Influence of temperature on the enzymic semisynthesis of human insulin by coupling and transpeptidation methods

Abstract

The influence of temperature of enzymic semisynthesis of human insulin ester was determined by using coupling and transpeptidation methods with trypsin and Achromobacter lyticus proteinase I as catalysts. The optimal reaction conditions were studied at the selected temperatures of 25, 12 and 4 degrees C. The results showed that the synthesis rates by both methods with trypsin increased as the temperature increased, but the final product yield correspondingly decreased. Therefore the reaction with trypsin should be done below 12 degrees C, preferably at 4 degrees C. This agrees well with the stability of trypsin at these temperatures. When the catalyst was Achromobacter lyticus proteinase I, no such complex temperature effects were observed, and the findings indicated that the reactions should be conducted below 37 degrees C for enzyme stability.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

“…Briefly, the initial capture of the IP precursor from cell-free supernatant was carried out by ion exchange chromatography to concentrate the IP. The IP was then treated with recombinant bovine trypsin expressed in P. pastoris [15] followed by transpeptidation [16] to produce two-chain functional insulin. The RP-HPLC step was specially developed and employed [17] to remove the glycosylated, product-related impurities.…”

A Simplified and Efficient Process for Insulin Production in Pichia pastoris

“…The insulin precursor was converted enzymatically into insulin ester via transpeptidation [18–20] in the presence of trypsin and an excess of O-t-butyl-L-threonine t-butyl ester (H-Thr(tBu)-OtBu) acetate salt. Trypsin mediates cleavage of the N-terminal spacer peptide and the Ala-Ala-Lys connecting linker peptide and the transesterification reaction where threonine ester is added to B-29 residue as previously described [6].…”

Two‐step transpeptidation of the insulin precursor expressed in Pichia pastoris to insulin ester via trypsin‐catalyzed cleavage and coupling

“…Low‐water content in the conversion reaction mixture was critical in achieving the high conversion ratio of the transpeptidation reaction. The addition of an aprotic organic solvent, such as 1,4‐butanediol, DMSO, or ( N , N )‐dimethyl acetamide was very helpful, and the pH value should be neutral or weakly acidic . However, high concentrations of organic solvent usually inhibited or reduced trypsin activity.…”