Glucan lyase producing 1,5-anhydrofructose

• US 5,695,970 A
• Filed: 04/18/1995
• Issued: 12/09/1997
• Est. Priority Date: 10/21/1992
• Status: Expired due to Fees

• Current Assignee: T&M Biopolymer AB
• Original Assignee: T&M Biopolymer AB
• Inventors: Pedersen, Marianne, Kenne, Lennart, Yu, Shukun
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Nashed, Nashaat T.
• Application Number:
  US08/416,709
• Time in Patent Office: 966 Days
• Field of Search: 435/232, 435/257.1, 435/105, 435/257.2, 536/23.2
• US Class Current: 435/105
• CPC Class Codes: C07H 3/10 Anhydrosugars, e.g. epoxides
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60

Abstract

The invention relates to an enzyme, exo-α-1,4-glucan lyase, capable of successively cleaving the terminal α-1,4-D-glucosidic bonds from the non-reducing ends of an α-1,4-glucan. The enzyme is selected from the group consisting of α-1,4-glucan lyase isolated from an alga and functional derivatives and analogs derived from a nucleotide sequence related to an alga α-1,4-glucan lyase. Further, a method of enzymatically cleaving the terminal α-1,4-D-glucosidic bonds from the non-reducing ends of an α-1,4-glucan, and the degradation product 1,5-anhydrofructose for use as an oxygen radical scavenger or anti-oxidant and/or as a sugar substitute, are disclosed. Additionally, a method of producing an enzyme capable of successively cleaving the terminal α-1,4-glucosidic bonds from the non-reducing ends of an α-1,4-glucan; an antibody which binds to the amino acid sequence of the enzyme, and a DNA or RNA probe which recognizes a nucleotide sequence coding for the enzyme, are described.

Claims

1. An isolated and purified exo-α

-1,4-glucan lyase enzyme selected from the group consisting of α

-1,4-glucan lyase isolated from an alga and its enzymatically active mutants.

2. An enzyme according to claim 1, wherein said alga is a red seaweed of the order Gigartinales.

3. An enzyme according to claim 2, wherein said red seaweed is chosen from the group consisting of Gracilariopsis lemaneiformis, Gracilaria verrucosa and Phyllophora truncata.

4. An enzyme according to claim 1, wherein said enzyme is in immobilized form.

5. An enzyme according to claim 1, wherein said enzyme has an amino acid sequence which comprises at least one of the following fragments with amino acid sequence of:

Fragment 1 (SEQ ID NO;

     1), Fragment 2 (SEQ ID NO;

     2), Fragment 3 (SEQ ID NO;

     3) or Fragment 4 (SEQ ID NO.

     4).

6. An enzyme according to claim 1, wherein said enzyme has an isoelectric point of around 3.9 and an approximate molecular weight of 111,000 Da by SDS-gel electrophoresis and 98,000 Da by gel filtration chromatography and the following amino acid composition determined as amino acid residues per molecule by amino acid analysis:

Asx 155, Thr 71, Ser 57, Gly 94, Pro 49, Gly 98, Ala 52, Cys 15, Val 70, Met 21, Ile 41, Leu 61, Tyr 62, Phe 52, His 16, Lys 37, Arg 48, and Trp not determined.

7. An enzyme according to claim 1, wherein said enzyme has a molecular weight of about 54,000 Da.

8. A method of cleaving the terminal α

-1,4-D-glucosidic bonds from the non-reducing ends of an α

-1,4-glucan, characterized in that said α

-1,4-glucan is brought into contact with an enzyme according to claim 1.

9. A method according to claim 8 where said α

-1,4-glucan comprises branched chains and said enzyme is used together with a polysaccharide debranching enzyme.

10. A method according to claim 8, wherein the degradation product comprises 1,5-anhydrofructose.

11. A method of producing the enzyme of claim 1 capable of successively cleaving the terminal α

-1,4-D-glucosidic bonds from the non-reducing ends of an α

-1,4-glucan, characterized in that it is isolated from an alga by extraction and purification in per se known manner, or it is produced and purified from a culture of transformed cells with the nucleotide sequence coding for an algol α

-1,4-glucan lyase.

12. A method according to claim 9, wherein the polysaccharide debranching enzyme is pullulanase.