Biotechnol. Prog. 1990, 6, 214-219
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Recovery of Clostridium thermosulfurogenes Produced @-Amylaseby (Hydroxypropy1)methylcellulose Partition Everson A. Miranda and Kris A. Berglund* Departments of Agricultural and Chemical Engineering, Michigan State University, East Lansing, Michigan 48824, and Michigan Biotechnology Institute, 3900 Collins Road, Lansing, Michigan 48909
A procedure for recovering Clostridium thermosulfurogenes produced 0-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropy1)methylcellulose. The 0-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. T h e process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethy1ene glycol)/dextran, the current approach appeared t o be biospecific.
I. Introduction Partition by an aqueous two-phase system is a separation method based on the difference in affinity of cell particles and macromolecules to two immiscible aqueous phases (Albertsson, 1986; Kula et al., 1982). Two immiscible liquid phases are formed by mixing aqueous solutions of two polymers or a polymer and a salt. However, a two-phase system composed of a liquid and a gel may be formed instead of two liquid phases under the correct conditions [e.g.,type of polymers, presence of salts, and concentration of salts and polymers (Albertsson, 1986)l. In the present work the term gel is used to describe a polymer phase that is substantially less fluid than the initial solution and may even contain some solid. Poly(ethylene glycol),dextran, and cellulose ethers (e.g., hydroxypropyl methylcellulose and methylcellulose) are examples of such polymers which produce a gel upon addition of salts (Dove and Mitra, 1986; Ganz, 1977; Kroner et al., 1982). No previous work in the literature about the use of these systems containing a gellike phase in recovery of biomolecules was found in an extensive literature search. The advantage of using these systems would be the addition of the desirable characteristics of precipitation to the partition process (e.g., more concentration), since the gel resembles a precipitate (Albertsson, 1986). The objective of the current work was to study the recovery of P-amylase produced by Clostridium thermosulfurogenes (C. thermosulfurogenes) from fermentation broth by partition to a cellulose ether gel. Cellulose ethers were chosen due to their structural similarity with the P-amylase basic substrate unit, amylose, with the potential of imparting specificity to the separation. 11. Materials and Methods 11.1. Enzyme Organism and Cultivation. /I-Amylase produced by the anaerobic bacterium C. thermosulfurogenes was first isolated and characterized by Hyun and Zeikus (1985a). Its thermostability and thermoactivity are attractive for use in processes to produce maltose syrups. The general mechanism for P-amylase synthesis by the bacteria, the ability of the enzyme to 8756-7938/90/3006-0214$02.50/0
0 1990 American
bind starch and to produce maltose syrups, its purification, and characterization of its molecular and biochemical properties have been reported elsewhere (Hyun and Zeikus, 1985b; Saha et al., 1988; Shen et al., 1988; Elankovan, 1989). The catabolic repression resistant mutant H-12-1derived from C. thermosulfurogenes wild-type strain 4B (ATCC 33743) was used. The culture was routinely grown a t 60 "C in 26-mL anaerobic pressure tubes, Bellco Glass, Inc., containing 10 mL of TYE medium supplemented with 0.5% soluble starch and a N2-CO2 (955) gas headspace according to Hyun and Zeikus (1985b). The culture was grown overnight in a carboy (18-L capacity) containing 16 L of TYE medium with either 0.5% maltose or partially hydrolyzed starch (maltodextrin) as substrate. It was transferred to a 250-L fermenter containing 180 L of the same medium used in the previous step for the final growth until stationary phase (18-22 h). 11.2. Broth Preparation. Cell removal and broth concentration were done under aerobic conditions. When maltose was used as the fermentation substrate, cells were removed from the broth by ultrafiltration in an Amicon hollow fiber ultrafiltration cartridge, Model H26MP0143, with a 100 000 molecular weight cutoff (MWCO) membrane. A 20-L batch of this broth (free of cells), subsequently referred to as maltose broth, was then concentrated to 800 mL by using an Amicon spiral ultrafiltration cartridge, Model SlOY30, with a 30000 MWCO membrane. Three aliquots of 500 mL were taken during the concentration. These aliquots were denoted by their concentration ratio: initial volume (20 L) divided by the volume a t the time of sampling. For the case where starch was the fermentation substrate, cell removal was done by centrifugation at 3000 rpm for 10 min in a Sorvall centrifuge, Model RC5B, and the concentration of the resulting broth, subsequently referred to as starch broth, was performed the same as that for the maltose broth case. 11.3. @-AmylasePreparation. Crude P-amylase preparation was obtained by precipitating maltose broth obtained as described above with ammonium sulfate (50% saturation) followed by suspension and dialysis of the
Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prcg., 1990,Vol. 6, No. 3
precipitate in acetate buffer (50 mM sodium acetate with 5 mM CaC12, pH 6.0). A purified preparation was obtained from the crude preparation by precipitation with 20% v/v ethanol and dialysis as above. Prior to use, each preparation was centrifuged in a clinical centrifuge and filtered through a Gelman Acrodisc 0.45-pm filter. The term “purified” does not mean that the enzyme was purified to homogeneity. It is only meant to indicate that this preparation is more pure than the crude one. 11.4. Cellulose Ethers. The cellulose ethers methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) were selected for this study due to their foodgrade status, ready availability, and moderate cost. The MC and HPMC used were the commercial products METHOCEL (Dow Chemical Co.). MC was of type A4M with nominal molecular weight of 85 000 and methoxyl degree of substitutions in the range of 1.6-1.9. Three types of HPMC were used: K15M, K4M, and KlOO M premium with nominal molecular weights of 15 000,85 000, and 250 000, respectively. These HPMC’s have methoxyl and hydroxypropyl molar substitutions in the range of 1.1-1.4 and 0.1-0.3, respectively. The solutions were prepared according to the manufacturer’s instructions [Handbook on Methocel Cellulose Ethers Products (Form 192-702-78);Dow Chemical Co.; Midland, MI.(1978)]: wetting and dispersing the particles in hot water and then adding cold buffer to dissolve the wetted particles. 11.5. Assays. @-Amylaseactivity was measured a t 60 OC with 2 % boiled starch in a pH 6.0 acetate buffer. The reducing sugars released by the enzymatic hydrolysis of starch were determined by the dinitrosalicylic acid method (Bernfeld, 1955). One unit of P-amylase activity was defined as 1 pmol of reducing sugars as maltose produced per minute under the above conditions. The total carbohydrates content was determined by the phenol-sulfuric acid method of Dubois and co-workers (1956) with MC A4M (nominal molecular weight 85 000) as the standard. Protein was determined by the method of Lowry and co-workers (1951) with bovine serum albumin as the standard. SDS-polyacrylamide slab gel electrophoresis was performed as described by Laemmli (1970). 11.6. Partition Experiments. The parameter used for evaluation of activity partition was “percentage partitioned“, defined as the percentage of total activity missing from the supernatant after partition (compared with the initial total activity). This is an indirect measurement of how much activity was partitioned to the pellet. “Percentage precipitated” was defined in a similar way to account for polymer or &amylase salted-out by ammonium sulfate. Preliminary experiments a t different enzyme/HPMC solution pH exhibited little effect of this variable on the partition. Therefore, pH 6.0 was selected for all other experiments. Experiments were carried out a t room temperature (21 “C), unless specified otherwise. The percentage of saturation a t 21 OC was used as the unit of concentration for ammonium sulfate in solution. Its solubility at this temperature is 757 g/L. Small-scale screening experiments were conducted in 1.5-mL Eppendorf tubes, using 500 pL of @-amylasepreparation and 500 pL of HPMC or MC solution at 2-fold the final desired concentration. After homogenization in a vortex for 10 s, the appropriate volume of saturated ammonium sulfate solution was added and the contents of the tubes were mixed again. After 10 min, the mixture was centrifuged at 12 000 rpm for 10 min in a microcentrifuge, Eppendorf Model 5415. The supernatant was separated from the pellet and filtered through a Milli-
