Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-05-18
2001-10-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S320100, C435S252310, C435S254110, C435S325000, C435S419000, C536S023100, C536S023200, C536S024100, C536S023400
Reexamination Certificate
active
06300115
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to recombinant pullulanases, expression constructs which encode the enzymes and drive their expression and secretion in heterologous hosts, microbial hosts transformed to contain the expression constructs, and methods of manufacturing recombinant pullulanases using the transformed hosts.
DESCRIPTION OF THE PRIOR ART
Many high-molecular weight carbohydrates, such as starch, are polymers of glucose in which the glucosidic units are joined by &agr;-1,6-glucosidic linkages or &agr;-1,4-glucosidic linkages. Depolymerization of these carbohydrates into smaller subunits is a common industrial process in the manufacture of various syrups, food additives, and chemicals, such as high maltose syrup, dextrose, and the like. Depolymerization of these complex carbohydrates is conventionally accomplished by enzymatic hydrolysis.
For example, the conventional process for manufacturing d-glucose (dextrose) calls for a two-stage enzymatic hydrolysis of starch. In the first stage, the starch is liquefied by treating it with an &agr;-amylase enzyme at a pH of between about 5.5 and 7.0 and at 104° C. or greater, whereby some of the &agr;-1,4-glucosidic bonds in the starch are hydrolyzed. In the second stage, the liquefact then is treated with one or more enzymes to hydrolyze the &agr;-1,6-glucosidic linkages and the remaining &agr;-1,4-glucosidic linkages, thereby producing the primarily dextrose syrup. This hydrolysis (or “saccharification”) is usually accomplished by the addition of a glucoamylase (1,4-&agr;-D-glucan glycohydrolase; EC 3.2.1.3), an enzyme which hydrolyzes both &agr;-1,4- and &agr;-1,6-glucosidic linkages. The presence of &agr;-1,6-glucosidic linkages in starch limits the maximum yield of dextrose in typical saccharifications because the formation of reversion products is much faster than the hydrolysis rate of the &agr;-1,6-glucosidic linkages. Glucoamylase-containing products may be supplemented with other enzymes that increase the rate of &agr;-1,6-glucosidic hydrolysis to maximize yield. Pullulanase (&agr;-dextrin 6-glucanohydrolase; EC 3.2.1.41) is one such enzyme.
Because of the large demand for these hydrolyzing enzymes, much work has been done to identify novel enzymes and to optimize their functionality at the elevated temperatures and acidic conditions required in many commercial manufacturing protocols.
For an early discussion on the production of a recombinant &agr;-amylase, see Zeman and McCrea (1985),
Cereal Foods World
30(11):777-780.
Mielenz et al., U.S. Pat. No. 4,493,893 (issued Jan. 15, 1985), describe the cloning of a thermostable &agr;-amylase from
Bacillus stearothermophilus
and expression of the &agr;-amylase in
B. subtilis
and
Escherichia coli.
Colson et al., U.S. Pat. No. 4,469,791 (issued Sep. 4, 1984) and U.S. Pat. No. 4,806,426 (issued Feb. 21, 1989), describe the production of &agr;-amylases in genetically-engineered microbes. In these patents, a gene encoding an &agr;-amylase was isolated from
B. megaterium
(NCIB No. 11568). Of particular note is the description of the plasmid pUB110 in the U.S. Pat. No. 4,806,426. The pUB110 plasmid can be used as a vector for insertion of the recombinant DNA sequences described below into bacterial hosts. As noted in the U.S. Pat. No. 4,806,426 the pUB110 plasmid has been found to be particularly stable and is replicated to high copy numbers in
B. subtilis
host cells.
Stephens et al., U.S. Pat. No. 4,769,327 (issued Sep. 6, 1988), describe vectors for the expression of fusion proteins in bacterial hosts. The vectors include promoter and leader sequences from the
B. licheniformis
&agr;-amylase gene.
Tomimura, U.S. Pat. No. 5,055,403 (issued Oct. 8, 1991), describes a thermoduric and aciduric pullulanase enzyme isolated from
B. naganoensis,
ATCC No. 53909. This pullulanase retains at least 50% of its pullulan-hydrolyzing activity for 232 hours in aqueous solution at 60° C., pH 4.5. (See also Tomimura et al. (1990),
Int. J. Syst. Bacteriol.
