MXPA00008430A - Modified forms of pullulanase - Google Patents

Abstract

The present invention relates to modified pullulanases useful in the starch industry. The present invention provides methods for producing the modified pullulanase, enzymatic compositions comprising the modified pullulanase, and methods for the saccharification of starch comprising the use of the enzymatic compositions.

Description

MODIFIED FORMS OF PULLULANASA FIELD OF THE INVENTION The present invention relates to modified forms of pullulanase that maintain the ability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond, to compositions comprising the modified pullulanases, to methods for making the modified pullulanase and to methods to use the modified pullulanase, especially for saccharification of starch.

BACKGROUND OF THE INVENTION Starch, the essential constituents of which are linear amylose polymers and branched amylopectin-glucose can be converted into simple sugars by an enzymatic process carried out in two stages: a stage of liquefaction of the starch and a stage of saccharification of liquefied starch. In order to obtain a high conversion level of the starch, pullulanase (EC 3.2.1.41, -dextrin-6-glucan hydrolase also called alpha-1,6-glucosidase) has been used to catalyze the hydrolysis of the alpha-linkages. 1, 6-glucosid? Cos. REF .: 12_450 The pullulanase enzymes in the art include those known to have optimal activity at acidic pH, as well as those known to have activity at alkaline pH. Pullulanases described in the art include pullulanase derived from a strain of Bacillus a cidopul l ulytius described for having an optimum activity at a pH of 4-5 at 60 ° C (U.S. Patent No. 4,560,651 ); pullulanase derived from Ba ci ll na gan s if described by having a maximum activity at a pH of about 5, measured at 60 ° C and a maximum activity at a temperature of about 62.5 ° C, measured at a pH of 4.5 (U.S. Patent No. 5,055,403); pullulanase derived from Ba ci ll us sectorramus described as having an optimum pH at 5.0 to 5.5 and an optimum temperature at 50 ° C (U.S. Patent No. 4,902,622); and pullulanase derived from Ba ci ll brevi s PL-1 described because it has activity at 4.5-5.5 at 60 ° C (JP 04/023985). Pullulanase can be used with glucoamylase or β-amylase for the production of syrups with high glucose content and high maltose content. In addition to increasing the yields of sugars, pullulanase reduces the reaction time allows high concentrations of substrate and a reduction of up to 50% in the use of glucoamylase (Bakshi et al., (1992) Biotechnology Letters vol 14 pp. 689-694).

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the surprising and unexpected discovery by the applicants that modified forms of pullulanase retain the ability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond. The present invention provides modified forms of pullulanase and methods for producing the modified pullulanase, especially in recombinant host microorganisms. The present invention further relates to enzymatic compositions comprising a modified form of pullulanase useful in the saccharification of starch and methods for the saccharification of starch comprising the use of the enzyme compositions.

The present invention is based, in part, on the discovery that when the pullulanase obtained from Ba ci l us derami fi ters was expressed recombinantly and cultured in Ba ci l l us In the case that the pullulanase produced was a mixture of modified forms, even the modified forms of pullulanase surprisingly retained the ability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond. The modified forms comprised pullulanase from S. derami fi tr truncated at the amino terminus, ie having an amino acid deletion from the amino terminus, and from B. derami fi ca n s that has additional amino acids in the amino terminus of the mature pullulanase. Therefore, in one aspect, the present invention provides modified pullulanase having an amino acid deletion from the amino terminus of a pullulanase obtainable from a gram-positive or a gram-negative microorganism while the modified pullulanase retains the capacity to catalyze the hydrolysis of an alpha-1 bond, 6-glucosidic. In another aspect, the present invention provides modified pullulanase having additional amino acids at the amino terminus of a pullulanase obtainable from a gram-negative or gram-positi microorganism or while the modified pullulanase retains the ability to catalyze the hydrolysis of a alpha-1, 6-glucosidic bond. The present invention it also encompasses amino acid variations of a pullulanase obtainable from a gram-negative or gram-positive microorganism insofar as the modified pullulanase retains the ability to catalyze the hydrolysis of an alpha-1,6-glucosidic. In one embodiment, the modified pullulanase is a modification of the pullulanase obtainable from the Klebsiella species. In another embodiment, the modified pullulanase is a modification of the pullulanase obtainable from the Bacillus species. In yet another embodiment, the modified pullulanase is a modification of the pullulanase obtainable from Bacillus, including, inter alia, B. subtilis, B. deramificans, B. stearothermophilus, B. naganoensis, B. flavocaldarius, B. acidopullulyticus, Bacillus sp APC -9603, B. sectorramus, B. cereus, B. fermus. In a preferred embodiment, the modified pullulanase is a modification of the pullulanase obtainable from B. deramificans having the designation T89.117D (LMG P-13056) deposited on June 21, 1993 under the Budapest treaty in the collection of LMG culture, University of Ghent, Microbiology Laboratory, KL Ledeganckstraat 35, B-9000 GKENT, Belgium In one embodiment, the modified pullulanase has a deletion of about 100 amino acids from the amino terminus of a pullulanase. In another embodiment, the modified pullulanase has a deletion of about 200 amino acids from the amino terminus of a pullulanase and even in another embodiment, the modified pullulanase has a deletion of about 300 amino acids from the amino terminus of a pullulanase. In a further embodiment, the modified pullulanase has a deletion of 98 amino acids from the amino terminus of the pullulanase obtainable from B. derami fi cans. In a further embodiment, the modified pullulanase has a deletion of about 102 amino acids from the amino terminus of the pullulanase obtainable from B. derami fi s s. In a further embodiment, the modified pullulanase has at least one additional amino acid in the amino terminus of the pullulanase obtainable from B. deramí fi cans. In another embodiment, the modified pullulanase has an additional amino acid residue, alanine, added to the amino terminus of pullulanase obtainable from B. derami fi cans. Modulated forms of pullulanase having a decrease in molecular weight provide the advantage of a higher specific activity (activity / unit weight) and therefore, less weight of pullulanase activity is required in a saccharification process to obtain results equivalent to the use of a pullulanase that occurs naturally, obtainable from or produced by a microorganism. The recombinant production of modified pullulanase as taught herein provides enzymatic compositions comprising at least 60% and at least 80% pullulanase activity. In one embodiment, the enzyme composition comprises at least one modified pullulanase. In another embodiment, the enzyme composition comprises more than one modified pullulanase. These enzyme compositions are advantageous for the starch processing industry because of their ability to produce a high glucose yield with respect to a shortened saccharification time without the loss of glucose yield associated with the products of the inversion reaction. Further, it was unexpectedly found that by using an enzymatic composition comprising 20% glucoamylase and 80% pullulanase, the mostly dissolved starting solids (DS) could be used in a saccharification process, thereby increasing the production capacity of the plant without an increase in capital investment. Additionally, saccharification in mostly dissolved solids increases the mechanical compression capacity, thus providing a more energy efficient process. In a modality, the present invention provides modified pullulanase produced by the method comprising the steps of obtaining a recombinant host cell comprising nucleic acid encoding mature pullulanase, culturing the host cell under conditions suitable for the production of modified pullulanase and optionally recovering pullulanase modified. In one embodiment, the host cell is Bacillus, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thupngiensis.