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pore Millex HV 0.45-pm filter. The filtrate was used for enzyme activity and total carbohydrates assays. To check if the enzyme could partition to the gel after its formation, a pellet of MC was prepared from 1 mL of 0.4 5% w/v MC solution by adding ammonium sulfate saturated solution up to 30% saturation. The pellet was washed twice with 1 mL of the salt solution a t 30% saturation, and then 1430 pL of prepurified @-amylasea t 30% salt saturation was added to it. The tube was shaken for 30 s and let stand at room temperature for 10 min, and after centrifugation the supernatant was filtrated and analyzed for activity. The possibility of separating the enzyme from the gel was checked by both HPMC reprecipitation and a backpartition experiment. In the first case, a pellet obtained by stirred batch partition (to be described later) of maltose broth (concentration ratio of 7) was dissolved in 100 mL of acetate buffer and the HPMC reprecipitated by ammonium sulfate a t 21 and 56 “C. The supernatant was separated by centrifugation and anaiyzed for enzyme activity and total carbohydrates. Back-partition experiments were selective extraction of the enzyme from a 6-amylase-containing pellet to solutions of different salt concentrations. P-Amylasecontaining pellets were prepared by adding 430 p L of saturated solution of ammonium sulfate to 500 pL of 1 % MC solution, mixing, and then adding 500 pL of enzyme solution. After 15 min under gentle mixing, the pellets were formed by centrifugation. The pellets were placed in ammonium sulfate solutions of different concentrations, called “equilibrium solutions”. Tubes containing the mixtures were shaken for 30 s and allowed to stand at room temperature overnight. The supernatant was centrifuged and analyzed for activity and total carbohydrates. To verify the importance of hydrophobic interaction on the partition process, experiments were performed with a protein well-known by its hydrophobicity, bovine albumin. The experimental conditions were 30% ammonium sulfate saturation and 0.2% w/v HPMC (loading of 10 mg of polymer to 1 mg of protein). Stirred batch experiments were done with maltose and starch broths with concentration ratios ranging from 1-17. HPMC and ammonium sulfate concentrations were 0.2% and 30% saturation, respectively. The broth pH was in the range of 5.1-5.2, and little change was observed after addition of HPMC. The experiments were carried out in a 50-mL beaker at room temperature, unless otherwise specified. Broth and 1% HPMC aliquots, 20 and 5 mL, respectively, were homogenized for 5 min. A 10.7-mL amount of ammonium sulfate saturated solution was then added dropwise, and mixing was continued for more 10 min. The gellike phase formed was separated from the supernatant by centrifugation a t 8000 rpm for 20 min (Sorval centrifuge Model RC5B), and the pellet was dissolved in 100 mL of acetate buffer. This solution was used in electrophoresis after concentration in a 10000 MWCO disc membrane with an Amicon ultrafiltration cell, Model 8010. The same solution was also used for the HPMC recovery study, enzyme activity, total carbohydrates, and protein assays. The volume of the supernatant was measured and the supernatant was used for activity assay. One aliquot of the supernatant was dialyzed, concentrated by ultrafiltration as above, and used for electrophoresis analysis. The effect of temperature on the partition of @-amylase from starch broth was investigated. The order of solution mixing had to be changed and HPMC solution
Biotechnol. Prog., 1990. Vol. 6 , No. 3
218 1101
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a" 60 Ammomum sulfate concentration (Percentage of saturation)
Figure 1. Comparison between partition of @-amylase(A),salting-out of @-amylase(O),and salting-outof HPMC ( 0 ) . Enzyme used: crude @-amylasepreparations. Conditions of experiments: enzyme activity = 33.0-80 units/mL; protein concentration = 0.7-2.4 mg/mL; HPMC concentration = 0.2-0.005% (w/v).
added a t 21 "C because HPMC spontaneously precipitates a t high temperatures (Greminger and Krumel, 1980). First, starch broth and ammonium sulfate solutions were equilibrated a t the desired experimental temperature and then added one to the other. At last, HPMC solution was added to the broth/salt mixture. Centrifugation was done in a prewarmed rotor. 111. Results 111.1. Experiments with 8-Amylase Preparations. Small-scale screening experiments with @-amylase preparations were performed prior to the experiments with fermentation broths. Addition of ammonium sulfate to the enzyme/cellulose ether solutions, under mixing, initially produced a cloudy suspension that changed to a clear suspension of filaments as the salt concentration was increased. Observation under 200X magnification revealed particles, strands twisted around each other in a helix form, and droplets. Figure 1shows results of the comparison between precipitation by ammonium sulfate and partition as separation techniques for @-amylase.Partition requires approximately 10% less salt saturation (36% less salt) than precipitation. Figure 1shows also that precipitation of HPMC took place a t lower salt concentrations than @-amylase partition and precipitation. Partition of the enzyme after formation of the gel phase was tested by contacting the enzyme solution a t 30% ammonium sulfate saturation with the MC pellet. Only 9 70 of the initial total activity remained in the supernatant. If only precipitation of the enzyme (salting-out) had occurred, the remaining activity would be 21% (Figure 1). Partition experiments with bovine albumin showed lower protein recovery than activity recovery for @-amylase.No more than 30% of the protein was present in the gel phase. Comparison of the two polymers, MC and HPMC, as partitioning agents at a concentration of 0.2% showed that they are equally effective in partitioning ,&amylase a t 30% ammonium sulfate saturation (98% partition). However, HPMC was more effective a t 20% salt saturation, giving 58% partition versus 34% for MC. The effect of HPMC loading (mass of polymer per mass of protein) on partition was evaluated, and the result is
0.0
0.5
1.0
1.5
2.0
HPMC loading (mg HPMC/mg protein)
Figure 2. HPMC loading for maximum partition. Enzyme used: crude @-amylasepreparation. Conditions of the experiments: enzyme activity = 36.7 units/mL; protein concentration = 1.2 mg/mL; ammonium sulfate concentration = 30% saturation. Bars indicate standard deviation (n = 3).
