- Email: [email protected]
Amylolytic enzymes from hyperthermophiles
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AMYLOLYTICENZYMES
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closely related ~-glucosidase from P. woesei possesses an even higher activity toward sucrose than to maltose. 1° Comparisons between the putative oz-glucosidases from different organisms will be facilitated greatly by the availability of the amino acid (gene) sequences of these enzymes.
Acknowledgments RMK acknowledges the Novartis Agricultural Biotechnology Research Institute (NABRI) (Research Triangle Park, NC), the U.S. Department of Agriculture, and the National Science Foundation for financial support of this work.
[1 7]
Amylolytic Enzymes
By
COSTANZO
BERTOLDO
from Hyperthermophiles and
GARABED
ANTRANIKIAN
Introduction In recent years, many hyperthermophilic organisms that are able to grow at temperatures up to the normal boiling point of water have been shown to utilize natural polymeric substrates as carbon and energy sources. These extreme thermophilic microorganisms, which belong to Archaea and Bacteria, can facilitate the enzymatic degradation of carbohydrates by producing heat-stable enzymes that are catalyically active above 100°. Such enzymes share the ability to hydrolyze the glycosidic bonds between two or more sugar molecules or between a carbohydrate and a noncarbohydrate moiety. The enzymatic hydrolysis and the modification of polysaccharides are of great interest in the field of food technology. The potential exploitation of this natural source of sugars is not only useful for glucose syrup production, but also for the synthesis of nonfermentable carbohydrates, anticariogenic agents, and antistaling agents in baking, a In most hyperthermophilic microorganisms, the activity levels of starch-hydrolyzing enzymes appear to be too low for biotechnological applications. The molecular cloning of the corresponding genes and their expression in heterologous hosts, however, circumvent the problem of insufficient expression in the natural host. The number of genes from hyperthermophiles encoding amylolytic enzymes that have been cloned and expressed in mesophiles has been increasing significantly. In most cases, the thermostable proteins expressed in mesophilic hosts maintain their thermostability, are correctly i D. Cowan, Tibtech 14, 177 (1996).
METHODS IN ENZYMOLOGY, VOL. 330
Copyright © 200l by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $35.00
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folded at low temperature, are resistant to host proteolysis, and can be purified easily using thermal denaturation of the mesophilic host proteins. The degree of enzyme purity obtained is generally suitable for most industrial applications. This article briefly discusses the enzymatic action and properties of starch-hydrolyzing enzymes and focuses only on those enzymes that have been isolated and characterized from extreme thermophilic and hyperthermophilic Archaea and Bacteria. It also briefly describes their potential biotechnological exploitation. Some of these aspects have been already presented. 2-8 Furthermore, this article describes the general methodology for gene identification and expression and the procedure for the purification of some amylolytic enzymes that have been successfully isolated and extensively studied in the authors' laboratory.
Amylolytic E n z y m e s Starch from cultivated plants represents a ubiquitous and easily accessible source of energy. In plant cells or seeds, starch is usually deposited in the form of large granules in the cytoplasm. Starch is composed exclusively of a-glucose units that are linked by ot-1,4- or ot-1,6-glycosidic bonds. The two high molecular weight components of starch are amylose (15-25%), a linear polymer consisting of tx-1,4-1inked glucopyranose residues, and amylopectin (75-85%), a branched polymer containing, in addition to a1,4-glycosidic linkages, ot-l,6-1inked branch points occurring every 17-26 glucose units, ot-Amylose chains, which are not soluble in water but form hydrated micelles, are polydisperse and their molecular weights vary from several hundred to thousands. The molecular weight of amylopectin may be as high as 100 million and in solution such a polymer has colloidal or micellar forms. Because of the complex structure of starch, cells require an appropriate combination of hydrolyzing enzymes for the depolymerization of starch to 2 G. Antranikian, in "Microbial Degradation of Natural Products" (G. Winkelmann,ed.), Vol. 2, p. 28. VCH, Weilheim,1992. 3R. Ladenstein and G. Antranikian, Adv. Biochem. Eng./Biotechnol. 61, 37 (1998). 4 C. Leuschner and G Antranikian, World J. Microbiol. Biotechnol. 11, 95 (1995). 5A. R0diger,A. Sunna, and G. Antranikian, in (D. Waldmann,ed.), "Carbohydrases:Handbook of Enzyme Catalysisin Organic Synthesis,"p. 946. VCH, Weilheim,1994. 6A. Sunna, M. Moracci, M. Rossi, and G. Antranikian, Extremophiles 1, 2 (1996). 7R. MOiler,G. Antranikian, S. Maloney,and R. Sharp, Adv. Biochem. Eng. Biotechnol. 61, 155 (1998). 8F. Niehaus, C. Bertoldo, M. Ktihler,and G. Antranikian, AppL Microbiol. Biotechnol. 51, 711 (1999).
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oligosaccharides and smaller sugars, such as glucose and maltose. They can be simply classified into two groups: endo-acting enzymes or endohydrolases and exo-acting enzymes or exohydrolases (Fig. 1). Endo-acting enzymes, such as a-amylase (a-l,4-glucan-4-glucanohydrolase; EC 3.2.1.1), hydrolyze linkages in the interior of the starch polymer in a random fashion, which
0Oo
gM~,#trAnsforme~
00• • glucoee
c~ maltmm
oooo
134rny~.o g~.coe.~m
c~
mJtouand
~.limit-dextrin
•
g~co~
~
o~
O 0 m~ose •
~-.-~
oo~
type l and
pullulan
I/
O-C)-O o~o maltotriose
~ ~
~,,
panose
punulm hydrolmetype~l% l Isopanose
FXG. 1. Schematic presentation of the action of amylolytic and pullulytic enzymes. Pullulanase type I also attacks a-l,6-glycosidic linkages in oligo- and polysaccharides. Pullulanase type II attacks, in addition, a-l,4-1inkages in various oligo- and polysaccharides.
272
SACCHAROLYTICENZYMES
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leads to the formation of linear and branched oligosaccharides. The sugarreducing groups are liberated in the a-anomeric configuration. Most starchhydrolyzing enzymes belong to the a-amylase family, which contains a characteristic catalytic (fl/a)s-barrel domain, a-Amylases belong to two families, number 13 and 57 of the glycosylhydrolase families.9 Family 13 has approximately 150 members from Eukarya and Bacteria. Family 57, however, has only three members from Bacteria and Archaea. Throughout the a-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. 1° On the structural level, there are to date no X-ray structures of amylolytic enzymes derived from hyperthermophiles. Exo-acting starch hydrolases include//-amylase, glucoamylase, a-glucosidase, and isoamylase. These enzymes attack the substrate from the nonreducing end, producing small and well-defined oligosaccharides, r Amylase (EC 3.2.1.2), also referred to as a-l,4-D-glucan maltohydrolase or saccharogen amylase, hydrolyzes a-l,4-glucosidic linkages to remove successive maltose units from the nonreducing ends of the starch chains, producing//-maltose by an inversion of the anomeric configuration of the maltose.//-Amylase belongs to family 14 of the glycosylhydrolases, having 11 members from Eukarya and Bacteria. Glucoamylase (EC 3.2.1.3) hydrolyzes terminal a-l,4-1inked-D-glucose residues successively from nonreducing ends of the chains, releasing//-Dglucose. Glucoamylase, which is typically a fungal enzyme, has several names: a-l,4-D-glucan hydrolase, amyloglucosidase, and y-amylase. Most forms of the enzyme can hydrolyze a-l,6-D-glucosidic bonds when the next bond in sequence is a-l,4. However, in vitro this enzyme hydrolyzes a-l,6and a-l,3-D-glucosidic bonds in other polysaccharides with high molecular weights. a-Glucosidase (EC 3.2.1.20), or a-D-glucoside glucohydrolase, attacks the a-l,4 linkages of oligosaccharides that are produced by the action of other amylolytic enzymes. Unlike glucoamylase, a-glucosidase liberates glucose with an a-anomeric configuration, a-Glucosidases are members of family 15 and the very diverse family 31 of the glycosylhydrolases.9 Isoamylase (EC 3.2.1.68), or glycogen 6-glucanohydrolase, is a debranching enzyme specific for a-l,6 linkages in polysaccharides, such as amylopectin, glycogen, and //-limit dextrin, but it is unable to hydrolyze the a-l,6 linkages in pullulan or branched oligosaccharide; therefore, it has limited action on alimit dextrin. 9B. Henrissat,Biochem. J. 280, 309 (1991). 10T. Kuriki and T. Imanaka,J. Biosci. Bioeng. 87, 557 (1999).
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Pullulytic Enzymes Pullulan is a linear a-glucan consisting of maltotriose units joined by a-l,6-glycosidic linkages and it is produced by Aureobasidium pullulans with a chain length of 480 maltotriose units. Enzymes capable of hydrolyzing a-l,6 glycosidic bonds in pullulan are defined as pullulanases (Fig. 1). On the basis of substrate specificity and product formation, pullulanases have been classified into two groups: pullulanase type I and pullulanase type II. Pullulanase type I (EC 3.2.1.41) specifically hydrolyzes the a-l,6 linkages in pullulan as well as in branched oligosaccharides, and its degradation products are maltotriose and linear oligosaccharides, respectively. Pullulanase type I is unable to attack a-l,4 linkages in a-glucans and belongs to family 13 of the glycosylhydrolases. Pullulanase type II, or amylopullulanase, attacks a-l,6-glycosidic linkages in pullulan and a-l,4 linkages in branched and linear oligosaccharides (Fig. 1). The enzyme has a multiple specificity and is able to fully convert polysaccharides (e.g., amylopectin) to small sugars (DP1, DP2, DP3; DP is the degree of polymerization) in the absence of other enzymes, such as a-amylase or fl-amylase. In contrast to the previously described pullulanases, pullulan hydrolases type I and type II are unable to hydrolyze a-l,6-glycosidic linkages in pullulan or in branched substrates. They can attack a-l,4-glycosidic linkages in pullulan-forming panose or isopanose. PuUulan hydrolase type I or neopullulanase (EC 3.2.1.135) hydrolyze pullulan to panose (a-6-D-glucosylmaltose). Pullulan hydrolase type II or isopullulanase (EC 3.2.1.57) hydrolyzes pullulan to isopanose (a-6-maltosylglucose) (Fig. 1). Finally, cyclodextrin glycosyltransferase (EC 2.4.1.19), or a-l,4-Dglucan a-4-D-(a-l,4-D-glucano)transferase, is an enzyme that is generally found in Bacteria and has been discovered in Archaea. This enzyme produces a series of nonreducing cyclic dextrins from starch, amylose, and other polysaccharides, a-, fl-, and ~/-cyclodextrins are rings formed by 6, 7, and 8 glucose units that are linked by a-l,4 bonds, respectively (Fig. 1).
Enzyme Production by Hypertherinophilic Microorganisms Hyperthermophilic Bacteria and Archaea that are able to grow on starch at temperatures over 70° have been isolated and the corresponding starchdegrading enzymes have been purified and characterized. In several cases, genes encoding these enzymes have been isolated, cloned, and overexpressed in heterologous hosts (Table I).
TABLE I STARCH-HYDROLYZING ENZYMES FROM EXTREMELY THERMOPHILIC AND HYPERTHERMOPHILIC ARCHAEA AND BACTERIA Enzyme properties a
Enzyme a-Amylase
Organism a
Desulfurococeus mucosus (85) Pyrococcus furiosus (100)
Pyrococcus sp. KOD1 P. woesei (100) Pyrodictium abyssi (98) Staphylothermus rnarinus
Optimal temperature 85 100 100
Optimal pH
Molecular mass (kDa)
5.5
74
6.5-7.5 7.0
Purified/cloned
129 68
90
6.5
49.5
100 100 100
5.5 5.0 5.0
68
Remarks
b --
Purificd/cloned/intracellular Purified/cloned/extracellular Purified/cloned/extracellular Purified/extracellular Crude extract Crude extract
(90)
Pullulanase type I
Pullulanase type It
Glucoamylase
CGTase ~x-Glucosidase
Sulfolobus solfataricus (88) Thermococcus celer (85) T. profundus DT5432 (80) T. profundus (80) T. aggregans (85) Dyctyoglomus thermophilum Rt46B.1 (73) Thermotoga maritima MSB8 (90) Fervidobacterium pennivorans Ven5 (75) T. maritima MSB8 (90) Thermus caldophilus GK24 (75) Desulfurococcus mucosus (88) P. woesei (100)
--
--
240
90 80
5.5 5.5
80 95 90
4.0-5.0 6.5 5.5
42
7.0
61
85-90
Extracellular --
42
-75
Purified/"Amy L" Cloned Purified/cloned/cytoplasmic fraction Purified/cloned/lipoprotein
80
6
90 75
6.0 5.5
93 (subunit) 65
Cloned/type I c Purified/ceU associated
85
5.0
74
Purified/cloned
100
6.0
90
P. abyssi (98) T. celer (85) Thermococcus litoralis (90) T. hydrothermalis (80)
100 90 98
9.0 5.5 5.5
119
95
5.5
128
T. aggregans (85) Thermoplasma acidophilum (60) Picrophilus oshimae (60) P. torridus (60) Thermococcus sp. (75) Thermococcus strain AN1 (80) Thermococcus hydrothermalls (80)
100 90
6,5 6.5
83 141
Purified/cloned/cell associated Crude extract Crude extract Purified/extraccUular/glycoprotein Purified/extracellular/glycoprotein Purified/cloned Purified
90 90 100 130
2.0 2.0 2.0
140 133 83 63
--
190 (93)
Crude extract Purified/cloned/"Amy S"
---
--
--
--
Purified/cloned
Purified Purified Purified Purified/extracellular/glycoprotein Cloned
Values in parentheses give the optimal growth temperature for each organism in degrees Celsius. b Not determined. c Unpublished results.
