CHARACTERIZATION AND CHEMICAL MODIFICATION OF PARTIALLY PURIFIED GLUCOAMYLASE PRODUCED FROM INDIGENOUS FUNGAL STRAIN HUMICOLA INSOLENS
Main Article Content
Keywords
Glucoamylase, chemical modification, characterization, Humicola insolens
Abstract
In the present study, GA was produced from Humicola insolens by submerged fermentation conditions, 45 °C temperature, pH 5.0, 4% substrate (w/v) and 5 % inoculum density for maximum extracellular enzyme production (1.002 U/mL) and protein (0.12921mg/mL). Enzyme was purified using ion exchange and gel filtration column, 10 fold purity with 7 % recovery was obtained. The apparent sub unit molecular weight was 57 kDa. The number of isoforms of the GA was determined by running PAGE. Optimum temperature for GA activity was 45ºC at pH 5.4. Ea of 26.52 kJ mol-1 was required for the formation of activated enzyme-substrate complex at 45 ºC. Kd at 65 ºC= 0.005423, ∆S = 0.273 kJ mol-1 K-1 , ∆G* = 70.95 kJ mol-1, ∆H=23.88 kJ mol-1 and its half-life =36 (min) at 55ºC. Enzyme was chemically modified by using succinic anhydride and aniline for the modification of amino group and surface carboxylic group. The data revealed that the modified forms of GA had deviation from the energy of activation than the native one. The amino group modified form has large value of Ea as compared to native enzyme whereas both forms, showed the same optimum pH, 6.2 at optimum temperatures and showed stability in pH range of 5.4-7.2. The pH range of modified Ani-60 and succi-130 was higher than that of native. The values of Vmax, Km and kcat (s-1) for native were 34.78 U mg-1 protein, 4.76 mg starch ml-1 and 32.75 s-1 at 45 ºC respectively. The specificity constant, kcat/Km was 6.87. For amino group modified GA, the values of Vmax, Km and kcat were 76.92 U mg-1 protein min-1, 2.17 mg starch ml-1 and 73.74 s-1 respectively. The specificity constant, kcat/Km was 31.74.
References
1. Adewale, P., Yancheshmeh, M. S., and Lam, E. (2022). Starch modification for non-food, industrial applications: Market intelligence and critical review. Carbohydrate Polymer 291:119590.
2. Apostolidi, M. E., Kalantzi S., Hatzinikolaou. D. G., Kekos, D. Mamma, D. (2020). Catalytic and thermodynamic properties of an acidic αamylase produced by the fungus Paecilomycesvariotii ATHUM 889. 3 Biotech, 10:311 (7).
3. Archer, D. B. (2000). Filamentous Fungi as Microbial Cell Factories for Food Use. Current Opinion Biotechnology 11, 134-1.
4. Ayodeji, O. S. Bamidele, Ayodele, O. K. and Ajele, J. O. (2017) Physicochemical and kinetic properties of a high salt tolerant Aspergillus flavus glucoamylase. Biocatalysis and Agricultural Biotechnology 9, 35-40.
5. Bhatti, H. N., Rashid, M. H., Nawaz, R., Asghar, M., Perveen, R. & Jabbar, A. (2007a) Purification and characterization of a novel glucoamylase from Fusarium solani. Food Chemistry, 103: 338-343.
6. Bhatti, H. N., Rashid, M. H., Asghar, M., Nawaz, R., Khalid, A. M. & Perveen, R., (2007b). Chemical modification results in hyperactivation and thermostabilization of Fusarium solani glucoamylase. Canadian Journal of Microbiology, 53(2): 177-185.
7. Bhatti, H. N., Rashid, M. H., Nawaz, R., Khalid, A. M., Asghar, M. & Jabbar, A.,(2007c). Effect of aniline coupling on kinetic and thermodynamic properties of Fusarium solani glucoamylase. Applied Microbiology & Biotechnology, 73(6): 1290-1298.
