WO1992002614A1

WO1992002614A1 - Novel thermostable pullulanases

Info

Publication number: WO1992002614A1
Application number: PCT/DK1991/000219
Authority: WO
Inventors: Garabed Antranikian; Per Linå JØRGENSEN; Mogens WÜMPELMANN; Steen Troels JØRGENSEN
Original assignee: Novo Nordisk A/S
Priority date: 1990-08-01
Filing date: 1991-07-31
Publication date: 1992-02-20
Also published as: EP0541676A1; CA2088592A1; FI930381A0; JPH05508997A; FI930381A

Abstract

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

NOVEL THERMOSTABLE PULLULANASES

TECHNICAL FIELD

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 homogenous 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 the preparation of the pullulanase components.

The invention further relates to the use of the pul- lulanases in starch converting processes, and to liquefaction and/or saccharification processes.

BACKGROUND ART

Thermostable pullulanases are known and isolated from e.g. Bacillus acidopullulyticus, and their use in industrial saccharification processes has been described, vide EP Patent Publication No. 63,909. To comply with the demands for more thermostable enzymes extensive search has proceeded. It is the purpose of this invention to provide novel pullulanases with improved thermostability.

BRIEF DISCLOSURE OF THE INVENTION

It has now been found that members of the genus Pyrococcus are able to produce novel pullulytic enzymes that show extraordinary thermostability as well as thermoactivity.

Accordingly, in its first aspect, the present invention provides a pullulanases having immunoσhemical proper¬ ties identical or partially identical to those of the pul- lulanases derived from Pyrococcus woesei, DSM No. 3773, or Pyrococcus furiosus. DSM No. 3638.

In another aspect, the invention provides pullula¬ nases that are characterized by having pH-optimum in the range of from pH 5 to 7, and temperature optimum in the range of from 85 to 115^βC, 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.

In a third aspect, the invention provides a process for the preparation of the pullulanases of the invention, 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. In a fourth aspect, the present invention provides 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. In a more specific aspect, the present invention provides 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. In a fifth aspect, the invention provides a high expression process for the preparation of the enzyme 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 as an autonomously replicating plasmid or integrated into the chromosome; cultivating the host organism under conditions leading to expression of the pullula¬ nase; and recovering of the pullulanase from the culture medium. In a sixth aspect, the invention is directed towards the use of a pullulanase of the invention in starch converting processes. In a more specific aspect, the present invention provides 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 of the invention and one or more enzymes selected from the group consisting of glucoamylase, α-glucosidase, β- amylase or other saccharifying enzymes. In another specific aspect, the invention provides 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 of the invention and a thermos¬ table α-amylase, and subsequent saccharification in the presence of one or more enzymes selected from the group consisting of glucoamylase, α-glucosidase, 3-amylase or other saccharifying enzymes, optionally together with a pullulanase.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the accompanying drawings, in which:

Fig. 1 shows the relation between temperature and the enzymatic activity of an enzyme according to the invention; Fig. 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^βC; A ιιo°C; ■ 120"C) ; and Fig. 4 shows a restriction map of plasmid pSJ933.

DETAILED DISCLOSURE OF THE INVENTION

The enzyme

The present invention provides novel pullulytic enzymes obtainable from members of the genus Pyrococcus. or mutants or variants thereof, or pullulytic enzymes having immunochemical properties identical or partially identical to those of a pullulanase obtainable from a strain of Pyrococcus. By an enzyme variant or mutated enzyme is meant an enzyme obtainable by alteration of the DNA nucleotide sequence of the parent gene or its derivatives. The enzyme variant or mutated enzyme may be expressed and produced when the DNA nucleotide sequence encoding the enzyme is inserted into a suitable vector in a suitable host organism. The host organism is not necessarily identical to the organism from which the parent gene originated.

The enzymes of the invention are valuable for use in starch converting processes, especially in combination with other enzymes for industrial conversion of starch into various sugars.

The pullulytic enzymes of the invention can be described by the following characteristics.

