Xylanase Immobilization In 2000s

Sugar Crush Detox

Natural Remedies for Food Cravings

Get Instant Access

Enzyme Immobilisatiobn

Fig. 1. Characterization of enzyme-polymer conjugate by (A) fluorescence spectroscopy difference spectrum of the immobilized and free enzymes and difference spectrum of the immobilized enzyme after subtracting the contribution of the polymer from the enzyme spectrum and (B) circular dichroic measurements. Xylanase was purified by affinity precipitation with Eudragit S-100, before being immobilized on Eudragit L-100. Equal amounts of protein were taken for (I) free as well as (II) immobilized enzyme. (From ref. 17 with permission from Elsevier.)

Fig. 1. Characterization of enzyme-polymer conjugate by (A) fluorescence spectroscopy difference spectrum of the immobilized and free enzymes and difference spectrum of the immobilized enzyme after subtracting the contribution of the polymer from the enzyme spectrum and (B) circular dichroic measurements. Xylanase was purified by affinity precipitation with Eudragit S-100, before being immobilized on Eudragit L-100. Equal amounts of protein were taken for (I) free as well as (II) immobilized enzyme. (From ref. 17 with permission from Elsevier.)

2. Materials

2.1. Assay of Xylanase Activity

1. Aspergillus niger xylanase (supplied as Pectinex 3XL, Novozymes, Bagsvaerd, Denmark)

2. Buffer 1: 0.05 mMsodium acetate buffer, pH 5.6.

3. Oat spelt xylan (Sigma Chemical Co., St. Louis, MO).

4. Dinitrosalicylic acid reagent (18).

2.2. Preparation of Eudragit L-100 Solution

1. Eudragit L-100 (Rohm Pharma GmbH, Weiterstadt, Germany).

4. Distilled water.

5. Pasteur pipet.

6. Small magnetic bar.

7. Magnetic stirrer.

2.3. Immobilization of Xylanase on Eudragit L-100

1. 0.1 NAcetic acid.

2. Buffer 2: 0.05 Msodium acetate buffer, pH 4.0.

3. Benchtop centrifuge with a rotor capable of accommodating 10-mL centrifuge tubes and achieving a minimum RCF value of 12,000g.

3. Methods

3.1. Assay of Xylanase Activity (19)

1. Incubate the enzyme sample (1 mL, appropriately diluted in Buffer 1) in a reaction mixture containing 1% (w/v) xylan suspension (in 1 mL Buffer 1) at 50°C (see Note 1).

2. Stop the reaction after 30 min by adding 1 mL dinitrosalicylic acid reagent (18) and immersing the test tubes in a boiling water bath. Cool the test tubes after boiling for 5 min, add 10 mL of distilled water, shake, and read the absorbance of the liberated reducing sugars at 540 nm. One enzyme unit liberates 1 |imol of xylose per minute at 50°C, pH 5.6. Because the immobilized enzyme is soluble at pH 5.6, its activity can also be measured in the same manner as that for the free enzyme.

3.2. Preparation of Eudragit L-100 Solution

1. Dissolve 100 g Eudragit L-100 in 40 mL of double distilled water by stirring the suspension on a magnetic stirrer and adding 3 M NaOH dropwise using a Pasteur pipet until the pH increases to 11.0 (see Note 2).

2. After the polymer dissolves completely, decrease the pH to 4.3 by adding 3 M HCl (see Fig. 2). Centrifuge the precipitated Eudragit, decant the supernatant and dissolve the precipitate by adding 40 mL of distilled water and increasing the pH to 7.0 with 3 M NaOH. Make up the total volume to 50 mL with distilled water. This solution is quite stable when stored at 4°C for a few weeks.

Fig. 2. Preparation of Eudragit L-100 solution for the immobilization of xylanase.

pH meter Magnetic stirrer

Fig. 2. Preparation of Eudragit L-100 solution for the immobilization of xylanase.

3.3. Immobilization of Xylanase on Eudragit L-100 (17) (see Note 3)

1. Add 130 to 1070 pL of Pectinex 3XL to 2.5 mL, 2% (w/v) Eudragit L-100 solution and make up the final volume to 4 mL with Buffer 1 (see Notes 4 and 5).

2. Incubate this solution for 1 h at 25°C. After 1 h, add 130 pL of 0.1 ^-acetic acid and let the suspension stand for 20 min (see Note 6).

3. Centrifuge the suspension at 12,000^ for 20 min at 25°C and collect the supernatant. Wash the precipitate with 4 mL of Buffer 2 and keep for 10 min at 25°C.

4. Centrifuge the suspension as before and collect the washing. Keep on washing the precipitate until no enzyme activity is detected in the washings (see Note 7).