8. Bhatti, H. N., Zia, A., Nawaz, R., Sheikh, M. N., Rashid, M. H. & Khalid, A. M. (2005). Effect of copper ions on thermostability of glucoamylase from Fusarium sp. International Journal of Agriculture and Biology,7(4): 585-587.
9. Bokhari, S. A., Afzal, A. J., Rashid, M. H., Rajoka, M. I. & Siddiqui, K. S. (2002). Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water miscible organic mixture. Biotechnology Progress, 18: 276-281.
10. Carrasco M, Alcaíno J, Cifuentes V, Baeza M. (2017) Purification and characterization of a novel cold adapted fungal glucoamylase. Microb Cell Fact. 2017;16:75.
11. Eyring, H. & Stearn, A.E., (1939). The application of the theory of absolute reaction rates to proteins. Chemical Reviews, 24: 253-270.
12. Fath, M., Fazaelipoor, M. H. (2015). Production of proteases in a novel trickling tray bioreactor. Waste Biomass Valoriz. 475–480
13. Guo, W., Yang, J., Huang, T., Liu, D., Liu, Q., Li, J., et al. (2021). Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification. Biotechnol. Biofuels 14:225.
14. Hashim, S. O. (2020). Starch-modifying enzymes. Adv. Biochem. Eng. Biotechnol. 172, 221–244.
15. Hakamada, Y., Hatada, Y., Koike, K., Yoshimatsu, T., Kawai, S., Kobayashi, T. & Ito, S. (2000). Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an alkaliphilic Bacillus strain. Bioscience, Biotechnology & Biochemistry, 64: 2281–2289.
16. Ichikawa, T., Tanaka, M., Watanabe, T., Zhan, S., Watanabe, A., Shintani, T., et al. (2021). Crucial role of the intracellular-glucosidase MalT in the activation of the transcription factor AmyR essential for amylolytic gene expression in Aspergillus oryzae. Biosci. Biotechnol. Biochem. 85, 2076–2083
17. Iqbal, Z., Rashid, M. H., Jabbar, A., Malana, M. A., Khalid, A. M. & Rajoka, M. I. (2003). Kinetics of enhanced thermostability of an extracellular glucoamylase from Arachniotus sp. Biotechnology Letters, 25(19): 1667-70.
18. Jabbar, A., Rashid, M. H., Javed, M. R., Perveen, R. & Malana, M. A. (2008). Kinetics and thermodynamics of a novel endoglucanase (CMCase) from Gymnoascella citrina produced under solid-state condition. Journal of Industrial Microbiology & Biotechnology, 35:515–524.
19. Jebor, M. A., Zahra M. A. & Baydaa A. H. (2014). Purification and characterization of the glucoamylase from Aspergillus niger. Inernational Journal of Current Microbioly and Applied Sciences (3(1): 63-75
20. Khajeh, K., Manesh, H. N., Ranjbar, B., Movahedi, A. M. & Gorgani, M. V. (2001). Chemical modification of lysine residues in Bacillus α-amylases: Effect on activity and stability. Enzyme and Microbial Technology, 28: 543-549.
21. Klotz, I. M., (1967). Methods of Enzymology, (11): 576-580.
22. Marlida, Y., Saari, N., Hassan, Z., Radu, S. & Baker, J. (2000). Purification and characterization of sago starch degrading glucoamylase from Acremonium sp. endophytic fungus. Food Chemistry, 71(2): 221-227.
23. Montes, F. J., Battanar, E., Catalan, J. & Galan, M.A., (1995). Kinetics and heat inactivation mechanism of D-amino oxidase. Process Biochemistry, 30: 217-224.
24. Munch, O., & Tritsch, D., (1990). Irreversible thermo inactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups. Biochemica & Biophysical Acta., 1041(2): 111-116.