40(2):123-125.)
A series of mutant precursors of
B. stearothermophilus
&agr;-amylase having altered leader peptides is described in Yamaguchi et al. (1993),
Biosci. Biotechnol. Biochem.
57(8):1384-1386. The mutated peptides were then expressed in
B. brevis.
This study found that more efficient extracellular production of the
B. stearothermophilus
&agr;-amylase in
B. brevis
was accomplished by introducing point mutations at positions between −6 and −4 of the leader sequence or by replacing the entire
B. stearothermophilus
&agr;-amylase leader sequence with the leader sequence of the
B. brevis
middle wall protein.
A similar study on the effects of leader peptide mutations on the expression of recombinant
B. stearothermophilus
&agr;-amylase in
E. coli
is presented in Suominen et al. (1995),
Microbiology
141:649-654.
B. stearothermophilus
&agr;-amylase has also been cloned into other microbial hosts, such as lactobacilli. See Cocconcelli et al. (1991),
Res. Microbiol.
142:643-652. Other references (largely cumulative to those discussed above) describing the cloning of &agr;-amylases from
B. stearothermophilus
include Jørgensen et al. (1991),
FEMS Microbiol. Lett.
77:271-276; Nakajima et al. (1985),
J. Bacteriol.
163(1):401-406; and Gray et al. (1986),
J. Bacteriol.
166(2):635-643.
Jørgensen et al. (1990),
Gene
96:37-41 describes a homologous in vivo recombination yielding a plasmid containing the leader sequence of a
B. licheniformis
&agr;-amylase-encoding gene operationally linked to the structural gene of
B. stearothermophilus
&agr;-amylase. When transformed into
B. subtilis,
this plasmid drives the expression of an &agr;-amylase which is reported as being identical to the mature &agr;-amylase secreted by wild-type
B. stearothermophilus.
PCT Application WO 93/10249 describes a construct containing a
B. licheniformis
&agr;-amylase promoter sequence operationally linked to the structural gene for
B. stearothermophilus
&agr;-amylase. When transformed into
B. subtilis,
the construct drives the expression of functional
B. stearothermophilus
&agr;-amylase.
U.S. Pat. No. 5,514,576 to Bower (issued May 7, 1996) describes the cloning of a rice pullulanase gene into a recombinant yeast host.
SUMMARY OF THE INVENTION
The immediate goal of the subject invention was to move the genetic information encoding the pullulanase described in U.S. Pat. No. 5,055,403 into a host organism which is better suited for commercial enzyme production. In so doing, it was discovered that constructs containing the native
B. naganoensis
promoter and leader sequences operationally linked to the pullulanase gene yielded negligible enzyme production. However, a construct containing the pullulanase coding sequence from
B. naganoensis
operationally linked to the promoter and leader sequences from the
B. stearothermophilus
&agr;-amylase gene was found to drive high level production of the
B. naganoensis
pullulanase when transformed into a suitable bacterial host.
A first embodiment of the invention is drawn to a DNA expression construct comprising, in 5′ to 3′ order: an &agr;-amylase promoter sequence derived from
B. stearothermophilus,
operationally linked to an &agr;-amylase leader sequence derived from
B. stearothermophilus,
operationally linked to a DNA sequence encoding a pullulanase derived from
B. naganoensis.
The construct drives the expression and secretion of the encoded pullulanase in heterologous hosts.
A second embodiment of the invention is a DNA expression construct as described above which further comprises a DNA sequence encoding an N-terminal fragment of
B. stearothermophilus
&agr;-amylase operationally disposed between the leader sequence and the DNA sequence encoding a pullulanase such that the expression construct expresses a fusion protein comprising N-terminal residues of
B. stearothermophilus
&agr;-amylase bonded to a pullulanase. Here, the construct drives the expression of a fusion protein which retains its pullulan-hydrolyzing activity.
A
Allen Larry N.
Brikun Igor A.
Brumm Phillip J.
Teague W. Martin
Achutamurthy Ponnathapu
DeWitt Ross & Stevens S.C.
Enzyme Bio-Systems Ltd.
Kerr Kathleen
Leone, Esq. Joseph T.
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