shown in Figure 2. A minimum of 0.2 mg of HPMC/mg of protein was required for maximum partition. At the protein concentration used (1.2 mg/mL), this loading corresponds to a HPMC concentration of 0.024% w/v. Table I displays the results of the experiments made with HPMC of different nominal molecular weights. The HPMC with the nominal molecular weight of 85 000 produced the highest partition among the three polymers studied. Resolubilization of the enzyme from the gel phase by back-partition with little polymer dissolution (average of 2.1% with standard deviation of 0.7%) was possible, as shown by Figure 3. Reprecipitation of HPMC a t two different temperatures, 2 1 and 56 "C, separated a t least 90% of the cellulose ether along with only 5 % of the total enzyme activity (Figure 4). However, only 5% ammonium sulfate saturation was required a t 56 "C against 15% a t 21 "C. This is the result of the shift of the partition and precipitation curves to the left by the increase in the temperature. Note that at 56 "C the partition and precipitation curves are more separated from each other than at 21 "C. 111.2. Experiments with Fermentation Broths. Stirred batch partition experiments were carried out with both types of broth at various levels of concentration (Table 11), and no @-amylasedenaturation was observed. The behavior of maltose broth was the same as that in the small-scale experiments, with practically 100% of the enzyme activity recovered with the pellets. @-Amylase from starch broth was not efficiently partitioned, with only an average of 29% of the activity found in the pellets a t most (concentration ratio of 3). Partition of the @-amylasefrom maltose broth achieved different purification, according to the broth ultrafiltration (Figure 5). The purification for the maltose broth with a 17-fold concentration ratio was qualitatively confirmed by SDS-polyacrylamide gel electrophoresis. Temperature increases improved enzyme partition from starch broth (Figure 6). Even though high levels of partition were not achieved, 25% of @-amylaseactivity was partitioned a t 75 "C while it was almost negligible at 2 1 "C (concentration ratio of 7). The maximum enzyme denaturation was around 10% at 75 "C.
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Biotechnol. Prog., 1990, Vol. 6, No. 3
Table I. Partition Dependence on the HPMC Nominal Molecular Weight. HPMC nominal percentage partitioned molec w t treatment 1 treatment 2 avb 15 OOO 85 OOO 250 000
66 85 75
68 85 72
63 85 78
a Enzyme used, crude preparation. Conditions of experiments: HPMC concentration = 0.2% (w/v); HPMC loading = 0.7; ammonium sulfate concentration = 25% saturation. * av is the average of treatments 1 and 2.
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Figure 3. Back-partition of @-amylase. @-Amylase-containing pellets were obtained by adding enzyme solution to MC pellets. These pellets were prepared with 1.0% MC solution and ammonium sulfate at 30% saturation.
'0 5 10 15 20 25 30 Ammonium sulfate concentration (Percentage of saturation)
Figure 4. Separation of @-amylasefrom HPMC. &Amylase partition (circles) and HPMC precipitation (triangles) at 21 OC (open symbols) and at 56 "C (closed symbols) from dissolved 8-amylase pellet. Pellet obtained by @-amylasepartition from maltose broth (17-fold concentrated) with 0.2% (w/v) HPMC and 30% ammonium sulfate saturation.
IV.Discussion Partition of @-amylaseto the gel phase formed by HPMC is a very efficient separation process. Yields of practically 100% were obtained with a purification ratio up to 100-fold. Recovery of the same enzyme by precipitation with ethanol, ammonium sulfate, and a combination of both precipitations gave yields in the range of 90-9596 and a purification ratio around 30-fold at most (Elankovan, 1990). Separation of the HPMC and the enzyme
from each other is accomplished by precipitation of the polymer at an ammonium sulfate concentration where only the enzyme is soluble. This also enables recovery of HPMC for reuse. HPMC loading required for maximum partition is low (0.2 mg of HPMC/mg of protein), which avoids the problem of high solution viscosity. Moreover, the salt concentration needed for partition is lower than conventional salting-out. It was shown that a temperature increase can further reduce the salt concentration needed for partition and enzyme/HPMC separation. Ninety percent of @-amylasecan be partitioned with a 20% saturated salt solution at 56 OC. This represents a 75% reduction in the salt requirement as compared with the optimum salting-out concentration of 50% saturation recommended by Elankovan (1990). Clearly, this advantage can only be realized in the recovery of thermophilic enzymes. Partition depends on the fermentation carbon source similar to the way that salting-out does: the enzyme produced with maltose separates well while the one produced with starch does not. Elankovan (1989)attributed this poor separation to binding of the @-amylaseto starch. The same phenomenon seems to affect partition. The significant partition at higher temperatures of @-amylaseproduced with starch is probably due to a higher starch hydrolysis rate. Molecules of enzyme could be free of the starch moiety and interact with HPMC. This possible competition between starch and HPMC by the enzyme, the fact that these polysaccharides have similar backbones, and the relative high purification obtained by partition, leads us to believe that there is a specificity in the partition process. This idea is supported by a companion work where two other amylases (an a-amylase and a pullulanase) were also partitioned by the HPMC gel phase while three non-amylase proteins (a glucose isomerase, a bovine albumin, and the B sub-unit of Escherichia coli toxin) were not. The interaction between HPMC and amylases may mimic amylases binding to starch. The partition does not seem to be a simple phenomenon. The gellike phase is, in fact, composed of three phases (particles, filaments, and droplets). Due to the use of a nonionic polysaccharide (HPMC) and high salt concentration, this partition method could potentially be controlled by hydrophobic interaction. Such interaction is known to be the mechanism responsible for protein binding to some polysaccharide columns, such as Sepharose 4B and cellulose, at high salt concentrations (Arakawa, 1986). However, the results of bovine albumin partition dismissed this possibility by the low recovery obtained. @-Amylasepartition takes place at ammonium sulfate concentrations intermediate to the concentration necessary to precipitate the enzyme and HPMC from their respective solutions. We see two possible explanations for this. First, it may be that HPMC and @-amylaseinteract with each other and form a complex that has solubility decreased by salt addition. Second, the enzyme may partition to the gel phase only after its formation. The larger shift to the left of the HPMC insolubilization curve than the shift of the @-amylasepartition at higher temperature supports the first idea. The better behavior of HPMC in comparison to MC at 20% salt saturation may be explained by the difference in methoxyl degree of substitution of HPMC (in the range of 1.1-1.4)and MC (in the range of 1.6-1.9), since it is known that a degree of substitution lower than 1.4 yields products with low water solubility (Greminger and Krumel, 1980).