25. Natael M. Wayllace, Mariana Martín, María V. Busi & Diego F. Gomez-Casat. 2023. Microbial glucoamylases: structural and functional properties and biotechnological uses World Journal of Microbiology and Biotechnology 39, (293)
26. Niaz, M., Ghafoor, M. Y., Jabbar, A., Wahid, A., Rasul, E., Ahmed, R. and Rashid, M. H. (2004). Properties of glucoamylase from a mesophilic fungus Arachniotus citrinus produced under solid-state growth condition. International Journal of Biotechnology, 1(2): 223-231.
27. Odibo, F. J. C. & Ulbrich-Hofmann, R. (2001). Thermostable α-amylase and glucoamylase from Thermomyces lanuginosus F1. Acta Biotechnology, 21:141–153.
28. Prajapati, V. S., Trivedi, U. B., & Patel, K. C. (2014). Kinetic and thermodynamic characterization of glucoamylase from Colletotrichum sp. KCP1. Indian journal of microbiology, 54, 87-93.
29. Rajoka, M. I. (2008). Kinetics and enhanced ethanol productivity using raw starch hydrolyzing glucoamylase from Aspergillus niger mutant produced in solid state fermentation Letters in Applied Microbiology, 39(1): 13-18.
30. Rashid, M. H. & Siddiqui, K. S., (1997). Thermodynamic and kinetic study of stability of the native and chemically modified ß-glucosidase from Aspergillus niger. Process Biochemistry, 33(2): 109-115.
31. Rashid, M. H., Shakoori, A. R., Rajoka, M. I. & Siddiqui, K. S. (1998). Monitoring of carboxyl group modification of ß-glucosidase from Aspergillus niger NIAB-280 by native enzyme mobility shift assay (NEMSA). Pakistan Journal of Zoology, 30(2): 99-104.
32. Riaz, M., Perveen, R., Javed, M. R., Nadeem, H. U. & Rashid, M. H. (2007). Kinetic and Thermodynamic properties of novel glucoamylase from Humicola sp. Enzyme Microbiology and Technology, 41: 558-564.
33. Saleem M., Rashid M. H., Jabbar A., Perveen R., Khalid A.M. & Rajoka M. I. (2005). Kinetic and thermodynamic properties of an immobilized endoglucanase from Arachniotus Citrinus. Process Biochemistry, 40: 849–55.
34. Siddiqui, K. S., Rashid, M. H. & Rajoka, M. I. (1997). Kinetic analysis of the active site of an intracellular ß-glucosidase from Cellulomonas biazotea. Folia Microbiol. 42: 53-58.
35. Siddiqui, K. S., Azhar, M. J., Rashid, M. H., Ghuri, T. M. & Rajoka, M.I., (1997a). Purification and the effect of Manganese ions on the activity carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea. Folia Microbiology, 42 (4): 303-311.
36. Siddiqui, K. S., Saqib, A. A. N., Rashid, M. H. & Rajoka, M. I. (2000). Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger. Enzyme and Microbial Technology, 27: 467–47.
37. 2-Souza, P.M. (2010) Application of microbial α-amylase in industry-A review. Brazilian Journal of Microbiology, 41, 850–861.
38. Urabe, I., Nanjo, H & Okada, H. (1973). Effect of Acetylation of Bacillus Subtilis Α-Amylase on the Kinetics of Heat Inactivation. Biochimica Et Biophysica Acta (Bba)-Enzymology, 302(1):73-79 ·
39. Vanier, N. L., El Halal, S. L. M., Dias, A. R. G., and da Rosa Zavareze, E. (2017). Molecular structure, functionality and applications of oxidized starches: A review. Food Chem. 221, 1546–1559.
40. Wang C, Yang L, Luo L, Tang S, Wang Q. (2020) Purification and characterization of glucoamylase of Aspergillus oryzae from Luzhou-flavour Daqu. Biotech Letters, 42:2345–55.