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AMYLOLYTICENZYMES
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Heat-Stable Amylases and Glucoamylases Extremely thermostable a-amylases have been characterized from Pyrococcus furiosus, 11 eyrococcus woesei, 12 and Thermococcus profundus. 13 Optimal temperatures for the activity of these enzymes are 100 °, 100°, and 80°, respectively. In addition, either amylase and/or pullulanase activities have been observed in hyperthermophilic Archaea of the genera Sulfolobus, Thermophilum, Desulfurococcus, Thermococcus, and Staphylothermus.14,15 Molecular cloning of the corresponding genes and their expression in heterologous hosts circumvent the problem of insufficient expression in the natural host. The gene encoding an extracellular a-amylase from P. furiosus has been cloned and the recombinant enzyme expressed in B. subtilis and Escherichia coli. 16,17This is the first report on the expression of an archaeal gene derived from an extremophile in a Bacillus strain. The high thermostability of the pyrococcal extracellular a-amylase (thermal activity even at 130°) in the absence of metal ions, together with its unique product pattern and substrate specificity, makes this enzyme an interesting candidate for industrial application. In addition, an intracellular a-amylase gene from P. furiosus has been cloned and sequenced. 18 It is interesting that the four highly conserved regions usually identified in a-amylases are not found in this enzyme, a-Amylases with lower thermostability and thermoactivity have been isolates from the archaea T. profundus, 19Pyrococcus sp. KOD 1,2° and the bacterium Thermotoga maritima. 21 Genes encoding these enzymes were successfully expressed in E. coli. Similar to the amylase from B. licheniformis, which is commonly used in liquefaction, the enzyme from T. maritima requires Ca 2+ for activity. Further investigations have shown that 11 R. Koch, K. Spreinat, K. Lemke, and G. Antranikian, Arch. Microbiol. 155, 572 (1991). 12 y. C. Chung, T. Kobayashi, H. Kanai, T. Akiba, and T. Kudo, Appl. Environ. Microbiol. 61, 1502 (1995). 13j. T. Lee, H. Kanai, T. Kobayashi, T. Akiba, and T. Kudo, J. Ferment. Bioeng. 82, 432 (1996). 14j. M. Bragger, R. M. Daniel, T. Coolbear, and H. W. Morgan, Appl. Microbiol. Biotechnol. 31, 556 (1989). 15F. Canganella, C. Andrade, and G. Antranikian, Appl. MicrobioL BiotechnoL 42, 239 (1994). J6 G. Dong, C. Vieille, A. Savchenko, and J. G. Zeikus, Appl. Environ. Microbiol. 63, 3569 (1997). 17 S. Jc~rgensen, C. E. Vorgias, and G. Antranikian, J. Biol. Chem. 272, 16335 (1997). 18 K. A. Laderman, K. Asada, T. Uemori, H. Mukai, Y. Taguchi, I. Kato, and C. B. Anfinsen, J. Biol. Chem. 268, 24402 (1993). 19 y. S. Kwak, T. Akeba, and T. Kudo, J. Ferment. Bioeng. 86, 363 (1998). 20 y. Tachibana, L. M. Mendez, S. Fujiwara, M. Takagi, and T. Imanaka, J. Ferment. Bioeng. 82, 224 (1996). 21 W. Liebl, I. Stemplinger, and P. Ruile, J. Bacteriol. 179, 941 (1997).
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SACCHAROLYTICENZYMES
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the extreme hyperthermophilic archaeon Pyrodictium abyssi can grow on various polysaccharides and also secretes a heat-stable amylase that is optimally active above 100° and has a wide functional pH range (unpublished results). Unlike ~x-amylase, the production of glucoamylase seems to be very rare in extremely thermophilic and hyperthermophilic Bacteria and Archaea. Among thermophilic anaerobic bacteria, glucoamylases have been purified and characterized from Clostridium thermohydrosulfuricum 39E, 22 Thermoanaerobacterium thermosaccharolyticurn DSM 571, 23 and Clostridium thermosaccharolyticurn. 24 The latter enzyme is optimally active at 70° and pH 5.0. It has been shown that the thermoacidophilic archaea Thermoplasma acidophilum, Picrophilus torridus, and Picrophilus oshimae produce heatand acid-stable glucoamylases. The purified archaeal glucoamylases are optimally active at pH 2 and 90°. Catalytic activity is still detectable at pH 0.5 and 100°. This represents the first report on the presence of glucoamylases in thermophilic Archaea (unpublished results). ot-Glucosidases have been found in thermophilic Archaea and Bacteria. An extracellular a-glucosidase from the thermophilic archaeon Thermococcus strain AN125 was purified and its molecular characteristics determined. The monomeric enzyme (60 kDa) is optimally active at 98°. The purified enzyme has a half-life around 35 min, which is increased to around 215 min in the presence of 1% (w/v) dithiothreitol (DTT) and 1% (w/v) bovine serum albumin (BSA). The substrate preference of the enzyme is pNP-o~D-glucoside > nigerose > panose > palatinose > isomaltose > maltose > turanose. No activity was found with starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose, and fl-gentiobiose. The enzyme was active at 130°. The gene encoding an o~-glucosidase from Thermococcus hydrothermalis 26 was cloned by complementation of a Saccharornyces cerevisiae deficiency maltase-deficient mutant strain. The cDNA clone isolated encodes an open reading frame (ORF) corresponding to a protein of 242 amino acids. The protein shows 42% identity to a Pyrococcus horikoshii unknown ORF, but no similarities were obtained with other polysaccharidase sequences.
22 H. H. Hyun and J. G. Zeikus, J. Bacteriol. 16,1, 1146 (1985). 23 D. Ganghofner, J. Kellermann, W. L. Staudenbauer, and K. Bronnenmeier, Biosci. Biotechnol. Biochem. 62, 302 (1998). 24 U. Specka, F. Mayer, and G. Antranikian, Appl. Environ. Microbiol. 57, 2317 (1991). 25 K. Piller, R. M. Daniel, and H. H. Petach, Protein Struct. Mol. Enzymol. 1, 197 (1996). 26 A. Galichet and A. Belarbi, FEBS Lett. 2, 188 (1999).
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AMYLOLYTICENZYMES
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Thermoactive Pullulanases and CGTases Thermostable and thermoactive pullulanases from extreme thermophilic microorganisms have been detected in Thermococcus celer, Desulfurococcus mucosus, Staphylothermus marinus, and Thermococcus aggregans. 15 Temperature optima between 90 and 105°, as well as remarkable thermostability, even in the absence of substrate and calcium ions, have been observed. Most thermoactive pullulanases identified to date belong to the type II group, which attack or-l,4- and ~-l,6-glycosidic linkages. They have been purified from P. furiosus and T. litoralis, 27 T. hydrothermalis, 28 and Pyrococcus strain E S 4 . 29 Pullulanases type II from P. furiosus and P. woesei have been expressed in E. coli. 30'31 The unfolding and refolding of pullulanase from P. woesei have been investigated using guanidinium chloride as the denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation, and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +_ 0.29 M for intrinsic fluorescence and 4.90 ___0.31 M for far-UV circular dichroism (CD) changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained on removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca 2+, which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca 2+ ions with a Ko of 0.080 _+ 0.010 /zM and a Hill coefficient H of 1.00 -2-_ 0.10. This cation significantly enhances the stability of the pullulanase against guanidinium chloride-induced unfolding. The refolding of pullulanase, however, was not affected by Ca2+. 32 Genes encoding pullulanases from T. hydrothermalis 33 and 7". aggregans (unpublished results) have been isolated and expressed in mesophilic hosts. The aerobic thermophilic bacterium Thermus caldophilus GK-24 produces a thermostable pullulanase of type I when grown on
2v S. H. Brown and R. M. Kelly, Appl. Environ. Microbiol. 59, 2614 (1993). 28 H. Gantelet and F. Duchiron, AppL Microbiol. Biotechnol. 49, 770 (1998). 29 j. W. Schuliger, S. H. Brown, J, A. Baross, and R. M. Kelly, Mol. Mar. Biol. Biotechnol. 2, 76 (1993). 30 A. R0diger, P. L. JCrgensen, and G. Antranikian, AppL Environ. Microbiol. 61, 567 (1995). 3~ G. Dong, C. Vieille, and J. G. Zeikus, Appl. Environ. Microbiol. 63, 3577 (1997). 32 R. M. Schwerdffeger, R. Chiaraluce, V. Consalvi, R. Scandurra, and G. Antranikian, Eur. J. Biochem. 264, 479 (1999). 33 M. Erra-Pujada, P. Debeire, F. Duchiron, and M. J. O. Donohue, J. Bacteriol. 181, 3282 (1999).
278
SACCHAROLYTICENZYMES
1171
starch. 34 This enzyme debranches amylopectin by attacking specifically ot1,6-glycosidic linkages. The pullulanase is optimally active at 75 ° and pH 5.5, is thermostable up to 90°, and does not require Ca 2+ for either activity or stability. The first debranching enzyme from an anaerobic thermophile was identified in the bacterium Fervidobacterium pennivorans Ven5 and finally cloned and expressed in E. coli. 35 In contrast to the pullulanase from P. woesei (specific to both a-l,6 and a-l,4 glycosidic linkages), pullulanase type II or amylopullulanase, the enzyme from F. pennivorans ven5 attacks exclusively the ot-l,6-glycosidic linkages in polysaccharides (pullulanase type I). This thermostable debranching enzyme leads to the formation of long chain linear polysaccharides from amylopectin. Thermostable CGTases are produced by the members of the genus Thermoanaerobacter and Thermoanaerobacterium thermosulfurigenes 36'37 and Anaerobranca bogoriae. 38 A CGTase has been purified from a newly isolated archaeon, Thermococcus sp. The optimum temperatures for starchdegrading activity and cyclodextrin synthesis are 110 and 90-100 °, respectively. This is the first report on the presence of a thermostable CGTase in a hyperthermophilic archaeon. 39 The ability of extreme thermophiles and hyperthermophiles to produce heat-stable glycosylhydrolases is summarized in Table I.
Methods
Cloning and Expression of Hyperthermophilic Amylolytic Enzymes in Mesophilic Hosts Recombinant starch-hydrolyzing enzymes from several extreme thermophilic and hyperthermophilic microorganisms have been expressed in E. coli as soluble cytosolic proteins. In general, the recombinant enzyme produced in mesophilic hosts maintains the properties of the wild-type enzyme. The gene encoding amylolytic enzyme can be identified and ampli34 C. H. Kim, O. Nashiru, and J. H. Ko, FEMS Microbiol. Lett. 138, 147 (1996). 35 C. Bertoldo, F. Duffner, P. L. J0rgensen, and G. Antranikian, Appl. Environ. Microbiol. 65, 2084(1999). 36 S. Pedersen, B. F. Jensen, L. Dijkhuizen, S. T. J0rgensen, and B. W. Dijkstra, Chemtech 12, 19 (1995). 37 R. Wind, W. Liebl, R. Buitlaar, D. Penninga, A. Spreinat, L. Dijkhuizen, and H. Bahl, Appl. Environ. Microbiol. 61, 1257 (1995). 38 S. Prowe, J. van de Vossenberg, A. Driessen, G. Antranikian, and W. Konnings, J. Bacteriol. 178, 4099 (1996). 39 y. Tachibana, A. Kuramura, N. Shirasaka, Y. Suzuki, T. Yamamoto, S. Fujiwara, M. Takagi, and T. Imanaka, Appl. Environ. Microbiol. 65, 1991 (1999).
