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MULTISTEP MUTAGENESIS FOR THE OVER-EXPRESSION OF CELLULASE IN HUMICOLA INSOLENS

MUHAMMAD MOHSIN JAVED^1*, IKRAM-UL-HAQ¹ AND IRFANA MARIYAM²

Abstract: The present study describes the improvement of a thermophilic fungal strain Humicola insolens for cellulase production. H. insolens TAS-13 has the potential to produce 1.00 U/ml/min CMC-ase, 0.43 U/ml/min FP-ase and 0.30 U/ml/min β-glucosidase, which was improved through UV and chemical mutagenesis like N-methyl-N-nitro-N-nitrosoguanidine (MNNG), nitrous acid (HNO₂), ethyl methyl sulphonate (EMS) or Ethidium bromide (EtBr). Two alternative mutation steps were carried out and mutants were screened for cellulase production. After first step mutation, 26 mutants were isolated and among them 5 best mutants (TAS_UV-4, TAS_NG-7, TAS_HN-4, TAS_EB-2 and TAS_EMS-1) were further screened on the basis of hypercellulolytic ability. These mutants gave 17.36, 41.17, 5.25, 23.07 & 29.07% CMC-ase, 18.87, 30.64, 6.52, 10.41 & 17.30% FP-ase and 19, 61.54, 62.02, 14.28 & 23.07% β-glucosidase, respectively more than their wild strain H. insolens TAS-13. When these mutant were further mutated alternatively with the same mutagens under the same conditions, a total of 33 mutants were picked up as second generation mutants. Among these, a mutant strain TAS-13_UV-4^NG-5 proved to be the best mutant, which produced 43.19% CMC-ase, 60.15% FP-ase and 59.78% β-glucosidase more than the first generation mutant TAS-13_UV-4. Furthermore, this mutant was highly stable upto many generations possessing cellulolytic ability.

¹Institute of Industrial Biotechnology, GC University, Lahore Pakistan

²Department of Chemistry, GC University, Lahore Pakistan

*Corresponding author: Email: mmj_bot@yahoo.com