Abstract:
A novel thermoacidophilic pullulanase (Tk-PUL) from hyperthermophilic archaeon  Thermococcus kodakaraensis  KOD1 is described here that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions. The gene encoding Tk-PUL was cloned and expressed in  E. coli  cells. The purified recombinant enzyme possesses the following properties;
       shows both pullulanase and α-amylase activities   displays highest activity at 95-100° C.   active over a broad pH range (3.0-8.5) with optimum working pH 3.5   stable for several hours at 90° C. and displays a half-life of 45 minutes at 100° C.   activity and stability are independent of calcium and other metal ions   hydrolyzes maltotriose       
 
     Moreover, recombinant Tk-PUL can be used for single step liquefaction and saccharification of corn starch (without any α-amylase or β-amylase) at pH 4.2 in the absence of calcium.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a continuation of U.S. patent application Ser. No. 13/765,481, entitled “Single Step Liquefaction and Saccharification of Corn Starch Using an Acidophilic, Calcium Independent and Hyperthermophilic Pullulanase” filed on Feb. 12, 2013, the contents of which are incorporated herein by reference in its entirety. 
     
    
     FIELD OF INVENTION 
       [0002]    The present invention is within the field of thermostable amylases. More specifically, the present invention relates to a novel hyperthermostable pullulanase having an acidic pH optima and no metal ion requirement. Moreover, it relates to methods of producing the pullulanase and use of this enzyme in simultaneous liquefaction and saccharification of starch. 
       DESCRIPTION OF THE PRIOR ART 
       [0003]    The most abundant storage polysaccharide i.e., starch is a polymer of anhydro glucose units. Its major utilization is in the production of sweeteners. These sweeteners may be solids like glucose (dextrose), maltose and fructose or they may be liquids like glucose syrup, maltose syrup and high fructose corn syrup. Glucose/glucose syrup is either used directly in the production of various foods or used as raw material in other biotechnological processes for the production of sorbitol, citric acid, amino acids and fuel ethanol (Crabb and Mitchinson, 1997; Crabb and Shetty, 1999; Ibeto et al., 2011). 
         [0004]    For production of glucose/glucose syrup corn starch is hydrolysed in two-steps, liquefaction and saccharification, which involve a number of starch hydrolyzing enzymes like α-amylase, glucoamylase/α-glucosidase and pullulanase. During liquefaction α-amylase splits starch into varying lengths of polysaccharides in the presence of calcium (40 ppm) at a temperature of 95-105° C. and pH about 6. During saccharification a-glucosidase and pullulanase are used that hydrolyze the polysaccharides to monosaccharides (glucose) at a temperature of 60-65° C. and pH close to 4.5 (Le´vêque et al., 2000). For liquefaction concentrated suspension of corn starch (30-35%) is prepared, pH is increased from 4.5 (natural pH of starch slurry) to 6.0 and calcium is added. α-amylase is then mixed and suspension is heated to 105-110° C. and held at this temperature for 5-10 minutes. The temperature is then lowered to 90-95° C. and liquefaction is completed at this temperature in 1-2 hours. For saccharification both pH and temperature of the liquefied starch are lowered to the optimal pH (4.5) and temperature (60-62° C.) of the saccharifying enzyme and process is completed under these conditions in 72-96 hours. 
         [0005]    Though pH adjustment before and after liquefaction step increases the cost of process but is necessary because currently available liquefying enzymes (α-amylases) are unable to work efficiently below pH 5.9 (Van der Maarel et al., 2002). Furthermore, for efficient amylolytic activity starch granules should be completely gelatinized which is only possible above 100° C. Therefore the starch-processing industry needs thermostable and acid-stable amylases to decrease the cost of glucose-production. They would help in completing the processes quickly and efficiently as wastage of chemicals in pH adjustment and time in cooling processes will be avoided. 
         [0006]    Another major problem for starch industry arises during conversion of glucose syrup to high fructose syrup. Starch liquefying enzymes require calcium for their activity and thermostability but this added calcium inhibits the activity of xylose isomerase (commonly known as glucose isomerase). This enzyme is used for isomersation of glucose to fructose syrup (Wang et al., 2007). Furthermore, calcium oxalate is produced as a waste product which deposits in the pipes and heat exchangers. This deposition chokes them and increases the production cost. With the development of calcium independent, thermostable and acid stable enzymes this problem can be solved. 
         [0007]    Thermostable amylases were previously isolated from  Bacillus  species especially from  B. licheniformis, B. amyloliquefaciens  and  B. subtilis  (Underkofler, 1976). TERMAMYL® (NOVO NORDISK A/S Denmark) is a thermostable α-amylase produced from  B. licheniformis.  It has an optimal temperature of 90° C. and requires additional calcium for its thermostability.  B. stearothermophilus  α-amylase disclosed in U.S. Pat. No. 4,284,722 shows superiority over  B. licheniformis  amylase in respect of lower pH optima but it is also not suitable for starch liquefaction below pH 5.0. 
