Enzymatic catalysis / Protein characterization

Pier carlo Montecucchi pcmontecucchi at compuserve.com
Sat Feb 17 15:21:56 EST 2001

TO: Dima Klenchin
FROM: Pier Carlo Montecucchi

Dear Dima:

Please find enclosed some general information about the instrument

(i)   Bioconversion of charged  penicillin G by penicillin G acylase
(ii)  Histidine decarboxylase reactor
(iii) Production of pure D-phenylglycine by penicillin G Acylase
(iv) Thrombin reactor [Thrombin digestion of Gst-fusion protein (Gst-Tat)
and subsequent purification of cleaved products
Tat, released upon thrombin hydrolysis, is harvested in a pure form in the
pH region 9 to 11; Gst in the pH region  <5
 (v) Trypsin reactor
 The tryptic digestion of a milk beta-casein releases the peptides with
different  biological activities
either directly [peptide beta(1-25) and beta (33-48) with anticariogenic
activity and peptide beta (114 - 169)]
or indirectly as a precursor [peptide beta (49-97) containing in its
sequence peptide beta (60-66) with casomorphine activity and peptide beta
(63-68) with antihypertensive activity]
(vi) Urease reactor
(vii) Purification of chicken liver basic FABS (fatty acid binding protein)

1) Isoelectric focusing: Immobilized pH Gradients:Theory and Methodology by
P.G. Righetti (1990), Elsevier, Amsterdam
2) Continuous enzymatic hydrolysis of beta-casein and isoelectric collection
of some of the biologically active peptides in an electric field by P.G.
Righetti, F. Nembri, A. Bossi and M. Mortarino, in: Biotechnol. Prog. (1997)
13 (3), 258 - 264
3) Isoelectrically trapped enzymatic bioreactors in a multimembrane cell
coupled to an electric field: theoretical modelling and experimental
validation with urease by F. Nembri, A. Bossi, S. Ermakov and P.G. Righetti,
in: Biotechnol. Bioengineer. (1997) 53, 110 - 119
4) Production of D-phenylglycine from racemic (D,L)-phenylglycine via
"isoelectrically trapped" penicillin G acylase by A. Bossi, M. Cretich and
P.G. Righetti, in: Biotechnol. Bioengineer. (1998) 60, 454 - 461
5) Electrically immobilized enzyme reactors: bioconversion of a charged
substrate: hydrolysis of penicillin G acylase by A. Bossi, S. Guerrera, P.G.
Righetti, in: Biotechnol. Bioengineer.(1998) 64, 383 - 391
6) Analysis of cross-linked human hemoglobin by conventional isoelectric
focusing, immobilized pH gradients, capillary electrophoresis, and mass
spectrometry by A. Bossi, M.J. Patel, E.J. Webb, M.A. Baldwin, R.J. Jacob,
A.L. Burlingame, P.G. Righetti, in: Electrophoresis (1999), 20, 2810 - 2817
7) Crystallization of chicken liver (basic) fatty acid binding protein after
purification in multicompartment electrolyzers with isoelectric membranes by
M. Parduca, A Bossi, L. Goldoni, Hugo L. Monaco, P.G. Righetti, in:
Electrophoresis (2000), 21, 0000 - 0000

In particular:

<< What if substrate or product happen to have pI close to that of the

It is possible to obtain tthe separation of isoelectric and isoionic protein
products with pIs that differ by 0.001 pH umits.


Pier Carlo Montecucchi

PS. Your E-mail address has problems.

"Dima Klenchin" <klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu> wrote in
message news:96htgi$2fc_006 at doit.wisc.edu...
> "Pier carlo Montecucchi" <pcmontecucchi at compuserve.com> wrote:
> :If you are working in the area of enzymatic catalysis / protein
> :characterization, probably you could have some interest to know the
> :potentialities of instrument M.I.E.R. (Multicompartment Immobilized
> :Reactor), an isoelectrically trapped enzyme reactor that integrates
> :catalytic conversion, product separation, product concentration and
> :recovery into a single operation. The instrument has been developed by
> :Italian company Talent (Trieste) and it is commercialized in U.S.A. by
> :Montegen.
> :
> :The instrument is modular and it is built to comprise 8 flow-through
> :(recycling) chambers.: one anodic, one cathodic, one for the enzyme
> :anfd five chambers for collecting any desired product of the enzyme
> :reaction, or cofactor or other material escaping the enzyme reaction
> :chamber.
> :The enzyme reaction operates under an electric field , which is used to
> :the enzyme confined inton the reaction chamber (via an isoelectric
> :mechanism) and to drive in and out of the reaction chamber any charged
> :substrate, product or cofactor.
> I can see several problems with such arrangement:
> 1. A lot of proteins are extremely unhappy at pH ~ pI
> 2. An optimal reaction pH might be very far from pI
> 3. What if substrate or product happen to have pI close
> to that of the enzyme?
>         - Dima

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