158
reasonably correctly predicted even though the complete effects of such modifications remain less successful. Many substitutions have been found to increase the
specificity of the enzyme for particular peptide links, whilst reducing it for others,
particularly for the glycine residue at the bottom of the P 1 cleft (Gly 166 ).
The oxygen produced by bleaches can inactivate subtilisin; this inactivation is
due to the oxidation of the methionine residue at position 222 (Met 222 ) to its sulphoxide. Met 222 lies adjacent to the active site Ser residue, which is necessary for
catalysis. Oxidation of Met 222 most likely prevents substrate access to the active site.
Engineered subtilisin in which the Met 222 residue was substituted with serine or
alanine was much less sensitive to oxygen than the native enzyme; however, these
enzymes demonstrated much reduced specific activities (only 53% and 35% specific activities, respectively, in comparison to their wild-type counterparts)
(Bradshaw and Purton 2012).
Lactate Dehydrogenase Lactate dehydrogenase, LDH (EC 1.1.1.27), catalyses the
transformation of lactate to pyruvic acid and back, as it converts NAD
+
to NADH and
back. The lactate dehydrogenase produced by Bacillus stearothermophilus was used
for enzyme engineering (Holmberg et al. 1999). The specificity of this enzyme can be
shifted from lactate to maleate by modifying (engineering) this enzyme. This specificity was found to be associated with the existence of glutamine at position 102 (Gln 102 ).
Replacement of Gln 102 with arginine resulted in change in specificity from lactate to
maleate. The engineered enzyme became specific to maleate to the same magnitude to
which the native enzymes have specificity for lactate. Meng et al. (2016) reported that
an NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842
can be rationally engineered to efficiently use both NADH and NADPH as cofactors.
These examples clearly demonstrate that enzyme engineering may be capable to create potential enzymes with novel substrate specificities.
α-Amylase The enzyme α-amylase (1,4-α-D-glucan glucanohydrolase, EC. 3.2.1.1)
catalyses the hydrolysis of α-(1,4) glycosidic linkages in starch and related polysaccharides to yield malto-oligosaccharides, such as maltotriose (G3), maltotetraose
(G4), maltopentaose (G5), and maltohexaose (G6). The production of maltooligosaccharides has attracted attention in food industry as they contribute desirable
changes to enhance flavour and physicochemical characteristics of food in addition
to many beneficial properties to human health. They are highly useful in beverages,
bakery products, confectionery, infant milk powders, yogurts, and dairy desserts.
However, the price of pure malto-oligosaccharides is exceptionally huge as the
chemical structure of malto-oligosaccharides is bigger than maltotriose and because
of its difficulty in production (Subramanian et al. 2012).
The enzyme AmyUS100 produced by Bacillus stearothermophilus has a primary
amino acid sequence identical to that of another enzyme AmyS produced by a different strain (B. stearothermophilus strain DN1792) of the same bacterium. Among
the 516 residues, only three residues varied in the mature protein, one of which was
in the terminal region that is not involved in catalysis. Both the amylases, AmyUS100
and AmyS, demonstrated identical optimum pH and temperature but differed in
their starch hydrolysis profile. Thus, it was implicated that the two amino acids
S.M. Basheer and S. Chellappan
reasonably correctly predicted even though the complete effects of such modifications remain less successful. Many substitutions have been found to increase the
specificity of the enzyme for particular peptide links, whilst reducing it for others,
particularly for the glycine residue at the bottom of the P 1 cleft (Gly 166 ).
The oxygen produced by bleaches can inactivate subtilisin; this inactivation is
due to the oxidation of the methionine residue at position 222 (Met 222 ) to its sulphoxide. Met 222 lies adjacent to the active site Ser residue, which is necessary for
catalysis. Oxidation of Met 222 most likely prevents substrate access to the active site.
Engineered subtilisin in which the Met 222 residue was substituted with serine or
alanine was much less sensitive to oxygen than the native enzyme; however, these
enzymes demonstrated much reduced specific activities (only 53% and 35% specific activities, respectively, in comparison to their wild-type counterparts)
(Bradshaw and Purton 2012).
Lactate Dehydrogenase Lactate dehydrogenase, LDH (EC 1.1.1.27), catalyses the
transformation of lactate to pyruvic acid and back, as it converts NAD
+
to NADH and
back. The lactate dehydrogenase produced by Bacillus stearothermophilus was used
for enzyme engineering (Holmberg et al. 1999). The specificity of this enzyme can be
shifted from lactate to maleate by modifying (engineering) this enzyme. This specificity was found to be associated with the existence of glutamine at position 102 (Gln 102 ).
Replacement of Gln 102 with arginine resulted in change in specificity from lactate to
maleate. The engineered enzyme became specific to maleate to the same magnitude to
which the native enzymes have specificity for lactate. Meng et al. (2016) reported that
an NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842
can be rationally engineered to efficiently use both NADH and NADPH as cofactors.
These examples clearly demonstrate that enzyme engineering may be capable to create potential enzymes with novel substrate specificities.
α-Amylase The enzyme α-amylase (1,4-α-D-glucan glucanohydrolase, EC. 3.2.1.1)
catalyses the hydrolysis of α-(1,4) glycosidic linkages in starch and related polysaccharides to yield malto-oligosaccharides, such as maltotriose (G3), maltotetraose
(G4), maltopentaose (G5), and maltohexaose (G6). The production of maltooligosaccharides has attracted attention in food industry as they contribute desirable
changes to enhance flavour and physicochemical characteristics of food in addition
to many beneficial properties to human health. They are highly useful in beverages,
bakery products, confectionery, infant milk powders, yogurts, and dairy desserts.
However, the price of pure malto-oligosaccharides is exceptionally huge as the
chemical structure of malto-oligosaccharides is bigger than maltotriose and because
of its difficulty in production (Subramanian et al. 2012).
The enzyme AmyUS100 produced by Bacillus stearothermophilus has a primary
amino acid sequence identical to that of another enzyme AmyS produced by a different strain (B. stearothermophilus strain DN1792) of the same bacterium. Among
the 516 residues, only three residues varied in the mature protein, one of which was
in the terminal region that is not involved in catalysis. Both the amylases, AmyUS100
and AmyS, demonstrated identical optimum pH and temperature but differed in
their starch hydrolysis profile. Thus, it was implicated that the two amino acids
S.M. Basheer and S. Chellappan