Directed evolution of glucoamylase to improve thermal stability

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McDaniel, Allison
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"Glucoamylase (EC, GA) is an [Alpha](1,4)-D-glucan glucohydrolase. It is an enzyme that catalyzes the hydrolysis of [Alpha]-1,4 glucosidic bonds from the nonreducing ends of starch to release [Beta]-D-glucose. Past research has revealed that the thermo-stability of glucoamylase can be increased by substituting amino acids at specific positions within the protein. In this work, the yeast host Saccharomyces cerevisiae was used to perform random recombination with the glucoamylase cDNA of several mutant lines to create new combinations of mutations. The most thermostable mutants were then selected and one was chosen for further mutagenesis using PCR and an error prone PCR mix to insert new mutations into the gene. Transformation into yeast, colony screening, and subsequent DNA sequencing gave four new mutations: Val88[right pointing arrow]Ile, Asp293[right pointing arrow]Ala, Tyr402[right pointing arrow]Phe, and Glu408[right pointing arrow]Lys. These four new mutations were combined with the most thermostable mutant from random recombination, RE15, to create the ""super"" mutant SRE15. Kinetic assays revealed that RE15 and SRE15 had increases in melting temperature (Tm) of 4.10C and 6.20C, respectively, over that of wild-type glucoamylase, which has a T[subscript m] of 68.80C. In addition, the two mutants also displayed an increase in [Delta]G, which was 3 kJ/mol for RE15 and 2.6 kJ/mol for SRE15 (at 650C). Other kinetic parameters such as K[subscript m1], k[subscript cat1] and V[subscript max] were similar for wild-type glucoamylase, RE15, and SRE15. Thoughtful analysis and comparison of mutant proteins in thermostability assays has led to several conclusions regarding the mutations present in SRE15. It was found that the four individual mutations by themselves in the wild-type back-ground did little to increase glucoamylase thermal stability. Yet, when these mutations were brought together into one enzyme, SRE15, increases in the T[subscript m] and [Delta]G were observed. It is thought that the four new mutations that were inserted into the RE15 gene may create a new network of interactions with the amino acids in the surrounding environment that leads to an increase in thermal stability."

Sat Jan 01 00:00:00 UTC 2005