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Table 11. Partition as a Function of Fermentation Substrate and Concentration Ratio. total activitv,b % pellet fermentn substrate concn ratio Tlc T2c avC TI maltose 1 95 97 96 8 2 108 108 108 5 3 93 94 94 5 7 98 98 98 5 17 107 108 108 5 starch 1 20 8 14 78 2 10 20 15 91 3 26 32 29 82 12 9 82 7 6 17 9 5 7 93 ~~
~~~
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supernatant T2 9 6 5 4 4 85 93 77 90 98
av 9 6 5 5 5 82 92 80 86 96
4 Conditions of experiments: enzyme activities = 5.0-29.2 units/mL and 13.1-127.5 units/mL for maltose and starch broth, respectively; protein concentration = 2.4-2.6 mg/mL for maltose broth (loading from 0.78 to 0.83 mg of HPMC/mg of protein); HPMC concentration = 0.2% (w/v); ammonium sulfate concentration = 30% saturation. Compared to total initial activity. T1, T2, and av means treatment 1, treatment 2, and average, respectively.
in Table I, it seems that there is an optimum HPMC molecular weight for the @-amylasepartition.
V. Conclusion A process for recovering C. thermosulfurogenes @-amylase from maltose fermentation broth was developed. It makes use of low quantities of a food-grade polymer and requires lower concentrations of ammonium sulfate than conventional salting-out. Therefore, it is suitable for largescale processing due to low chemical consumption, high recovery and high purity of the final product, and low waste generation. Its specificity can also be extended to other amylases.
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Figure 5. 0-Amylase purification achieved by partition from maltose broth with 0.2% w/v HPMC and 30% ammonium sulfate saturation as function of broth concentration. lOo"'--'''~"
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Acknowledgment We acknowledge the Michigan Biotechnology Institute for financial support and for use of its facilities, the Conselho Nacional de Desenvolvimento Cientifico & Tecnolbgico, CNPq,Brazil, forsupp0rtingE.A.M. withaschoiarship, and the Dow Chemical Co. for donating the METHOCEL products.
70
Temperature ("C)
F i g u r e 6. Effect of temperature increase in @-amylasepartition from starch broth (7-fold concentrated): (A)pellet; (0) supernatant; (A) total recovery. Conditions of experiments: HPMC concentration = 0.2% (w/v); ammonium sulfate concentration = 30% saturation.
The nominal molecular weight of HPMC influences the partition of @-amylase,contrary to what was expected since it is known that gelation and precipitation properties for commercial HPMC have little dependence on this variable (Sarkar, 1979). On the basis of the results shown
Literature Cited Albertsson, P.-A. Partition of Cell Particles and Macromolecules; John Wiley and Sons: New York, 1986; p 346. Arakawa, T. Thermodynamic analysis of the effect of concentrated salts on protein interaction with hydrophobic and polysaccharide columns. Arch. Biochem. Biophys. 1986,248 (l), 101-105. Bernfeld, P. Amylases CY and @. Methods Entymol. 1955, 1, 149-150. Dove, G. B.; Mitra, G. Recovery of proteins from polyethylene glycol-water solution by salt partition. ACS Symp. Ser. 1986, 314,93-108. Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A,; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956,28 (3), 350-356. Elankovan, P. Ph.D. Dissertation, Michigan State University, East Lansing, MI, 1990. Ganz, A. J. In Food Colloids; Graham, H. D., Ed.; AVI: Westport, CT, 1977; pp 383-417. Greminger, G. K.; Krumel, K. L. In Handbook of Water Soluble Gums and Resins; Davidson: R. L., Ed.; McGraw-Hill: New York, 1980; p p 3/1-3/25. Hyun, H. H.; Zeikus, J. G. General biochemical characterization of thermostable extracellular @-amylasefrom ClostridEnviron. Microbiol. 1985a, ium thermosulfurogenes. Appl. .. 49 (5), 1162-1167. Hvun. H. H.: Zeikus. J. G. Regulation and genetic enhancement of glucoamylase a n i pullulanase production in Clostridium thermohydrosulfuricum. J . Bacteriol. 1985b, 164 (3), 1146-1152.
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Kroner, K. H.; Hustedt, H.; Kula, M.-R. Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale. Biotechnol. Bioeng. 1982,24,1015-1045. Kula, Ma-R.;Kroner, K. H.; Hustedt, H. Advances in Biochemical Engineering, Reaction Engineering; Springer-Verlag: New York, 1982;pp 73-117. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970,227, 680-685. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 1951,193, 265-275.
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Saha, B. C.; LeCureux, L. W.; Zeikus, J. G. Raw starch adsorption-desorption of a thermostable @-amylasefrom Clostridium thermosulfurogenes. Anal. Biochem. 1988, 175, 569572. Sarkar, N. Thermal gelation properties of methyl and hydroxypropyl methylcellulose. J . Appl. Polym. Sci. 1979,24,10731087. Shen, G.-J.; Saha, B. C.; Lee, Y.-E.; Bhatnagar, L.; Zeikus, J. G. Purification and characterization of a novel thermostable @-amylasefrom Clostridium thermosulfurogenes. Biochem. J. 1988,254,835-840. Registry No. p-Amylase, 9000-91-3; (hydroxypropy1)methylcellulose, 9004-65-3.
