Immobilized cycloaliphatic peptide acyltransferase and preparation method and uses thereof

Abstract

Disclosed in the present invention are an immobilized cycloaliphatic peptide acyltransferase and a preparation method and use thereof. The cycloaliphatic peptide acyltransferase is immobilized on a carrier; the cycloaliphatic peptide acyltransferase is derived from natural or artificial mutants or variants thereof, or can be obtained by introducing a foreign cyclic acyltransferase gene and transforming thereafter; the material of the carrier is selected from an inorganic carrier or a polypropylene resin carrier. Also disclosed in the present invention are the preparation method for the immobilized cycloaliphatic peptide acyltransferase and uses thereof.

1 . An immobilized cycloaliphatic peptide acyltransferase. 
     
     
         2 . The immobilized cycloaliphatic peptide acyltransferase according to  claim 1 , wherein cycloaliphatic peptide acyltransferase is immobilized on a carrier; and the carrier is selected from inorganic carrier, or porous, hydrophilic enzyme carrier. 
     
     
         3 . The immobilized cycloaliphatic peptide acyltransferase according to  claim 2 , wherein, based on the total weight of the inorganic carrier, the content of SiO 2  is more than 50 wt %, and the content of Al 2 O 3  is more than 1 wt %. 
     
     
         4 . The immobilized cycloaliphatic peptide acyltransferase according to  claim 2 , wherein the porous hydrophilic enzyme carrier is selected from the following: a enzyme-carrier in which polymethacrylate is used as matrix with bonded epoxide or containing amino functional groups. 
     
     
         5 . The immobilized cycloaliphatic peptide acyltransferase according to  claim 1 , wherein the immobilized cycloaliphatic peptide acyltransferase is used to catalyze deacylation of acyl of R′ in the compound of formula I, thereby obtaining the compound of formula II, 
       
         
           
           
               
               
           
         
         wherein R 1  is an acyl, R 2  is a hydroxy or acyloxy, R 3  is a hydrogen or hydroxy, R 4  is a hydrogen or hydroxy, R 5  is a hydrogen or a hydroxy sulfonyloxy, and R 6  is a hydrogen or carbamoyl. 
       
     
     
         6 . The immobilized cycloaliphatic peptide acyltransferase according to  claim 1 , wherein the compound of formula I is the compound of formula Ia or Ib or a pharmaceutically acceptable salt thereof: 
       
         
           
           
               
               
           
         
       
     
     
         7 . A method for preparing the immobilized cycloaliphatic peptide acyltransferase according to  claim 1 , comprising the following steps:
 mixing a solution containing free cycloaliphatic peptide acyltransferase with a carrier to obtain the immobilized cycloaliphatic peptide acyltransferase according to  claim 1 .   
     
     
         8 . The method according to  claim 7 , wherein said cycloaliphatic peptide acyltransferase is derived from natural or artificial mutants, or variants thereof and can be obtained through transformation by introducing foreign genes of cycloaliphatic peptide acyltransferase. 
     
     
         9 . The method according to  claim 7 , wherein the solution containing free cycloaliphatic peptide acyltransferase is obtained from the following manner: culturing a strain, so as to obtain the solution containing free cycloaliphatic peptide acyltransferase; or culturing the strain, and then breaking the cell wall of obtained hyphae, so as to obtain the solution containing free cycloaliphatic peptide acyltransferase. 
     
     
         10 . The method according to  claim 9 , wherein the strain belongs to  Actinoplanes  or  Streptomyces.   
     
     
         11 . The method according to  claim 7 , wherein the carrier is selected from inorganic carriers, or porous, hydrophilic enzyme carriers. 
     
     
         12 . The method according to  claim 7 , wherein the mixing ratio of the free cycloaliphatic peptide acyltransferase to the carrier is 10-1000 unites (u), preferably 20-600 units (u) of enzyme vs one gram of carrier. 
     
     
         13 . The method according to  claim 7 , wherein the pH value of the solution containing free cycloaliphatic peptide acyltransferase is 4-9. 
     
     
         14 . The method according to  claim 7 , wherein the temperature for mixing the solution containing free cycloaliphatic peptide acyltransferase with the carrier is 0-80° C.; preferably, 20-35° C. 
     
     
         15 . The method according to  claim 7 , wherein after mixing the solution containing free cycloaliphatic peptide acyltransferase with the carrier, the free cycloaliphatic peptide acyltransferase is separated from the cycloaliphatic peptide acyltransferase binding to the carrier, so as to obtain the immobilized cycloaliphatic peptide acyltransferase. 
     
     
         16 . A method for preparing the compound of formula II, comprising the following steps:
 (a) mixing the compound of formula I with a buffer solution, so as to obtain solution 1;   (b) mixing solution 1 with the immobilized cycloaliphatic peptide acyltransferase according to  claim 1  for conducting deacylation reaction, so as to give the compound of formula II;   
       
         
           
           
               
               
           
         
         wherein R 1  is an acyl, R 2  is a hydroxy or acyloxy, R 3  is a hydrogen or hydroxy, R 4  is a hydrogen or hydroxy, R 5  is a hydrogen or a hydroxy sulfonyloxy, and R 6  is a hydrogen or carbamoyl. 
       
     
     
         17 . The method according to  claim 16 , wherein the ratio of the immobilized cycloaliphatic peptide acyltransferase to the compound of formula I is 0.01-10 u/g. 
     
     
         18 . The method according to  claim 16 , wherein the pH value of the buffer in step (a) is 4-9. 
     
     
         19 . The method according to  claim 16 , wherein the buffer in step (a) is selected from one or more of the group consisting of sodium citrate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer and Tris-HCl buffer. 
     
     
         20 . The method according to  claim 16 , wherein the temperature for deacylation reaction in step (b) is 20-70° C. 
     
     
         21 . The method according to  claim 16 , wherein, in step (b), upon deacylation, the immobilized cycloaliphatic peptide acyltransferase is separated from the product-containing reaction solution to give the compound of formula II. 
     
     
         22 . The method according to  claim 21 , wherein the method for separating the immobilized cycloaliphatic peptide acyltransferase from the product-containing reaction solution includes filtration or centrifugation.