41. Maniglia, B. C., Castanha, N., Le-Bail, P., Le-Bail, A., and Augusto, P. E. D. (2021). Starch modification through environmentally friendly alternatives: A review. Crit. Rev. Food Sci. Nutr. 61, 2482–2505.
42. Xian L, Wang F, Luo X, Feng Y. L, Feng J. X (2015) Purifcation and characterization of a highly efcient calcium-independent α-amylase from Talaromycespinophilus 1–95. Plose One 10(3).
2. Apostolidi, M. E., Kalantzi S., Hatzinikolaou. D. G., Kekos, D. Mamma, D. (2020). Catalytic and thermodynamic properties of an acidic αamylase produced by the fungus Paecilomycesvariotii ATHUM 889. 3 Biotech, 10:311 (7).
3. Archer, D. B. (2000). Filamentous Fungi as Microbial Cell Factories for Food Use. Current Opinion Biotechnology 11, 134-1.
4. Ayodeji, O. S. Bamidele, Ayodele, O. K. and Ajele, J. O. (2017) Physicochemical and kinetic properties of a high salt tolerant Aspergillus flavus glucoamylase. Biocatalysis and Agricultural Biotechnology 9, 35-40.
5. Bhatti, H. N., Rashid, M. H., Nawaz, R., Asghar, M., Perveen, R. & Jabbar, A. (2007a) Purification and characterization of a novel glucoamylase from Fusarium solani. Food Chemistry, 103: 338-343.
6. Bhatti, H. N., Rashid, M. H., Asghar, M., Nawaz, R., Khalid, A. M. & Perveen, R., (2007b). Chemical modification results in hyperactivation and thermostabilization of Fusarium solani glucoamylase. Canadian Journal of Microbiology, 53(2): 177-185.
7. Bhatti, H. N., Rashid, M. H., Nawaz, R., Khalid, A. M., Asghar, M. & Jabbar, A.,(2007c). Effect of aniline coupling on kinetic and thermodynamic properties of Fusarium solani glucoamylase. Applied Microbiology & Biotechnology, 73(6): 1290-1298.
8. Bhatti, H. N., Zia, A., Nawaz, R., Sheikh, M. N., Rashid, M. H. & Khalid, A. M. (2005). Effect of copper ions on thermostability of glucoamylase from Fusarium sp. International Journal of Agriculture and Biology,7(4): 585-587.
9. Bokhari, S. A., Afzal, A. J., Rashid, M. H., Rajoka, M. I. & Siddiqui, K. S. (2002). Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water miscible organic mixture. Biotechnology Progress, 18: 276-281.
10. Carrasco M, Alcaíno J, Cifuentes V, Baeza M. (2017) Purification and characterization of a novel cold adapted fungal glucoamylase. Microb Cell Fact. 2017;16:75.
11. Eyring, H. & Stearn, A.E., (1939). The application of the theory of absolute reaction rates to proteins. Chemical Reviews, 24: 253-270.
12. Fath, M., Fazaelipoor, M. H. (2015). Production of proteases in a novel trickling tray bioreactor. Waste Biomass Valoriz. 475–480
13. Guo, W., Yang, J., Huang, T., Liu, D., Liu, Q., Li, J., et al. (2021). Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification. Biotechnol. Biofuels 14:225.
14. Hashim, S. O. (2020). Starch-modifying enzymes. Adv. Biochem. Eng. Biotechnol. 172, 221–244.
15. Hakamada, Y., Hatada, Y., Koike, K., Yoshimatsu, T., Kawai, S., Kobayashi, T. & Ito, S. (2000). Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an alkaliphilic Bacillus strain. Bioscience, Biotechnology & Biochemistry, 64: 2281–2289.