214
Recovery of Clostridium thermosulfurogenes Produced @-Amylaseby (Hydroxypropy1)methylcellulose Partition Everson A. Miranda and Kris A. Berglund* Departments of Agricultural and Chemical Engineering, Michigan State University, East Lansing, Michigan 48824, and Michigan Biotechnology Institute, 3900 Collins Road, Lansing, Michigan 48909
A procedure for recovering Clostridium thermosulfurogenes produced 0-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropy1)methylcellulose. The 0-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. T h e process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethy1ene glycol)/dextran, the current approach appeared t o be biospecific.
I. Introduction Partition by an aqueous two-phase system is a separation method based on the difference in affinity of cell particles and macromolecules to two immiscible aqueous phases (Albertsson, 1986; Kula et al., 1982). Two immiscible liquid phases are formed by mixing aqueous solutions of two polymers or a polymer and a salt. However, a two-phase system composed of a liquid and a gel may be formed instead of two liquid phases under the correct conditions [e.g.,type of polymers, presence of salts, and concentration of salts and polymers (Albertsson, 1986)l. In the present work the term gel is used to describe a polymer phase that is substantially less fluid than the initial solution and may even contain some solid. Poly(ethylene glycol),dextran, and cellulose ethers (e.g., hydroxypropyl methylcellulose and methylcellulose) are examples of such polymers which produce a gel upon addition of salts (Dove and Mitra, 1986; Ganz, 1977; Kroner et al., 1982). No previous work in the literature about the use of these systems containing a gellike phase in recovery of biomolecules was found in an extensive literature search. The advantage of using these systems would be the addition of the desirable characteristics of precipitation to the partition process (e.g., more concentration), since the gel resembles a precipitate (Albertsson, 1986). The objective of the current work was to study the recovery of P-amylase produced by Clostridium thermosulfurogenes (C. thermosulfurogenes) from fermentation broth by partition to a cellulose ether gel. Cellulose ethers were chosen due to their structural similarity with the P-amylase basic substrate unit, amylose, with the potential of imparting specificity to the separation. 11. Materials and Methods 11.1. Enzyme Organism and Cultivation. /I-Amylase produced by the anaerobic bacterium C. thermosulfurogenes was first isolated and characterized by Hyun and Zeikus (1985a). Its thermostability and thermoactivity are attractive for use in processes to produce maltose syrups. The general mechanism for P-amylase synthesis by the bacteria, the ability of the enzyme to 8756-7938/90/3006-0214$02.50/0
0 1990 American
bind starch and to produce maltose syrups, its purification, and characterization of its molecular and biochemical properties have been reported elsewhere (Hyun and Zeikus, 1985b; Saha et al., 1988; Shen et al., 1988; Elankovan, 1989). The catabolic repression resistant mutant H-12-1derived from C. thermosulfurogenes wild-type strain 4B (ATCC 33743) was used. The culture was routinely grown a t 60 "C in 26-mL anaerobic pressure tubes, Bellco Glass, Inc., containing 10 mL of TYE medium supplemented with 0.5% soluble starch and a N2-CO2 (955) gas headspace according to Hyun and Zeikus (1985b). The culture was grown overnight in a carboy (18-L capacity) containing 16 L of TYE medium with either 0.5% maltose or partially hydrolyzed starch (maltodextrin) as substrate. It was transferred to a 250-L fermenter containing 180 L of the same medium used in the previous step for the final growth until stationary phase (18-22 h). 11.2. Broth Preparation. Cell removal and broth concentration were done under aerobic conditions. When maltose was used as the fermentation substrate, cells were removed from the broth by ultrafiltration in an Amicon hollow fiber ultrafiltration cartridge, Model H26MP0143, with a 100 000 molecular weight cutoff (MWCO) membrane. A 20-L batch of this broth (free of cells), subsequently referred to as maltose broth, was then concentrated to 800 mL by using an Amicon spiral ultrafiltration cartridge, Model SlOY30, with a 30000 MWCO membrane. Three aliquots of 500 mL were taken during the concentration. These aliquots were denoted by their concentration ratio: initial volume (20 L) divided by the volume a t the time of sampling. For the case where starch was the fermentation substrate, cell removal was done by centrifugation at 3000 rpm for 10 min in a Sorvall centrifuge, Model RC5B, and the concentration of the resulting broth, subsequently referred to as starch broth, was performed the same as that for the maltose broth case. 11.3. @-AmylasePreparation. Crude P-amylase preparation was obtained by precipitating maltose broth obtained as described above with ammonium sulfate (50% saturation) followed by suspension and dialysis of the
Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prcg., 1990,Vol. 6, No. 3
precipitate in acetate buffer (50 mM sodium acetate with 5 mM CaC12, pH 6.0). A purified preparation was obtained from the crude preparation by precipitation with 20% v/v ethanol and dialysis as above. Prior to use, each preparation was centrifuged in a clinical centrifuge and filtered through a Gelman Acrodisc 0.45-pm filter. The term “purified” does not mean that the enzyme was purified to homogeneity. It is only meant to indicate that this preparation is more pure than the crude one. 11.4. Cellulose Ethers. The cellulose ethers methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) were selected for this study due to their foodgrade status, ready availability, and moderate cost. The MC and HPMC used were the commercial products METHOCEL (Dow Chemical Co.). MC was of type A4M with nominal molecular weight of 85 000 and methoxyl degree of substitutions in the range of 1.6-1.9. Three types of HPMC were used: K15M, K4M, and KlOO M premium with nominal molecular weights of 15 000,85 000, and 250 000, respectively. These HPMC’s have methoxyl and hydroxypropyl molar substitutions in the range of 1.1-1.4 and 0.1-0.3, respectively. The solutions were prepared according to the manufacturer’s instructions [Handbook on Methocel Cellulose Ethers Products (Form 192-702-78);Dow Chemical Co.; Midland, MI.(1978)]: wetting and dispersing the particles in hot water and then adding cold buffer to dissolve the wetted particles. 11.5. Assays. @-Amylaseactivity was measured a t 60 OC with 2 % boiled starch in a pH 6.0 acetate buffer. The reducing sugars released by the enzymatic hydrolysis of starch were determined by the dinitrosalicylic acid method (Bernfeld, 1955). One unit of P-amylase activity was defined as 1 pmol of reducing sugars as maltose produced per minute under the above conditions. The total carbohydrates content was determined by the phenol-sulfuric acid method of Dubois and co-workers (1956) with MC A4M (nominal molecular weight 85 000) as the standard. Protein was determined by the method of Lowry and co-workers (1951) with bovine serum albumin as the standard. SDS-polyacrylamide slab gel electrophoresis was performed as described by Laemmli (1970). 