In a preferred embodiment, the Bacillus cell is B. licheniformis comprising a first gene encoding the Carlsberg protease and a second gene encoding endoGluC, the first and / or second gene encoding the protease (s) ( s) that has been altered in the Bacillus species such that the protease activity is essentially eliminated and the nucleic acid coding for the mature pullulanase can be obtained from B. deramificans. In an alternative embodiment, the present invention provides methods for the production of a modified pullulanase in a recombinant host cell comprising the steps of obtaining a recombinant microorganism comprising nucleic acid encoding a modified pullulanase, culturing the microorganism under conditions suitable for production of the modified pullulanase and optionally recover the modified pullulanase, produced. In one embodiment, the host cell is a gram-negative or gram-positive microorganism. In another embodiment, the host cell is a Bacillus including, inter alia, B. subtilis, B. licheniformis, B. lentus, B. lentus, B. brevis, B. stearothermophilus, B. alkalsphilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thurmgiens. In another embodiment, the Bacillus cell is B. lichenlformis comprising a first gene encoding the Carlsberg protease and a second gene coding for endoGluC, the first and / or second gene encoding the protease (s). ) which has been altered in the Bacillus species such that the protease activity and the nucleic acid coding for the modified pullulanase which is a modification of the pullulanase obtainable from B. deramificans are substantially eliminated. The present invention also provides a nucleic acid comprising a polynucleotide sequence encoding the modified pullulanase. In one embodiment, the nucleic acid has at least 70% identity, at least 80% identity, at least 90% identity or at least 95% identity to the polynucleotide sequence shown in SEQ ID NO: 1, which encodes for the pullulanase obtainable from B. deramificans. The present invention also provides expression vectors and host microorganisms comprising nucleic acid encoding a modified pullulanase of the present invention.

The present invention provides an enzymatic composition comprising at least one modified pullulanase of the present invention. In one embodiment, the enzymatic composition comprises multiple modified forms of pullulanase. In another embodiment, the composition additionally comprises an enzyme selected from the group consisting of glucoamylase, alpha-amylase, beta-amylase, alpha-glucosidase, isoamylase, cyclomaltodextrin, glucotransferase, beta-gl'ucanase, glucose-isomerase, enzymes of saccharification, and / or enzymes that cleave glycosidic bonds. In a preferred embodiment, the enzyme composition comprises a modified pullulanase and glucoamylase. In one embodiment, glucoamylase is derived from a strain of Aspergi l l us. In another embodiment, glucoamylase is derived from a strain of Aspergillus, including, but not limited to, Aspergillus n i ger, Aspergi l l us a wamori and Aspergi l lus foe ti dus. The enzyme composition can be in a solid form or a liquid form. In one embodiment of the present invention, the enzyme composition comprises at least 60% modified pullulanase and in the other embodiment, the composition comprises minus 30% modified pullulanase. The present invention also provides a process for saccharification of starch, wherein the process allows reduced concentrations of saccharification inversion by-products, which comprises the step of contacting the liquefied, aqueous starch with an enzymatic composition comprising pullulanase. modified. In a modality, the process also includes the steps of heating the liquefied starch, and recovering the product. In one embodiment of the process, the enzymatic composition additionally comprises glucoamylase. In another embodiment of the process, the contacting is at a pH of approximately less than or equal to 7.0 and greater than or equal to 3 and in yet another, the pH is approximately 4.2. In a further embodiment of the process, the heating is at a temperature range of between 55 and 65 ° C. In another embodiment, the temperature is approximately 60 degrees.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1E illustrate the nucleic acid (SEQ ID NO: 1) encoding the mature amino acid sequence (SEQ ID NO: 2) of the pullulanase obtainable from Ba ci l l us. Figures 2A-2D are an alignment of the amino acid sequences of the pullulanase obtainable from B. derami fi cans (designated pullseqsig. seq. PRO), B. s ubti l i s (designated subpull.seq.pro), and K. pneumonia (designated klebpnseqsig.seq.pro) showing the conserved domains and the variability of the amino terminus of these pullulanases. This alignment also includes the signal sequences for the respective pullulanases. Figures 3A-3C illustrate a fermentation time course and the various modified pullulanase species that are formed during fermentation. Peak 1 designates the mature pullulanase of B. deram i fi ca n s having a molecular weight of 105 kD; peak 2 designates the modified pullulanase having a deletion of 102 amino acids from the amino terminus of the mature pullulanase of B. derami fi s; and peak 3 designates the modified pullulanase having a deletion of 98 amino acids from the amino terminus as measured by normal HPLC analysis. Figure 3A illustrates the fermentation for 37 hours. Figure 3B illustrates the fermentation during 60 hours. Figure 3C illustrates the fermentation for 70 hours. Figures 4A-4D illustrate the stability of the modified pullulanase species as a function of pH as measured by normal HPLC analysis. Figure 4A illustrates the pullulanase stability at 24 hours at a pH of 4.5 at room temperature. Figure 4B illustrates pullulanase stability at 24 hours at a pH of 5.5 at room temperature. Figure 4C illustrates the pullulanase stability at 24 hours at a pH of 6.5 at room temperature. Figure 4D illustrates the pullulanase stability at 96 hours at a pH of 4.5 at room temperature. Figures 5A-5C illustrate the effect of enzyme compositions comprising various concentrations of pullulanase and glucoamylase in the final glucose yield and the formation of disaccharides during the saccharification time. The solid line refers to an enzymatic mixture comprising 80% pullulanase activity (including modified pullulanase having a deletion of 98 amino acids from the amino terminus of B. deram i fi s s, modified pullulanase having a deletion of 102. amino acids from the amino terminus of B. derami fi cans; mature pullulanase from B. derami fi s and mature pullulanase from B. derami fi cans that has an additional amino acid (alanine) at the amino terminus) and 20% glucoamylase (20:80). The dashed line refers to an enzymatic composition comprising a mixture of enzymes comprising 75% glucoamylase obtainable from Aspergi l. Us sp. and 25% mature pullulanase obtainable from B. derami fi cans (75:25). The solid line with squares refers to disaccharides formed with the enzyme mixture comprising an activity of 20% glucoamylase and 80% pullulanase as described above (20:80) during the saccharification time and the dotted line with circles refers to di-saccharides formed with 75:25 during the saccharification time. The left X axis is the% glucose yield and the right X axis is the% disaccharide. Figure 5A refers to the saccharification process using 0.550 liters of enzyme composition per metric ton of dissolved solids; Figure 5B refers to the saccharification process using 0.635 liters of enzyme composition per metric ton of dissolved solids; Figure 5C is refers to the saccharification process using 0.718 liters of enzyme composition per metric ton of dissolved solids. This figure illustrates that an enzymatic 20:80 composition is capable of increasing the final glucose yield without an increase in the initial or undesirable formation of disaccharides. Figure 6 illustrates the effect of dissolved solids (% w / w) (Y axis) on final glucose yield during saccharification of liquefied starch using 20:80, 75:25, and 100% glucoamylase enzyme compositions to 0.55 liters of enzyme per metric ton of dissolved solids. Line A is the enzymatic composition 20:80 described in Figures 5A-5C; line B is the enzyme composition 75:25 and line C is an enzymatic composition comprising 100% glucoamylase.

