Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

8
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2614-2621 Vol. 59, No. 8 0099-2240/93/082614-08$02.00/0 Copyright © 1993, American Society for Microbiology Characterization of Amylolytic Enzymes, Having Both ox-1,4 and ao-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis STEPHEN H. BROWNt AND ROBERT M. KELLYt* Center of Marine Biotechnology, University of Maryland, Baltimore, Maryland 21202, and Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218 Received 4 November 1992/Accepted 29 May 1993 Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcusfuriosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca2", and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca2+ also affected substrate binding, as evidenced by a decrease in Km for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the a-1,6 linkages in pullulan, a-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller a-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopuliulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide- metabolizing systems possessed by these two organisms. A characteristic of a number of extremely thermophilic, anaerobic heterotrophic archaea is the capability to utilize complex saccharolytic substrates, such as starch and glyco- gen, as carbon and energy sources. Pyrococcus furiosus (10), a marine hyperthermophile which grows optimally at 98°C, is probably the best-characterized member of the thermophilic archaea. P. furiosus grows heterotrophically by metabolizing a variety of complex substrates (3, 10). The metabolism of these substrates begins with their hydrolysis by a series of extracellular amylolytic enzymes, comprising amylase and pullulanase activities, which were first de- scribed in crude form (4). Subsequently, highly thermostable amylases possessing hydrolytic activity against a-1,4 link- ages in starch, amylose and other polysaccharides have been purified from P. furiosus (16) and a related organism, Pyro- coccus woesei (15). The hydrolysis products resulting from the action of extracellular hydrolases are transported into the cell by an as yet uncharacterized mechanism, where they are acted upon by an intracellular a-glucosidase (7), produc- ing glucose, which feeds into a modified version of the Entner-Doudoroff pathway termed pyroglycolysis, proposed by Mukund and Adams (23) and substantiated by Schafer and Schonheit (34). This pathway is centered around a tungsten-based acetaldehyde oxidoreductase (23) and, as a result, is actuated in the presence of sufficient levels of tungsten in the growth medium (35). This organism, which can also utilize peptides as carbon and energy sources, apparently prefers saccharides, given the significant de- * Corresponding author. t Present address: Novo-Nordisk Biotech, Inc., Davis, CA 95616- 4880. t Present address: Department of Chemical Engineering, Campus Box 7905, North Carolina State University, Raleigh, NC 27695- 7905. crease in expressed proteolytic activity in the presence of saccharides (35). The ability to digest complex saccharides is common to several heterotrophic extreme thermophiles. For example, Thermococcus litoralis is an extremely thermophilic marine archaebacterium with an optimal growth temperature of 90°C (1, 25). It resembles P. furiosus in many aspects of growth and metabolism, although it has not been as well characterized. We have determined that T. litoralis produces a series of amylolytic enzymes in response to the presence of a-1,4-linked saccharides in the growth medium, much as P. furiosus does (4a). Presumably, these enzymes perform the same functions as the Pyrococcus enzymes, generating smaller saccharides which are consequently metabolized to meet carbon and energy requirements, perhaps by a series of reactions resembling the pyroglycolytic pathway. In addition to their importance in understanding the phys- iological and bioenergetic features of high-temperature mi- croorganisms, amylolytic enzymes with high levels of ther- mostability have technological importance. The utilization of microbial amylolytic enzymes in the production of sweeten- ers from starch has allowed the development of processes which produce saccharide syrups of high quality in relatively high yields (11). Several steps in these processes are per- formed at elevated temperatures, necessitated by the poor solubility of starch at low temperatures and the desire to avoid microbial contamination. Because starch is composed of glucose units joined by both a-1,4 and a-1,6 linkages, a number of enzymes with differing substrate specificities are required to complete the hydrolysis. The need to identify starch-hydrolyzing enzymes with increased thermal stability and, perhaps, new substrate hydrolysis patterns has led researchers to screen thermo- philic bacteria for the production of enzymes with these characteristics. One especially interesting development has 2614

Transcript of Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

Page 1: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2614-2621 Vol. 59, No. 80099-2240/93/082614-08$02.00/0Copyright © 1993, American Society for Microbiology

Characterization of Amylolytic Enzymes, Having Both ox-1,4and ao-1,6 Hydrolytic Activity, from the Thermophilic

Archaea Pyrococcus furiosus and Thermococcus litoralisSTEPHEN H. BROWNt AND ROBERT M. KELLYt*

Center ofMarine Biotechnology, University ofMaryland, Baltimore, Maryland 21202, and Department ofChemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218

Received 4 November 1992/Accepted 29 May 1993

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilicarchaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcusfuriosus (optimal growthtemperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da byelectrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the sameconditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and aretherefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in thepresence of 5 mM Ca2", and under these conditions, enzyme activity could be measured at temperatures of upto 130 to 140°C. The addition ofCa2+ also affected substrate binding, as evidenced by a decrease in Km for bothenzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in additionto the a-1,6 linkages in pullulan, a-1,4 linkages in amylose and soluble starch. Neither enzyme possessedactivity against maltohexaose or other smaller a-1,4-linked oligosaccharides. The enzymes from T. litoralis andP. furiosus appear to represent highly thermostable amylopuliulanases, versions of which have been isolatedfrom less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.

A characteristic of a number of extremely thermophilic,anaerobic heterotrophic archaea is the capability to utilizecomplex saccharolytic substrates, such as starch and glyco-gen, as carbon and energy sources. Pyrococcus furiosus(10), a marine hyperthermophile which grows optimally at98°C, is probably the best-characterized member of thethermophilic archaea. P. furiosus grows heterotrophically bymetabolizing a variety of complex substrates (3, 10). Themetabolism of these substrates begins with their hydrolysisby a series of extracellular amylolytic enzymes, comprisingamylase and pullulanase activities, which were first de-scribed in crude form (4). Subsequently, highly thermostableamylases possessing hydrolytic activity against a-1,4 link-ages in starch, amylose and other polysaccharides have beenpurified from P. furiosus (16) and a related organism, Pyro-coccus woesei (15). The hydrolysis products resulting fromthe action of extracellular hydrolases are transported intothe cell by an as yet uncharacterized mechanism, where theyare acted upon by an intracellular a-glucosidase (7), produc-ing glucose, which feeds into a modified version of theEntner-Doudoroff pathway termed pyroglycolysis, proposedby Mukund and Adams (23) and substantiated by Schaferand Schonheit (34). This pathway is centered around atungsten-based acetaldehyde oxidoreductase (23) and, as aresult, is actuated in the presence of sufficient levels oftungsten in the growth medium (35). This organism, whichcan also utilize peptides as carbon and energy sources,apparently prefers saccharides, given the significant de-