Physical-chemical properties

The enzymes of the invention are active in a very broad temperature and pH range. The enzymes possess pullulytic activity in a temperature range of from 40 to above lSO'C, showing temperature optimum in the range of 85 to 115°C, more specifically in the range of 100 to 110°C. The enzymes possess pullulytic activity in a pH range of from pH 3.5 to above 8, showing pH optimum in the range of from pH 5 to 7, more specifically pH 5.5 to pH 6.5. At pH 8 approximately 55% of pullulytic activity are detectable. After 4 hours at 100°C the pullulanases show essentially no loss of pullulytic activity. After 24 hours at 100°C a residual activity of 65% is detect¬ able. After 1 hour at 110°C a residual activity of approxi¬ mately 10% is detectable. Addition of metal ions is not required for catalytic activity. The enzyme is inhibited by α- and 0-cyclodextrins.

The enzymes examined specifically attack the α-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 are also attacked by the pullulanases. Dextran on the other hand is not hydrolysed by the pullulanase of the invention. Unlike the pullulanases from other bacteria, the pullulanases are also capable of hydrolysing α-1,6-linkages in small substrates such as panose; products are maltose and glucose.

Immunochemical properties

The pullulanase of the invention 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. The immunochemical 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 I muno- precipitation-in-Gel Techniques; Blackwell Scientific Publica¬ tions (1983), chapters 5 and 14. The terms "antigenic identity" and "partial antigenic identity" are described in the same book, chapters 5, 19 and 20.

Monospecific antiserum is generated, according to the above mentioned method, by immunizing rabbits with the purified pullulanase of the invention. The immunogen is mixed with Freund*s adjuvant and injected subcutaneously into rabbits every second week. Antiserum is obtained after a total immuniz¬ ation period of 8 weeks, and immunoglobulin is prepared therefrom as described by N. H. Axelsen, supra.

Preparation of the pullulanase

The pullulanases of the invention can be prepared by 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.

The species P. woesei and P. furiosus are represen¬ tative members of the genus Pyrococcus. A representative strain of P. woesei is available from DSM, No. 3773, and a represen¬ tative strain of P. furiosus is available from DSM, No. 3638. Members of the hyperthermophilic archaebacteria Pyrococcus exhibit growth optimum between 80 and 105°C, and at pH values between 4.5 and 7.5, and are capable of growing on various complex media containing polysaccharides such as starch, glycogen, dextrin and oligosaccharides. It has been demonstrated that during growth of these bacteria, extremely thermoactive intracellular and extracellular pullulanases are produced.

In order to obtain a fermentation method with better industrial applications, a process for the preparation of a pullulanase has been developed that accomplishes continuous gassing of the nutrient medium. It was found that continuous gassing of the nutrient medium stimulates the enzyme produc¬ tion. In addition, a more defined nutrient medium for the growth of Pyrococcus sp. has been developed. Unlike other media described, this medium is not turbid, and it does not contain elementary sulphur. Optimal growth and enzyme production in this medium can be obtained by continuous gassing with N₂/C0₂ or N₂. Examples of compositions of such media appear from Tables 2 and 3.

Table 1

I-complex nutrient medium containing elementary sulphur (per litre) :

Adjust pH to 6.3-6.5. Temperature 90-100"C. Gassing with H₂/C0₂; 80/20,

*Trace element solution (per litre)

1.5 g 3.0 g 500 mg

1 g

100 mg

180 mg

10 mg

25 g

CaCl₂ x 2 H₂0 10 mg

Adjust pH to 7.0

Table 2 Il-complex nutrient medium without elementary sulphur (per litre) : (NH₄)₂S0₄ 1.3 g 250 mg 30.0 g

1.4 g 50 mg

38 mg

Na₂Se0₃ x 5 H₂0 5 μM

*Trace element solution (see below) 10 ml

Tryptone i g

Yeast extract i g

Starch 2 g

Resazurin 1 mg

DL-malate 13.4 g NaHC0₃ l g

Cysteine x HC1 500 g Adjust to pH 6.2 - 6.5. Temperature 90 - 100°c. Gassing with N₂/C0₂; 80/20,

