Use of alpha-1,4-glucan lyase for preparation of 1,5-D-anhydrofructose

Abstract

A method of preparing the sugar 1,5-D-anhydrofructose is described. The method comprises treating an α-1,4-glucan with an α-1,4-glucan lyase wherein the enzyme is used in substantially pure form. In a preferred embodiment, if the glucan contains links other than and in addition to the α-1,4-links, the α-1,4-glucan lyase is used in conjunction with a suitable reagent that can break the other links.

1 . A method of preparing the sugar 1,5-D-anhydrofructose comprising treating an α-1,4-glucan with the enzyme α-1,4-glucan lyase characterised in that enzyme is used in substantially pure form.  
     
     
         2 . A method according to  claim 1  wherein if the glucan contains links other than and in addition to the α-1,4-link the α-1,4-glucan lyase is used in conjunction with a suitable reagent that can break the other links.  
     
     
         3 . A method according to  claim 2  wherein the glucan is starch and a hydrolase, preferably a glucanohydrolase, is used in conjunction with the α-1,4-glucan lyase.  
     
     
         4 . A method according to  claim 2  or  claim 3  wherein the hydrolase is at least one of pullanase or isoamylase.  
     
     
         5 . A method according to any preceding claim wherein the α-1,4-glucan lyase is bound to a support or, more preferably, is in a dissolved form.  
     
     
         6 . A method according to any preceding claim wherein the enzyme is isolated from either a fungus, preferably  Morchella costata  or  Morchella vulgaris,  or from a fungally infected algae, preferably  Gracilariopsis lemaneiformis  or from algae alone, preferably  Gracilariopsis lemaneiformis.    
     
     
         7 . A method according to  claim 6  wherein the enzyme is isolated and/or further purified from the fungus or from the fungally infected algae or from algae alone using a gel that is not degraded by the enzyme.  
     
     
         8 . A method according to  claim 7  wherein the gel is based on dextrin or derivatives thereof, preferably the gel is a cyclodextrin—more preferably beta-cyclodextrin.  
     
     
         9 . A method according to any of the preceding claims wherein the enzyme comprises the amino acid sequence SEQ. ID. No. 1. or the amino acid sequence SEQ. ID. No. 2 or the amino acid sequence SEQ. ID. No. 5. or the amino acid sequence SEQ. ID. No. 6, or any variant thereof.  
     
     
         10 . A method according to any preceding claim wherein the enzyme is obtained from the expression of a nucleotide sequence coding for the enzyme.  
     
     
         11 . A method according to  claim 10  wherein the nucleotide sequence is a DNA sequence.  
     
     
         12 . A method according to  claim 11  wherein the DNA sequence comprises a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No. 8.  
     
     
         13 . The method according to  claim 3  or any claim dependent thereon wherein the starch is used in high concentration—such as up to about 25% solution.  
     
     
         14 . The method according to any one of the preceding claims wherein the substrate is treated with the enzyme in the presence of a buffer.  
     
     
         15 . The method according to any one of  claims 1  to  13  wherein the substrate is treated with the enzyme in the presence of at least substantially pure water.  
     
     
         16 . The method according to any one of the preceding claims wherein the substrate is treated with the enzyme in the absence of a co-factor.  
     
     
         17 . The method according to any one of the preceding claims wherein the enzyme is used in combination with amylopectin or dextrin.  
     
     
         18 . A method of preparing the sugar 1,5-D-anhydrofructose comprising treating an α-1,4-glucan with the enzyme α-1,4-glucan lyase characterised in that enzyme comprises the amino acid sequence SEQ. ID. No. 1. or the amino acid sequence SEQ. ID. No. 2 or the amino acid sequence SEQ. ID. No. 5. or the amino acid sequence SEQ. ED. No. 6, or any variant thereof.  
     
     
         19 . The sugar 1,5-D-anhydrofructose when prepared by the method of the present invention.  
     
     
         20 . The use of a reagent that can increase the hydrophobicity of the reaction medium to increase the stability and activity of the GL enzyme.  
     
     
         21 . Use of AF as an anti-oxidant.  
     
     
         22 . Use of AF as a sweetener.