* Corresponding author.t Present address: Novo-Nordisk Biotech, Inc., Davis, CA 95616-

4880.t Present address: Department of Chemical Engineering, Campus

Box 7905, North Carolina State University, Raleigh, NC 27695-7905.

crease in expressed proteolytic activity in the presence ofsaccharides (35).The ability to digest complex saccharides is common to

several heterotrophic extreme thermophiles. For example,Thermococcus litoralis is an extremely thermophilic marinearchaebacterium with an optimal growth temperature of90°C (1, 25). It resembles P. furiosus in many aspects ofgrowth and metabolism, although it has not been as wellcharacterized. We have determined that T. litoralis producesa series of amylolytic enzymes in response to the presence ofa-1,4-linked saccharides in the growth medium, much as P.furiosus does (4a). Presumably, these enzymes perform thesame functions as the Pyrococcus enzymes, generatingsmaller saccharides which are consequently metabolized tomeet carbon and energy requirements, perhaps by a series ofreactions resembling the pyroglycolytic pathway.

In addition to their importance in understanding the phys-iological and bioenergetic features of high-temperature mi-croorganisms, amylolytic enzymes with high levels of ther-mostability have technological importance. The utilization ofmicrobial amylolytic enzymes in the production of sweeten-ers from starch has allowed the development of processeswhich produce saccharide syrups of high quality in relativelyhigh yields (11). Several steps in these processes are per-formed at elevated temperatures, necessitated by the poorsolubility of starch at low temperatures and the desire toavoid microbial contamination. Because starch is composedof glucose units joined by both a-1,4 and a-1,6 linkages, anumber of enzymes with differing substrate specificities arerequired to complete the hydrolysis.The need to identify starch-hydrolyzing enzymes with

increased thermal stability and, perhaps, new substratehydrolysis patterns has led researchers to screen thermo-philic bacteria for the production of enzymes with thesecharacteristics. One especially interesting development has

2614

Page 2: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

AMYLOLYTIC ENZYMES FROM THERMOPHILIC ARCHAEA 2615

been the discovery of individual enzymes which are able tohydrolyze both a-1,6 linkages in pullulan and starch ando-1,4 linkages in starch. Since these enzymes have bothamylase and pullulanase activities, the term amylopullula-nase has been coined to categorize them (33). Amylopullu-lanases are produced by a number of moderately thermo-philic bacterial species, such as Bacillus sp. strain 3183 (32),Thennus aquaticus (29), Thermus sp. strain AMD33 (24),Clostridium thermohydrosulfuricum (20, 21, 31), Clostridiumthermosulfurogenes EM1 (37), Thermoanaerobium sp.

strain Tok6-B1 (27), and Thermoanaerobacter sp. strainB6A (30), and the industrial potential of some of theseenzymes is under evaluation (33).

Reported here are the purification and characterization ofthe enzyme responsible for pullulan hydrolysis by crudeenzyme preparations of P. furiosus. In addition to its hydro-lytic activity against the a-1,6 linkages in pullulan, thispullulanase can attack a-1,4 linkages in starch and amylose.A similar enzyme has been purified from T. litoralis, and thecharacteristics of this enzyme are also described. The dualsubstrate specificity of these enzymes indicates that theybelong to the class of amylolytic enzymes termed amylopul-lulanases (described above), which have been found previ-ously in association with less-thermophilic organisms. Theextremely high level of thermostability exhibited by theseenzymes, coupled with their ability to attack both types ofthe glycosidic linkages found in starch, may lead to improve-ments in the industrial starch hydrolysis process. The de-scription of these activities in two thermophilic marinearchaea is further evidence of the significance of polysac-charide hydrolysis to the metabolism of bacteria from ex-

treme thermal environments.

MATERIALS AND METHODS

Bacterial strains and culture conditions. P. furiosus DSM3638 and T. litoralis DSM 5473 were obtained from theDeutsche Sammlung von Mikroorganismen, Braunschweig,Federal Republic of Germany. Cells were cultured in an

artificial seawater-based medium supplemented with 0.1%yeast extract and 0.5% tryptone, with 0.4% of a commercialmaltooligosaccharide mixture added as an inducer. Artificialseawater was modified from the formulation of Kester et al.(13) and contained (per liter): NaCl, 15.0 g; Na2SO4, 2.0 g;MgCl2- 6H20, 2.0 g; CaCl2- 2H20, 0.50 g; NaHCO3, 0.25 g;K2HPO4, 0.10 g; KBr, 50 mg; H3HO3, 20 mg; KI, 20 mg;Fe(NH4)2(SO4)2, 15 mg; Na2WO4 2H20, 3 mg; andNiCl2 6H20, 2 mg. Carbohydrate and phosphate were

added after sterilization. Inocula were routinely grown inclosed bottles (125 ml to 2 liters), and anaerobic conditionswere obtained by heating the medium to 98°C in an oil-filledbath for 30 to 90 min, sparging with nitrogen, and addingNa2S .9H20 (0.5 g/liter) from a 50-g/liter stock solution.Prior to inoculation, cultures were cooled to either 85°C (T.litoralis) or 90°C (P. furiosus).

Large-scale (350-liter) cultures were grown in a NewBrunswick Scientific fermentor under nitrogen sparge (ap-proximately 5 liter/min) at 250 rpm. The temperature was

maintained at 85 + 2°C (T. litoralis) or 90 + 2°C (P. furiosus)by manual addition of steam to the jacket. Titanium(III)nitrilotriacetate (22) (final concentration, 50 p,M) was used as

the reductant for large-scale fermentations. Growth was

monitored by epifluorescence microscopy with acridine or-

ange stain, as well as by measuring the optical density at 600nm (OD16w). Fermentations were stopped in the late expo-nential phase by cooling to 7 to 10°C. Cell densities of about

108/ml were routinely achieved with P. furiosus (OD6w, 0.4),while densities of 6 x 108 to 8 x 108/ml were commonlyfound with T. litoralis (OD6w, 1.0). Cells were harvestedwith a Sharples centrifuge.

