Production, purifi cation, and characterization of -amylase by bacillus subtilis and its mutant derivates
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Enzymes involved in the hydrolysis of polyfructans are of
interest both for fundamental studies and for industrial appli-
cations. Especially inulin is of growing interest as a renewable
carbohydrate raw material for biotechnology. Two aspects are
of main importance: (i) production of pure fructose syrups,
so-called high-fructose inulin syrups, by enzymatic hydrolysis
of inulin (43) and (ii) direct fermentation of inulin by employ-
ing inulinase-producing microbes in order to synthesize various
products such as ethanol or aceton-butanol (20, 21).
b-D-Fructofuranosidases are usually classified upon their
ability to hydrolyze levan (levanases), inulin (inulinases), and
also the disaccharide sucrose (sucrases and invertases). How-
ever, many of these enzymes are capable of hydrolyzing more
than one type of these substrates. Inulinases (or inulases)
which are specific for inulin have been isolated only from
Jerusalem artichoke tubers (7, 12), whereas levanases which
are specific for levan have been isolated from bacteria only.
Examples are the levanases of Streptococcus salivarius KTA-19
(38) and Actinomyces viscosus ATCC 19246 (16). Conversely, a
variety of nonspecific b-D-fructofuranosidases have been found
in bacteria, yeasts, and fungi. For example, inulinases and
levanases which are capable of hydrolyzing inulin, levan, and
sucrose have been isolated from Bacillus subtilis (17), Actino-
myces viscosus ATCC 15987 (24), Streptococcus mutans (5),
Kluyveromyces fragilis (35), Chrysosporium pannorum (46, 47)
and Penicillium sp. strain (27). Enzymes active on inulin and
sucrose but not on levan have been found in filamentous fungi,
among them the b-fructofuranosidases (I to III) from Aspergil-
lus niger (42), the F2 inulinase from C. pannorum (47), and the
PII inulinase from Aspergillus niger (26). The inulinase II from
Arthrobacter ureafaciens (41) splits levan and inulin, but no
sucrose-hydrolyzing activity could be detected. However, un-
specific b-fructosidases which are active on levan and sucrose
but not on inulin have not been described yet.
Most inulinases and levanases from microorganisms which
were investigated in more detail have been found to be exoen-
zymes (11). They attack the inulin or levan molecules from the
fructose end and liberate fructose as the sole reaction product.
Exoinulinases or exolevanases are incapable of hydrolyzing
melezitose (3-O-a-D-glucopyranosyl-b-D-fructofuranosyl-a-D-
glucopyranoside), a trisaccharide with the same terminal con-
figuration as inulin (36). In melezitose the centrally located
fructose is protected from terminal hydrolysis by the second
glycosyl residue. Only endoinulinases are capable of hydrolyz-
ing melezitose. Most enzymes of this type have been isolated
and characterized from fungi, among them the endoinulinases
from Aspergillus ficuum (10) and the endoinulinase from C.
pannorum (47).
Most microbial levanases and inulinases which have been
purified and characterized in more detail were isolated from
yeasts (31, 32, 35, 36) and filamentous fungi (10, 11, 26, 27, 46,
47), whereas only a few bacterial enzymes have been purified
and characterized so far (5, 16).
The enzyme levanase from B. subtilis is a b-D-fructofurano-
sidase capable of hydrolyzing levan, inulin, and sucrose (17, 22,
23, 33, 34). B. subtilis levanase was first described by Kunst et
al. (17) and assigned as levanase because specific activity on
levan, inulin, sucrose, and raffinose was observed. However,
according to the Avigad and Bauer classification (2), it should
be assigned as a nonspecific b-fructofuranosidase to distin-
guish it from true levanases (specific 2 3 6 activity). Levanase
has been partially purified (22) and characterized with crude
protein extracts (17). Levanase expression in B. subtilis is
tightly regulated (23), and detectable amounts of enzyme are
found only with regulatory mutants (sacL mutants). The struc-
tural gene coding for levanase has been cloned in Escherichia
coli (13), sequenced, and characterized in detail (22, 33, 34).
