Characterisation of thermostable dextranases from micro-organisms for commercial application
thesisposted on 06.12.2017, 13:14 by Christine Fay Galea
Currently, when dextranase is used in the sugar factory, modifications are required to normal factory operating conditions in order to achieve cost effective dextran 'removal'. The major change involves the decrease in primary heater temperature from 75°C to about 60°C. A number of processing problems result from this temperature change. Therefore, it was proposed to develop a thermostable dextranase which was active and stable at 75°C. The use of a high temperature dextranase would also overcome the problems associated with operating the primary heater at the lower temperature. Existing culture collections, thermal environments and sites within raw sugar factories were used as the sources of microbial isolates screened for thermostable dextranase producers. Based on the amount of enzyme produced at elevated temperatures, five bacterial strains ( SRI 2125, SRI 2128, AB11A, RT364 and DP17) were selected as the most promising sources of thermostable dextranases.These isolates were grown to pure strain using standard microbial techniques. Thereafter, broth culture of these strains was undertaken to produce sufficient crude extracellular dextranase for liquid assay. However, compared to fungal isolates, the amount of dextranase produced by the bacterial isolates was low. Several assays to measure micro-quantities of dextranase activity were developed or modified to assess the thermal stability of the enzymes. Temperature and pH profiles of the crude extracts were determined using the PAHBAH assay to obtain a rapid and sensitive measure of optimal conditions required for enzymic activity. The subsequent development of the micro-haze test allowed the thermal stability and activity to be reliably assessed under simulated factory conditions. Both new and existing enzymic assays were utilised to determine the activity and thermal stability of the most cost-effective commercial dextranase currently available i.e. the C. gracile dextranase. This enzyme was found to exhibit optimum activity at 55-60°C (pH 5.0) under all assay conditions. However, thermal stability at temperatures above 65°C was very low. A comparative assessment of the commercial potential of new thermostable dextranases was possible using the C. gracile dextranase as the 'bench mark'. In addition, detailed studies on the C. gracile dextranase were performed to determine the physico-chemical characteristics of the enzyme(s) in the commercial preparation. The dextranase was separated into five distinct components by electrophoresis of the native enzyme. However, each of these components were found to possess a similar (if not identical) molecular size. Partial separation of these components was achieved using chromatofocusing. Each fraction obtained exhibited endo-dextranase activity..Crude preparations of the five bacterial dextranases were found to exhibit optimal activity between 63 and 75°C (depending on the assay conditions and the method employed for activity measurement). The specific activities of these crude dextranase preparations were very low (0.021 to 0.68 mole min-¹mg-¹) compared to the crude extracts obtained from fungal sources such as C. gracile. The presence of endo-dextranase activity was confirmed by determining the activity against cane dextran under simulated factory conditions. The dextranase produced by the RT364 isolate was selected for purification investigations as it was the most active in the crude form. Several standard techniques for protein purification were utilised and the purity of the resulting preparations established by electrophoretic analysis. The active dextranase in the fraction of highest purity and activity was shown to represent in excess of 50 per cent of the protein in the preparation. Using this estimate, a final specific activity for the purified thermostable dextranase from RT364 was calculated to be about 20 mo1e min-¹mg-¹. At this level, the specific activity of the RT364 enzyme is approximately 150 times less than that of the commercial C..gracile dextranase measured its optimum at temperature of 55°C. On this basis, the commercial potential of the thermostable dextranase from the RT364 isolate appears to be limited. To date, the specific activities of purified forms of the remaining four thermostable dextranases have not been determined. It is possible that one of these isolates may be the source of a thermo-stable dextranase with greater commercial potential. However, further investigations with these enzymes to establish their commercial viability would require high developmental costs in relation to the very small market (the Australian sugar industry) currently available for these enzymes.