CA2088592A1 - Thermostable pullulanases - Google Patents

Abstract

2088592 9202614 PCTABS00010 This invention is within the field of thermostable enzymes. More specifically, the present invention relates to novel thermostable pullulanases obtainable from members of the genus Pyrococcus and to processes for the preparation of these enzymes. The invention also relates to recombinantly produced pullulanase preparations consisting essentially of a homogeneous pullulanase component, the DNA encoding the pullulanase being derived from the genome of a member of the genus Pyrococcus. Moreover, the invention relates to high expression processes for preparation of the pullulanase components devoid of any contaminating enzyme activities.
The invention further relates to the use of the pullulanases in starch converting processes, and to liquefaction and/or saccharification processes.

Description

~92/02614 2 0 8 ~ 5 9X PCT/DKgl/002t9 QYEL~HE~MO~ABLE PU~L~NA~B~

~CHNICAL FIELD

This inven~ion is within the field of thermostable enæymes. More specifically~ the present invention relates to 5 novel thermostable pullulanases ohtainable from members of the genus Pyrococcus and to processes for th~ preparation of these enz~mes.
The invention also relates ~o recombinantly produced pullulanase preparations consisting essentially of a homcgenous lO pullulanase component, the ~NA encoding the pullulanase being derived from the genome v~ a member of the genus Pyrococcus.
Moreover, the invenkion relates to high expression processes for the prepar~tion of the pullulanase components.
The invention further relates to the use of the pul-~: 15 lulanases in starch c~nverting processes, and to liquefaction and/or saccharification processes.

B~C~ROUND ART

e ~ ostable:~pullulanases are known and isolated f~omeOg.~ Bacillus~aç~d~y~ cus, and their use in industrial ~; 20 ~accharif1cation~;processes~has been described, vide EP Patent P~bliaation No.~:63~909.~ To comply~with the demands for more thermost~le~enzymes~extensive search has proceeded. It is the purpose~of~ this~ invention:to provide novel pullulanases with improved;thermos:tabi1ity.~

25 ~ ~ BRIEF DISC~05~RE~OF ~NE INVEN~ION

It has~ now:been found that members of the ~enus rococcus are:~ab1e to produce novel pullulytic enzymes that ow e~tr~ordinary~;the~most:ability as well as thermoactivity:.
; According~ly, ~in its first asp~ct, the present 30 invent~ion provides a pullulanases~having immunochemical proper-ties identi~ or~partial1y 1dentical to those of the pul-WO92~02614 , PCT/DK91/002~

2 0 ~ ~5 92 2 lulanases derived from Pyrococcus woesei/ DSM No. 3773, orPyro~occus furiosus, DSM No. 3638.
In another aspect, the invention provides pullula-nases that are characteriæed by having pH-optimum in the range 5 of from pH 5 to 7, and temperature optimum in the range of from 85 to 115C, a residual activity of more than 90% aft~r 4 hours at 100C, and more than 30% after 20 minutes at 110C, measured after incubation in the absence of substrate and calcium.
In a third aspect, the invention provides a process 10 for the preparation of the pullulanases of the invention, which process comprises culti~ation of a pullulanase producing strain o~ Pyrococcus in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
lSIn a fourth aspect, the present invention proYides a - recombinantly produced pullulanase preparation consisting essentially of a~homogenous pullulanase component, the DNA
encoding the pullulanase being derived from the genome of a : member of the genu~ Pyrococcus. In a more specific asp~ct, the 20 pre~ent inventiQn provides a pullulanase prepa~ation consisting essentially of a~homogenous pullulanase cGmponent~ which has ~ immunochemi~al properties: identical or partially identica~ to ;~: thos~ of the pullulanases derived from Pyrococcus wQesei, DSM
No. 3773~ or Pyrococcus furiosus, DSM No. 3638.
25~ In a::;~f~i~th aspect, the inven~ion provides a high expreæsion proàess fo~ the~preparation of the enzyme comprising ; isolating a DNA~fragment ~encoding the pullulanase; combining ~he:DNA fra~ment with an appropriate expression signal in an appropriate plasmid~vector; introducing the plasmid vector into : 30 an appropriate host either as an autonomously replicating plasmid or integrated:into~the chromosome; cultivating the host ; organism under conditions leading to expression of the pullula-n~se;~ ~nd recovering of~ the :pullulanase from the culture : medium.
35In a sixth aspect, the invention is directed towards :~ ` the use o~ a pullulanasa of the~invention in starch converting processes. In a more specific aspect, the present invention : :, ~.~92/02614 2 ~ ~ ~ 5 9 2 PCT/D~g1/00219 provides a process for converting starch into syrups containin~
glucose and/or maltose, which process comprises conducting the saccharification of starch hydrolysates in the presence of a pullulanase of the invention and one or more enz~nes selected 5 from the yroup consisting of glucoamylase, ~-glucosidase t ~-amylase or other saccharifying enæymes. In another specific aspect, the in~ention provides a process for converting starch into syrups containing glucose and/or maltose, which process comprises conducting a simultaneous liquefaction/debranching in 10 the presence of a pullulanase of ~he invention and a the~mos-table ~-amylase, and subse~uent saccharificatlon in the presence of one or more enzymes selected from the group consisting of glucoamylase/ ~-glucosidase, ~-amylase or other saccharifying enzymes, optionally together wikh a pullulanase~

BRI~F DESCRIPTTON OF DRAWINGS

The present invention is ~urther illustrated by ref~rence to the accompanying drawingsl in which:
Fig. 1 shows the relation between t~mperature and the ~: : : enzymatic acti~ity of an enzyme according to the invention;
: 20 FigO 2 shows the relation between pH and the .en-zymatic activity~of an enzyme according to the invention;
Fig.~ 3 shows the time coarse of the enzymatic activity of an enzyme of the invention at various temperatures 100~C; 110C;~ 120C); and : : 25 Fig.:4~shows~a restriction map of plasmid pS3933.

