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.
Porcine insulin can be enzymatically converted into human insulin by two different methods. In condensation reactions catalyzed by trypsin' and protease I from Achromobacter Zyticus, L-threonine tert-butyl ester can be coupled to desalanine (B-30)-porcine insulin (DAI) to obtain an ester derivative of human insulin. In the other method, the terminal alanine residue of the B chain in porcine insulin is exchanged for amides and esters of L-threonine in a single transpeptidation step catalyzed by either t r y p~i n ,~.~ carboxypeptidase Y,' or Achromobacter protease I.6 The protected insulin derivative obtained from the enzymecatalyzed reactions is subsequently deblocked in a one-step chemical procedure which yields human insulin.For efficient use of enzymes in industry, continuous operation of the enzyme-catalyzed process is desirable. This can be achieved by using immobilized enzymes, which are known to be stable, reusable, and also easily recoverable.Achromobacter protease I attached to polyglutamic-acid-SiOz can be used for semisynthesis of human insulin ester by coupling DAI with T~~-O B U ' .~, 'Several attempts at semisynthesis at 37°C have been made using immobilized trypsin prepared by various procedures, but all were unsu~cessful.~ Recent work' has shown that the semisynthesis of human insulin ester by trypsin should be done below 12°C because of the enzyme instability. Therefore, the previous failure of the semisynthesis with trypsin might have been due to the reaction temperature of 37°C or room temperature. Thus, the present study was undertaken to investigate whether the immobilized trypsin which is commercially available can be used for semisynthesis of human insulin ester at 4°C or not. MATERIALS AND METHODSPorcine Zn-insulin (crystals, 25 U/mg) was obtained from Calbiochem-Behring (Los Angeles, CA). Benzoyl-Larginine ethyl ester (BAEE) was obtained from Protein Research Foundation, (Minoh, Osaka, Japan). Immobilized trypsin (T-4019, TPCK-treated, attached to beaded agarose, 80 U/mL packed gel), carboxypeptidase A (type *To whom correspondence should be addressed. Thr-OBu' was prepared according to the usual chemical method.'0," The DAI was prepared by digestion of porcine insulin with carboxypeptidase A by a modification of the method of Schmitt and Gattner." The DAI content of the preparation was determined to be more than 90% from the amino acid analysis.The enzymatic activity of immobilized trypsin was determined using BAEE as substrate. Esterase activity was measured by the formation of hydroxamic acid-ferric complex from the residual ~ubstrate.'~ The reaction mixture contained 0.5 mL of lOmM substrate in 50mM phosphate buffer (pH 7.4) and 0.5 mL enzyme suspension containing a suitable amount of packed gel (immobilized trypsin) in 50mM phosphate buffer (pH 7.4) was incubated at 37°C with stimng. After 30 min reaction, 1.5 mL of a mixture of equal volumes of 3.5M sodium hydroxide and 2M NH,OH . HCl were added to the reaction mixture to stop further hydrolysis, and the reaction mixture was kept ...
The synthetic proteinase inhibitor, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), strongly suppressed activation of Clr at 37"C, causing 50% inhibition at 0.03 mM. To clarify whether the inhibitor was incorporated into the active site of intermediary Clr formed during the incubation, determination of the active site was tried using this inhibitor. Consequently, release of amidinonaphthol equimolar with the amount of Clr used was observed in the early period of incubation, in which the activation to Clrwas about 5%. These results indicate that intermediary Clr already has a complete active site.
It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobucter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.
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