MODIFIED ß-GALACTOSIDASE

Abstract

The usefulness of β-galactosidases derived from Bacillus circulans is further enhanced. A modified β-galactosidase in which one or more amino acids selected from the group consisting of proline 182 (P182), tyrosine 187 (Y187), serine 188 (S188), tryptophan 405 (W405), alanine 406 (A406), glutamine 407 (Q407), tyrosine 449 (Y449), threonine 483 (T483), serine 512 (S512), serine 531 (S531), threonine 533 (T533), serine 534 (S534), asparagine 550 (N550), glutamine 551 (Q551), tryptophan 593 (W593), tyrosine 598 (Y598), proline 602 (P602), proline 604 (P604), tyrosine 609 (Y609), lysine 612 (K612), and tyrosine 615 (Y615), or an amino acid(s) corresponding thereto, has/have been substituted by other amino acid in a β-galactosidase consisting of the amino acid sequence of any of SEQ ID NOs. 1 to 4 or an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any of SEQ ID NOs. 1 to 4.

1 - 27 . (canceled) 
     
     
         28 . A modified β-galactosidase consisting of an amino acid sequence that has at least 98% sequence identity to the amino acid sequence of SEQ ID NO. 1, 2, 3, or 4, wherein the modified β-galactosidase includes one or more amino acid substitutions selected from the group consisting of proline at position 182 (P182), serine at position 188 (S188),tryptophan at position 405 (W405), glutamine at position 407 (Q407), tyrosine at position 449 (Y449), threonine at position 483 (T483), serine at position 531 (S531), asparagine at position 550 (N550), proline at position 602 (P602), proline at position 604 (P604), tryptophan at position 85 (W85), phenylalanine at position 91 (F91), tyrosine at position 103 (Y103), tyrosine at position 134 (Y134), tyrosine at position 153 (Y153), proline at position 182 (P182), tryptophan at position 186 (W186), tyrosine at position 192 (Y192), tyrosine at position 295 (Y295), histidine at position 345 (H345), glycine at position 349 (G349), phenylalanine at position 402 (F402), glutamine at position 407 (Q407), glycine at position 479 (G479), glutamic acid at position 480 (E480), lysine at position 482 (K482), threonine at position 483 (T483), arginine at position 484 (R484), glycine at position 485 (G485), valine at position 488 (V488), tyrosine at position 496 (Y496), asparagine at position 514 (N514), asparagine at position 515 (N515), tyrosine at position 516 (Y516), aspartic acid at position 517 (D517), tyrosine at position 519 (Y519), threonine at position 533 (T533), tyrosine at position 547 (Y547), tyrosine at position 549 (Y549), tyrosine at position 555 (Y555), threonine at position 573 (T573), tyrosine at position 598 (Y598), tyrosine at position 605 (Y605), tyrosine at position 606 (Y606), tyrosine at position 609 (Y609), phenylalanine at position 616 (F616), phenylalanine at position 624 (F624), tryptophan at position 648 (W648), and tryptophan at position 650 (W650), or an amino acid(s) corresponding thereto, relative to SEQ ID NO. 1, 2, 3, or 4, wherein the modified β-galactosidase produces oligosaccharides in which the content of trisaccharides and higher saccharides is approximately equal and the content of tetrasaccharides is smaller, relative to the case of the parent wild-type enzyme. 
     