11.6. Partition Experiments. The parameter used for evaluation of activity partition was “percentage partitioned“, defined as the percentage of total activity missing from the supernatant after partition (compared with the initial total activity). This is an indirect measurement of how much activity was partitioned to the pellet. “Percentage precipitated” was defined in a similar way to account for polymer or &amylase salted-out by ammonium sulfate. Preliminary experiments a t different enzyme/HPMC solution pH exhibited little effect of this variable on the partition. Therefore, pH 6.0 was selected for all other experiments. Experiments were carried out a t room temperature (21 “C), unless specified otherwise. The percentage of saturation a t 21 OC was used as the unit of concentration for ammonium sulfate in solution. Its solubility at this temperature is 757 g/L. Small-scale screening experiments were conducted in 1.5-mL Eppendorf tubes, using 500 pL of @-amylasepreparation and 500 pL of HPMC or MC solution at 2-fold the final desired concentration. After homogenization in a vortex for 10 s, the appropriate volume of saturated ammonium sulfate solution was added and the contents of the tubes were mixed again. After 10 min, the mixture was centrifuged at 12 000 rpm for 10 min in a microcentrifuge, Eppendorf Model 5415. The supernatant was separated from the pellet and filtered through a Milli-
215
pore Millex HV 0.45-pm filter. The filtrate was used for enzyme activity and total carbohydrates assays. To check if the enzyme could partition to the gel after its formation, a pellet of MC was prepared from 1 mL of 0.4 5% w/v MC solution by adding ammonium sulfate saturated solution up to 30% saturation. The pellet was washed twice with 1 mL of the salt solution a t 30% saturation, and then 1430 pL of prepurified @-amylasea t 30% salt saturation was added to it. The tube was shaken for 30 s and let stand at room temperature for 10 min, and after centrifugation the supernatant was filtrated and analyzed for activity. The possibility of separating the enzyme from the gel was checked by both HPMC reprecipitation and a backpartition experiment. In the first case, a pellet obtained by stirred batch partition (to be described later) of maltose broth (concentration ratio of 7) was dissolved in 100 mL of acetate buffer and the HPMC reprecipitated by ammonium sulfate a t 21 and 56 “C. The supernatant was separated by centrifugation and anaiyzed for enzyme activity and total carbohydrates. Back-partition experiments were selective extraction of the enzyme from a 6-amylase-containing pellet to solutions of different salt concentrations. P-Amylasecontaining pellets were prepared by adding 430 p L of saturated solution of ammonium sulfate to 500 pL of 1 % MC solution, mixing, and then adding 500 pL of enzyme solution. After 15 min under gentle mixing, the pellets were formed by centrifugation. The pellets were placed in ammonium sulfate solutions of different concentrations, called “equilibrium solutions”. Tubes containing the mixtures were shaken for 30 s and allowed to stand at room temperature overnight. The supernatant was centrifuged and analyzed for activity and total carbohydrates. To verify the importance of hydrophobic interaction on the partition process, experiments were performed with a protein well-known by its hydrophobicity, bovine albumin. The experimental conditions were 30% ammonium sulfate saturation and 0.2% w/v HPMC (loading of 10 mg of polymer to 1 mg of protein). Stirred batch experiments were done with maltose and starch broths with concentration ratios ranging from 1-17. HPMC and ammonium sulfate concentrations were 0.2% and 30% saturation, respectively. The broth pH was in the range of 5.1-5.2, and little change was observed after addition of HPMC. The experiments were carried out in a 50-mL beaker at room temperature, unless otherwise specified. Broth and 1% HPMC aliquots, 20 and 5 mL, respectively, were homogenized for 5 min. A 10.7-mL amount of ammonium sulfate saturated solution was then added dropwise, and mixing was continued for more 10 min. The gellike phase formed was separated from the supernatant by centrifugation a t 8000 rpm for 20 min (Sorval centrifuge Model RC5B), and the pellet was dissolved in 100 mL of acetate buffer. This solution was used in electrophoresis after concentration in a 10000 MWCO disc membrane with an Amicon ultrafiltration cell, Model 8010. The same solution was also used for the HPMC recovery study, enzyme activity, total carbohydrates, and protein assays. The volume of the supernatant was measured and the supernatant was used for activity assay. One aliquot of the supernatant was dialyzed, concentrated by ultrafiltration as above, and used for electrophoresis analysis. The effect of temperature on the partition of @-amylase from starch broth was investigated. The order of solution mixing had to be changed and HPMC solution
Biotechnol. Prog., 1990. Vol. 6 , No. 3
218 1101
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Figure 1. Comparison between partition of @-amylase(A),salting-out of @-amylase(O),and salting-outof HPMC ( 0 ) . Enzyme used: crude @-amylasepreparations. Conditions of experiments: enzyme activity = 33.0-80 units/mL; protein concentration = 0.7-2.4 mg/mL; HPMC concentration = 0.2-0.005% (w/v).
added a t 21 "C because HPMC spontaneously precipitates a t high temperatures (Greminger and Krumel, 1980). First, starch broth and ammonium sulfate solutions were equilibrated a t the desired experimental temperature and then added one to the other. At last, HPMC solution was added to the broth/salt mixture. Centrifugation was done in a prewarmed rotor. 111. Results 111.1. Experiments with 8-Amylase Preparations. Small-scale screening experiments with @-amylase preparations were performed prior to the experiments with fermentation broths. Addition of ammonium sulfate to the enzyme/cellulose ether solutions, under mixing, initially produced a cloudy suspension that changed to a clear suspension of filaments as the salt concentration was increased. Observation under 200X magnification revealed particles, strands twisted around each other in a helix form, and droplets. Figure 1shows results of the comparison between precipitation by ammonium sulfate and partition as separation techniques for @-amylase.Partition requires approximately 10% less salt saturation (36% less salt) than precipitation. Figure 1shows also that precipitation of HPMC took place a t lower salt concentrations than @-amylase partition and precipitation. Partition of the enzyme after formation of the gel phase was tested by contacting the enzyme solution a t 30% ammonium sulfate saturation with the MC pellet. Only 9 70 of the initial total activity remained in the supernatant. If only precipitation of the enzyme (salting-out) had occurred, the remaining activity would be 21% (Figure 1). Partition experiments with bovine albumin showed lower protein recovery than activity recovery for @-amylase.No more than 30% of the protein was present in the gel phase. Comparison of the two polymers, MC and HPMC, as partitioning agents at a concentration of 0.2% showed that they are equally effective in partitioning ,&amylase a t 30% ammonium sulfate saturation (98% partition). However, HPMC was more effective a t 20% salt saturation, giving 58% partition versus 34% for MC. The effect of HPMC loading (mass of polymer per mass of protein) on partition was evaluated, and the result is
0.0
0.5
1.0
1.5
2.0
HPMC loading (mg HPMC/mg protein)
Figure 2. HPMC loading for maximum partition. Enzyme used: crude @-amylasepreparation. Conditions of the experiments: enzyme activity = 36.7 units/mL; protein concentration = 1.2 mg/mL; ammonium sulfate concentration = 30% saturation. Bars indicate standard deviation (n = 3).