DETAILED DESCRIPTION Definitions The term "pullulanase" as used herein refers to any enzyme that has the ability to cleave the alpha-1,6-glucosidic bond in starch to produce anuloses of Straight chain. These enzymes are preferably classified in CEC 3.2.1.41 and include neopululanases. As shown in Figures 2A-2D, there are regions of similarity between the pullulanases obtainable from gram-positive and gram-negative microorganisms. The amino acid alignment of pullulanase obtainable from Bacillus derami fi can s with the pullulanase obtainable from K. pneumonia and B. ubilis reveals that when the conserved domains are aligned, the amino terminus is not associated with the domains preserved is of variable length. As used herein, the term "modified" when referring to pullulanase means a pullulanase enzyme in which the conserved domains are retained while any amino acid length in the amino terminal portion of the amino acid sequence presented naturally not associated with the conserved domains has been altered by a deletion of these amino acid residues or by the addition of at least one amino acid to the amino terminus while the modified pullulanase retains the ability to catalyze the hydrolysis of an alpha-1 bond, 6-glucosidic. The Deletion in the amino-terminal amino acids of a pullulanase can be of variable length, but it is at least three amino acids in length and deletion can only occur at the beginning of the first conserved domain that in B. derami fi can is tyrosine in the amino acid residue 310 as shown in Figures 1A-1E. In one embodiment, the deletion is about 100 amino acids from the amino terminus of the mature pullulanase. In another embodiment, the deletion is about 200 amino acids from the amino terminus of the mature pullulanase, and in another embodiment, the deletion is about 300 amino acids from the amino terminus of the mature pullulanase. In a preferred embodiment, the modification is a deletion of 98 amino acids from the amino terminus of B. derami fi ca n s. In yet another embodiment, the deletion is 102 amino acids from the amino terminus of B. derami fi s s. In a further embodiment, the modification is an addition of at least 1 amino acid to the amino terminus of the mature pullulanase that occurs naturally from Bacillus derami fi ters. In another preferred embodiment, the amino acid residue, alanine, is added to the amino terminus of the mature pullulanase. As used herein, the term "mature" refers to a protein that includes the N-terminal amino acid residue found after the site of natural incision of the signal sequence. As illustrated in Figures 2A-2D, the pullulanase of B. derami fi cans and pullulanase from K. pneumonia are examples of pullulanases that have similarities in the length of the amino terminus to the beginning of the first conserved domain (which in B. derami fi s is amino acid residue 310, tyrosine). The pullulanase from B. s ub ti l i s is an example of a pullulanase having a shorter length of amino acid residues up to the beginning of the first conserved domain as shown in Figure 2B. As used herein, "nucleic acid" refers to a sequence of nucleotides or polynucleotides, and fragments or portions thereof, and to a DNA or RNA of genomic or synthetic origin which may be double-stranded or single-stranded, whether it represents the homosense or antisense strand. As used herein, "amino acid" refers to peptide or protein sequences or portions thereof.

The present invention encompasses polynucleotides having at least 70%, at least 80%, at 90% and at least 95% identity to the polynucleotide coding for the pullulanase of B. derami fi ca ns, as well as polynucleotides encoding a Pullulanase activity capable of hybridizing the nucleic acid coding for the pullulanase of B. derami fi s under conditions of high severity. The terms "isolated" or "purified" as used herein refer to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present invention relates to the discovery that the pullulanase recombinantly produced in a Ba ci us host is modified but still unexpectedly maintains the ability to catalyze the hydrolysis of an alpha-1, 6-bond. -glucos? d? co. Modification of the recombinantly produced pullulanase product appears to result from poor processing of the signal sequence by a signal peptidase as well as susceptibility to extracellular proteolytic processing. The Modulated pullulanase is used to produce compositions and methods useful in the starch industry.

Pullulanase Sequences The present invention encompasses any modified pullulanase that retains the ability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond. A variety of pullulanases have been described in the art, including those obtainable from gram-positive microorganisms, - as well as gram-negative microorganisms, or those produced naturally thereof. The microorganisms that naturally produce pullulanase include, but are not limited to, B. deram i fí cans (which has the designation T89.117D in the LMG culture collection, University of Ghent, Microbiology Laboratory, KL Ledeganckstraat 35, B-9000 Ghent, Belgium) the nucleic acid sequence (SEQ ID NO: 1) and of amino acids (SEQ ID NO: 2) which is described in Figures 1A-1E; B. na gan oen s i s (American Type Culture Collection, accession number ATCC 53909), described in U.S. Patent No. 5,056,403 issued October 8, 1991; B. acidopullulyticus (National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, NCIB 11607, NCIB 11610, NCIB 11611, NCIB 11636, NCIB 11637, NCIB 11639, NCIB 11638, NCIB 11647, NCIB 11777), described in the patent of the United States No. 4,560,651, issued December 24, 1985; B. ramus sector (Fermentation Research Institute, Agency of Industrial Science and Technology, l-3, Higashi 1- chome, Yatabe-machi, Tsukuba-gun, Ibaraki 305 Japan FERM BP-1471), described in the United States Patent No. 4,902,622, issued February 20, 1998; Bacillus FERM BP-4204 described in U.S. Patent No. 5,387,516 issued February 7, 1995; B. stearothermophilus (SWISS-PROT id NEPU_BACST ac P38940); B. cereus var. mycoides (IFO 300) described in Y. Takasaki et al., 1976, Agrie. Biol. Chem. 40: 1515; B. fermus (IFO 3330); Klebsiella pneumonia, Patent of the United States No. - 3,897,305 (SWISS-PROT id PULA_KLEPN ac P07206 and ATCC 15050; Klebsiella aerogenes (SWISS-PROT id PULA_KLEAE ac P07811); Thermoanaerobium brockii (ATCC No. 33075), U.S. Patent No. 4,628,028; Streptomyces sp. Described in M. Yagisawa et al., 1972, J. Ferment Technolo. 50: 572; Caldicellulosiruptor saccharolyticus described in Albertson et al., 1997, Biochimica et Biophysica Acta 1354: 35-39; Intermediate Escherity Ueda et al., 1967, Applied Microbiology vol. 15: 492 U.S. Patent No. 3,716,455 (issued in 1973) St eptococcus mites Walker 1968, Biochem. J., vol. 108: 33; Streptomyces (Ueda et al., 1971, J. Ferment, Tech. Vol. 49: 552); Flavochromogenes, as described in U.S. Patent No. 4, .902,622; Fia voba cter i um esteromaticum Japanese Patent Application Kckoku 18826/1973; Cytophaga United States Patent No. 3,790,446 issued in 1974; Lactobacillus, Micrococcus, Nocardia, Staphylococcus, Azo tobactger, Sarcina Patent of England 11260418, United States Patent No. 3,827,940 issued in 1974; and Actinomycetes U.S. Patent No. 3,741,873 issued in 1973. Any pullulanase known in the art comprising the conserved pullulanase regions as shown in Figures 2A-2D can be modified to have deletions or additions to the amino terminus while the Modulated pullulanase maintains the ability to catalyze the hydrolysis of an alpha-1,6-glucosidic bond.

A nucleic acid sequence encoding a pullulanase can be obtained from a microorganism through hybridization technology using the nucleic acid sequences encoding the conserved pullulanase domains (as shown in Figures 2A-2D ) as primers and / or probes. (U.S. Patent No. 5,514,576; Southern, E. 1979, Methods Enzymol, 68: 152-176; Saiki, et al., 1988, Science 239: 487-491). In a mode described herein for the pullulanase of B. derami fi can s, the naturally occurring nucleic acid (SEQ ID NO: 1) coding for a mature pullulanase was introduced into B. l i ch in i forms that has the suppression of the Carlsburg protease (Jacobs et al., 1985, Nucleic Acid Research 13: 8913-8926) and endoGluC proteases (Kakudo et al., 1992, Journal of Bio. Vol. 267: 23782-23788), the B. li ch eni forms comprising the nucleic acid encoding the mature pullulanase was cultured under conditions suitable for expression of the nucleic acid and suppression of the expressed pullulanase. The protease suppressions of B. l i ch eni formi s were made through techniques known per se. those skilled in the art. Through the fermentation process, the expressed pullulanase was extracellularly cleaved into multiple pullulanase species that maintain the ability to catalyze the hydrolysis of alpha-1, 6-glucosidic bonds. The multiple species are a pullulanase that has a deletion of the first 98 amino acid residues from the amino terminus and initiating into glutamic acid, a pullulanase that has a deletion of the first 102 amino acid residues from the amino terminus (and starting at the glutamic acid), of a pullulanase having the addition of at least one amino acid residue to the amino terminus of the mature pullulanase, together with the mature pullulanase as shown in Figures 1A-1E. As shown in Example II, it appears that the extracellular cleavage in the multiple species may be due to a protease activity in the fermentation broth. In an alternative embodiment of the present invention, the nucleic acid encoding a mature pullulanase is genetically engineered to create a modified pullulanase having an amino acid deletion at the amino terminus or having amino acids added to the amino terminus. The The genetically modified pullulanase is introduced into a host cell, preferably a Ba cylus host cell, which is cultured under conditions suitable for the expression and secretion of the modified pullulanase. The nucleic acid encoding a mature pullulanase can be a sequence that occurs naturally, a variant form of nucleic acid or from any source, whether natural, synthetic or recombinant. Regional sequence homologies in the starch degradation enzymes have been described in Janse et al., (1993) Curr. Genet 24: 400-407. Janse describes regions conserved in α-amylases that are involved in substrate binding, catalysis, and calcium binding. An amino acid alignment of the pullulanases of B. deramifi cans, B. subti li s, and K. pn e umon í a is shown in Figures 2A-2D. When the homologies in starch degradation enzymes were compared by Janse et al, four conserved regions, regions 1, 2, 3, and 4, were identified. Three of these regions were associated with specific functions found in enzyme degradation enzymes. starch: region 1: DVVINH; region 2: GFRLDAAKH; and region 4: FVDVHD.

