Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1994-10-04
2001-02-06
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S099000, C435S101000, C435S210000, C435S193000, C435S822000
Reexamination Certificate
active
06184001
ABSTRACT:
TECHNICAL FIELD
This invention relates to a thermostable cyclodextrin glycosyl transferase (CGTase), to a method for producing it, to a microbial strain capable of producing it, and to processes of using a thermostable CGTase for liquefying starch and for producing cyclodextrin, glucose and ethanol.
BACKGROUND ART
Cyclodextrins, also known as Schardinger dextrins, are starch-derived cyclic compounds containing six, seven, or eight &agr;-D-glucopyranose residues linked by &agr;-1,4 bonds. They are known as &agr;-, &bgr;-, or &ggr;-cyclodextrin depending on the number of glucose residues, 6, 7 or 8, respectively. These cyclized molecules have neither a non-reducing nor reducing end-group. The cyclic nature of these compounds produces a cavity which is predominantly hydrophobic enabling the formation of inclusion complexes with a number of compounds. This complexation ability is of particular interest to the food, agrochemical, cosmetic, and pharmaceutical industries for: taste masking, stabilization, increasing solubility, powdering, color masking, and emulsification, to name a few possible uses.
A starch solution can be degraded into cyclodextrin by enzymes known as cyclodextrin glycosyl transferases (1,4-alpha-D-glucan 4-alpha-D-(1,4-alpha-D-glucano)transferase, E.C. 2.4.1.19), hereinafter termed cyclodextrin glycosyl transferase or CGTase. The CGTase enzymes degrade the liquefied starch by catalyzing cyclization and disproportionation reactions.
Typically, cyclodextrin has been prepared heretofore by variations of the method described by E. B. Tilden and C. S. Hudson (J. American Chemical Society) 64:1432[1942], which method involves treating liquefied starch with a cyclodextrin glycosyl transferase (CGTase) enzyme from
Bacillus macerans.
All variations of this process have a number of disadvantages. First, since the CGTase is not sufficiently thermostable to be used above the gelatinization temperature of starch, the starch must be pretreated, e.g., with an &agr;-amylase, to solubilize the starch. It is important that the starch be liquefied to a relatively low DE (Dextrose Equivalent), so after conduct of the starch liquefaction process, the treating agent, normally an &agr;-amylase, must be inactivated, to obtain good cyclodextrin yield. Second, the
Bacillus macerans
CGTase is not sufficiently stable to be used at elevated temperatures, and consequently, the enzymatic cyclodextrinization process is carried out at about 50° C., where it is subject to possible microbial contamination. Third, conversion of starch to cyclodextrin (at 50° C., pH 7.0) by the
Bacillus macerans
CGTase requires extended reaction time, before reasonable yields are achieved.
Importantly, reactions catalyzed by CGTase which cleave the starch molecule can generate a desirable viscosity reduction in liquefied starch solutions by lowering the average molecular weight of the dextrin in the solution (without, in the instance of the CGTase, generating reducing sugars). The CGTase enzymes previously known to the art were produced by such microorganisms as
Bacillus macerans, Bacillus circulans, Bacillus stearothermophilus, Bacillus megaterium, Bacillus ohbensis,
alkalophilic Bacillus sp.,
Micrococcus luteus, Micrococcus varians,
and
Kiebsiella pneumoniae.
Unfortunately, none of the CGTase enzymes produced by these microorganisms seem to be sufficiently thermostable for use in industrial-scale for a combination of starch liquefaction and cyclodextrin production at temperatures sufficiently elevated to avoid possible microbial contamination.
Enzymatic liquefaction of aqueous starch slurry is widely practiced as the first step in converting starch to dextrose (glucose). To a great extent the starch industry has adopted the liquefaction process of U.S. Pat. No. 3,921,590. Typical conditions are jet cooking at 105° C. for 5 minutes, followed by a 90 minute hold at 95° C., at a starch concentration of 35% DS (dry substance), by weight. The enzyme used in this process is Termamyl™ (product of Novo Industri A/S), an &agr;-amylase from
Bacillus licheniformis.