     
         29 . The modified β-galactosidase according to  claim 28 , wherein the amino acid substitution for proline at position 182 (P182) is leucine; the amino acid substitution for serine at position 188 (S188) is isoleucine; the amino acid substitution for tryptophan at position 405 (W405) is alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, asparagine, serine, or threonine; the amino acid for glutamine at position 407 (Q407) is glycine or arginine; the amino acid substitution for tyrosine at position 449 (Y449) is isoleucine, glutamine, arginine, or serine; the amino acid substitution for threonine at position 483 (T483) is glutamine; the amino acid substitution for serine at position 512 (S512) is phenylalanine, threonine, or valine; the amino acid substitution for serine at position 531 (S531) is proline; the amino acid substitution for asparagine at position 550 (N550) is leucine, arginine, or serine; the amino acid substitution for proline at position 602 (P602) is leucine, asparagine, or glutamine; the amino acid substitution for tryptophan at position 85 (W85) is glycine; the amino acid substitution for phenylalanine at position 91 (F91) is alanine or isoleucine; the amino acid substitution for tyrosine at position 103 (Y103) is glycine; the amino acid substitution for tyrosine at position 134 (Y134) is glycine; the amino acid substitution for tyrosine at position 153 (Y153) is glycine; the amino acid substitution for proline at position 182 (P182) is tryptophan or tyrosine; the amino acid substitution for tryptophan at position 186 (W186) is methionine; the amino acid substitution for tyrosine at position 192 (Y192) is glycine; the amino acid substitution for tyrosine at position 295 (Y295) is glycine; the amino acid substitution for histidine at position 345 (H345) is glycine; the amino acid substitution for glycine at position 349 (G349) is tryptophan; the amino acid substitution for phenylalanine at position 402 (F402) is glycine; the amino acid substitution for glutamine at position 407 (Q407) is isoleucine, leucine, methionine, valine, or tyrosine; the amino acid substitution for glycine at position 479 (G479) is leucine, proline, or glutamine; the amino acid substitution for glutamic acid at position 480 (E480) is asparagine; the amino acid substitution for lysine at position 482 (K482) is methionine or valine; the amino acid substitution for threonine at position 483 (T483) is glycine or serine; the amino acid substitution for arginine at position 484 (R484) is phenylalanine, isoleucine, methionine, asparagine, or glutamine; the amino acid substitution for glycine at position 485 (G485) is aspartic acid, asparagine, glutamine, or threonine; the amino acid substitution for valine at position 488 (V488) is glycine, isoleucine, leucine, arginine, serine, threonine, tryptophan, or tyrosine; the amino acid substitution for tyrosine at position 496 (Y496) is glycine; the amino acid substitution for asparagine at position 514 (N514) is glutamic acid, leucine, or lysine; the amino acid substitution for asparagine at position 515 (N515) is alanine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, proline, or valine; the amino acid substitution for tyrosine at position 516 (Y516) is tryptophan; the amino acid substitution for aspartic acid at position 517 (D517) is lysine or tryptophan; the amino acid substitution for tyrosine at position 519 (Y519) is glycine; the amino acid substitution for threonine at position 533 (T533) is glutamic acid; the amino acid substitution for tyrosine at position 547 (Y547) is glycine; the amino acid substitution for tyrosine at position 549 (Y549) is glycine; the amino acid substitution for tyrosine at position 555 (Y555) is glycine; the amino acid substitution for threonine at position 573 (T573) is glutamic acid; the amino acid substitution for tyrosine at position 598 (Y598) is histidine or asparagine; the amino acid substitution for tyrosine at position 605 (Y605) is cysteine, aspartic acid, histidine, isoleucine, lysine, proline, arginine, or threonine; the amino acid substitution for tyrosine at position 606 (Y606) is alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, or serine; the amino acid substitution for tyrosine at position 609 (Y609) is glutamic acid, asparagine, or serine; the amino acid substitution for phenylalanine at position 616 (F616) is glycine; the amino acid substitution for phenylalanine at position 624 (F624) is glycine; the amino acid substitution for tryptophan at position 648 (W648) is glycine; and the amino acid substitution for tryptophan at position 650 (W650) is glycine. 
     
     
         30 . A polynucleotide encoding the modified β-galactosidase of  claim 28 . 
     
     
         31 . A recombinant DNA comprising the polynucleotide of  claim 30 . 
     
     
         32 . A microorganism carrying the recombinant DNA of  claim 31 . 
     
     
         33 . An enzyme preparation comprising the modified β-galactosidase of  claim 28 . 
     
     
         34 . A method for producing an oligosaccharide, the method comprising contacting the modified β-galactosidase of  claim 28  with a disaccharide, an oligosaccharide, or a polysaccharide having at least one of β-1,3-bond, β-1,4-bond, β-1,6-bond, or a combination thereof.