         [0008]    Another important liquid sweetener, maltose syrup, is a concentrated and purified solution containing major proportion of maltose with respect to other saccharides but maltose content not less than 30% of the solution on dry basis. The preparation of maltose syrup by the action of a combination of amylolytic enzymes has previously been disclosed in U.S. Pat. No. 3,565,765 (maltogenic amylase and pullulanase), U.S. Pat. Nos. 3,795,584 and 3,804,715 (betα-amylase and alpha-1,6 glucosidas/pullulanase), U.S. Pat. No. 3,791,865 (betα-amylase and amylo-1,6 glucosidase), U.S. Patent No. 3,549,496 ( Bacillus polymyxa  amylase and glucoamylase), U.S. Pat. Nos. 3,832,285; 4,032,403; 3,996,107; 3,998,696 and 4,113,509 (alpha amylase and betα-amylase). There is no report of using a single enzyme for simultaneous liquefaction and saccharification. 
         [0009]    During past three decades hyperthermophilic archaea attracted the researchers because their enzymes show extreme thermostability. Recently, several hyperthermostable amylolytic enzymes have been reported from  Pyrococcus furiosus, Pyrococcus woesei  (U.S. Pat. No. 5,370,997),  Thermococcus litoralis  (Brown and Kelly, 1993),  Thermococcus aggregans  (Canganella et al., 1994) and  Thermococcus kodakaraensis  KOD1 (Murakami et al., 2006). The genes encoding some of these enzymes have been cloned. For example intra and extracellular α-amylases from  P. furiosus  (Laderman et al., 1993; Dong et al., 1997),  Pyrococcus sp.  KOD1 (Tachibana et al., 1996),  T. profundus  (Lee et al., 1996),  Sulfolobus acidocaldarius  (Kobayashi et al., 1996a),  Sulfolobus solfataricus  (Kobayashi et al., 1996b) and  T. aggregans  (Niehaus et al., 2000). α-amylase from  P. furiosus  (U.S. Pat. No. 5,370,997) has been reported to be independent of calcium requirement with pH optima between 4.0-6.0. 
         [0010]    We describe here a novel pullulanase (Tk-PUL) from hyperthermophilic anaerobic archaeon  Thermococcus kodakaraensis  KOD1 that is capable of simultaneous liquefaction and saccharifaction of starch slurry at 90° C. and pH 4.2 in the absence of calcium or any other metal ion without the addition of any liquefying α-amylase and saccharifying β-amylase. 
       BRIEF SUMMARY OF THE INVENTION 
       [0011]    A 2298 by nucleotide sequence coding for Tk-PUL was identified in the genome of  T. kodakaraensis  KOD 1, amplified by polymerase chain reaction and cloned in expression pET-21 a(+) (SEQ ID NO. 1). The amino acid sequence of Tk-PUL shared only a 62% or less identity with already known sequences of amylolytic enzymes (SEQ ID NO. 2). Maximum identity (62%) was with pullulan hydrolase III from  T. aggregans.  Recombinant Tk-PUL was produced in  E. coli  and purified to apparent homogeneity on SDS-PAGE. Specific activity of purified Tk-PUL was 70.5 U/mg using pullulan as a substrate. Molecular mass of Tk-PUL was found to be 84402.053 Da by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Size exclusion chromatography revealed that the recombinant Tk-PUL was a monomer. The recombinant enzyme possessed both pullulanase and α-amylase activities. Highest activities were observed at 95-100° C. Although the enzyme was active over a broad pH range (3.0-8.5), the pH optimum for both activities was 3.5 (in acetate buffer) and 4.2 (in citrate buffer). Tk-PUL was stable for several hours at 90° C. Half-life at 100° C. was 45 minutes (when incubated either at pH 6.5 or 8.5). The enzyme was also stable over a pH range of 4.2-8.5. Calcium ions were not required for activity and stability of recombinant Tk-PUL. Addition of divalent cations such as Mg +2 , Mn +2 , Co +2  and Zn +2  had no effect on the enzyme activity while Ni +2 , Cu +2  and Fe +2  exhibited slight inhibitory effect. 
         [0012]    One embodiment of the present invention comprises a novel pullulan hydrolase type III (Tk-PUL) comprising an amino acid sequence having at least 65%, 85%, 90%, 98%, or 99% homology to the amino acid sequence of SEQ ID NO:2; a molecular mass of 84.4 kDa; both pullulanase and α-amylase activities; a temperature optimum between 95-100° C. (at pH 4.2 and 6.5); a pH optimum 3.5 (in acetate buffer) and 4.2 (in citrate buffer); a residual activity of 90% after 10 hours incubation (in the absence of substrate) at 90° C.; a half-life of 45 minutes at 100° C. (when incubated in the absence of substrate either at pH 6.5 or 8.5); an independence of calcium ions for activity and stability; an ability to hydrolyze maltotriose into maltose and glucose; and an ability to hydrolyze α-1,4 glycosidic linkages in pullulan in addition to α-1,6 linkages. 
         [0013]    Another embodiment comprises a composition comprising an isolated peptide molecule having at least 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO 2. 
         [0014]    Another embodiment comprises the pullulan hydrolase encoded by SEQ ID NO 1, as well as expression vectors and host cells for expressing the encoded protein. 
         [0015]    Another embodiment of the present invention comprises a process for the simultaneous liquefaction and saccharification of starch comprising adding the pullulan hydrolase type III sequence having at least 65%, 85%, 90%, 98%, or 99% homology to the amino acid sequence of SEQ ID NO:2 to a starch solution, adjusting the temperature of the solution to about 100° C. for 10 minutes; lowering the temperature to 90° C. until both liquefaction and saccharification are complete; and wherein the pullulan hydrolase performs both pullulanase and α-amylase activities. 
         [0016]    In a further embodiment the process can be carried out at a pH of about 4.2 throughout the process and both liquefaction and saccharification are carried out without the addition of calcium or any other metal ions. 
         [0017]    Another embodiment comprises a process wherein liquefaction of a starch solution proceeds in the presence of the pullulan hydrolase described above at a pH of about 4.2 in the absence of calcium followed by saccharification of the liquefied starch by  Aspergillus niger  glucoamylase without pH adjustment. 
