Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / Bioethanol production from lignocellulosic biomass is seen as an alternative source of energy. However, large-scale production of bioethanol from lignocellulosic biomass is still not feasible due to the high cost of cellulase and lack of cellulases with a high specific activity that can act on crystalline cellulose. The study aimed at screening for microbial cellulolytic enzymes using a proteomic approach. The objectives were to screen for microbial cellulases with a high specific activity and separate the cellulolytic enzymes using a combination of zymography and two dimensional (2-D) gel electrophoresis followed by tryptic digestion, matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) and bioinformatics analysis. Fungal and bacterial isolates were cultured in M9 minimal and Mandel media for a period of 168 hours at 60°C and 30°C with cellobiose and Avicel as carbon sources. Microbial cells were separated from the supernatants through centrifugation and the crude enzymes from the cultures were used for the determination of cellulase activity, zymography, SDS-PAGE and two-dimensional gel electrophoresis. Five isolates, with lytic action on carbon sources studied were a bacterial strain, (BARK) and fungal strains (VCFF1, VCFF14, VCFF17 and VCFF18). Peak cellulase production by the isolates was found to be 3.8U/ml, 2.09U/ml, 3.38U/ml, 3.18U/ml and 1.95U/ml, respectively. Beta-glucosidase zymography resulted in a dark brown band and clear zones against a dark background for endoglucanase. Affinity precipitation of the VCFF17 isolate’s crude enzyme resulted in seven glycoside hydrolases with a carbohydrate binding module (CBM). The presence of the CBM in the glycoside hydrolases produced by the VCFF17 confer the isolate’s potential to be used in the hydrolysis of plant biomass for bioethanol production. Two-dimensional gel protein maps resulted in the separation and quantitative expression of different proteins by the microbial isolates. MALDI-TOF analysis and database search showed that the expressed proteins in this study closely relate to different glycoside hydrolases produced by other microbial species (Hypocrea jecorina, Emericella nidulans, Trichoderma pseudokoningii and Trichoderma koningii). BARK, VCFF1, VCFF14, VCFF17 and VCFF18 showed great potential as cellulolytic enzyme producers for bioethanol production. The BARK isolate exhibited the highest beta glucosidase activity. The isolates studied may benefit the industry in reducing the costs associated with bioethanol production in consolidated bioprocessing system. / National Research Foundation (NRF)