Enzymes from high-temperature microorganisms

Enzymes from high-temperature microorganisms Robert M. Kelly and Stephen H. Brown

North Carolina State University, Raleigh and Novo-Nordisk Biotech Inc., Davis, USA

Enzymes derived from microorganisms growing at extreme temperatures are of biotechnological use as highly thermostable biocatalysts and should provide insight into the intrinsic basis of protein stability. So far, only DNA polymerases from these organisms have been put to commercial use, although the application of other classes of highly thermostable enzymes is being considered. Problems in the cultivation of high-temperature microorganisms and in the production of their enzymes still hampers progress in this field.

Current Opinion in Biotechnology 1993, 4:188-192

Introduction

Even though it has been known for some time that microorganisms inhabit geothermal sites and thrive at high temperatures, the biotechnological roles that microorganisms from such extreme environments might play are, for the most part, still being considered [1,2",3",4"',5",6"']. Enzymes with high thermostability have been considered for use in applications ranging from starch processing to the modification of nucleic acids. However, many candidate enzymes are produced by high temperature microorganisms that are obligate anaerobes and rapidly generate sulfidic gases during growth, and attempts to overproduce these enzymes in more suitable host organisms are still in their infancy. Thus, the technological prospects for these enzymes are still unclear although the list of potential applications is expanding.

In this review, organisms with optimal growth temperatures above 75°C will be considered; all organisms with optimal growth temperatures at or above this point are designated extreme thermophiles. Hyperthermophiles will be defined as microorganisms with optimal growth temperatures at or above 90°C. Most organisms that prosper at high temperatures phylogenetically fall into the Archaea (formerly, the Archaebacteria), according to the proposed classification scheme of Woese et al. [7], and are distinct from the Bacteria and Eucarya.

Microbial physiology

Although new thermophites continue to be identified and characterized, the frequency of such reports has diminished in recent years, as has the diversity (based on metabolic characteristics as well as 16S rRNA and 23S rRNA sequencing [8]) of the organisms isolated [9]. This is because of at least three factors. First, recent sampling of geothermal sites, both terrestrial and marine, has resulted in the isolation of strains simi-

lar to those already reported; the isolation techniques used tend to pick up metabolic characteristics related to isolates already identified. Second, modern taxon- omy of novel organisms and those that are difficult to cultivate requires extensive effort and expense, so that many potentially interesting isolates take some consid- erable time to be characterized and classified. Third, increasing technological interest in the enzymes from high temperature microorganisms has created intellec- tual property issues that tend to restrict open discussion of current research efforts.

Nonetheless, reporting of newly discovered isolates continues. For example, Jannasch et al. [10"] report three new hyperthermophilic isolates, including a Py- rococcus strain incapable of utilizing saccharides for growth, as well as one isolate that does not obli- gately require sulfur for growth. The last characteristic is important but uncommon among high temperature microorganisms as it allows cultivation of the microbe in conventional fermentation equipment [11]. Pledger and Baross [12"] have also reported a heterotrophic archaeum, designated ES4, that has been shown to grow at temperatures up to 110°C. Preliminary reports of several other isolates can be found in the abstracts from the International Conference of Thermophiles (Interna- tional Conference on Thermophiles: Science and Tech- nology, Reykjavik, Iceland, August 23-26, 1992).

Central to the determination of potential commercial opportunities for highly thermostable enzymes is a better understanding of the physiology of the source microorganisms. In this regard, recent attention has focused on Pyrococcus furiosus, an anaerobic het- erotroph with an optimal growth temperature of approximately 100"C [13]. Mukund and Adams [14"1 pos- tulated that a novel glycolytic pathway exists in this organism, which they termed pyroglycolysis. This pathway, a modification of the Entner-Doudoroff pathway, is apparently centered on a tungsten-dependent ac- etaldehyde oxidoreductase, and utilizes a ferredoxin

Abbreviations PCR--polymerase chain reaction; TF--thermophilic factor.