         [0018]    Tk-PUL was able to hydrolyze a variety of substrates including cyclodextrins and smaller linear oligosaccharides such as maltoheptaose to maltotriose. Final products of hydrolysis (from long chain and small chain saccharides) consisted of a mixture of maltotriose, maltose and glucose. Recombinant Tk-PUL displayed the novel property to hydrolyze maltotriose into maltose and glucose. The end product of pullulan hydrolysis was a mixture of maltotriose, maltose, panose and isomaltose. Tk-PUL is, therefore, proposed as pullulan hydrolase type III though it was previously annotated as pullulanase type II in the genome of  T. kodakaraensis.    
         [0019]    Tk-PUL was able to produce maltose syrup (containing more than 50% of mono, di- and tri-saccharides) from the hydrolysis of corn starch at 90° C. and glucose syrup (containing mor than 90% glucose). It is important to note that the experiments were conducted throughout at pH 4.2 (the natural pH of starch slurry) in the absence of calcium. The recombinant Tk-PUL efficiently liquefied the corn starch in the absence of any liquefying α-amylase. In addition it was able to saccharify (in the absence of β-amylase) the liquefied starch into a mixture of maltotriose, maltose and glucose. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1 : Schematic diagram of recombinant plasmid PulN-pET representing restriction enzymes that have recognition sites within the pullulanase gene. 
           [0021]      FIG. 2 : Nucleotide (top) and deduced amino acid (below) sequences of Tk-PUL. A signal peptide of 17 amino acids is double underlined. 
           [0022]      FIG. 3 : Alignment of Tk-PUL with other archaeal pullulanases. Gaps are shown by dashes, identical residues are shown in white with black background and similar residues are shown in black with gray background. The sequences used in alignment were: T kod, Tk-PUL from  Thermococcus kodakaraensis  KOD1 accession number Q5JID9, T agg, Pullulan hydrolase type III from  T. aggregans  accession number Q9P9A0, T gam, Pullulan hydrolase type III from  T. gammatolerans  accession number C5A115, T AM4, pullulanase type II from  Thermococcus  sp. AM4 accession number B7R259, P cal, Pullulanase from  Pyrobaculum calidifontis  accession number A3MUT4, D kam, pullulanase from  Desulfurococcus kamchatkensis  accession number B8D2L1, D muc, pullulanase from  D. mucosus  accession number Q9HHB0. 
           [0023]      FIG. 4 : Regions conserved among pullulanases and other amylolytic enzymes. Three acidic residues essential for catalytic activity are marked by #, identical residues are shown in white with black background and similar residues are shown in black with gray background. Swiss-Prot accession numbers of sequences were used i.e., Q5JID9 (Tk-PUL); Q9P9A0 (Pullulan hydrolase type III form  T. aggregans ); Q9HHB0 (Pullulanases from  D. mucosus ); P32818 (Maltogenic α-amylase from  B. cidopullulyticus ); P29964 (Cyclomaltodextrin hydrolase from  Thermoanaerobacter ethanolicus ); Q08751 (Neopullulanase from  Thermoactinomyces vulgaris ); P38940 (Neopullulanase from  B. stearothermophilus ); Q57482 (Neopullulanase from  Bacillus  sp.); Q45490 (Maltogenic amylase from  G. stearothermophilus ); Q819G8 (Neopullulanase from  Bacillus cereus ). 
           [0024]      FIG. 5   a : Graphical presentation of pullulanase activity possessed by recombinant Tk-PUL at various pH values in sodium citrate (□), sodium acetate () and sodium phosphate (▪) buffers. Each buffer was used at a concentration of 50 mM. 
           [0025]      FIG. 5   b : Graphical presentation of α-amylase activity possessed by recombinant Tk-PUL at various pH values in sodium citrate (□), sodium actetate () and sodium phosphate (▪) buffers. Each buffer was used at a concentration of 50 mM. 
           [0026]      FIG. 6 : Graph representing the stability of recombinant Tk-PUL at various pH values over various intervals of time. Buffers used were 50 mM sodium citrate pH 4.2 (□), 50 mM sodium acetate pH 6.5 (⋄) and 50 mM Tris-Cl pH 8.5(). 
           [0027]      FIG. 7 : Graph representing relative pullulanase activity at various temperatures. (a) Activity in sodium citrate buffer pH 4.2. (b) Activity in sodium acetate buffer pH 6.5. 
           [0028]      FIG. 8 : Graph representing the relative α-amylase activity at various temperatures. (a) Activity in sodium citrate buffer pH 4.2. (b) Activity in sodium acetate buffer pH 6.5. 
           [0029]      FIG. 9 : Stability of Tk-PUL at 90° C. in buffers of various pH values. 50 mM buffers used were sodium citrate pH 4.2 (▴), sodium acetate pH 6.5 () and Tris-Cl pH 8.5 (♦). 
           [0030]      FIG. 10 : Stability of Tk-PUL at 100° C. in sodium acetate pH 6.5 (▪) and Tris-Cl pH 8.5 (). Each buffer was used at a final concentration of 50 mM. 
           [0031]      FIG. 11 : Time course release of oligosaccharides from 1% (w/v) corn starch by the action of recombinant Tk-PUL. Upper panel represents sugars released as percentage of the total products against hydrolysis time. Lower panel shows sugars as percent of their maximum (released) against hydrolysis time. DP stands for degree of polymerization while DP7 to DP1 are representing maltoheptaose to glucose, respectively. 
           [0032]      FIG. 12   a : HPLC profile showing liquefaction of 25% (w/v) corn starch by the action of recombinant Tk-PUL. 
           [0033]      FIG. 12   b : HPLC profile showing liquefaction of 25% (w/v) corn starch by the action of α-amylase from  B. licheniformis ; Termamyl 120L. 
           [0034]      FIG. 13   a : HPLC profile showing final saccharide composition of glucose syrup produced from corn starch liquefied by the action of recombinant Tk-PUL. The highest peak with retention time 20.7 minutes is representing glucose. 