DETAILED DI8CLOSURE OF THE I~VB~TIO~

The~enzvme The present invention ~provides novel pullulytic snæymes obtainable from members of the genus Pyrococcus, or 30 mutants or variants~ thereof, or pullulytic enzymes having : immunochemical properties identical or partially identical to th~se of a pullulanase obtainable from a strain of Py~rococcus.
: : ::
:

WO92/0261q PCT/DK91/00214 2U~5 Y2 By an enzyme variant or mutated enzyme is meant an enz~ne obtaina~le by alteration of the DNA nucleotide sequence o~ the parent gene or its derivatives. The enzyme variant or mutated enzyme may be expressed and produced when the DNA
5 nucl~otide sequence encoding the enzyme is inserted into a suitable vector in a suitable host organ.ism. The host organism i5 not necessarily identical to the organism from which the parent gene originated.
The enæymes of the invention are ~aluable for use in 10 starch converting processes, especially in combination with other e~æymes for industrial conversion of starch into various sugars .
The pullulytic enzymes of the invention can be de~cribed by the following characteristics.

15 Phvsical-chemlcal Properties The enzymes of the invention are acti~e in a ~ery broad temperature and pH range. The enzymes possess pullulytic acti~ity in a temperature range of from 40~to above 130C, showing temperature optimum in the ranye of 85 to 115~C; more 20 speci~ically in the range of 100 to 110C. The enæymes possess pUllulytic activity in a pH range of from pH 3.5 to above 8, showing pH optimum in the range of from pH S to 7, more speci~ically pH 5.5 to pH 6.5. At pH 8 approximately 55% of , pullulytic activity are detectable. After 4 hours at 100C the : 25 pullula~ases show essentially no loss of pUllUlytiG activity.
After 24 hour~ at 100C a residual activity of 65% is detect-!~ ~ . a~le- A~ter 1 hour at 110C a residual activity of approxi-mately 10% is detectahle. Addition of metal ions is not : requi.r~d for catalytic activity. The enzyme is inhibited by ~-`30 and ~-cyclodextrins.
The enzymes examined specifically attack the ~ 1,6-: glycosidic linkages in pullulan in a random fashion, forming ~: DP3, DP6 and DP9. (DP: degree of polymerization). Branched ; : oligosaccharides with low molecular weight ars also attacked by 35 the pullulanases. Dextran on the other hand is not hydrolysed ~092/026l4 ~ 592 PCT/DK91/00219 by the pullulanase of the invention. Unlike the pullulanases from other bacteria, the pullulanases are also capable of hydrolysing ~-1,6~1inkages in small substrates such as panose;
products are maltose and glucose.

5 Immunoch mical proPerties The pullulanase of the .inven~ion has immunochemical properties identical or p~rtially identical to those of the pullulanases derived from Pyrococcus woesei, DSM ~o. 3773, or P~3~ ys ~uriosus, DSM No. 3638.
The immuno¢hemical properties can be determined by immunological cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double im-munodiffusion procedure or by tandem crossed immunoelectro~
phoresis according to N. H. Axelsen; Handbook of Immuno-15 precipitation-in-Gel Techniques; Blackwell Scientific Publica-tions ~1983~, chapters ~ and 14. The terms "antigenic identity"
and "partial antigenic iden~ity" are descxibed in the same book, chapters 5,:19 and 20.
Mon~specific antiserum is generated, accordiny to the 20 above mentioned method, by immunizing rabbits with the puri~ied pullulanase of the invention. The immunogen is mixed with Freund's adjuvant and injected subcutaneously in~o rabbits every second week. Antiserum is obtained after a total immuniz-ation period of 8 weeks, and immunoglobulin is prepared 25 there~rom as described by::N. H. Axelsen, supra.

Prep~_rationio~i~the pullulanase The pullulanases of the invention can be prepared by ~` ~: cultivation of a pullulanase producing strain of Pyroc ccus in a suitable nutrient medium, containing carbon and nitrogen : `
30 sources:~and inorganic salts, followed by recovery of the : desired enzyme.
~ The species ~P. woesei and P. furiosus are represen-- tative members of the genus PYrococcus. ~ representative strain .

WO92/02614 2 0 8 ~ ~ 9 2 PCT/DK91/0021~

of P. woesei is available from DSM, No. 3773, and a represen-tative strain o~ P. furiosus is available from DSM, No. 363~o Members of the hyperthermophilic archaebacteria Pyrococcus exhibit growth optimum between 80 and 105C, and at 5 pH ~alues between 4.5 and 7.5, and are capable of growing on Yarious complex media containing polysaccharides such as starch, glycogen, dextrin and oligosaccharides~ It has been demonstrated that during growth o~ these bacteria, extremely thermoactive intracellular and extracellular pullulanases are 10 produc~d.
In order to obtain a fermentation method with better industrial applications, a process for the preparation of a pullulanase has ~een developed that accomplishes continuous gassing of the nutrient medium. It was found that continuous 15 gas5ing of the nutrient medium stimulates the enzyme produc-tion. In addition, a more defined nutrient medium for the growth of Pyrococcus s~. has been developed. Unlike other media d~scribed, this medium is not turbid, and it does not contain elementary sulphur. Optimal growth and enzyme production in 20 this medium can be obtained by conkinuous gassing with N2/CO2 or N2.. Examples of compositions of such media appear from Tabl s 2 and 3. q ~:~ TabIe l I-complex nutrient medium containing elementary 25 sulphur (per litre):
Sea salt (Wiegand,~Krefeld~ FRG)30 g : I ~ KH2P4 ~ . 1.5~g : NiC12 x 6H2O 2 mg *Trace element solution (see below)lO ml 30 : :Sulphur (powdered) lO g ; Yeast:extract l g Peptone ~ ~ 2 g ;; Starch ; . 2 g esazurln 1 mg 35 Na2S x 9~2 500 mg :

. .. ...... . .

W~92/02614 2 ~ 8 ~ ~ 9 ~ Pcr/DK91/oo219 Adjust pH to 6.3-6.5.
Temperature ~0-lOO~C.
Gassin~ with H2/C02; 80/20.

*Trace element solution (per litre):
Titriplex I (Merck) l.5 g ~gS04 x 7H20 3.0 y ~nS04 x 2H20 500 mg NaCl l g FeS0~, x 7H20 j lO0 my CaCl2 x 2HzO lO0 mg ZnS04 x 7H20 180 mg CuS04 X 5H20 10 mg KAl (S04) 2 10 mg H3BO3 lO mg Na2MoO4 x 2H20 lO mg NiCl2 x 6H20 25 mg CaClz x 2 H20 lO mg Adjust pH to 7.0 ,.