188 © Current Biology Ltd ISSN 0958-1669

Enzymes from high-temperature microorganisms Kelly and Brown 189

instead of NAD+ as an electron acceptor. Sch/ifer and Sch6nheit [15"] have verified the existence of this pathway in P. furiosus and the requirement for tungsten was demonstrated in the chemostat studies of Schicho et al. [16].

Attempts to improve cultivation techniques for high temperature microorganisms continue. Rudiger et al. [17"] showed that the growth of Pyrococcus woesei at 90°C in a dialysis reactor led to an approximately three- fold improvement in yield, based on cell density. How- ever, large-scale culture of hyperthermophilic, sulfur- requiring anaerobes must still be done in glass-lined fermentors or alternatively in glass vessels by continuous culture [18]. As many of these organisms are isolated from deep-sea environments, cultivation systems that function under high levels of hydrostatic or hyperbaric pressure and temperature may prove to be necessary to uncover important physiological and biochemical characteristics. Clark and co-workers [19,20"] describe such a system and demonstrate the signifi- cance of pressure effects for the growth of the deep-sea hyperthermophile, ES4 [12"]. Hydrostatic pressure was found to stimulate growth three-fold more than hyperbaric pressure (at 500 arm and 95°C) for ES4. High hydrostatic pressure (500 atm) resulted in a 40% higher growth rate than low hydrostatic pressure (35 arm). Vessel construction (glass versus stainless steed also affected ES4 growth.

Nucleic acid modifying enzymes

The increasingly wide-spread use of thermostable DNA polymerases in the polymerase chain reaction (PCR) represents the most significant application yet for enzymes from high temperature microorganisms. Al- though versions of the Thermus aquaticus DNA polymerase are still the most widely used, other thermostable polymerases have also been commercial- ized recently. A DNA polymerase from the hyperthermophilic archaeum Thermococcus litoralis [21], named Vent TM DNA polymerase (New England Biolabs Inc., Beverly, Massachusetts) [22], has a reported half-life of 6.7 hours at 95"C, compared to approximately one hour for the T. aquaticus enzyme. DNA polymerases from two strains of Pyrococcus are also available com- mercially: Pfu polymerase from the type strain isolated by Fiala and Stetter (Stratagene Inc., La Jolla) [13] and Deep Vent TM DNA polymerase from Pyrococcus GB- D, isolated by Jannasch et al. (New England Biotabs Inc.) [10]. Both Pyrococcus polymerases have half- lives in excess of 20 hours at 95°C. Lundberg et al. [23"] showed that the Pfu polymerase has an associated 3' to 5' exonuctease activity which gives a 10-fold improvement in fidelity over the Taq DNA polymerase after a 105-fold amplification; a genetic assay to meas- ure fidelity in vitro was developed for this comparative study which involved the amplification and cloning of the lacIOZa gene sequences.

In a related development that may prove to be important in efforts to clone and express genes for enzymes from the thermophilic archaea, Perler et al. [24-.] found

introns in the gene encoding the DNA polymerase from T. litoralis. The gene, expressed in Escherichia coli, was split by two intervening sequences that formed a single open reading frame with the surrounding ex- ons. They suggested that these intervening sequences are removed by protein splicing instead of mRNA splicing. Introns are certainly not characteristic of genes in the thermophilic archaea; for example, Zwickl, Fabry, Bogedain, Haas and Hensel [25"] found no such prob- lem in cloning and expressing the P. woesei gene encoding the glyceraldehyde-3-phosphate dehydrogenase in E. coil