           [0035]      FIG. 13   b : HPLC profile showing final saccharide composition of glucose syrup produced from corn starch liquefied by the action of α-amylase from  B. licheniformis;  Termamyl 120L. The highest peak with retention time 20.7 minutes is representing glucose 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Cloning of Tk-Pul Gene 
       [0036]    The hyperthermophilic archaeal strain KOD1, isolated form Kodakara Island, Kagoshima, Japan, was cultured at 85° C. and cells were obtained. Chromosomal DNA of strain KOD1 was prepared by the method of Ramakrishnan and Adams (Ramakrishnan and Adams, 1995). A 2298 by open reading frame (ORF) coding for a pullulanase type II of GH13 family was identified in the genome of  T. kodakaraensis  KOD1. A set of primers (5′-CATATGAGCGGATGTATCTCGGAGAGCAACG-3′ (SEQ ID NO 3, corresponding to 5′ end of gene) and 5′-GAAGCGGGGGTCAACCCCGCTCAAG-3′ (SEQ ID NO 4, corresponding to 3′ end of the gene) was synthesized. Restriction site of enzyme NdeI (CATATG) was introduced in the forward primer. The gene was amplified by polymerase chain reaction (PCR) using this pair of primers and genomic DNA of  T. kodakaraensis  KOD1 as template. PCR mixture (50 μL) composed of  T. kodakaraensis  KOD1 genomic DNA (100 ng), 1× PCR buffer (0.01% Tween 20, 20 mM (NH4)2SO4, 75 mM Tris-Cl pH 8.8 at 25° C.), KCl (50 mM), MgCl2 (2 mM), deoxyribonucleoside triphosphates (dNTPs, 250 μM), forward and reverse primers (100 pmol each), and Taq DNA polymerase (5 units). DNA was amplified in Eppendorf Master Cycler. PCR conditions were: initial denaturation at 94° C. for 2 minutes; followed by 30 cycles of: denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds, extension at 72° C. for 90 seconds; and final extension at 72° C. for 10 minutes. After completion of PCR the amplified product was analyzed by gel electrophoresis and purified. The amplified DNA fragment was then ligatd into T/A cloning vector pTZ57R/T (Fermentas) and used to transform  E. coli  DH5α cells. This recombinant plasmid containing pullulanase gene was named PulN-pTZ. Recombinant plasmid PulN-pTZ was digested with NdeI and BamHI to liberate pullulanase gene which was purified and subsequently ligated between NdeI and BamHI restriction sites of pET-21a(+) (Novagen). The resulting recombinant expression vector was named PulN-pET. Restriction map of PulN-pET is shown in  FIG. 1 . 
       DNA Sequence Analysis 
       [0037]    The presence of pullulanase gene in recombinant expression vector was confirmed by DNA sequencing using CEQ800 Beckman Coulter sequencing system. Multiple sequence alignment was performed by using ClustalW in BioEdit Sequence Alignment Editor (Hall, 1999). 
       Gene Expression and Purification Of Recombinant Tk-PUL 
       [0038]      E. coli  BL21 CodonPlus(DE3)-RIL cells were transformed using PulN-pET expression vector and grown in LB medium till OD600 reached to 0.4. Gene expression was then induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) and growth was continued at 37° C. with shaking at 100 rpm. After 4.5 hours of induction, cells were harvested by centrifugation at 13,000 rpm for 10 minutes at 4° C. Cell pallet was washed with 50 mM Tris-Cl pH 8.0 and re-suspended in the same buffer. Harvested cells were disrupted by sonication and cell debris was removed by centrifugation at 20,000 rpm for 10 minutes at 4° C. Supernatant thus obtained was heated at 80° C. for 30 minutes to denature heat labile host cell ( E. coli ) proteins that were removed by centrifugation. Tk-PUL obtained in the supernatant was precipitated by fractional ammonium sulfate precipitation (0-20%, 20-40% and 40-60%). Precipitates obtained after 40% and 60% ammonium sulfate saturation were pooled, dialyzed and fractionated by anion exchange chromatography using Res Q-6mL column on Fast Protein Liquid Chromatography (FPLC) system, AKTA Purifier (GE Healthcare). The column was equilibrated with 50 mM Tris-Cl pH 8.0 before loading the protein sample. Elution of the proteins bound to the column was done by a linear gradient of 0 to 1 M sodium chloride solution (prepared in 50 mM Tris-Cl pH 8.0. Fractions containing recombinant Tk-PUL were pooled and dialyzed against 50 mM Tris-Cl pH 8.0 
       Enzyme Activity Assay 
       [0039]    Pullulanase activity of recombinant Tk-PUL was measured in terms of the amount of reducing sugars librated upon incubation of the enzyme with pullulan. Maltose was used as a standard for reducing sugars. In a standard assay mixture, 125 μL of 0.5% (w/v) pullulan in 50 mM sodium citrate buffer (pH 4.2) were mixed with 125 μL of properly diluted (0.8-1.2 U/mL) enzyme (in the same buffer) and incubated at 90° C. for 10 minutes. The reaction was stopped by quenching in ice water and reducing ends released were determined by dinitrosalicylic acid (DNS) method (Bernfeld, 1955). Reducing groups released by the non-enzymatic factors were corrected by preparing enzyme blank sample (assay mixture without enzyme) and substrate blank sample (assay mixture without substrate). One unit for pullulanase activity was defined as the amount of enzyme that released 1 μmole of reducing sugars (in terms of maltose) in one minute under standard assay conditions. Protein concentration was estimated by Coomassie dye-binding assay (Bradford, 1976) using Quick Start™ Bradford Protein assay kit (Bio-Rad Laboratories, Inc., CA, USA). Bovine serum albumin was used as a standard for protein quantification. α-amylase activity of recombinant Tk-PUL was measured by the same procedure but replacing pullulan with 1% (w/v) starch as substrate. 
       Effect of Ph and Temperature on the Enzyme Activity 
