(1 -> 3, 1 -> 4)-beta-glucanase of enhanced stability

Abstract

A modified cereal (1→3,1→4)-β-glucanase is produced by the method of single point substitution in a native cereal (1→3,1→4)-β-glucanase enzyme, whereby the substitution: a) maintains enzyme specificity by conserving the active site groove of the native cereal (1→3,1→4)-β-glucanase enzyme; and b) effects increased thermostability over the native cereal (1→3,1→4)-β-glucanase enzyme by: i) replacing glycine by proline or alanine in helices of the cereal (1→3,1→4)-β-glucanase enzyme, in order to stiffen the enzyme amino acid chain and reduce entropy of the unfolded enzyme; ii) attaching negatively charged residues to N-termini of helices in the native cereal (1→3,1→4)-β-glucanase enzyme; iii) introducing ion pairs into the native cereal (1→3,1→4)-β-glucanase enzyme, to increase binding energy in the folded enzyme; iv) replacing lysine by arginine in the cereal (1→3,1→4)-β-glucanase enzyme, and thereby preventing lysine glycation and increasing hydrogen bonding with other parts of the enzyme; v) replacing, by glycine, an amino acid in the native cereal (1→3,1→4)-β-glucanase enzyme in which the main chain torsion angle about the N and C α atoms is greater than 0°; or vi) creating cysteine pairs in the native cereal (1→3,1→4)-β-glucanase enzyme which can form disulphide bonds across the C and N terminals.

1 . A plant (1→3,1→4)-β-glucanase enzyme of enhanced thermostability and/or pH stability, said enzyme being modified by transfer of a non-homologous protein sequence of different substrate specificity.  
     
     
         2 . A plant (1→3,1→4)-β-glucanase in which the amino acid sequence of said enzyme: 
 (a) is modified to comprise structural elements of plant (1→3)-β-glucanase, said structural elements conferring improved heat stability;  
 (b) is modified at sites other than the active site to stabilise helices, to increase binding energy of the folded protein, to increase hydrogen bonding, and/or to prevent glycation; or  
 (c) is modified by creating cysteine pairs which can form disulphide bonds across the C and N terminals.  
 
     
     
         3 . A (1→3,1→4)-β-glucanase according to  claim 2 , in which two or more of the modifications (a) to (c) are present.  
     
     
         4 . A (1→3,1→4)-β-glucanase according to  claim 1  comprising the structural framework of the enzyme (1→3)-β-glucanase and elements of the catalytic site of (1→3,1→4)-β-glucanase.  
     
     
         5 . An enzyme according to  claim 4  additionally comprising modification (b) and/or modification (c) of  claim 2 .  
     
     
         6 . A (1→3,1→4)-β-glucanase according to  claim 1 , in which the amino acid sequence of (1→3,1→4)-β-glucanase isoenzyme EII is modified to comprise one or more substitutions selected from the group consisting of: 
 Ala 14 Ser  
 Ala 15 Arg  
 Thr 17 Asp  
 Lys 23 Arg  
 Lys 28 Arg  
 Asn 36 Asp  
 Gly 44 Arg  
 Gly 45 Asn  
 Gly 53 Asp  
 Gly 53 Glu  
 Lys 74 Arg  
 Gln 78 Arg  
 Ala 79 Pro  
 Lys 82 Arg  
 Ala 95 Asp  
 Gly 97 Pro  
 Phe 85 Tyr  
 Lys 107 Arg  
 Gly 111 Ala  
 Gly 119 Pro  
 Lys 122 Arg  
 Ser 128 Arg  
 Gly 133 Ala  
 Gly 145 Asn  
 Gly 152 Thr  
 Pro 153 Asp  
 Gln 156 Arg  
 Asn 162 Gly  
 Gly 185 Asn  
 Ala 191 Pro  
 Gly 193 Ala  
 Gly 199 Pro  
 Ala 200 Gly  
 Gly 202 Thr  
 Gly 219 Glu  
 Lys 220 Arg  
 His 221 Ala  
 Gly 223 Ala  
 Ser 224 Pro  
 Lys 227 Arg  
 Gly 238 Ala  
 Gly 239 Gln  
 Ala 242 Gly  
 Gly 260 Glu  
 Pro 267 Arg  
 Gly 268 Glu  
 Gly 286 Ala  
 Gly 286 Asp  
 Gln 289 Arg  
 Met 298 Lys  
 His 300 Pro  
 subject to the proviso that the following ion pairs must both be substituted:  
                                                 Ala 15 Arg   and   Asn 36 Asp         Thr 17 Asp   and   Met 298 Lys         Ala 95 Asp   and   Ser 128 Arg         Pro 153 Asp   and   Gln 156 Arg         Lys 227 Arg   and   Gly 268 Arg         Gly 152 Thr   and   His 221 Ala.                                                
 