Proteolytic enzymes

A number of high temperature microorganisms have been shown to be proteolytic. P. furiosus, for example, produces several proteolytic species [26-28], including a 66 kDa, SDS-resistant serine protease that is highly thermostable and highly regulated [29]. Klinge- berg et al. [30] describe the properties of proteases from five thermophilic Archaea. Burlini et al. [31] recently isolated a chymotrypsin-like serine protease from Sul- folobus sulfataricus that exhibited high levels of thermostability. The potential applicability of these enzymes remains to be investigated. There are still problems in generating sufficient amounts of enzyme for evaluation, although there has been some success in cloning and expressing thermophilic proteases. Lin et al. [32] were able to clone and express Thermopsin, a heat stable acid protease from Sulfolobus acidocaldar- ius, as inclusion bodies in the cytosol o f E. coli as well as in insect cells. Among these possible applications of thermostable proteases is their use for cleaning ul- trafiltration membranes fouled during whey processing [33"].

Saccharide-modifying enzymes

A range of inducible intracellular and extracellular amylolytic (starch-degrading) enzymes are produced by heterotrophic archaea such as P. furiosus [34-36], P. woesei [37], and ES4 [38"]. For example, the extracellular (z-amylase formed by P. woesei will hydrolyze (z-1,4 glucosidic linkages in starch and glycogen, pro- ducing oligosaccharides as small as maltose [36]. ES4 produces an extracellular enzyme with hydrolytic activity against both (z-1,4 and (z-1,6 glucosidic linkages, leading to its classification as an amylopullulanase [38"]. Thermotoga maritima, a thermophilic bacterium growing optimally at 80"C, produces a thermostable 4-(z-glucanotransferase which has been cloned and expressed in E. coli [39]. Oligosaccharides produced by these enzymes may be transported into the cell and acted upon by intracellular (z-glucosidases, such as that produced by P. furiosus [35]. This enzyme is able to hydrolyze small oligosaccharides to glucose, facilitating their utilization in catabolic pathways, such as the pyroglycolytic pathway (see above). All of these enzymes have temperature optima near or above 100°C, which may lead to improvements in conventional starch conver- sion processes.

190 Biochemical engineering

Members of the genus Thermotoga produce several enzymes able to degrade and metabolize plant polysac- charides, such as xylan and cellulose. A Fijian Ther- motoga isolate designated FjSS3-B.1 constitutively produces an extracellular xylanase [40"]. The purified enzyme has a half-life of 90 rain at 95°C, 8 min at 100°C, and less than 2 rain at 105°C in the free state. Immo- bilization on controlled pore glass increases the half- life at 105°C to 10rain, indicating that even highly thermostable enzymes may show stability gains on im- mobilization. Xylanases have been shown to increase the efficiency of pulp bleaching in paper production, and a xylanase with a high degree of thermostability would be useful for this application [2"]. This Ther- motoga isolate also secretes a cellobiohydrolase with a temperature opt imum of 105°C, representing the most thermostable cellulase reported to date [41]. As part of its xylan-metabolizing system, Thermotoga maritima produces an intracellular xylose (glucose) isomerase [42]. Enzymes of this type are used in the production of high fructose corn syrup as they are able to isomer- ize glucose syrups to a temperature-dependent equilibrium mixture of glucose and fructose. The T. maritima isomerase is optimally active at about 100°C, and the increased isomerization temperature allows the production of syrups with elevated equilibrium fructose concentrations, resulting in a significant process advan- tage over less stable enzymes.

Protein thermostability

Given the fact that the intrinsic basis of protein stability is not well understood for mesophilic systems, it is not surprising that so little is understood sti l l about the factors that lead to protein stability at elevated temperatures. While some clues may arise from inspection of the amino acid sequence [25], the expectations that sequence comparisons among homologous proteins with vastly different temperature optima would be fruitful have not been realized [43]. In fact, rules favoring increased thermostability, deduced from the analysis of amino acid substitutions, have not always been reinforced upon examination of high temperature proteins. In some cases, there has been progress in elucidating the factors responsible for extreme levels of thermostability. Rubredoxin, a small, 6.8 kDa electron transport protein, from P. furiosus has been studied by Adams and co-workers [44-]. Although amino acid sequence comparisons be tween rubredoxin and its less thermostable counterparts have revealed little, rubredoxin is distinct in lacking an amino-terminal methionine. As a result, an alanine at the amino ter- minus is free to bind to other residues in the protein's a-sheet (confirmed by protein NMR studies), thus pre- venting unraveling at high temperatures. Several thermostable amylolytic enzymes from hype~hermophi les are large (140 kDa) monomers that exhibit activity over an approximately 100°C temperature range; for example, an (x-glucosidase from P. furiosus has a constant activation energy over an 80°C range suggesting that rigidity might play a role in stabilization [11]. Colonna and co-workers [45",46"] studied the propylamine trans-