       [0040]    Effect of pH and temperature on pullulanase and α-amylase activities of recombinant Tk-PUL was studied using purified enzyme and same assay procedure (Bernfeld, 1955). For pH study buffers used were, 50 mM sodium citrate (pH 2.5-4.5), 50 mM sodium acetate (pH 3.25-6.5) and 50 mM sodium phosphate (pH 6.5-8.5). pH was adjusted at room temperature. To measure the effect of temperature on the enzymatic activity, assay mixtures were prepared either in 50 mM sodium citrate buffer (pH 4.2) or in 50 mM sodium acetate pH 6.5 and incubated for 10 minutes at temperatures from 40 to 120° C. An oil bath was used for temperatures above 90° C. and incubations were performed in tightly screw capped Hungate tubes to prevent boiling of the samples. 
       Ph Stability of Recombinant Tk-Pul 
       [0041]    The pH stability of recombinant Tk-PUL was studied at 4° C. in buffers of various pHs (50 mM sodium citrate pH 4.2; 50 mM sodium acetate pH 6.5 and 50 mM Tris-Cl pH 8.5). The purified recombinant enzyme was diluted (0.04 mg/mL final concentration) in respective buffer and incubated at 4° C. for 56 hours. Aliquots were withdrawn at regular intervals (8 hourly) and the pH stability was studied by measuring residual pullulanase activity (in terms of reducing sugars released as maltose) using DNS method (Bernfeld, 1955). 
       Thermostability of Recombinant Tk-Pul 
       [0042]    For thermostability analysis the purified enzyme was diluted (40 μg/mL final concentration) in 50 mM buffers of various pH values (sodium citrate pH 4.2, sodium acetate pH 6.5 and Tris-Cl pH 8.5) and incubated at 90° C. and 100° C. All incubations were performed in tightly screw capped Hungate tubes to prevent boiling of the samples. At various interval of time, samples (50 μL˜2 μs protein) were taken, centrifuged for clarification and tested for residual pullulanase activity by standard assay method (Bernfeld, 1955). 
       Effect of Metal Ions on Recombinant Tk-PUL 
       [0043]    For this study purified recombinant Tk-PUL was extensively dialyzed against 10 mM EDTA in 50 mM Tris-Cl pH 8.0. Properly diluted enzyme (1.7 U/mL, final concentration) was mixed with metal ions (either 50 μM or 5 mM, final concentrations) and incubated at 60° C. for 15 minutes. Samples were withdrawn and pullulanase activity was examined by routine assay method (Bernfeld, 1955). 
       Substrate Specificity and Characterization of the Hydrolysis Products 
       [0044]    Substrate preference and relative hydrolysis rates of various polysaccharides (pullulan, starch, glycogen, amylose, amylopectin, dextrin, and cyclodextrins) were determined by incubating each of them (at a final concentration of 0.25% w/v) with recombinant Tk-PUL. Substrate solutions were prepared in 50 mM sodium citrate buffer (pH 4.2) and after adding purified enzyme (0.15 U≈2.2 μs protein) incubated at 90° C. for 2 to 30 minutes. The hydrolysis rate (μ moles of reducing sugars (maltose)/min.mL) of these substrates was measured after every 2 minutes by DNS method (Bernfeld, 1955). For characterization of oligosaccharides (obtained in hydrolysis products) incubations were done under similar conditions for upto 16 hours. The products were analyzed by High Performance Liquid Chromatography (HPLC) on Aminex HPX-42A column (Bio-Rad, USA) at 85° C. Peaks of chromatography were detected by differential refractive index detector (S 3580) on HPLC system (Sykam GmbH, Germany). 
       Application of Recombinant Tk-PUL in the Production of Maltose and Glucose Syrups 
       [0045]    For production of maltose syrup corn starch was suspended in 0.1 M sodium citrate buffer according to desired concentration (1%, 12% or 30% w/v). After addition of recombinant Tk-PUL the pH was adjusted to 4.2 or to the desired value. The slurry was heated at 100° C. (boiling water) for 5-10 minutes and then shifted to water bath at 90° C. for simultaneous liquefaction and saccharification. Commercial α-amylase from  B. licheniformis;  Termamyl 120 L (Sigma) was used as control for starch liquefaction under similar conditions. Samples were taken at specified intervals and oligosaccharide profile was analyzed by HPLC on Aminex HPX-42A column. 
         [0046]    For the production of glucose syrup saccharification of liquefied starch was done by cooling it to 60° C. (after 2 hours incubation with recombinant Tk-PUL at 90° C.), adding 1.29 U of commercial glucoamylase from  Aspergillus niger  per gram of starch; AMG 300L (Sigma) and continuing incubation at 60° C. for up to 96 hours. Before addition of glucoamylase the pH (6.5) of starch liquefied by Termamyl 120L was lowered to 4.5. Analysis of degree of saccharification over time was performed by HPLC on Aminex HPX-42A column. 
       Results  
     Nucleotide and Protein Sequences 
       [0047]    Nucleotide sequence of the cloned gene was determined by using Beckman Coulter CEQ™ 8000 Genetic Analysis System. The sequence thus obtained was in accordance with the gene sequence SEQ ID NO 1 (available at sequence databases) of  T. kodakaraenis  pullulananse. The full length gene consisted of 2298 nucleotides encoding a protein of 765 amino acids (SEQ ID NO 2). A signal peptide of 17 amino acids (shown as double underlined in  FIG. 2 ) was predicted using SignalP 3.0 software (Bendtsen et al., 2004) and was excluded during primer designing for PCR amplification. 
       Sequence Analysis of Tk-PUL 
       [0048]    Multiple sequence alignment ( FIG. 3 ) revealed that the amino acid sequence of Tk-PUL has only 62% or less identity with already known sequences of amylolytic enzymes. Maximum identity (62%) was observed with already characterized pullulan hydrolase III from  T. aggregans  (Table 1). Four highly conserved regions that are common in almost all amylolytic enzymes (Nakajima et al., 1986) were also identified in Tk-PUL sequence ( FIG. 4 ). Three acid residues (Asp 418 , Asp 601  and Glu 534 ) crucial for catalytic activity were also conserved. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Percent identity between amino acid sequence of 
               
