Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-02-14
2002-03-12
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S200000, C435S201000
Reexamination Certificate
active
06355467
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to saccharification enzymes (i.e., enzymes that hydrolyze polysaccharides into constituent sugars) obtainable from hyperthermophilic bacteria, and processes for producing them. In particular, this invention relates to the following types of thermostable enzymes: amylases, pullulanases, &agr;-glucosidases and &bgr;-glucosidases.
Hyperthermophilic bacteria, i.e. bacteria which thrive on temperatures around the boiling point of water, are found in the depths of the ocean close to geothermal springs. Since these bacteria live in high temperature environments, their enzymes, which are essential to sustaining life processes, such as digestion and respiration, must be able to function at such extreme temperature conditions. Enzymes in common mesophilic bacteria (i.e., organisms that can grow at intermediate temperatures compared to the upper and lower extremes for all organisms) degenerate rapidly at these high temperatures.
Proteins which can function at high temperatures can be extremely advantageous for use in a number of industries. For example, soda syrup, laundry detergent, and many pharmaceuticals contain, or are manufactured by, enzymes extracted from microbes. If enzymes from hyperthermophilic bacteria were used in place of the enzymes commonly used today, the processes to make these products could be performed at higher temperatures. Higher temperatures speed up reactions and prevent contamination by fungi and common bacteria. Alternatively, lesser amounts of the enzymes from hyperthermophilic bacteria might be required to sustain enzymatic processes under current temperature conditions where the thermostability of such enzymes correlates with a longer useful life under those conditions.
A number of microorganisms that are capable of growth at or above 100° C. (i.e., hyperthermophiles) have been isolated from several terrestrial and marine environments and are of considerable scientific interest. (See Kelly, R. M., and J. W. Deming, 1988, “Extremely Thermophilic Archaebacteria: Biological and Engineering Considerations”, Chem. Engr. Prog. 4:47-62; and Wiegel J., and L. G. Ljungdahl, 1986, “The Importance of Thermophilic Bacteria In Biotechnology,” CRC Crit. Rev. Microbiol. 3:39-107.) However, previously it has not been possible to take full advantage of the utility potential of these organisms, in large part because of a lack of understanding of their growth and metabolic characteristics.
Detailed study of specific enzymes from hyperthermophiles is just beginning, and the few known reports on such enzymes that have been published so far all appeared in 1989. For example, it has been shown (Pihl, T. D. et al., 1989, Proc. Nat. Acad. Sci. 86:138-141) that an extremely thermostable hydrogenase isolated from
Pyrodictium brockii
is immunologically related to the comparable enzyme in the
Bradyrhizobium japonicum,
a mesophile. Adams and coworkers have described several distinctive characteristics of a hydrogenase (Bryant, F. O. and Adams, M. W. W., 1989, J. Biol. Chem. 264:5070-5079) and a ferredoxin (Aono, S., et al., 1989, J. Bacteriol. 171:3433-3439) from the hyperthermophilic bacterium,
Pyrococcus furiosus.
In the broad context of the present invention, saccharification is the process of degradation of a polysaccharide into its basic constituents which are simple sugars. Complex polysaccharides, such as cellulose (in plant cell walls), chitin (in the outer coverings of insects and crustaceans), glycogen (in animal cells) and starch (in plant cells) are all macromolecular polymers of simple sugars, mainly D-glucose, coupled by various chemical linkages into linear and cross-linked or branched networks. Thus, to degrade such polymeric substrates into their simplest sugar components requires a variety of saccharification enzymes having different specificities with respect to the type of sugar (e.g, five or six carbon ring), the positions of linkages between those sugars [e.g., carbon 1 to carbon 4 (i.e., 1,4 linkage) or 1,6 linkage] and the isomeric configurations of those linkages (e.g., &agr;-1,4 or &bgr;-1,4).
Starch, for example, is a highly branched polymer essentially composed of &agr;-D-glucose coupled by both &agr;-1,4- and &agr;-1,6- glycosidic linkages. The current industrial processes of digestion of starch into simple sugars (e.g., in the making of corn syrup sweeteners) illustrate many problems with the enzymes that are presently available for large scale digestion of polysaccharides. Two enzymatic steps are applied in the hydrolysis of starch to &agr;-D-glucose. Although the entire process may be considered saccharification in the broad context of this application, the two steps are commonly known as liquification and saccharification. Liquification involves the use of an &agr;-amylase to hydrolyze starch granules which have been slurried in water and gelatinized by heat to allow the enzyme to attack. This process is designed to reduce the viscosity of the hydrolysate and produce a high yield of low molecular weight polysaccharides, primarily dextrins. In the saccharification step, these dextrins are then further hydrolyzed to glucose by another enzyme activity which may be a classified as a glucoamylase. The commercial objective in the overall starch hydrolysis process is to obtain a hydrolysate with maximum glucose content (DX), at least 96% of the dry weight of starch.
A recent comparison of &agr;-amylases (i.e., 1,4-&agr;-D-glucanohydrolases) that are available for commercial scale starch liquification (Shetty, J. K. and Allen, W. G., 1988, Cereal Foods World 33:929-934) pointed out deficiencies in the industry standard, the
Bacillus licheniformis
&agr;-amylase, even though it does meet present operational and performance demands of starch processors. Areas for improvement include: reducing demands for chemicals, ion exchange processing, and other product refining; reducing by-product and color formation; and improving glucose yield and product quality.
Control of pH is critical to the efficiency and operation of current commercial starch liquification processes. Values of lower than pH 6.2 to 6.4 decrease the reaction rate and the stability of the
Bacillus licheniformis
&agr;-amylase. At higher. pH values, color and by-product formation during liquefaction increase with increasing pH. The pH of the refined starch slurry that enters the enzyme liquefaction process from the wet milling operation is normally in the range of 4.0-5.0. For liquification by the
Bacillus licheniformis
&agr;-amylase, base must be added to increase the pH of the starch slurry to the range of 6.2-6.4. After liquefaction, acid must be added for efficient saccharification, typically by a glucoamylase at pH 4.2-4.5. Therefore, the ability to perform liquefactions at a lower pH would decrease the demand for chemically adjusting pH prior to and after liquefaction. It would also reduce color and by-product formation, and lower refining requirements and costs.
An additional problem with
Bacillus licheniformis
&agr;-amylase is that it is a metalloprotein, requiring calcium ion for maximum stability of the enzymatically active configuration. Calcium binding increases resistance to denaturation at extremes of temperature and pH, but the ion is not involved in the starch hydrolysis reaction. Calcium salts are normally added to starch slurry for enzymatic liquification at, for example, 80 mg calcium ion/kg enzyme. Addition of calcium ion reduces enzyme cost but increases product refining cost. Therefore, a more stable enzyme would reduce the amount of calcium ion that must be added for enzyme stabilization and would thereby reduce costs of its removal later. Further, a more stable enzyme would reduce the required amount of the enzyme itself, under current reaction conditions; or, alternatively, higher temperatures could be used to shorten reaction times.
A principal reason that the starch industry has difficulties in achieving 96% DX is that a major by-product in the current starch-to-glucose processes is maltulose, a
Brown Stephen H.
Costantino Henry R.
Kelly Robert M.
Hobbs Ann S.
Johns Hopkins University
Marx Irene
Venable
LandOfFree
Saccharification enzymes from hyperthermophilic bacteria and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Saccharification enzymes from hyperthermophilic bacteria and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Saccharification enzymes from hyperthermophilic bacteria and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2820692