*Trace element solution (per litre)

MnCl₂ x 4 H₂0 100 mg

CoCl₂ X 6 H₂0 200 mg

NiCl₂ X 6 H₂0 100 mg

ZnCl₂ 100 mg caCl₂ X 2 H₂o 50 mg

CuS0₄ X 2 H₂0 50 mg

Table 3 Ill-complex nutrient medium without elementary sulphur (per litre) :

(NH₄) ₂S0₄ 1.3 g MgS0₄ X 7 H₂0 250 mg NaCl 30.0 g KH₂P0₄ 1.4 g CaCl₂ 50 mg FeS0₄ X 7 H₂0 38 mg Na₂Se0₃ x H₂0 5 μM

*Trace element solution (see below) 10 ml Tryptone i g

Yeast extract i g Starch i g Resazurin 1 mg Cysteine x HC1 500 mg Adjust to pH 6.2 - 6.5. Temperature 90 - 100°C. Gassing with N₂/C0₂; 80/20. *Trace element solution (per litre) :

MnCl₂ x 4 H₂0 100 mg

CoCl₂ x 6 H₂0 200 mg

NiCl₂ x 6 H₂0 100 mg ZnCl₂ 100 mg

CaCl₂ x 2 H₂0 50 mg

CuS0₄ x 2 H₂0 50 mg

Na₂Mo0₄ x 2 H₂0 50 mg

Recombinant DNA technology

Experiments with strains of Pyrococcus maintain that they produce pullulanases in scanty amounts, and production methods based on cultivation of these organisms inevitably lead to very low yields. Moreover, investigations with enzymes from these organisms have shown that they produce complex mixtures of various single pullulanase components and accordingly preparations obtainable from these organisms have a relatively low specific activity.

These facts set back the practical exploitation of production methods based on cultivation of strains of Pyrococ- cus. The low yield and the difficulty of optimising the production of single components in multiple enzyme systems make it difficult to implement industrial cost-effective production of pullulanase preparations, and their actual use is hampered by difficulties arising from the need to employ rather large quantities of the enzymes to achieve the desired enzymatic effect.

These drawbacks may be remedied by using high yield processes allowing single-component enzyme preparations with high specific activity to be obtained. Therefore, it is a further object of the present invention to provide a recom¬ binantly produced pullulanase preparation consisting essential¬ ly of a homogenous pullulanase component, and a high expression process for the preparation of this single-component prepara¬ tion. Sinσle-component preparations

The present invention provides recombinantly produced pullulanase preparations consisting essentially of a homogenous pullulanase component. The DNA encoding the pullulanase component can be derived from any member of the genus Pyrococ¬ cus. Preferably, the DNA encoding the pullulanase component is derived from a strain of P. woesei or P. furiosus. A represen¬ tative strain of P. woesie is available from DSM, with No. 3773, and a representative strain of P. furiosus is available from the same institute with No. 3638.

The pullulanase preparations of the invention consist essentially of a homogenous pullulanase component, which has immunochemical properties (vide e.g. N. H. Axelsen, op. cit.) identical or partially identical to those of the pullulanases derived 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 least 20 PU/ g of protein. A method for determining the pullulytic activity and a definition of PU are cited in Example 8.

The pullulanase preparation of the invention is a mono-component preparation. Furthermore, the pullulanase com¬ ponent has an apparent molecular weight of 95 kD. These features are determined in Example 9 of the present specifi¬ cation.

The pullulanase preparation of the invention has a pH optimum 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 preparation of the invention has a temperature optimum in the range of from 85 to 115°C, more specifically 95 to 115°C, determined as in Example 7.

The pullulanase preparation of the invention has a thermal stability, measured as residual activity, after 30 minutes at 100°C of more than 80%, preferably more than 90%; after 2 hours at 100°C of more than 70%, preferably more than 80%; and after 4 hours at 100°C of more than 60%, preferably more than 70%, yet more preferably more than 80%; as determined in Example 7.