Table 2 II-complex nutrient medium without elementary sulphur (per litre): `
(NH4)2S04 l.3 g MgS04 x 7 H20 250 mg NaCl ~ 30.0 g KH2P4 1.4 CaCl2 50 mg FeS04 x 7 H20 38 mg NazSeO3 x~5 HzO 5 ~M
: *Trace:element solution (see below)lO ml :
: ~0 : : Tryptone l g :~ Yeast extract l g Starch 2 g ~: ~ Resazurln l mg ~: DL-malate 13.4 g :: :

WC> 92/02614 2 0 8 8 ~ 9 2 PCT/DI~91/00219~

NaHCO3 1 g Cysteine x HCl 500 mg Adjust to pH 6~2 - 6.5.
Temperature ~ 0 - 10 0 C .
5 Gassing with N2/C02; 80/20.

~Trac:e element solution (per litre):

MnCl2 x 4 H20 100 mg ~::0C12 x 6 H20 2 00 mg NiC12 x 6 H2~ 100 mg 10 ZllClj~ 100 mg CaCl2 x 2 H20 5 0 mg CUS04 X 2 H20 5 0 mg Na2M04 x 2 HzO 5 0 mg Table 3 III-complex nutrient mediumwithoutelementary ulphur (per li~re): ~
~.
~NH4)2S04 1.3 g MgS04 x 7 HzO 2 5 0 mg NaCl 30 . 0 g KH PO~ g ; CaCl ~ 50 mg FeS04: x 7 H20 38 mg ~: : Na25eo3 x 5 H20 5 ~M
~ *Trace element ~olution (see below) lO i ml 2 5 Tryptone ~ 1 g Yeast extract ~ 1 g Starch ~ 1 g Resazurin : 1 mg Cysteine x HCl 500 mg

3 0 Adj ust ~to pH 6 . 2 ~- 6 . 5 .
Temperature 90 - lûQC.
~;assing wlth N2/C02; 80/20.

~0 gu026lq 2 0 S ~ ~ 9 2 PCr/~91~00z~9 *Trace element solution (per litre):
MnCl2 x 4 H20 100 mg CoCl2 x 6 H20 200 mg NiC12 x 6 H20 100 mg ZnCl2 100 mg CaCl2 x 2 H20 50 mg CUS4 X 2 ~2 50 mg Na2MoO4 X 2 H20 5 0 mg Recombinant DNA technoloqy Experiments with strains of PYrococcus maintain ~hat they produce pullulanases in scanty amounts, and production methods based on cultivation of these oryanisms inevitably lead to ~ery low yields. Moreover, investig~tions with enzymes from these organisms have shown that they produce complex mixtures ~ 15 of various single pullulanase components. and accordingly :~ preparations obtainable from these organisms ~ave a relatively low specific activity.
:These facts set baak the practical~exploita~ion of : production methods based on cultivation of strains of ~3yaa~=
20 cus. The low :yield and the difficulty of optimising ~he produotion of single components in multiple enzyme systems make it di:fiault to implemen~ industrial cost-effective production of pullulanase~preparations, and their actual use is hampered by difficulties arising;from the need to employ rather large : 25 ~uantities of the enzymes to achieve the desired enzym~tic e~f~ct.
These drawbacks may~be remedied by using high~yield : processes allowing s~ingle-cvmponent:enzyme preparations with high;- specific ~ctivity: t~ be~:obtained. Therefore, it i5 a :3~ fur~her: objec~ of ~the present invention to provide a recom-inantly:~produaed~pullulanase~preparation consisting essential-ly~o~a~homogen~us pullulanase component, and a high expression process~ for the prepa~ration of this single-component prepara-~ : tion. ':

:. : ~ ~ : ::

WO9Z/026l4 PCT/DK91/002~.g~
2088~i92 Sinale-com~onent pre~rations The present in~ention prov.ides recombinantly produced pullulanase preparations consisting essentially of a homogenous pullulanase component~ The DNA encoding the pullulanase 5 a~mponent can be derived from any member of the genus PyroQ oc-CU5. Pre~erably, the DNA encoding tha pullulanase ~omponent is derived from a strain of P. woesei or P. furiosus. A represen-tative ~train of P. woesie is available from DSM, with No.
3773, and a representative strain Of ~ YEi~ is available 10 from the ~ame institute with No. 363B~
The pullulanase preparations of the invention consist essentially of a homogenous pu1lulanase component, which has immunochemical properties (vide e.g. N. H. Axelsen, op. cit.) identical or partially id~ntical to those of the pullulanases ~5 deri~ed from P~ woesei, DSM No. 3773, or P. furiosus, DSM No.
: 3638.
Preferably the pullulanase preparation of the invention has a pullulytic activity of at least 6 pullulanase units (PU)/mg of protein, more preferred at l~ast 20 PU/mg of 20 protein. A method for determininy the pulluly~ic activity ~nd a definition of PU are cited in Example 8.
The pullulanase preparation of the inv~ention is a mcno-component preparation. Furthermore, the pullulanase com-ponent has an apparent molecular weight of 95 k~. These : 25 f~ature~are determined in Example 9 of the present speci~i-cation.
, ~:~ The~pullulanase preparation of the invention has a pH
, j~ o~timum in the~ range of from pH 4.5 to 6.0, more specifically of from pH 4.5 to 5.5, determined as in Example 7~
~ The pullulanase prepara~ion of the invention has a temperature optimum in the range of from 85 to 115~C, more ::
specifically 9~ to 115C, determined as ~in ~xample 7.
, The pullulanase preparation of the invention has a thermal stability, measured as residual activity, after 30 :35 minut~s at 100C of more than 80~, preferably more than 90%;
: :a~ter 2 hours at 100C of more than 70~, preferably more than :

W~92/02614 2 0 ~ 8 5 9 2 PCT~DK91/00219 80%; and after 4 hours at 100C of more than 6~%, prefera~ly more than 70%, yet more preferably more than B0%; as determined in Example 7.
Investigations in relation to substrate specificity 5 show that the pullulanase preparation of the invention degrades pullulan almost exclusively to maltotriose, panose is degraded almost exclusively to glucose and maltose, and soluble starch is degraded to hydrolysing products covering all gluco~e oligo~
mers, i.e. glucose, maltose, glucotriose, etc n The rate of 10 pullulan hydrolysis is significan~ly the fastest.