shown in Figure 2. A minimum of 0.2 mg of HPMC/mg of protein was required for maximum partition. At the protein concentration used (1.2 mg/mL), this loading corresponds to a HPMC concentration of 0.024% w/v. Table I displays the results of the experiments made with HPMC of different nominal molecular weights. The HPMC with the nominal molecular weight of 85 000 produced the highest partition among the three polymers studied. Resolubilization of the enzyme from the gel phase by back-partition with little polymer dissolution (average of 2.1% with standard deviation of 0.7%) was possible, as shown by Figure 3. Reprecipitation of HPMC a t two different temperatures, 2 1 and 56 "C, separated a t least 90% of the cellulose ether along with only 5 % of the total enzyme activity (Figure 4). However, only 5% ammonium sulfate saturation was required a t 56 "C against 15% a t 21 "C. This is the result of the shift of the partition and precipitation curves to the left by the increase in the temperature. Note that at 56 "C the partition and precipitation curves are more separated from each other than at 21 "C. 111.2. Experiments with Fermentation Broths. Stirred batch partition experiments were carried out with both types of broth at various levels of concentration (Table 11), and no @-amylasedenaturation was observed. The behavior of maltose broth was the same as that in the small-scale experiments, with practically 100% of the enzyme activity recovered with the pellets. @-Amylase from starch broth was not efficiently partitioned, with only an average of 29% of the activity found in the pellets a t most (concentration ratio of 3). Partition of the @-amylasefrom maltose broth achieved different purification, according to the broth ultrafiltration (Figure 5). The purification for the maltose broth with a 17-fold concentration ratio was qualitatively confirmed by SDS-polyacrylamide gel electrophoresis. Temperature increases improved enzyme partition from starch broth (Figure 6). Even though high levels of partition were not achieved, 25% of @-amylaseactivity was partitioned a t 75 "C while it was almost negligible at 2 1 "C (concentration ratio of 7). The maximum enzyme denaturation was around 10% at 75 "C.
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Biotechnol. Prog., 1990, Vol. 6, No. 3
Table I. Partition Dependence on the HPMC Nominal Molecular Weight. HPMC nominal percentage partitioned molec w t treatment 1 treatment 2 avb 15 OOO 85 OOO 250 000
66 85 75
68 85 72
63 85 78
a Enzyme used, crude preparation. Conditions of experiments: HPMC concentration = 0.2% (w/v); HPMC loading = 0.7; ammonium sulfate concentration = 25% saturation. * av is the average of treatments 1 and 2.
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Figure 3. Back-partition of @-amylase. @-Amylase-containing pellets were obtained by adding enzyme solution to MC pellets. These pellets were prepared with 1.0% MC solution and ammonium sulfate at 30% saturation.
'0 5 10 15 20 25 30 Ammonium sulfate concentration (Percentage of saturation)
Figure 4. Separation of @-amylasefrom HPMC. &Amylase partition (circles) and HPMC precipitation (triangles) at 21 OC (open symbols) and at 56 "C (closed symbols) from dissolved 8-amylase pellet. Pellet obtained by @-amylasepartition from maltose broth (17-fold concentrated) with 0.2% (w/v) HPMC and 30% ammonium sulfate saturation.
IV.Discussion Partition of @-amylaseto the gel phase formed by HPMC is a very efficient separation process. Yields of practically 100% were obtained with a purification ratio up to 100-fold. Recovery of the same enzyme by precipitation with ethanol, ammonium sulfate, and a combination of both precipitations gave yields in the range of 90-9596 and a purification ratio around 30-fold at most (Elankovan, 1990). Separation of the HPMC and the enzyme
from each other is accomplished by precipitation of the polymer at an ammonium sulfate concentration where only the enzyme is soluble. This also enables recovery of HPMC for reuse. HPMC loading required for maximum partition is low (0.2 mg of HPMC/mg of protein), which avoids the problem of high solution viscosity. Moreover, the salt concentration needed for partition is lower than conventional salting-out. It was shown that a temperature increase can further reduce the salt concentration needed for partition and enzyme/HPMC separation. Ninety percent of @-amylasecan be partitioned with a 20% saturated salt solution at 56 OC. This represents a 75% reduction in the salt requirement as compared with the optimum salting-out concentration of 50% saturation recommended by Elankovan (1990). Clearly, this advantage can only be realized in the recovery of thermophilic enzymes. Partition depends on the fermentation carbon source similar to the way that salting-out does: the enzyme produced with maltose separates well while the one produced with starch does not. Elankovan (1989)attributed this poor separation to binding of the @-amylaseto starch. The same phenomenon seems to affect partition. The significant partition at higher temperatures of @-amylaseproduced with starch is probably due to a higher starch hydrolysis rate. Molecules of enzyme could be free of the starch moiety and interact with HPMC. This possible competition between starch and HPMC by the enzyme, the fact that these polysaccharides have similar backbones, and the relative high purification obtained by partition, leads us to believe that there is a specificity in the partition process. This idea is supported by a companion work where two other amylases (an a-amylase and a pullulanase) were also partitioned by the HPMC gel phase while three non-amylase proteins (a glucose isomerase, a bovine albumin, and the B sub-unit of Escherichia coli toxin) were not. The interaction between HPMC and amylases may mimic amylases binding to starch. The partition does not seem to be a simple phenomenon. The gellike phase is, in fact, composed of three phases (particles, filaments, and droplets). Due to the use of a nonionic polysaccharide (HPMC) and high salt concentration, this partition method could potentially be controlled by hydrophobic interaction. Such interaction is known to be the mechanism responsible for protein binding to some polysaccharide columns, such as Sepharose 4B and cellulose, at high salt concentrations (Arakawa, 1986). However, the results of bovine albumin partition dismissed this possibility by the low recovery obtained. @-Amylasepartition takes place at ammonium sulfate concentrations intermediate to the concentration necessary to precipitate the enzyme and HPMC from their respective solutions. We see two possible explanations for this. First, it may be that HPMC and @-amylaseinteract with each other and form a complex that has solubility decreased by salt addition. Second, the enzyme may partition to the gel phase only after its formation. The larger shift to the left of the HPMC insolubilization curve than the shift of the @-amylasepartition at higher temperature supports the first idea. The better behavior of HPMC in comparison to MC at 20% salt saturation may be explained by the difference in methoxyl degree of substitution of HPMC (in the range of 1.1-1.4)and MC (in the range of 1.6-1.9), since it is known that a degree of substitution lower than 1.4 yields products with low water solubility (Greminger and Krumel, 1980).
