1
ISSN 0027-1314, Moscow University Chemistry Bulletin, 2018, Vol. 73, No. 1, pp. 1–6. © Allerton Press, Inc., 2018.
Published in Russian in Vestnik Moskovskogo Universiteta, Seriya 2: Khimiya, 2018, No. 2, pp. 70–77.
Rational Design of Practically Important Enzymes
V. I. Tishkov
a, b, c,
*, A. A. Pometun
b, c
, A. V. Stepashkina
b, c
, V. V. Fedorchuk
a, c
, S. A. Zarubina
a, c
,
I. S. Kargov
a, b, c
, D. L. Atroshenko
a, b
, P. D. Parshin
a, b
, M. D. Shelomov
a, b
, R. P. Kovalevski
a, b
,
K. M. Boiko
c
, M. A. Eldarov
c
, E. D’Oronzo
d
, S. Facheris
d
, F. Secundo
d
, and S. S. Savin
a, b
a
Department of Chemistry, Moscow State University, Moscow, 119991 Russia
b
Innovations and High Technologies MSU Ltd, Moscow, 109559 Russia
c
Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology”
of the Russian Academy of Sciences, Moscow, 119071 Russia
d
Istituto di Chimica del Riconoscimento Molecolare, CNR, Milan, 20131 Italy
*e-mail: vitishkov@gmail.com
Received January 9, 2018
Abstract⎯Majority of native enzymes are poorly applicable for practical usage: that is why different methods
of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of
genome sequencing and various modern approaches in protein engineering allow one to identify protein of
interest and to improve the enzyme properties for a particular process. This review describes the results on
development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate
dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharo-
myces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively),
have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to
have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as
good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of
SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH)
yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability.
New generation of PseFDH preparations with the coenzyme specificity changed from NAD
+
to NADP
+
have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type
recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase
(DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino
acid replacements have been shown to exert an additive effect, improving both kinetic parameters and
increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned.
α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Struc-
tural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The struc-
ture of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants
of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and character-
ized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.
Keywords: formate dehydrogenase, D-amino acid oxidase, penicillin acylase, α-amino acid ester hydrolase,
rational design, kinetic properties, stability
DOI: 10.3103/S0027131418020153
INTRODUCTION
The absolute number of enzyme-based biocatalysts
is currently produced by means of genetic engineering.
Highly productive recombinant strains – super-pro-
ducers of target enzymes – are required to lower the
production costs. In addition, the directed improve-
ments of the properties of natural enzymes have to be
preliminary performed to optimize the process under
development.
In this laboratory we carry out systematic studies of
practically important enzymes by means of gene clon-
ing and protein engineering. This review summarizes
the recent achievements in design of novel forms of for-
mate dehydrogenase, D-amino acid oxidase, α-amino
acid ester hydrolase, and penicillin acylase.
Formate Dehydrogenase
Cloning and protein engineering of formate dehy-
drogenase (FDH, EC 1.2.1.2) from various sources are
described in our reviews published in 2004, 2006 and
2011 [1–3]. Formate dehydrogenase genes from bac-
teria Pseudomonas sp.101 (PseFDH) and Mycobacte-
rium vaccae N10 (MycFDH), yeast Candida boidinii
@Phil_Robichaud
@deepthiw
@JoseServera