Cell supernatants were concentrated from 350 liters toabout 2 liters with a Pellicon ultrafiltration system (MilliporeCorp., Bedford, Mass.) with a 30,000-Da cutoff polysulfonemembrane and further concentrated to approximately 500 mlwith a Minitan ultrafiltration system (Millipore) equippedwith a 30,000-Da cutoff polysulfone membrane. During thislast concentration step, both enzyme preparations werediafiltered with 50 mM Tris-HCl, pH 8.0, containing 0.04%NaN3. After diafiltering, concentrated supernatants werefilter sterilized and stored at room temperature to protectagainst potential cold denaturation, although, subsequently,no evidence for cold denaturation was found.Enzyme purification. All enzyme purification procedures

were performed at room temperature under aerobic condi-tions unless otherwise stated. Chromotography columnswere controlled with a fast protein liquid chromatography(FPLC) system (Pharmacia, Uppsala, Sweden). Both the T.litoralis and P. furiosus enzymes were purified by essentiallythe same procedure.Each concentrated supernatant was applied to a column (5

by 22 cm) of Q Sepharose Fast Flow (Pharmacia) preequil-ibrated with 50 mM Tris-HCl, pH 8.0. The column waswashed with the same buffer and eluted with 5 bed volumesof a linear NaCl salt gradient (0 to 1.0 M) in the same buffer.Fractions containing pullulanase activity were pooled,0.04% NaN3 was added, and the preparation was stored atroom temperature. K2HPO4 was added to pooled fractionsto a concentration of 5 mM, and this material was applied toa column (5 by 22 cm) containing ceramic hydroxyapatite(American International Products, Natick, Mass.), previ-ously equilibrated with 50 mM Tris-HCl, pH 8.0, containing5 mM K2HPO4 and 0.04% NaN3. The column was washedwith 2 bed volumes of the same buffer, and adsorbed enzymewas eluted with 5 bed volumes of a linear gradient of 0 to 0.5M K2HPO4 in 50mM Tris-HCl, pH 8.0. Fractions containingpullulanase activity were pooled, concentrated to 20 to 30ml, and dialyzed overnight against 50 mM acetate-aceticacid buffer, pH 5.6, containing 0.15 M NaCl and 0.04%NaN3; this was done to potentially increase the binding ofthe enzyme to the affinity system used in the subsequentstep.

a-Cyclodextrin was previously demonstrated to be a com-petitive inhibitor of P. furiosus pullulanase activity (4) andwas also found to inhibit T. litoralis pullulanase activity (datanot shown). This material was used as an affinity ligand andwas coupled to Toyopearl AF-Epoxy-650 M resin (Toso-Haas, Philadelphia, Pa.) by the following procedure assuggested by the manufacturer. a-Cyclodextrin (4.5 g) wasdissolved in 80 ml of 0.1 M NaOH, and 5 g of hydrated resinwas added. This mixture was gently agitated by rocking at45°C for 16 h. The resin was washed with, in succession,distilled water, 1 M NaCl, and distilled water. Residualepoxy groups were blocked by incubation with 1 M ethanol-amine for 12 h at room temperature. The resin was washedthoroughly with distilled water and packed into a column(1.6 by 10 cm).

This a-cyclodextrin affinity column was washed with 50mM acetate buffer, pH 5.6, containing 0.15 M NaCl and0.04% NaN3, and the dialyzed material from the hydroxyap-atite column was applied (in several portions). The columnwas washed with 1 bed volume of the same buffer and againwith the acetate buffer supplemented to 1 M NaCl. Enzyme

VOL. 59, 1993

Page 3: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

2616 BROWN AND KELLY

was eluted with 50 mM acetate buffer (pH 5.6)-0.15 MNaCl-0.04% NaN3 containing 0.5% a-cyclodextrin. Frac-tions containing pullulanase activity were pooled, concen-trated approximately fivefold with an Amicon stirred cellwith a YM 30 membrane (Amicon, Beverly, Mass.), anddialyzed overnight at room temperature in 50 mM Tris-HCl,pH 8.0, containing 0.15 M NaCl and 0.04% NaN3.

Dialyzed activity pools from the a-cyclodextrin columnwere applied in 0.5-ml portions to a HiLoad 16/60 Superdex200 preparation grade gel filtration column (Pharmacia) andeluted with the same buffer used for dialysis. Active frac-tions were pooled, dialyzed against 50 mM Tris-HCl, pH 8.0,containing 0.04% NaN3, sterile filtered, and stored at roomtemperature. Portions of each enzyme were dialyzed for 48 hagainst 50 mM Tris-HCl, pH 8.0, containing 1 mM EDTAand 0.04% NaN3 and then against buffer without EDTA (twochanges). The EDTA-treated enzyme samples were used instudies on the effect of metal ions as described below.

Determination of enzyme activity. Amylase and pullulanaseactivities were determined by measuring the amount ofreducing sugar released during incubation with starch andpullulan, respectively. Soluble starch (2%) was autoclavedfor 10 min prior to use in enzyme assays to facilitatedissolution. The substrate, 0.5 ml of 2% soluble starch or 2%pullulan, was added to 0.5 ml of 0.1 M acetate buffer, pH 5.6.From 10 to 100 ,lI of enzyme solution was added, and thesamples were incubated at 98°C for 30 min. The reaction wasstopped by cooling the mixture on ice, and the amount ofreducing sugar released was determined by the dinitrosali-cylic acid method (2). Sample blanks were used to correctfor nonenzymatic release of reducing sugar. The effect ofcalcium on enzyme activity was determined by adding 5 mMCa2+ to the assay mixture. One unit of enzyme activity wasdefined as the amount of enzyme that released 1 ,umol ofreducing sugar (as glucose standard) per min under the assayconditions specified. Enzyme samples were diluted appro-priately to maintain linearity over the assay interval used.Amylase and pullulanase assays conducted at high tem-

peratures (>100°C) were performed in sealed, stirred 1-mlReacti-Vials with a Reacti-Therm heating-stirring module(Pierce, Rockford, Ill.). After the reaction mixture hadreached the assay temperature, enzyme was injectedthrough a resealable Tuf-bond Teflon-silicone disc. The vialswere held at the assay temperature for 30 min and cooled inan ice bath with stirring before determination of reducingsugar released.The effect of pH on enzyme activity was determined (with