Investigations in relation to substrate specificity 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 glucose oligo- ers, i.e. glucose, maltose, glucotriose, etc. The rate of pullulan hydrolysis is significantly 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 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 as an autonomously replicating plasmid or integrated into the chromo- some; cultivating the host organism under conditions leading to expression of the pullulanase; and recovering of the pul¬ lulanase from the culture medium.

Recombinant DNA techniques and methods are known in the art and may readily be carried out by persons skilled in the art.

The pullulanase component of the pullulanase prepara¬ tion of the invention is producible by a species of Pyrococcus. Preferred species are P. woesei and P. furiosus. A DNA fragment encoding the pullulanase component or a precursor thereof may for instance be isolated by establishing a cDNA or genomic library of a pullulanase producing organism, such as e.g. P. woesei. DSM No. 3773, or P. furiosus, DSM No. 3638, and screening for positive clones by conventional procedures such as hybridization to oligonucleotide probes synthesized on the basis of the full or partial amino acid sequence of the pullulanase, or by selecting for clones expressing the appro¬ priate pullulytic activity, or by selecting for clones produc¬ ing a protein which is reactive with an antibody against a native pullulanase component. Screening of appropriate DNA sequences and construc¬ tion of vectors may be carried out by standard procedures known in the art.

Once selected the DNA sequence may be inserted into a suitable replicable expression vector comprising appropriate promotor, operator and terminator sequences permitting the pullulanase to be expressed in a particular host organism, as well as an origin of replication enabling the vector to replicate in the host organism in question.

The resulting expression vector may then be trans- formed into a suitable host cell, such as an Escherichia coli, or a member of the genii Bacillus. Asper illus, or Strep- tomyces. If E. coli is used as a host organism, suitable plasmids are pBR322 and pACYC, or derivatives thereof. If a Bacillus sp. is used as a host organism, a suitable plasmid will be pUBllO; pC194; or pE194. A suitable Bacillus sp. will be B. subtilis, B. licheniformis, B. amyloliquefaciens or B. lentus.

The medium used to cultivate the transformed host may be any conventional medium suitable for growing the cells in 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 been found that it is possible to purify the pullulanase in a most remarkable way. Accordingly, 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, causing denaturation of the native proteins, followed by centrifugation of the fermentation broth, and recovery of the pullulanase from the supernatant. 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, 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. after 2 to 5 minutes, the pullulanase component of the inven¬ tion is stable to boiling for several hours, cf. the determina- tion of the thermal stability in Example 7. Hereby the vast majority 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 completed, i.e. recombinant pullulanase has been recovered without any contaminating enzyme activities.

Starch converting: processes

Due to substrate specificity and high thermostabi¬ lity, in the absence of metal ions, the pullulanases of the invention are suitable for use in combination with other enzymes for the industrial conversion of starch into various sugars. Being a debranching enzyme, pullulanases from Pyrococ¬ cus are especially advantageous for use in saccharification processes, following liquefaction of starch by thermostable α- amylases that retain substantial parts of their activity at saccharification conditions. When α-amylase activity is present during saccharification, accumulation of low molecular weight, branched oligosaccharides can occur, lowering the final yield in the saccharification process. Pullulanase with sufficient thermostability hitherto known in the art does not have any activity towards these low molecular weight substances (Promo- zy e^®, Novo Nordisk) .

Saccharification processes can be carried out by conventional means and essentially as described in EP Patent Publication No. 63,909. Preferred enzyme dosages are in the range of 1-100 μg of pullulanase per g dry substance, more preferred 1-20 μg/g dry substance.

The high thermostability of pullulanases of the invention also makes these suitable for use in simultaneous liquefaction/debranching processes, the advantage of this unusual combination of enzyme activities (α-amylolytic and pullulytic) being that a further reduction 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 enzyme dosages are in the range of 1-100 μg of pul¬ lulanase per g dry substance, more preferred 1-20 μg/g dry substance. Other conditions are as for conventional liquefac¬ tion and saccharification processes, vide e.g. US Patent No. 3,912,590, EP Patent Publications Nos. 252,730 and 63,909, and International Patent Application WO 90/11352.