     
     
         7 . A (1→3,1→4)-β-glucanase according to  claim 4  in which amino acids in the loops forming the sides and bottom of the active site cleft of (1→3)-β-glucanase GII are replaced by corresponding amino acids from (1→3,1→4)-β-glucanase EII, as follows:  
       
         
           
                 
                 
               
                     
                 
                     
                 
                   residue 
                   8 Ile → Ser, 
                 
                   residue 
                   34 Phe → Ala, 
                 
                   residue 
                   208 Ala → Thr, 
                 
                   residue 
                   209 Met → Thr, 
                 
                   residue 
                   189-191 Gln-Pro-Gly → Asn-Ala-Ser 
                 
                   residue 
                   128-137 Ile-Arg-Phe-Asp-Glu-Val-Ala-Asn-Ser-Phe → Val-Ser- 
                 
                     
                   Gln-Ala-Ile-Leu-Gly-Val-Phe-Ser (SEQ. ID NO: 1), 
                 
                     
                 
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
                
               
            
           
         
         residue 171-179 Phe-Ala-Tyr-Arg-Asp-Asn-Pro-Gly-Ser→Leu-Ala-Trp-Ala-Tyr-Asn-Pro-Ser-Ala (SEQ. ID NO: 2) and  
         residue 283-291 Thr-Gly-Asp-Ala-Thr-Glu-Arg-Ser-Phe→Asp-Ser-Gly-Val-Glu-Gln-Asn-Trp (SEQ. ID NO: 3)  
       
     
     
         8 . A (1→3,1→4)-β-glucanase according to  claim 6  comprising one or more of the following substitutions: 
 Gly 53 Asp  
 Gly 53 Glu  
 Thr 17 Asp; Met 298 Lys  
 Ala 95 Asp; Ser 128 Arg  
 Lys 122 Arg  
 Lys 23 Arg  
 Lys 74 Arg  
 Gly 44 Arg  
 Gly 223 Ala  
 Ala 89 Pro  
 Phe 85 Tyr  
 
     
     
         9 . A (1→3,1→4)-β-glucanase according to any one of  claims 6  to  8 , additionally comprising the mutation 189-191 Gln-Pro-Gly-→Asn-Ala-Ser.  
     
     
         10 . A (1→3,1→4)-β-glucanase according to  claim 6  comprising the substitution Lys 122→Arg and/or the substitution Phe 85→Tyr.  
     
     
         11 . A DNA molecule whose sequence encodes a (1→3,1→4)-β-glucanase according to any one of  claims 1  to  10 .  
     
     
         12 . A plasmid comprising a DNA sequence according to  claim 11 .  
     
     
         13 . An expression vector comprising a DNA sequence according to  claim 11 .  
     
     
         14 . A transgenic plant comprising a DNA sequence according to  claim 11 .  
     
     
         15 . A transgenic plant according to  claim 14 , selected from the group consisting of barley, wheat, rice, and maize.  
     
     
         16 . A transgenic plant according to  claim 15 , which is barley.  
     
     
         17 . A process selected from the group consisting of malting, brewing and stockfeed processing, comprising the step of: 
 (a) using barley expressing the (1→3,1→4)-β-glucanase of any one of  claims 1  to  10  as a starting material; or    (b) adding the (1→3,1→4)-β-glucanase of any one of  claims 1  to  10  to a grain to be processed.    
     
     
         18 . A composition for use in malting, brewing, or stockfeed processing, comprising the (1→3,1→4)-β-glucanase of any one of  claims 1  to  10 , together with a carrier acceptable for use in processing of beverages or of stockfeeds.  
     
     
         19 . A beverage produced using a composition according to  claim 18 .  
     
     
         20 . A stockfeed produced using a composition according to  claim 18 .  
     
     
         21 . Grain produced by a transgenic plant according to  claim 15 .  
     
     
         22 . Barley grain produced by a transgenic barley according to  claim 16.