pullulan as the substrate) by following the above protocolwith substitution of the appropriate buffer and in the absenceof added metal. For a pH range of 4.0 to 5.6, 0.1 M acetatebuffer was used, while 0.1 M sodium phosphate buffer wasused in the pH range of 5.7 to 8.0.Enzyme activity assays were also conducted with the

artificial substrates 4-nitrophenyl-a-D-maltotrioside (PG3)and 4-nitrophenyl-a-D-maltoheptoside (PG7). In this case,enzyme activity was measured by the release ofp-nitrophe-nol (PNP) in a heated spectrophotometer system which hasbeen described previously (7). In order to allow assays attemperatures of up to 120°C, an 80:20 mix of triethyleneglycol-water was substituted for the 50:50 ethylene glycol-water mixture used earlier. Sample blanks were used tocorrect for nonenzymatic release of PNP. One unit ofenzyme activity was defined as the amount of enzymereleasing 1 ,umol of PNP per min under the specified assayconditions. The effect of metals on the rate of PNP releasewas determined by adding 5 mM Mg2+, 5 mM Ca2+, or 5 mM

Sr"9 to the reaction mixture prior to enzyme addition. TheMichaelis-Menten constants Km and Vinxwere measured at98°C in the absence or presence of 5 mM Ca2+ by performingthe PNP-linked assay with decreasing concentrations ofsubstrate. Parameters were calculated from initial rate datawith the Enzfitter software package (Elsevier-BIOSOFT,Cambridge, United Kingdom).

This system was also used to measure the half-lives ofboth enzymes at 120°C in the absence and presence of themetals listed above. The substrate solution was heated to120°C, and enzyme was added to the cuvette through arubber septum. Absorbance readings were taken by com-puter every 5 s after addition, and the absorbance-versus-time data were fit to a first-order decay model to allowcalculation of the decay constants.Other techniques. The products released through hydroly-

sis of starch, pullulan, glycogen, and various oligosaccha-rides by each enzyme were determined by thin-layer chro-motography. Enzyme samples were incubated in thepresence of each substrate in 0.1 M acetate buffer, pH 5.6, at90°C for 24 h, and products were analyzed by the method ofHansen (12).Sodium dodecyl sulfate-polyacrylamide gel electrophore-

sis (SDS-PAGE) of protein samples was conducted with thediscontinuous system of Laemmli (18). Samples were pre-pared for SDS-PAGE by boiling for 4 min in the presence of1% SDS, 80 mM dithiothreitol, and 100 mM Tris-HCI (pH7.5). Proteins were visualized by staining with 0.18% amidoblack-0.004% Coomassie brilliant blue in a 35% metha-nol-7% acetic acid solution. Molecular masses were esti-mated with standard markers (Life Technologies, Inc.,Gaithersburg, Md.). Glycoproteins were detected after SDS-PAGE by the periodic acid-Schiff method (9). Protein con-centrations were measured with the Pierce Coomassie re-agent, with bovine serum albumin (Pierce) as the standard.

Chemicals. Soluble starch, pullulan, glycogen, a-cyclo-dextrin, and Schiff reagent were obtained from Sigma (St.Louis, Mo.). The maltooligosaccharide mixture was ob-tained from Pfanstiehl Laboratories, Waukegan, Ill. PG3,PG7, and other oligosaccharides were supplied by Boehr-inger Mannheim, Indianapolis, Ind. Tryptone and yeastextract were obtained from Marcor Development Corp.,Hackensack, N.J. All other chemicals were of reagent grade.

RESULTS

Enzyme purification. T. litoralis and P. furiosus bothproduce an extracellular pullulan-hydrolyzing activity whengrown in the presence of a-1,4-linked oligosaccharides andpolysaccharides. A commercially available maltooligosac-charide mixture, consisting of maltotriose through maltode-caose, was used as the inducer for large-scale enzymeproduction. By using this mixture as an inducer rather thanpolysaccharides such as starch, residual nonmetabolizedsaccharide could be removed by diafiltering the culturesupernatant, decreasing background reducing-sugar levels inenzyme activity assays.The results from the purification of the pullulan-hydrolyz-

ing activities produced by T. litoralis and P. furiosus areshown in Tables 1 and 2, respectively. Both organismsproduced a pullulanase which, when purified, also displayedsignificant hydrolytic activity against starch. In the case of T.litoralis, this enzyme appeared to be the predominant extra-cellular amylolytic enzyme produced under these cultureconditions, as no amylase or pullulanase activities wereobserved in column fractions other than those containing the

APPL. ENvIRON. MICROBIOL.

Page 4: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

AMYLOLYTIC ENZYMES FROM THERMOPHILIC ARCHAEA 2617

TABLE 1. Purification of amylopullulanase from T. litoralis

Step ~~~~Protein Aciiy()Sp act P/A PurificationStep (mg) Activity (U) (U/mg) ratiob factor (fold)

Culture supernatant (from 350 liters) 2,090 2,271 (1,259) 1.09 (0.6) 1.82 1.0 (1.0)Q-Sepharose 330 980 (606) 2.97 (1.8) 1.61 2.7 (3.0)Ceramic hydroxyapatite 200 860 (456) 4.30 (2.3) 1.89 4.0 (4.6)et-Cyclodextrin affinity 8.4 374 (227) 44.53 (27.0) 1.65 41.0 (45.0)Superdex 200 gel filtration 2.04 118 (71) 57.7 (34.8) 1.66 53.0 (58.0)

a Values in parentheses refer to amylase (starch-hydrolyzing) activity.b P/A ratio, pullulanase/amylase activity ratio.

purified enzyme. P. furiosus produces, in addition to theenzyme purified here, an extracellular a-amylase which hasno hydrolytic activity against pullulan (16). However, thisactivity is baseline separated from the pullulanase activity onthe Q-Sepharose ion-exchange column, resulting in the dropin amylase activity after this column shown in Table 2.The pullulanase-to-amylase ratio was essentially constantthroughout the course of the purification for the T. litoralisenzyme, and this behavior was also observed with theenzyme from P. furiosus after the initial ion-exchange col-umn step.Each enzyme was judged to be homogeneous, as they

appeared as single bands on SDS-PAGE. The molecularmass of the T. litoralis was estimated at 119,000 Da bySDS-PAGE and 125,000 Da by gel filtration on the Superdex200 column. The P. furiosus enzyme was found to be 110,000Da by SDS-PAGE and 135,000 Da by gel filtration. Bothenzymes were positively stained by the periodic acid-Schiffmethod, indicating that they contained bound saccharidesand were therefore glycoproteins.Enzyme characterization. The amylopullulanases from P.