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AMYLOLYTIC ENZYMES
279
fled by polymerase chain reaction (PCR) using conserved sequences that are known for amylolytic enzymes. 18,3t In many cases, it is possible to detect a starch-hydrolyzing activity after a phenotypical screening of a genomic library, created by shotgun cloning. By using sensitive screening methods, it is possible to detect positive clones producing very low levels of thermostable enzymes. By employing this technique, it is possible to identify new genes expressing novel hydrolytic enzymes. Such procedures have been used to isolate heat-stable a-amylases, t6,17,2°,21 as well as pullulanase type II from the archaeon P. woesei3° and pullulanase type I from the anaerobic bacterium F. pennivorans Ven5. 35 Figure 2 outlines the clone selection procedure used to isolate these genes, which is based on the secretion of amylolytic/pullulytic activity from recombinant cells and the detection of enzymatic activity by producing a clear halo around the colonies. The next section focuses on the general methodology used to clone amylolytic and pullulytic enzymes from extreme thermophilic Archaea and Bacteria. Screening of Genomic Library. Chromosomal DNA is isolated from thermophilic Archaea and Bacteria according to Pitcher e t al., 4° and 100 tzg of DANN is partially digested with 20 U of Sau3A for 10 rain. The digestion is terminated by a phenol:chloroform extraction, and the DNA is ethanol precipitated, size fractionated on a sucrose gradient and after fragments between 3 and 7 kb are pooled. A plasmid library is constructed in the vector pSJ9334~ and the host strain E. coli MC100 using the following method. The cloning vector is digested with BamHI, and a 3.8-kb fragment is purified from an agarose gel. Approximately 0.75 tzg of vector fragment is ligated to 4 /xg of size-fractionated chromosomal DNA and used to transform E. coli MC100 by electroporation. Red dyed substrate (amylopectin or pullulan) is made by suspending 50 g substrate (Hayashibara Biochemical Laboratories) and 5 g Cibachron Rot B (Ciba Geigy) in 500 ml 0.5 M NaOH and incubating at room temperature under constant stirring for 16 hr. The pH is adjusted to 7.0 with 4 N H2SO4. The dyed substrates are precipitated under constant stirring on the addition of 600 ml ethanol, harvested by centrifugation, and then resuspended in 500 ml distilled water. This precipitation procedure is repeated three times and the final dyed substrate is resuspended in 500 ml of distilled water and autoclaved. Reddyed substrate is added to solid medium at a concentration of 1% (v/v). Positive clones expressing active o~-amylase or pullulanase can be identified by forming a clear halo around the colonies. Halos are formed when the
40 D. G. Pitcher, N. A. Saunders, and R. J. Owen, Lett. Appl. Microbiol. 8, 151 (1989). 41 G. Antranikian, P. L. JCrgensen, M. Wtimpelmann, and S. T. JCrgensen, Patent WO 92/ 02614.
280
SACCHAROLYTIC ENZYMES Chromosomal DNA Partial Sau3A
[ 17]
Plasmid pSJ1678 BamHI digest
digestion
3-7 kb fragments isolated
3.8 kb fragment isolated
Ligation Electroporation into E. eoli Plating on red-dye substrate plates
Replica plating and incubation at 37° overnight.
l,u u - o v
]
~
'
I
Isolation of amylolytic reeombinants (5 among 10,000) FIO. 2. Outline of the methodology applied to isolate, clone, and detect DNA fragments of chromosomal DNA from hyperthermophilic Archaea and Bacteria exhibiting amylolytic or pullulytic activity.
[17]
AMYLOLYTICENZYMES
281
dye amylopectin or pullulan complex is attacked by the enzyme, causing the release of the dye. Escherichia coli transformants are plated on LB agar containing 10/zg! ml chloramphenicol and, after 16 hr of incubation at 37°, approximately 14,000 colonies are observed. These are replica plated onto a new set of LB plates containing 2% agar, 6 /~g chloramphenicol/ml, and 1% dyed amylopectin or pullulan and grown overnight at 37°. The plates are then incubated at 60° for 4 hr, and the positive clones are identified by halo formation. E. coli clones expressing the recombinant extracellular a-amylases and pullulanase are grown aerobically at 37° in LB medium 42 containing 10 /zg/ml chloramphenicol. The plasmid containing the gene of interest is isolated using a kit from Qiagen (Hilden, Germany). Gene Expression and Characterization
Using the just mentioned method, we were able to isolate E. coIi clones containing the a-amylase gene from P. furiosus, 16 the pullulanase type II (amylopullulanase) from P. woesei, 3° and the debranching enzymes (pullulanase type I) from F. pennivorans. 35 This section reports on the expression and characterization of the gene encoding pullulanase type I from F. pennivorans Ven5 and the purification of the recombinant enzyme after expression in E. coll. After shogun cloning, E. coil clone PL2125 producing thermostable pullulanase type I from F. pennivorans Ven5 is further characterized. The entire 8.1-kb insert is sequenced in both directions and three large open reading frames (ORFs) are identified. ORF1 is confirmed as encoding a pullulanase by subcloning into pUC18 and observing activity of the E. coli transformants on red-dyed pullulan plates. The subcloning is performed by carrying out PCR amplification using the Expand Long Template PCR System (Boehringer Mannheim) using the following temperature profile: 94°, 2 rain and 30 cycles of 94°; 10 sec, 45°; 45 sec, 68°, 4 min. Cloning of PCRamplified fragments is carried out using the TA cloning kit (Invitrogen). Pullulanase activity can be seen after overnight growth of the positive clones on LB medium containing red-dyed pullulan at 37° and after heat treatment at 70 ° for 16 hr. The plasmid pSE420 containing the IPTGinducible trc promoter (Invitrogen) is used for~expression. The pullulanase gene of F. pennivorans Ven5 (pulA) is 2550 bp in length and encodes a protein of 849 amino acids with a predicted molecular mass of 96.6 kDa before processing. A Shine-Dalgarno-like sequence of A G G A G G is present at positions -10 to -15 from the ATG site. The 42 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
282
SACCHAROLYTICENZYMES
[ 171
G + C content of puIA is 41.9%. A signal sequence of 28 amino acids is present with a cleavage site between the amino acids Ala and Glu. This was confirmed by N-terminal sequencing of the mature pullulanase isolated from F. pennivorans Ven5 (ETELIIHYHRW). The pulA gene is subcloned without its signal sequence and overexpressed in E. coli (under the control of the trc promoter). It is interesting to note that this clone, E. coli FD748, produces two proteins (93 and 83 kDa) with pullulanase activity. This is not due to proteolytic hydrolysis of the recombinant mature protein but due to the presence of a second start site (TTG) identified 128 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG). The TTG translation initiation codon was then mutated to produce only the mature 93-kDa protein. Mutation of the leucine codon, TTG, was carried out by PCR according to the method described by Nelson and Long. 43 The primers are as follows: mutation primer, GTG GCT c T r A C A A G G A A T AG; nonsense, CGA TCG ATC G A G G A T CCT TA; reverse + nonsense, CGA TCG ATC G A G G A T CCT TAT T A A TFA CCT TTG TAC ATT ACC; and forward, ATA A A C ATG TCG G A A ACA G A G CTG A T r ATC. The PCR product is cloned into pCR2.1 and the mutation is confirmed by sequencing. The fragment containing the mutation is digested with AfllII and BamHI and cloned into pSE420/NcoI and BamHI. The mutation is again confirmed by sequencing. Pullulanase-containing clones are detected on pullulan-red agar plates. E. coli FD748 containing the pullulanase cloned without the signal peptide is cultivated in LB or TB medium 42 containing 100 /xg/ml ampicillin and induced with 1 mM IPTG, when the absorbance of the culture at 600 nm is approximately 0.8. The pullulanase expression level of this clone is 40 times higher than that from E. coli PL2125.
Purification of Recombinant Pullulanase from E. coli FD748 In general, heat treatment of the cell extract is ideal for the purification of heat-stable recombinant proteins. All purification steps are performed at room temperature. E. coli cells (10 g) expressing pullulanase type I from F. pennivorans Ven5 are washed with 25 mM Tris-HCl, pH 7.4 (buffer A), and then resuspended with 50 ml of the same buffer. Cells are disrupted by sonication, and cell debris is removed by centrifugation for 20 min at 30,000g. The supernatant is heat treated at 75 ° for 60 min and the denatured host proteins are pelleted by centrifugation (15 min at 30,000g). The pullulanase remains in the clear supernatant. The specific activity of the purified 43 R. M. Nelson and G. L. Long, Anal Biochem. 180, 147 (1989).
[ 17]
AMYLOLYTICENZYMES
283
recombinant pullulanase of F. pennivorans Ven5 expressed in E. coli FD748m (rpulA) is 3 U/mg (Table II). The pullulanase preparation after heat treatment is applied to a /3cyclodextrin-epoxy-activated Sepharose column (1 × 10 cm) equilibrated in 50 mM sodium acetate buffer, pH 6.0 (buffer C). The column is washed stepwise with 50 ml of buffer C and then with the same buffer containing 1 M NaCI until no absorbance at 280 nm is detectable. Pullulanase activity is eluted with 1% pullulan in buffer C, containing 1 M NaC1. Active fractions are pooled, concentrated by ultrafiltration (cutoff 10 kDa), and dialyzed against 1000 volumes of buffer A. The protein solution is then applied to a Mono Q HR 5/5 column (Pharmacia LKB, Freiburg, Germany), which is equilibrated with buffer B, and elution is carried out with the same buffer at a flow rate of 0.5 ml/min. After anion-exchange chromatography on Mono Q, a single protein band is observed. The recombinant full-length pullulanase is purified 25-fold with a specific activity of 75 U/mg and a final yield of about 11.7%. (Table II). The pure enzyme on SDS-PAGE has a molecular mass of 93 kDa. The estimated molecular weight of the native recombinant pulA, calculated in a native gradient gel electrophoresis, is 190 kDa. Accordingly, the enzyme, which is optimally active at 80° and pH 6.0, is a homodimer. /3-Cyclodextrin-Sepharose affinity chromatography is a suitable step to purify pullulanases because cyclodextrins are competitive inhibitors of these enzymes. The/3-cyclodextrin-Sepharose affinity matrix is prepared by coupling /3-cyclodextrin of epoxy-activated Sepharose 6B according to the following protocol following the instructions of the manufacturer (Affinity Chromatography, Pharmacia Fine Chemicals, Uppsala, Sweden). The ep-
T A B L E II PURIFICATION OF RECOMBINANT PULLULANASEFROM E. coli FD748m CLONEa
Step Crude extract Heat treatment (1 hr at 75 °) /3-Cyclodextrin-Sepharose Mono Q
Total protein (rag)
Total activity (U) h
747 172
2268 2184
16.2 3.56
873.6 267
Specific activity (U/mg)
Yield (%)
Purification (-fold)
3 12.7
100 96
4.2
53.9 75
23 11.7
17.9 25
After the aerobic growth of E. coli at 37 °, a 5-liter culture was centrifuged (6 g of ceils wet weight) and the ceils were disrupted by sonication. b One unit of pullulanase catalyzes the formation of 1/zmol of reducing sugars/min under the defined conditions; maltose was used as a standard.
284
SACCHAROLYTICENZYMES
[ 17]
oxy-activated Sepahrose 6B (5 g) is washed with distilled water (1 liter) and reswelled in 0.1 M carbonate buffer, pH 11. To this gel suspension, 40 ml of/3-cyclodextrin (40/zmol x g resin) is added and mixed. The mixture is left under agitation on a shaker (60 rpm) at 40 ° for 16 hr. Excess ligand is removed by washing with carbonate buffer (pH 11) and then with 0.1 M phosphate buffer, pH 8. The remaining epoxy groups are blocked with 1 M ethanolamine dissolved in 0.1 M phosphate buffer, pH 8. After incubation for 16 hr, the resulting/3-cyclodextrin-Sepharose is washed with water and then equilibrated with 50 mM sodium acetate buffer, pH 5.5. Analytical Methods
Assay for Amylolytic/Pullulytic Enzymes Amylolytic/pullulytic activity is determined by measuring the amount of reducing sugars released during incubation with a substrate. To 50 ~1 of l% (w/v) substrate (starch for amylolytic and pullulan for pullulytic activity) dissolved in 50 mM sodium acetate buffer (pH 6.0), 25 or 50/~1 of enzyme solution is added, and the samples are incubated at different temperatures for 10 to 60 min. The reaction is stopped by cooling on ice, and the amount of reducing sugars released is determined by the dinitrosalicylic acid (DNS) method. During starch degradation, monomeric or oligomeric sugars are released. The reducing ends of the released sugars bind covalently to (DNS) at 100°, forming a red complex. The absorption of this complex is determined photometrically at 546 nm and is linearly proportional to the amount of reducing sugars. One unit (U) is defined as the amount of enzyme required to liberate 1/xmol of reducing sugar (with maltose as standard) per minute at a temperature, which is optimal for enzymatic activity, a-Amylase or pullulanase is measured in 50 mM sodium acetate buffer at pH 6.0 and 0.5% (w/v) substrate (starch or pullulan). In order to calculate the enzyme concentration (U/ml), the following equation is used: AEFV U/ml - - t where AE is extinction at 546 nm against a blank value, F is the factor from the maltose standard curve (1.02), V is the factor considering the sample volume employed in the assay,* and t is the incubation time at optimal temperature in minutes. * Units should be calculated for l - m l samples. Sample blanks are extremely important and are used to correct for the nonenzymatic release of reducing sugars that can take place u n d e r assay conditions.
