الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 138 |  |  |
| | | from | 138 | | |
Abstract
The Folin Ciocalteu assay is specific to determine the cucurbitacin E and D-glucose due to presence of the functional group in the free state and possible to interact with components of Folin Ciocalteu. These functional groups are responsible for the reducing properties of the enzyme products.
The presence of the substrate without the fungal growth showed increase more than Blank, Curvularia only with folin ragent due to presence of cucurbitacin E glucoside which reacts with folin ragent giving coloured complex. On the other hand, presence of substrate with algal growth showed the highest concentration with folin reagent due to presence of the enzyme activity which catalyzed splitting the sugar from substrate giving cucurbitacin E which produce darker colour with folin.
Factors affecting β-glucosidase activity :
- Effect of pH and temperatureon enzyme activity
The effect of pH on the β-glucosidases was determined over a pH range of 4–10. β-glucosidase exhibited a pH optimum of 8.0. It was concluded that the optimal pH of the enzyme was 5.0.
- Effect of different incubation times on the enzyme activity
It can be seen that enzyme activity was linear with time at least up to 20 min reaction time, after which the linearity of the reaction was not presented. It is also noticed that the enzyme activity expressed in term of enzyme units at each reaction time was higher at 60°C incubation temperature for all reaction times used.
- Effect of different substrate concentrations on the β-glucosidase activity
It was showed that the increase of substrate concentration leads to a gradual increase in the cucurbitacin E production and reached its maximum value at a substrate concentration of 5 mg. After this point, the increase in substrate concentration would not increase the velocity, which means all the enzyme active sites were bound to substrate and converted to enzyme-substrate complex.
- Effect of different enzyme concentrations on the β-glucosidase activity
It was showed that the increase of enzyme concentration leads to a gradual increase in the cucurbitacin E production and reached its maximum value at concentration of 0.2 mg. After this point, the increase in enzyme concentration would not increase the velocity.
- Effect of different incubation temperature on the β-glucosidase activity
Within a wide temperature ranged between 10–70ºC, the enzymatic activity gradually increased by increasing temperature up to 30ºC, at which the maximum enzyme activity was achieved. Temperature above 30ºC resulted in loss in the enzyme activity.
- Effect of metals on the β-glucosidase activity
Some metals such as Cu, Zn and Ni caused inhibition of the enzyme activity. The three of the investigated heavy metals inhibited the enzyme activity in acetate buffer. At metal concentrations of 0.6 mM, Zn and Ni reduced the enzyme activity by 25-30% under optimal pH conditions (pH 5-5.2). Under the same conditions, Cu showed an even more pronounced inhibitory effect than Zn and Ni. Presence of Pb caused toxicity of the enzyme. The presence of monovalent and divalent metal ions Na+, K+, Ca2+ and Mg2+ also positively influenced the activity of β-glucosidase.
- The effect of sulfate, sulfite and sulfide on β-glucosidase activity
The enzyme activity was maximal in the absence of sulfate and the activity generally decreased as the concentration of the sulfate was increased. Sulfate therefore appeared to be an inhibitor of β-glucosidase activity. Sulfite at an optimal concentration of led to an increase in the enzyme activity compared to when no sulfite was added. Sulfide was a potent inhibitor of β-glucosidase activity.
The data showed after 1 hr of the reaction beginning that concentration of the steroidal portion increase with the increasing the enzyme concentration till reach 1.5 mg / ml of the enzyme concentration. After that the product concentration remained unchanged even though with increasing the enzyme concentration. This indicated that the substrate concentration was insufficient to consume the enzyme completely. After 4 hr of the reaction beginning that concentration of the steroidal portion increase with the increasing the enzyme concentration till reach 0.6 mg / ml after that the product concentration remained unchanged till the point 1 mg / ml. The product concentration re-increased with increasing the enzym concentration till the point 1.5 mg / ml after that the product concentration remained unchanged. This indicated that 4 hr of the reaction beginning sufficient to decrease the enzyme activity and the enzyme tried to retain its activity with increasing the enzyme concentration so the product re-increased slowly after 1 mg / ml of the enzyme concentration. This confirmed the concept which showed that enzyme concentration and reaction time vital factors affecting the reaction efficiency.