Increased thermostability of Asn182 → Ala mutant Aspergillus awamori glucoamylase

Abstract

Asn182 --> Ala Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae had a first-order thermodeactivation coefficient 40% that of wild-type glucoamylase at pH 4.5 between 60 degrees and 65 degrees C, caused by the elimination of an Asn-Gly sequence subject to deamidation and eventual chain breakage. Above 70 degrees C, and at pHs 3.5 and 5.5, thermodeactivation coefficients of wild-type and mutant enzymes were roughly equal, because the fastest deactivation mechanism was no longer deamidation. The mutation had little effect on the enzyme's optimal pH for activity and subsite map, or on the glucose yield from starch dextrin hydrolysis. During enzyme production by yeast fermentation, highest cell densities and activities of wild-type and mutant glucoamylases were attained after a period of glucose starvation, followed by a second addition of glucose. (c) 1994 John Wiley & Sons, Inc.

Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase fromBacillus licheniformisRT7PE1 and Its Characteristics

“…The values of BLA here are markedly lower, therefore, up to 95°C, the enzyme was reasonably thermostable. These values are also significantly lower than those reported on thermostable mutant of Aspergillus awamori glucoamylase [24]. When enthalpy and entropy values for inactivation were calculated at each temperature, Δ S * had again negative values (Table 3(b)).…”

“…The culture had altered the values of both entropy and enthalpy of activation for irreversible inactivation of enzyme as observed for thermostabilized enzymes [24]. Actually thermal inactivation occurs in two steps as follows: $\begin{array}{c}N⟷U⟶I,\end{array}$ where N is the native, U is the unfolded enzyme which could be reversibly refolded upon cooling, and I is the inactivated enzyme formed after prolonged exposure to heat and therefore can not be recovered on cooling.…”

“…The thermal denaturation of enzymes is accompanied by the disruption of noncovalent linkages, including hydrophobic interactions, with concomitant increase in the enthalpy of activation [24]. The opening up of the enzyme structure is accompanied by an increase in the disorder, randomness or entropy of activation.…”

“…The enzyme had 44.8 kJ mol −1 and −155.6 J·mol −1  K −1 Δ H * (enthalpy of deactivation) and Δ S * (entropy of deactivation) values respectively. The values of Δ S * and Δ H * of β -glucosidase from a thermophilic culture of Aspergillus wentii [24] were 125 kJ mol −1 and 65 J·mol −1  K −1 , respectively. The values of BLA here are markedly lower, therefore, up to 95°C, the enzyme was reasonably thermostable.…”

Protein asparagine deamidation prediction based on structures with machine learning methods

“…All test proteins are distinct from the training set. It consists of 81 Asn residues on 3 high-resolution protein structures including an anthrax protective antigen, an angiogenin, and a glucoamylase [26–30]. The test set was prepared the same way as the training set.…”