Cellulose degrading organisms are being employed for the conversion of native cellulosic materials into soluble sugars. Cellulases are commercially important enzymes and have applications in various eco-friendly processes. For enhanced cellulase production, a high priority has been given to several approaches including chemical mutations, UV irradiations and genetic engineering. Genetically improved strains have been employed in a number of applications including animal feed, pharmaceutical and textile industries. Cellulolytic microorganisms in their typical habitat in nature probably depend on the phenomenon of catabolite repression and act as a control on cellulase synthesis. Cellulase biosynthesis severely limits the ability of wild type cultures to produce cellulase on a commercial scale and hampers its application in the biomass conversion process. Various methods have been developed to obtain catabolite repression resistant mutants. The potential industrial utilization of cellulose will require organisms that have high cellulase activity and are no longer under repressive control. Purified cellulases from improved mutants that exhibit high temperature stability, activity over a wide range of pH under non-conventional conditions are of great interest and are employed in a variety of industrial applications.  In the present investigations, Carboxymethyl cellulase (CMCase) of a catabolite repression resistant mutant strain of Pseudomonas (PsCCRRNT₉) when subjected to partial purification by (NH₄)₂SO₄ precipitation, a 15.65-fold purification was observed and was later on characterized.  Two protein bands with molecular weights 53.0 and 85.1 were detected during SDS-PAGE analysis.  Further investigations revealed that the CMCase from this strain is active over a wide range of pH (6-10) and exhibited maximum activity at temperature 50 ⁰C.  Of different metal ions investigated, CaCl₂.2H₂O was found to enhance the CMCase activity at 5 mM and 10 mM concentrations.  CMCase activity was completely inhibited at 10 mM concentration of HgCl₂.

References

1. Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and molecular biology reviews.2002Sep;66(3):506-77. https://doi.org/10.1128/MMBR.66.3.506-577.2002
2. Mostafa YS, Alamri SA, Hashem M, Nafady NA, Abo-Elyousr KA, Mohamed ZA. Thermostable cellulase biosynthesis from Paenibacillus alvei and its utilization in lactic acid production by simultaneous saccharification and fermentation. Open Life Sciences. 2020 Jan 1;15(1):185-97. https://doi.org/10.1515/biol-2020-0019
3. Bhat M. Cellulases and related enzymes in biotechnology. Biotechnology advances. 2000 Aug 1;18(5):355-83.    https://doi.org/10.1016/S0734-9750(00)00041-0
4. Wood TM, Wilson CA, McCrae SI. Synergism between components of the cellulase system of the anaerobic rumen fungus Neocallimastix frontalis and those of the aerobic fungi Penicillium pinophilum and Trichoderma koningii in degrading crystalline cellulose. Applied microbiology and biotechnology. 1994 Apr;41(2):257-61. https://doi.org/10.1007/BF00186969
5. Vyas A, Vyas D. Isolation and screening of cellulases producing fungi from diverse habitats. J. Basic Appl. Mycol. 2005;4:138-41
6. Sukumaran RK, Christopher M, Kooloth-Valappil P, Sreeja-Raju A, Mathew RM, Sankar M, Puthiyamadam A, Adarsh VP, Aswathi A, Rebinro V, Abraham A. Addressing challenges in production of cellulases for biomass hydrolysis: Targeted interventions into the genetics of cellulase producing fungi. Bioresource Technology. 2021 Jan 29:124746.  https://doi.org/10.1016/j.biortech.2021.124746
7. Priyanka P, Yuvraj C, Farha S, Aranganathan V. Isolation of cellulose degrading fungi from soil and optimization for cellulase production using carboxy methyl cellulose. International Journal of Life science and Pharma Research. 2017 Jan 1;7(1):56-60.
8. Kuhad RC, Gupta R, Singh A. Microbial cellulases and their industrial applications. Enzyme research. 2011;2011. https://doi:10.4061/2011/280696
9. Labudova I, Farkaš V. Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase. FEMS microbiology letters. 1983 Oct 1;20(2):211-5.  https://doi.org/10.1111/j.1574-6968.1983.tb00119.x
10. Kotchoni OD, Shonukan OO, Gachomo WE. Bacillus pumilus BpCRI 6, a promising candidate for cellulase production under conditions of catabolite repression. African journal of biotechnology. 2003;2(6):140-6.  https://www.ajol.info/index.php/ajb/article/view/14787
11. Aristidou A, Penttilä M. Metabolic engineering applications to renewable resource utilization. Current Opinion in Biotechnology. 2000 Apr 1;11(2):187-98. https://doi.org/10.1016/S0958-1669(00)00085-9
12. Oksanen T, Pere J, Paavilainen L, Buchert J, Viikari L. Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases. Journal of Biotechnology. 2000 Feb 29;78(1):39-48.  https://doi.org/10.1016/S0168-1656(99)00232-1
13. Suto M, Tomita F. Induction and catabolite repression mechanisms of cellulase in fungi. Journal of bioscience and bioengineering. 2001 Jan 1;92(4):305-11.  https://doi.org/10.1016/S1389-1723(01)80231-0
14. Berg B, Hofsten BV, Pettersson G. Growth and cellulase formation by Cellvibrio fulvus. Journal of Applied Bacteriology. 1972 Jun;35(2):201-14.  https://doi.org/10.1111/j.1365-2672.1972.tb03691.x
15. Montenecourt BS, Eveleigh DE. Production and characterization of high yielding cellulase mutants of Trichoderma reesei. TAPPI J. 1979 Mar;28:101-8.