[ 17]
AMYLOLYT1CENZYMES
285
Gel Electrophoresis
In order to determine the size of the native enzyme, native polyacrylamide gels containing a gradient of 5 to 27% polyacrylamide are prepared as described by Koch et aL44 Gels are run at 300 V for 24 hr at 4. High molecular weight marker proteins (Pharmacia Biotech) are used as standards. In order to examine the subunit composition of the pullulanase, protein samples are also analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (12% S D S - P A G E ) as described by Laemmli 45 after heating the samples at 100° for 5 min. Low molecular weight marker proteins (Pharmacia Biotech) are used as standards. Following native and SDS-PAGE, the proteins are stained with Coomassie blue. Zymogram staining for pullulytic activity is performed according to Furegon et al. 46 The red dye-pullulan, prepared as described previously, is incorporated in the resolving native or SDS gel at a final concentration of 1% (v/v). For electrophoresis, samples are diluted in buffer according to Laemmli. 45 After electrophoresis performed at room temperature under 20 mA, the gels are immersed in 100 ml of buffer at the optimum pH for the amylolytic activity preheated at 70° and incubated with gentle agitation at the same temperature in a water bath. When SDS gels are used, the buffer is changed after the first 10 min of incubation. After a period of time that varies according to the activity of the enzyme assayed, pullulanase is detectable as a yellow band on a red background (Fig. 3). When the desired intensity of the yellow band is reached, the reaction is stopped by immersion of the gel in 7.5% (v/v) acetic acid. Amylolytic activity is detected as follows: after rinsing the native or SDS gel with water, it is soaked in 50 mM sodium acetate (buffer C) containing 1% starch (w/v) at 4° for 60 min. The gel is further incubated at the optimum temperature required for enzymatic activity in buffer C. After 2 min of incubation, the gel is soaked in a solution containing 0.15% (w/v) iodine and 1.5% (w/v) potassium iodide solution until clear bands on a dark brown-blue background become visible (Fig. 4). The stained gels are stored in 10% (v/v) of ethanol at room temperature. Characterization of Hydrolysis Product
Hydrolysis products arising after the action of amylolytic enzymes on various linear and branched polysaccharides are analyzed by high-perfor4,~R. Koch, F. Canganella, H. Hippe, K. D. Jahnke, and G. Antranikian, AppL Environ. Microbiol. 63, 1088 (1997). 45 U. K. Laemli, Nature 227, 680 (1970). 46 L. Furegon, A. Curioni, and D. B. A. Peruffo, Anal Biochem. 221, 200 (1994).
286
SACCHAROLYTIC ENZYMES
1
[ 17]
2 kDa
- 94
- 67
- 45
- 30
FIG. 3. SDS-PAGE of a partially purified pullulanase from Desulfurococcus mucosus. The halo indicating pullulytic activity (lane 1) was obtained by loading 20/zg of protein onto SDS-PAGE, followed by electrophoresis, removal of SDS, and staining for heat-stable pullulytic activity. Lane 2, molecular markers. Protein bands were then detected with Coomassie blue (0.1%).
mance liquid chromatography (HPLC) with an Aminex HPX-42A column (300 by 78 mm) (Bio-Rad, Hercules CA). A refractometer is used to detect the peaks. Double distilled water is used as the mobile phase at a flow rate of 0.3 ml/min. Various oligosaccharide peaks (DP7 to DP1) are eluted between 9 and 18 min. The purified pullulanase is incubated at 65 ° with 0.5% (w/v) pullulan, starch, glycogen, amylopectin, maltodextrin, panose, and 0.2% (w/v) amylose. Samples are withdrawn at different time intervals, and the reaction is stopped by incubation on ice. Figure 5 shows the mode of action of pullula-
[ 171
AMYLOLYTICENZYMES kDa
1
2
287
3
440210 140-
7-
FIG. 4. Native polyacrylamide gel (left) with corresponding zymogram (right) of the purified recombinant a-amylase from Pyrococcus woesei. For the zymogram, 20 mU of a-amylase activity was loaded onto a native gel followed by electrophoresis and staining for heat-stable a-amylase activity as described in the text. Lane 1, molecular marker; lane 2, purified aamylase from Pyrococcus woesei; lane 3, zymogram.
nase type I from F. pennivorans Ven5. The thermostable pullulanase hydrolyzes more than 98% of pullulan after 1 hr of incubation at 80° (Fig. 5a). The hydrolysis pattern, after its action on pullulan, reveals the complete conversion of pullulan to maltotriose in an endo-acting fashion by attacking the a-l,6-glycosidic linkages. In order to confirm that the hydrolysis product from pullulan is maltotriose (possessing o~-l,4-glycosidic linkages) and not panose or isopanose (possessing two a-l,4- and o~-l,6-glycosidic linkages), incubation of the products of pullulan hydrolysis is performed in the presence of o~-glycosidase from yeast. The formation of glucose as the main product confirms the formation of maltotriose (and not panose) from pullulan (Fig. 5b). No degradation of amylose is observed after 16 hr of incubation at 65 ° with the recombinant pullulanase demonstrating the low affinity of the purified enzyme to a-l,4-glycosidic linkages. In contrast to this, incubation of amylose with purified recombinant pullulanase type II at 100° from P. woesei 3° leads to the formation of oligosaccharides of different degrees of polymerization and glucose, thus indicating activity toward a-l,6- and o~-l,4-glycosidic linkages (Fig. 5c). After 72 hr of incubation with the purified rpulA, very low levels of maltose and maltotriose are detectable in the
288
[ 171
SACCHAROLYTIC E N Z Y M E S )pn
c~
Pullulan
o~
DP3
Dpl
Dpl
Dp3
Control
1h
Opn
Amylose
Maltotrlose+ 16h + a-glucosldase (x-glucooldase
Pullulanl6 h
2h
d
Starch ~pn
Dpn
Dpn Dpn
ID
Dpn |
Control
16h
Amylopectin
e
Op6 - 1
2 h with PulhlhuIIImDlype |1
from P. wofp~
Control
16h
48h
Glvcoqen
Dpn
pn
Dpn
Dpn
Control
16h
~h
Control
Dpn
16h
48 h
FIG. 5. HPLC analysis of hydrolysis products formed after incubation of the purified recombinant pullulanase from Fervidobacteriumpennivorans Ven5 in the presence of different substrates: (a) 0.5% pullulan, (d) 0.5% starch, (e) 0.5% amylopectin, and (f) 0.5% glycogen. Samples were incubated at 65° and aliquots were withdrawn and analyzed on an Aminex HPX 42-A column for oligosaccharides at different time intervals. (b) The hydrolysis product formed after incubation of 0.5% pullulan with pullulanase from F. pennivorans Ven5 (16 hr pullulan) and 0.5% maltotriose was incubated with commercial a-glucosidase and then analyzed by HPLC. (c) HPLC analysis of hydrolysis products formed after incubation of 0.2% amylose in the presence of recombinant pullulanases from F. pennivorans Ven5 (65° for 16 hr) and from P. woesei(75° for 2 hr). DP, degree of polymerization; DP1, glucose; DP2, maltose; and SO o n .
h y d r o l y s i s p r o d u c t o f s o l u b l e s t a r c h (Fig. 5d), a m y l o p e c t i n (Fig. 5e), a n d g l y c o g e n (Fig. 5 0 . A c c o r d i n g to t h e s e results, rpulA a t t a c k s specifically ot1,6 l i n k a g e s o f puUulan a n d b r a n c h e d o l i g o s a c c h a r i d e s a n d is classified as p u l l u l a n a s e t y p e I.
[ 171
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Biotechnological Relevance The finding of extremely thermostable starch-hydrolyzing enzymes such as amylases and pullulanases that are active under similar conditions will significantly improve the industrial starch bioconversion process, i.e., liquefaction, saccharification, and isomerization. Because of the lack of novel thermostable enzymes that are active and stable above 100° and at acidic pH values, the bioconversion of starch to glucose and fructose has to be performed under various conditions. This multistage process (step 1: pH 6.0-6.5, 95-105°; step 2: pH 4.5, 60-62°; step 3: pH 7.0-8.5, 55-60 °) is accompanied by the formation of undesirably high concentrations of salts. In the final step, where high fructose syrup is produced, salts have to be removed by expensive ion exchangers. In addition, by using robust starchmodifying enzymes from hyperthermophilic microorganisms, innovative and environmentally friendly processes can be developed aimed at the formation of products of high added value from native starch for the food industry. New and enhanced functionality can be obtained by changing the structural properties of starch. In order to prevent retrogradation, starchmodifying enzymes can be used at a higher temperature. The use of extremely thermostable amylolytic enzymes can lead to valuable products, which include innovative starch-based materials with gelatine-like characteristics and defined, linear dextrins that can be used as fat substitutes, texturizers, aroma stabilizers, and prebiotics. 1,47 CGTases are used for the production of cyclodextrins that can be used as a gelling, thickening, or stabilizing agent in jelly desserts, dressing, confectionery, and dairy and meat products. Because of the ability of cyclodextrins to form inclusion complexes with a variety of organic molecules, cyclodextrins improve the solubility of hydrophobic compounds in aqueous solution. This is of interest for pharmaceutical and cosmetic industries. Cyclodextrin production is a multistage process in which starch is first liquefied by a heat-stable amylase, and in the second step a less-thermostable CGTase from Bacillus sp. is used. Due to the low stability of the latter enzyme, the second step must run at lower temperatures. The application of heat-stable CGTase in jet cooking, where temperatures up to 105° are achieved, will allow liquefaction and cyclization to take place in one step. 48
47 W. D. Crabb and C. Mitchinson, Tibtech 15, 349 (1997). 48 B. E. Norman and S. T. J0rgensen, Denpun Kagaku 39, 101 (1992).
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[18] Cellulolytic Enzymes from Thermotoga Species B y WOLFGANG LIEBL
Introduction
/3-Glucans are important natural polymers consisting of/3-glycosidically linked glucose residues. These polysaccharides mostly fulfill structural roles. The /3-1,4-glucan cellulose and mixed-linkage /3-1,4/1,3-glucans, such as barley/3-glucan and lichenan, occur as cell wall components of higher plants and lichens, and /3-1,3-glucans are found in the cell walls of yeast and filamentous fungi and as structural and storage polysaccharide of the marine macroalga Laminaria saccharina (laminarin). Thermostable enzymes for the degradation of these types of/3-glucans and/or their corresponding genes have been isolated from different strains of the heterotrophic strictly anaerobic bacterial genus Thermotoga.a-l° The following is an overview of /3-glucan-cleaving enzymes found in Thermotoga species and describes the preparation and characteristics of an endoglucanase and a/3-glucosidase of Thermotoga maritima. /3-Gluca_n-Degrading Enzymes of Thermotoga Species In T. maritima, a hyperthermophile with an optimum growth temperature of 80°, enzymes capable of the hydrolysis of/3-1,4-glucans and mixed1 W. Liebl, P. Ruile, K. Bronnenmeier, K. Riedel, F. Lottspeich, and I. Greif, Microbiology 142, 2533 (1996). 2 K. Bronnenmeier, A. Kern, W. Liebl, and W. Staudenbauer, Appl. Environ. MicrobioL 61, 1399 (1995). 3 O. N. Dakhova, N. E. Kurepina, V. V. Zverlov, V. A. Svetlichnyi, and G. A. Velikodvorskaya, Biochem. Biophys. Res. Cornmun. 194, 1359 (1993). 4 L. D. Ruttersmith and R. M. Daniel, Biochem. J. 277, 887 (1991). 5 L. D. Ruttersmith and R. M. Daniel, Biochim. Biophys. Acta 1156, 167 (1993). 6 J.-D. Bok, D. A. Yernool, and D. E. Eveleigh, Appl. Environ. Microbiol. 64, 4774 (1998). 7 J.-D, Bok, S. K. Goers, and D. E. Eveleigh, in "Enzymatic Conversion of Biomass for Fuels Production" (M. E. Himmel, J. O. Baker, and R. P. Overend, eds.), p. 54. American Chem. Society, Washington, DC, 1994. 8 D. E. Eveleigh, J. D. Bok, H. E1-Dorry, S. EI-Gogary, K. Elliston, A. Goyal, C.. Waldron, R. Wright, and Y.-M. Wu, Appl. Biochem. Biotech. 51152, 169 (1995). 9 V. V. Zverlov, I. Y. Volkov, T. V. Velikodvorskaya, and W. Schwarz, Microbiology 143, 1701 (1997). ~oV. V. Zverlov, I. Y. Volkov, T. V. Velikodvorskaya, and W. Schwarz, Microbiology 143, 3537 (1997).
