Cellulose is the most abundant biopolymer on earth, and is the major component of urban waste. Thus cellulose must be seen as a very significant renewable source of chemical foodstocks when fossil fuels become restricted

Daniel, Roy M.

Morgan, Hugh W.

Sharrock, Keith R.

Sissons, Christopher H.

English

Name not available

Cellulases from extremely thermophilic bacteria

Research Commons@Waikato

Cellulases FromExtremely ThermophilicBacteriaK.R. Sharrock*, C.H. Sissons, R.M. Daniel,H.W. MorganDepartment of Biological Sciences,University of WaikatoOriginally presented at the 1982 Conference at whichKeith Sharrock won the Student Paper Competition.INTRODUCTIONCellulose is the most abundant biopolymer on earth,and is the major component of urban waste. Thuscellulose must be seen as a very significant renewablesource of chemical foodstocks when fossil fuels become restricted.Cellulose is a 1-4 - linked glucose polymer with acomplex secondary and tertiary structure.1 The glucosemolecules exhibit alternating orientation so that thebasic repeating unit is cellobiose.The polyglucose chains align to form microfibrils.These are ordered structures so that about 70% of theirlength can be termed "crystalline".1 In these areas thepolyglucose chains are cross-linked by one hydrogenAftergraduating B.Sc Hons in AgriculturalBotany at the University of Wales, Aberystwythin 1965, he completed his Ph.D. in SoilMicrobiology at the University of Guelph,Canada in 1970. He was awarded an ICIPostdoctoral fellowship to work at theh'Iacaulay Institute for Soil ResearchAberdeen prior to taking up the lectureshipin Waikato in 1972.Chris Sissonsjoined the Thermophile Groupof the University of Waikato in 1981 as aResearch Fellow, working on cellulases. Hegraduated in Biochemistry MiSc. atVictoria University of Wellington in 1965,with a thesis on insecticide detoxificationand Ph.D. in Cell Biology at AucklandUniversity in 1971, studying cellfree proteinsyn thesis in yeast and including a yearlecturing in the Biochemistry Department atAuckland. He then spent six years doingpost-doctoral research on enzyme synthesisin the yeast cell cycle in the Zoology Dept. atEdinburgh University and four years at.Ruakura as a NRAC Facial Eczemafellowstudying mycotoxin detoxjfication.bond per glucose. In addition, there are two intra-chairihydrogen bonds per glucose. The gaps between adjacent polyglucose chains are too smalito allow enzymesto penetrate the crystal, and even the exposed exteriorchains are difficult to break up. This is because severalbonds must be broken simultaneously; the 1-4 gIuco-sidic bond, and also three hydrogen bonds, if a glucosemolecule is to be removed to create a "nick" in the chain.Thus the intervening amorphous regions, where someirregularities opening the structure may already exist,are initially the targets for hydrolytic enzymes.Most cellulose occurs naturally in a lignin-cellulose -hemicellulose complex which is often extremely resistant to hydrolysis. Lignin is the plant's equivalent ofinvolved a study of nitrogen fixation andtranslocation in Coriaria arborea tutu.During 1980 he wasa TeachingFellow in theBotany Department, Canterbury University,and completed his LTc'L `Pe/formers, on theviolin. In 1981 he returned to WaikatoUniversity to join the thermophile researchgroup.Roy Daniel is Senior Lecturer in Biochemistly at the University of Waikato. He graduated in `hemistry, B.Sc. Hons at theUniversity ofLeicester in 1965 and Ph.D. inBiochemistry in 1968 at the same university.After working as a Postdoctoralfellow at theAgriculture Research Unitfor Nitrogen Fixation at the University of Sussex and theDivision ofPlantlndusny. SIRQ c'anberra,he took up a lectureship in theDepartnientofCell Biology, University of Glasgow in 1971.He has been at Waikato since 1975.Hugh Morgan is Senior Lecturer in Biological Sciences at the University of Waikato.Keith Sharrock, a D.Phil. student ofWaikato University, is studying the biochemistry ofcellulasesfrom extremely therm -ophilic bacteria. He gmduated B.Sc. frWaikato in 1976 and M.Sc. `Hons, in 1979at the same university. His MSc. research62/June 1 983/Chemistry In New Zealandconcrete, and cellulose fibres act as the internal reinforcing. Woody materials generally require some pretreatment to open up the lignin-cellulose-hemicellulosecomplex sufficiently for effective enzymic hydrolysis ofthe cellulose. We have centred our studies on theenzymic hydrolysis of pure cellulose as an initial step inunderstanding cellulose hydrolysis in the natural lignincellulose-hemicellulose complex.SOURCES OF CELLULASEMost of the cellulolytic enzymes available for commercial purposes are of fungal origin. More recently athermophi I ic anaerobic bacterium Clostridium therinocellumgrowth