Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy...
Reexamination Certificate
1999-08-18
2003-01-14
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
C435S183000, C435S195000, C435S200000, C435S204000, C435S205000, C435S207000, C435S208000, C435S267000, C435S274000
Reexamination Certificate
active
06506592
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a novel nucleic acid coding for a thermostable glycosyl hydrolase, alpha-glucosidase, from the archaeon
Sulfolobus solfataricus
and a novel enzyme coded by its gene, malA. It also relates to bacteria and plants transformed with hyperthermophilic alpha-glucosidase and a method for preparing the recombinant alpha-glucosidase by use of the same. The present invention further relates to a method of using the transgenic thermostable glycosyl hydrolases for the bioprocessing of plant carbohydrates.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are, in part, respectively grouped in the appended List of References.
A variety of industries, such as food and chemical, employ hydrolases for the production of glucose, sucrose and other sugars. High value is placed on thermostability and thermoactivity of enzymes for use in the bioprocessing of starch into maltodextrins, glucose, fructose and various other sugars.
One hydrolase, alpha-glucosidase, is defined by the International Union of Biochemistry as an enzyme which hydrolyzes glucose oligomers to glucose. Alpha-glucosidase and other glycosidase genes from non-thermophilic eukaryotic and eubacterial organisms have been cloned and characterized (Hermans, et al., 1991). In addition, the major, endogenous, soluble alpha-glucosidase from
Sulfolobus solfataricus
has been purified and characterized. Although it has not, to date, been cloned, the native enzyme hydrolyzes sucrose and liberates glucose from maltose. It has a temperature optimum in excess of 100° C., with a prolonged half life at elevated temperatures, and is resistant to proteolysis and denaturants including chaotropes, detergents and aliphatic alcohols (Rolfsmeier and Blum, 1995). Another hydrolase from
S. solfataricus,
beta-glycosidase, has been characterized (Haseltine, et al., 1999(a)).
Plant-derived polysaccharides are used in enormous quantities as foods (sugar) and, following conversion, in other specialty and value added chemicals such as ethanol. Consequently, these materials are recognized as essential and are traded as commodities. Indeed, key measures of inflation are strongly influenced by the value assigned to such commodities. Sugar (glucose) is produced by bioprocessing of plant starch from plants including corn, sugar beets and sugar cane. Incremental improvements in sugar production reflect two basic strategies, increasing the yield of polysaccharide on a per plant mass basis and increasing the conversion of endogenous starch into sugar. Where the former approach results from advances in plant breeding, the latter approach initiated the introduction of microbial gene products as cell-free bioprocessing components. Key among these additives are two enzymes, alpha-amylase and gluco-amylase, which are employed as commodity materials. Alpha-amylase is an endo-acting enzyme which debranches starch yielding dextrin. Glucoamylase is an exo-acting enzyme which serially hydrolyzes dextrin subunits into free glucose. Plant starch hydrolysis requires hydration to enable enzyme action, consequently the introduction of water is a critical first step and is conventionally performed by heating and extrusion. Cost effective bioprocessing necessitates rapid throughput therefore, a thermostable alpha-amylase is commonly added to enable immediate debranching of hydrated starch. However, both the pH and the temperature of the bioprocessing reactors must thereafter be adjusted to create near-neutral and reduced temperatures to support activity of the commercial glucoamylase. As a result, a source of transgenic, thermostable glycosyl hydrolases would be an important contribution to the starch processing industry due to their activity at high temperatures and extreme stability at room temperatures, which offers extended shelf life. Although glycosyl hydrolases occur among the hyperthermophillic archaea (Costantino, et al., 1990; Rolfsmeir and Blum, 1995; Haseltine, et al., 1999(a)), their culture as a source of enzyme is complicated by their extreme growth requirements. Furthermore, expression of archaeal enzymes in heterologous hosts is complicated by the altered environment in which expression occurs and difficulty with translation and post translational processing (Trent, et al., 1991). Over production of archaeal proteins in plants would require that the protein fold properly in plants which, for thermostable proteins, may require chaperones not normally present in plants. To date, no archaeal alpha-glucosidase genes have been cloned or expressed recombinantly.
SUMMARY OF THE INVENTION
The present invention relates to a novel nucleic acid coding for a thermostable glycosyl hydrolase, alpha-glucosidase, from the archaeon
Sulfolobus solfatoricus
and a novel enzyme coded by its gene, malA. It also relates to bacteria and plants transformed with hyperthermophilic alpha-glucosidase and a method for preparing the recombinant alpha-glucosidase by use of the same. The present invention further relates to a method of using the transgenic thermostable glycosyl hydrolases for the bioprocessing of plant carbohydrates.
The malA gene of the present invention has been identified, cloned and expressed in bacteria and plants. The distribution and associated activity of the malA gene in the three most common members of the Sulfolobus genus have been determined.
The purified alpha-glucosidase produced by the recombinant malA gene, provides an extremely thermostable enzyme which is able to hydrolyze a surprisingly wide range of polysaccharides to glucose. Substrates include starch and maltodextrins indicating that the enzyme acts as both an alpha-amylase and an alpha-glucosidase. Additionally, sucrose is hydrolyzed to invert sugar by the malA gene product, indicating the enzyme is also a sucrase.
Extracts of transgenic plants of the present invention expressing thermostable glycosyl hydrolases have been prepared. The plant extracts are able to undergo “autohydrolysis” of plant polysaccharide into glucose and other sugars. The recombinant glycosyl hydrolases expressed in the transgenic plants are nontoxic in plant tissues because the enzymes are inactive at temperatures supporting plant growth and therefore do not significantly interfere with normal metabolism in the living plant. Thus, regulated plant promoters are not required and constitutive promoters are sufficient. When desired, the transgenic plant tissue can be heated to activate the enzymes, causing in situ processing of storage carbohydrates to glucose and other small molecules by the recombinant plant extract, itself. This obviates the need to add separately produced commodity enzymes to plant material, as is the current method used in industry.
Accordingly, this invention comprises the following aspects:
1) An isolated nucleic acid molecule encoding thermostable alpha-glucosidase comprising the nucleotide sequence of SEQ ID NO:1.
2) An expression vector comprising the nucleotide sequence of SEQ ID NO: 1.
3) Transformed host cells comprising the nucleotide sequence of SEQ ID NO: 1.
4) Transformed host cells expressing the recombinant amino acid sequence of SEQ ID NO:2.
5) Methods of preparing the transformed hosts of 4).
6) Methods for extracting and purifying heterologous, thermostable glycosyl hydrolases from transformed host cells.
7) Characterization of recombinant glycosyl hydrolase enzymes, including their useful chemical and physical properties and utility for industrial applications.
8) Novel recombinant enzyme of 7) comprising the amino acid sequence of SEQ ID NO:2.
9) Transgenic plant cells expressing thermostable glycosyl hydrolases that are capable of autohydrolysis (in situ processing) of substrate into glucose and other small molecules.
REFERENCES:
patent: 4729788 (1988-03-01), Hutchins et al.
patent: 4997665 (1991-03-01), Grethlein
patent: 5102992 (1992-04-01), Glasser et al.
patent: 5196069 (1993-03-0
Achutamurthy Ponnathapu
Board of Regents of the University of Nebraska
Rao Manjunath N.
Rothwell Figg Ernst & Manbeck
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