Novel rhamnogalacturonan hydrolases

Abstract

Novel isolated enzymes exhibiting rhamnogalacturonan hydrolase activity and belonging to a glycosyl hydrolase family other than family 28 are obtained and cloned from bacterial strains, for example, Bacillus licheniformis, Bacillus subtilis, Bacillus halodurans, Bacillus agaradhaerens, Caldicellulosiruptor, Streptomyces and Sorangium.

What is claimed is:  
     
         1 . An isolated enzyme exhibiting rhamnogalacturonan hydrolase activity wherein the enzyme belongs to a glycosyl hydrolase family other than family 28.  
     
     
         2 . The enzyme according to  claim 2 , which is obtained from a microbial strain belonging to Bacteria.  
     
     
         3 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence having amino acids 1-621 of the sequence of SEQ ID NO:2, or  
 ii) comprises an analogue of the polypeptide defined in i) which is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         4 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 3 .  
     
     
         5 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence having amino acids 1-620 of the sequence of SEQ ID NO:4, or  
 ii) comprises an analogue of the polypeptide defined in i) which is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         6 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 5 .  
     
     
         7 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence having amino acids 1-620 of the sequence of SEQ ID NO:6, or  
 ii) comprises an analogue of the polypeptide defined in i) which is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         8 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 7 .  
     
     
         9 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence comprising amino acids 1-471 of the sequence of SEQ ID NO:8, or  
 ii) comprises an analogue of the polypeptide defined in i) of which a part is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         10 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 9 .  
     
     
         11 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence comprising amino acids 1-170 of the sequence of SEQ ID NO:10, or  
 ii) comprises an analogue of the polypeptide defined in i) of which a part is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         12 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 11 .  
     
     
         13 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence comprising amino acids 1-112 of the sequence of SEQ ID NO:12, or  
 ii) comprises an analogue of the polypeptide defined in i) of which a part is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         14 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 13 .  
     
     
         15 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence having amino acids 1-655 of the sequence of SEQ ID NO:14, or  
 ii) comprises an analogue of the polypeptide defined in i) which is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         16 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 15 .  
     
     
         17 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence having amino acids 1-631 of the sequence of SEQ ID NO:16, or  
 ii) comprises an analogue of the polypeptide defined in i) which is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         18 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 17 .  
     
     
         19 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence having amino acids 1-389 of the sequence of SEQ ID NO:18, or  
 ii) comprises an analogue of the polypeptide defined in i) which is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         20 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 19 .  
     
     
         21 . The enzyme according to  claim 1  which 
 i) comprises a polypeptide comprising an amino acid sequence comprising amino acids 1-169 of the sequence of SEQ ID NO:20, or  
 ii) comprises an analogue of the polypeptide defined in i) of which a part is at least 75% homologous with the polypeptide, or  
 iii) comprises a mutant or variant of the polypeptide which is derived from the polypeptide by substitution, deletion or addition of one or several amino acids or  
 iv) is immunologically reactive with a polyclonal antibody raised against the polypeptide in purified form.  
 
     
     
         22 . An isolated polynucleotide molecule encoding a polypeptide exhibiting rhamnogalacturonan hydrolase activity of  claim 21 .  
     
     
         23 . An isolated enzyme exhibiting rhamnogalacturonan hydrolase activity, exhibiting the characteristics of: 
 a) cleaving a rhamnogalacturonan backbone, wherein the rhamnoses are left as the non-reducing ends; and    b) a relative activity towards hairy regions from apples of at least 30% at pH 8.    
     
     
         24 . An isolated enzyme exhibiting rhamnogalacturonan hydrolase activity, exhibiting the characteristics of: 
 a) cleaving a rhamnogalacturonan backbone, wherein the rhamnoses are left as the non-reducing ends; and    b) a relative activity towards hairy regions from apples of at least 30% at pH 9.    
     
     
         25 . A method of reducing the viscosity of a plant material, which method comprises treating the plant material with the enzyme of  claim 1  under suitable conditions for the viscosity to be reduced.  
     
     
         26 . An animal feed composition comprising plant material rich in pectic substance and the rhamnogalacturonan hydrolase of  claim 1 .  
     
     
         27 . A method for increasing the digestibility of an animal feed, comprising adding the rhamnogalacturonan hydrolase of  claim 1  to one or more of soy, pea or rapeseed, or other material derived from Fabales or Cruciferaceae.  
     
     
         28 . A method for reducing the viscosity of an animal feed, comprising adding the rhamnogalacturonan hydrolase of  claim 1  to one or more of soy, pea or rapeseed, or other material derived from Fabales or Cruciferaceae.  
     
     
         29 . A detergent composition comprising the enzyme according to  claim 1  and a surfactant.  
     
     
         30 . The enzyme according to  claim 1  which comprises at least one amino acid sequence segment selected from the group of amino acid sequence segments consisting of 
 a) NIRAGAHTQF(M or L)VYD(F or L)DGDGKAE;  
 b) YGNRVDRFLAG;  
 c) YGNRVDRFLAGXAYLDG;  
 d) AGQGNH(N or S)LS(I or V)ADVDGDGKDEII; and  
 e) AGQGNH(N or S)L(S or A)(I or V)ADVDGDGKDEII.  
 
     
     
         31 . The enzyme according to  claim 1  which comprises at least one amino acid sequence segment selected from the group of amino acid sequence segments consisting of 
 a) EVRDATIGLL;  
 b) NNYVVGNPI; and  
 c) DADRTNRA.