The following examples further illustrate the present invention.

EXAMPLE 1

Cultivation of P. woesei and P. furiosus

P. woesei, DSM No. 3773, and P. furiosus , DSM No. 3638, respectively, were cultivated in a Ill-complex nutrient medium as defined in Table 3, in a 300 1 stainless steel fermentor. Cells were harvested after 14 - 24 hours of growth at 90°C. Approximately 70 - 120 g (wet weight) of cells were obtained from 300 1 of cell suspension.

In addition to the intracellular enzyme, pullulanase activity is also detectable in the culture supernatant. No significant difference is observed between the intracellular 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 French press at a pressure of 8 MPa. The suspension was then incubated with DNAse and RNAse at 37 ° C for 3 hours. Cell debris was separated from the enzyme containing supernatant by centrifugation at 12,000 xg for 20 minutes. The enzyme sample, which contains both pul¬ lulanase and amylase, was then dialysed in the same buffer. The amylase activity was gradually reduced after this step, because it is irreversibly inactivated in the absence of its substrate (starch or dextrin) . After this step the sample (60 ml) contains around 102 U of pullulanase (1700 U/l) and 59 U of amylolytic activity (990 U/l) . The specific activity of the 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 was measured according to the following procedure: 50 μl of enzyme sample were added to 200 μl of a solution containing 0.5% pullulan and 50 mM sodium acetate, pH 6.0. The mixture was incubated at 100"C for 5, 15 or 30 minutes. Incubation was terminated by transferring the mixture to an ice/water bath (0°C). After further addition of 250 μl of reagent A (see below) , the mixture was incubated at 100°C for 5 minutes. 2.5 ml deionized water were added and absorbance at 546 nm was measured.

Reagent A:

3,5 dinitrosalicylic acid 1.0 g 2 N NaOH 10 ml

K,Na - tartrate x H₂0 30.0 g

Deionized water ad 100 ml

1 unit (U) is defined as the amount of enzyme which under the conditions described above liberates 1 μmol/min. of reducing sugar measured against maltose as standard. α-amylolytic activity

α-amylase activity was measured according to the following procedure: To 250 μl of sodium acetate buffer (50 mM pH 5.5) containing 1% (w/v) starch, up to 100 μl of enzyme solution was added and incubation was conducted at 95°C for 30 and 60 min. The activity of 1 U of α-amylase is defined as that amount of enzyme which liberates 1 μmol of reducing sugar per min with maltose as a standard.

EXAMPLE 2

Purification of the pullulanase

O-Sepharose chromatography: 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, at a flow rate of 3 ml/min. , and using a NaCl gradient (0-500 mM) . 10 ml fractions were collected. The major fraction containing pullulytic activity was eluted with 360-400 mM NaCl. A minor peak was also eluted with 470-485 mM NaCl. After three runs the fractions containing pullulytic activity were pooled and concentrated using 10,000 Da cut-off membranes (total volume 16.5 ml containing 1.4 mg/ml) . Cf. Table 4 for further data.

Mono 0 chromatography (FPLC) : The concentrated sample was then applied onto a Mono Q column (FPLC; HR 5/5) at a flow rate of 1 ml/min. 2 ml were used per run. The buffer used for equilibration and elution was 20 mM potassium phosphate, pH 7.0. Fractions containing pullulytic activity were eluted after applying a NaCl gradient (200-500 mM) . The major fraction was eluted with 360-420 mM NaCl. After eight runs the fractions containing pullulytic activity were pooled and concentrated. After this step the pullulanase was purified 15 fold. Cf. Table 4 for further data. Gel filtration FPLC: The concentrated sample (200 μl each, 680 μg/ml) from the previous step was applied to gel filtration using a Superose 12 column. The buffer used was 50 mM sodium acetate (100 mM NaCl, pH 5.5). Proteins were eluted

5 at a flow rate of 0.4 ml/min. Cf. Table 4 for further data.

