159
(Asn 315 and Val 450 ) were involved in the catalysis (Ben Ali et al. 2006). Site-directed
mutagenesis helped in the production of three mutants from AmyUS100:
AmyUS100-D (with mutation N315D), AmyUS100-G (with mutation V450G), and
AmyUS100-D/G (with double mutation N315D/V450G). Among the three, the
V450G mutation did not change the starch hydrolysis profile, whereas the introduction of both substitutions strongly affected the hydrolysis profile, and the main end
products shifted from G6/G5 to G3/G2 (Ben Ali et al. 2006). The α-amylase from
B. amyloliquefaciens was engineered by site-directed mutagenesis, and a modified
optimum pH activity profile was exhibited by the variants thus produced. The variants improved the volume of the bread as compared to the parent bacterial α-amylase
(Danielsen and Lundqvist 2008).
Xylanase The enzyme xylanase (EC 3.2.1.8) degrades hemicellulose, one of the
major components of plant cell walls by breaking the linear polysaccharide β-1,4xylan into xylose. One of the best stabilizations ever achieved by means of directed
evolution strategy of enzyme engineering (increase in melt temperature, Tm, of
more than 30 °C) was reported by Palackal et al. (2004). Xylanase was obtained by
screening 50,000 plaques from a complex environmental DNA library derived from
fresh bovine manure. The application of site-saturation mutagenesis and screening
of nearly 70,000 clones helped in the identification of nine interesting mutations,
which, when combined, increased the Tm by 34.2 °C (Palackal et al. 2004).
Enzyme immobilization can be considered as a physical method to improve the
efficiency of enzymes, which are mainly used in industrial applications, e.g., in food
and beverage industry (Mohamad et al. 2015; Ribeiro et al. 2010; Sharma et al. 2017).
Naringinase Naringinase, an enzymatic heterodimeric complex composed of two
subunits, α-L-rhamnosidase (EC. 3.2.1.40) and β-D-glucosidase (EC. 3.2.1.21), is
generally acquired from fungi. The use of naringinase provides a valuable choice
for the removal of excess bitter taste of some citrus fruit juices, such as grapefruit,
in the beverage industry. Naringin (a flavonoid that gives such flavour to the juice)
is hydrolysed by naringinase to naringenin, glucose, and rhamnose. Despite its usefulness, the huge manufacturing cost of naringinase restricts its use on an industrial
scale. Busto et al. (2007) obtained biocatalytically active beads of naringinase from
Aspergillus niger by entrapping the enzyme in a polymeric matrix consisting of
poly (vinyl alcohol) hydrogel, which was cryostructured in liquid nitrogen. Due to
this strategy, the optimum temperature of naringinase derivative raised from 60 up
to 70 °C. This experiment proves that increased operational stability of an enzyme
can be provided by the stabilization of its tertiary structure (Busto et al. 2007).
Puri and co-workers used wood chips, an unusual immobilization support, to
describe an exceptional rate of hydrolysis using naringinase (but still with inhibition
by-products) (Puri et al. 2005). Wood chips, activated with glutaraldehyde, was
used to covalently immobilize the naringinase obtained from Penicillium sp. Studies
showed that when a small amount (10 UI) of the immobilized enzyme (with 120%
of recovered activity) was incubated with freshly prepared kinnow mandarin juice
(50 mL), a maximum of 76% hydrolysis of naringin was obtained in 1 h. The
enzyme preparation also exhibited a more acidic optimum pH (from 4.5 to 3), which
6 Enzyme Engineering
(Asn 315 and Val 450 ) were involved in the catalysis (Ben Ali et al. 2006). Site-directed
mutagenesis helped in the production of three mutants from AmyUS100:
AmyUS100-D (with mutation N315D), AmyUS100-G (with mutation V450G), and
AmyUS100-D/G (with double mutation N315D/V450G). Among the three, the
V450G mutation did not change the starch hydrolysis profile, whereas the introduction of both substitutions strongly affected the hydrolysis profile, and the main end
products shifted from G6/G5 to G3/G2 (Ben Ali et al. 2006). The α-amylase from
B. amyloliquefaciens was engineered by site-directed mutagenesis, and a modified
optimum pH activity profile was exhibited by the variants thus produced. The variants improved the volume of the bread as compared to the parent bacterial α-amylase
(Danielsen and Lundqvist 2008).
Xylanase The enzyme xylanase (EC 3.2.1.8) degrades hemicellulose, one of the
major components of plant cell walls by breaking the linear polysaccharide β-1,4xylan into xylose. One of the best stabilizations ever achieved by means of directed
evolution strategy of enzyme engineering (increase in melt temperature, Tm, of
more than 30 °C) was reported by Palackal et al. (2004). Xylanase was obtained by
screening 50,000 plaques from a complex environmental DNA library derived from
fresh bovine manure. The application of site-saturation mutagenesis and screening
of nearly 70,000 clones helped in the identification of nine interesting mutations,
which, when combined, increased the Tm by 34.2 °C (Palackal et al. 2004).
Enzyme immobilization can be considered as a physical method to improve the
efficiency of enzymes, which are mainly used in industrial applications, e.g., in food
and beverage industry (Mohamad et al. 2015; Ribeiro et al. 2010; Sharma et al. 2017).
Naringinase Naringinase, an enzymatic heterodimeric complex composed of two
subunits, α-L-rhamnosidase (EC. 3.2.1.40) and β-D-glucosidase (EC. 3.2.1.21), is
generally acquired from fungi. The use of naringinase provides a valuable choice
for the removal of excess bitter taste of some citrus fruit juices, such as grapefruit,
in the beverage industry. Naringin (a flavonoid that gives such flavour to the juice)
is hydrolysed by naringinase to naringenin, glucose, and rhamnose. Despite its usefulness, the huge manufacturing cost of naringinase restricts its use on an industrial
scale. Busto et al. (2007) obtained biocatalytically active beads of naringinase from
Aspergillus niger by entrapping the enzyme in a polymeric matrix consisting of
poly (vinyl alcohol) hydrogel, which was cryostructured in liquid nitrogen. Due to
this strategy, the optimum temperature of naringinase derivative raised from 60 up
to 70 °C. This experiment proves that increased operational stability of an enzyme
can be provided by the stabilization of its tertiary structure (Busto et al. 2007).
Puri and co-workers used wood chips, an unusual immobilization support, to
describe an exceptional rate of hydrolysis using naringinase (but still with inhibition
by-products) (Puri et al. 2005). Wood chips, activated with glutaraldehyde, was
used to covalently immobilize the naringinase obtained from Penicillium sp. Studies
showed that when a small amount (10 UI) of the immobilized enzyme (with 120%
of recovered activity) was incubated with freshly prepared kinnow mandarin juice
(50 mL), a maximum of 76% hydrolysis of naringin was obtained in 1 h. The
enzyme preparation also exhibited a more acidic optimum pH (from 4.5 to 3), which
6 Enzyme Engineering