RecombinantlY Produced pullulanases To obtain the pullulanase as a mono-component preparation recombinant DNA technique has been employed. A
process of the invention preferably is a high expression lS proces5 comprising isolating a DNA fragment encoding the pullulanase; combining the DNA fragment with an appropriate ; expression signal in an appropriate plasmid vector; introducing :~ the plasmid ve~tor into an appropriate host either as an autonomously replicating plasmid or integrated into the chromo-2Q some; cultiYating the host organism under conditions leadiny~to : ~ expression of thb pullulanase; and recovering of the pul-lulanase from the:culture medium.
Recomblnant DMA techniques and methods are known in , ~: the art and may~readily be carried out by p~rsons skilled in 25 the art. ~ ~ ~
The pullulanase component of the pullulanase prepara ti~n of the inv~ntlon is producible by a species of Pyrococcus.
Preferred~species are P. w_esei. and P. furiosusO A DNA fragmPnt e~ncodlng~the pullulanase component or a precursor thereof may 30 for :instance be isolated:by establishlng a cDNA or geno~ic : library: of a pullulanase: producing srganism, such as e.g. P.
woesel,~ DSM No. 3773, or P. furiosus, DSM No. 3638, and ~:~` ` scr~ening for positive clones by conYentional procedures such : as hybridization~to oligonucleotide probes synthesized on the ~` 35 basis of the full~ or partial amino acid sequence o~ the : ~ ~: : :

WOg2/02614 PCT/DK91/00219 ~5~q~l'2 pullulanase, or by selecting for clones expressing the appro-priate pullulytic activity, or by selec~ing for clones produc-ing a protein which is reactive with an an~ibody against a native pullulanase component.
Screening o~ appropriate DNA seguences and construc-tion of ~ectors may be carried out by standard procedures known in the art.
Once selected the DNA se~uence may be inserted into a suitable replicable expression vector comprising appropriate 10 promotor, operakor and terminator sequences permitting the pullul~nase to be expressed in a particular host organism, as well as an origin of replica~ion enabling the vector to replicate in the host organism in question.
The resulting expression vector may then be trans~
15 formed into a suitable host cell, such as an Escherichia coli, or a m~mber of the genii Bacillus, As~_rqillus, or Strep-tomYces. If E~_ 2~1~ is used as a host organism, suitable plasmids are pBR322 and pACYC, or derivatives there~f. If a Bacil~us sp. is used as a host organism, a suitable plasmid 20 ~ill be pUBl10; pC194; or pEl94. ~ suitable Ba~illus sP. will be B. æubtilis, B. licheni~formis, B. amyloliquefaciens or B.
lentus. ~ , The medium u~ed to cultivate the transformed host may b2 any conventional medium suitable fsr growin~ the cell~ în 25 question.
Other growth conditions may be chosen in accordance with the principles of the known art.
Due to the thermostability of the pullulanase ~, component obtained by the process of the invention it has now 30 been found that it is possible to purify the pullulanase in a most remarkable way. ~ccordingly,~ in a preferred embodiment, the invention provides a process for the preparation of the pullulanase, the process comprising purification of the ` expressed pullulanase by boiling the fermentation broth, 35 causing denaturation of the native proteins, followed by centrifugation of~ the fermentation broth, and recovery of the ; pullulanase from the supernatant.

W092/02614 20~S5g2 PCT/DK91/00219 While the culture medium is heated to boiling, the native proteins, i.e. the proteins and polypeptides originating from the host organism other than the pullulanase component of the invention, are denatured. The pullulanase component, 5 however, being an extremely thermostable enzyme, easily tolerates such treatment. While the native proteins are denatured in a few seconds, above all in a few minutes, e.g.
a~ter 2 to 5 minutes, the pullulanase component of the inven-tion is stable to boiling for several hours, cf. the determina-1~ tion o~ the thermal stability in Example 7. Hereby the vastmajority of the proteins present in the culture medium, including intracellular proteins as a consequence of lysation, can easily be separated off by centrifugation, and in terms of enzymatic activity the purification procedure has been 15 completed, i.e. recombinant pullulanase has been recovered without any contaminating enzyme acti~ities.

Starch convertinq Processes Due to substrate specificity and high thermostabi-lity, in the absence of metal ions/ the pullulanases of the 20 invention are suitable for use in combination with ot~er enzymes for the industrial conversion of starch into various sugars. Being a debranching enæyme, pullulanases from Pyrococ-cus are especially advantageous for use in saccharification processes, ~ollowing liquefaction of starch by thermostable ~
25 amylases that retain substantial parts of their activity at saccharification conditions. When:~-amylase activity is present dur,ing saccharification, accumulation of low molecular.weight, branched oligosaccharides can occur, lowering the final yield in the saccharification process. Pullulanase with sufficient ~30 the~mostability hitherto known in the art does not have any :~activity towards these low molecular weight substances ~Promo-zym~, Novo Nordisk).
Saccharification processes can be carried out by conventional means and essentially as described in EP Patent 35 Publication No. 63,909. Preferred enzyme dasages are in the W092/02614 2 0 8 ~ 5 9 2 PCT/DKg1/002l~.

range of 1-100 ~g of pullulanase per g dry substance, ~ore preferred 1-20 ~g/g dry substance.
The high thermostability of pullulanases of the invention also makes these su.itable for use in simultaneous 5 liquefaction/debranching processes, the advantage of this unusual combination of enzyme activities (~-amylolytic and pullulytic) being that a further r~duction of fluid viscosity in the starch slurry can be achieved hereby, allowing for an increase in the starch concentration during saccharification.
In simultaneous liquefaction/debranching processes preferred enzy~e dosages are in the range of 1-100 ~g of pul-lulanase per g dry substance, more preferred 1-20 ~g/g dry substance. Other conditions are as for conventional li~uefac~
tion and saccharification processes, vide e.g. US Patent No.
15 3,912,590, EP Patent Publica~ions Nos. 252,730 and 63,90g, and International Patent Application WO 90/11352.
The following examples fur~her illustrate the present invention.