16. Ichikawa, T., Tanaka, M., Watanabe, T., Zhan, S., Watanabe, A., Shintani, T., et al. (2021). Crucial role of the intracellular-glucosidase MalT in the activation of the transcription factor AmyR essential for amylolytic gene expression in Aspergillus oryzae. Biosci. Biotechnol. Biochem. 85, 2076–2083
17. Iqbal, Z., Rashid, M. H., Jabbar, A., Malana, M. A., Khalid, A. M. & Rajoka, M. I. (2003). Kinetics of enhanced thermostability of an extracellular glucoamylase from Arachniotus sp. Biotechnology Letters, 25(19): 1667-70.
18. Jabbar, A., Rashid, M. H., Javed, M. R., Perveen, R. & Malana, M. A. (2008). Kinetics and thermodynamics of a novel endoglucanase (CMCase) from Gymnoascella citrina produced under solid-state condition. Journal of Industrial Microbiology & Biotechnology, 35:515–524.
19. Jebor, M. A., Zahra M. A. & Baydaa A. H. (2014). Purification and characterization of the glucoamylase from Aspergillus niger. Inernational Journal of Current Microbioly and Applied Sciences (3(1): 63-75
20. Khajeh, K., Manesh, H. N., Ranjbar, B., Movahedi, A. M. & Gorgani, M. V. (2001). Chemical modification of lysine residues in Bacillus α-amylases: Effect on activity and stability. Enzyme and Microbial Technology, 28: 543-549.
21. Klotz, I. M., (1967). Methods of Enzymology, (11): 576-580.
22. Marlida, Y., Saari, N., Hassan, Z., Radu, S. & Baker, J. (2000). Purification and characterization of sago starch degrading glucoamylase from Acremonium sp. endophytic fungus. Food Chemistry, 71(2): 221-227.
23. Montes, F. J., Battanar, E., Catalan, J. & Galan, M.A., (1995). Kinetics and heat inactivation mechanism of D-amino oxidase. Process Biochemistry, 30: 217-224.
24. Munch, O., & Tritsch, D., (1990). Irreversible thermo inactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups. Biochemica & Biophysical Acta., 1041(2): 111-116.
25. Natael M. Wayllace, Mariana Martín, María V. Busi & Diego F. Gomez-Casat. 2023. Microbial glucoamylases: structural and functional properties and biotechnological uses World Journal of Microbiology and Biotechnology 39, (293)
26. Niaz, M., Ghafoor, M. Y., Jabbar, A., Wahid, A., Rasul, E., Ahmed, R. and Rashid, M. H. (2004). Properties of glucoamylase from a mesophilic fungus Arachniotus citrinus produced under solid-state growth condition. International Journal of Biotechnology, 1(2): 223-231.
27. Odibo, F. J. C. & Ulbrich-Hofmann, R. (2001). Thermostable α-amylase and glucoamylase from Thermomyces lanuginosus F1. Acta Biotechnology, 21:141–153.
28. Prajapati, V. S., Trivedi, U. B., & Patel, K. C. (2014). Kinetic and thermodynamic characterization of glucoamylase from Colletotrichum sp. KCP1. Indian journal of microbiology, 54, 87-93.
29. Rajoka, M. I. (2008). Kinetics and enhanced ethanol productivity using raw starch hydrolyzing glucoamylase from Aspergillus niger mutant produced in solid state fermentation Letters in Applied Microbiology, 39(1): 13-18.
30. Rashid, M. H. & Siddiqui, K. S., (1997). Thermodynamic and kinetic study of stability of the native and chemically modified ß-glucosidase from Aspergillus niger. Process Biochemistry, 33(2): 109-115.
31. Rashid, M. H., Shakoori, A. R., Rajoka, M. I. & Siddiqui, K. S. (1998). Monitoring of carboxyl group modification of ß-glucosidase from Aspergillus niger NIAB-280 by native enzyme mobility shift assay (NEMSA). Pakistan Journal of Zoology, 30(2): 99-104.
32. Riaz, M., Perveen, R., Javed, M. R., Nadeem, H. U. & Rashid, M. H. (2007). Kinetic and Thermodynamic properties of novel glucoamylase from Humicola sp. Enzyme Microbiology and Technology, 41: 558-564.