5. Estimate the amount of xylanase activity in the supernatant and washing.

6. Dissolve the enzyme-bound polymer precipitate in 4 mL of Buffer 1 (see Note 8) and estimate the activity of the immobilized enzyme (as described in Subheading 3.1.; see Notes 9 and 10).

7. In this system, the preparation obtained by starting with 200 pL of Pectinex 3XL was found to give a maximum immobilization efficiency of 0.60 (17). For characterization and applications of this conjugate see ref. 17.

4. Notes

1. Preincubate the enzyme, Buffer 1, and the substrate separately at 50°C for 10 min before starting the assay.

2. Initially, it is necessary to raise the pH to above 10.0 for dissolution of a fresh sample of Eudragit powder. After a clear solution has been obtained, pH 7.0 is more than adequate to dissolve a Eudragit precipitate formed at pH 4.3.

3. Eudragit is a co-polymer of methyl methacrylate and methacrylic acid. The ratio of ester to free carboxyl groups is 1:1 in the case of Eudragit L-100 and 2:1 in the case of Eudragit S-100. Often, these two show different binding affinities with a chosen protein. In the present protocol, Eudragit L-100 has been used. Eudragit S-100 could be successfully used for purification of xylanase by selective precipitation (17). This illustrates a general rule: modification of a polymer or use of related polymers may turn out to be suitable matrices for immobilization, even though the original choice may not show an adequate binding constant.

4. One of the major applications of xylanases is in paper pulp bleaching. It is therefore desirable that the xylanase being used does not contain significant cellulase activity. An interesting and useful aspect of this protocol is that cellulase activity (present in Pectinex 3XL) does not bind to the polymer and hence is not present in the immobilized form (17).

5. Another application of xylanase is in the food industry. Eudragit L-100 constitutes a safe matrix because it is a nontoxic and food-grade polymer. In fact, it is an enteric polymer.

6. This bioconjugate exploits the fact that Eudragit (and the bioconjugate) show pH-responsive reversible solubility. However, Eudragit can also be precipitated by other stimuli. Depending upon the stimulus used during noncovalent immobilization, the binding constant of the protein may change dramatically (20). This can be exploited as another parameter for obtaining the bioconjugate.

7. Irrespective of whether covalent or noncovalent immobilization is carried out, some molecules are almost always more loosely bound than other molecules of the same protein. It is therefore desirable to check the stability of the bioconjugate under process conditions (e.g., pH, ionic strength, temperature, presence of a detergent, or a substrate/product analog).

8. Presence of Eudragit lowers the pH of the solution. It is therefore necessary to adjust the pH to 5.5 with a small volume of 3 MNaOH to dissolve the precipitate completely.

9. The final activity expressed by the polymer-bound enzyme is critically dependent upon the polymer concentration and enzyme load at the time of preparation. This should be optimized for individual systems.

10. The thermal stability and/or pH optimum of the enzyme may change upon immobilization. The bioconjugate should be evaluated for such parameters before actual application.

Acknowledgments

Financial support from the Department of Science and Technology, the Department of Biotechnology, and Council for Scientific and Industrial Research, and all Government of India organizations, is gratefully acknowledged.

References

1. Taniguchi, M, Tanahashi, S., and Fujii, M. (1990) Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme. Appl. Microbiol. Biotechnol. 33, 629-632.

2. Fujii, M. and Taniguchi, M. (1991) Application of reversibly soluble polymers in bioprocessing. Trends Biotechnol. 9, 191-196.

3. Roy, I., Sharma, S., and Gupta, M. N. (2003) Smart biocatalysts: design and applications. Adv. Biochem. Eng. Biotechnol. 86, 159-189.

4. Roy, I., and Gupta, M. N. (2004) Repeated enzymatic hydrolysis of polygalacturonic acid, chitosan and chitin using a novel reversibly-soluble pecti-nase with the aid of K-carrageenan. Biocatal. Biotransformation (in press)

5. Hoffman, A.S. (2000) Bioconjugation of intelligent polymers and recognition proteins for use in diagnostics and affinity separations. Clin. Chem. 46, 1478-1486.

6. Roy, I. and Gupta, M. N. (2003) Smart polymeric materials: emerging biochemical applications. Chem. Biol. 10, 1161-1171.

7. Tyagi, R., Roy, I., Agarwal, R. and Gupta, M.N. (1998) Carbodiimide coupling of enzymes to the reversibly soluble-insoluble polymer Eudragit S-100. Biotechnol. Appl. Biochem. 28, 201-206.

8. Carlsson, J., Janson, J.-C., and Sparrman, M. (1989) Affinity chromatography. In: Protein Purification: Principles, High Resolution Methods and Applications, (Janson, J. C. and Ryden, L., eds.), Wiley-VCH, New York, pp. 275-329.