ferase from Solfolobus solfataricus and concluded that its thermal stability is a balance be tween an overall increase in flexibility with temperature and a corre- sponding decrease in the number of accessible surface residues buried by folding.

Protein folding at elevated temperatures (both in vivo and in vitro) is a subject of great interest. Schultes and Jaenicke [47"q recently reported the isolation of the in vitro folding intermediates of a glyceraldehyde-3-phosphate dehydrogenase from T. maritima and characterized aspects of the folding pathway. Trent et al. [48], having identified the major heat shock protein of Sulfolobus shibatae [49"q, which they designated as thermophilic factor 55 (TF55), went on to postu- late that TF55 is a member of a new class of molecular chaperones. The TF55 complex binds to unfolded polypeptides in vitro and has ATPase activity. Further- more, the TF55 complex is highly homologous to t- complex polypeptide-1, which i s purported to play a role in mitotic spindle formation in yeast.

It remains to be seen if the study of proteins from high temperature microorganisms will reveal more than can be learned from studies of mesophilic proteins, but the t remendous differences in temperature optima may make certain thermostabilizing features more evident. Because of the high temperatures at which these proteins denature and their high temperature optima, modifications to current biophysical instrumentation and techniques applied to the study of high temperature proteins will be necessary [11].

Conclusion

The scientific study and biotechnological evaluation of temperature-resistant enzymes is still in its infancy. The tools of molecular biology should enable sufficient quantities of interesting enzymes to be produced for further study. Continuing efforts to explore geothermal environments for novel organisms should be en- couraged, as should studies of the associated microbial physiology. By the time this subject is next reviewed, there well may be several success stories, both scientific and technological, relating to high temperature microorganisms.

Acknowledgments

RM Kelly acknowledges the support of the National Science Foun- dation (BCS-9011583) and the North Carolina Biotechnology Cen- ter.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest • . of outstanding interest

1. BORMAN S: Bacteria That F l our i sh A b o v e 100"C Could Benefit Industrial P r o c e s s i n g . Chem Eng News 1991, 69:31-34.

Enzymes from high-temperature microorganisms Kelly and Brown 191

2. ZAMOST BL, NIELSEN, STARNES RL: Thermostable Enzytnes for Industrial Applications. J Industr Microbiol 1991, 8:72-82.

3. ADAMS ~ , KELLY RM (EDS): Biocatalysts at Extreme Tem- peratures. American Chemical Society Symposium Series No. 498; 1992.

First collection of reviews focusing on biocatalysis at very high temperature.

4. COOLBEAR T, DANIEL RM, MORGAN HXXf: The Enzymes f r o m ,, E x t r e m e T h e r m o p h i l e s . Bacter ia l Sources, Thermosta-

bilities a n d Indus t r ia l Relevance. Adv Biochem Eng 1992, 45:57-98.

Excellent summary of enzymes from high temperature organisms with a focus on bacteria growing at about 75"C.

5. LUDLOW JM, CLARK DS: E n g i n e e r i n g Considerations for the Application o f Extremophiles in Biotechnology. Crit Rev Biotechnol 1991, 10:321-345.

Very thorough review of all biotechnological aspects of ex- tremopbAlic organisms, not restricted to high temperatures.

