Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina

Pugnière, Martine; Skalli, A.; Ma, Che; Previero, A.

doi:10.1002/prot.340010205

Cited by 19 publications

(1 citation statement)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…reverse micelles (Lfithi and Luisi 1984), powdered- (Klibanov 1986), immobilized- (Pugniere et al 1986;Reslow et al 1987) or PEG-modified enzyme. Thus, peptide synthesis catalysed by subtilisin as a suspended powder, adsorbed on celite, and modified with PEG was carried out in 1,1,1-trichloroethane under comparable enzyme hydration conditions.…”

Section: Introductionmentioning

confidence: 99%

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Ferjancic¹,

Puigserver²,

Gaertner³

1990

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Subtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents. The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions. The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch. Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture. The high affinity of benzyloxycarbonyl-L-serine-methyl ester as compared to benzyloxycarbonyl-L-phenylalanine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase. The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-L-serine-L-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents. This also resulted in a lower stability of PEG-subtilisin at high temperature.

show abstract

Section: Introductionmentioning

confidence: 99%

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Ferjancic¹,

Puigserver²,

Gaertner³

1990

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

Protein immobilization to alumina supports: I. Characterization of alumina‐organophosphate ligand interactions and use in the attachment of papain

Hyndman

Flynn

Lever³

et al. 1992

Biotech & Bioengineering

View full text Add to dashboard Cite

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support.

show abstract

Protein immobilization to alumina supports: II. Papain immobilization to alumina via organophosphate linkers

Hyndman

Burrell²,

Lever³

et al. 1992

Biotech & Bioengineering

View full text Add to dashboard Cite

Particulate aluminum oxides (alumina) were examined as supports for the immobilization of the proteolytic enzyme papain. Two alumina supports termed C1 and CPC were derivatized using organic phosphate linkers to create free carboxyl groups using a two-step process. Papain binding to these derivatized aluminas was performed using the water soluble carbodiimide 1-ethyl-3-(dimethylaminopropyl) carbodiimide. Reactions were optimal at 10 mM carbodiimide. The immobilized protein showed similar kinetic constants when compared to the solution protein. The pH dependence and thermal stability were essentially identical. The immobilized papain showed a blue shift in the intrinsic fluorescence emission maxima. Papain modified with the active site-specific fluorescent probe acrylodan showed overlapping emission maxima. These results are interpreted as retention of the hydrophobic environment of the active site with a perturbation in the structure of the rest of the protein caused by its association with the negatively charged surface.

show abstract

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina

Cited by 19 publications

References 13 publications

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Protein immobilization to alumina supports: I. Characterization of alumina‐organophosphate ligand interactions and use in the attachment of papain

Protein immobilization to alumina supports: II. Papain immobilization to alumina via organophosphate linkers

Contact Info

Product

Resources

About