Abstract:
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.
