ENZYMES HAVING PULLULANASE ACTIVITY

Abstract

The present invention relates to thermostable pullulanases useful for industrial and scientific purposes. The present invention provides methods for producing the modified pullulanase, enzymatic compositions comprising the modified pullulanase, and methods for use of the enzymatic compositions.

Claims

1. An isolated, synthetic, or recombinant nucleic acid encoding a polypeptide having pullulanase activity, selected from the group consisting of: (a) a nucleic acid encoding a polypeptide having pullunase activity comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:1; (b) a nucleic acid encoding a polypeptide having pullulanase activity comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:1, or a fragment thereof, wherein the fragment encodes a polypeptide having pullulanase activity; (c) a nucleic acid sequence encoding a polypeptide having pullulanase activity comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2; (d) a nucleic acid sequence encoding a polypeptide having pullulanase activity comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, or a fragment thereof, wherein the fragment has pullulanase activity; (e) the nucleic acid of (a), (b), (c), or (d) encoding a polypeptide having pullulanase activity but lacking a signal sequence or a carbohydrate binding module; (f) the nucleic acid of (a), (b), (c), (d) or (e) encoding a polypeptide having pullulanase activity, and further comprising a heterologous sequence; (g) the nucleic acid sequence of (g), wherein the heterologous sequence comprises a sequence encoding a heterologous signal sequence, carbohydrate binding module, catalytic domain (CD), or a combination thereof, or the heterologous signal sequence, carbohydrate binding module, or catalytic domain (CD) is derived from another pullulanase enzyme, or a non-pullulanase enzyme; or (h) a nucleic acid sequence fully complementary to (a), (b), (c), (d), (e), (f), or (g).

2. The isolated, synthetic, or recombinant nucleic acid of claim 1, wherein the pullulanase activity comprises the cleavage of both alpha-1,6 and alpha-1,4 bonds.

3. The isolated, synthetic, or recombinant nucleic acid of claim 1, wherein the pullulanase activity comprises type II pullulanase activity.

4. The isolated, synthetic, or recombinant nucleic acid of claim 1, wherein the pullulanase activity is thermostable and or thermotolerant.

5. An isolated, synthetic, or recombinant polypeptide having pullulanase comprising (a) an amino acid sequence having at least 50% identity, or complete sequence identity to sequence of SEQ ID No. 2; (b) having the amino acid sequence encoded by the nucleic acid of claim 1; (c) the amino acid sequence of (a) or (b), and comprising at least one conservative amino acid residue conservative substitutions; (d) the amino acid sequence of (a) or (b) or (c) or a fragment thereof with pullulanase activity.

6. The isolated, synthetic, or recombinant polypeptide of claim 5, where the pullulanase activity is thermostable.

7. The isolated, synthetic, or recombinant polypeptide of claim 5 wherein the polypeptide retains an pullulanase activity under conditions comprising a temperature range of between about 37 C. to about 84 C., or between about 55 C. to about 85 C., or between about 70 C. to about 84 C.

8. The isolated, synthetic, or recombinant polypeptide of claim 5, where the pullulanase activity is thermotolerant.

9. The isolated, synthetic, or recombinant nucleic acid of claim 5, wherein the polypeptide retains an pullulanase activity after exposure to a temperature in the range from greater than 37 C. to about 84 C., from greater than 55 C. to about 84 C.

10. A method of hydrolyzing a starch linkage comprising contacting a substance containing the starch with a polypeptide of claim 5, and sequences substantially identical thereto.

11. A detergent additive comprising with a polypeptide of claim 5.

12. A method of producing ethanol comprising use of the polypeptide of claim 5.

13. The method of claim 12, wherein the polypeptide is capable of digesting limit dextrans.

14. The method of claim 10 or 11 or 12, further comprising addition of an pullulanase or a combination thereof.

15. A detergent composition comprising the polypeptide of claim 5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

[0019] FIG. 1 is a chromatogram displaying the results of a digestion using the pullulanase of SEQ ID No.: 1 & 2 with a 1% corn starch substrate at 50 degrees Celcius, and as further described in Example 1.

[0020] FIG. 2 is a chromatogram displaying the results of a digestion using the pullulanase of SEQ ID No.: 1 & 2 with a 1% pullulan at 50 degrees Celcius, and as further described in Example 1.

[0021] FIG. 3 is a chromatogram displaying the results of a digestion using the pullulanase of SEQ ID No.: 1 & 2 with a 1% corn starch substrate at 75 degrees Celcius, and as further described in Example 1.