16. Shibata N, Kakeshita H, Igarashi K, Takimura Y, Shida Y, Ogasawara W, Koda T, Hasunuma T, Kondo A. Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose. Biotechnology for biofuels. 2021 Dec;14(1):1-6. https://doi.org/10.1186/s13068-021-01887-0
17. Berghem LE, Pettersson LG. The mechanism of enzymatic cellulose degradation: Purification of a cellulolytic enzyme from Trichoderma viride active on highly ordered cellulose. European Journal of Biochemistry. 1973 Aug;37(1):21-30.  https://doi.org/10.1111/j.1432-1033.1973.tb02952.x
18. Berghem LE, Pettersson LG, Axiö?Fredriksson UB. The Mechanism of Enzymatic Cellulose Degradation: Purification and Some Properties of Two Different 1,4?β?Glucan Glucanohydrolases from Trichoderma viride. European journal of biochemistry. 1976 Jan;61(2):621-30. https://doi.org/10.1111/j.1432-1033.1976.tb10058.x
19. Hurst PL, Nielsen J, Sullivan PA, Shepherd MG. Purification and properties of a cellulase from Aspergillus niger. Biochemical Journal. 1977 Jul 1;165(1):33-41. https://doi.org/10.1042/bj1650033
20. Hong SW, Hah YC, Maeng PJ, Jeong CS. Purification and mode of action of low molecular weight β-1, 4-glucan glucanohydrolase from Trichoderma koningii. Enzyme and microbial technology. 1986 Apr 1;8(4):227-35. https://doi.org/10.1016/0141-0229(86)90093-1
21. Lachke AH, Srinivasan MC, Deshmukh SS, Deshpande MV. Strain selection criteria for Penicillium funiculosum in enzymic hydrolysis of lignocellulosics. Biotechnology letters. 1987 Feb;9(2):147-50.  https://doi.org/10.1007/BF01032756
22. Sahasrabudhe NA, Lachke AH, Ranjekar PK. Characterization of the purified multifunctional cellulase component of Penicillium funiculosum. Biotechnology letters. 1987 Dec;9(12):881-6.  https://doi.org/10.1007/BF01026203
23. Bhat KM, McCrae SI, Wood TM. The endo-(1→4)-β-d-glucanase system of Penicillium pinophilum cellulase: Isolation, purification, and characterization of five major endoglucanase components. Carbohydrate research. 1989 Jul 15;190(2):279-97.  https://doi.org/10.1016/0008-6215(89)84131-X
24. Bhat KM, Hay AJ, Claeyssens M, Wood TM. Study of the mode of action and site-specificity of the endo-(1→4)-β-d-glucanases of the fungus Penicillium pinophilum with normal, 1-3H-labelled, reduced and chromogenic cello-oligosaccharides. Biochemical journal. 1990 Mar 1;266(2):371-8.  https://doi.org/10.1042/bj2660371
25. Claeyssens M, Aerts G. Characterisation of cellulolytic activities in commercial Trichoderma reesei preparations: an approach using small, chromogenic substrates. Bioresource Technology. 1992 Jan 1;39(2):143-6. https://doi.org/10.1016/0960-8524(92)90133-I
26. Schülein M. Enzymatic properties of cellulases from Humicola insolens. Journal of biotechnology. 1997 Sep 16;57(1-3):71-81. https://doi.org/10.1016/S0168-1656(97)00090-4
27. Patel N, Ajit KG. Isolation of potential extracellular cellulase producer and determination of cellulase production efficiency with various raw substrates.(2021). Int. J. Life Sci. Pharma Res.;11(2):L131-134. https://doi.10.22376/ijpbs/lpr.2021.11.2.L131-134
28. Huang XP, Monk C. Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov. World journal of Microbiology and Biotechnology. 2004 Feb;20(1):85-92.  https://doi.org/10.1023/B:WIBI.0000013316.12730.e7
29. Chen PJ, Wei TC, Chang YT, Lin LP. Purification and characterization of carboxymethyl cellulase from Sinorhizobium fredii. Botanical Bulletin of Academia Sinica. 2004 Apr 1;45. https://ejournal.sinica.edu.tw/bbas/content/2004/2/Bot452-02.html
30. Khyami-Horani H. Characterization and Cellulase Synthesis. Some Thermotolerant Bacilli from Jordan. PhD Thesis. University of Heriot-Watt, Edinburgh, Scotland. 1991.