Additional analysis of five pullulanase type I sequences by Albertson et al., (1997, Biochimica et Biophysica Acta 1354: 35-39) revealed other regions conserved among a group of gram-positive and gram-negative pullulanases. These include regions called DPY, A, B, C, D, E, and YNWGY. Two regions, the DPY and the YNWGY were identified as being characteristic of true pullulanases. The conserved regions A-E are closely aligned with the ß-leaf elements as defined for the amylases. further, two other conserved regions close to the N-terminus of pullulanase, referred to as Y and VWAP in Figures 2A-2D, indicate the limits of amino acid truncations in the N-terminus of pullulanases in general. This prediction is based on the lack of additional, conserved regions of identity among the known pullulanases beyond the Y region as one that proceeds to the N-terminus. Due to the size heterogeneity of the known pullulanases, the N-terminal regions beyond the Y region can vary between about 100-300 amino acids. For the pullulanase of B. derami fi ca ns, a truncation of 309 amino acids will leave the first region intact conserved (and in amino acid residue 310 in Figures 1A-1E).

Pullulanase from B. deramificans The mature pullulanase from B. deramificans comprises the amino acid sequence (? EQ ID NO: 2) shown in Figures 1A-1E. The following description of the characteristics refers to the mature pullulanase of B. deramificans. The pullulanase of B. deramificans has an isoelectric point between 4.1 and 4.5, is heat stable and active over a wide range of temperatures. The pullulanase of B. deramificans is active at an acidic pH. This pullulanase is capable of catalyzing the hydrolysis of the alpha-1,6-glucosidic bonds present in both amylopectin and pululan. It disintegrates the pululan in maltrotiosa and amylopectin in amylose. The polysaccharide swarm, which is a polymer of maltotriose units connected together by - Alpha-1, 6-links can be obtained from Aureobas idium pullulans (Pullaria pullulans) by the procedure of Ueda et al., Applied Microbiology, 11, 211-215 1963). The pullulanase from B. deramificans hydrolyzes Amylopectin to form oligosaccharides (maltooligosaccharides). During this hydrolysis, the formation of oligosaccharides composed of approximately 13 glucose units is observed (degree of polymerization of 13, this molecule is also called "chain" A), followed by the formation of oligosaccharides consisting of approximately 47 glucose units ( polymerization of 47, this molecule is also called "B chain"). The oligosaccharides with the A and B chains are defined with reference to D.J. MANNERS ("Structural Analysis of Starch Components by Debranching Enzymes" in "New Approaches to Research on Cereal Carbohydrates", Amsterdam, 1985, pages 45-54) and B.E. ENEVOLDSEN ("Aspects of the fine structure of starch" in "New Approaches to research on Cereal Carbohydrates", Amsterdam, 1985, pages 55-60). The pullulanase from B. derami fi fe hydrolyzes the amylopectin of the potato. This hydrolysis can be carried out with an aqueous suspension of amylopectin in the presence of pullulanase with the conditions of optimum pullulanase activity, that is, at a temperature of about 60 ° C and at a pH of about 4.3. The pullulanase of B. derami fí cans cataliza the maltose condensation reaction to form tetraholosides (oligosaccharides having 4 glucose units). The pullulanase of B. derami fi can s has a half-life of about 55 hours, measured at a temperature of about 60 ° C in a buffered solution at a pH of 4.5 and in the absence of a substrate. The half-life means that the pullulanase shows a relative enzymatic activity of at least 50%, measured after an incubation of 55 hours at a temperature of about 60CC in a buffered solution at a pH of about 4.5 and in the absence of substrate. Pullulanase from B. derami fi tas is heat stable at an acidic pH and shows a relative enzymatic activity of at least 55%, measured after a 40-hour incubation at a temperature of 60 ° C in a buffered solution at a pH of about 4.5 in the absence of the substrate. It shows a relative enzymatic activity of at least 70%, measured after a 24-hour incubation under these same conditions. Relative enzymatic activity means the relation between the enzymatic activity measured in the course of a test carried out under the given pH, temperature, substrate and conditions of duration, and the maximum enzymatic activity measured during the course of this same test, the enzymatic activity that is measured starting from the hydrolysis of pullulan and the maximum enzymatic activity which is arbitrarily set at a value of 100. The pullulanase of B. derami fi thas is also stable over a wide range of acid pH values. With the conditions described below, it is active at a pH greater than or equal to 3. In fact, the pullulanase from B. derami fi tas shows a relative enzymatic activity of at least 85%, measured after a 60 minute incubation at a temperature of about 60CC in the absence of substrate and in a pH range greater than or equal to about 3.5. With the conditions described below, it is active at a pH of less than or equal to 7. In reality, the pullulanase of B. derami fi can s shows an enzymatic activity of at least 85%, measured after a 60-minute incubation at a temperature of approximately 60 ° C in the absence of the substrate in a pH range less than or equal to about 5.8. It preferably shows a relative enzymatic activity of more than 90%, measured in a pH range of about 3.8 and about 5 under these same conditions. The pullulanase of B. derami fi s s develop optimal enzymatic activity, measured at a temperature of approximately 60 ° C, in a pH range greater than 4.0. It develops an optimal enzymatic activity, measured at a temperature of approximately 60 ° C, in a pH range of less than 4.8. The pullulanase of B. deram i fi s s preferably develop an optical enzymatic activity, measured at a temperature of about 60CC, at a pH of about 4.3. Additionally, it develops an optimal enzymatic activity, measured at a pH of approximately 4.3, in a temperature range between 55 and 65 ° C, and more particularly at 60 ° C. The pullulanase of B. derami fi reta develops an enzymatic activity of more than 80% of the maximum enzymatic activity (the maximum enzymatic activity measured at a temperature of 60 ° C and a pH of 4.3) in a pH range of between about 3.8 and about 4.9 at a temperature of about 60 ° C. Strain T 89.117D from Ba ci ll derami fi thas has been deposited in the collection called BELGIUM COORDINATED COLLECTIONS OF MICROORGANISM (LMC culture collection, University of Ghent, Laboratory of Microbiology - KL Ledegans straat 35, B - 9000 GHENT, Belgium ) in accordance with the Budapest Treaty under number LMG P-13056 on June 21, 1993.

II. Expression Systems The present invention provides host cells, expression methods and systems for the production and secretion of modified pullulanase in gram-positive microorganisms and gram-negative microorganisms. In one embodiment, a host cell is genetically engineered to comprise a nucleic acid encoding a modified pullulanase. In another embodiment, the host cell is genetically engineered to comprise the nucleic acid encoding a mature or full-length pullulanase, which in the culture produces a modified pullulanase. In a Preferred embodiment, the host cell is a member of the genus Bailusus that has been modified to have a mutation or deletion of the endogenous proteases.