6. DANSON MJ, HOUGH DW, LUNT GG (EDs): Archaebacteria: • , Biochemistry and Biotechnology. Biochemical Society Sym-

posium No. 58; 1992. This paper, along with [1,2",3",4"',5"], provides a good overview of possible applications of enzymes from high temperature microorganisms as well as information about their physiology.

7. WOESE CR, KANDLER O, WHEELIS ML: Towards a Natural System o f Organ i sms : P r o p o s a l for the Dora*ins Ar- chaea , Bacter ia a n d Eucarya. Proc Natl Acad Sci USA 1990, 87:4576.

8, KJEMS J, LAP, SEN N, DALGAARD JZ, GARRETT R_A, STETrER KO: Phylogenetie Relationships Amongst the Hyperther- mophi l ic Archaea Determined f r o m Part ia l 23S rRNA Gene Sequences . Syst Appl Microbtol 1992, 15:203-208.

9. ADAMs MWW, PARK J-B, MUKUND S, BLAMEY J, KELLY RM: Enzymes from sulfur-Dependent Extremely Ther- mophi l ic Organ i sms . In Btocatalysis at Extreme Temper- atures. Edited by Adams MXVW and Kelly RM. American Chemical Society Symposium Series No. 498; 1992:4-22.

10. JANNASCH HW, WmSEN CO, MOLYNF.AUX SJ, LANGWORTHY TA: Compara t i ve Phys io log ica l Studies on Hyperther- mophl l ic Archaea I so la ted f r o m Deep-sea Hot Vents w i t h Emphasis on PyrococCus Strain GB-D. Appl En- viron Mtcrobiol 1992, 58:3472-3481.

A good description not only of some novel isolates but also of the process involved in obtaining and classifying microorganisms from geothermal environments.

11. KELLY RM, BROWN SH, BLUMENTALS, ADAMS MW'~V: Char- ac te r i za t ion of Enzymes from High Temperature Bac- teria. In Biocatalysts at Extreme Temperatures. Edited by Adams MWW and Kelly RM. American Chemical Society Symposium Series No. 498; 1992:23--41.

12. PLEDGER RJ, BAROSS JA: Prellmln~ry Description and Nu- • . t r i t iona l Charac t e r i za t ion of a Chemoorganotrophic

A r c h a e b a c t e r i u m G r o w i n g at Temperatures of up to l l0"C Isolated from a Submarine Hydrothertn~l Vent E n v i r o n m e n t . J Gen Microbtol 1991, 137:203-211.

This paper describes a hyperthermophilic organism from a new submarine geological formation.

13. FIALA G, STETIXR KO: Pyrococcus furiosus, Sp. Nov., Represents a Novel Genus of Marine Heterotrophic Arc.haebacter ia Growing Optimally at 100"C. Arch Mi- crobiol 1986, 145:56-61.

14. MUKUND S, ADAMS MWW: The Novel T u n g s t e n - I r o n - Sulfur P r o t e i n of the HyperthermophUic Archaebac- terium, Pyrococcus furiosus, is an Aldehyde Ferre- dox in Oxidoreductase: Evidence for its Participation in a U n ique Glycolytic Pathway. J Biol Chem 1991, 266:14208-14216.

Description of the characteristics of a novel tungsten-based enzyme and its role in a unique glycolytic pathway found in a hyperthermophilic organism,

15. S c ~ e g T, SCH(3NHEIT P:Maltose Fermentation to Acetate, CO 2 and H 2 in the Anaerobic Hyperthermophilic Ar- chaeon Pyrococcus furiosus: Evidence for Operation of a Novel Sugar Fermentation Pathway. Arch Mtcrobiol 1992, 158:1134-1139.

Detailed study of a putative pyroglycolytic pathway.

16. SCHICHO RN, SNOWDEN LJ, NIUKUND S, PARK J-B, ADAMS MWW, KELLY RM: I n f l u e n c e of Tungsten on Metabolic Patterns in Pyrococcus furiosus, a H y p e r t h e r m o p h i l i c Archaeum. Arch Microbiol 1993, in press.

