Chapter 29
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Starch Liquefaction with a Highly Thermostable Cyclodextrin Glycosyl Transferase from Thermoanaerobacter Species R. L. Starnes, C. L. Hoffman, V. M. Flint, P. C. Trackman, D. J. Duhart, and D. M. Katkocin Entotech, Inc., 1497 Drew Avenue, Davis, CA 95616-4880
A novel process is proposed for enzymatically liquefying starch with a highly thermostable cyclodextrin glycosyl transferase (CGTase). Conditions are described for liquefying 35% dry solids starch at pH 4.5 without added calcium, followed by saccharification to 96% dextrose. The CGTase is produced extracellularly by Thermoanaerobacter sp. ATCC 53,627, a thermophilic obligate anaerobe. This enzyme has a pH optimum activity observed at pH 3.5. The temperature optimum is 95°C. In the absence of starch and calcium, 95% of the activity remains after incubation at 80°C and pH 5.0 for 40 minutes. This CGTase shows the highest stability reported for any CGTase to date. These remarkable properties enable liquefaction of starch at pH 4.5, resulting in a process that can operate at the saccharifying pH of 4.5, thus eliminating the need for pH adjustment.
The production of high fructose syrup from starch requires three steps called liquefaction, saccharification, and isomerization. Liquefaction is generally accomplished with an alpha-amylase at a pH of approximately 6.0-6.5 followed by saccharification at a pH of approximately 4.5 with an amyloglucosidase. As a result of this pH incompatibility, the starch industry has long sought starch liquefaction enzymes capable of operating at the saccharifying pH of 4.5 in order to eliminate the need for pH adjustment of the starch as it arrives in the plant. The availability of these enzymes would provide significant process advantages with regard to saving costs and reducing undesirable by-products. The starch industry has adopted standard conditions for liquefaction. These conditions constitute a short-term jet-cooking treatment of a 35-40% dry solids (DS) starch slurry at 105°C for 5 minutes, known as gelatinization or primary liquefaction, followed by a 90-minute hold at 95°C, known as dextrinization or secondary liquefaction Alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) have conventionally been employed in starch liquefaction. Alpha-amylases hydrolyze the alpha-l,4-glucosidic linkages of the starch, producing maltodextrins. The alpha-amylases 0097-6156/91/0460-0384$06.00/0 © 1991 American Chemical Society Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
29. STARNESETAL
Starch Liquefaction
385
commercially used today are produced by Bacillus licheniformis and Bacillus stearothermophilus. Starch can also be degraded by a group of enzymes known as cyclodextrin glycosyl transferases [CGTase, 1,4-alpha-D-glucan 4-alpha-D-(l ,4-alpha-D-glucano)-transferase, E.C. 2.4.1.19]. The CGTase enzymes degrade starch by catalyzing cyclization, coupling, and disproportionation reactions as shown below (2): glc
cyclization * » glc (n-x) + χ coupling
n
(1)
χ = 6 alpha-cyclodextrin χ = 7 beta-cyclodextrin χ = 8 gamma-cyclodextrin glc
disproportionation + glc , glc (m+x) + glc(n-x)
m
(2)
n
The cyclization reactions produce cyclodextrins (also known as Schardinger dextrins) which are cyclic molecules comprised of six, seven, or eight alpha-D-glucopyranose residues linked by alpha-1,4 bonds (3,4). They are known as alpha-, beta-, or gammacyclodextrin depending on the number of glucose residues, 6, 7, or 8, respectively. These cyclized molecules have neither a non-reducing nor reducing end-group. Since CGTases can degrade starch by catalyzing cyclization and disproportionation reactions, a CGTase characterized by a high thermostability similar to the alpha-amylases employed by the starch industry should be able to solubilize the starch. In general, CGTases have lower pH optima than