33. Saleem M., Rashid M. H., Jabbar A., Perveen R., Khalid A.M. & Rajoka M. I. (2005). Kinetic and thermodynamic properties of an immobilized endoglucanase from Arachniotus Citrinus. Process Biochemistry, 40: 849–55.
34. Siddiqui, K. S., Rashid, M. H. & Rajoka, M. I. (1997). Kinetic analysis of the active site of an intracellular ß-glucosidase from Cellulomonas biazotea. Folia Microbiol. 42: 53-58.
35. Siddiqui, K. S., Azhar, M. J., Rashid, M. H., Ghuri, T. M. & Rajoka, M.I., (1997a). Purification and the effect of Manganese ions on the activity carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea. Folia Microbiology, 42 (4): 303-311.
36. Siddiqui, K. S., Saqib, A. A. N., Rashid, M. H. & Rajoka, M. I. (2000). Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger. Enzyme and Microbial Technology, 27: 467–47.
37. 2-Souza, P.M. (2010) Application of microbial α-amylase in industry-A review. Brazilian Journal of Microbiology, 41, 850–861.
38. Urabe, I., Nanjo, H & Okada, H. (1973). Effect of Acetylation of Bacillus Subtilis Α-Amylase on the Kinetics of Heat Inactivation. Biochimica Et Biophysica Acta (Bba)-Enzymology, 302(1):73-79 ·
39. Vanier, N. L., El Halal, S. L. M., Dias, A. R. G., and da Rosa Zavareze, E. (2017). Molecular structure, functionality and applications of oxidized starches: A review. Food Chem. 221, 1546–1559.
40. Wang C, Yang L, Luo L, Tang S, Wang Q. (2020) Purification and characterization of glucoamylase of Aspergillus oryzae from Luzhou-flavour Daqu. Biotech Letters, 42:2345–55.
41. Maniglia, B. C., Castanha, N., Le-Bail, P., Le-Bail, A., and Augusto, P. E. D. (2021). Starch modification through environmentally friendly alternatives: A review. Crit. Rev. Food Sci. Nutr. 61, 2482–2505.
42. Xian L, Wang F, Luo X, Feng Y. L, Feng J. X (2015) Purifcation and characterization of a highly efcient calcium-independent α-amylase from Talaromycespinophilus 1–95. Plose One 10(3).
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Aug 22, 2024
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Farhana Yasmin, Haq Nawaz Bhatti, & Abdul Jabbar. (2024). CHARACTERIZATION AND CHEMICAL MODIFICATION OF PARTIALLY PURIFIED GLUCOAMYLASE PRODUCED FROM INDIGENOUS FUNGAL STRAIN HUMICOLA INSOLENS. Journal of Population Therapeutics and Clinical Pharmacology, 31(8), 3420-3434. https://doi.org/10.53555/3z6kpx37
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Author Biographies
Farhana Yasmin
Department of Chemistry, Faculty of Science, Governament College University, Faisalabad, Punjab, Pakistan, farhanascholar@gmail.com
Haq Nawaz Bhatti
Department of Chemistry, Faculty of Science, University of Agriculture, Faisalabad, Punjab, Pakistan, Hnbhatti2005@yahoo.com
Abdul Jabbar
Department of Chemistry, Faculty of Science, Government College University, Punjab, Pakistan.
How to Cite
Farhana Yasmin, Haq Nawaz Bhatti, & Abdul Jabbar. (2024). CHARACTERIZATION AND CHEMICAL MODIFICATION OF PARTIALLY PURIFIED GLUCOAMYLASE PRODUCED FROM INDIGENOUS FUNGAL STRAIN HUMICOLA INSOLENS. Journal of Population Therapeutics and Clinical Pharmacology, 31(8), 3420-3434. https://doi.org/10.53555/3z6kpx37