METHODS IN ENZYMOLOGY.VOL. 330
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closely related ~-glucosidase from P. woesei possesses an even higher activity toward sucrose than to maltose. 1° Comparisons between the putative oz-glucosidases from different organisms will be facilitated greatly by the availability of the amino acid (gene) sequences of these enzymes.
Acknowledgments RMK acknowledges the Novartis Agricultural Biotechnology Research Institute (NABRI) (Research Triangle Park, NC), the U.S. Department of Agriculture, and the National Science Foundation for financial support of this work.
[1 7]
Amylolytic Enzymes
By
COSTANZO
BERTOLDO
from Hyperthermophiles and
GARABED
ANTRANIKIAN
Introduction In recent years, many hyperthermophilic organisms that are able to grow at temperatures up to the normal boiling point of water have been shown to utilize natural polymeric substrates as carbon and energy sources. These extreme thermophilic microorganisms, which belong to Archaea and Bacteria, can facilitate the enzymatic degradation of carbohydrates by producing heat-stable enzymes that are catalyically active above 100°. Such enzymes share the ability to hydrolyze the glycosidic bonds between two or more sugar molecules or between a carbohydrate and a noncarbohydrate moiety. The enzymatic hydrolysis and the modification of polysaccharides are of great interest in the field of food technology. The potential exploitation of this natural source of sugars is not only useful for glucose syrup production, but also for the synthesis of nonfermentable carbohydrates, anticariogenic agents, and antistaling agents in baking, a In most hyperthermophilic microorganisms, the activity levels of starch-hydrolyzing enzymes appear to be too low for biotechnological applications. The molecular cloning of the corresponding genes and their expression in heterologous hosts, however, circumvent the problem of insufficient expression in the natural host. The number of genes from hyperthermophiles encoding amylolytic enzymes that have been cloned and expressed in mesophiles has been increasing significantly. In most cases, the thermostable proteins expressed in mesophilic hosts maintain their thermostability, are correctly i D. Cowan, Tibtech 14, 177 (1996).
METHODS IN ENZYMOLOGY, VOL. 330
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folded at low temperature, are resistant to host proteolysis, and can be purified easily using thermal denaturation of the mesophilic host proteins. The degree of enzyme purity obtained is generally suitable for most industrial applications. This article briefly discusses the enzymatic action and properties of starch-hydrolyzing enzymes and focuses only on those enzymes that have been isolated and characterized from extreme thermophilic and hyperthermophilic Archaea and Bacteria. It also briefly describes their potential biotechnological exploitation. Some of these aspects have been already presented. 2-8 Furthermore, this article describes the general methodology for gene identification and expression and the procedure for the purification of some amylolytic enzymes that have been successfully isolated and extensively studied in the authors' laboratory.
Amylolytic E n z y m e s Starch from cultivated plants represents a ubiquitous and easily accessible source of energy. In plant cells or seeds, starch is usually deposited in the form of large granules in the cytoplasm. Starch is composed exclusively of a-glucose units that are linked by ot-1,4- or ot-1,6-glycosidic bonds. The two high molecular weight components of starch are amylose (15-25%), a linear polymer consisting of tx-1,4-1inked glucopyranose residues, and amylopectin (75-85%), a branched polymer containing, in addition to a1,4-glycosidic linkages, ot-l,6-1inked branch points occurring every 17-26 glucose units, ot-Amylose chains, which are not soluble in water but form hydrated micelles, are polydisperse and their molecular weights vary from several hundred to thousands. The molecular weight of amylopectin may be as high as 100 million and in solution such a polymer has colloidal or micellar forms. Because of the complex structure of starch, cells require an appropriate combination of hydrolyzing enzymes for the depolymerization of starch to 2 G. Antranikian, in "Microbial Degradation of Natural Products" (G. Winkelmann,ed.), Vol. 2, p. 28. VCH, Weilheim,1992. 3R. Ladenstein and G. Antranikian, Adv. Biochem. Eng./Biotechnol. 61, 37 (1998). 4 C. Leuschner and G Antranikian, World J. Microbiol. Biotechnol. 11, 95 (1995). 5A. R0diger,A. Sunna, and G. Antranikian, in (D. Waldmann,ed.), "Carbohydrases:Handbook of Enzyme Catalysisin Organic Synthesis,"p. 946. VCH, Weilheim,1994. 6A. Sunna, M. Moracci, M. Rossi, and G. Antranikian, Extremophiles 1, 2 (1996). 7R. MOiler,G. Antranikian, S. Maloney,and R. Sharp, Adv. Biochem. Eng. Biotechnol. 61, 155 (1998). 8F. Niehaus, C. Bertoldo, M. Ktihler,and G. Antranikian, AppL Microbiol. Biotechnol. 51, 711 (1999).
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oligosaccharides and smaller sugars, such as glucose and maltose. They can be simply classified into two groups: endo-acting enzymes or endohydrolases and exo-acting enzymes or exohydrolases (Fig. 1). Endo-acting enzymes, such as a-amylase (a-l,4-glucan-4-glucanohydrolase; EC 3.2.1.1), hydrolyze linkages in the interior of the starch polymer in a random fashion, which
0Oo
gM~,#trAnsforme~
00• • glucoee
c~ maltmm
oooo
134rny~.o g~.coe.~m
c~
mJtouand
~.limit-dextrin
•
g~co~
~
o~
O 0 m~ose •
~-.-~
oo~
type l and
pullulan
I/
O-C)-O o~o maltotriose
~ ~
~,,
panose
punulm hydrolmetype~l% l Isopanose
FXG. 1. Schematic presentation of the action of amylolytic and pullulytic enzymes. Pullulanase type I also attacks a-l,6-glycosidic linkages in oligo- and polysaccharides. Pullulanase type II attacks, in addition, a-l,4-1inkages in various oligo- and polysaccharides.
272
SACCHAROLYTICENZYMES
[ 17]
leads to the formation of linear and branched oligosaccharides. The sugarreducing groups are liberated in the a-anomeric configuration. Most starchhydrolyzing enzymes belong to the a-amylase family, which contains a characteristic catalytic (fl/a)s-barrel domain, a-Amylases belong to two families, number 13 and 57 of the glycosylhydrolase families.9 Family 13 has approximately 150 members from Eukarya and Bacteria. Family 57, however, has only three members from Bacteria and Archaea. Throughout the a-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. 1° On the structural level, there are to date no X-ray structures of amylolytic enzymes derived from hyperthermophiles. Exo-acting starch hydrolases include//-amylase, glucoamylase, a-glucosidase, and isoamylase. These enzymes attack the substrate from the nonreducing end, producing small and well-defined oligosaccharides, r Amylase (EC 3.2.1.2), also referred to as a-l,4-D-glucan maltohydrolase or saccharogen amylase, hydrolyzes a-l,4-glucosidic linkages to remove successive maltose units from the nonreducing ends of the starch chains, producing//-maltose by an inversion of the anomeric configuration of the maltose.//-Amylase belongs to family 14 of the glycosylhydrolases, having 11 members from Eukarya and Bacteria. Glucoamylase (EC 3.2.1.3) hydrolyzes terminal a-l,4-1inked-D-glucose residues successively from nonreducing ends of the chains, releasing//-Dglucose. Glucoamylase, which is typically a fungal enzyme, has several names: a-l,4-D-glucan hydrolase, amyloglucosidase, and y-amylase. Most forms of the enzyme can hydrolyze a-l,6-D-glucosidic bonds when the next bond in sequence is a-l,4. However, in vitro this enzyme hydrolyzes a-l,6and a-l,3-D-glucosidic bonds in other polysaccharides with high molecular weights. a-Glucosidase (EC 3.2.1.20), or a-D-glucoside glucohydrolase, attacks the a-l,4 linkages of oligosaccharides that are produced by the action of other amylolytic enzymes. Unlike glucoamylase, a-glucosidase liberates glucose with an a-anomeric configuration, a-Glucosidases are members of family 15 and the very diverse family 31 of the glycosylhydrolases.9 Isoamylase (EC 3.2.1.68), or glycogen 6-glucanohydrolase, is a debranching enzyme specific for a-l,6 linkages in polysaccharides, such as amylopectin, glycogen, and //-limit dextrin, but it is unable to hydrolyze the a-l,6 linkages in pullulan or branched oligosaccharide; therefore, it has limited action on alimit dextrin. 9B. Henrissat,Biochem. J. 280, 309 (1991). 10T. Kuriki and T. Imanaka,J. Biosci. Bioeng. 87, 557 (1999).
[ 171
AMYLOLYTICENZYMES
273
Pullulytic Enzymes Pullulan is a linear a-glucan consisting of maltotriose units joined by a-l,6-glycosidic linkages and it is produced by Aureobasidium pullulans with a chain length of 480 maltotriose units. Enzymes capable of hydrolyzing a-l,6 glycosidic bonds in pullulan are defined as pullulanases (Fig. 1). On the basis of substrate specificity and product formation, pullulanases have been classified into two groups: pullulanase type I and pullulanase type II. Pullulanase type I (EC 3.2.1.41) specifically hydrolyzes the a-l,6 linkages in pullulan as well as in branched oligosaccharides, and its degradation products are maltotriose and linear oligosaccharides, respectively. Pullulanase type I is unable to attack a-l,4 linkages in a-glucans and belongs to family 13 of the glycosylhydrolases. Pullulanase type II, or amylopullulanase, attacks a-l,6-glycosidic linkages in pullulan and a-l,4 linkages in branched and linear oligosaccharides (Fig. 1). The enzyme has a multiple specificity and is able to fully convert polysaccharides (e.g., amylopectin) to small sugars (DP1, DP2, DP3; DP is the degree of polymerization) in the absence of other enzymes, such as a-amylase or fl-amylase. In contrast to the previously described pullulanases, pullulan hydrolases type I and type II are unable to hydrolyze a-l,6-glycosidic linkages in pullulan or in branched substrates. They can attack a-l,4-glycosidic linkages in pullulan-forming panose or isopanose. PuUulan hydrolase type I or neopullulanase (EC 3.2.1.135) hydrolyze pullulan to panose (a-6-D-glucosylmaltose). Pullulan hydrolase type II or isopullulanase (EC 3.2.1.57) hydrolyzes pullulan to isopanose (a-6-maltosylglucose) (Fig. 1). Finally, cyclodextrin glycosyltransferase (EC 2.4.1.19), or a-l,4-Dglucan a-4-D-(a-l,4-D-glucano)transferase, is an enzyme that is generally found in Bacteria and has been discovered in Archaea. This enzyme produces a series of nonreducing cyclic dextrins from starch, amylose, and other polysaccharides, a-, fl-, and ~/-cyclodextrins are rings formed by 6, 7, and 8 glucose units that are linked by a-l,4 bonds, respectively (Fig. 1).
Enzyme Production by Hypertherinophilic Microorganisms Hyperthermophilic Bacteria and Archaea that are able to grow on starch at temperatures over 70° have been isolated and the corresponding starchdegrading enzymes have been purified and characterized. In several cases, genes encoding these enzymes have been isolated, cloned, and overexpressed in heterologous hosts (Table I).
TABLE I STARCH-HYDROLYZING ENZYMES FROM EXTREMELY THERMOPHILIC AND HYPERTHERMOPHILIC ARCHAEA AND BACTERIA Enzyme properties a
Enzyme a-Amylase
Organism a
Desulfurococeus mucosus (85) Pyrococcus furiosus (100)
Pyrococcus sp. KOD1 P. woesei (100) Pyrodictium abyssi (98) Staphylothermus rnarinus
Optimal temperature 85 100 100
Optimal pH
Molecular mass (kDa)
5.5
74
6.5-7.5 7.0
Purified/cloned
129 68
90
6.5
49.5
100 100 100
5.5 5.0 5.0
68
Remarks
b --
Purificd/cloned/intracellular Purified/cloned/extracellular Purified/cloned/extracellular Purified/extracellular Crude extract Crude extract
(90)
Pullulanase type I
Pullulanase type It
Glucoamylase
CGTase ~x-Glucosidase
Sulfolobus solfataricus (88) Thermococcus celer (85) T. profundus DT5432 (80) T. profundus (80) T. aggregans (85) Dyctyoglomus thermophilum Rt46B.1 (73) Thermotoga maritima MSB8 (90) Fervidobacterium pennivorans Ven5 (75) T. maritima MSB8 (90) Thermus caldophilus GK24 (75) Desulfurococcus mucosus (88) P. woesei (100)
--
--
240
90 80
5.5 5.5
80 95 90
4.0-5.0 6.5 5.5
42
7.0
61
85-90
Extracellular --
42
-75
Purified/"Amy L" Cloned Purified/cloned/cytoplasmic fraction Purified/cloned/lipoprotein
80
6
90 75
6.0 5.5
93 (subunit) 65
Cloned/type I c Purified/ceU associated
85
5.0
74
Purified/cloned
100
6.0
90
P. abyssi (98) T. celer (85) Thermococcus litoralis (90) T. hydrothermalis (80)
100 90 98
9.0 5.5 5.5
119
95
5.5
128
T. aggregans (85) Thermoplasma acidophilum (60) Picrophilus oshimae (60) P. torridus (60) Thermococcus sp. (75) Thermococcus strain AN1 (80) Thermococcus hydrothermalls (80)
100 90
6,5 6.5
83 141
Purified/cloned/cell associated Crude extract Crude extract Purified/extraccUular/glycoprotein Purified/extracellular/glycoprotein Purified/cloned Purified
90 90 100 130
2.0 2.0 2.0
140 133 83 63
--
190 (93)
Crude extract Purified/cloned/"Amy S"
---
--
--
--
Purified/cloned
Purified Purified Purified Purified/extracellular/glycoprotein Cloned
Values in parentheses give the optimal growth temperature for each organism in degrees Celsius. b Not determined. c Unpublished results.
