The sucrose is a natural sweetener, traditionally, used in human nourishment due to its pleasant taste, nutritious value and low cost production. The sugar cane is one of the most important sucrose source, containing until 20% wt sucrose (Glazer and Nikaido, 1995). Sucrose hydrolysis produces equal quantities of fructose and glucose resulting in a mixture named inverted sugar, which has higher edulcorant power. The inverted sugar is incorporated more easily in industrial preparations and is sweeter than sucrose (Chou and Jasovsky, 1993). Even though the Brazilian sucrose production is the largest in the world, its production of inverted sugar is not sufficient for the Brazilian demand. However, due to the sucrose low market value, the research on methods to produce inverted sugar from sugarcane sucrose has increased in interest.

Acid and enzymatic hydrolysis have been identified as chemical and biochemical ways to sucrose inversion (disaccharide) into glucose and fructose (soluble monosaccharide). The acidity produced in the acid hydrolysis may be caused by an acid direct action or a H⁺ cationic resin liberation. However, syrups obtained in this process are highly coloured due to the extreme reaction conditions (pH and temperature) (Arruda and Vitole, 1999) leading to hydrometil furfural formation (HMF).

However microorganisms do not release the produced enzyme to the medium, keeping it jointed to periplasmic cell space. Therefore, the biomass utilization of fungus Cladosporium cladosporiodes, as a natural support for invertase (auto-immobilization), is a low cost technology with good results. This offers great operational stability to the enzyme, allowing it to be reused without significant activity losses (Costaglioli et al. 1997; CoutinhoFilho et al. 1999).

The bioprocess production of inverted sugar, as any other biotechnological process, is very complex due to the number of variables involved. Hence, a balanced environment with optimum temperature, pH, agitation and aeration is necessary to reach a good productivity (Blanch and Clark, 1997).

The factorial planning technique allows the correlation between the independent variables and dependent ones by a minimum number of assays. Factorial planning method associated to a response surface analysis is the statistic tool based theory (Netoet al. 1995). By this planning technique, it is possible to evaluate the independent variables influence on the dependent variables, as well as the interaction between both.

Hence, this work objective is to apply the factorial planning method to determine the optimum conditions to sucrose enzymatic hydrolyses, using the auto-immobilized enzyme in the fungus Cladosporium cladosporioides cellular mass, and to determine the kinetic parameters K_M and V_max.

The fungi Cladosporium cladosporioides strain URM 4331 was obtained from the culture collection of the Mycology Department of the Universidade Federal de Pernambuco, Brazil. Its fungi form can be observed in Figure 1a. The fungus grew under a constant 300 rpm agitation, at ambient temperature of 28 ± 2ºC during 20 days. The applied agitation allowed the "pellets" formation with an average diameter of 0.132 cm, which can be observed in Figure 1b. These "pellets" were the supports for the enzyme used as biocatalyst on the hydrolysis process.

The enzymatic hydrolysis experiments were carried out in discontinued systems (batch system), with and without agitation, using substrate solutions of sucrose (50 g/l of concentration) prepared using a buffer solution consisting of sodium acetate 0.1 M, pH 4 and 6. For the hydrolysis experiments, 3 g-cells pellets were incubated in 50 ml erlenmeyers flasks containing 30 ml of sucrose solution. As invertase is auto-immobilized in the fungus, it was necessary to determine the fungus mass concentration that optimizes the hydrolysis process.

The factorial planning led to the identification of the variables with more influence in the sucrose hydrolysis process by the enzymatic route. The optimum operational condition was: temperature of 70ºC, pH 6 and moderate agitation. These conditions agree with other works mentioned in the literature, which uses agitation below 300 rpm, pH between 4.5 and 6 and temperatures around 60ºC (Blanch and Clark, 1997; Chávez et al. 1997).

For the kinetics applied of the auto-immobilized invertase in the fungus Cladosporium cladosporioides, the Michaelis-Menten kinetic model represents satisfactorily this enzyme behaviour. The value of K_M obtained for auto-immobilized enzyme was higher than those for soluble ones mentioned in literature. This result can be related to the mass transport resistance imposed by cellular structure, which does not occur with the soluble invertase.

In spite of the diffusive limitations of cellular structure, the cell acts as the immobilization support of the enzyme. This immobilization seems to improve the invertase operational stability which justifies the use of these cells in industrial bioreactors instead of the soluble invertase.

References

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