HEAT TOLERANT ALKALINE PHOSPHATASE

1 . A heat tolerant bacterial alkaline phosphatase enzyme polypeptide, said polypeptide comprising:

(a) the polypeptide

(SEQ ID NO: 2)

ESSNGQSKNLIVLIGDGMGPAQVSAARYFQQHX 1 NNINSLNLDPYYVGQATTYADRGEDGG

HIVSGIX 2 TSSAX 3 AGTAX 4 ATGNKTX 5 NAAISVSNEDVSRPFASVX 6 EAAELSGKSTGLVTTARI

THATPAVYX 7 SHVRX 8 RDNENAIAFQYLDSGIDVLLGGGESFFVTX 9 EEKGKRNDKNLLPEFE

AKGYKVVKX 10 GQSLKSLSAKDAKVLGLFGGSHIX 11 YVPDRSDETPSLAEMTSKALEILSTNE

NGFAIMIEGGRIDHAX 12 HANDFPTMVQEX 13 LX 14 FDEAFKVAIDFAKKDGNTSVVVTADHET

GGLSLX 15 RDNIYELNVDLWNKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQY

ILDGDGSSYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTRIATA

SAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGGKYQAANGTATISGMS

GTITVDLNAKKAKLSGNSSX 16 ITIDVDNDVLYLPLTAFSQITGQTLKWDALSERIMLK 

wherein:

X 1 is I or L,

X 2 is I,

X 3 is A,

X 4 is I,

X 5 is N,

X 6 is H or F,

X 7 is G,

X 8 is N,

X 9 is N,

X 10 is N,

X 11 is P,

X 12 is A,

X 13 is M, V, T or E,

X 14 is A or E,

X 15 is G or A, and

X 16 is E;

(b) the polypeptide:

 (SEQ ID NO: 3)

ESSNGQSKNLIVLIGDGMGPAQVSAARYFQQHINNINSLNLDPYYVGQATTYADRGEDGGHI

VSGIVTSSASAGTALATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVTTARITHAT

PAVYASHVRNRDNENAIAFQYLDSGIDVLLGGGESFFVTNEEKGKRNDKNLLPEFEAKGYK

VVKTGQSLKSLSAKDAKVLGLFGGSHIAYVPDRSDETPSLAEMTSKALEILSTNENGFAIMIE

GGRIDHAGHANDFPTMVQEALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIY

ELNVDLWNKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGSSYKRE

GGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTRIATASAEVLGVDLKK

ATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGGKYQAANGTATISGMSGTITVDLNAKK

AKLSGNSSSITIDVDNDVLYLPLTAFSQITGQTLKWDALSERIMLK; 

or

(c) a variant of (a) or (b), wherein the variant of the alkaline phosphatase enzyme polypeptides retains phosphatase activity which is not significantly different from heat tolerant mutant enzyme SEQ ID NO:1.

2 . The heat tolerant bacterial alkaline phosphatase enzyme polypeptide of claim 1 , denoted APHT, said polypeptide comprising:

 (SEQ ID NO: 1)

ESSNGQSKNLIVLIGDGMGPAQVSAARYFQQHINNINSLNLDPYYVGQATTYADRGEDGGHIVSGIITSS

AAAGTAIATGNKTNNAAISVSNEDVSRPFASVHEAAELSGKSTGLVTTARITHATPAVYGSHVRNRDNEN

AIAFQYLDSGIDVLLGGGESFFVTNEEKGKRNDKNLLPEFEAKGYKVVKNGQSLKSLSAKDAKVLGLFGG

SHIPYVPDRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAAHANDFPTMVQEMLAFDEAFKVAI

DFAKKDGNTSVVVTADHETGGLSLGRDNIYELNVDLWNKQKNSSESLVSALNEAKTIADVKKIVSDNTWI

TDLTNEEAQYILDGDGSSYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTRIAT

ASAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGGKYQAANGTATISGMSGTITVDLN

AKKAKLSGNSSEITIDVDNDVLYLPLTAFSQITGQTLKWDALSERIMLK.