fiuriosus and T. litoralis had similar activity-pH profiles, withoptima centered at about pH 5.5 and 20% of maximal activityat pHs 4.3 and 7.8 (data not shown). These results are inagreement with earlier observations of the effect of pH onthe activity of crude pullulanase preparations from P. furio-sus (4).The relative hydrolytic activities of each enzyme towards

starch, pullulan, and glycogen at 98°C are shown in Table 3.Enzyme activities were measured at pH 5.6 in the absenceand presence of 5 mM Ca2". Both enzymes have a higherspecific activity against pullulan than against starch, andglycogen is hydrolyzed relatively slowly. The effect of Ca2"on enzyme activity at this temperature differs depending onthe substrate; the starch-hydrolyzing activity of the T. lito-ralis enzyme is inhibited by about 14%, while the P. funiosusenzyme is inhibited by about 30%. In contrast, the pullulan-hydrolyzing activity of both enzymes is stimulated by about25% in the presence of Ca2". The hydrolytic activity of both

enzymes against glycogen is also slightly inhibited by Ca2"at 98°C.The effect of temperature and Ca2" ions on enzyme

activity is illustrated in Fig. 1 and 2. For both enzymes, thepresence of Ca2' has a significant positive effect on enzymeactivity at temperatures above 120°C, extending the range atwhich activity can be measured up to 130 to 140°C. Thesetemperatures are well above those allowing growth of eitherorganism, and they are also significantly above those pres-ently used in the industrial starch hydrolysis process (11).Model substrate experiments. The activities of the T.

litoralis and P. furiosus enzymes against the model sub-strates PG3 and PG7 versus temperature are shown in Fig. 3and 4. For these substrates, enzyme activities were mea-sured in the absence of added metal and in the presence of 5mM Ca2 , Mg2+, or Sr2'. Magnesium and strontium werechosen because they represent the next-smallest and next-largest divalent cations, respectively, relative to calcium inthe periodic table. The presence of Mg2+ reduces the ther-moactivity of both enzymes below the level seen in theabsence of added metal. The effects of Sr2+ and Ca2+ areapproximately equivalent up to about 110 to 115°C, at whichpoint the presence of Ca2+ has a more favorable effect onenzyme activity. Also, the effect of temperature on theactivity of both enzymes against the two substrates issomewhat different. Activities against the PG3 substrateincrease gradually over the whole temperature range, whilethe activities against the PG7 substrate are much lower attemperatures of up to 100°C and then increase sharply.Perhaps the increased enzyme flexibility, which presumablyoccurs at higher temperatures, is required to accommodatethe larger PG7 substrate into the active site.The activity-enhancing effect of Ca2+ was further ex-

plored by determining the Michaelis-Menten parameters ofeach enzyme towards the PG3 substrate at 98°C in theabsence and presence of 5 mM Ca2+. The results of theseexperiments are listed in Table 4. Calcium has a slightenhancing effect on the Vm.. of both enzymes against thePG3 substrate. The effect on the Kms of both enzymes is

TABLE 2. Purification of amylopullulanase from P. furiosusa

Step Protein Activty (U) Sp act P/A Purification(mg) (U/mg) ratio factor (fold)

Culture supematant (from 350 liters) 1,274 1,211 (9,682)" 0.95 (7.6) 0.13 1.0 (1.0)Q-Sepharose 133 632 (442) 4.75 (3.3) 1.43 5.0 (0.4)Ceramic hydroxyapatite 54.6 363 (227) 6.65 (4.2) 1.60 7.0 (0.6)a-Cyclodextrin affinity 2.7 145 (86) 53.20 (31.9) 1.67 56 (4.2)Superdex 200 gel filtration 1.25 97 (61) 77.5 (48.8) 1.59 82 (6.4)

a See Table 1, footnotes a and b.bIncludes a-amylase activity not associated with amylopullulanase. See text for additional discussion.

VOL. 59, 1993

Page 5: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

2618 BROWN AND KELLY

TABLE 3. Specific polysaccharide hydrolysis rates ofamylopullulanases from T. litoralis and P. furiosus

Sp acta (U/mg)Substrate

P. fwiosus enzyme T. litoralis enzyme

Starch-Ca2+ 48.8 34.8+Ca2+ 34.1 29.9

Pullulan-Ca2+ 77.5 45.6+Ca2+ 97.0 57.7

Glycogen-Ca2+ 1.0 2.4+Ca2+ 0.8 2.2

aAt 980C.

more striking. When calcium is absent, the Km of the T.litoralis enzyme is approximately 1/10 the value for the P.furiosus enzyme. In the presence of 5 mM Ca2 , the Km ofthe T. litoralis enzyme is approximately half its value in theabsence of Ca2 , while the Km of the P. furiosus enzyme isreduced to approximately 1/10 of its original value. There-fore, at least one aspect of the calcium effect involves afacilitating effect on substrate binding. The difference in theKm values of the two enzymes might indeed reflect differ-ences in substrate-binding behavior and, therefore, enzymeactive-site structure. However, the observation that calciumion addition has a more pronounced effect on the Km of theP. furiosus enzyme could also indicate that the T. litoralisenzyme still has some calcium bound to it that was notremoved by EDTA treatment.

bo

C-)a,

04

a.'

C)1

0.

90

80

70

60

50

40

30

20

10

0

60

50

40

30

20

10

0 _40

Temperature (OC)

FIG. 1. Effect of temperature on the pullulan (A)- and starch(B)-hydrolyzing activities of amylopullulanase from T. litoralis.Open symbols, no metal added; solid symbols, 5 mM Ca2' added.

120

,-100

> 80a.'c)-: eO04

00a 20

90t_ o

g 80

70

60

.> 50

., 40

0 3030

>' 20

10

04 160

Temperature (IC)FIG. 2. Effect of temperature on the pullulan (A)- and starch

(B)-hydrolyzing activities of amylopullulanase from P. fturiosus.Open symbols, no metal added; solid symbols, 5 mM Ca2+ added.