17. RUDIGER A, OGBONNA JC, MARKL H, Ar,rrgANIKIAN G: Effect of Gassing, Agitation, Supplementation a n d Dialysis on the Growth of an Extremely Thermophil ic Ar- chaeon Pyrococcus woesei. Appl Microbiol Biotechnol 1992, 37:501-504.

This paper describes attempts to improve growth yields for a hyperthermophile. Low growth yields have been at the center of problems for those interested in the study and application of high temperature enzymes.

18. BROWN SH, KELLY RM: cu l t i va t i on Techn iques for Hyper- thermophil ic Archaebacterim Continuous Culture of Pyrococcus furiosus at Temperatures Near 100"C. Appl Environ Mtcrobiol 1989, 55:2086-2088.

19. NELSON CM, SCHUPPENHAUER MR, CLARK DS: Effects of Hyperbaric Pressure on a Deep-Sea A r c h a e b a c t e r i u m in Sta inless Steel and Glass-Lined Vessels. Appl Environ Microbiol 1991, 57:3576-3580.

20. NELSON CM, SCHUPPENHAUER MR, CLARK DS: H i g h - P r e s s u r e , , High Temperature Bioreactor for Comparing Effects

of Hyperbaric and Hydrostatic Pressure o n Bacterial Growth . Appl Environ Mtcrobiol 1992, 58:1789-1793.

.As many high temperature microorganisms are isolated from deep sea, high pressure locales, specialized equipment is needed to re- create in situ conditions. Such a device is detailed in this paper.

21. BELKIN S, JANNASCH H30g: A N e w Extremely Thermophil ic Sulfur Reducing Heterotrophic Mar ine Bacter ium, Arch Mtcrobiol 1985, 141:181-186.

22. MATFILA P, KORPELA J, TENKANEN T, PITKXNEN K: Fideli ty of DNA Synthesis by the Thermococcus litoralis DNA Polymerase - - An Extremely Heat Stable En- zytne w i t h P r o o f r e a d i n g Activity. Nucleic Acids Res 1991, 19:4967--4973.

23. LUNDBERG KS, SHOEMAKER DD, ADAMS MWW, SHORT JM, SORGE JA, MATHUR EJ: High-Fidel i ty Amplification Us- ing a Thermostable DNA Polymerase Isolated from Pyrococcus f u r i o s u s . Gene 1991, 108:1-6.

First report of a DNA polymerase isolated from an organism with an optimum growth temperature of approximately 100"C.

24. PERLER FB, COMB DG, JACK WE, MORAN IS, QLANG B, KUCERA o, RB, BENNER J, SLATKO BE, NWANKWO DO, HEMPSTEAD SK,

ET AL.: I n t e r v e n i n g Sequences i n a n Archaea DNA Poly- merase Gene. Proc Natl Acad Sci USA 1992, 89:5577-5581.

This paper provides some insight into problems that may occur in the doning and expression of high temperature enzymes and the need to consider introns.

25. ZWICKL P, FABRY S, BOGEDAIN C, HAAS A, HENSEL R: • . G lyce ra ldehyde -3 -Phospha te D e h y d r o g e n a s e f r o m the

Hyperthermophil ic Archaebacterium Pyrococcus woesei Character'mation o f the Enzyme, Cloning and Se- quencing of the Gene, and Expression i n Escbericbia coli. J Bacteriol 1990, 172:4329-4338.

First detailed report of the gene sequence of a high temperature enzyme and analysis of possible motifs important for high levels of thermostability.

26. BLUMENTALS Ii, ROBINSON AS, KELLY RM: Character iza- t i on o f S o d i u m Dodecyl su l fa te -Res is tan t Pro teoly t ic

192 Biochemical engineering

Activity in the Hyper thermophi l ic Archaebacter ium Pyrococcus f u r i o s u s . Appl Environ Microbtol 1990, 56:1255-1262.