Inactivation of a Protease in a Host Cell The production of an expression host cell incapable of producing a naturally occurring protease requires the replacement and / or inactivation of the gene that occurs naturally from the genome of the host cell . In a preferred embodiment, the mutation is a non-reverting mutation. One method for mutating the nucleic acid encoding a protease is to clone the nucleic acid or part thereof, modify the nucleic acid for sequence directed mutagenesis and reintroduce the mutated nucleic acid into the cell in a plasmid. By homologous recombination, the mutated gene can be introduced into the chromosome. In the host cell of origin, the result is that the nucleic acid that occurs naturally and the mutated nucleic acid are located in tandem on the chromosome. After a second Upon recombination, the modified sequence is left in the chromosome having thus effectively introduced the mutation into the chromosomal gene for the progeny of the host cell of origin. Another method to inactivate the proteolytic activity of the protease is through the deletion of the copy of the chromosomal gene. In a preferred embodiment, the entire gene is deleted, the deletion occurring in such a way as to render reversal impossible. In another preferred embodiment, partial deletion occurs, with the proviso that the nucleic acid sequence left from the chromosome is too short for homologous recombination with a plasmid-encoded metalloprotease gene. In another preferred embodiment, the nucleic acid encoding the catalytic amino acid residues is deleted. The suppression of the protease of the naturally occurring microorganism can be carried out as follows. A protease gene that includes 5 'and 3' subregions is isolated and inserted into a cloning vector. The coding region of the protease gene is deleted from vector in vitro, leaving behind a sufficient amount of the 5 'and 3' flanking sequences to provide homologous recombination with the gene that occurs naturally in the host cell of origin. The vector is then transformed into the host cell. The vector is integrated into the chromosome via homologous recombination in the flanking regions. This method leads to a strain in which the protease gene has been deleted. The vector used in an integration method is preferably a plasmid. A selectable marker may be included to allow easy identification of the desired, recombinant microorganisms. Additionally, as will be appreciated by one of ordinary skill in the art, the vector is a preferentially one that can be selectively integrated into the chromosome. This can be achieved by introducing an inducible origin of replication, for example, a temperature-sensitive origin in the plasmid. By culturing the transformants at a temperature at which the origin of replication is sensitive, the replication function of the plasmid is reactivated, thereby providing a means for selection of chromosomal members. Members can be selected to grow at high temperatures in the presence of the selectable marker, such as an antibiotic. The integration mechanisms are described in WO 88/06623. Integration by the Campbell-like mechanism can take place in the 5 'flanking region of the protease gene, resulting in a protease-positive strain carrying the complete plasmid vector on the chromosome at the pullulanase locus. Since legitimate recombination will give different results, it will be necessary to determine whether the entire gene has been deleted, such as through nucleic acid sequencing or restriction maps. Another method to inactivate the naturally occurring protease gene is to mutagenize the copy of the chromosomal gene by transforming a microorganism with oligonucleotides that are mutagenic. Alternatively, the professed, chromosomal gene can be replaced with a mutant gene by homologous recombination. The present invention encompasses Bacillus host cells that have deletions or J protease mutations, such as deletions or mutations in apr, npr, epr, mpr, isp and / or bpf and / or others known to those skilled in the art. The description regarding the suppression of protease (s) in the gram-positive microorganism, Ba ci l lus, can be found in U.S. Patent Nos. 5,264,366; 5,585,253; 5,620,880 and in European Patent No. EP 0369 817 Bl. An assay for the detection of mutants comprises culturing the host cell of Bacillus in a medium containing a protease substrate and measuring the appearance or lack thereof of the zone of clearance of halo from the colonies. Host cells that have an inactive protease will exhibit little or no halo around the colonies.

III. Production of modified pullulanase For the production of modified pullulanase in a host cell, an expression vector comprising at least one copy of nucleic acid encoding a modified pullulanase, and preferably comprising multiple copies, is transformed into the host cell low suitable conditions for the expression of the modified pullulanase. According to the present invention, the polynucleotides encoding a modified pullulanase, or fusion proteins or homologous polynucleotide sequences encoding amino acid variants of the modified pullulanase (while the variant retains the ability to catalyze the hydrolysis of a linkage alpha-1, 6-glucosidic), can be used to generate recombinant DNA molecules that direct their expression in host cells. A host cell can be a gram-positive or gram-negative cell. In a modality, the host cell corresponds to the genus Bacillus. In another embodiment, the Bacillus host cell includes B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalopinus, B. amyloliquefaciens, B. coagulans, B.circulans, B. lautus and Bacillus thu ingiensis. In a preferred embodiment, the gram-positive host cell is Bacillus licheniformis. As will be understood by those skilled in the art, it may be advantageous to produce polynucleotide sequences that possess codons that do not occur naturally. The codons Preferred by a particular gram-positive host cell (Murray ER et al., (1989) Nuc.Aids Res. 17: 477-508) can be selected, for example, to increase the rate of expression or to produce recombinant RNA transcripts which have the desirable properties, such as a longer half-life, in which the transcripts produced from the naturally occurring sequence. The altered sequences of pullulanase polynucleotides that can be used according to the invention include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide encoding the same or a functionally equivalent modified pullulanase. As used herein, a "deletion" is defined as a change in the sequence of either the nucleotides or amino acids in which one or more nucleotides or -resides of amino acids are absent, respectively. As used herein an "insertion" or "addition" is that change in a sequence of nucleotides or amino acids that has resulted in the addition of one or more nucleotides or residues of amino acids, respectively, compared to the modified pullulanase that occurs naturally. As used herein, "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. The encoded protein can also show deletions, insertions or substitutions of amino acid residues that produce an absent change and results in a functionally equivalent pullulanase. Deliberate amino acid substitutions can be made based on similarity in the plurality, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues while the variant retains the ability to modulate secretion. For example, negatively charged amino acids include aspartic acid and glutamic acid; the positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine, phenylalanine and tyrosine. The polynucleotides encoding a modified pullulanase of the present invention can be managed to modify the cloning, processing and / or expression of the gene product. For example, mutations can be introduced using techniques that are well known in the art, for example, sequence directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change the codon preference, as example. In one embodiment of the present invention, a polynucleotide encoding a modified polymerase can be added to a heterologous sequence to encode a fusion protein. A fusion protein can also be managed to contain an incision site located between the modified nucleotide sequence of the modified pullulanase and the sequence of heterologous proteins, so that the modified pullulanase can be cleaved and purified from the heterologous fusion partner.

IV. Vector sequences The expression vectors used to expressing the pullulanases of the present invention in the host microorganisms comprise at least one promoter associated with a modified pullulanase, promoter that is functional in the host cell. In one embodiment of the present invention, the promoter is the wild-type promoter for the selected pullulanase and in another embodiment of the present invention, the promoter is heterologous to the pullulanase, but still functional in the host cell. In one embodiment of the present invention, the nucleic acid encoding the modified pullulanase is stably integrated into the genome of the microorganism. In a preferred embodiment, the expression vector contains a multiple cloning site cartridge that preferably comprises at least one restriction endonuclease site unique to the vector, to facilitate manipulation of the nucleic acid. In a preferred embodiment, the vector also comprises one or more selectable markers. As used herein, the term "selectable marker" refers to a gene capable of expression in the host microorganism that allows ease of selection of those hosts that contain the vector. The examples of these selectable markers include, but are not limited to, antibiotics, such as erythromycin, actinomycin, chloramphenicol and tetracycline.