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Heat-Stable Amylases and Glucoamylases Extremely thermostable a-amylases have been characterized from Pyrococcus furiosus, 11 eyrococcus woesei, 12 and Thermococcus profundus. 13 Optimal temperatures for the activity of these enzymes are 100 °, 100°, and 80°, respectively. In addition, either amylase and/or pullulanase activities have been observed in hyperthermophilic Archaea of the genera Sulfolobus, Thermophilum, Desulfurococcus, Thermococcus, and Staphylothermus.14,15 Molecular cloning of the corresponding genes and their expression in heterologous hosts circumvent the problem of insufficient expression in the natural host. The gene encoding an extracellular a-amylase from P. furiosus has been cloned and the recombinant enzyme expressed in B. subtilis and Escherichia coli. 16,17This is the first report on the expression of an archaeal gene derived from an extremophile in a Bacillus strain. The high thermostability of the pyrococcal extracellular a-amylase (thermal activity even at 130°) in the absence of metal ions, together with its unique product pattern and substrate specificity, makes this enzyme an interesting candidate for industrial application. In addition, an intracellular a-amylase gene from P. furiosus has been cloned and sequenced. 18 It is interesting that the four highly conserved regions usually identified in a-amylases are not found in this enzyme, a-Amylases with lower thermostability and thermoactivity have been isolates from the archaea T. profundus, 19Pyrococcus sp. KOD 1,2° and the bacterium Thermotoga maritima. 21 Genes encoding these enzymes were successfully expressed in E. coli. Similar to the amylase from B. licheniformis, which is commonly used in liquefaction, the enzyme from T. maritima requires Ca 2+ for activity. Further investigations have shown that 11 R. Koch, K. Spreinat, K. Lemke, and G. Antranikian, Arch. Microbiol. 155, 572 (1991). 12 y. C. Chung, T. Kobayashi, H. Kanai, T. Akiba, and T. Kudo, Appl. Environ. Microbiol. 61, 1502 (1995). 13j. T. Lee, H. Kanai, T. Kobayashi, T. Akiba, and T. Kudo, J. Ferment. Bioeng. 82, 432 (1996). 14j. M. Bragger, R. M. Daniel, T. Coolbear, and H. W. Morgan, Appl. Microbiol. Biotechnol. 31, 556 (1989). 15F. Canganella, C. Andrade, and G. Antranikian, Appl. MicrobioL BiotechnoL 42, 239 (1994). J6 G. Dong, C. Vieille, A. Savchenko, and J. G. Zeikus, Appl. Environ. Microbiol. 63, 3569 (1997). 17 S. Jc~rgensen, C. E. Vorgias, and G. Antranikian, J. Biol. Chem. 272, 16335 (1997). 18 K. A. Laderman, K. Asada, T. Uemori, H. Mukai, Y. Taguchi, I. Kato, and C. B. Anfinsen, J. Biol. Chem. 268, 24402 (1993). 19 y. S. Kwak, T. Akeba, and T. Kudo, J. Ferment. Bioeng. 86, 363 (1998). 20 y. Tachibana, L. M. Mendez, S. Fujiwara, M. Takagi, and T. Imanaka, J. Ferment. Bioeng. 82, 224 (1996). 21 W. Liebl, I. Stemplinger, and P. Ruile, J. Bacteriol. 179, 941 (1997).
276
SACCHAROLYTICENZYMES
[ 171
the extreme hyperthermophilic archaeon Pyrodictium abyssi can grow on various polysaccharides and also secretes a heat-stable amylase that is optimally active above 100° and has a wide functional pH range (unpublished results). Unlike ~x-amylase, the production of glucoamylase seems to be very rare in extremely thermophilic and hyperthermophilic Bacteria and Archaea. Among thermophilic anaerobic bacteria, glucoamylases have been purified and characterized from Clostridium thermohydrosulfuricum 39E, 22 Thermoanaerobacterium thermosaccharolyticurn DSM 571, 23 and Clostridium thermosaccharolyticurn. 24 The latter enzyme is optimally active at 70° and pH 5.0. It has been shown that the thermoacidophilic archaea Thermoplasma acidophilum, Picrophilus torridus, and Picrophilus oshimae produce heatand acid-stable glucoamylases. The purified archaeal glucoamylases are optimally active at pH 2 and 90°. Catalytic activity is still detectable at pH 0.5 and 100°. This represents the first report on the presence of glucoamylases in thermophilic Archaea (unpublished results). ot-Glucosidases have been found in thermophilic Archaea and Bacteria. An extracellular a-glucosidase from the thermophilic archaeon Thermococcus strain AN125 was purified and its molecular characteristics determined. The monomeric enzyme (60 kDa) is optimally active at 98°. The purified enzyme has a half-life around 35 min, which is increased to around 215 min in the presence of 1% (w/v) dithiothreitol (DTT) and 1% (w/v) bovine serum albumin (BSA). The substrate preference of the enzyme is pNP-o~D-glucoside > nigerose > panose > palatinose > isomaltose > maltose > turanose. No activity was found with starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose, and fl-gentiobiose. The enzyme was active at 130°. The gene encoding an o~-glucosidase from Thermococcus hydrothermalis 26 was cloned by complementation of a Saccharornyces cerevisiae deficiency maltase-deficient mutant strain. The cDNA clone isolated encodes an open reading frame (ORF) corresponding to a protein of 242 amino acids. The protein shows 42% identity to a Pyrococcus horikoshii unknown ORF, but no similarities were obtained with other polysaccharidase sequences.
22 H. H. Hyun and J. G. Zeikus, J. Bacteriol. 16,1, 1146 (1985). 23 D. Ganghofner, J. Kellermann, W. L. Staudenbauer, and K. Bronnenmeier, Biosci. Biotechnol. Biochem. 62, 302 (1998). 24 U. Specka, F. Mayer, and G. Antranikian, Appl. Environ. Microbiol. 57, 2317 (1991). 25 K. Piller, R. M. Daniel, and H. H. Petach, Protein Struct. Mol. Enzymol. 1, 197 (1996). 26 A. Galichet and A. Belarbi, FEBS Lett. 2, 188 (1999).
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AMYLOLYTICENZYMES
277
Thermoactive Pullulanases and CGTases Thermostable and thermoactive pullulanases from extreme thermophilic microorganisms have been detected in Thermococcus celer, Desulfurococcus mucosus, Staphylothermus marinus, and Thermococcus aggregans. 15 Temperature optima between 90 and 105°, as well as remarkable thermostability, even in the absence of substrate and calcium ions, have been observed. Most thermoactive pullulanases identified to date belong to the type II group, which attack or-l,4- and ~-l,6-glycosidic linkages. They have been purified from P. furiosus and T. litoralis, 27 T. hydrothermalis, 28 and Pyrococcus strain E S 4 . 29 Pullulanases type II from P. furiosus and P. woesei have been expressed in E. coli. 30'31 The unfolding and refolding of pullulanase from P. woesei have been investigated using guanidinium chloride as the denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation, and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +_ 0.29 M for intrinsic fluorescence and 4.90 ___0.31 M for far-UV circular dichroism (CD) changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained on removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca 2+, which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca 2+ ions with a Ko of 0.080 _+ 0.010 /zM and a Hill coefficient H of 1.00 -2-_ 0.10. This cation significantly enhances the stability of the pullulanase against guanidinium chloride-induced unfolding. The refolding of pullulanase, however, was not affected by Ca2+. 32 Genes encoding pullulanases from T. hydrothermalis 33 and 7". aggregans (unpublished results) have been isolated and expressed in mesophilic hosts. The aerobic thermophilic bacterium Thermus caldophilus GK-24 produces a thermostable pullulanase of type I when grown on
2v S. H. Brown and R. M. Kelly, Appl. Environ. Microbiol. 59, 2614 (1993). 28 H. Gantelet and F. Duchiron, AppL Microbiol. Biotechnol. 49, 770 (1998). 29 j. W. Schuliger, S. H. Brown, J, A. Baross, and R. M. Kelly, Mol. Mar. Biol. Biotechnol. 2, 76 (1993). 30 A. R0diger, P. L. JCrgensen, and G. Antranikian, AppL Environ. Microbiol. 61, 567 (1995). 3~ G. Dong, C. Vieille, and J. G. Zeikus, Appl. Environ. Microbiol. 63, 3577 (1997). 32 R. M. Schwerdffeger, R. Chiaraluce, V. Consalvi, R. Scandurra, and G. Antranikian, Eur. J. Biochem. 264, 479 (1999). 33 M. Erra-Pujada, P. Debeire, F. Duchiron, and M. J. O. Donohue, J. Bacteriol. 181, 3282 (1999).
278
SACCHAROLYTICENZYMES
1171
starch. 34 This enzyme debranches amylopectin by attacking specifically ot1,6-glycosidic linkages. The pullulanase is optimally active at 75 ° and pH 5.5, is thermostable up to 90°, and does not require Ca 2+ for either activity or stability. The first debranching enzyme from an anaerobic thermophile was identified in the bacterium Fervidobacterium pennivorans Ven5 and finally cloned and expressed in E. coli. 35 In contrast to the pullulanase from P. woesei (specific to both a-l,6 and a-l,4 glycosidic linkages), pullulanase type II or amylopullulanase, the enzyme from F. pennivorans ven5 attacks exclusively the ot-l,6-glycosidic linkages in polysaccharides (pullulanase type I). This thermostable debranching enzyme leads to the formation of long chain linear polysaccharides from amylopectin. Thermostable CGTases are produced by the members of the genus Thermoanaerobacter and Thermoanaerobacterium thermosulfurigenes 36'37 and Anaerobranca bogoriae. 38 A CGTase has been purified from a newly isolated archaeon, Thermococcus sp. The optimum temperatures for starchdegrading activity and cyclodextrin synthesis are 110 and 90-100 °, respectively. This is the first report on the presence of a thermostable CGTase in a hyperthermophilic archaeon. 39 The ability of extreme thermophiles and hyperthermophiles to produce heat-stable glycosylhydrolases is summarized in Table I.
Methods
Cloning and Expression of Hyperthermophilic Amylolytic Enzymes in Mesophilic Hosts Recombinant starch-hydrolyzing enzymes from several extreme thermophilic and hyperthermophilic microorganisms have been expressed in E. coli as soluble cytosolic proteins. In general, the recombinant enzyme produced in mesophilic hosts maintains the properties of the wild-type enzyme. The gene encoding amylolytic enzyme can be identified and ampli34 C. H. Kim, O. Nashiru, and J. H. Ko, FEMS Microbiol. Lett. 138, 147 (1996). 35 C. Bertoldo, F. Duffner, P. L. J0rgensen, and G. Antranikian, Appl. Environ. Microbiol. 65, 2084(1999). 36 S. Pedersen, B. F. Jensen, L. Dijkhuizen, S. T. J0rgensen, and B. W. Dijkstra, Chemtech 12, 19 (1995). 37 R. Wind, W. Liebl, R. Buitlaar, D. Penninga, A. Spreinat, L. Dijkhuizen, and H. Bahl, Appl. Environ. Microbiol. 61, 1257 (1995). 38 S. Prowe, J. van de Vossenberg, A. Driessen, G. Antranikian, and W. Konnings, J. Bacteriol. 178, 4099 (1996). 39 y. Tachibana, A. Kuramura, N. Shirasaka, Y. Suzuki, T. Yamamoto, S. Fujiwara, M. Takagi, and T. Imanaka, Appl. Environ. Microbiol. 65, 1991 (1999).