~X~MP~E 1 20 Cultivation of P. woesei and P. furiosus P woesei, DSM No. 3773, and P. furiosus~ DSM No.
3638, respectively, were cultivated in a III-complex nutrient medium as defined in Table 3, in a 300 l stainless steel fermentor. Cells were harvested after 14 - 24 hours of growth 25 at 90~. Approximately 70 ~ 120 g (wet weight) of cells were obtained frqm 3:00 1 of cell suspension~ !
In addition to the intracellular enzyme, pullulanase : activity is also de~ectable in the culture supernatant. No significant difference is observed between the intracellular 30 and the extracellular pullulanases. The supernatant was : concentrated using 20,000 cut-off membranes.
Cells were mixed with 50 ml of 50 mM acetate buffer, pH 6.0, and cells were ruptured by F~ench press at a pressure of 8 MPa. The suspension was then incubated with DNAse and ~92/02614 2 0 X ~ 5 9 ~ PCT/DK91/00219 RNAse at 37C for 3 hours. Cell debris was separated from the ~nzyme containi.ng supernatant by centri~ugation at l~,000 xg for 20 minutes. The enzyme sample, which contains both pul-lulanase and amylase, was then dialysed in the same buf~er. The 5 amylase activity was gradually reduced after thîs s~ep, because it is irreversibly inactivated in the ab~ence of its substrate (starch or dextrin). After this step the sampl~ (~0 ml) ~ontains around 102 U of pullula~ase (1700 U/l) a~d 5g U of amylolytic ac~ivity (990 U/l). The specific acti~ity of the 10 enzymes was therefore 0.07 U/mg of pullulanase and 0.04 U/mg of amylase. The presence of oxygen did not influence the activity of these enzymes.

Assay for pullulytic activity Pullulanase activity w~s measured according to the lS following procedure: 50 ~l of enzyme sample were added to 200 ~1 of a solution containing 0.5% pullulan and 50 mM sodium acetate, pH 6Ø The mixture was incubated at l00C for 5, 15 or 30 minutes~ Incubation was terminated by transferring the . .
mixture to an i~e/water bath (0C). After further addition of 20 250 ~l of reagent A (see below), the mixture was incubated~at lO0C for 5 minutes. 2.5 ml deionized water were addsd and absorbance at 546 nm was measured.
`
eaaenk A:
3,5 dinitrosalicylic acid l~0 g 2 N NaOH lO ml K,Na tartrate x H2O ~ 30.0 g Deionized water ad lOO ml 1 unit ~(U) is defined as the amount of enzyme which under the conditions described a~ove liberates l ~mol/min. of 30 redycing sugar measured against maltose as standard.

4 ~ 0 8 ~ ~ ~ 2 PCT/DK91/~021~!~

~-amvlolytic activitY

~ -amylase activity was measured according to the ollowing procedure: To 250 ~1 of sodiium acetate buffer (50 mM
pH 5.5) containing 1% (w/v) starch, up to 100 ~1 of enzyms

5 solution w~s added and incubation was conducted at 95C for 30 and 60 min. The activity of 1 U of ~-amylase i5 defined as that amount of enzyme which ~iberates 1 ~ol of reducing ~ugar per min with maltose as a standard, 10 Purification of the pullulanase 0-Sepharose chromatoqr.aphy: 20 ml of cell-free extract (46 mg of proteins), obtained according to Example 1, were applied onto a Q-Sepharose Fast Flow column (5 x 40 cm) and proteins were eluted using 20 mM tris;HCl buffer, pH 8.0, 15 at a flow rate of 3 ml/min~, and using a NaCl gradient (0 500 ~;: mM). 10 ml fractions were collected. The ~major fraction contai~ing pullulytic activity was eluted with 360-400 mM NaCl.
A minor peak was also eluted with 470-485 mM NaCl. After t~ree runs the fr~ctions containin~ pullulytic activity were pooled 20 and concentrated using 10,000 Da cut-off me~branes (total volume 16.5 ml containing 1.4 mg/ml). Cf. Table 4 ~or further data.

Mono Q c~romato~raphy (FPLC): The concentrated sample was then applliqd onto a Mono Q column ~FPLC; HR 5/5) at a flow 25 rate of 1 ml/min. 2 ml were used per run. The buffer used for : equilibration and elution~was 20 mM potassium phosphate, pH
7Ø Fractions containing pullulytic activity were eluted after : applying a NaCl gradient (200-500 mM). The major fraction was e}uted with 360-420 mM NaCl. After eight runs the fractions 30 containing pullulytic activity were pooled and concentrated.
; After this step the pullulanase was purified 15 fold. Cf. Table ~ : 4 for further data.

~092/02614 2 ~ 9 Z PC~/DK91/00219 Gel filtration FPLC: The concentrated sample (200 ~l each, 680 ~g/ml) ~rom the previous s~ep was applied to gel filtxation using a Superose 12 column. The buffer used was 50 mM sodium acetate (lO0 mM NaCl, pH 5.5). Proteins were eluted 5 at a f~ow rate of 0.4 ml/min. Cf. Table 4 for further data.

~.

Purification of the Pullulanase STEP Volume Total Total Specific Purification Yield protein activity activityfactor (ml~ (mg) (U) (~/mg~ ~%) _ Cell extract60 1380 102 0.07 l lO0 Q-Sepharose 16.5 23.0 8.0 0.34 5 7.8 15 Mono Q 1.5 l.I l.l 1.06 15 l.0 Superose 12 ll 0.07 0.45 6.30 90 0.4 To lO g of cells ~0 ml of 50 mM sodium acetate buffer, pH 5.5, was ad~ed, and cells were ruptured using French press.

: EX~MPLE 3 : : .
. ._ Ç5~y~ Li3~QDL~of: the ~ullulanase _obtained a~co din~ to ExamPles l-2.: ~ .
:: : :
:~:: :Temperature optimum~

The enz~yme~ activity was measured betwe~n 40 ~and 25 130Co The buffer~used was 50 mM sodium acetate, pH 575. Enzyme ass~y was performed~for 5 and 15: minutes. The result is presented in Fig:.~l. ;
~:: :The enzymes:possess pullulytic activity in a tempera-:ture ra~ge of :from 40 to above~ l30C, showing temperature :30 optimum in the:range~of 85 to 115C, more specifically in the ranye of 100 ~o~110C.
:
:::::

W092/02614 . PCT/DK91/OQ21~s~

2 0 8 8 S ~ 2 18 pH optimum The enzyme activi~y was measured between pH 3.5 and 8Ø The buffer used contained potassium acetate, potassium phosphate and ~ris, ~0 mM of each. The assay was performed at 5 9SC for 5 and 15 minutes. The result is presented in Fig~ 2.
The enzymes possess pullulytic activity in a pH range of from pH 3.5 to above 8, showing pH optimum in the range of fxom pH 5 to 7, more speci~ically pH 5.5 to pH 6.5. At pH
approximately 55% of pullulytic activity are detectable.