31. Chatterjee U, Sanwal GG. Purification and properties of a protein from Lantana camara activating Cuscuta reflexa cellulase. Phytochemistry. 1999 Oct 1;52(3):361-6. https://doi.org/10.1016/S0031-9422(99)00223-X
32. Onsori H, Zamani MR, Motallebi M, Zarghami N. Identification of over producer strain of endo-ß-1, 4-glucanase in Aspergillus Species: Characterization of crude carboxymethyl cellulase. African Journal of Biotechnology. 2005 Mar 24;4(1):26-30. https://www.ajol.info/index.php/ajb/article/view/15047
33. Reese ET, Mandels M. Enzymic hydrolysis of cellulose and its derivatives. In Cellulose 1963 (pp. 139-143).
34. Ghose TK. Measurement of cellulase activities. Pure and applied Chemistry. 1987 Jan 1;59(2):257-68.
35.  
36. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry. 1959 Mar 1;31(3):426-8.  https://doi.org/10.1021/ac60147a030
37. Lowry OH, Rosebrough NJ, Farr AL. Randall RJ. Protein measurement with the phenol folin reagent, J Biol Chem. 1951; 193:265.
38. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature. 1970 Aug;227(5259):680-5. https://www.nature.com/articles/227680a0
39. Gaur R, Tiwari S. Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07. BMC biotechnology. 2015 Dec;15(1):1-2. DOI 10.1186/s12896-015-0129-9.
40. Sadhu S, Saha P, Sen SK, Mayilraj S, Maiti TK. Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillus strain isolated from cow dung. Springer Plus. 2013 Dec;2(1):1-10. https://doi.org/10.1186/2193-1801-2-10.
41. Siddiqui KS, Azhar MJ, Rashid MH, Rajoka MI. Stability and identification of active-site residues of carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea. Folia microbiologica. 1997 Aug;42(4):312-8. https://doi.org/10.1007/BF02816941
42. Eckert K, Schneider E. A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases. European journal of biochemistry. 2003 Sep;270(17):3593-602. https://doi.org/10.1046/j.1432-1033.2003.03744.x
43. Shankar T, Sankaralingam S, Balachandran C, Chinnathambi A, Nasif O, Alharbi SA, Park S, Baskar K. Purification and characterization of carboxymethylcellulase from Bacillus pumilus EWBCM1 isolated from earthworm gut (Eudrilus eugeniae). Journal of King Saud University-Science. 2021Jan1;33(1):101261. https://doi.org/10.1016/j.jksus.2020.101261
44. Fullbrook PD. Practical applied kinetics.  In: Godfrey T, West S (Eds.), Industrial-Enzymology, 2^nd edition, Stockton Press, New York, 1996; 483-540.
45. Singh J, Batra N, Sobti RC. A highly thermostable, alkaline CMCase produced by a newly isolated Bacillus sp. VG1. World Journal of Microbiology and Biotechnology. 2001 Nov;17(8):761-5. https://doi.org/10.1023/A:1013564717107
46. Immanuel G, Dhanusha R, Prema P, Palavesam A. Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. International Journal of Environmental Science & Technology. 2006 Dec 1;3(1):25-34. https://doi.org/10.1007/BF03325904
47. 46.       Goel N, Patra R, Verma SK, Sharma PC. Purification and characterization of cellulase from Pseudomonas sp. isolated from waste dumping site soil. Journal of Applied Biotechnol Bioeng. 2019;6(3):118-24.          DOI: 10.15406/jabb.2019.06.00183 
48. Ramachandran LK, Witkop B. The interaction of mercuric acetate with indoles, tryptophan, and proteins. Biochemistry. 1964 Nov 1;3(11):1603-11.   https://doi.org/10.1021/bi00899a001
49. Mehta A, Singh A, Ferosh S, Mehta P. Effect of cultural factors on cellulase activity of Micrococcus luteus-2, Vibrio metschnikovii and Chromobacterium sp. Indian Journal of Microbiology. 2004; 44:47-52.
50. Mandels M, Reese ET. Inhibition of cellulases and β-glucosidases. In Advances in enzymic hydrolysis of cellulose and related materials 1963 Jan 1 (pp. 115-157). Pergamon.
51. Saxena S, Bahadur J, Varma A. Effect of cobalt and nickel on growth and carboxymethyl cellulase activity of Cellulomonas spp. BioMetals. 1992 Dec 1;5(4):209-12. https://doi.org/10.1007/BF01061219
52. Sankaralingam S, Palpperumal S, Harinathan B, Kathiresan D, Shankar T, Sundarapandian S. Screening and characterization of cellulase by Bacillus megaterium isolated from marine sediments and its antimicrobial activity. Bol. Latinoam. Caribe Plant. Med. Aromat. 2018;17(5):646-53. DOI: 10.5829/idosi.wasj.2018.646.653    
53. Dar RA, Saba I, Shahnawaz M, Sangale MK, Ade AB, Rather SA, Qazi PH. Isolation, purification and characterization of carboxymethyl cellulase (CMCase) from endophytic Fusarium oxysporum producing podophyllotoxin. Adv Enzyme Res. 2013 Dec 9;1:91-6. http://dx.doi.org/10.4236/aer.2013.14010.