As described above, several experiments were conductedto determine the effect of metals on the half-lives of bothenzymes at 120°C in the presence of the PG3 and PG7substrates. Representative results from these experimentsare shown in Table 5. At 120°C, no activity can be measuredin either the absence of metal or the presence of Mg2+,indicating that metal ions with the correct properties arerequired for stability at high temperatures. For both en-zymes, it appears that Ca2+ is a more effective stabilizerthan Sr2"-the half-lives are approximately doubled in thepresence of Ca2+. The results with the P. furiosus enzymeindicate that the PG7 substrate has a greater stabilizing effectthan the PG3 substrate (half-life of 12 min versus 1.1 min).This may be the result of additional interactions that occur asthe larger substrate binds to the enzyme's active site. Also,under identical substrate and metal conditions, the enzymefrom P. furiosus exhibits a longer half-life than the T.litoralis enzyme, implying that the P. furiosus enzyme issomewhat more thermostable.

Substrate hydrolysis patterns. The substrate hydrolysispatterns of the T. litoralis and P. fiuiosus enzymes weredetermined by thin-layer chromatography. Neither enzymehas detectable hydrolytic activity against a-1,4-linked oli-gosaccharides ranging from maltose (DP2) through malto-hexaose (DP6). Amylose, starch, and glycogen are hydro-lyzed in an endo fashion to form a series of oligosaccharidesas small as glucose, with the majority of product in the DP4to DP6 range. The release of small oligosaccharides fromamylose confirms the a-1,4 hydrolytic activity of both en-zymes. Each enzyme attacks only the ox-1,6 linkages inpullulan, resulting in the formation of maltotriose (DP3).Both PG3 and PG7 are hydrolyzed in an exo fashion, withrelease of PNP and the unhydrolyzed oligosaccharide. The

APPL. ENvIRON. MICROBIOL.

L40

Page 6: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

AMYLOLYTIC ENZYMES FROM THERMOPHILIC ARCHAEA

120

ooioS 100

80

P- 604.3

c)a

a 40

C)0-

0 20P3uz

3o

0

N

4c._

._

c)0a)

94

.I)

o _100 _

80 _

60 _

40 -

20 -

O40 50 60 70 80 90 100 110 120 130

Temperature (°C)FIG. 3. Effect of temperature and metal ions (5 mM) on the

activity of T. litoralis amylopullulanase against the model substratesPG3 (A) and PG7 (B). Symbols: E, Mg2"; S, no metal added; A,

Sr2+; *, Ca2

exo attack pattern on the PNP-linked saccharides is puzzlingin view of the lack of activity against the DP2 through DP6substrates. It may be that the PNP-saccharide link mimicseither an internal a-1,4 starch linkage or the a-1,6 linkagefound in pullulan.

DISCUSSION

The determination that amylopullulanases are associatedwith thermophilic archaea extends the phylogenetic range ofthese enzymes. However, without detailed sequence data, itis not possible to determine how closely related any of theseenzymes are to each other. Certainly, the enzymes from T.litoralis and P. furiosus (along with the enzyme from ES4,another thermophilic archaebacterium [36]) are the mostthermoactive and thermostable versions of these enzymesreported to date. This is not surprising in view of theelevated optimal growth temperatures of these two organ-isms.

Substrate gel overlay experiments conducted previously(4) showed the presence of multiple bands of pullulanaseactivity in crude enzyme samples from P. fuiosus composedof both supernatant and cell extract. The relationship be-tween these bands and the amylopullulanase purified here isnot known, although it could represent at least one of theearlier bands. It should be noted that the number and relativemobility of bands detected by overlay gels varied somewhatdepending on the particular enzyme sample being examined,and these bands may represent proteolytic fragments oraggregates rather than discrete enzyme species. As both P.furiosus and T. litoralis exhibit intracellular pullulanaseactivity (data not shown), another possibility is that more

to 140

1120A

100 110

*00

60

403

200

to 140

120

40

20

00

~ ~ ~ ~ eprtue(C

FIG. 4. Effect of temperature and metal ions (5 mM) on theactivity ofP. fiviosus amylopullulanase against the model substratesPG3 (A) and PG7 (B). Symbols: Ml Mg +; *, no metal added; A,

2,

Sr2+; *, Ca +.

than one pullulanase is produced. Experiments with anti-serum raised against the T. litoralis enzyme were unsuccess-ful in resolving this issue, apparently because of low anti-body titers and poor specificity (data not shown).The occurrence of bound sugar on these enzymes is

somewhat surprising, since bacterial proteins appear to beonly rarely glycosylated. However, glycosylation is notunprecedented for amylopullulanases, as the enzyme pro-duced by Clostridium thernohydrosulfuricum has been dem-onstrated to contain bound sugars (21, 31). The particularrole that glycosylation may serve in this case is not clear.Glycosylation of eukaryotic proteins often plays a role inprotein folding, stability, and protection from proteolyticdegradation (8); all of these functions may be important here.The presence of calcium ions has a positive effect on the

thermostability of other amylopullulanases in addition tothose reported here, including the enzymes from a Thermo-anaerobium sp. (27), a Thermus sp. (24), and C. thermohy-drosulfuricum (31). The thermal stability of a-amylases isalso typically enhanced by Ca2+ (5, 14). The reasons forthese effects are not known, although metal ions often act assalt or ion bridges between two adjacent amino acids. The

TABLE 4. Effect of Ca2+ on Michaelis-Menten parameters forthe PG3 substrate

Km (mM) Vm. (U/mg)Enzyme aa

_ca2+ +ca2+ -Ca2+ +ca2+

T. litoralis 0.067 0.037 46.3 60.1P. furiosus 0.664 0.077 78.4 96.0

VOL. 59, 1993 2619

Page 7: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

2620 BROWN AND KELLY

TABLE 5. Effect of substrate and metal on the stability ofamylopullulanases from T. litoralis and P. funiosus

Enzyme Substrate Divalent ka (10' s-) t1 (min)cation Ic(1- ) 12m)

T. litoralis PG7 Ca2+ 2.3 5.0PG7 Sr2+ 4.2 2.8

P. furiosus PG7 Ca2+ 1.0 12.0PG7 Sr2+ 1.8 6.5PG3 Ca2+ 10.3 1.1

a k, first-order decay constant.

binding of calcium ions has been shown to increase thea-helical structure of Bacillus amyloliquefaciens ot-amylase,leading to elevated stability (26), and a similar effect may beat work here. In contrast, the thermostability of the P.furiosus and Pyrococcus woesei extracellular amylases is notaffected by calcium (15, 16). It does appear that a minimumion size may be necessary to stabilize the T. litoralis and P.funiosus amylopullulanases, as the presence of magnesiumions, which are smaller than calcium ions, has a significantnegative effect on enzyme stability, perhaps because theyare too small to form the "bridge." Calcium ions wereshown to have an additional effect on substrate binding, asevidenced by the decrease in the Km towards the PG3substrate for both enzymes. Whether this effect is due toenzyme conformational changes which modify the catalyticsite or results from direct participation of calcium in thesubstrate-binding reactions has not been determined.