alpha-amylases. The low pH optima of these enzymes should eliminate the need for pH adjustment prior to saccharification thereby providing a major process improvement. Known producers of CGTase include Bacillus mascerans (7), Bacillus megaterium (8), Bacillus ohbensis (9), alkalophilic Bacillus sp. (10-14), Bacillus amyloliquefacien (15 Bacillus subtilis (16), Klebsiella oxytoca (17), and Micrococcus sp. (18). However, none of these CGTase enzymes are sufficiently thermostable for use in industrial starch liquefaction. Novel CGTases possessing this high thermostability property have now been discovered in a group of thermophilic anaerobic microorganisms belonging to the genus Thermoanaerobacter. The ability of one of these enzymes from Thermoanaerobacter sp. ATCC 53627 to liquefy starch is reported. The cyclodextrin-producing ability of this enzyme has previously been reported (Starnes, R.L.; Flint, V.M.; Katkocin, D.M. Cyclodextrin Production with a Highly Thermostable Cyclodextrin Glycosyl Transferase from Thermoanaerobacter sp. presented at the Fifth International Symposium on Cyclodextrins, Paris, France, March 27-30, 1990). Materials and Methods Enzymes. Termamyl, AMG, and Dextrozyme were obtained from Novo Nordisk Bioindustrials, Inc., Danbury, CT. Bacillus stearothermophilus alpha-amylase was supplied by Enzyme Bio-Systems Ltd., Englewood Cliffs, NJ. CGTase Production. Strain ATCC 53627 was cultured for 40 hours at 70 °C in a prereduced liquid medium under argon at pH 7.0 comprised of the following components in grams/liter: Maltrin M-100, 5.0; KH^PO^ 2.0; K H P 0 , 6.0; NaCl, 1.0; (NH^SO,, 2.5; MgS0 .7H 0, 0.5; CaCU.2H 80 ο
"35
\\
Φ
oc 40
\
20 0«-fc-
20
30
40
\
50 60 Temperature °C
Figure 4. Thermostability of CGTase relative to Termamyl and Bacillus stearothermophilus alpha-amylase at 80° C, pH 5.0.
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
100
ENZYMES IN BIOMASS CONVERSION
390
Table I. Liquefying Activity and Dextrose Yields after Saccharification Enzyme
pH
Amylase* Amylase Amylase Amylase Amylase Amylase
4.5 4.5 5.0 5.0 5.5 5.5
CGTase CGTase CGTase CGTase CGTase CGTase Amylase Termamyl
Ca+ +
% Dextrose
DE
Liquefied
+ + + -
Not determinable Not determinable 4.78 Not determinable 9.78 5.58
No No Yes** No Yes Yes
Not determinable Not determinable 96.0 Not determinable 95.8 95.9
4.5 4.5 5.0 5.0 5.5 5.5
+ + + -
0.51 0.44 0.73 0.69 1.10 0.83
Yes Yes Yes Yes Yes Yes
96.0 96.0 95.6 95.5 95.7 95.8
5.8 6.2
+ +
13.6 14.4
Yes Yes
96.8 96.4
* Bacillus stearothermophilus ** Rated as liquefied, but very viscous. Confirmation of the liquefying ability of the CGTase at pH 4.5 in the absence of added C a was obtained by measuring the viscosity reduction as a function of enzyme dosage. Termamyl at pH 6.2 and B. stearothermophilus alpha-amylase at pH 5.8 were run as controls. All the primary liquefactions were at 105°C, except those with CGTase at 0.446 and 0.223 Phadebas units/gram DS were at 100°C to compensate for the lower enzyme dosage. Secondary liquefactions were all performed at 90° C for 4 hours (Table II). Flow curves for these liquefactions at an enzyme dose of 4.46 Phadebas units/gram DS starch showing torque reading versus drive rotation speed at 60°C demonstrated a negligible increase in the rate at which torque reading increases as drive rotation speed is increased for liquefactions involving Termamyl and B. stearothermophilus alpha-amylase (Figure 5). There is a slight increase in this rate for liquefaction with CGTase. As the CGTase dose is decreased from 4.46 to 0.223 Phadebas units/gram DS starch, there is a corresponding increase in the torque measurements. + +
Table II. Viscosity Measurements Dosage Phadebas Added Enzyme U/gDS
Primary Ca+ +
CGTase 0.223 CGTase 0.446 CGTase 0.892 CGTase 2.23 4.46 CGTase 4.46 Termamyl 40 ppm 4.46 Ba amylase 40 ppm * measurement not possible at this speed
pH
Viscosity Liq. temp.