10 Thermostability The enz~mes were incubated in a 50 mM sodium acetate buffer, pH S.5, without substrate and metal ions. APter 6 hours of incubation at 100C no loss of enzyme activity was observed.
After 12 hours, 24 hours and 48 hours at lO0C around 91%, 65%
15 and 35% of enzyme acti~ity was measured, respecti~ely. At llOC
: 44% of enzyme activity was measured after 20 minutes and 10% of ~ ~ enzyme activity was measured after 1 hour. At 120C lO~ of : enzym~ activity was detected after 5 minutes. The results are ~ presented in Fig. 3.

.
20 S8~la3~Q Dec~ Lisl~Y

1% (w/~3 of substrate was incubated in 50 mM sodium acetate buffer, pH 6.0, i.n the presence of 1.3 U/ml of a pul-: lulanase preparation . In~ubation was conducted at lO0C.
: $amples of 5Q0 ~l were taken an~ treated with an ion exchanger : 2S ~Serdolit MB), and analyzed by HPLC (carbohydrate column Aminex :
HPX-42A).
Pullulan was hydrolysed~ in an endo fashion forming DP3, DP6 and DP9. After l hour 80% of DP3 were formed. In order to identify DP3~ as maltotriose;~it was ~incubat:ed with an ~-30 glucosidase from ~yeast. DP3 was converted completely toglucose. Consequently, it is evident that maltotriose was :

vvo 92/U2614 2 0 8 ~ ~i 9 2 pcr/DK91/oo219 liberated by the action of pullulanase. Dextran was not attacked by th~ enzyme system.
G2 G2-~-cyclodex~rin (obtained from Novo Nordisk A/S, Denmark) was attacked. Maltose was formed at high concentra-5 tionc, In order to find the smallest substrate containing 1,6-linkage, the partially purified pullulanase was incubated with panose. Interestingly, and unlike the pullulanases ~rom known organisms, panose was also a~ack~d, ~ut at a slow rate.
10 The products of hydrolysis were DPl and DP2. ~P~ was identified ~y TLC as maltose~ Therefore, the ~-1,6-linkages in panose are also hydrolysed ~y the enzyme.
In addition to these substrates the pullulanases were also capable of attacking branchPd oligosaccharides ~h~t were 15 prepared in the laboratory by incubation of starch with a-amylase. The branched oligosaccharides formed were then separated using a Bio-Gel column. More than 80% of the branched oligosaccharides were degraded to ~P5 or lower~
:
: ~XAMPLE 4 20 Clonina of ~ 3n~one from PYrococcus woesei ; ~ Pyrococcus woesei chromosomal DNA was isolated according to ~ (1989); Let~. Appl. Microbiol., 8t ~ 151-156; and partially digested with Sau3A. 100 ~g of P. woesei : ~ : DNA was digested:with 20 units of Sau3A for 10 min. at 37C.
25 The digestion was terminated by phenol:chloroform extraction ~and the DNA was ethanol precipitated. Ligation was performed by usin~ chromosomal!DNA: pSJ933;(digested by BamHI, the larger fragment of 5.8 kb was isolated) with a ratio of 1:3, using 4 g of DN~/10 ~1 and~adding 2 units:of T4 ligase and incubating 30 ~t room tempera~ure (25~) ~or 4 hours. (The plasmid pSJ~33 i5 - deposited in the~E. coll strain~SJ989 at NCIMB, a map of the plasmid is shown in Fig. 4). ~ strain MC1000 was trans-: formed with:the ligated DNA and~plated on Luria broth plus 2%
agar containing l0 ~g/ml chIoramphenicol and incubated at 37C.
35 After 16 hours of incubation approximately 14,000 chloramphéni-WO92/02614 2 0 ~ ~ ~ Y 2 PCT/DK91/0021~

col resistant colonies were observed on the plates. Thesecolonies were replica plated onto a new set of Luria broth plates containing ~% agar, 6 ~g/ml chloramphenicol and 0.1%
dyed pullulan and grown overnighk. (Dyed pullulan: 50 g 5 pullulan (Hayashiba Biochemical Laboratories) and 5 g of Cibachron Rot B (Ciba Geigy) are suspended in 500 ml 0.5 M
NaOH, and the mixture is inoubated under co~stant stirring at room temperature for 16 hours. pH is adjusted to 7.0 with 4N
H2SO4. The dyed pullulan is thereafter harvested by centrifuga-10 tion, and the pullulan is washed 3 times with distilled waterand resuspended in an appropriate volume of distilled water).
The plates wsre then incubated at 60C for 4 hours, and around one of the colonies a halo appeared resulting from degradation of the dyed pullulan. The corresponding colony on the first set 15 of ~uria broth plates was isolated and analyzed for plasmid content. The isolated colony PL2118 was grown in 10 ml Luria broth, and the plasmid was isolated by the method described by Kieser et al~ (Plasmid 12:19, 1984). The plasmid was analyzed by restriction mapping and showed an insert of Pyrococcus DNA
~0 of approximately 4.5 kb.

~X~MPLE 5 Cultivatlon example The strain a~cording tG Example 4 was cultivated in ; a 2 1 fermenter of Porton type; pH was maintained at 6.5-7.0, 25 temperature at 37C, aeration at 1.1 l/min., and agitation at 1000 rpm.
Inloculation was made by resuspending culture in physiological salt solution grown on LB agar slant containing

6 mgjl chloramphenicol overnight at 37C. Harvest ~as mad~ at 30 0 ~ours from inoculation.
The cultivation was conducted as a batch fermentation on the following substrate:~
:

~: -:

~092/02614 2 ~ ~ S 5 9 2 PCT/DK91/00219 ~lycerol 60 g/l Tryptone (Bacto) 27 g/l Yeast extract ~Bacto) 4 g/l NaCl 1~3 g/l K2HPO4 5-3 g/l ~I2P4 1.3 g/l K2SO4 3'5 g/l CaClz~2H2O 17 mg/l MgSO~'7H2O 0.67 g/1 Mikrosoyl) 20 ml/l Pluronic 1 ml/l pH adjusted to 7.0, substrate autoclaved in the fermenter for 40 minutes at 121C.
After sterilization, 15 g glucose in 50 ml and 6 mg 15 chloramphenicol were added per litre o~ substrate.