optimum 60-65°C has been shown by overseasgroups 28 to produce cellulases capable of completelyhydrolysing crystalline cellulose. Since these enzymespossess specific activities which are similar to those ofthe best fungal cellulases but are more heat-stable, 67they are potentially of commercial interest. This beingso, it seemed worthwhile to look for a source of evenmore thermostable cellulases, in extreme thermophilesi.e. organisms with growth optima above 65°C.ENZYMIC DEGRADATION OF CELLULOSENo single enzyme has yet been discovered that aloneis capable of hydrolysing crystalline cellulose to anygreat extent. Fungal systems have been most intensively studied, and they are often based upon the concertedaction of two or three types of enzyme, endocellulase,exocellulase probably physically associated andglucosidase. 8-10 Many fungi have been shown to produce several different enzymes vithin each of thesebasic types, 910 so the term `cellulase" refers to anenzyme complex, not a single enzyme.HOT POOLS SCREENED FOR CELLULOLYTICBACTERIAThe first step in our study was to find extremelythermophilic bacteria that produce cellulases. We wereparticularly interested in cellulases with high thermalstability and high specific activity. The level of cellulaseproduction was notcorisideredto be so importantatthisstage since the yield of enzyme bythe bacterium qan begenetically raised. Thermal stability and specific activity, being characteristics of the enzyme itself, will beextremely difficult to improve.Water and sediments were collected from about 50natural thermal sites and bacteria from them weregrown at 75°C in cellulose-containing enrichment media,both aerobically and anaerobically. After six and 21days growth we subjected the cultures to the firstscreening step. This involved assay of the cell-freesupernatants for endocellulases. Only eight culturesfulfilled this requirement. All were anaerobic.The second screening tested forthe abilityto degradecrystalline cellulose, since the ultimate objective was tofind bacteria capable of degrading natural cellulosicmaterials with a minimum of pretreatment. Two of thecultures, 1P8 and TP1O, could be seen to consumecrystalline cellulose from their growth media, and assays on the cell-free culture supernatants confirmedthat they were producing cellulases capable of hydrolysing crystalline cellulose to reducing sugars.Specific activities of the cellulases of both crudeculture supernatants compared well with that of thecellulase we obtained from C. ther,nocellum. We includedC. thermocellu,n as a reference in most of our comparisons,although it had to be grown at 60°C whereas ourbacteria grew at 75°C.Apart from TP8 and TP1 0, none of the other culturesproduced enzymes that would release significant q uanties of reducing sugar from crystalline cellulose.Stage 3 of the screening involved a comparison of thethermal stabilities of the supernatant endocellulaseactivities Figs 1.. These exhibited half-lives at 85°C offive hours, 20 minutes and less than five minutes fromTP8, TP1Q and C. therniocellum respectively. Thus TP8was selected for further study.Some of the bacteria which we rejected after stage 2of the screen subsequently proved to have extremelystable endocellulases, with half-lives at 85°C In excessof 20 hours. Unfortunatelythese bacteria are not able toattack crystalline cellulose by themselves.Fig 1 Thermal stabilities of endocellulasesCulture supernatants were incubated at 85°C andsamples taken atthe times indicated were assayed at 75°Cfor remaining endocellulaseCMCase.'3ISOLATION OF BACTERIA IN PURE CULTUREIn order to study the cellulases of TP8 further, it wasnecessary to isolate the celluloytic bacteria in pureculture. Cellulolytic anaerobes are renowned for thedifficulties they present in the face of any effort made toisolatethem and thosefrom the TP8 culture proved tobe no exceptions. We had additional problems in dealing with extreme thermophiles in that agar dries out ormelts at their growth temperatures. Eventually we developed a system which allowed isolation and cultivation from individual colonies, each of which originatedfrom a single bacterium. Two different celluloytic strainswere isolated from TP8. The more stable endocellulasewas produced by a strain which didn't attack crystallinecellulose to any extent. The other strain was consideredmore worthy offurtherstudy since it produced enzymescapable of hydrolysing crystalline cellulose to reducingsugars.SEPARATION AND CHARACTERISATION OFCELLULASE COMPONENTSPurification and study of the cellulases from thisisolate has only just begun, and so far we have preliminary information only.Enzymes from the supernatant of 80 litres of cultureHours at 85°CChemistry in New Zealand/June/i 983/63were concentrated by ammonium sulphate precipitation,redissolved and washed over a 10,000 M.W. cut-offultra-filter. The concentrate was then applied to a G-1 00Sephadex column and the fractions collected weresubjected to four different assays Fig. 2."CMCase" and A vicelase' One nmol.glucose equivalents.min."B-glucosidase: 130 pinol. p-nitrophenoLmin'Plate-clearing assay. Units arbitraiy, based on antilog, of cleareddiameter around wells in C'MC-agar.'4Optical density at 280 nm was measured to roughlymonitor protein concentration, and showed two majorpeaks. The first contained proteins of the void volume,and the second appeared much later and tailed considerably.