Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-11-24
2001-07-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S205000, C435S254100
Reexamination Certificate
active
06255084
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a thermostable glucoamylase suitable for, e.g., starch conversion, e.g., for producing glucose from starch. The present invention also relates to the use of said thermostable glucoamylase in various processes, in particular in the saccharification step in starch convention processes.
BACKGROUND OF THE INVENTION
Glucoamylases (1,4-&agr;-D-glucan glucohydrolase, EC 3.2.1.3) are enzymes which catalyze the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules.
Glucoamylases are produced by several filamentous fungi and yeasts, including
Aspergillus niger
and
Aspergillus awamori.
Commercially, the glucoamylases are used to convert corn starch which is already partially hydrolyzed by an &agr;-amylase to glucose. The glucose may further be converted by glucose isomerase to a mixture composed almost equally of glucose and fructose. This mixture, or the mixture further enriched with fructose, is the commonly used high fructose corn syrup commercialized throughout the world. This syrup is the world's largest tonnage product produced by an enzymatic process. The three enzymes involved in the conversion of starch to fructose are among the most important industrial enzymes produced.
One of the main problems existing with regard to the commercial use of glucoamylase in the production of high fructose corn syrup is the relatively low thermal stability of glucoamylases, such as the commercially available
Aspergillus niger
glucoamylase (i.e., (sold as AMG by Novo Nordisk A/S). The commercial Aspergillus glucoamylase is not as thermally stable as &agr;-amylase or glucose isomerase and it is most active and stable at lower pH's than either &agr;-amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH.
U.S. Pat. No. 4,247,637 describes a thermostable glucoamylase having a molecular weight of about 31,000 Da derived from
Talaromyces duponti
suitable for saccharifying a liquefied starch solution to a syrup. The glucoamylase is stated to retain at least about 90% of its initial glucoamylase activity when held at 70° C. for 10 minutes at pH 4.5.
U.S. Pat. No. 4,587,215 discloses a thermostable amyloglucosidase derived from the species
Talaromyces thermophilus
with a molecular weight of about 45,000 Da. The disclosed amyloglucosidase (or glucoamylase) loses its enzymatic activity in two distinct phases, an initial period of rapid decay followed by a period of slow decay. At 70° C. (pH=5.0) the half-life for the fast decay is about 18 minutes with no measurable loss of activity within an hour in the second phase of decay. Bunni L et al., (1989), Enzyme Microb. Technol., Vol. 11, p. 370-375. concerns production, isolation and partial characterization of an extracellular amylolytic system composed of at least one form of &agr;-amylase and one form of an &agr;-glucosidase produced by
Talaromyces emersonii
CBS 814.70. Only the &agr;-amylase is isolated, purified and characterized.
BRIEF DISCLOSURE OF THE INVENTION
The present invention is based upon the finding of a novel thermostable glucoamylase suitable for use, e.g., in the saccharification step in starch conversion processes.
The terms “glucoamylase” and “AMG” are used interchangeably below.
The thermal stability of the glucoamylase of the invention is measured as T
½
(half-life) using the method described in the “Materials and Methods” section below.
The inventors of the present invention have isolated, purified and characterized a thermostable glucoamylase from a strain of
Talaromyces emersonii
now deposited with the Centraalbureau voor Schimmelcultures under the number CBS 793.97.
When applied to a protein, the term “isolated” indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e., “homologous impurities” (see below)).
It is preferred to provide the protein in a greater than 40% pure form, more preferably greater than 60% pure form. Even more preferably it is preferred to provide the protein in a highly purified form, i.e., greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
The term “isolated enzyme” may alternatively be termed “purified enzyme”.
The term “homologous impurities” means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originates from the homologous cell, from where the polypeptide of the invention is originally obtained.
The isolated glucoamylase has a very high thermal stability in comparison to prior art glucoamylases, such as the
Aspergillus niger
glucoamylase (available from Novo Nordisk A/S under the trade name AMG). The T½ (half-life) was determined to be about 120 minutes at 70° C. (pH 4.5) as described in Example 2 below. The T½ of the recombinant
T. emersonii
AMG expressed in yeast was determined to be about 110 minutes as described in Example 12.
Therefore, in the first aspect the present invention relates to an isolated enzyme with glucoamylase activity having a T
½
(half-life) of at least 100 minutes in 50 mM NaOAc, 0.2 AGU/ml, pH 4.5, at 70° C.
In the second aspect the invention relates to an enzyme with glucoamylase activity comprising one or more of the partial sequences shown in SEQ ID Nos. 1-6 or the full length enzyme shown in SEQ ID NO: 7 or an enzyme with glucoamylase activity being substantially homologous thereto.
The term “partial sequence” denotes a partial polypeptide sequence which is comprised in a longer polypeptide sequence, wherein said longer polypeptide sequence is having the activity of interest.
The invention also relates to the cloned DNA sequence encoding the glucoamylase of the invention.
Further, the invention also relates to a process of converting starch or partially hydrolyzed starch into a syrup containing, e.g., dextrose, said process including the step of saccharifying starch hydrolyzate in the presence of a glucoamylase of the invention.
It is an object of the invention to provide a method of saccharifying a liquefied starch solution, wherein an enzymatic saccharification is carried out using a glucoamylase of the invention.
Furthermore, the invention relates to the use of a glucoamylase of the invention in a starch conversion process, such as a continuous starch conversion process. In an embodiment of the continuous starch conversion process it includes a continuous saccharification step.
The glucoamylase of the invention may also be used in processes for producing oligosaccharides or specialty syrups.
Finally, the invention relates to an isolated pure culture of the microorganism
Talaromyces emersonii
CBS 793.97 or a mutant thereof capable of producing a glucoamylase of the invention.
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patent: Re. 32153 (1986-05-01), Tamura et al.
patent: 4247637 (1981-01-01), Tamura et al.
patent: 4536477 (1985-08-01), Katkocin et al.
patent: 4587215 (1986-05-01), Hirsh
patent: 4628031 (1986-12-01), Zeikus et al.
patent: 0 135 138 A2 (1985-03-01), None
patent: 0 255 124 A2 (1988-02-01), None
patent: 292 609 (1988-11-01), None
patent: WO 86/01831 (1986-03-01), None
L. Bunni et al., “Production, Isolation and Partial Characterization of an Amylase System Produced byTalaromyces EmersoniiCBS 814.70”, Enzyme Microb. Technol., 1989, vo. 11, Jun. pp. 370-375.
Abstract of Japanese Patent Application No. 60/145905 Jul. 1985.
Abstract of Japanese Patent No. 63039577 A Feb. 1998 and Japanese Patent No. JP 92025794 B, May 1992.
Hata et al., Agric. Biol. Chem., vol. 55, pp. 941-949 (1991).
Ventura et al., Applied and Environmental Microbiology, vol. 61, pp. 399-402 (1995).
Hata et al., SWISS-PROT Accession AMVG-ASPOR, Jun. 1994.
GCG—GeneSeq Accession No. Q04731 Oct. 1990.
PIR Accession No. JQ1346 Jun. 1992.
GCG—GeneSeq Accession No. Q26053 Dec. 1992.
GCG—GeneSeq Accession No. P40212 Jan. 1992.
Lehmbeck Jan
Nielsen Bjarne Rønfeldt
Nielsen Ruby Ilum
Lambiris Esq. Elias J.
Novozymes A/S
Prouty Rebecca E.
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