V. Transformation A variety of host cells can be used for the production of the modified pullulanase including bacterial, fungal, mammalian and insect cells. General transformation procedures are taught in Current Protocols in Molecular Biology (vol.1, edited by ausubel et al., John Wiley &Sons, Inc. 1987, Chapter 9) and include calcium phosphate methods, transformation using DEAE- Dextran and electroporation. Plant transformation methods are taught in Rodriguez (WO 95/14099, published May 26, 1995). In a preferred embodiment, the host cell is a gram-positive microorganism and in another preferred embodiment, the host cell is Bacillus. In a further preferred embodiment, the Ba ci l l us host is Ba ci l l l l i i i n i form i s. In one embodiment of the present invention, the nucleic acid encoding a pullulanase The modified of the present invention is introduced into a host cell via an expression vector capable of replicating with the Bacillus host cell. Replication plasmids suitable for Bacillus are described in Molecular Biological Methods for Bacillus, Ed. Harwood and Cutting, John Wiley & Sons, 19909, incorporated in this way expressly by reference; see chapter 3 in plasmids. Replication plasmids suitable for B. subtilis are listed on page 92. In another embodiment, the nucleic acid encoding a modified pullulanase of the present invention is stably integrated into the genome of the host microorganism. Preferred host cells are gram-positive host cells. Another preferred host is Bacillus. Bacillus host cells include B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis. A preferred host is Bacillus subtilis. Another preferred host is B. licheniformis. Several strategies have been described in the literature for the direct cloning of DNA in Bacillus. The Rescue transformation of the plasmid marker comprises the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (Contente et al., Plasmid 2: 555-571 (1979); Haima et al., Mol. Gen. Genet 223: 185-191 (1990), Weinrauch et al., J. Bacteriol., 154 (3): 1077-1087 (1983), and Weinrauch et al., J. Bacteriol. 169 (3): 1205-1211 ( 1987)). The incoming donor plasmid recombines with the homologous region of the "auxiliary" plasmid resident in a process that mimics chromosomal transformation. Transformation by protoplast transformation is described for B. subtilis in Chang and Cohe, (1979) Mol. Gen. Genet 168: 111-115; for B. megaterium in Vorobjeva et al., (1980) FEMS Microbiol. Letters 7: 261-263; for B. amyloliquefaciens in Smith et al., (1986) Appl. and Env. Microbiol. 51: 634; for B. thuringiensis in Fisher et al, (1981) Arch. Microbiol, 139: 213-217; for B. sphaericus in McDonald (1984) J. Gen.

Microbiol. 130: 203; and B. larvae in Bakhiet et al., (1985), Appl. Environ Microbiol. 49: 577), Mann et al., (1986), Current Microbiol, 13: 131-135) reports the transformation of Bacillus protoplasts and 4 Holubova, (1985) Folia Microbiol, 30:97) describes methods for introducing DNA into protoplasts using DNA containing liposomes.

SAW. Identification of Transformants If a host cell has been transformed with a modified gene or a gene occurs naturally encoding a pullulanase activity, detection of the presence / absence of marker gene expression can suggest whether the gene of interest is present. However, your expression must be confirmed. For example, if the nucleic acid coding for a modified pullulanase is inserted into a marker gene sequence, the recombinant cells containing the insert can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the nucleic acid encoding pullulanase under the control of an individual promoter. The expression of the marker gene in response to induction or selection also indicates the expression of pullulanase. Alternatively, the host cells containing the sequence of Coding for a modified pullulanase and expressing the protein can be identified by a variety of procedures known to those skilled in the art. These methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassays or immunoassay techniques that include membrane-based, solution-based, or chunk-based technologies for the detection and / or quantification of nucleic acid. or protein. The presence of the pullulanase polynucleotide sequence in a host microorganism can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of the pullulanase polynucleotide sequences.

VIII. Pullulanase Activity Assay There are several assays known to those skilled in the art for detecting and measuring pullulanase activity. An enzymatic pullulanase unit of B. derami fi fe (PUN) is defined as the amount of enzyme which, at a pH of 4.5, at a temperature of 60 degrees C and the presence of pullulanase, catalyzes the release of reducing sugars at a rate of 1 μM glucose equivalent per minute. Pullulanase activity can be measured in the presence or absence of substrate. In one aspect, the pullulanase activity can be measured in the presence of the substrate according to the following protocol. 1 ml of a 1% concentrated solution of polyulane is incubated in a 50 nM acetate buffer at pH 4.5 at 60 ° C for 10 minutes. To this is added 0.1 ml of a pullulanase solution corresponding to an activity of 0.2 and 1 PUN / ml. The reaction is stopped for 15 minutes by the addition of 0.4 ml of 0.5 M NaOH. The released reducing sugars are analyzed by the method of SOMOGYI-NELSON [J. Biol. Chem., 153 (1944) pages 375-380; and J. Biol. Chem., 160 (1945), pages 61-68]. Another method can be used to analyze pullulanase. The enzymatic reaction in the presence of polulan is carried out according to the above test conditions, and then stopped by the addition of sulfuric acid (0.1 N). The polulan hydrolysis products are then subjected to HPLC chromatography (BIO-RAD HPX-87H column, the mobile phase is 10 mM H2SO) in order to Separate the various constituents. The amount of maltotriose formed is estimated by measuring the area of the peak obtained. The so-called de-branching activity, ie the hydrolysis of the α-1,6-glucosidic bonds present in the amylopectm, can be quantified by the increase in the blue coloration caused, in the presence of iodine, by the release of anulose from amylopectma. The de-branching enzymatic activity is measured according to the following protocol. 0.4 ml of a 1% concentrated amylopectin solution containing a 50 mM acetate buffer at pH 4.5 is incubated at 60 ° C for 10 minutes. The reaction is initiated by the addition of 0.2 ml of pullulanase, and stopped after 30 minutes by the addition of 0.4 ml of 0.3 M HCl. Then 0.8 ml of a concentrated solution at 0.0025% (v / v) of iodine is added. 0.2 ml of this reaction mixture and the optical density is measured at 565 nm. A preferred method is described in Example IV and depends on a colorimetric method that uses a soluble red-polyulane substrate for the determination of pullulanase activity. As the pullulanase enzyme hydrolyzes the substrate, the soluble fragments of the substrate obtained in the reaction solution are released. The substrate is then precipitated with an ethanol solution and the supernatants are evaluated for the color intensity with the spectrophotometer. In this test, the degree of color intensity is proportional to the enzymatic activity.

VIII. Secretion of Recombinant Proteins A means for determining secretion levels of a modified pullulanase in a host microorganism and detecting secreted proteins includes, using either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS). These and other assays are described, inter alia, in Hampton R et al., (1990, Serological Methods, a Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al., (1983, J. Exp Med 158 : 1211). A wide variety of brands and conjugation techniques are known to those skilled in the art and can be used in various assays of amino acids and nucleic acid. A means for producing labeled hybridization or PCR probes to detect specific sequences of polynucleotides includes oligoloking, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the nucleotide sequence or any portion thereof, can be cloned into a vector for the production of an mRNA probe. These vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by the addition of an appropriate mRNA polymerase such as T7, T3 or SP6 and labeled nucleotides. Several companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison Wl), and US Biochemical Corp (Cleveland OH) supply commercial equipment and protocols for these procedures. Suitable indicator molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminic, or chromogenic agents as well as substrates, co-factors, inhibitors, magnetic particles, and the like. Patents that teach the use of these trademarks include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins can be produced as shown in U.S. Patent No. 4,816,567 incorporated herein by reference.

IX. Protein Purification Host cells transformed with polynucleotide sequences encoding the modified pullulanase can be cultured under conditions suitable for the expression and recovery of pullulanase from the cell culture. The protein produced by a recombinant gram-positive host cell comprising a mutation or suppression of endogenous protease activity will segregate in the culture medium. Other recombinant constructs can bind the polynucleotide sequences of the modified pullulanase to a nucleotide sequence coding for a polypeptide domain that will facilitate the purification of soluble proteins (Kroll DJ et al., (1993) DNA Cell Biol 12: 441-53 ). These purification facilitation domains include, without limitation, metal chelation peptides such as histidine-t-rytophan modules that allow the purification of immobilized metals (Porth J (1992) Protein Expr Purif 3: 263-281), protein A domains that allow purification in immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (Immunex Corp. Seattle WA). The inclusion of a cleavable linker sequence such as factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and the heterologous protein can be used to facilitate purification.