: 1~Mikrosoy:
G/L
Na3 citrate la ,, Borax (Na2B4O7 l0H2O)l.O
MnSO4 H2O
FeSO4 7H2O : 2.0 50~5H2o~ 0.2 --~

BaC12~2H2O 0~1 :
ZnC12 : :~ 0.2 :~ : 25 ~ ~(N~4)~4M724 4H2Q ; 0.l ; ~ ~ EXAMPLE:6 Purification ex~a~ e ~:~
The harvested culture broth prepared according to Example 5~was c~entrifuged;~or 30:min. at 4000 rpm/servall H
30 ~OO0A~in~a Servall RC-3B centrifuge.
The pellet was resuspended with SO mM Tris puffer, pH
~; : 7~0 to a final volume:of 10% of~culture bro~h volume. Lysozyme WAS added to 2 g/l~ final concentration, upon which incubation at 40C for 1 h ~ollowed by treatment~of:the:suspension with an W092/02614 PCT/DK91/002~2 2088S9~

Ultratorrax/type TPl8/10;18N;170W, su~merged into the suspen-sion, for 3 minutes.
The suspension was suhsequently set to boil for 1 h and centrifuged at 9000 rpm/Servall GS3 at 4C for 30 minutes 5 ~n a Servall RC-5B centrifuge.
The pullulanase activity in the supern~ant was concentrated 2.8 times by ultrafiltration on a DDS/GR 81PP
membrane at room tempera~ure (5mM NaN3 added prior to ultrafil-tra~ion).

~O EXAM~E 7 Characterization The pullulanase sample prepared according to Example 6 was submitted to characterization of the pullulanase activity in terms of pH-optimum, temperature optimum, thermal stability 15 and substrate speci~icity.

Substrate specifiGity The pullulanase sample was incubated as follows:

Puffer: 50 mM NaAc, pH 5.0; final concentration~
Substrate: 2~ w/v pullulan, panose or soluble starch 29 (Merck); final concentr~tion.
Temperature: 60C.
Time: : 2~ hours.
Amount of pullu-lanase sample 25 added to incuba on mixture: 5-50% v/v.
: Enzyme reaction :
~ stopped by: Rapid cooling at 0C~

:: From T1C analysis it was evident that pullulan was 30 degraded almost exclusively to maltotrios~, panose was de~raded : almost exclusively :to glucose~and maltose, soluble starch was degraded covering all glucose ~oligomers starting from D~1 and .

~092/02614 2 ~ 8 ~ 5 '3 2 PCT/DKg1/00219 upwards, as products of hydrolysis. The rate of pullulan hydrolysis was significantly the fastest.

Thermal stability Small quantities of the pullulanase sample, sealed in 5 glass amp~les, were incubated ~t 12~C for 10, 30, 90 min. or at 100C for 30 min., 1, 2, 4 hours and rapidly cooled upon incubation. Remaining pullulanase activity at 60C, pH 50 w~s determined as described in Example ~.

~ Rem~1nina Pullulanase ACtiVitY
_ _ _ Preincubation temperature (C) 121 100 Preincubation time (minutes) 10 30 go 30 120 240 _~
15 Activity (%) 45 0 0 97 83 86 _ ~ _ ~ .
Pullulanase activity at 60C was determined as described in Example 8, except that ~arlous incubation :~ 20 pH/buffers were used.

Buffers:
: pH 3.5-5.5: 0.05 M NaAc ~final concentration): ~-buffer : pH 5.5-7.5: 0~025 M Na3 citrate and 0.025 M KH2P04 ~final concentration): CP-buffer .
~: 25 - - -Incubatlon pH 3.5 qi.55.5 5.5 6~57.5 Buffèr j ! A A A CP CP CP
~ctivity (%) ~ : 5 91100 ~l00 63 3 ~ .
:~ : : : :
: 30 TemE~raturQ optimum Pullulanase activity at~ pH 5.0 was determined as described in Example 8, except that various incubation tempera-tures w re used. ~ ~

W092/02614 PCl/~K91/flO21~
2 0 8 ~ 5 ~3 24 Incubation ~emperature (C) 60 70 80 90 100 105 121 ~cti~i~y (%) 18 38 41 54 9~ 100 48 __ EXAMP~E ~

Method of ~ullulanase actlvity analys s 1 ml of 4% w/v pullulan in 0.1 M NaAcl pH 5.0, isadded ko 1 ml o~ diluted enzyme solution (the pullulanase sample is diluted in order to produce a m~ximum of 0.2 g 10 glucose eguivalents/l in the incubation mixture after addition of Na2CO3 buffer) at o D C ~
One ml of the incubation mixture ~olume is incubated at 60C for 30 minutes. One ml of the incubation ~ixture volume i5 incubated at 0C for 30 minutes. To each portion 1.5 ml of 15 0.5 M Na2CO3 buffer, pH 10.0, are added, and the mixture is rapidly cooled.
The amount of reducing ~arbohydrate is determ,ined in :~ ~ both samples by the Nelson/Somogoi method (Melson,N. ~1944); J.
Biol. Chem.; ~, 375~80; and Ssmoqsi, M. (1952): J. Biol.
20 Chem.; 195, 1~-23). After subtracting ~he blind value (0C) the activity is expressed as ol qluc _e equivalents formed ~ ... ~
min. x 1 culture broth ~PU/l) : .EXANP~E~9 25 MW determination :
The molecular weight of the pullulanase of the invention was determined by SDS PAGE conducted according to Pharmacia Phast System flle No. 110, on Phastgel Gradient 8-25.
A molecular weight of 95,000 ( t/- ~0, 000~ was determined~
~ ~ The identity between the pullulanase and the ~ =
95,000 band was established by overlayering the gel with 0~1%
dyed pullulan and~incubating at ~0C for 4 hours. Only one band with pullulanase activity was detected.

