Method for modifying transglutaminases from microorganisms

Abstract

The present invention relates to a method for designing and preparing mutant transglutaminases on the basis of the three-dimensional structure of MTG derived from Streptoverticillium mobaraense (MTG), and the mutant MTG thus prepared. The present invention provides a method for modifying MTG on the basis of the three-dimensional structure, and transglutaminase having reactivity on the substrate improved by the method. In the present invention, the binding site of MTG for the substrate is extrapolated based on the three-dimensional structure obtained by X-ray crystal structure analysis of MTG crystals, and the mutant transglutaminases are designed and produced by replacing, inserting or deleting amino acid residues positioned at the substrate-binding site of the transglutaminase.

What is claimed is:  
     
         1 . A method for designing and preparing a mutant transglutaminase by extrapolating the binding site of a transglutaminase derived from  Streptoverticillium mobaraense  (MTG) for the substrate on the basis of the three-dimensional structure obtained by X-ray crystal structure analysis and replacing, inserting or deleting amino acid residues positioned at the extrapolated substrate-binding site of the transglutaminase.  
     
     
         2 . A mutant MTG (excluding a mutant MTG wherein Asp 1 is deleted) having modified substrate specificity, in which at least one of the following positions (residues positioned within 20 Å from active residue Cys 64  and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequence according to SEQ ID No: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296,-297 and 300-304.  
     
     
         3 . A mutant transglutaminase having transglutaminase activity and modified substrate specificity, wherein when the alignment of the sequence of said mutant transglutaminase with the MTG amino acid sequence according to SEQ ID NO: 2 is carried out, at least one of the positions corresponding to the following positions (residues positioned within 20 Å from active residue Cys 64  and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequence according to SEQ ID No: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296-297 and 300-304.  
     
     
         4 . A mutant transglutaminase having transglutaminase activity and modified substrate specificity, wherein when the alignment of the three-dimensional structure with the MTG amino acid sequence according to SEQ ID NO: 2 is carried out by treading method, at least one of the positions corresponding to the following positions (residues positioned within 20 Å from active residue Cys 64  and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequences according to SEQ ID No: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296-297 and 300-304.  
     
     
         5 . A mutant MTG (excluding a mutant MTG from which Asp 1  is deleted) or mutant transglutaminase having modified substrate specificity, wherein a negative charge is reduced by modifying an acidic amino acid residue or an amino acid residue adjacent to the acidic amino acid located at the position described in any one of  claims 2  to  4   
     
     
         6 . A gene encoding the mutant MTG or the mutant transglutaminase according to any one of  claims 2  to  4 .  
     
     
         7 . A recombinant DNA containing the gene according to  claim 6 .  
     
     
         8 . A microorganisms containing the recombinant DNA according to  claim 7 .  
     
     
         9 . A method for producing a mutant MTG or a mutant transglutaminase, which comprises culturing the microorganisms according to  claim 8  and collecting the mutant MTG or the mutant transglutaminase.  
     
     
         10 . A monoclinic MTG crystals belonging to P2 1  space group.