Characterisation and role of sugarcane invertase with special reference to neutral invertase.

Thesis (Ph.D.)-University of Natal, Durban, 2000.The relationship between extractable invertase activities and sucrose accumulation in the sugarcane (Saccharum spp. hybrids) culm and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid invertase (SAI), cell wall bound acid invertase (CWA) and neutral invertase (NI) from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature culm tissue of the same cultivar. Sugarcane NI had a higher specific activity than SAI (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane culm. CWA was also present in significant quantities in both immature and mature tissue. Sugarcane NI was partially purified from mature sugarcane culm tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer. Most of the activity elutes as a monomer of native Mr ca 60 kDa. The enzyme displays typical hyperbolic saturation kinetics for sucrose hydrolysis. It has a Km of 9.8 mM for sucrose and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for sucrose to be 62.5 kJ.mol-1 below 30°C and -11.6 kJ.mol-1 above 30°C. Sugarcane NI is inhibited by its products, with fructose being a more effective inhibitor than glucose. Sugarcane NI is significantly inhibited by HgCI2, AgNO-3, ZnCI2, CuSO4 and CoCI2 but not by CaCI2, MgCI2 or MnCI2. Sugarcane NI showed no significant hydrolysis of cellobiose or trehalose. When radiolabelled fructose was fed to sugarcane internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. A combination of continuous feeding and pulse chase experiments was used to investigate the in vivo contribution of the invertases and the compartmentation of sugars. Sucrose is synthesised at a rate greater than the rate of breakdown at all stages of maturity in sugarcane culm tissue. The turnover time of the total cytosolic label pool is longer for internode three than internode six. A higher vacuolar:cytosolic sugar molar ratio than previously assumed is indicated. Developmentally, the greatest change in carbon allocation occurs from internodes three to six. The main competing pools are the insoluble and neutral fractions. As the tissue matures, less carbon is allocated to the insoluble and more to the neutral fraction. The neutral fraction consists mainly of sucrose, glucose and fructose. The compartmented nature of sugarcane storage parenchyma carbohydrate metabolism results in a system that is complex and difficult to investigate. A computer based metabolic flux model was developed to aid in the interpretation of timecourse labelling studies. A significant obstacle was the global optimization of the model, while maintaining physiologically meaningful flux parameters. Once the vacuolar:cytosolic molar ratio was increased, the model was able to describe the internode three and six labelling profiles. The model results were in agreement with experimental observation. An increase in the rate of sucrose accumulation was observed with tissue maturation. Only the internode three glucokinase activity was greater than the experimentally determined limit. The rate was however physiologically feasible and may reflect the underestimation of the in vivo rate. SAI and NI contributed to sucrose hydrolysis in internode three but not in internode six. The rates in internode six were set to fixed low values to enable the model to fit the experimental data. This does not however preclude low levels of in vivo SAI and NI activity, which would prove significant over a longer time period. The flow of label through the individual pools, which comprise the experimentally measured composite pools could be observed. This provides insight into the sucrose moiety label ratio, SPS:SuSy sucrose synthesis ratio, and the rate of 14CO2 release. The model provides a framework for the investigation and interpretation of timecourse labelling studies of sugarcane storage parenchyma