~ 92/02614 2 0 8 8 5 9 2 Pcr/DKg1/~0219 As the DNA insert from Pyrococclls is only 4 ~ 5 kb, it is not likely that the VNA insert encoàes for more than one pullulanase ..

~ ~ :

. .

~ v , .

~ . . , ~ .

Claims (28)

1. A pullulanase, characterized by having immuno-chemical properties identical or partially identical to those of the pullulanases derived from Pyrococcus woesei, DSM No.
3773, or Pyrococcus furiosus, DSM No. 3638.

2. A pullulanase, characterized by having the following properties:
(a) pH optimum in the range of from pH 5 to 7;
(b) temperature optimum in the range of from 85 to 115°C;
(c) a residual activity of more than 90% after 4 hours at 100°C, and more than 30% after 20 minutes at 110°C, measured after incubation in the absence of substrate and calcium.

3. The pullulanase of claim 2, having .alpha.-1,6-bond cleavage activity towards panose.

4. A pullulanase of either of claims 2-3, being ob-tainable from a strain of Pyrococcus, or a mutant or a variant thereof.

5. The pullulanase of claim 4, being obtainable from Pyrococcus woesei, DSM No. 3773, or Pyrococcus furiosus, DSM
No. 3638.

6. A process for the preparation of a pullulanase according to any of claims 1-5, which process comprises cultivation of a pullulanase producing strain of Pyrococcus in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.

7. The process of claim 6, comprising cultivation of a pullulanase producing strain of Pyroccus in a nutrient medium that does not contain elementary sulphur, and that is not turbid, the cultivation being accomplished under continuous gassing with N2/CO2 or N2.

8. A process according to either of claims 6-7, in which a strain of P. woesei or P. furiosus is cultivated.

9. The process of claim 8, in which P. woesei, DSM
No. 3773, or P. furiosus, DSM No. 3638, or a mutant or a variant thereof, is cultivated.

10. A recombinantly produced pullulanase preparation consisting essentially of a homogenous pullulanase component, the DNA encoding the pullulanase being derived from the genome of a member of the genus Pyrococcus.

11. A pullulanase preparation consisting essentially of a homogenous pullulanase component, which has immunochemical properties identical or partially identical to those of the pullulanases derived from Pyrococcus woesei, DSM No. 3773, or Pyrococcus furiosus, DSM No. 3638.

12. A pullulanase preparation of either of claims 10-11, the pullulanase component having a pullulytic activity of at least 6 U/mg of protein, more preferred at least 20 U/mg of protein.

13. A pullulanase preparation of any of claims 10-12, showing a substantially homogenous band determined by SDS PAGE, with an apparent MW of 95 kD, said band possessing pullulytic activity.

14. A pullulanase preparation of any of claims 10-13, the pullulanase component having pH optimum in the range of 4.5 to 6.0; temperature optimum in the range of 85 to 115°C; and a residual activity of more than 60% after 4 hours at 100°C.

15. A pullulanase preparation of any of claims 10-14, being able to degrade pullulan to maltotriose; panose to glucose and maltose; and starch to various glucose oligomers from DP1 and upwards.

16. A pullulanase preparation according to any of claims 10-15, the pullulanase component being producible by a species of Pyrococcus, e.g. P. woesei or P. furiosus.

17. The pullulanase preparation of claim 16, the pullulanase component being producible by P. woesei, DSM No.
3773, or P.furiosus, DSM No. 3638.

18. A high expression process for preparation of a pullulanase according to any of claims 10-17, the process comprising isolating a DNA fragment encoding the pullulanase;
combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector; introducing the plasmid vector into an appropriate host either an autonomously replicating plasmid or integrated into the chromosome; culti-vating the host organism under conditions leading to expression of the pullulanase; and recovering of the pullulanase from the culture medium.

19. The process of claim 18, in which the host organism is an Escherichia coli, or a member of the genii Bacillus, Aspergillus, or Streptomyces.

1 20. The process of claim 18, in which the plasmid is pBR322 or pACYC, or a derivative hereof, and the host organism is E. coli.

21. The process of claim 18, in which the plasmid is pUB110; pC194; or pE194, and the host organism is a Bacillus sp...

22. The process of claim 21, in which the host organism is B. subtilis, B. licheniformis, B. amyloliquefaciens or B. lentus.

23. The process of claim 10, further comprising purification of the expressed pullulanase by boiling the fermentation broth, causing denaturation of the native pro-teins, followed by centrifugation of the fermentation broth, and recovering the pullulanase from the supernatant devoid of any contaminating enzyme activities.

24. The use of a pullulanase according to any of claims 1-5, or a pullulanase preparation according to any of claims 10-17, in starch converting processes.

25. A process for converting starch into syrups containing glucose and/or maltose, which process comprises conducting the saccharification of starch hydrolysates in the presence of a pullulanase according to any of claims 1-5, or a pullulanase preparation according to any of claims 10-17, and one or more enzymes selected from the group consisting of glucoamylase, .alpha.-glucosidase, .beta.-amylase, or other saccharifying enzymes.

26. The starch converting process according to claim 25, in which the saccharification is conducted in the pH-range of from 4.0 to 6.0 and at a temperature in the range of from 60 to 80°C, the pullulanase dosage being 1-100 µg/g dry substance.

27. A process for converting starch into syrups containing glucose and/or maltose, which process comprises conducting a simultaneous liquefaction/debranching in the presence of a pullulanase according to any of claims 1-5, or a pullulanase preparation according to any of claims 10-17, and a thermostable .alpha.-amylase, and subsequent saccharification in the presence of one or more enzymes selected from the group consisting of glucoamylase, .alpha.-glucosidase, .beta.-amylase, or other saccharifying enzymes, optionally together with a pullulanase.

28. The starch converting process according to claim 27, in which the simultaneous liquefaction/debranching is conducted on a starch slurry containing 25-45% w/w dry sub-stance, in a pH range of from 4.0 to 6.0 and at a temperature in the range of from 95 to 130°C, the pullulanase dosage being 1-100 µg/g dry substance and the thermostable .alpha.-amylase dosage being 1-20 µg/g dry substance.