interest both for fundamental studies and for industrial appli-
cations. Especially inulin is of growing interest as a renewable
carbohydrate raw material for biotechnology. Two aspects are
of main importance: (i) production of pure fructose syrups,
so-called high-fructose inulin syrups, by enzymatic hydrolysis
of inulin (43) and (ii) direct fermentation of inulin by employ-
ing inulinase-producing microbes in order to synthesize various
products such as ethanol or aceton-butanol (20, 21).
b-D-Fructofuranosidases are usually classified upon their
ability to hydrolyze levan (levanases), inulin (inulinases), and
also the disaccharide sucrose (sucrases and invertases). How-
ever, many of these enzymes are capable of hydrolyzing more
than one type of these substrates. Inulinases (or inulases)
which are specific for inulin have been isolated only from
Jerusalem artichoke tubers (7, 12), whereas levanases which
are specific for levan have been isolated from bacteria only.
Examples are the levanases of Streptococcus salivarius KTA-19
(38) and Actinomyces viscosus ATCC 19246 (16). Conversely, a
variety of nonspecific b-D-fructofuranosidases have been found
in bacteria, yeasts, and fungi. For example, inulinases and
levanases which are capable of hydrolyzing inulin, levan, and
sucrose have been isolated from Bacillus subtilis (17), Actino-
myces viscosus ATCC 15987 (24), Streptococcus mutans (5),
Kluyveromyces fragilis (35), Chrysosporium pannorum (46, 47)
and Penicillium sp. strain (27). Enzymes active on inulin and
sucrose but not on levan have been found in filamentous fungi,
among them the b-fructofuranosidases (I to III) from Aspergil-
lus niger (42), the F2 inulinase from C. pannorum (47), and the
PII inulinase from Aspergillus niger (26). The inulinase II from
Arthrobacter ureafaciens (41) splits levan and inulin, but no
sucrose-hydrolyzing activity could be detected. However, un-
specific b-fructosidases which are active on levan and sucrose
but not on inulin have not been described yet.
Most inulinases and levanases from microorganisms which
were investigated in more detail have been found to be exoen-
zymes (11). They attack the inulin or levan molecules from the
fructose end and liberate fructose as the sole reaction product.
Exoinulinases or exolevanases are incapable of hydrolyzing
melezitose (3-O-a-D-glucopyranosyl-b-D-fructofuranosyl-a-D-
glucopyranoside), a trisaccharide with the same terminal con-
figuration as inulin (36). In melezitose the centrally located
fructose is protected from terminal hydrolysis by the second
glycosyl residue. Only endoinulinases are capable of hydrolyz-
ing melezitose. Most enzymes of this type have been isolated
and characterized from fungi, among them the endoinulinases
from Aspergillus ficuum (10) and the endoinulinase from C.
pannorum (47).
Most microbial levanases and inulinases which have been
purified and characterized in more detail were isolated from
yeasts (31, 32, 35, 36) and filamentous fungi (10, 11, 26, 27, 46,
47), whereas only a few bacterial enzymes have been purified
and characterized so far (5, 16).
The enzyme levanase from B. subtilis is a b-D-fructofurano-
sidase capable of hydrolyzing levan, inulin, and sucrose (17, 22,
23, 33, 34). B. subtilis levanase was first described by Kunst et
al. (17) and assigned as levanase because specific activity on
levan, inulin, sucrose, and raffinose was observed. However,
according to the Avigad and Bauer classification (2), it should
be assigned as a nonspecific b-fructofuranosidase to distin-
guish it from true levanases (specific 2 3 6 activity). Levanase
has been partially purified (22) and characterized with crude
protein extracts (17). Levanase expression in B. subtilis is
tightly regulated (23), and detectable amounts of enzyme are
found only with regulatory mutants (sacL mutants). The struc-
tural gene coding for levanase has been cloned in Escherichia
coli (13), sequenced, and characterized in detail (22, 33, 34).
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