               
                 Tk-PUL and that of other amylolytic enzymes. 
               
             
          
           
               
                   
                 Percent identity with Tk-PUL sequence 
               
             
          
           
               
                 Amylolytic enzyme and its source 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
               
               
                   
               
             
          
           
               
                 1. 
                 Tk-PUL 
                 100 
                 62 
                 38.3 
                 21.7 
                 21.3 
                 21.3 
                 21.3 
                 20.2 
                 20.2 
                 19.5 
               
               
                   
                 ( T. kodakaraensis ) 
               
               
                 2. 
                 Pullulan hydrolase type III 
                   
                 100 
                 41.3 
                 21.8 
                 20.7 
                 22 
                 21 
                 21.2 
                 20.2 
                 19.8 
               
               
                   
                 ( T. aggregans ) 
               
               
                 3. 
                 Pullulanases 
                   
                   
                 100 
                 24.5 
                 23.7 
                 25 
                 24.1 
                 22.2 
                 22.8 
                 21.6 
               
               
                   
                 ( D. mucosus ) 
               
               
                 4. 
                 Maltogenic α-amylase 
                   
                   
                   
                 100 
                 43.8 
                 41.3 
                 57.4 
                 55.7 
                 58.7 
                 55.7 
               
               
                   
                 ( Bacillus cidopullulyticus ) 
               
               
                 5. 
                 Cyclomaltodextrin hydrolase 
                   
                   
                   
                   
                 100 
                 47.7 
                 47.7 
                 44.8 
                 46.7 
                 45.7 
               
               
                   
                 ( T. ethanolicus ) 
               
               
                 6. 
                 Neopullulanase 
                   
                   
                   
                   
                   
                 100 
                 45.9 
                 42.1 
                 45.1 
                 43.4 
               
               
                   
                 ( T. vulgaris ) 
               
               
                 7. 
                 Neopullulanase 
                   
                   
                   
                   
                   
                   
                 100 
                 57.7 
                 69.6 
                 59.5 
               
               
                   
                 ( B. stearothermophilus ) 
               
               
                 8. 
                 Neopullulanase 
                   
                   
                   
                   
                   
                   
                   
                 100 
                 60.5 
                 58.6 
               
               
                   
                 ( Bacillus  sp.) 
               
               
                 9. 
                 Maltogenic amylase 
                   
                   
                   
                   
                   
                   
                   
                   
                 100 
                 64 
               
               
                   
                 ( G. stearothermophilus ) 
               
               
                 10. 
                 Neopullulanase 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 100 
               
               
                   
                 ( B. cereus ) 
               
               
                   
               
               
                 Swiss-prot accession numbers of sequences used were, 1, Q5JID9; 2, Q9P9A0; 3, Q9HHB0; 4, P32818; 5, P29964; 6, Q08751; 7, P38940; 8, Q57482; 9, Q45490; 10, Q819G8. 
               
             
          
         
       
     
       Purification of Recombinant Tk-PUL 
       [0049]    Recombinant Tk-PUL was purified to apparent homogeneity on SDS-PAGE. The purified pullulanase after Resource Q column showed 11.19-fold higher specific activity (70.5 U/mg) than that of crude extract (total cell lysate having specific activity 6.3 U/mg). Overall yield after purification was 89.2%. 
       pH Optimum for the Enzyme Activity 
       [0050]    The highest activities (pullulanse and α-amylase) were observed at pH 3.5 (in acetate buffer,  FIG. 5   a  and  FIG. 5   b ) while in citrate buffer maximum activities were observed at pH 4.25. 
       pH Stability of Tk-PUL 
       [0051]    The recombinant Tk-PUL displayed 84.47%, 77.47% and 56.86% of the maximal activities after 56 hour incubation (at 4° C.) at pH values 8.5, 6.5, and 4.2, respectively ( FIG. 6 ). These results indicated that the enzyme is more stable in alkaline pH though it has highest activity in acidic pH. 
       Optimum Temperature for Activity of Recombinant Tk-PUL 
       [0052]    The maximal pullulanase and α-amylase activities of recombinant Tk-PUL were observed at 100° C. in case of acetate buffer (pH 6.5) while in case of citrate buffer (pH 4.2) the maximal activities were observed at 95° C. Even at 120° C. the enzyme displayed more than 50% of the maximal activities ( FIGS. 7 and 8 ). 
       Effect of Metal Ions on the Activity of Recombinant Tk-PUL 
       [0053]    No increase in activity of Tk-PUL was observed in the presence of calcium (0-5 mM, final concentration in assay mixture) which indicated that unlike other amylolytic enzymes, recombinant Tk-PUL does not depend on calcium for its activity (Table 2). The activity of Tk-PUL was also not affected by the presence of Mg +2 , Mn +2 , Co +2  and Zn +2  while Ni +2 , Cu +2  and Fe +2  had inhibitory effect at 5 mM concentration but no effect at 0.05 mM concentration as shown in Table 3. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Pullulanase activity in the presence and absence of calcium. 
               
             
          
           
               
                   
                 [CaCl 2 ] 
                 Relative activity 
               
               
                   
                 (mM) 
                 (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 0.00 
                 100.00 
               
               
                   
                 0.02 
                 104.12 
               
               
                   
                 0.05 
                 104.47 
               
               
                   
                 0.10 
                 97.92 
               
               
                   
                 0.50 
                 103.76 
               
               
                   
                 1.00 
                 104.12 
               
               
                   
                 5.00 
                 97.74 
               
               
                   
                   
               
               
                   
                 Purified recombinant Tk-PUL was extensively dialyzed against 10 mM EDTA in 50 mM Tris-Cl pH 8.0. Properly diluted enzyme (1.7 U/mL, final concentration) was mixed with various concentrations of calcium chloride and incubated at 60° C. for 15 minutes. Samples were withdrawn and pullulanase activity was examined by DNS method (Bernfeld, 1955). 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Pullulanase activity in the presence of various concentrations of metal ions. 
               
