was due to denaturation effect of these temperatures.

Fig. 6. Thermal stability of polymers entrapped polygalacturonases with the comparison of free polygalacturonase at (A) 30 °C, (B) 40 °C and (C) 50 °C with respect to different time intervals (means ± S.E., n = 6).

3.7 Kinetic parameters of polymers entrapped polygalacturonases

The kinetic parameters such as K_m and V_max of polymers entrapped polygalacturonases were estimated by performing the enzyme assay using different concentration of citrus pectin under standard assay conditions. The K_m and V_maxvalues were calculated from the plot of 1/[V] against 1/[S]. The K_m value of polygalacturonase was slightly increased from 1.017 mg ml⁻¹ to 1.055, 1.042 and 1.033 mg ml⁻¹, and V_max was apparently decreased from 23800 μM min⁻¹ to 11410, 19392 and 20921 μM min⁻¹ after entrapment within calcium alginate beads, polyacrylamide gel and agar-agar, respectively (Fig. 7). The increased of K_m and decreased V_max values of polymers entrapped polygalacturonases could be due to steric obstruction of active site of enzyme by support, decrease of enzyme flexibility required for binding of substrate or diffusion limitation of substrate and product to move in and out of the support. However, polyacrylamide caused less diffusion limitation for mass transfer of substrate and product within the polymer as compared to agar-agar and calcium alginate beads and showed K_m and V_max values very close to free polygalacturonase.

Fig. 7. Kinetic parameters of polymers entrapped polygalacturonase with the comparison of free polygalacturonase. (A) Michaelis–Menten and Lineweaver–Burk plots of free polygalacturonase. (B) Michaelis–Menten and Lineweaver–Burk plots of calcium beads entrapped polygalacturonase. (C) Michaelis–Menten and Lineweaver–Burk plots of polyacrylamide gel entrapped polygalacturonase. (D) Michaelis–Menten and Lineweaver–Burk plots of agar-agar entrapped polygalacturonase.

3.8 Recycling efficiency of polymers entrapped polygalacturonases

Recycling is one of the key qualities of immobilized enzyme system for the financial feasibility. The recycling efficiency of polymers entrapped polygalacturonases was determined by measuring the enzymatic activity of same immobilized polygalacturonases in 10 continuous cycles. After every cycle the polymers immobilized polygalacturonases were washed with buffer (0.05 M glycine–NaOH buffer of pH 10) and new substrates were added for next cycle. Polyacrylamide gel and agar-agar entrapped polygalacturonases showed higher recycling efficiency and retained 22% and 15% of their initial activities during 10th cycle, respectively as compared to calcium alginate beads entrapped polygalacturonase which lost its complete activity after 7th cycle (Fig. 8). The lost of enzymatic activities of polymers entrapped enzymes could be due to leakage of enzyme from polymers matrixes during washing after the end of each cycle or it could be due to the conformational changes by repeatedly reusing of the enzyme.

Fig. 8. Recycling efficiency of polymers entrapped polygalacturonases in batch reactions. The first cycle is considered as 100% (means ± S.E., n = 6).

4 Conclusion

Polymers play significant role in different aspects of biological sciences and current study calcium alginate, polyacrylamide gel and agar-agar matrix were used to immobilize polygalacturonase by entrapment technique. The thermal stability of polygalacturonase was improved after immobilization and polymers immobilized enzymes showed higher tolerance against different temperatures as compared to soluble enzyme. The polymers entrapped polygalacturonases also exhibited good reusability. Immobilization of polygalacturonase through entrapment method within different polymers is a simple method to enhance the catalytic properties of enzyme. In term of immobilization efficiency and reusability, these approaches might have potential applications in various food and textile industries. Further, significant research work will require in order evaluating the feasibility of this approach under different industrial processes.

Acknowledgements

Authors gratefully acknowledge the financial support from KIBGE, University of Karachi, Karachi, Pakistan.

