دانلود رایگان مقاله انگشت نگاری آنزیم های پلی ساکارید-هیدرولیز

Abstract

We present three case studies, based on literature data, in which the “fingerprinting” method of determining the relative specificities of an enzyme is applied to branched reaction schemes. The first case study involves the hydrolysis of maltoheptaose by a β-amylase and shows that the fingerprinting method can be applied to schemes involving processivity. The analysis reveals that the native β-amylase has a 1.26-fold preference for attacking maltoheptaose by the processive route over the non-processive route, but that for a mutant enzyme this preference is 0.18-fold. The second case study involves the hydrolysis of β-1,6-N-acetylglucosamine oligomers by DispersinB. Our set of relative specificity constants is more consistent with the results of initial rate experiments than is the set that the authors obtained by fitting a pseudo-first order model to their data. The third case study involves the hydrolysis of galacturonic acid oligomers by an endopolygalacturonase. This enzyme can catalyze a total of 11 different reactions with a mixture of tri-, tetra-, penta-, hexa- and heptagalacturonates. We determined the relative specificity constants for these 11 reactions. The fingerprinting method has advantages over the methods that have been previously used to determine specificity constants for branched reaction schemes, being able to use a single experimental reaction profile for determination of all relative specificity constants.

5. Conclusions

Mathematical models are important tools for guiding the development of processes for the hydrolysis of polysaccharides. In order to use these mathematical models, it is necessary not only to have correctly formulated equations, but also to have good estimates of the parameters of the model. Models of the saccharification of polysaccharides will need to include descriptions of the later stages ofthe reaction, when the enzymes are attacking oligosaccharides, in complex, branched reaction schemes. The preferences of an enzyme for the various reactions that it can catalyze within such reaction schemes depend on the length of the oligomer and on the position of the bond within the oligomer. In the current work, we have demonstrated that the fingerprinting method of determining relative specificity constants can be applied to branched reaction schemes. This method is simpler to apply than methods that have been used to date, such as those based on the determination of initial rates and the bond cleavage frequency method: Estimates of all relative specificity constants can be obtained from one complete time course experiment.