Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-12-07
2001-12-11
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S200000
Reexamination Certificate
active
06329186
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a glucoamylase variant of a parent glucoamylase, a DNA sequence encoding the variant glucoamylase and a process using such variant enzyme for hydrolyzing starch.
More specifically, the present invention relates to a glucoamylase variant having improved thermostability.
BACKGROUND OF THE INVENTION
Glucoamylase (1,4-&agr;-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme which catalyzes 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, with those from Aspergillus being commercially most important.
Commercially, the glucoamylase enzyme is used to convert corn starch which is already partially hydrolyzed by an &agr;-amylase to glucose. The glucose is further 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 exist with regard to the commercial use of glucoamylase in the production of high fructose corn syrup is the relatively low thermal stability of glucoamylase. 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.
SUMMARY OF THE INVENTION
Thus, the object of the present invention is to improve properties of enzymes with glucoamylase activity, in particular to improve the thermal stability of such enzymes.
It has surprisingly been found that it is possible to significantly enhance the thermal stability of an enzyme with glucoamylase activity by linking a peptide extension to the N-terminal of the enzyme.
Consequently, in a first aspect the invention relates to a variant of a parent glucoamylase, which has a peptide extension at the N-terminus.
In the present context the term “peptide extension” is intended to indicate that a stretch of one or more consecutive amino acid residues has been added to the N-terminal end of the parent (mature) glucoamylase.
The term “mature glucoamylase” is used in its conventional meaning, i.e., to indicate the active form of the glucoamylase resulting after posttranslational and postsecretional processing (to trim glycosylation and remove N and/or C-terminal sequences, such as pre- and pro-peptide sequences) by the producer organism in question. More specifically this means that amino acid sequences such as the pre- and pro-peptide sequences, if present, have been removed from the initially translated glucoamylase, i.e., the unprocessed glucoamylase. A mature glucoamylase encompassed by the present definition is a glucoamylase cut (processed) by Tripeptidyl amino peptidase (TPAP), which cuts the
A. niger
glucoamylase (see SEQ ID NO: 1) at position 3, i.e., between Leu and Asp.
The term “parent glucoamylase” is intended to indicate the glucoamylase to be modified according to the invention. The parent glucoamylase may be a naturally-occurring (or wild type) glucoamylase or may be a variant thereof prepared by any suitable means. For instance, the parent glucoamylase may be a variant of a naturally-occurring glucoamylase which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring glucoamylase, typically in the structural part of the glucoamylase.
In other aspects the invention relates to a DNA sequence encoding a glucoamylase variant as defined above, a DNA construct, a recombinant expression comprising a DNA sequence of the invention, and a host cell harbouring a DNA sequence of the invention or a vector of the invention.
The glucoamylase variant of the invention may conveniently be used in a process for converting starch and accordingly, in yet other aspects the invention relates to a process for converting starch or partially hydrolyzed starch into syrup containing dextrose, said process including the step saccharifying starch hydrolyzate in the presence of a glucoamylase variant of the invention.
In final aspects, the invention provides a method for improving the thermostability of parent glucoamylase by making an extension at the N-terminus.
The inventors of the present invention have provided a number of improved variants of a parent glucoamylase with improved thermostability. The improved thermal stability is obtained by linking a peptide extension to a parent glucoamylase. This will be described in details below.
DETAILED DESCRIPTION OF THE INVENTION
Peptide Extension
As stated above it has surprisingly been found that a glucoamylase variant, in particular with improved thermostability, may be achieved when an appropriate peptide extension can be found at the N-terminus of the parent glucoamylase. The present invention is based on this finding.
In the context of the present invention “..can be found at the N-terminal..” means that the mature glucoamylase has a peptide extension at the N-terminal. In one embodiment the peptide extension is native to the parent glucoamylase, i.e., before posttranslational processing to remove the pro- and/or pre-sequence. Thus, the peptide extension may be the pre- and/or pro-sequence of the unprocessed parent glucoamylase, which is normally removed or cut off after expression and posttranslational processing.
In another embodiment the extension is a peptide at the N-terminal identical to the peptide sequence normally being cut of by the donor cell during processing, e.g., the pre- and/or pro-sequence. In most cases the peptide extension is different from the pre- and/or pro-sequence. This will be described further below.
In the case of the extension is linked to the N-terminal it may be done by means of any well-known protein engineering methods in the art.
The term “a glucoamylase variant with improved thermostability” means in the context of the present invention a glucoamylase variant, which has a higher T
½
(half-time) or residual enzymatic activity after a fix incubation period than the corresponding parent glucoamylase. The determination of thermostability, e.g., T½ and residual activity, is described below in the Materials and Method section.
The term “an appropriate peptide extension” is used to indicate that the peptide extension to be used is one, which is capable of effecting an improved thermostability as defined above. The “appropriateness” of the peptide extension may be checked by a comparative analysis of the thermostability of a modified glucoamylase variant to which the peptide extension has been linked and of the corresponding parent glucoamylase, respectively. The thermostability may, e.g., be determined by any suitable technique such as the thermostability assay described in the present application.
It is presently believed that the capability of the peptide extension of providing the desired effect such as improved thermostability depends on, e.g., the identity of the parent glucoamylase to be modified, the structure (including length) of the peptide extension, the impact of the peptide extension on the structure of the entire glucoamylase variant enzyme, the nature or functionality of amino acid residues of the peptide extension, etc. A prerequisite for the peptide extension being capable of providing the desired effect is, of course, that the glucoamylase variant containing the peptide extension is expressible in a suitable host organism. The following general considerations may be of relevance for the design of a suitable peptide extension:
Length of peptide extension: It has been found that peptide extensions containin
Bojsen Kirsten
Nielsen Rønfeldt Bjarne
Pedersen Henrik
Svendsen Allan
Vind Jesper
Garbell Jason I.
Lambiris Elias T.
Novozymes A/S
Prouty Rebecca E.
Rao Manjunath N.
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