9. Roy, I., Sardar, M., and Gupta, M. N. (2003) Evaluation of a smart bioconjugate of pectinase for chitin hydrolysis. Biochem. Eng. J. 16, 329-335.

10. Sardar, M., Roy, I. and Gupta, M. N. (2003) A smart bioconjugate of alginate and pectinase with unusual biological activity towards chitosan. Biotechnol. Prog. 19, 1654-1658.

11. Ding, Z. L., Chen, G. H., and Hoffman, A. S. (1996) Synthesis and purification of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. Bioconjug. Chem. 7, 121-126.

12. Ding, Z. L., Chen, G. H., and Hoffman, A. S. (1998) Unusual properties of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. J. Biomed. Mater. Res. 39, 498-505.

13. Morris, J. E., Hoffman, A. S., and Fisher, R. R. (1993) Affinity precipitation of proteins by polyligands. Biotechnol. Bioeng. 41, 991-997.

14. Shimoboji, T., Ding, Z., Stayton, P. S., and Hoffman, A. S. (2001) Mechanistic investigation of smart polymer-protein conjugates. Bioconjug. Chem. 12, 314-319.

15. Ito, Y., Sugimura, N., Kown, O. H., and Imanishi, Y. (1999) Enzyme modification by polymers with solubilities that change in response to photoirradiation in organic media. Nature Biotechnol. 17, 73-75.

16. Shimoboji, S., Larenas, E., Fowler, T., Kulkarni, S., Hoffman, A. S., and Stayton, P.S. (2002) Photoresponsive polymer-enzyme switches. Proc. Natl. Acad. Sci. USA 99, 16,592-16,596.

17. Sardar, M., Roy, I., and Gupta, M. N. (2000) Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit™ L-100. Enzyme Microb. Technol. 27, 672-679.

18. Miller, G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428.

19. Bailey, M. J., Biely, P., and Poutanen, K. (1992) Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257-270.

20. Guoqiang, D., Batra, R., Kaul, R., Gupta, M. N., and Mattiasson, B. (1995) Alternative modes of precipitation of Eudragit S-100: A potential ligand carrier for affinity precipitation of proteins. Bioseparation 5, 339-350.

21. Fujimura, M., Mori, T., and Tosa, T. (1987) Preparation and properties of soluble-insoluble immobilized proteases. Biotechnol. Bioeng. 29, 747-752.

22. Dominguez, E., Nilsson, M., and Hahn-Hagerdal, B. (1988) Carbodiimide coupling of P-galactosidase from Aspergillus oryzae to alginate. Enzyme Microb. Technol. 10, 606-610.

23. Vazquez-Duhalt, R., Tinoco, R., D'Antonio, P., Topoleski, L. D. T., and Payne, G. F. (2001) Enzyme conjugation to the polysaccharide chitosan: smart biocatalysts and biocatalytic hydrogels. Bioconjug. Chem. 12, 301-306.

24. Willner, I. and Rubin, S. (1993) Reversible photoregulation of the activities of proteins. Reactive Polym. 21, 177-181.

25. Okumura, K., Ikura, K., Yoshikawa, M., Sasaki, R., and Chiba, H. (1984) Preparation of soluble-insoluble interconvertible enzymes: Enzyme polymerised as1-casein conjugates. Agric. Biol. Chem. 48, 2435-2440.

26. Mondal, K., Roy, I., and Gupta, M. N. (2003) K-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase. Protein Expr. Purif. 32, 151-160.

27. Chen, J. P. and Chang, K. C. (1994) Immobilization of chitinase on a reversibly soluble-insoluble polymer for chitin hydrolysis. J. Chem. Technol. Biotechnol. 60, 133-140.

28. Ito, Y., Kotoura, M., Chung, D. J., and Imanishi, Y. (1993) Trypsin modification by vinyl polymers with variable solubilities in response to external signals. Bioconjug. Chem. 4, 358-361.

29. Nath, N. and Chilkoti, A. (2003) Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide. Anal. Chem. 75, 709-715.

30. Brahim, S., Narinesingh, D., and Guiseppi-Elie, A. (2002) Bio-smart hydrogels: co-joined molecular recognition and signal transduction in biosensor fabrication and drug delivery. Biosens. Bioelectron. 17, 973-981.

Was this article helpful?

0 0
Appetite Antidote

Appetite Antidote

Discover How You Can Free Yourself From  Uncontrolled Habits And Get Your Eating Under Control Once And For All! This Book Is One Of The Most Valuable Resources In The World When It Comes To Ways To Reclaime Your Rightful Body. Sound eating isn't about rigid nutrition doctrines, staying unrealistically skinny, or depriving yourself of the foods you adore.

Get My Free Ebook


Responses

  • PEONY
    What does L stands for in Eudragit L100?
    8 years ago

Post a comment