Fermentation studies and genetic modification of cellobiohydrolase I gene of mutated aspergillus niger and pencillium citrinum isolated from sawdust (Mitragyna ciliata)
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos
The need for utilizing renewable resources to meet the future demand for fuel and other value added products has increased the attention on lignocellulose, the most abundant and renewable resource in the world. Lignocellulose is degraded by lignocellulolytic enzymes produced by fungi and bacteria. Mutagenic agents can be used to achieve improvement of these strains. However, there has been a challenge in improving and optimizing fermentation process which in most cases has been carried out independently. The aim of this study was to hyper produce cellulase from fungi in order to enhance lignocellulosic wastes biodegradation. Raw Abura sawdust (Mitragyna ciliata) was collected from Okobaba sawmill, Ebute-meta, Lagos. It was pretreated mechanically and chemically using ammonium hydroxide. The lignocellulosic and proximate compositions of both the raw and pretreated sawdust types were determined. Fungi were isolated from decomposing wood wastes. They were genotyped by amplifying the internally transcribed spacer (ITS) regions on their DNA, sequencing the amplicons and analyzing the sequences. Two fungal strains with best cellulolytic potential were selected and co-cultured. They were genetically modified for enhanced cellulase production using ultraviolet rays. The optimal conditions for the effective production of cellulase by both wild and mutant strains of the fungi were investigated. The solid-state fermentation of these mutants was optimized in order to further enhance the production of cellulase. The enzyme from both the wild and mutant strains were partially purified and characterized. RNA of the fungal mutants was extracted from their mycelia. The extracted RNA was reverse transcribed to complementary DNA (cDNA). Selected genes coding for cellulase were amplified and the amplicons were sequenced. The points of mutation were identified from the sequence and the sequences of the mutants were aligned and compared with referenced sequences which included those of Trichoderma reesei and Phanerochaete chrysosporium using MEGA5 and Jalview softwares. The sequences were also translated to amino acid and in-silico X-ray crystallography structure of the active site of the enzyme was constructed. The hemicellulose content of the sawdust reduced significantly upon pretreatment (32.70 ± 2.20% to 19.80 ± 1.30%) while its cellulose content increased (48.11 ± 1.60% to 64.94 ± 1.20%). Proximate analysis revealed that moisture (6.30 ± 0.60% to 3.60 ± 1.30%) and crude fibre contents (62.20 ± 3.40% to 52.80 ± 2.20%) reduced significantly upon pretreatment. Aspergillus niger and Penicillium citrinum were selected out of four fungi genotyped which included Trichosporon asahii and Penicillium corylophylum. Co-culturing was more efficient in the biodegradation of sawdust. Aspergilus niger mutant strain had a 2.1-fold and 2.4-fold increase in carboxymethylcellulase (CMCase) and filter paper cellulase (FPase) production respectively more than the wild strain while Penicillium citrinum had a 1.8-fold and 2.1-fold increase in CMCase and FPase production. However, optimized fermentation of A. niger mutant produced a 7.4-fold and 7.6-fold higher increase in CMCase and FPase production more than the wild strain while P. citrinum mutant produced a 5.3-fold and 5.8-fold increase (CMCase and FPase). Purified cellulase from wild A. niger had a catalytic efficiency of 0.305M-1s-1 while its mutant had a catalytic efficiency of 0.429M-1s-1. Purified cellulase from wild P. citrinum had a catalytic efficiency of 0.858M-1s-1 while its mutant had a catalytic efficiency of 1.036M-1s-1. Bioinformatics analysis of the sequence of the cbh1 gene of the A. niger mutant showed that it had strong similarity with compared industrially beneficial fungi; Trichoderma reesei and Phanerochaete chrysosporium. Amino acid residues in its active site had low hydropathy index. Predicted structure of cellulase from A. niger mutant revealed that alteration occurred in the β-pleated sheets of the enzyme. Genetic modification of Aspergillus niger and Penicillium citrinum and optimizing their solid-state fermentation resulted in enhanced cellulase enzyme production.