[ 17]
AMYLOLYTIC ENZYMES
279
fled by polymerase chain reaction (PCR) using conserved sequences that are known for amylolytic enzymes. 18,3t In many cases, it is possible to detect a starch-hydrolyzing activity after a phenotypical screening of a genomic library, created by shotgun cloning. By using sensitive screening methods, it is possible to detect positive clones producing very low levels of thermostable enzymes. By employing this technique, it is possible to identify new genes expressing novel hydrolytic enzymes. Such procedures have been used to isolate heat-stable a-amylases, t6,17,2°,21 as well as pullulanase type II from the archaeon P. woesei3° and pullulanase type I from the anaerobic bacterium F. pennivorans Ven5. 35 Figure 2 outlines the clone selection procedure used to isolate these genes, which is based on the secretion of amylolytic/pullulytic activity from recombinant cells and the detection of enzymatic activity by producing a clear halo around the colonies. The next section focuses on the general methodology used to clone amylolytic and pullulytic enzymes from extreme thermophilic Archaea and Bacteria. Screening of Genomic Library. Chromosomal DNA is isolated from thermophilic Archaea and Bacteria according to Pitcher e t al., 4° and 100 tzg of DANN is partially digested with 20 U of Sau3A for 10 rain. The digestion is terminated by a phenol:chloroform extraction, and the DNA is ethanol precipitated, size fractionated on a sucrose gradient and after fragments between 3 and 7 kb are pooled. A plasmid library is constructed in the vector pSJ9334~ and the host strain E. coli MC100 using the following method. The cloning vector is digested with BamHI, and a 3.8-kb fragment is purified from an agarose gel. Approximately 0.75 tzg of vector fragment is ligated to 4 /xg of size-fractionated chromosomal DNA and used to transform E. coli MC100 by electroporation. Red dyed substrate (amylopectin or pullulan) is made by suspending 50 g substrate (Hayashibara Biochemical Laboratories) and 5 g Cibachron Rot B (Ciba Geigy) in 500 ml 0.5 M NaOH and incubating at room temperature under constant stirring for 16 hr. The pH is adjusted to 7.0 with 4 N H2SO4. The dyed substrates are precipitated under constant stirring on the addition of 600 ml ethanol, harvested by centrifugation, and then resuspended in 500 ml distilled water. This precipitation procedure is repeated three times and the final dyed substrate is resuspended in 500 ml of distilled water and autoclaved. Reddyed substrate is added to solid medium at a concentration of 1% (v/v). Positive clones expressing active o~-amylase or pullulanase can be identified by forming a clear halo around the colonies. Halos are formed when the
40 D. G. Pitcher, N. A. Saunders, and R. J. Owen, Lett. Appl. Microbiol. 8, 151 (1989). 41 G. Antranikian, P. L. JCrgensen, M. Wtimpelmann, and S. T. JCrgensen, Patent WO 92/ 02614.
280
SACCHAROLYTIC ENZYMES Chromosomal DNA Partial Sau3A
[ 17]
Plasmid pSJ1678 BamHI digest
digestion
3-7 kb fragments isolated
3.8 kb fragment isolated
Ligation Electroporation into E. eoli Plating on red-dye substrate plates
Replica plating and incubation at 37° overnight.
l,u u - o v
]
~
'
I
Isolation of amylolytic reeombinants (5 among 10,000) FIO. 2. Outline of the methodology applied to isolate, clone, and detect DNA fragments of chromosomal DNA from hyperthermophilic Archaea and Bacteria exhibiting amylolytic or pullulytic activity.
[17]
AMYLOLYTICENZYMES
281
dye amylopectin or pullulan complex is attacked by the enzyme, causing the release of the dye. Escherichia coli transformants are plated on LB agar containing 10/zg! ml chloramphenicol and, after 16 hr of incubation at 37°, approximately 14,000 colonies are observed. These are replica plated onto a new set of LB plates containing 2% agar, 6 /~g chloramphenicol/ml, and 1% dyed amylopectin or pullulan and grown overnight at 37°. The plates are then incubated at 60° for 4 hr, and the positive clones are identified by halo formation. E. coli clones expressing the recombinant extracellular a-amylases and pullulanase are grown aerobically at 37° in LB medium 42 containing 10 /zg/ml chloramphenicol. The plasmid containing the gene of interest is isolated using a kit from Qiagen (Hilden, Germany). Gene Expression and Characterization
Using the just mentioned method, we were able to isolate E. coIi clones containing the a-amylase gene from P. furiosus, 16 the pullulanase type II (amylopullulanase) from P. woesei, 3° and the debranching enzymes (pullulanase type I) from F. pennivorans. 35 This section reports on the expression and characterization of the gene encoding pullulanase type I from F. pennivorans Ven5 and the purification of the recombinant enzyme after expression in E. coll. After shogun cloning, E. coil clone PL2125 producing thermostable pullulanase type I from F. pennivorans Ven5 is further characterized. The entire 8.1-kb insert is sequenced in both directions and three large open reading frames (ORFs) are identified. ORF1 is confirmed as encoding a pullulanase by subcloning into pUC18 and observing activity of the E. coli transformants on red-dyed pullulan plates. The subcloning is performed by carrying out PCR amplification using the Expand Long Template PCR System (Boehringer Mannheim) using the following temperature profile: 94°, 2 rain and 30 cycles of 94°; 10 sec, 45°; 45 sec, 68°, 4 min. Cloning of PCRamplified fragments is carried out using the TA cloning kit (Invitrogen). Pullulanase activity can be seen after overnight growth of the positive clones on LB medium containing red-dyed pullulan at 37° and after heat treatment at 70 ° for 16 hr. The plasmid pSE420 containing the IPTGinducible trc promoter (Invitrogen) is used for~expression. The pullulanase gene of F. pennivorans Ven5 (pulA) is 2550 bp in length and encodes a protein of 849 amino acids with a predicted molecular mass of 96.6 kDa before processing. A Shine-Dalgarno-like sequence of A G G A G G is present at positions -10 to -15 from the ATG site. The 42 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
282
SACCHAROLYTICENZYMES
[ 171
G + C content of puIA is 41.9%. A signal sequence of 28 amino acids is present with a cleavage site between the amino acids Ala and Glu. This was confirmed by N-terminal sequencing of the mature pullulanase isolated from F. pennivorans Ven5 (ETELIIHYHRW). The pulA gene is subcloned without its signal sequence and overexpressed in E. coli (under the control of the trc promoter). It is interesting to note that this clone, E. coli FD748, produces two proteins (93 and 83 kDa) with pullulanase activity. This is not due to proteolytic hydrolysis of the recombinant mature protein but due to the presence of a second start site (TTG) identified 128 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG). The TTG translation initiation codon was then mutated to produce only the mature 93-kDa protein. Mutation of the leucine codon, TTG, was carried out by PCR according to the method described by Nelson and Long. 43 The primers are as follows: mutation primer, GTG GCT c T r A C A A G G A A T AG; nonsense, CGA TCG ATC G A G G A T CCT TA; reverse + nonsense, CGA TCG ATC G A G G A T CCT TAT T A A TFA CCT TTG TAC ATT ACC; and forward, ATA A A C ATG TCG G A A ACA G A G CTG A T r ATC. The PCR product is cloned into pCR2.1 and the mutation is confirmed by sequencing. The fragment containing the mutation is digested with AfllII and BamHI and cloned into pSE420/NcoI and BamHI. The mutation is again confirmed by sequencing. Pullulanase-containing clones are detected on pullulan-red agar plates. E. coli FD748 containing the pullulanase cloned without the signal peptide is cultivated in LB or TB medium 42 containing 100 /xg/ml ampicillin and induced with 1 mM IPTG, when the absorbance of the culture at 600 nm is approximately 0.8. The pullulanase expression level of this clone is 40 times higher than that from E. coli PL2125.
Purification of Recombinant Pullulanase from E. coli FD748 In general, heat treatment of the cell extract is ideal for the purification of heat-stable recombinant proteins. All purification steps are performed at room temperature. E. coli cells (10 g) expressing pullulanase type I from F. pennivorans Ven5 are washed with 25 mM Tris-HCl, pH 7.4 (buffer A), and then resuspended with 50 ml of the same buffer. Cells are disrupted by sonication, and cell debris is removed by centrifugation for 20 min at 30,000g. The supernatant is heat treated at 75 ° for 60 min and the denatured host proteins are pelleted by centrifugation (15 min at 30,000g). The pullulanase remains in the clear supernatant. The specific activity of the purified 43 R. M. Nelson and G. L. Long, Anal Biochem. 180, 147 (1989).
[ 17]
AMYLOLYTICENZYMES
283
recombinant pullulanase of F. pennivorans Ven5 expressed in E. coli FD748m (rpulA) is 3 U/mg (Table II). The pullulanase preparation after heat treatment is applied to a /3cyclodextrin-epoxy-activated Sepharose column (1 × 10 cm) equilibrated in 50 mM sodium acetate buffer, pH 6.0 (buffer C). The column is washed stepwise with 50 ml of buffer C and then with the same buffer containing 1 M NaCI until no absorbance at 280 nm is detectable. Pullulanase activity is eluted with 1% pullulan in buffer C, containing 1 M NaC1. Active fractions are pooled, concentrated by ultrafiltration (cutoff 10 kDa), and dialyzed against 1000 volumes of buffer A. The protein solution is then applied to a Mono Q HR 5/5 column (Pharmacia LKB, Freiburg, Germany), which is equilibrated with buffer B, and elution is carried out with the same buffer at a flow rate of 0.5 ml/min. After anion-exchange chromatography on Mono Q, a single protein band is observed. The recombinant full-length pullulanase is purified 25-fold with a specific activity of 75 U/mg and a final yield of about 11.7%. (Table II). The pure enzyme on SDS-PAGE has a molecular mass of 93 kDa. The estimated molecular weight of the native recombinant pulA, calculated in a native gradient gel electrophoresis, is 190 kDa. Accordingly, the enzyme, which is optimally active at 80° and pH 6.0, is a homodimer. /3-Cyclodextrin-Sepharose affinity chromatography is a suitable step to purify pullulanases because cyclodextrins are competitive inhibitors of these enzymes. The/3-cyclodextrin-Sepharose affinity matrix is prepared by coupling /3-cyclodextrin of epoxy-activated Sepharose 6B according to the following protocol following the instructions of the manufacturer (Affinity Chromatography, Pharmacia Fine Chemicals, Uppsala, Sweden). The ep-
T A B L E II PURIFICATION OF RECOMBINANT PULLULANASEFROM E. coli FD748m CLONEa
Step Crude extract Heat treatment (1 hr at 75 °) /3-Cyclodextrin-Sepharose Mono Q
Total protein (rag)
Total activity (U) h
747 172
2268 2184
16.2 3.56
873.6 267
Specific activity (U/mg)
Yield (%)
Purification (-fold)
3 12.7
100 96
4.2
53.9 75
23 11.7
17.9 25
After the aerobic growth of E. coli at 37 °, a 5-liter culture was centrifuged (6 g of ceils wet weight) and the ceils were disrupted by sonication. b One unit of pullulanase catalyzes the formation of 1/zmol of reducing sugars/min under the defined conditions; maltose was used as a standard.
284
SACCHAROLYTICENZYMES
[ 17]
oxy-activated Sepahrose 6B (5 g) is washed with distilled water (1 liter) and reswelled in 0.1 M carbonate buffer, pH 11. To this gel suspension, 40 ml of/3-cyclodextrin (40/zmol x g resin) is added and mixed. The mixture is left under agitation on a shaker (60 rpm) at 40 ° for 16 hr. Excess ligand is removed by washing with carbonate buffer (pH 11) and then with 0.1 M phosphate buffer, pH 8. The remaining epoxy groups are blocked with 1 M ethanolamine dissolved in 0.1 M phosphate buffer, pH 8. After incubation for 16 hr, the resulting/3-cyclodextrin-Sepharose is washed with water and then equilibrated with 50 mM sodium acetate buffer, pH 5.5. Analytical Methods
Assay for Amylolytic/Pullulytic Enzymes Amylolytic/pullulytic activity is determined by measuring the amount of reducing sugars released during incubation with a substrate. To 50 ~1 of l% (w/v) substrate (starch for amylolytic and pullulan for pullulytic activity) dissolved in 50 mM sodium acetate buffer (pH 6.0), 25 or 50/~1 of enzyme solution is added, and the samples are incubated at different temperatures for 10 to 60 min. The reaction is stopped by cooling on ice, and the amount of reducing sugars released is determined by the dinitrosalicylic acid (DNS) method. During starch degradation, monomeric or oligomeric sugars are released. The reducing ends of the released sugars bind covalently to (DNS) at 100°, forming a red complex. The absorption of this complex is determined photometrically at 546 nm and is linearly proportional to the amount of reducing sugars. One unit (U) is defined as the amount of enzyme required to liberate 1/xmol of reducing sugar (with maltose as standard) per minute at a temperature, which is optimal for enzymatic activity, a-Amylase or pullulanase is measured in 50 mM sodium acetate buffer at pH 6.0 and 0.5% (w/v) substrate (starch or pullulan). In order to calculate the enzyme concentration (U/ml), the following equation is used: AEFV U/ml - - t where AE is extinction at 546 nm against a blank value, F is the factor from the maltose standard curve (1.02), V is the factor considering the sample volume employed in the assay,* and t is the incubation time at optimal temperature in minutes. * Units should be calculated for l - m l samples. Sample blanks are extremely important and are used to correct for the nonenzymatic release of reducing sugars that can take place u n d e r assay conditions.