X. Uses of the present invention Modulated Pullulanase A modified pullulanase of the present invention finds use in various industries including the food industry, the pharmaceutical industry and the chemical industry. A modified pullulanase can be used in the baking as an "anti-rancid" agent, ie as an additive to prevent the bread from becoming rancid during storage, or in the fermentation during the production of low-calorie beers. The Pullulanase can also be used in the production of low calorie foods in which amylose is used as a substitute for fats. Pullulanase can be used, for example, to clarify fruit juices. For food applications, pullulanase can be immobilized on a support. The techniques for the immobilization of enzymes are well known to an expert. Pullulanase can also be used to hydrolyze amylopectin and to form oligosaccharides starting from this amylopectin. Pullulanase can also be used to form tetraholosides starting from maltose. Pullulanase can also be used to condense mono- or oligo-saccharides creating alpha-1, 6-glucosidic type bonds. Pullulanase can be used for the liquefaction of starch. A modified pullulanase can be used in the same way as its respective, unmodified form. A modified pullulanase, which in the unmodified form has activity under alkaline conditions, will retain activity under alkaline conditions. A modified pullulanase that in the unmodified form has low activity Acid conditions, will retain the activity ba or acid conditions. A modified, particular pullulanase will be formulated according to the proposed uses. Stabilizers or preservatives can also be added to the enzyme compositions comprising a modified pullulanase. For example, a modified pullulanase can be stabilized by the addition of propylene glycol, ethylene glycol, glycerol, starch, polyulan, a sugar, such as glucose or sorbitol, a salt, such as sodium chloride, calcium chloride, potassium sorbate, benzoate. of sodium, a mixture of two or more of these products. The enzyme compositions according to the invention may also comprise one or more different enzymes. These enzymes include, but are not limited to, glucoamylase, alpha-amylase, beta-amylase, alpha-glucosidase, isoamylase, cyclomaltodextpna, glucotransferase, beta-glucanase, glucose-isomerase, saccharification enzymes, and enzymes that cleave glycosidic bonds or a mixture of two or more of these. In a preferred embodiment, the enzymatic composition comprises a modified pullulanase of the present invention at 80% and a glucoamylase at twenty %. The manner and method for carrying out the present invention can be more fully understood by those skilled in the art by reference to the following examples, examples which are not intended in any way to limit the scope of the present invention or the directed claims. to the same EXAMPLES Example I: Example I illustrates the production of a modified pullulanase as described herein. The nucleic acid sequence encoding a pullulanase is modified by recombinant DNA techniques such as enzymatic amplification directed by a PCR primer, normal to the DNA with a thermostable DNA polymerase (Saiki, RK, et al., 1988, Science 239 : 487-491) and fusion techniques by PCR (Fleming, AB et al., Appl. In vi ron Mi crobi ol. 61, 3775-3780). The DNA encoding the desired modified pullulanase is fused to the C-terminus and a signal sequence, preferably a signal sequence of the host microorganism. This construction clones and transforms into a host cell, such as, B. sutilis or B. licheniformis, and is grown under normal fermentation conditions. The modified pullulanase is purified from the fermentation broth and titrated for activity.

EXAMPLE II Example II describes the modified forms of pullulanase obtained by culturing the recombinant host cell of B. licheniformis comprising the nucleic acid encoding a mature pullulanase from B. deramificans wherein the host cell has a deletion of the Carlsburg proteases. and endoGluC. B. licheniformis was cultured under normal fermentation conditions in a complete medium. The fermentation broth was subjected to normal HPLC analysis and the results are shown in Figures 3A-3C illustrating a time course of the various species of the modified pullulanase formed during the fermentation process. Peak 1 designates the mature pullulanase from B. deramificans which has a molecular weight of 105 kD; peak 2 designates the modified pullulanase having the deletion of 102 amino acids from the amino terminus of the mature pullulanase of B. derami fi thas; and point 3 designates mature pullulanase having a deletion of 98 amino acids from the amino terminus as measured by normal HPLC analysis. The modified pullulanase species having an additional amino acid in the mature sequence are not detectable by HPLC analysis but were detected in the nucleic acid sequencing. Figures 3A-3C illustrate that during the fermentation time, the peak 1 corresponding to the mature pullulanase of B. derami fi s decreases while the peaks 2 and 3 are increased. Figures 4A-4D illustrate the stability of the modified pullulanase produced in the fermentation of B li ch in iformi s that has a suppression of Carlsburg and endoGluC proteases. The B. The formulation comprising the nucleic acid coding for a mature B derami fi cant was cultured under conditions suitable for the expression and suppression of the modified pullulanase and the fermentation broth was adjusted to a pH of 4.5.5. 5 and 6.5 at room temperature. The modified pullulanase was more stable at a pH of 4 5.

Example III Example III describes the saccharification process comprising the enzymatic compositions comprising different percentages of pullulanase. Enzyme compositions comprising either 20% glucoamylase activity: 80% modified pullulanase (20:80) or 75% glucoamylase activity: 25% pullulanase (75:25) were tested in the saccharification process at a concentration of 0.550, 0.635 and 0.718 liters of the enzymatic composition per metric ton of dissolved solids. As shown in Figures 5A-5C, an enzymatic composition comprising an activity of 20% glucoamylase and 80% pullulanase is capable of increasing the final glucose yield without an increase in the undesirable formation of disaccharides. Additionally, the absolute concentration of the enzyme composition of 20:80 can be increased without the undesirable increase in the formation of disaccharides that occurs with the enzyme composition 25:75 or the glucoamylase alone.

Example IV Example IV describes an assay for the determination of the activity of a modified pullulanase of the present invention. This assay is based on a colorimetric method that uses a soluble red-pululan substrate for the determination of pullulanase activity.

Preparation of Reagents A 200 mM sodium acetate buffer pH 5.0 with / Acarbose (density - 1.010) was prepared by weighing 16.406 g of anhydrous sodium acetate or 27.21 g of sodium acetate trihydrate and dissolving in 900 ml of deionized water (DI). ) in a 1 L graduated cylinder when shaking with a magnetic stir bar. The pH was adjusted to 5.0 with glacial acetic acid. 0.300 g of Acarbose was added to the solution and allowed to dissolve. The volume was brought to 1000 mL with DI water and mixed.

Preparation of the red-polulan substrate at 2 1.00 g of the red-pululan substrate was weighed and dissolved in 50 mL of buffer of sodium acetate by stirring with a magnetic stirring bar for about 20-30 minutes. This solution is stable for 2 weeks when stored at 4 ° C.

Preparation of a working standard Using positive displacement pipettes, a 1:10 dilution of the pullulanase standard was prepared. The assigned activity of the standard was 195.9 ASPU / ml. The following work concentrations were prepared from the standard of the concentrated solution of 1:10.

Sample preparation For a control, Optimax L-300 was used MA7EC191 PU Bl 3-19A available from Genencor International. The control was diluted 1: 1000 in sodium acetate buffer. All samples were diluted in sodium acetate buffer to obtain final reaction absorbances that fall in the calibration curve. The sample was placed at room temperature. A minimum of 100 μl of the sample was used for the initial dilution.

Test Procedure Two 250 μl of each normal work, control and sample concentration were placed in two appropriately labeled microcentrifuge tubes. 250 μl of the 2% substrate solution was added to each tube with a repeating pipette and a Combitip set of 12.5 ml in 1. The samples were vortexed for 3 seconds and incubated at 40 ° C for 20 minutes. The samples were removed from the water bath and immediately 1.0 ml of 95% EtOH was added to the samples in the same order as before. A repeating pipette and a Combitip set of 12.5 or 50 ml were used in 4 or 1. The samples were vortexed for 3 seconds. The samples were incubated at room temperature for 5-10 minutes, then centrifuged for ten minutes in a benchtop centrifuge. The supernatant standards and samples were eluted in a 510 nm spectrophotometer using 1.5 m tubes. (The spectrophotometer was zeroed with 95% EtOH).