             
          
           
               
                   
                 Metal ion concentration (mM) 
               
             
          
           
               
                   
                 0 
                 0.050 
                 5 
               
             
          
           
               
                 Metal ion used 
                 Relative activity (%) 
               
               
                   
               
             
          
           
               
                 Mg +2   
                 100 
                 98.5 
                 95 
               
               
                 Mn +2   
                 100 
                 102 
                 104 
               
               
                 Co +2   
                 100 
                 107 
                 94 
               
               
                 Zn +2   
                 100 
                 100 
                 94 
               
               
                 Ni +2   
                 100 
                 97 
                 89.5 
               
               
                 Cu +2   
                 100 
                 99 
                 47.8 
               
               
                 Fe +2   
                 100 
                 91.5 
                 32.7 
               
               
                 Ca +2   
                 100 
                 102 
                 103 
               
               
                   
               
               
                 Purified recombinant Tk-PUL was extensively dialyzed against 10 mM EDTA in 50 mM Tris-Cl pH 8.0. Properly diluted enzyme (1.7 U/mL, final concentration) was mixed with metal ions (either 50 μM or 5 mM, final concentrations) and incubated at 60° C. for 15 minutes. Samples were withdrawn and pullulanase activity was examined by DNS method (Bernfeld, 1955). 
               
             
          
         
       
     
       Thermostability of Tk-PUL 
       [0054]    The enzyme was highly stable at 90° C. (in the absence of substrates) in buffers of pH 6.5 and pH 8.5. Almost no loss of activity was seen even after 4 hours of incubation at this temperature. After 10 hours of incubation at 90° C., 90.36% and 76.49% of the maximal activity was found at pH 6.5 and pH 8.5, respectively ( FIG. 9 ). The half-life of Tk-PUL was 45 minutes at 100° C. (in the absence of substrates) in both buffers ( FIG. 10 ). The half-life of Tk-PUL in pH 4.2 buffer at 90° C. (in the absence of substrates) was 100 minutes as shown in  FIG. 9 . 
       Substrate Preference and Relative Hydrolysis Rate 
       [0055]    Besides pullulan (having 100% hydrolysis rate), the most preferred substrate by the Tk-PUL was γ-cyclodextrin. The other carbohydrates were hydrolyzed in the following preference order; y-cyclodextrin (75.65%) &gt;potato starch (60.13%) &gt;amylose (45.58%) &gt;corn starch (40.74%) &gt;dextrin (42.68%) &gt;amylopectin (37.02%)&gt;glycogen (25.86%) &gt;β-cyclodextrin (4.83%). 
         [0056]    End products analysis on HPLC revealed that Tk-PUL was able to hydrolyze cyclodextrins (α, β and γ) which are commonly known as competitive inhibitors of pullulanases. Smaller linear oligosaccharides from maltoheptaose to maltotriose were also hydrolyzed but at a slower rate. Final products of hydrolysis (from long chain and small chain saccharides) comprised of a mixture of maltotriose, maltose and glucose with predominant concentrations of maltose. It is worth mentioning that recombinant Tk-PUL was also able to hydrolyze maltotriose into maltose and glucose. Pullulan was hydrolyzed to a mixture of maltotriose, maltose, panose and isomaltose indicating that the enzyme hydrolyzes α-1,4 glycosidic linkages in pullulan in addition to α-1,6 linkages. On the basis of this fact Tk-PUL is proposed as pullulan hydrolase type III though it was previously annotated as pullulanase type II in the genome of  T. kodakaraensis.    
         [0057]    Recombinant Tk-PUL hydrolyzes the pullulan so efficiently that within 10 minutes more than 98% pullulan was converted to trisaccharides (maltotriose/panose) in the presence of 2.6 U≈40 μg of the enzyme when 0.25% w/v pullulan (final concentration) was used. To our knowledge none of the previously reported enzymes could hydrolyze the pullulan so efficiently. Data regarding the amount of enzyme utilized in the end product analysis experiments are also missing in previous reports. Recombinant Tk-PUL was also able to subsequently hydrolyze maltotriose into maltose and glucose. This unique feature of hydrolyzing trisaccharide maltotriose to maltose and glucose was also not reported previously. 
         [0000]    Application of Tk-PUL in the Production of Maltose Syrup from Corn Starch 
       Time Course Hydrolysis of Corn Starch 
       [0058]    In order to analyze the suitability of Tk-PUL in the production of glucose and maltose syrups time course hydrolysis of corn starch was performed. Purified Tk-PUL (1.6 mg of protein per gram of starch on dry basis) was incubated with 1% (w/v) starch (final concentration) in 0.1M sodium citrate buffer pH 4.2 at 90° C. At regular intervals samples were taken and saccharide composition was analyzed by HPLC on Aminex HPX-42A column. Starch was completely hydrolyzed to maltohexaose and smaller oligosaccharides within 6 hours and after 48 hours of incubation maltose concentration of the reaction mixture reached to 56% as represented in Table 4 and  FIG. 11 . These results indicated that Tk-PUL can be a candidate for application in starch industry for the production of maltose syrup even in the absence of α-amylase (required for liquefaction of starch) and β-amylase (maltogenic enzyme for saccharification). 
         [0000]    Production of Maltose Syrup from 12% Starch 
         [0059]    Purified Tk-PUL (0.67 mg/g starch on dry basis) was added to 12% (w/v) starch slurry (final concentration in 0.1 M sodium citrate buffer pH 4.2) and incubated first at 100° C. for 10 minutes and then at 90° C. for upto 72 hours. Saccharide composition was analyzed at regular intervals on Aminex HPX-42A column. After 72 hours of incubation maltose was the predominant sugar (26.2%) and more than 58% of the hydrolysates were oligosaccharides from DP4-DP1 (Table 5). 
         [0000]                                                                                                                  TABLE 5                   Production of maltose syrup from 12% (w/v) corn starch.            Time   Products concentration (%)            (Min)   DPn   DP7+   DP7   DP6   DP5   DP4   DP3   DP2   DP1                    0   100   0   0   0   0   0   0   0   0       2   81.6   0   7.0   0   1.8   1.3   3.0   5.3   0       3   77.8   0   5.6   2.2   2.3   1.7   3.6   6.3   0.5       5   71.2   1.7   5.6   2.8   3.0   2.6   4.8   8.1   0.2       48   24.4   7.0   3.5   5.2   7.3   9.4   18.2   23.7   1.3       72   16.8   7.7   3.7   5.5   7.9   10.2   19.8   26.4   2.0               Purified Tk-PUL (0.67 mg/g starch on dry basis) was added to 12% (w/v) starch slurry (final concentration in 0.1M sodium citrate buffer pH 4.2) and incubated first at 100° C. for 10 minutes and then at 90° C. for upto 72 hours. Saccharide composition was analyzed at regular intervals on Aminex HPX-42A column.       DP standsfor degree of polymerization while DP7 to DP1 are representing maltoheptaose to glucose, respectively.            
Production of Maltose Syrup from 30% Starch
 