[0022] FIG. 4 is SEQ ID No.: 1, the deoxyribonucleic acid (DNA) sequence of the present invention.

[0023] FIG. 5 is SEQ ID No.: 2, the amino acid sequence of the present invention.

EXAMPLES

Example 1

[0024] The pullulanase of the present invention (as embodied by SEQ ID No.: 1 & 2) was tested against 1% solids using an aliquot from a trial cell lysate. The results shown in FIGS. 1, 2, and 3, show that the pullulanase of the present invention is able to cleave both alpha-1,6 and alpha-1,4 bonds. As displayed in FIG. 2, the pullulanase of the present invention (as embodied by SEQ ID No. 1 & 2) was assayed with 1% pullulan at 50 degrees Celsius. As displayed in FIG. 1, the pullulanase of the present invention (as embodied by SEQ ID NO 1 & 2) was assayed with 1% corn starch at 50 degrees Celcius. As displayed in FIG. 3, the pullulanase of the present invention (as embodied by SEQ ID No. 1 & 2) was assayed with 1% corn starch at 75 degrees Celsius. As indicated in FIGS. 1, 2, and 3, the enzyme is a type II pullulanase, as the reaction products (major peaks) are glucose, maltose, and maltotriose. Additionally, the peak 2 product was confirmed to be maltose and not isomaltose, while the peak 3 product was confirmed to be maltotriose and not panose.

Example 2

[0025] The pullulanase of the present invention (as embodied by SEQ ID No. 1 & 2) melting point or thermal denaturation was determined using differential scanning calorimetry. The Tm of the present invention (as embodied by SEQ ID No. 1 & 2) is 84 degrees Celsius.

DETAILED DESCRIPTION

[0026] The present invention relates to a pullulanase enzyme, polynucleotides encoding the enzymes, methods of making and using these polynucleotides and polypeptides. The invention is directed to novel polypeptides having pullulanase activity, nucleic acids encoding them. The polypeptides of the invention can be used in a variety of commercial, medical, and industrial contexts. The polypeptides of the invention can be used as, e.g., an additive for a detergent, for processing foods and for chemical synthesis utilizing a reverse reaction, saccharification of starch, liquefaction of starch, production of high-maltose corn syrup, production of high-fructose corn syrup, starch processing, ethanol production, production of cyclodextrins, and production of low-calorie beer, in the baking industry, as well as dental plaque control.

[0027] In one aspect of the invention the pullulanase is a type II pullulanase or is capable of cleaving both alpha-1,6 and alpha-1,4 bonds. In another aspect of the invention the pullulanase of the present invention is capable of cleaving both alpha-1,6 and alpha-1,4 of pullulan yielding glucose, maltose, and maltotriose. In a further embodiment of the present invention the pullulanase of the present invention is thermostable and or thermotolerant. In a further embodiment of the invention the pullulanase of the present invention is active at 75 degrees Celsius. In a further embodiment of the invention, the pullulanase of the invention is capable of saccharification of starch at higher temperatures then currently employed, thereby reducing processing times, and increasing yields due to lessening the rate of retrogradation of materials resulting in drops in temperature. In a further embodiment of the present invention the pullulanase is capable of enhancing ethanol or glucose production by digesting limit dextrans that an amylase cannot digest.

[0028] In a further embodiment of the present invention the pullulanase of the present invention is coupled with an amylase enzyme.

[0029] In one aspect, the nucleic acid encodes at least one polypeptide having pullulanase activity.

[0030] Synthetic nucleic acids (including oligonucleotides), polypeptides or proteins of the invention include those prepared by any chemical synthesis, e.g., as described, below.

[0031] The phrases nucleic acid or nucleic acid sequence includes oligonucleotides, nucleotides, polynucleotides, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic, recombinant or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA such as miRNA or siRNA, ribonucleoproteins (e.g., iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156.

[0032] Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.

[0033] The term gene includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability. Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).

[0034] The invention provides isolated and recombinant nucleic acids, including expression cassettes such as expression vectors encoding the polypeptides of the invention. The invention provides probes comprising or consisting of nucleic acids of the invention. The invention also includes methods for discovering new pullulanase sequences using the nucleic acids of the invention. The invention also includes methods for inhibiting the expression of pullulanase genes, transcripts and polypeptides using the nucleic acids of the invention.

[0035] The nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. In practicing the methods of the invention, homologous genes can be modified by manipulating a template nucleic acid, as described herein. The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.