M.S.

Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2013 / The production of cellulase is a key factor in the hydrolysis of cellulosic materials and it is essential to make the process economically viable. Cellulases are the most studied multi- enzyme complex and comprise of endo-glucanases (EG), cellobiohydrolases (CBH) and β- glucosidases (BGL). The complete cellulase system; comprising CBH, EG and BGL components thus acts synergistically to convert crystalline cellulose to glucose. Cellulases are currently the third largest industrial enzyme worldwide. This is due to their wide applications in cotton processing, paper recycling, juice extraction, as detergent enzymes and additives in animal feed. In this study, production of cellulase by five fungal isolates (BTU 251-BTU 255) isolated from mushrooms, was investigated and optimised. Internal transcribed spacer regions (ITS1 and ITS4) were applied to identify the five fungal microorganisms. Isolates were identified as follows: BTU 251 as Aspegillus niger,BTU 253 as Penicillium polonicum, and BTU 255 as Penicillium polonicum. Cellulase was produced in shake flask cultures using Mandel’s mineral solution medium and Avicel as a carbon source. Cellulase activity was tested using 3, 5-Dinitrosalicylic acid assay and zymography, A. niger BTU 251 showed five activity bands ranging from 25- 61 kDa had an average nkat of 7000. Cultures from BTU 252 were the least active with an average nkat/ml of 200 and one activity band of 25 kDa. P. polonicum BTU 253 showed three activity bands ranging between 45 and 60 kDa and had an average nkat/ml of 2200. BTU 254 showed five activity bands ranging from 22- 116 kDa and had average nkat of 350. P. polonicum BTU 255 produced the highest cellulase activity of 8000 nkat/ml and with three activity bands estimated at 45-60 kDa on zymography. The optimal temperature for activity of the cellulases was between 55-70°C and enzymes were most active within a pH range of 4-6. Optimal pH for production of cellulases by P. polonicum BTU 255, P. polonicum BTU 253 and A. niger BTU 251 was 4 while optimal temperature for production of the cellulases was between 50-55°C. Total cellulase activity was determined using Whatman No.1 filter paper as a substrate and β- glucosidase production was determined in polyacrylamide gels using esculin as a substrate. In the hydrolysis of crystalline cellulose (Avicel), a combination of A. niger BTU 251 and P. polonicum BTU 255 (1:1), (1:9), (1:3), and (1:2) produced maximum glucose as follows: 1:1 (0.83g/L), 1:9 (10.4g/L), 1:3 (0.77g/L) and 1:2 (0.73g/L). Cellulases from P. polonicum BTU 255 were partially purified using affinity precipitation and analysed using MALDI- TOF/TOF. Peptide sequences of P. polonicum obtained from MALDI-TOF/TOF analysis were aligned by multiple sequence alignment with C. pingtungium. Conserved regions were identified using BLAST anaylsis as sequences of cellobiohydrolases. More research is required in producing a variety of cellulases that are capable of hydrolysing crystalline cellulose, the current study contributes to possible provision of locally developed combinations of cellulases that can be used in the production of bioethanol.

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.