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Table 11. Partition as a Function of Fermentation Substrate and Concentration Ratio. total activitv,b % pellet fermentn substrate concn ratio Tlc T2c avC TI maltose 1 95 97 96 8 2 108 108 108 5 3 93 94 94 5 7 98 98 98 5 17 107 108 108 5 starch 1 20 8 14 78 2 10 20 15 91 3 26 32 29 82 12 9 82 7 6 17 9 5 7 93 ~~
~~~
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supernatant T2 9 6 5 4 4 85 93 77 90 98
av 9 6 5 5 5 82 92 80 86 96
4 Conditions of experiments: enzyme activities = 5.0-29.2 units/mL and 13.1-127.5 units/mL for maltose and starch broth, respectively; protein concentration = 2.4-2.6 mg/mL for maltose broth (loading from 0.78 to 0.83 mg of HPMC/mg of protein); HPMC concentration = 0.2% (w/v); ammonium sulfate concentration = 30% saturation. Compared to total initial activity. T1, T2, and av means treatment 1, treatment 2, and average, respectively.
in Table I, it seems that there is an optimum HPMC molecular weight for the @-amylasepartition.
V. Conclusion A process for recovering C. thermosulfurogenes @-amylase from maltose fermentation broth was developed. It makes use of low quantities of a food-grade polymer and requires lower concentrations of ammonium sulfate than conventional salting-out. Therefore, it is suitable for largescale processing due to low chemical consumption, high recovery and high purity of the final product, and low waste generation. Its specificity can also be extended to other amylases.
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Figure 5. 0-Amylase purification achieved by partition from maltose broth with 0.2% w/v HPMC and 30% ammonium sulfate saturation as function of broth concentration. lOo"'--'''~"
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Acknowledgment We acknowledge the Michigan Biotechnology Institute for financial support and for use of its facilities, the Conselho Nacional de Desenvolvimento Cientifico & Tecnolbgico, CNPq,Brazil, forsupp0rtingE.A.M. withaschoiarship, and the Dow Chemical Co. for donating the METHOCEL products.
70
Temperature ("C)
F i g u r e 6. Effect of temperature increase in @-amylasepartition from starch broth (7-fold concentrated): (A)pellet; (0) supernatant; (A) total recovery. Conditions of experiments: HPMC concentration = 0.2% (w/v); ammonium sulfate concentration = 30% saturation.
The nominal molecular weight of HPMC influences the partition of @-amylase,contrary to what was expected since it is known that gelation and precipitation properties for commercial HPMC have little dependence on this variable (Sarkar, 1979). On the basis of the results shown
Literature Cited Albertsson, P.-A. Partition of Cell Particles and Macromolecules; John Wiley and Sons: New York, 1986; p 346. Arakawa, T. Thermodynamic analysis of the effect of concentrated salts on protein interaction with hydrophobic and polysaccharide columns. Arch. Biochem. Biophys. 1986,248 (l), 101-105. Bernfeld, P. Amylases CY and @. Methods Entymol. 1955, 1, 149-150. Dove, G. B.; Mitra, G. Recovery of proteins from polyethylene glycol-water solution by salt partition. ACS Symp. Ser. 1986, 314,93-108. Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A,; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956,28 (3), 350-356. Elankovan, P. Ph.D. Dissertation, Michigan State University, East Lansing, MI, 1990. Ganz, A. J. In Food Colloids; Graham, H. D., Ed.; AVI: Westport, CT, 1977; pp 383-417. Greminger, G. K.; Krumel, K. L. In Handbook of Water Soluble Gums and Resins; Davidson: R. L., Ed.; McGraw-Hill: New York, 1980; p p 3/1-3/25. Hyun, H. H.; Zeikus, J. G. General biochemical characterization of thermostable extracellular @-amylasefrom ClostridEnviron. Microbiol. 1985a, ium thermosulfurogenes. Appl. .. 49 (5), 1162-1167. Hvun. H. H.: Zeikus. J. G. Regulation and genetic enhancement of glucoamylase a n i pullulanase production in Clostridium thermohydrosulfuricum. J . Bacteriol. 1985b, 164 (3), 1146-1152.
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Kroner, K. H.; Hustedt, H.; Kula, M.-R. Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale. Biotechnol. Bioeng. 1982,24,1015-1045. Kula, Ma-R.;Kroner, K. H.; Hustedt, H. Advances in Biochemical Engineering, Reaction Engineering; Springer-Verlag: New York, 1982;pp 73-117. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970,227, 680-685. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 1951,193, 265-275.
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Saha, B. C.; LeCureux, L. W.; Zeikus, J. G. Raw starch adsorption-desorption of a thermostable @-amylasefrom Clostridium thermosulfurogenes. Anal. Biochem. 1988, 175, 569572. Sarkar, N. Thermal gelation properties of methyl and hydroxypropyl methylcellulose. J . Appl. Polym. Sci. 1979,24,10731087. Shen, G.-J.; Saha, B. C.; Lee, Y.-E.; Bhatnagar, L.; Zeikus, J. G. Purification and characterization of a novel thermostable @-amylasefrom Clostridium thermosulfurogenes. Biochem. J. 1988,254,835-840. Registry No. p-Amylase, 9000-91-3; (hydroxypropy1)methylcellulose, 9004-65-3.