         [0060]    Purified Tk-PUL (1 mg/g starch on dry basis) was incubated with 30% (w/v) starch under similar conditions as described above and saccharide composition was similarly analyzed by HPLC on Aminex HPX-42A column. More than 50% of the hydrolysis products were consisting of DP1-DP3 with major proportion of maltose (25.4%) as analyzed after 96 hours of incubation (as shown in Table 6). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Production of maltose syrup from 30% (w/v) corn starch. 
               
             
          
           
               
                 Time 
                 Products concentration (%) 
               
             
          
           
               
                 (Hrs.) 
                 DPn 
                 DP7+ 
                 DP7 
                 DP6 
                 DP5 
                 DP4 
                 DP3 
                 DP2 
                 DP1 
               
               
                   
               
             
          
           
               
                 0 
                 100 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 2 
                 79.1 
                 2.3 
                 3.2 
                 2.3 
                 2.1 
                 1.8 
                 3.6 
                 5.6 
                 0 
               
               
                 3 
                 77.4 
                 2.2 
                 3 
                 2.3 
                 2.2 
                 2.1 
                 4 
                 6.5 
                 0.3 
               
               
                 8 
                 54.3 
                 6.2 
                 4.3 
                 4.5 
                 4.7 
                 4.9 
                 8.4 
                 12.2 
                 0.5 
               
               
                 10 
                 49.2 
                 6.6 
                 4.6 
                 5 
                 5.3 
                 5.7 
                 9.5 
                 13.6 
                 0.5 
               
               
                 48 
                 11.4 
                 10.8 
                 4.5 
                 6.4 
                 9.5 
                 11.1 
                 20.5 
                 24.6 
                 1.2 
               
               
                 96 
                 11.2 
                 8.3 
                 4 
                 5.7 
                 9.4 
                 11.1 
                 21.6 
                 25.4 
                 3.3 
               
               
                   
               
               
                 Purified Tk-PUL (1 mg/g starch on dry basis) was added to 30% (w/v) starch slurry (final concentration in 0.1M sodium citrate buffer pH 4.2) and incubated first at 100° C. for 10 minutes and then at 90° C. for upto 96 hours. Saccharide composition was analyzed at regular intervals on Aminex HPX-42A column. 
               
               
                 DP stands for degree of polymerization while DP7 to DP1 are representing maltoheptaose to glucose, respectively. 
               
             
          
         
       
     
       Application of Tk-PUL in the Production Glucose Syrup 
     Liquefaction of Corn Starch 
       [0061]    For liquefaction of corn starch commercial α-amylase from  B. licheniformis;  Termamyl 120L was used as control. Tk-PUL and control enzyme (equivalent to 70 pullulanase U/g dry starch) were added into 25% (w/v) starch slurry. Calcium at a final concentration of 2 mM was additionally added in control experiment (containing Termamyl 120L) but not in Tk-PUL containing experiment. pH of the slurry in control was adjusted to 6.0 while for Tk-PUL it was adjusted to 4.2. Both the mixtures were incubated at 100° C. for 10 minutes and then at 90° C. HPLC analysis of hydrolysis products after 2 hours of incubation revealed that oligosaccharide profile obtained by the action of Tk-PUL was different from that obtained by the action of commercial enzyme (Termamyl 120L). Maltotriose and maltose (DP3 and DP2) were the predominant saccharides among low molecular weight products obtained by the action of Tk-PUL (after 2 hours) while non-significant amounts of these sugars were observed in the starch hydrolyzed by the action of Termamyl 120L ( FIG. 12   a  and  FIG. 12   b ). 
       Saccharification of Liquefied Corn Starch 
       [0062]    For saccharification the liquefied starch was cooled to 60° C. and commercial glucoamylase from  Aspergillus niger;  AMG 300L was added (1.29 U/g starch). pH of the starch liquefied by Termamyl 120L was lowered to 4.5 while starch liquefied with Tk-PUL required no pH adjustment (already at 4.2). Both the mixtures were then incubated at 60° C. for further 24 hours. Final saccharide composition in Tk-PUL treated sample (liquefied starch) was significantly similar to that observed in the sample treated with commercial α-amylase from  B. licheniformis.  These results are shown in Table 7,  FIG. 13   a  and  FIG. 13   b . 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Saccharide composition of glucose syrup produced 
               
               
                 by the action of recombinant Tk-PUL and 
               
               
                 commercial α-amylase from  B. licheniformis . 
               
             
          
           
               
                 Liquefying Enzyme used 
                 DP1 
                 DP2 
                 DP3 
                 DP4+ 
               
               
                   
               
               
                 Termamyl 120L 
                 96.7 
                 2.2 
                 0.3 
                 0.8 
               
               
                 (α-amylase from  B. licheniformis ) 
                   
                   
                   
                   
               
               
                 Tk-PUL 
                 92.9 
                 4.9 
                 0.8 
                 1.4 
               
               
                   
               
               
                 25% (w/v) starch slurry was liquefied either by Termamyl 120L (at pH 6.0 in the presence of 2 mM calcium) or by Tk-PUL (at pH 4.2 in the absence of calcium) at 100° C. for 10 minutes and then at 90° C. for 2 hours. Saccharification was performed by commercial glucoamylase from  Aspergillus   niger ; AMG 300L at 60° C. DP1, DP2 and DP3 represent glucose, maltose and maltotriose, respectively.