4.5 4.5 4.5 4.5 4.5 6.2
100°C 100°C 105°C 105°C 105°C
5.8
105°C
105OC
centipose * *
208 66.9 42.4
41.6 34.1
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
29.
STARNESETAL.
Starch Liquefaction
391
The viscosity achieved by the CGTase at pH 4.5 is very similar to the viscosities produced by Termamyl at pH 6.2 and B. stearothermophilus alpha-amylase at pH 5.8 as measured at a drive speed of 32 (Table Π). Reducing the CGTase dose to 2.23 Phadebas units still yielded acceptable viscosity. These results establish that the CGTase can liquefy starch under simulated industrial conditions of high starch but at a pH; i.e., 4.5 where conventional alpha-amylases do not function. Jet-Cooking. Industrially, heating of the starch during primary liquefaction is accomplished by jet-cooking. Consequently, survival of the CGTase during jet-cooking is important in order to be suitable for industrial use. Thermophilic anaerobic bacteria are poor enzyme-producers. It was, therefore, necessary to clone the gene into a suitable expression host in order to produce a sufficient quantity of enzyme for jet cooking. The CGTase gene was expressed in E. coli HB101 using pBR322 as the vector. Restriction mapping of the recombinant plasmid revealed a DNA fragment 12.8 kbp in size had been inserted into the EcoRl site of pBR322. Deletion analysis with the restriction enzyme Bam HI showed the CGTase gene was located on a 6.0 kbp BamHI-BamHI fragment. Characterization of the CGTase enzyme revealed it was indistinguishable from the native CGTase with regard to pH and temperature optima, molecular weight, and thermostability. The CGTase has a molecular weight roughly 50% larger than other reported CGTases. However, initial sequencing of the gene revealed that the molecular weight is very close to other known CGTases. The larger molecular weight obtained by SDS-PAGE suggests the high thermostability of this enzyme prevents complete reduction-denaturation. The CGTase was produced by culturing the recombinant E. coli strain in Luria-Bertani medium containing tetracycline (15 mg/liter) at 37°C for 24 hours. The CGTase was recovered by lysis of the cells. A 35% DS corn starch slurry (30 lb.) was liquefied with the recombinant CGTase at pH 4.5 without added Ca++ at a dosage of 8.92 Phadebas units/gram DS starch by jetting at 105°C for 5 minutes (primary liquefaction) followed by a hold at 95°C for 2 hours or 9(PC for 4 hours (secondary liquefaction). During secondary liquefaction at 95°C or 90°C, a rapid reduction in viscosity was observed. At 90°C, the viscosity reduction was monitored over time with a Nametre viscometer. The results demonstrated there was a rapid reduction in viscosity to 400 centipoise χ gm/cm^ by 7 minutes into secondary liquefaction (Figure 6). The action patterns of the liquefied starches following secondary liquefaction demonstrated the characteristic cyclodextrin action pattern at both temperatures. DE values were < 1.0 indicating the absence of reducing end-groups consistent with the mechanism of a CGTase. The liquefied starch solutions were saccharified with AMG and Dextrozyme at pH 4.5, 60°C for 48 hours (0.18AG/gram DS). The results (Table III) demonstrated a good yield of dextrose in all cases with minor amounts of DP (degree of polymerization) products. The highest yield was achieved with secondary liquefaction at 95°C and saccharification with Dextrozyme. Sediment volumes were less than 1 ml in all cases based on a 100 ml volume. Similar saccharification results were achieved with starch liquefied with the CGTase at a dose of 4.46 Phadebas units/gram DS starch (data not shown). Initial optimization of the CGTase dose requirements suggests that the dose can be lowered to 3.58 Phadebas units per gram DS without compromising viscosity reduction and dextrose yields upon saccharification. This dosage enables the CGTase to be competitive with Termamyl in enzymatic liquefaction. The thermostable CGTase produced by Thermoanaerobacter sp. ATCC 53,627 is able to liquefy starch at pH 4.5 under standard industrial conditions. It is, therefore, unnecessary to pH adjust the dextrin solution prior to saccharification as is normally done in the industry today. Since there is no need for pH adjustment, significant process advantages are realized. There is a substantial cost improvement with regard to chemicals, ion-exchange media, charcoal, etc. Also, unwanted by-product formation; e.g., maltulose, colored products, base-catalyzed products are reduced. Consequently, these advantages will translate into real savings to the starch industry.
