PROTEIN ENGINEERING OF THE RELATIVE SPECIFICITY OF GLUCOAMYLASE FROM ASPERGILLUS-AWAMORI BASED ON SEQUENCE SIMILARITIES BETWEEN STARCH-DEGRADING ENZYMES
Mr. Sierks et B. Svensson, PROTEIN ENGINEERING OF THE RELATIVE SPECIFICITY OF GLUCOAMYLASE FROM ASPERGILLUS-AWAMORI BASED ON SEQUENCE SIMILARITIES BETWEEN STARCH-DEGRADING ENZYMES, Protein engineering, 7(12), 1994, pp. 1479-1484
Aspergillus glucoamylase catalyzes hydrolysis of D-glucose from non-re
ducing ends of starch with an similar to 300-fold (k(cat)/K-m) prefere
nce for the alpha-1,4- over the alpha-1,6-glucosidic linkage determine
d using the substrates maltose and isomaltose. It is postulated that a
s most amylolytic enzymes act on either the alpha-1,4- or alpha-1,6-li
nkages, sequence comparison between active-site regions should enable
the correlation of the substrate bond specificity with particular resi
dues at key positions. Therefore, the already high bond-type selectivi
ty in Aspergillus glucoamylase could theoretically be augmented furthe
r by three single mutations, Ser119--> Tyr, Gly183--> Lys and Ser184--
> His, in two separate active-site regions. These mutants all had slig
ht increases in activity as compared with the wild-type enzyme towards
the alpha-1,4-linked maltose; this was due to lower K-m values as wel
l as small decreases in activity towards isomaltose. This latter decre
ase in activity was a result of higher K-m values and a decrease in k(
cat) for the Ser184--> His mutant. As a consequence, the selectivity o
f the three glucoamylase mutants for alpha-1,4- over alpha-1,6-linked
disaccharides is enhanced 2.3- to 3.5-fold. In addition, the introduct
ion of a cationic side chain in Gly183--> Lys and Ser184--> His glucoa
mylase, broadens the optimal pH range for activity towards acidic as w
ell as alkaline conditions.