Liquefaction is performed at pH about 6.0, followed by saccharification with glucoamylase at a pH of approx 4.5-5.0.
The art has long sought starch liquefaction enzymes capable of liquefying at pH 4.5, in order to eliminate the need for intermediate pH adjustment. &agr;-amylase from
Bacillus stearothermophilus
has been suggested for this purpose, but data in this specification show that it does not liquefy well at pH as low as 4.5. U.S. Pat. No. 4,578,532 and U.S. Pat. No. 4,613,570 disclose aciduric &agr;-amylases from Clostridium, but data in said patents show that their stability at 100° C. or above at pH 4.5 is insufficient.
OBJECT OF THE INVENTION
It is an object of this invention to provide a cyclodextrin glycosyl transferase with sufficient heat stability to be used for CD production at 60° C. or higher, where the risk of microbial infection is slight, and even to be used for starch liquefaction above 90° C., where the starch is fully gelatinized. It is also an object of the invention to provide an enzyme capable of liquefying starch at pH 4.5 and a temperature above 100° C.
It is also an object of the invention to provide a method of using a thermostable CGTase for producing cyclodextrin. Other objects of the invention are the provision of a method of producing said thermostable enzyme and a microbial strain capable of producing it. It is a further object to provide a process using said enzyme to produce CD at 60° C. or higher and a process using said enzyme for starch liquefaction at a pH around 4.5-5.0.
SUMMARY OF THE INVENTION
The inventors have isolated a number of thermophilic obligate anaerobic strains that produce CGTases of surprising heat stability.
Accordingly, in its first aspect the invention provides a cyclodextrin glycosyl transferase, characterized in that it is native to a strain of Thermoanaerobacter or Thermoanaerobium, has a temperature optimum measured at pH 5.0 of about 95° C.; a pH optimum of about 5.0; and a residual activity after 40 minutes incubation at 80° C. and pH 5.0 of about 95% in the absence of starch and Ca
++
.
A second aspect of the invention provides a method for producing a cyclodextrin glycosyl transferase (CGTase) comprising cultivation of a CGTase producing strain of Thermoanaerobacter or Thermoanaerobium under anaerobic conditions, or cultivation of a transformed host organism containing the appropriate genetic information therefrom under aerobic conditions, in a suitable nutrient containing medium and thereafter recovering CGTase from the fermentation medium.
A third aspect of the invention provides a biologically pure culture of a strain of Thermoanaerobacter or Thermoanaerobium, characterized by the ability to produce cyclodextrin glycosyl transferase, and by being non-motile.
A fourth aspect of the invention provides a starch liquefaction process which comprises subjecting an aqueous starch slurry to enzymatic liquefaction in the presence of said cyclodextrin glycosyl transferase at a pH in the range of about 4.0 to 5.5 preferably at a temperature exceeding about 100° C.
A fifth aspect of the invention provides a process for producing cyclodextrin which comprises enzymatically treating a starch hydrolysate solution with said cyclodextrin glycosyl transferase, at a temperature of above 60° C. and thereafter recovering a cyclodextrin product from the reaction mixture.
Finally, a sixth aspect of the invention provides a process for producing cyclodextrin which comprises enzymatically treating an aqueous starch slurry with the cyclodextrin glycosyl transferase of claim
1
at a temperature of above about 100° C. and at a pH in the range of 4.0-5.5, preferably essentially without addition of a calcium salt, thereafter holding the resulting syrup at a temperature in the range of 80°-90° C. for not more than about 28 hours, the syrup being in the range of 20-30 DS during at least part of said hold period, and then recovering a cyclodextrin product from the reaction mixture.
Furthe
Lambiris Esq. Elias J.
Lilling Herbert J.
Novozymes A/S
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