Table 4

Purification of the pullulanase

STEP Volume Total Total Specific Purification Yield 10 protein activity activity factor

(ml) (mg) (U) (U/mg) (%)

Cell extract 60

Q-Sepharose 16.5 15 Mono Q 1.5 Superose 12 11

To 10 g of cells 50 ml of 50 mM sodium acetate buffer, pH 5.5, was added, and cells were ruptured using French press.

20 EXAMPLE 3

Characterisation of the pullulanase obtained according to Examples 1-2.

Temperature optimum

The enzyme activity was measured between 40 and 25130^βC. The buffer used was 50 mM sodium acetate, pH 5.5. Enzyme assay was performed for 5 and 15 minutes. The result is presented in Fig. 1.

The enzymes possess pullulytic activity in a tempera¬ ture range of from 40 to above 130°C, showing temperature 30 optimum in the range of 85 to 115°C, more specifically in the range of 100 to 110°C. pH optimum

The enzyme activity was measured between pH 3.5 and 8.0. The buffer used contained potassium acetate, potassium phosphate and tris, 50 mM of each. The assay was performed at 595°C 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 from pH 5 to 7, more specifically pH 5.5 to pH 6.5. At pH 8 approximately 55% of pullulytic activity are detectable.

0 Thermostability

The enzymes were incubated in a 50 mM sodium acetate buffer, pH 5.5, without substrate and metal ions. After 6 hours of incubation at 100°C no loss of enzyme activity was observed. After 12 hours, 24 hours and 48 hours at 100°C around 91%, 65% and 35% of enzyme activity was measured, respectively. At 110°C 44% of enzyme activity was measured after 20 minutes and 10% of enzyme activity was measured after 1 hour. At 120°C 10% of enzyme activity was detected after 5 minutes. The results are presented in Fig. 3.

Substrate specificity

1% (w/v) of substrate was incubated in 50 mM sodium acetate buffer, pH 6.0, in the presence of 1.3 U/ml of a pul¬ lulanase preparation. Incubation was conducted at 100°C. Samples of 500 μl were taken and treated with an ion exchanger (Serdolit MB) , and analyzed by HPLC (carbohydrate column Aminex HPX-42A) .

Pullulan was hydrolysed in an endo fashion forming DP3, DP6 and DP9. After 1 hour 80% of DP3 were formed. In order to identify DP3 as maltotriose it was incubated with an α- glucosidase from yeast. DP3 was converted completely to glucose. Consequently, it is evident that maltotriose was liberated by the action of pullulanase. Dextran was not attacked by the enzyme system.

G2 G2-/3-cyclodextrin (obtained from Novo Nordisk A/S, Denmark) was attacked. Maltose was formed at high concentra- tions.

In order to find the smallest substrate containing 1,6-linkage, the partially purified pullulanase was incubated with panose. Interestingly, and unlike the pullulanases from known organisms, panose was also attacked, but at a slow rate. The products of hydrolysis were DPI and DP2. DP2 was identified by TLC as maltose. Therefore, the α-1,6-linkages in panose are also hydrolysed by the enzyme.

In addition to these substrates the pullulanases were also capable of attacking branched oligosaccharides that were prepared in the laboratory by incubation of starch with α- amylase. The branched oligosaccharides formed were then separated using a Bio-Gel column. More than 80% of the branched oligosaccharides were degraded to DP5 or lower.

