Analysis of cellulose degrading enzymes in genetically modified sugarcane juice using capillary electrophoretic techniques

• Main Author: Gupta, Ruchi
• Other Authors: Grieve, Bruce
• Published: University of Manchester 2011
• Subjects: 662.6692
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.553308

Genetically modified (GM) sugarcane expressing cellobiohydrolase (CBH), endo-β-glucosidase (EG) and β glucosidase (BG) have a potential to provide low cost enzymes required for commercially viable production of ethanol from waste such as bagasse. The expression of enzymes in GM sugarcane is likely to be dependent on the variety of the plant and its growing conditions. In this project, reliable, rapid and easy-to-use methods capable of on-site analysis of cellulases in every batch of sugarcane juice (arriving hourly) were developed. Firstly, a capillary zone electrophoresis (CZE) method was devised for the separation and quantification of BG, CBH and EG, which were purified from sugarcane juice via ultrafiltration, in a single run. Under optimised conditions (15 mM phosphate buffer at pH 7.75), the analysis was achieved in 10 min. The linear dynamic range and the limit of detection of the developed method for the quantification of enzymes were suitable for monitoring their expected concentration in GM sugarcane juice. A novel isotachophoresis (ITP) method was then formulated for the purification of cellulases from sugarcane juice. An electrolyte system comprising a leading electrolyte of 10 mM formic acid at pH 9.0 and a terminating electrolyte of 10 mM β-Alanine was devised. The use of a simple front cutting method removed a majority of interfering species in the juice, thereby resulting in the formation of a distinct zone of desired proteins. In comparison to techniques such as ultrafiltration and liming, the loss of desired proteins was lower when the sample was prepared using ITP. A procedure for collecting fractions prepared via ITP was also formulated. The time required for sample clean-up of proteins in 10, 5 and 2 times diluted juice via the developed ITP method is ~30 min, ~45 min and ~60 min respectively. In other words, the enzymes in two-times diluted sample can be purified in ~60 min following which their concentration can be measured in ~10 min. This implies that the developed methods can be used to analyse sugarcane batches which are expected to arrive approximately every hour. The type of sugarcane and the agronomic conditions are also likely to affect the glycosylation of cellulases. The glycosylation plays a significant role in determining the enzyme activity. Hence, for the first time, CZE assays for the analysis of the glycoforms of CBH in an intact form were developed in this work. The developed methods were capable of analysing the glycoforms without the necessity of buffer additives or enzyme labelling protocols. The separation of the glycoforms of CBH was achieved using 15 mM, pH 4.5 and 50 mM, pH 7.5 phosphate electrolytes in a fused silica and a neutral capillary respectively. As the migration times of the glycoforms of CBH were highly repeatable in the latter method, its use is recommended for routine testing of glycosylation-mediated microheterogeneities in CBH. Finally, it is believed that the sample preparation and the analysis methods described in this thesis could serve as the basic operating principles of a microfluidic chip (called an Integrated Cellulase Unit, ICU). It is also envisioned that a cartridge containing multiple ICUs could be located at the end of the direct insertion probe to analyse cellulases at various points in the bagasse-to-bio-ethanol process.