Thermostability experiments indicate that the amylopullu-lanase from P. furiosus is somewhat more thermostable thanthe enzyme from T. litoralis. This result corresponds withthe higher optimal growth temperature of P. furiosus andshows that thermostability differences may be observedbetween enzymes whose parent organisms differ in optimalgrowth temperature by only 8 to 10°C.The substrate hydrolysis patterns of the T. litoralis and P.

furiosus amylopullulanases differ from those reported for theenzymes from C. thermosulfurogenes (37), Thermoanaero-bium sp. strain Tok6-B1 (27), and Thernus sp. strainAMD33 (24) in that each of these three enzymes willhydrolyze linear oligosaccharides as small as maltotetraose(DP4). Unfortunately, no information is available on whetherthese other enzymes display an exo attack pattern on PNP-linked oligosaccharides. The a-amylase from P. woesei hasno activity against oligosaccharides smaller than DP6 andonly a small amount of activity against DP7, yet it alsoproduces small oligosaccharides from starch, amylose, andglycogen (15). Both P. funiosus and T. litoralis produceintracellular a-glucosidases which are able to hydrolyzeoligosaccharides as large as DP6 completely to glucose(unpublished data). Therefore, it appears that these organ-isms produce extracellular amylopullulanases (and, in thecase of P. furiosus, an a-amylase) which hydrolyze a-1,4-and a-1,6-linked glucose polysaccharides to form a series ofoligosaccharides, which are then transported into the cell byan unknown mechanism. These oligosaccharides are actedupon by the intracellular ao-glucosidases to produce glucose,which most likely feeds into a pyroglycolytic-type pathway(23, 25). The original source of the extracellular polysaccha-rides is not known, although we have previously speculatedthat glycogens from either marine animals (6) or othermarine archaea (17) are likely candidates. We have demon-strated the production of a similar spectrum of amylolyticenzymes in a number of thermophilic archaea from both

marine and terrestrial sites (3a), illustrating the importanceof polysaccharide metabolism in these environments.

It has not yet been determined whether the amylopullula-nases from P. fnuiosus and T. litoralis are able to hydrolyzeboth a-1,6 and a-1,4 linkages at a single active site or utilizemultiple active sites. The amylopullulanases from Therno-anaerobium sp. strain Tok6-B1 and C. thernohydrosulfuri-cum have been shown to possess a single active site, theformer by chemical modification of the active site (28) andthe latter by substrate competition experiments (19). Addi-tional experimentation will be necessary to resolve this issuefor the two enzymes reported here.Although the amylopullulanases from T. litoralis and P.

furiosus certainly have the thermostability necessary forapplication in the industrial starch hydrolysis process, fur-ther evaluation will be required to determine their actualutility. Specifically, the ability of these enzymes to improveglucose yields and reduce the time required for the liquefac-tion or saccharification step must be demonstrated underindustrial conditions. These two amylopullulanases may alsobe useful in the production of specialty corn sweeteners, anapplication which has been suggested previously for less-thermostable amylopullulanases (33), and their unique prop-erties may allow the development of other novel saccharidehydrolysis processes.

ACKNOWLEDGMENTS

This work was supported in part through grants from the NationalScience Foundation (BCS-9011583 and BCS-9007762) and through aURI training grant from the Office of Naval Research.We thank J. Shiloach (Biotechnology Unit, National Institutes of

Health) and E. Sybert and M. Connor (Department of ChemicalEngineering, University of Maryland-College Park) for help andadvice with large-scale cultivation.

REFERENCES1. Belkin, S., and H. W. Jannasch. 1985. A new extremely ther-

mophilic sulfur reducing heterotrophic marine bacterium. Arch.Microbiol. 142:181-186.

2. Bernfeld, P. 1955. Amylases a and P. Methods Enzymol.1:149-158.

3. Blumentals, I. I., S. H. Brown, R. N. Schicho, A. K. Skaja, H. R.Costantino, and R. M. Kelly. 1990. The hyperthermophilicarchaebacterium Pyrococcus furiosus. Development of cultur-ing protocols, perspectives on scaleup, and potential applica-tions. Ann. N.Y. Acad. Sci. 589:301-314.

3a.Brown, S. H. 1992. Ph.D. thesis, Johns Hopkins University,Baltimore.

4. Brown, S. H., H. R. Costantino, and R. M. Kelly. 1990.Characterization of amylolytic enzyme activities associatedwith the hyperthermophilic archaebacterium Pyrococcusfuio-sus. Appl. Environ. Microbiol. 56:1985-1991.

4a.Brown, S. H., and R. M. Kelly. Unpublished data.5. Brumm, P. J., R. E. Hebeda, and W. M. Teague. 1988. Purifi-

cation and properties of a new, commercial, thermostableBacillus stearothermophilus a-amylase. Food Biotechnol. 2:67-80.

6. Corlis, J. B., J. Dymond, L. I. Gordon, J. M. Edmond, R. P. vonHerzen, R. D. Ballard, K. Green, D. Williams, A. Bainbridge, K.Crane, and T. H. van Andel. 1979. Submarine thermal springs onthe Galapagos rift. Science 203:1073-1083.

7. Costantino, H. R., S. H. Brown, and R. M. Kelly. 1990.Purification and characterization of an a-glucosidase from ahyperthermophilic archaebacterium, Pyrococcus furiosus, ex-hibiting a temperature optimum of 105 to 115°C. J. Bacteriol.172:3654-3660.

8. Elbein, A. D. 1991. The role of N-linked oligosaccharides inglycoprotein function. Trends Biotechnol. 9:346-352.

9. Fairbanks, G., T. L. Steck, and D. F. H. Wallach. 1971.

APPL. ENvIRON. MICROBIOL.

Page 8: Characterization of Amylolytic Enzymes, Having Both α-1, 4 and α-1 ...

AMYLOLYTIC ENZYMES FROM THERMOPHILIC ARCHAEA 2621

Electrophoretic analysis of the major polypeptides of the humanerythrocyte membrane. Biochemistry 10:2606-2613.

