C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/56 (2006.01) C12N 9/34 (2006.01) C12S 3/12 (2006.01) C13K 1/06 (2006.01)
Patent
CA 2209577
Aspergillus awamori glucoamylase [1,4-.alpha.-D-glucan glucohydrolase, EC 3.2:1.3] (GA) catalyzes the release of glucose from the non-reducing ends of starch and related oligosaccharides. GA is used in, and defines the rate limiting step of, the commercial production of high glucose syrups. GA is rapidly and irreversibly inactivated at temperatures above 65°C. Based on the known GA structure and homology with other GA's, four proline substitution mutations were constructed and expressed in Saccharomyces cerevisiae. These mutations were designed to increase GA thermal stability by decreasing conformational entropy of unfolding. None of the mutations decreased enzyme activity. Three of the mutations, Asp293-Pro, Asp345-Pro and Glu408-Pro, either decreased or did not significantly alter GA. However, one of the mutations, Ser30-Pro, significantly decreased the rate of irreversible thermal inactivation when analyzed between 65° and 80°C. At 70°C a 1.9-fold decrease in thermal inactivation rate coefficients was seen and the activation energy for thermal inactivation (.DELTA..DELTA.G~) was increased by 2.2 kJ/mol. relative to wild-type GA. Additionally, when the Ser30-Pro mutation was combined with a previously characterized stabilizing mutation (Gly137-Ala, which increased the.DELTA..DELTA.G~ by 1.3 kJ/mol. at 70°C) a 4.0 kJ/mol increase in .DELTA..DELTA.G~ relative to wild-type GA at 70°C was seen without loss of enzyme activity.
Allen Martin J.
Chen Hsiu-Mei
Coutinho Pedro M.
Fang Tsuei-Yun
Ford Clark F.
Iowa State University Research Foundation
Iowa State University Research Foundation Inc.
Moffat & Co.
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