[ 17]
AMYLOLYT1CENZYMES
285
Gel Electrophoresis
In order to determine the size of the native enzyme, native polyacrylamide gels containing a gradient of 5 to 27% polyacrylamide are prepared as described by Koch et aL44 Gels are run at 300 V for 24 hr at 4. High molecular weight marker proteins (Pharmacia Biotech) are used as standards. In order to examine the subunit composition of the pullulanase, protein samples are also analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (12% S D S - P A G E ) as described by Laemmli 45 after heating the samples at 100° for 5 min. Low molecular weight marker proteins (Pharmacia Biotech) are used as standards. Following native and SDS-PAGE, the proteins are stained with Coomassie blue. Zymogram staining for pullulytic activity is performed according to Furegon et al. 46 The red dye-pullulan, prepared as described previously, is incorporated in the resolving native or SDS gel at a final concentration of 1% (v/v). For electrophoresis, samples are diluted in buffer according to Laemmli. 45 After electrophoresis performed at room temperature under 20 mA, the gels are immersed in 100 ml of buffer at the optimum pH for the amylolytic activity preheated at 70° and incubated with gentle agitation at the same temperature in a water bath. When SDS gels are used, the buffer is changed after the first 10 min of incubation. After a period of time that varies according to the activity of the enzyme assayed, pullulanase is detectable as a yellow band on a red background (Fig. 3). When the desired intensity of the yellow band is reached, the reaction is stopped by immersion of the gel in 7.5% (v/v) acetic acid. Amylolytic activity is detected as follows: after rinsing the native or SDS gel with water, it is soaked in 50 mM sodium acetate (buffer C) containing 1% starch (w/v) at 4° for 60 min. The gel is further incubated at the optimum temperature required for enzymatic activity in buffer C. After 2 min of incubation, the gel is soaked in a solution containing 0.15% (w/v) iodine and 1.5% (w/v) potassium iodide solution until clear bands on a dark brown-blue background become visible (Fig. 4). The stained gels are stored in 10% (v/v) of ethanol at room temperature. Characterization of Hydrolysis Product
Hydrolysis products arising after the action of amylolytic enzymes on various linear and branched polysaccharides are analyzed by high-perfor4,~R. Koch, F. Canganella, H. Hippe, K. D. Jahnke, and G. Antranikian, AppL Environ. Microbiol. 63, 1088 (1997). 45 U. K. Laemli, Nature 227, 680 (1970). 46 L. Furegon, A. Curioni, and D. B. A. Peruffo, Anal Biochem. 221, 200 (1994).
286
SACCHAROLYTIC ENZYMES
1
[ 17]
2 kDa
- 94
- 67
- 45
- 30
FIG. 3. SDS-PAGE of a partially purified pullulanase from Desulfurococcus mucosus. The halo indicating pullulytic activity (lane 1) was obtained by loading 20/zg of protein onto SDS-PAGE, followed by electrophoresis, removal of SDS, and staining for heat-stable pullulytic activity. Lane 2, molecular markers. Protein bands were then detected with Coomassie blue (0.1%).
mance liquid chromatography (HPLC) with an Aminex HPX-42A column (300 by 78 mm) (Bio-Rad, Hercules CA). A refractometer is used to detect the peaks. Double distilled water is used as the mobile phase at a flow rate of 0.3 ml/min. Various oligosaccharide peaks (DP7 to DP1) are eluted between 9 and 18 min. The purified pullulanase is incubated at 65 ° with 0.5% (w/v) pullulan, starch, glycogen, amylopectin, maltodextrin, panose, and 0.2% (w/v) amylose. Samples are withdrawn at different time intervals, and the reaction is stopped by incubation on ice. Figure 5 shows the mode of action of pullula-
[ 171
AMYLOLYTICENZYMES kDa
1
2
287
3
440210 140-
7-
FIG. 4. Native polyacrylamide gel (left) with corresponding zymogram (right) of the purified recombinant a-amylase from Pyrococcus woesei. For the zymogram, 20 mU of a-amylase activity was loaded onto a native gel followed by electrophoresis and staining for heat-stable a-amylase activity as described in the text. Lane 1, molecular marker; lane 2, purified aamylase from Pyrococcus woesei; lane 3, zymogram.
nase type I from F. pennivorans Ven5. The thermostable pullulanase hydrolyzes more than 98% of pullulan after 1 hr of incubation at 80° (Fig. 5a). The hydrolysis pattern, after its action on pullulan, reveals the complete conversion of pullulan to maltotriose in an endo-acting fashion by attacking the a-l,6-glycosidic linkages. In order to confirm that the hydrolysis product from pullulan is maltotriose (possessing o~-l,4-glycosidic linkages) and not panose or isopanose (possessing two a-l,4- and o~-l,6-glycosidic linkages), incubation of the products of pullulan hydrolysis is performed in the presence of o~-glycosidase from yeast. The formation of glucose as the main product confirms the formation of maltotriose (and not panose) from pullulan (Fig. 5b). No degradation of amylose is observed after 16 hr of incubation at 65 ° with the recombinant pullulanase demonstrating the low affinity of the purified enzyme to a-l,4-glycosidic linkages. In contrast to this, incubation of amylose with purified recombinant pullulanase type II at 100° from P. woesei 3° leads to the formation of oligosaccharides of different degrees of polymerization and glucose, thus indicating activity toward a-l,6- and o~-l,4-glycosidic linkages (Fig. 5c). After 72 hr of incubation with the purified rpulA, very low levels of maltose and maltotriose are detectable in the
288
[ 171
SACCHAROLYTIC E N Z Y M E S )pn
c~
Pullulan
o~
DP3
Dpl
Dpl
Dp3
Control
1h
Opn
Amylose
Maltotrlose+ 16h + a-glucosldase (x-glucooldase
Pullulanl6 h
2h
d
Starch ~pn
Dpn
Dpn Dpn
ID
Dpn |
Control
16h
Amylopectin
e
Op6 - 1
2 h with PulhlhuIIImDlype |1
from P. wofp~
Control
16h
48h
Glvcoqen
Dpn
pn
Dpn
Dpn
Control
16h
~h
Control
Dpn
16h
48 h
FIG. 5. HPLC analysis of hydrolysis products formed after incubation of the purified recombinant pullulanase from Fervidobacteriumpennivorans Ven5 in the presence of different substrates: (a) 0.5% pullulan, (d) 0.5% starch, (e) 0.5% amylopectin, and (f) 0.5% glycogen. Samples were incubated at 65° and aliquots were withdrawn and analyzed on an Aminex HPX 42-A column for oligosaccharides at different time intervals. (b) The hydrolysis product formed after incubation of 0.5% pullulan with pullulanase from F. pennivorans Ven5 (16 hr pullulan) and 0.5% maltotriose was incubated with commercial a-glucosidase and then analyzed by HPLC. (c) HPLC analysis of hydrolysis products formed after incubation of 0.2% amylose in the presence of recombinant pullulanases from F. pennivorans Ven5 (65° for 16 hr) and from P. woesei(75° for 2 hr). DP, degree of polymerization; DP1, glucose; DP2, maltose; and SO o n .
h y d r o l y s i s p r o d u c t o f s o l u b l e s t a r c h (Fig. 5d), a m y l o p e c t i n (Fig. 5e), a n d g l y c o g e n (Fig. 5 0 . A c c o r d i n g to t h e s e results, rpulA a t t a c k s specifically ot1,6 l i n k a g e s o f puUulan a n d b r a n c h e d o l i g o s a c c h a r i d e s a n d is classified as p u l l u l a n a s e t y p e I.
[ 171
AMYLOLYTICENZYMES
289
Biotechnological Relevance The finding of extremely thermostable starch-hydrolyzing enzymes such as amylases and pullulanases that are active under similar conditions will significantly improve the industrial starch bioconversion process, i.e., liquefaction, saccharification, and isomerization. Because of the lack of novel thermostable enzymes that are active and stable above 100° and at acidic pH values, the bioconversion of starch to glucose and fructose has to be performed under various conditions. This multistage process (step 1: pH 6.0-6.5, 95-105°; step 2: pH 4.5, 60-62°; step 3: pH 7.0-8.5, 55-60 °) is accompanied by the formation of undesirably high concentrations of salts. In the final step, where high fructose syrup is produced, salts have to be removed by expensive ion exchangers. In addition, by using robust starchmodifying enzymes from hyperthermophilic microorganisms, innovative and environmentally friendly processes can be developed aimed at the formation of products of high added value from native starch for the food industry. New and enhanced functionality can be obtained by changing the structural properties of starch. In order to prevent retrogradation, starchmodifying enzymes can be used at a higher temperature. The use of extremely thermostable amylolytic enzymes can lead to valuable products, which include innovative starch-based materials with gelatine-like characteristics and defined, linear dextrins that can be used as fat substitutes, texturizers, aroma stabilizers, and prebiotics. 1,47 CGTases are used for the production of cyclodextrins that can be used as a gelling, thickening, or stabilizing agent in jelly desserts, dressing, confectionery, and dairy and meat products. Because of the ability of cyclodextrins to form inclusion complexes with a variety of organic molecules, cyclodextrins improve the solubility of hydrophobic compounds in aqueous solution. This is of interest for pharmaceutical and cosmetic industries. Cyclodextrin production is a multistage process in which starch is first liquefied by a heat-stable amylase, and in the second step a less-thermostable CGTase from Bacillus sp. is used. Due to the low stability of the latter enzyme, the second step must run at lower temperatures. The application of heat-stable CGTase in jet cooking, where temperatures up to 105° are achieved, will allow liquefaction and cyclization to take place in one step. 48
47 W. D. Crabb and C. Mitchinson, Tibtech 15, 349 (1997). 48 B. E. Norman and S. T. J0rgensen, Denpun Kagaku 39, 101 (1992).
290
SACCHAROLYTICENZYMES
[ 181
[18] Cellulolytic Enzymes from Thermotoga Species B y WOLFGANG LIEBL
Introduction
/3-Glucans are important natural polymers consisting of/3-glycosidically linked glucose residues. These polysaccharides mostly fulfill structural roles. The /3-1,4-glucan cellulose and mixed-linkage /3-1,4/1,3-glucans, such as barley/3-glucan and lichenan, occur as cell wall components of higher plants and lichens, and /3-1,3-glucans are found in the cell walls of yeast and filamentous fungi and as structural and storage polysaccharide of the marine macroalga Laminaria saccharina (laminarin). Thermostable enzymes for the degradation of these types of/3-glucans and/or their corresponding genes have been isolated from different strains of the heterotrophic strictly anaerobic bacterial genus Thermotoga.a-l° The following is an overview of /3-glucan-cleaving enzymes found in Thermotoga species and describes the preparation and characteristics of an endoglucanase and a/3-glucosidase of Thermotoga maritima. /3-Gluca_n-Degrading Enzymes of Thermotoga Species In T. maritima, a hyperthermophile with an optimum growth temperature of 80°, enzymes capable of the hydrolysis of/3-1,4-glucans and mixed1 W. Liebl, P. Ruile, K. Bronnenmeier, K. Riedel, F. Lottspeich, and I. Greif, Microbiology 142, 2533 (1996). 2 K. Bronnenmeier, A. Kern, W. Liebl, and W. Staudenbauer, Appl. Environ. MicrobioL 61, 1399 (1995). 3 O. N. Dakhova, N. E. Kurepina, V. V. Zverlov, V. A. Svetlichnyi, and G. A. Velikodvorskaya, Biochem. Biophys. Res. Cornmun. 194, 1359 (1993). 4 L. D. Ruttersmith and R. M. Daniel, Biochem. J. 277, 887 (1991). 5 L. D. Ruttersmith and R. M. Daniel, Biochim. Biophys. Acta 1156, 167 (1993). 6 J.-D. Bok, D. A. Yernool, and D. E. Eveleigh, Appl. Environ. Microbiol. 64, 4774 (1998). 7 J.-D, Bok, S. K. Goers, and D. E. Eveleigh, in "Enzymatic Conversion of Biomass for Fuels Production" (M. E. Himmel, J. O. Baker, and R. P. Overend, eds.), p. 54. American Chem. Society, Washington, DC, 1994. 8 D. E. Eveleigh, J. D. Bok, H. E1-Dorry, S. EI-Gogary, K. Elliston, A. Goyal, C.. Waldron, R. Wright, and Y.-M. Wu, Appl. Biochem. Biotech. 51152, 169 (1995). 9 V. V. Zverlov, I. Y. Volkov, T. V. Velikodvorskaya, and W. Schwarz, Microbiology 143, 1701 (1997). ~oV. V. Zverlov, I. Y. Volkov, T. V. Velikodvorskaya, and W. Schwarz, Microbiology 143, 3537 (1997).
METHODS IN ENZYMOLOGY.VOL. 330
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