References

1. • [1]
   • L. Ceci, J. Lozano
   • Determination of enzymatic activities of commercial pectinases for the clarification of apple juice
   • Food Chem., Volume 61, 1998, pp. 237–241
   • Article
      | 
      PDF (162 K)
      | 
     View Record in Scopus
     Citing articles (52)
2. • [2]
   • R.S. Jayani, S. Saxena, R. Gupta
   • Microbial pectinolytic enzymes: a review
   • Process Biochem., Volume 40, 2005, pp. 2931–2944
   • Article
      | 
      PDF (243 K)
      | 
     View Record in Scopus
     Citing articles (372)
3. • [3]
   • G.S. Hoondal, R.P. Tiwari, R. Tiwari, N. Dahiya, Q.K. Beg
   • Microbial alkaline pectinase and their industrial application
   • Appl. Microbiol. Biotechnol., Volume 59, 2002, pp. 409–418
   • View Record in Scopus
     Citing articles (210)
4. • [4]
   • D.R. Kashyap, P.K. Vohra, S. Chopra, R. Tewari
   • Applications of pectinases in the commercial sector: a review
   • Bioresour. Technol., Volume 77, 2001, pp. 215–227
   • Article
      | 
      PDF (153 K)
      | 
     View Record in Scopus
     Citing articles (431)
5. • [5]
   • R.A. Sheldon
   • Enzyme immobilization: the quest for optimum performance
   • Adv. Synth. Catal., Volume 349, 2007, pp. 1289–1307
   • View Record in Scopus
      | 
     Full Text via CrossRef
     Citing articles (805)
6. • [6]
   • J. Minshull, S. Govindarajan, T. Cox, J.E. Ness, C. Gustafsson
   • Engineered protein function by selective amino acid diversification
   • Methods, Volume 32, 2004, pp. 416–427
   • Article
      | 
      PDF (161 K)
      | 
     View Record in Scopus
     Citing articles (8)
7. • [7]
   • S.A. Costa, T. Tzanov, A.F. Carneiro, A. Paar, G.M. Gubitz, A. Cavaco-Paulo
   • Studies of stabilization of native catalase using additives
   • Enzyme Microb. Technol., Volume 30, 2002, pp. 387–391
   • Article
      | 
      PDF (63 K)
      | 
     View Record in Scopus
     Citing articles (49)
8. • [8]
   • K. Khajeh, B. Ranjbar, H. Naderi-Manesh, A.E. Habibi, M. Nemat-Gorgani
   • Chemical modification of bacterial α-amylases: changes in tertiary structures and the effect of additional calcium
   • Biochim. Biophys. Acta, Volume 1548, 2001, pp. 229–237
   • Article
      | 
      PDF (338 K)
      | 
     View Record in Scopus
     Citing articles (40)
9. • [9]
   • Z. Bílková, J. Mazurvá, J. Churácek, D. Horák, J. Turková
   • Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support
   • J. Chromatogr. A, Volume 852, 1999, pp. 141–149
   • Article
      | 
      PDF (299 K)
      | 
     View Record in Scopus
     Citing articles (13)
10. • [10]
    • M.L. Buga, S. Ibrahim, A.J. Nok
    • Physico-chemical characteristics of immobilized polygalacturonase from Aspergillus niger (SA6)
    • Afr. J. Biotechnol., Volume 9, 2010, pp. 8934–8943
    • View Record in Scopus
      Citing articles (6)
11. • [11]
    • M.A. Esawy, A.A. Gamal, Z. Kamel, A.S. Ismail, A.F. Abdel-Fattah
    • Evaluation of free and immobilized Aspergillus niger NRC1ami pectinase applicable in industrial processes
    • Carbohydr. Polym., Volume 92, 2013, pp. 1463–1469
    • Article
       | 
       PDF (651 K)
       | 
      View Record in Scopus
      Citing articles (9)
12. • [12]
    • M.E. Carrin, L. Ceci, J.E. Lozano
    • Effects of pectinase immobilization during hollow fiber ultrafiltration of apple juice
    • J. Food Process Eng., Volume 23, 2000, pp. 281–298
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (6)
13. • [13]
    • K. Liu, G. Zhao, B. He, L. Chena, L. Huanga
    • Immobilization of pectinase and lipase on macroporous resin coated with chitosan for treatment of whitewater from papermaking
    • Bioresour. Technol., Volume 123, 2012, pp. 616–619
    • Article
       | 
       PDF (437 K)
       | 
      View Record in Scopus
      Citing articles (19)
14. • [14]
    • K. Liu, X. Li, X. Li, B. He, G. Zhao
    • Lowering the cationic demand caused by PGA in papermaking by solute adsorption and immobilized pectinase on chitosan beads