-glucosidase12 eluted as a single sharp peak ofactivity. Endocellulase activity was measured by twomethods. The first, denoted the "CMCase" assay, involved incubation of column fractions with sodium carboxymethyl cellulose and measurement of the reducingsugars produced.13 The second was a plate-clearingassay, presumably dependent upon shortening of thecarboxymethyl cellulose molecules14. The plate-clearingassay was therefore more sensitive to centrally-actingendocellulase than was the "CMCase" reducing sugarassay. Both assays produced two broad peaks of activity, but the relative magnitudes of the first and secondpeaks were reversed, suggesting that the endocellulase in the second peak was the more centrally-acting.The fractions were also incubated with Avicel, which iscrystalline cellulose, and the reducing sugars producedwere measured;'5 This resulted in a single peak. Therewas °Avicelase" activity only in the first "CMCase" peak.Thus it appears that there are at least three differentenzymes involved in cellulose hydrolysis bythis bacterium. One has 13-glucosidase activity, and has a molecularweight of about 55,000.There may be a single enzyme or group of enzymes,each exhibiting both exo- and endocellulase activity,since crystalline cellulose and CMC hydrolysis peakedin the same fractions. Alternatively, each of these activities may be due to separate enzymes of similar molecularweights.The second protein peak had endocellulase but noexocellulase activity. Its late appearance from the columncould suggest an extremely low molecular weight <2000, but more probably it was retarded by binding toSephadex. Such binding was expected since at 75°Cthe enzyme concentrate had been shown to hydrolyseSephadex G-1 00 which is a dextran almost as efficientlyas it hydrolysed crystalline cellulose. However, no detectable release of reducing sugars from Sephadexoccurred during incubation at room temperature overnight. In preliminary work with a Biogel acrylamidepacked column the second endocellulase peak wasabsent, which may suggest that its molecular weight isactually quite similar to that of the first endocellulase,and that their differing affinities for Sephadex allowedtheir separation.FUTURE WORKFurther fractionation techniques such as affinity andion exchange chromatography and isoelectric focussingshould allow purification of the individual componentsof the cellulase complex. The components will then becharacterized independently and investigated for theirpossible co-operative action.ACKNOWLEDGMENTSWe wish to express our gratitude to the followingorganisations for finanacial support in this project:New Zealand Energy Research and Development Council,British Petroleum N.Z. for C.H.S. and DevelopmentFinance Corporation of New Zealand for K.R.S.Ourthanks alsoto Cohn Monkfor his excellent technicalassistance.REFERENCES1. L.T. Fan, Y.E. Lee and D.H. Beardmore,Adv. Biochem.Eng., 14, 101, 1980.2. E.A. Johnson, M. Sakajoh, G. Halhiwell A. Madia andAL. Demain,Appl. Environ, Microbiol, 43 11251982.3. N. Ait, N. Creuzet, J. Cattaneo, Biochem. Biophys. Res.Comm. 90, 537, 1979.4. A. Shin myo, D.V. Garcia-Martinez and A.L. Demain.J.Appl. Biochem., 1, 202 1979.5. B.H. Lee and T.H. Blackburn,Appl. Microbiol., 30, 3461975.6. T.K. Ng and J.G. Zeikus, AppL Environ. MicrobioL, 42,231 1981.7. M. Gritzahi, and R.D. Brown, Adv. Chem. Ser., 181, 2371979.8. M.G. Shepherd, Chemistry in NZ., 45. 92 1 981.9. T.M. Wood and S.l. McCrae,Adv. Chem. Ser., 181, 181-209 1 979.10. C.S. Gong, M.R. Ladisch, G.T. Tsao, Adv. Chem. Ser.,181, 261 1979.11. R.E. Hungate,Bacteriol Rev., 14, 11950.12. C.C. Tong, A.L. Cole and M.G. Shepherd, Biochem. L191, 83 1980.13. P.J. Weimer and J.G. Zeikus,Appl. Environ. Microbial.,33, 289, 1977.14. L. 1'Iankin and S.L. Anagnostakis,J. Gen. Microbiol, 98,109 1977.15. G.L. Miller, Anal. Chem., 31, 426 1959.Fig 2 Elution profile for cellulases and-glucosidase on a Sephadex G-100 columnAll assays at 75°C, with units as follows:64/June 1983/Chemistry in New Zealand

Cellulases from extremely thermophilic bacteria

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