Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-11-01
2001-10-30
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C536S023200
Reexamination Certificate
active
06309872
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated polypeptides having glucoamylase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
High dextrose and fructose syrups are made by enzymatic saccharification of liquefied starch. The saccharification is achieved by means of a dextrose-forming exo-amylase known as exo-1,4-alpha-D-glucosidase (glucoamylase or amylogulcosidase). The enzyme hydrolyzes 1,4-as well as 1,6-alpha-linkages in starch. During hydrolysis, glucose units are removed in a step-wise manner from the non-reducing ends of the substrate molecule, whereas the 1,6-alpha-linkages found in branched dextrins are broken down relatively slowly. Maltotriose and maltose are hydrolyzed at a lower rate than higher oligosaccharides. Glucoamylases are also used to lower the carbohydrate content of beer.
Thielavia terrestris
is a thermophilic filamentous fungus that can grow at low pH of 4.5 and elevated temperature of 40-45° C. (WO 96/02653). The ascomycete has a wide geographic distribution and a homothallic mating behavior (Mouchacca, 1997,
Cryptogamie Mycol.
18: 19-69). Several enzymes are known to be produced by Thielavia including xylanase, glucanase, cellulase, glucosidase, mannanase, and galactanase.
There is a need in the art for new sources of glucoamylase with improved properties of thermostability.
It is an object of the present invention to provide improved polypeptides having glucoamylase activity and nucleic acid encoding the polypeptides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having glucoamylase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 75% identity with amino acids 20 to 630 of SEQ ID NO:2;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) nucleotides 411 to 2581 of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides 411 to 2581 of SEQ ID NO:1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(c) a variant of the polypeptide having an amino acid sequence of SEQ ID NO:2 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(d) a fragment of (a) or (b) that has glucoamylase activity; and
(f) a polypeptide having with physicochemical properties of (i) a pH optimum in the range between about pH 3 and about pH 7, determined at 20° C. in the presence of maltotriose; (ii) a temperature optimum in the range of from about 20° C. to about 70° C., determined at pH 4 in the presence of maltotriose; and (iii) a residual activity of 32%, relative to initial activity, at pH 4.3 after 20 minutes at 70° C. in the absence of maltotriose.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
Golightly Elizabeth J.
Rey Michael W.
Nashed Nashaat T.
Novozymes Biotech, Inc
Starnes Robert L.
Walicka Malgorzata A.
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