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
ENZYMES IN BIOMASS CONVERSION
392 30
25
0.446U/G DS
Termamyl Thermoanaerobacter sp. CGTase Bacillus stearothermophilus Amylase
0.223U/G DS
4.46U/G DS Î46U/G DS 4.46U/G DS 10
20
30 40 Drive Rotation Speed
50
60
70
Figure 5. Flow diagrams for CGTase as a function of dose at 60° C.
2000
E κ 0
o. 1
φ ϋ ο ο
>
5 Time, Minutes
6
Figure 6. Viscosity reduction during secondary liquefaction at 90° C, pH 4.5.
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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STARNESETAL
Starch Liquefaction
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Table ΙΠ. Saccharifications From Cyclodextrin Glycosyl Transferase Jetting % Yield Sediment Enzvme Dextrose 95°C Secondary Liquefaction Dextrozyme 95.87 AMG 95.09 90°C Secondary Liquefaction Dextrozyme 95.37 AMG 95.36
DP2
DP3
DP4+
Voîume.ml
2.44 2.27
0.39 0.36
1.30 2.28
0.5 ml 0.5 ml
3.34 3.21
0.40 0.38
0.89 1.05
0.5 ml 0.5 ml
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Novo Industri A/S, U. S. Patent 3,912,590, 1975. Bender, H. Adv. in Biotechnol.Pocesses 1986, 6, 31. French, D.; Rundle, D.E. J. Amer. Chem. Soc. 1942, 64, 1651. Freudenberg, K.; Cramer, F. Z. Naturforsch 1948, B3, 464. Tilden, E.B.; Hudson, C.S. J. Bacteriol. 1942, 43, 527. Pongsawasdi, P.; Yagisawa, M. Agric.Biol.Chem. 1988, 52, 1099. Kitahata, S.; Okada S. J. Jap. Soc. Starch Sci. 1982, 29, 7. Kitahata, S.; Okada, S. Agric. Biol. Chem. 1974, 12, 2413. Yagi, Y.; Sato, M.; Ishikura, T. J. Jap. Soc. Starch Sci. 1986, 33, 144. Nakamura, N.; Horikoshi, K. Agric. Biol. Chem. 1976, 40, 1785. Kanedo T.; Hamamoto, T.; Horikoshi, K. J. Am. Microbiol. 1988, 134, 97. Kimura, K.; Takano, T.; Yamani, K. Appl. Microbiol. Biotechnol. 1987, 26, 149. Schmid, G.; Englbrecht, Α.; Schmid, D. In Proceedings of the Fourth International Symposium on Cyclodextrins; Huber, O.; Szejtli, J. Eds.; pp 71-76, Kluwer Academic: Munich, Germany, 1988. Schmid, G.; Huber, O.S.; Eberle, H.J. Proceedings of the Fourth International Symposium on Cyclodextrins; Huber, O.; Szejdi, J. Eds.; pp 87-92, Kluwer Academic: Munich, Germany, 1988. Yu, E.K.C.; Aoki, H.; Misawa, M. Appl. Microbiol. Biotechnol. 1988, 28, 377. Kato, T.; Horikoshi, K. J. Jap. Soc. Starch Sci. 1986, 2, 137. Yagi, Y.; Kuono, K.; Inui, T., United States Patent No. 4,317,881, 1982. Binder, F.; Huber, O.; Bock, A. Gene 1986, 47, 269. Stayn, Α.; Granum, P.E. Carb. Res. 1970, 75, 243. Kitahata, S.; Okada, S. J. Jap. Soc. Starch Sci. 1982, 29, 13.
RECEIVED November 6, 1990
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
