Enzymatic semisynthesis of [LeuB30] insulin.
Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.
D000421:Alcaligenes / Q000201:enzymology; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D007202:Indicators and Reagents; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D007700:Kinetics; D012697:Serine Endopeptidases; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D007202:Indicators and Reagents; D007328:Insulin; C030314:insulin, Leu(B30)-; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; C036813:lysyl endopeptidase