EXAMPLE 4

Cloning of a pullulanase gene from Pyrococcus woesei

Pyrococcus woesei chromosomal DNA was isolated according to Pitcher et al. (1989); Lett. Appl. Microbiol., 8_, 151-156; and partially digested with Sau3A. 100 μg of P. woesei DNA was digested with 20 units of Sau3A for 10 min. at 37 ° C. The digestion was terminated by phenol:chloroform extraction and the DNA was ethanol precipitated. Ligation was performed by using chromosomal DNA: pSJ933 (digested by BamHI, the larger fragment of 5.8 kb was isolated) with a ratio of 1:3, using 4 μg of DNA/10 μl and adding 2 units of T4 ligase and incubating at room temperature (25°C) for 4 hours. (The plasmid pSJ933 is deposited in the E. coli strain SJ989 at NCIMB, a map of the plasmid is shown in Fig. 4) . E. coli strain MC1000 was trans¬ formed with the ligated DNA and plated on Luria broth plus 2% agar containing 10 μg/ml chloramphenicol and incubated at 37^βC. After 16 hours of incubation approximately 14,000 chlorampheni- col resistant colonies were observed on the plates. These colonies were replica plated onto a new set of Luria broth plates containing 2% agar, 6 μg/ml chloramphenicol and 0.1% dyed pullulan and grown overnight. (Dyed pullulan: 50 g 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 incubated under constant stirring at room temperature for 16 hours. pH is adjusted to 7.0 with 4N H₂S0₄. The dyed pullulan is thereafter harvested by centrifuga- tion, and the pullulan is washed 3 times with distilled water and resuspended in an appropriate volume of distilled water) . The plates were then incubated at 60°C 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 of Luria 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 of approximately 4.5 kb.

EXAMPLE 5

Cultivation example

The strain according to Example 4 was cultivated in a 2 1 fermenter of Porton type; pH was maintained at 6.5-7.0, temperature at 37°C, aeration at 1.1 1/min., and agitation at 1000 rpm.

Inoculation was made by resuspending culture in physiological salt solution grown on LB agar slant containing 6 mg/1 chloramphenicol overnight at 37°C. Harvest was made at 50 hours from inoculation.

The cultivation was conducted as a batch fermentation on the following substrate: Glycerol Tryptone (Bacto) Yeast extract (Bacto) NaCl 5 K₂HP0₄

KH₂P0₄ κ₂so₄

CaCl₂'2H₂0 MgS0₄ '7H₂0 10 Mikrosoy^1>

Pluronic

pH adjusted to 7.0, substrate autoclaved in the fermenter for 40 minutes at 121°C.

After sterilization, 15 g glucose in 50 ml and 6 mg 15 chloramphenicol were added per litre of substrate.

¹⁾Mikrosoy:

G/L

Na₃ citrate 10

Borax (Na₂B₄0₇ '10H₂0) 1.0

20 MnS0₄'H₂0 0.5

FeS0₄"7H₂0 2.0

CUS0₄"5H₂0 0.2

BaCl₂"2H₂0 0.1

Zncl₂ 0.2

25 (NH₄)₄M0₇0₂₄'4H₂0 0.1

EXAMPLE 6

Purification example

The harvested culture broth prepared according to Example 5 was centrifuged for 30 min. at 4000 rpm/servall H 306000A in a Servall RC-3B centrifuge.

The pellet was resuspended with 50 mM Tris puffer, pH 7.0 to a final volume of 10% of culture broth volume. Lysozyme was added to 2 g/1 final concentration, upon which incubation at 40°C for 1 h followed by treatment of the suspension with an Ultratorrax/type TP18/10;18N;170W, submerged into the suspen¬ sion, for 3 minutes.

The suspension was subsequently set to boil for 1 and centrifuged at 9000 rpm/Servall GS3 at 4"C for 30 minutes in a Servall RC-5B centrifuge.

The pullulanase activity in the supernatant was concentrated 2.8 times by ultrafiltration on a DDS/GR 81PP membrane at room temperature (5mM NaN₃ added prior to ultrafil¬ tration) .

EXAMPLE 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 and substrate specificity.

Substrate specificity

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 (Merck) ; final concentration.

Temperature: 60°C.

Time: 24 hours.

Amount of pullu¬ lanase sample added to incuba¬ tion mixture: 5-50% v/v.

Enzyme reaction stopped by: Rapid cooling at 0°C.