Calculations Using normal concentrations and Correlation absorbances (subtracting the absorbance in white), a calibration curve is developed with a computer spreadsheet, programmable calculator, or graph paper. The curve must be linear over the range of normal concentrations with a correlation coefficient ® of 0.998 or greater. The test pressure should fall between 5-10% CV. For liquids: u / ml = (u / ml of the normal curve) * (sample illusion). It is noted that in relation to this date, the best method known by the applicant to carry out the present invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (51)

1. A modified pullulanase, characterized in that it is able to catalyze the hydrolysis of an alpha-1,6-glucosidic bond.

2. The modified pullulanase according to claim 1, characterized in that the pullulanase is a modification of a pullulanase obtainable from a gram-positive or gram-negative microorganism.

3. The modified pullulanase according to claim 2, characterized in that the gram-positive microorganism includes B. subtilis, B. deramificans, B. stearothermophilus, B. naganoensis, B. flavocaldarius, B. acidopullulyticus, Bacillus sp APC-9603, B secto ramus, B. cereus, and B. fermus.

4. The modified pullulanase according to claim 2, characterized in that the gram-negative microorganism includes Klebsiella pneumonia and Klebs iella aerogenes.

5. The modified pullulanase according to claim 3, characterized in that the pullulanase of B. deramifi ca n s has the designation T89.117D in the LMG culture collection.

6. The modified pullulanase according to claim 1, characterized in that the modification is a deletion of amino acids from the amino terminus of approximately 100 amino acids.

7. The modified pullulanase according to claim 1, characterized in that the modification is an amino acid deletion from the amino terminus of approximately 200 amino acids.

8. The modified pullulanase according to claim 1, characterized in that the modification is a deletion of amino acids from the amino terminus of approximately 300 amino acids.

9. The pullulanase modified according to the rei indication 6, characterized in that the suppression is 98 amino acids from the amino terminus of the pullulanase of B. derami fi cans.

10. The modified pullulanase according to claim 6, characterized in that the deletion is 102 amino acids from the amino terminus of the pullulanase of B. derami fi can s.

11. The modified pullulanase according to claim 1, characterized in that the modification is an addition of at least one amino acid to the amino terminus of the amino acid sequence of the mature pullulanase.

12. The modified pullulanase according to claim 11, characterized in that the pullulanase can be obtained from B. derami fi ca n s and the additional amino acid at the amino terminus is an alanine.

13. Modulated pullulanase characterized in that it is produced by the method comprising the steps of obtaining a recombinant host cell comprising a nucleic acid encoding mature pullulanase,, culturing the host cell under conditions suitable for the production of modified pullulanase and optionally recover the modified pullulanase.

14. The modified pullulanase according to claim 13, characterized in that the nucleic acid encoding mature pullulanase has at least 70% identity to the polynucleotide sequence as shown in SEQ ID NO: 1.

15. The modified pullulanase according to claim 13, characterized in that the host cell is B. li ch in i forms comprising a first gene encoding the Carlsberg protease and a second gene encoding the endo-Glu-C protease, the first and / or second gene encoding the protease (s) s) that has been altered such that the protease activity is essentially eliminated.

16. A nucleic acid characterized in that it comprises a polynucleotide sequence encoding a modified pullulanase of claim 1.

17. The nucleic acid according to claim 16, characterized in that it has at least 70% identity to the polynucleotide sequence shown in SEQ ID NO: 1.

18. The nucleic acid according to claim 16, characterized in that the polynucleotide sequence is as shown in SEQ ID NO: 1.

19. An expression vector, characterized in that it comprises the nucleic acid of claim 16.

20. A host microorganism characterized in that it comprises the expression vector of claim 19.

21. The host microorganism according to claim 20, characterized in that the microorganism is a Bacillus.

22. The host microorganism according to claim 21, characterized because Bacillus includes B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliqu faciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.

23. A method for the production of a modified pullulanase in a host cell, characterized in that it comprises the steps of: a) obtaining a recombinant host cell comprising the nucleic acid encoding a modified pullulanase; and b) culturing the microorganism under conditions suitable for the production of the modified pullulanase.

24. The method according to claim 23, characterized in that it further comprises the step of: c) recovering the modified pullulanase.

25. The method according to claim 23, characterized in that the host cell is a Bacillus that includes Bacillus includes B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.

26. The method according to claim 25, characterized in that the Bacillus host cell is B. licheniformis.

27. An enzymatic composition, characterized in that it comprises a modified pullulanase.

28. The enzymatic composition according to claim 27, characterized in that the modified pullulanase has an amino acid deletion from the amino terminus of about 100 amino acids.

29. The enzymatic composition according to claim 27, characterized in that the modified pullulanase has a deletion of amino acids from the amino terminus of about 200 amino acids.

30. The enzymatic composition of according to claim 27, characterized in that the modified pullulanase has an amino acid deletion from the amino terminus of about 300 amino acids.

31. The enzymatic composition according to claim 27, characterized in that the modified pullulanase has an amino acid suppression as shown in SEQ ID NO: 2 starting at the amino acid residue 99, a glutamic acid.

32. The enzymatic composition according to claim 27, characterized in that the modified pullulanase has the amino acid sequence as shown in SEQ ID NO: 2 starting at the amino acid residue 103, a glutamic acid.

33. The enzymatic composition according to claim 27, characterized in that it comprises an enzyme selected from the group consisting of glucoamylase, alpha-amylase, beta-amylase, alpha-glucosidase, isoamylase, cyclomaltodextrin, glucotransferase, beta-glucanase, glucose-isomerase, saccharification enzymes and / or enzymes that cleave glycosidic linkages.

34. The composition according to claim 27, characterized in that it also comprises a glucoamylase.

35. The composition according to claim 34, characterized in that the glucoamylase can be obtained from a strain of Aspergi II us.

36. The composition according to claim 35, characterized in that the strain of Aspergi l l us includes Aspergi l l us ni ger, Aspergill us a wamori and Aspergi l l us foe tidus.

37. The composition according to claim 27, characterized in that the composition is in a solid form.

38. The composition according to claim 27, characterized in that the composition is in a liquid form.

39. The composition according to claim 27, characterized in that it comprises at least 60% of modified pullulanase.

40. The composition according to claim 27, characterized in that it comprises at least 80% modified pullulanase.

41 A process for saccharification of starch, where the process allows reduced concentrations of saccharification by-products, characterized in that it comprises the step of contacting the liquefied, aqueous starch with an enzyme composition comprising the modified pullulanase.

42. The process according to claim 41, wherein the modified pullulanase has a suppression of up to about 100 amino acids, up to about 200 amino acids or up to about 300 amino acids from the amino terminus of the pullulanase obtainable from a gram-negative or gram-positive microorganism .

43. The process according to claim 41, characterized in that it also comprises the steps of heating the liquefied starch, and optionally recovering the product.

44. The process according to claim 41, characterized in that the enzyme composition additionally comprises the glucoamylase.

45. The process according to claim 41, characterized in that the enzyme composition comprises at least 80% modified pullulanase.

46. The process according to claim 41, characterized in that the contacting is at a pH of approximately less than or equal to 7.0 and greater than or equal to 3.

47. The process according to claim 41, characterized in that the pH is about 4.2.

48. The process in accordance with the claim 41, characterized in that the heating is at a temperature range between 55 and 65 ° C.

49. The process according to claim 41, characterized in that the temperature is about 60CC.

50. B. i chen i formi s, characterized in that it comprises a nucleic acid encoding a modified pullulanase wherein B. li ch eniformi s comprises a first gene coding for the Carlsberg protease and a second gene coding for the endo-Glu-C protease, the first and / or second gene coding for the protease (s) that it has been altered such that protease activity is essentially eliminated.

51. B. l i ch eni formi, characterized in that it comprises a nucleic acid encoding a mature pullulanase where B. li cheniformis comprises a first gene that codes for the Carlsberg protease and a second gene that encodes for the endo-Glu-C protease, the first and / or second gene encoding the protease (s) that has been altered such that protease activity is essentially eliminated.