27. EGGEN R, GEERLING A, WATTS J, DEVOS WM: Characteri- zation of Pyrolysin, a Hyperthermoactive Serine Pro- tease f rom the Archaebacter ium Pyrococcus f u r i o s u s . ~ S Microbtol Left 1990, 71:17-20.

28. CONNAmS H, COWAN DA, SHARP RJ: Heterogeneity of Proteases f rom the Hyper thermophUic Archaebac- t e r ium Pyrococcus f u r t o s u s . J Gen Microbiol 1991, 137:1193-1199.

29. SNOWDEN LJ, BLUMENTALS II, KELLY RM: Regulation of Proteolysis in Pyrococcus f u r i o s u s , a Hyperther- mophi l ic Archaebacterium, Appl Environ Microbtol 1992, 58:1134-1141.

30. KLINGEBERG M, HASHWA F, ~ I K I A N G: Propert ies o f Extremely Thermostable Proteases f rom Anaerobic Hyper thermophi l ic Bacteria. Appl Microbiol Btotechnol 1991, 34:715-719.

31. BURLINI N, MAGNANI P, VILLA A, MACCHI F, TORTORA P, GUERR1TORE A: A Heat-Stable Serine Protease f rom the Extreme Thermophi l ic Archaebacter ium Sulfolobus sol fatar icus . Biochim Biophys Acta 1992, 1122:283-292.

32. LIN XL, LIU NIT, TANG J: Heterologous Express ion of Thermopsin , a Heat-Stable Acid Protease. Enzyme Mi- crobiol Technol 1992, 14:696-701.

33. COOLBEAR T, MONK C, PEEK K, MORGAN HW, DANIEL RM: Laboratory-Scale Investigations into the Use of Ex- t r e m e l y Thermostable Proteinases for Cleaning Ultra- f i l trat ion Membranes Fouled During Whey Process ing J Memh Sci 1992, 67:93-101.

Assessment of how a high temperature enzyme (in this case, a protease) might be used in membrane-processing operations.

34. BROWN SH, COSTANTINO HR, KELLY RM: Characterization of Amylolytic Enzyme Activities Associated wi th the Hyper thermophi l ic Archaebacter ium Pyrococcus f u - r iosus . Appl Environ Microhiol 1990, 56:1985-1991.

35. COSTANTINO HR, BROWN SH, KELLY RM: Purification and Characterization of an 0~-Glucosidase f rom a Hyper- thermophi l ic Archaebacterium, Pyrococcus f u r i o s u s , l~.~rhlbiting a Temperature Opt imum of 105 to 115"C. J Bacteriol 1990, 172:3654-3660.

36. KOCH R, ZABLOWSKI, SPREINAT A, ANTRANIKIAN G: Extremely Thermostable Amylolytic Enzyme f r o m the Archaebac- t e r ium Pyrococcus f u r i o s u s . FEMS Microbiol Lett 1990, 71:21-26.

37. KOCH R, SPREINAT A, LEMKE K, ANTRANIKIAN G: Purification and Propert ies of a Hyperthermoact ive tx-Amylase f r o m the Archaebacter ium Pyrococcus woesei . Arch Microbtol 1991, 155:752-758.

38. SCHULIGER fW, BROWN SH, BAROSS JA, KELLY RM: PurlS- cation and Characterization of a Novel Amylolytic Enzyme f rom ES4, A Marine Hyper thermophi l ic Ar- chaeum, Mol Mar Biol Btotechnol 1993, in press.

The features of amylopullulanase enzyme activity are provided. En- zyme activity above 140"C is reported in the presence of divalent cations.

39. LIEBL W, FEIL R, GABELSCBERGERJ, KELLERMANN J, SCIILEIFER K-H: Purification and Characterization of a Novel Ther- mostable 4-¢-Glucanotransferase o f Thermotoga mar- / t ima Cloned in Escher icbia c o i l Eur J Biochem 1992, 207:81-88.