From TLC analysis it was evident that pullulan was degraded almost exclusively to maltotriose, panose was degraded almost exclusively to glucose and maltose, soluble starch was degraded covering all glucose oligomers starting from DP, and upwards, as products of hydrolysis. The rate of pullulan hydrolysis was significantly the fastest.

Thermal stability

Small quantities of the pullulanase sample, sealed in glass ampules, were incubated at 121°C for 10, 30, 90 min. or at 100^βC for 30 min., 1, 2 , 4 hours and rapidly cooled upon incubation. Remaining pullulanase activity at 60 " C, pH 50 was determined as described in Example 8.

% Remaining Pullulanase Activity

pH optimum

Pullulanase activity at 60°C was determined as described in Example 8, except that various incubation pH/buffers were used.

Buffers: pH 3.5-5.5: 0.05 M NaAc (final concentration): A-buffer pH 5.5-7.5: 0.025 M Na₃ citrate and 0.025 M KH₂P0₄ (final concentration) : CP-buffer

Incubation pH 3.5 4.5 5.5 5.5 6.5 7.5 Buffer A A A CP CP CP Activity (%) 5 91 100 100 63 3

Temperature optimum

Pullulanase activity at pH 5.0 was determined as described in Example 8, except that various incubation tempera¬ tures were used. Incubation temperature (°C) 60 70 80 90 100 105 121 Activity (%) 18 38 41 54 98 100 48

5 EXAMPLE 8

Method of pullulanase activity analysis

1 ml of 4% w/v pullulan in 0.1 M NaAc, pH 5.0, is added to 1 ml of diluted enzyme solution (the pullulanase sample is diluted in order to produce a maximum of 0.2 g 10 glucose equivalents/1 in the incubation mixture after addition of Na₂C0₃ buffer) at 0°C.

One ml of the incubation mixture volume is incubated at 60°C for 30 minutes. One ml of the incubation mixture volume is incubated at 0°C for 30 minutes. To each portion 1.5 ml of 150.5 M Na₂C0₃ buffer, pH 10.0, are added, and the mixture is rapidly cooled.

The amount of reducing carbohydrate is determined in both samples by the Nelson/Somogoi method (Nelson,N. (1944) ; J.

Biol. Chem. ; 153, 375-80; and Somogoi. M. (1952); J. Biol. 20 Chem.; 195. 19-23) . After subtracting the blind value (0°C) the activity is expressed as

umol glucose equivalents formed min. x 1 culture broth (PU/1)

EXAMPLE 9

25 MW determination

The molecular weight of the pullulanase of the invention was determined by SDS PAGE conducted according to Pharmacia Phast System file No. 110, on Phastgel Gradient 8-25. A molecular weight of 95,000 (+/- 10,000) was determined.

30 The identity between the pullulanase and the MW =

95,000 band was established by overlayering the gel with 0.1% dyed pullulan and incubating at 60 C for 4 hours. Only one band with pullulanase activity was detected. As the DNA insert from Pyrococcus is only 4.5 kb, it is not likely that the DNA insert encodes for more than one pullulanase.

Claims

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

53773, 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 10 115"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.

15 3. The pullulanase of claim 2, having α-l,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.

0 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 5 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 0 a pullulanase producing strain of Pyrococcus in a nutrient medium that does not contain elementary sulphur, and that is not turbid, the cultivation being accomplished under continuous gassing with N₂/C0₂ 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 DP., and upwards.

5 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.

10 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

15 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

20 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.

20. The process of claim 18, in which the plasmid is 25 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 pUBllO; 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. amylolicruefaciens 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, α-glucosidase, /3-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 α-amylase, and subsequent saccharification in the presence of one or more enzymes selected from the group consisting of glucoamylase, α-glucosidase, 3-amylase, or othe saccharifying enzymes, optionally together with a pullulanase

28. The starch converting process according to clai 27, in which the simultaneous liquefaction/debranching i 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 temperatur in the range of from 95 to 130°C, the pullulanase dosage bein 1-100 μg/g dry substance and the thermostable α-amylase dosag being 1-20 μg/g dry substance.