3 . The heat tolerant bacterial alkaline phosphatase enzyme polypeptide of claim 1 , said polypeptide comprising:

(SEQ ID NO: 3)

ESSNGQSKNLIVLIGDGMGPAQVSAARYFQQHINNINSLNLDPYYVGQATTYADRGEDGGHIVS

GIVTSSASAGTALATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVTTARITHATPAVY

ASHVRNRDNENAIAFQYLDSGIDVLLGGGESFFVTNEEKGKRNDKNLLPEFEAKGYKVVKTGQS

LKSLSAKDAKVLGLFGGSHIAYVPDRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAGHA

NDFPTMVQEALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIYELNVDLWNKQKN

SSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGSSYKREGGYNAVISKRLLVGW

SGHGHSAVDVGVWAYGPIADKVKGQIDNTRIATASAEVLGVDLKKATADLQSKYLYPKFKINR

NKEVLFPAKPLAEALGGKYQAANGTATISGMSGTITVDLNAKKAKLSGNSSSITIDVDNDVLYLP

LTAFSQITGQTLKWDALSERIMLK

4 . The heat tolerant bacterial alkaline phosphatase enzyme polypeptide of claim 1 , said polypeptide comprising the polypeptide of any of SEQ ID NOs: 18-73.

5 . The alkaline phosphatase enzyme polypeptide of claim 1 , further comprising an N terminal leader sequence.

6 . The alkaline phosphatase enzyme polypeptide of claim 5 , wherein the N terminal leader sequence is a sequence of 2 to 10 amino acids and wherein the leader sequence does not alter the heat tolerance or the enzymatic activity of the phosphatase.

7 . The alkaline phosphatase enzyme polypeptide of claim 5 , wherein the N terminal leader sequence is selected from SEQ ID NO: 10, 13, 16 and 17.

8 . A feed additive or feed composition comprising the mutant alkaline phosphatase enzyme polypeptide of claim 1 .

9 . The feed additive or feed composition of claim 8 , comprising the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 1, 2, 3 or 18-73.

10 . The feed additive or feed composition of claim 8 , comprising the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 2 or of SEQ ID NO: 1.

11 . (canceled)

12 . The feed additive or feed composition of claim 8 , comprising the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 1, 2, 3 or 18-73 in an amount of 4,000 IU/kg, 10,000 IU/kg, 12,000 IU/kg, 20,000 IU/kg, 40,000 IU/kg, up to 40,000 IU/kg, 100,000 IU/kg, or up to 200,000 IU/kg.

13 . (canceled)

14 . A composition comprising the mutant alkaline phosphatase enzyme polypeptide of claim 1 and an additive or diluent or carrier.

15 . The additive or composition of claim 8 , further comprising an anti-inflammatory agent, molecule or cytokine or further comprising an anti-bacterial and/or an anti-infective agent.

16 . (canceled)

17 . A method for reducing bacterial lipopolysaccharides (LPS) in the intestine, reducing inflammation particularly bacterial-mediated inflammation, reducing production of pro-inflammatory cytokines, particularly in the intestine, improving intestinal permeability markers, and/or improving or resulting in more efficient nutrient utilization in an animal, comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of claim 1 .

18 . The method of claim 17 , wherein production or expression of pro-inflammatory cytokine TNFα and/or of IL-6 is reduced in the intestine of an animal.

19 . The method of claim 17 , comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 1, 2, 3 or 18-73.

20 . The method of claim 17 , comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 2 or of SEQ ID NO: 1.

21 . (canceled)

22 . (canceled)

23 . (canceled)

24 . (canceled)

25 . (canceled)

26 . A method for increasing intestinal alkaline phosphatase production or activity in an animal, for reducing bacterial lipopolysaccharides (LPS) in the intestine, for reducing production of pro-inflammatory cytokines, or for reducing intestinal inflammation in an animal comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of claim 1 .

27 . The method of claim 26 , comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 1, 2, 3 or 18-73.

28 . The method of claim 26 , comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 2 or of SEQ ID NO: 1.

29 . (canceled)

30 . A method for increasing intestinal alkaline phosphatase production or activity in an animal, for reducing bacterial lipopolysaccharides (LPS) in the intestine, for reducing production of pro-inflammatory cytokines, or for reducing intestinal inflammation in an animal comprising administering to said animal the additive or composition of claim 8 .

31 . The method of claim 30 , comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 1, 2, 3 or 18-73.

32 . The method of claim 30 , comprising administering to said animal the mutant alkaline phosphatase enzyme polypeptide of SEQ ID NO: 2 or SEQ ID NO: 1.

33 . (canceled)