40. SIMPSON HD, HAUFLER UR, DANIEL RM: An Extremely Thermostable Xylan~se f r o m the Thermophi l ic Eubac- t e r ium Tbermotoga. Biochem J 1991, 277:413-417.

41. RUTtE~M1TH LD, DANIEL RM: Thermostuble Cellobio- hydrolase f rom the Thermophi l ie Eubacterium Ther- motoga sp. Strain FJSS3-B.1 - - Purification and Prop° erties. Biochem J 1991, 277:887-890.

42. BROWN SH, SJOHOLM C, KELLY RM: Purification and Characterization o f a Highly Thermostable Glucose Isomerase Produced by the Extremely Thermophi l ic Eubacterium, Thermotoga mari t ima. Biotechnol Bioeng 1993, in press

43. REHABER V, JAENICKE R: Stability and Reconsti tution of D-Glyceraldehyde-3-Phosphate Dehydrogenase f rom the Hyper thermophi l ic Eubacterium T h e r m o t o g a mar- i t ima . J Biol Chem 1992, 267:10999-11006.

44. ADAMS MWW: Novel Iron-Sulfur Centers in Metalloen- zymes and Redox Proteins f rom Extremely Ther- mophih 'c Bacteria. Adv Inorg Chem 1992, 38:341-396.

A good review of metalloenzymes from high temperature bacteria with some focus on possible mechanisms of extreme thermostability.

45. FACCHIANO F, RAGONE R, PORCELLI M, CACCIAPUCm G, COLONNA G: Effect o f Temperature o n the Propy- I~m|rte Transferase f r o m Sulfolobus so l fa taraicus , an Extreme Thermophil ic Archaebacterium. 1. Conforma- t ional Behavior of the OHgomeric Enzyme in Solution. Eur J Biochem 1992, 204:473-482.

See [46"].

46. RAGONE R, FACCHIANO F, CACCIAPUOTI G, PORCELLI M, COLONNA G: Ef~oL-~t of Temperature o n the Propy- l a i n | h e Transferase f r o m Sulfolobus sol fa taraicus , an Extreme Thermophi l ic Archaebacteriun~ 2. De- naturat ion and Structural Study. Eur J Biochem 1992, 204:483-490.

These two papers [45",46"] provide a fairly detailed biochemical assessment of thermostability issues as they relate to an enzyme from an extreme thermophile.

47. SCHULTES V, JAENICKE R: Folding Intermediates of • . Hyper thermophi l ic D-Glyceraklehyde-3-Phosphate De-

h y d r o g e n a s e f rom Tbermotoga mar i t ima are Trapped at Low Temperature. FEBS Lett 1991, 290:235-238.

This paper describes one of the first efforts to understand protein folding for enzymes from high temperature organisms.

48. TRENT JD, OS]PIUK J, PINKAU T: Acquired Thermoto l er - a n e e and Heat Shock in the Extremely Thermophi l ie Archaebacter ium Sulfolobus sp. Strain B12. J Bactertol 1990, 172:1478-1484.

49. TRENT JD, NIMMESGERN E, WALL JS, HAR'IX F-U, HORWICH AL: • . A Molecular Chaperone f rom a Thermophi l ic Archae-

bacter ium is Related to the Eukaryotic Protein T-Com- p lex Polypeptide-1. Nature 1991, 354:490-493.

This paper proposes that high temperature organisms also utilize chaperones in protein folding and that there might be a link to eukaryotic systems.

RM Kelly, Department of Chemical Engineering, North Carolina State University, Box 7905, Raleigh, NC 27695-7905, USA.

SH Brown, Novo-Nordisk Biotech Inc., 1445 Drew Avenue, Davis, CA 95616-4880, USA.

Documents

Enzymes from high-temperature microorganisms