    • Carbohydr. Polym., Volume 82, 2010, pp. 648–652
    • Article
       | 
       PDF (226 K)
       | 
      View Record in Scopus
      Citing articles (16)
15. • [15]
    • T. Li, S. Li, N. Wang, L. Tain
    • Immobilization and stabilization of pectinase by multipoint attachment onto an activated agar-gel support
    • Food Chem., Volume 109, 2008, pp. 703–708
    • Article
       | 
       PDF (176 K)
       | 
      View Record in Scopus
      Citing articles (36)
16. • [16]
    • H.U. Rehman, S.A.U. Qader, A. Aman
    • Polygalacturonase: production of pectin depolymersing enzyme from Bacillus licheniformis KIBGE IB-21
    • Carbohydr. Polym., Volume 90, 2012, pp. 387–391
    • Article
       | 
       PDF (354 K)
       | 
      View Record in Scopus
      Citing articles (18)
17. • [17]
    • H.U. Rehman, A. Aman, A. Silipo, S.A.U. Qader, A. Molinaro, A. Ansari
    • Degradation of complex carbohydrate polymer: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support
    • Food Chem., Volume 139, 2013, pp. 1081–1086
    • Article
       | 
       PDF (719 K)
       | 
      View Record in Scopus
      Citing articles (19)
18. • [18]
    • H.U. Rehman, A. Aman, R.R. Zohra, S.A.U. Qader
    • Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support
    • Carbohydr. Polym., Volume 102, 2014, pp. 622–626
    • Article
       | 
       PDF (1128 K)
       | 
      View Record in Scopus
      Citing articles (12)
19. • [19]
    • G.L. Miller
    • Use of dinitrosalicylic acid reagent for determination of reducing sugars
    • Anal. Chem., Volume 31, 1959, pp. 426–428
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (11996)
20. • [20]
    • P. Shah, N. Sridevi, A. Prabhune, V. Ramaswamy
    • Structural features of penicillin acylase adsorption on APTES functionalized SBA-15
    • Microporous Mesoporous Mater., Volume 116, 2008, pp. 157–165
    • Article
       | 
       PDF (708 K)
       | 
      View Record in Scopus
      Citing articles (59)
21. • [21]
    • Q. Zhao, J. Sun, H. Ren, Q. Zhou, Q. Lin
    • Horseradish peroxidase immobilized in macroporous hydrogel for acrylamide polymerization
    • J. Polym. Sci. Part A: Polym. Chem., Volume 46, 2008, pp. 2222–2232
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (19)
22. • [22]
    • S.A.U. Qader, A. Aman, N. Syed, S. Bano, A. Azhar
    • Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4
    • Ital. J. Biochem., Volume 56, 2007, pp. 158–162
    • View Record in Scopus
      Citing articles (11)
23. • [23]
    • F. Kara, G. Demirel, H. Tümtürk
    • Immobilization of urease by using chitosan-alginate and poly(acrylamide-co-acrylic acid)/kappa-carrageenan supports
    • Bioprocess Biosyst. Eng., Volume 29, 2006, pp. 207–211
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (35)
24. • [24]
    • Z. Lei, Q. Jiang
    • Synthesis and properties of immobilized pectinase onto the macroporous polyacrylamide microspheres
    • J. Agric. Food Chem., Volume 59, 2011, pp. 2592–2599
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (16)
25. • [25]
    • S. Phadtare, P. Parekh, A. Gole, M. Patil, A. Pundle, A. Prabhune, M. Sastry
    • Pencillin G acylase-fatty lipid biocomposite films show excellent catalytic activity and long term stability/reusability
    • Biotechnol. Prog., Volume 18, 2002, pp. 483–488
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (17)
26. • [26]
    • A. Bahrami, P. Hejazi
    • Electrostatic immobilization of pectinase on negatively charged AOT–Fe₃O₄nanoparticles
    • J. Mol. Catal. B: Enzym., Volume 93, 2013, pp. 1–7
    • Article
       | 
       PDF (1049 K)
       | 
      View Record in Scopus
      Citing articles (10)
27. • [27]
    • D. Andriani, C. Sunwoo, H.W. Ryu, B. Prasetya, D. Park
    • Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material
    • Bioprocess Biosyst. Eng., Volume 35, 2012, pp. 29–33
    • View Record in Scopus
       | 
      Full Text via CrossRef
      Citing articles (17)

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