10. Fiala, G., and K. 0. Stetter. 1986. Pyrococcusfuriosus sp. nov.represents a novel genus of marine heterotrophic archaebacteriagrowing optimally at 100°C. Arch. Microbiol. 145:56-60.

11. Goldstein, W. E. 1990. Enzymes in starch processing andbaking, p. 92-102. In W. Gerhartz (ed.), Enzymes in industry.VCH Publishers, New York.

12. Hansen, S. A. 1975. Thin-layer chromatographic method foridentification of oligosaccharides in starch hydrolysates. J.Chromatogr. 105:388-390.

13. Kester, D. R., I. W. Duedall, D. N. Connors, and R. M.Pytkowicz. 1967. Preparation of artificial seawater. Limnol.Oceanogr. 12:176-178.

14. Kindle, K. L. 1983. Characteristics and production of thermo-stable ca-amylase. Appl. Biochem. Biotechnol. 8:153-170.

15. Koch, R., A. Spreinat, K. Lemke, and G. Antranikian. 1991.Purification and properties of a hyperthermoactive a-amylasefrom the archaebacterium Pyrococcus woesei. Arch. Microbiol.155:572-578.

16. Koch, R., P. Zablowski, A. Spreinat, and G. Antranikian. 1990.Extremely thermostable amylolytic enzyme from the archae-bacterium Pyrococcus furiosus. FEMS Microbiol. Lett. 71:21-26.

17. Konig, H., R. Skorko, W. Zillig, and W. D. Reiter. 1982.Glycogen in thermoacidophilic archaebacteria of the generaSulfolobus, Thennoproteus, Desulfurococcus and Thermococ-cus. Arch. Microbiol. 132:297-303.

18. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

19. Mathupala, S., B. C. Saha, and J. G. Zeikus. 1990. Substratecompetition and specificity at the active site of amylopullula-nase from Clostridium thermohydrosulfuricum. Biochem. Bio-phys. Res. Commun. 166:126-132.

20. Melasniemi, H. 1987. Characterization of a-amylase and pullu-lanase activities of Clostridium thermohydrosulfuncum. Bio-chem. J. 246:193-197.

21. Melasniemi, H. 1988. Purification and some properties of theextracellular a-amylase-pullulanase produced by Clostridiumthennohydrosulfiuricum. Biochem. J. 250:813-818.

22. Moench, T. T., and J. G. Zeikus. 1983. An improved preparationmethod for a titanium(III) media reductant. J. Microbiol. Meth-ods 1:199-202.

23. Mukund, S., and M. W. W. Adams. 1991. The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium,Pyrococcusfuriosus, is an aldehyde ferredoxin oxidoreductase:evidence for its participation in a unique glycolytic pathway. J.Biol. Chem. 266:14208-14216.

24. Nakamura, N., N. Sashihara, H. Nagayama, and K. Horikoshi.1989. Characterization of pullulanase and a-amylase activities

of a Thennus sp. AMD33. Starch/Staerke 41:112-117.25. Neuner, A., H. W. Jannasch, S. Belkin, and K. 0. Stetter. 1990.

Thermococcus litoralis sp. nov., a new species of extremelythermophilic marine archaebacteria. Arch. Microbiol. 153:205-207.

26. Oh, H. S., K. H. Kim, S. W. Suh, and M. U. Choi. 1991.Spectroscopic and electrophoretic studies on structural stabilityof a-amylase from Bacillus amyloliquefaciens. Korean Bio-chem. J. 24:158-167.

27. Plant, A. R., R. M. Clemens, R. M. Daniel, and H. W. Morgan.1987. Purification and preliminary characterization of an extra-cellular pullulanase from Thermoanaerobium Tok6-B1. Appl.Microbiol. Biotechnol. 26:427-433.

28. Plant, A R, R. M. Clemens, H. W. Morgan, and R. M. Daniel.1987. Active-site and substrate-specificity of Thermoanaero-bium Tok6-B1 pullulanase. Biochem. J. 246:537-541.

29. Plant, A. R., H. W. Morgan, and R. M. Daniel. 1986. A highlystable pullulanase from Thermus aquaticus YT-1. Enzyme Mi-crob. Technol. 8:668-672.

30. Saha, B. C., R L. Lamed, C. Y. Lee, S. P. Mathupala, and J. G.Zeikus. 1990. Characterization of an endo-acting amylopullula-nase from Thermoanaerobacter strain B6A. Appl. Environ.Microbiol. 56:881-886.

31. Saha, B. C., S. P. Mathupala, and J. G. Zeikus. 1988. Purifica-tion and characterization of a highly thermostable novel pullu-lanase from Clostidium thermohydrosulfuricum. Biochem. J.252:343-348.

32. Saha, B. C., G. J. Shen, K. C. Srivastava, L. W. LeCureux, andJ. G. Zeikus. 1989. New thermostable a-amylase-like pullula-nase from thermophilic Bacillus sp. 3183. Enzyme Microb.Technol. 11:760-764.

33. Saha, B. C., and J. G. Zeikus. 1989. Novel highly thermostablepullulanase from thermophiles. Tibtech 7:234-238.

34. Schafer, T., and P. Schonheit. 1992. Maltose fermentation toacetate, CO2 and H2 in the anaerobic hyperthermophilic ar-chaeon Pyrococcus furiosus: evidence for the operation of anovel sugar fermentation pathway. Arch. Microbiol. 158:188-202.

35. Schicho, R N., L. J. Snowden, S. Mukund, J.-B. Park,M. W. W. Adams, and R. M. Kelly. 1993. Influence of tungstenon metabolic patterns in Pyrococcus fuiiosus, a hyperthermo-philic archaeum. Arch. Microbiol. 159:380-385.

36. Schuliger, J. W., S. H. Brown, J. A. Baross, and R. M. Kelly.Purification and characterization of a novel amylolytic enzymefrom ES4, a marine hyperthermophilic archaeum. Mol. Mar.Biol. Biotechnol., in press.

37. Spreinat, A., and G. Antranikian. 1990. Purification and prop-erties of a thermostable pullulanase from Clostridium thermo-sulfurogenes EM1 which hydrolyses both a-1,6 and a,1,4-glycosidic linkages. Appl. Microbiol. Biotechnol. 33:511-518.

VOL. 59, 1993