„Babeș-Bolyai” University of Cluj Napoca

Faculty of Chemistry and Chemical Engineering

Enzymatic and cellular catalysis

in organic synthesis
from Nature to Industry through Lab scale

Professor Dr. ing. Florin Dan IRIMIE

http://www.chem.ubbcluj.ro/BIO/CENTRU/index.php

Riobamba, October 29, 2018

International Congress
BIOCATALYST:
”An enzyme or enzyme complex
consisting of, or derived from, an
organism or cell culture (in cell-free
or whole-cell forms) that catalyses
metabolic reactions in living organisms
and/or substrate conversions in
various chemical reactions”.
IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold
Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell
Scientific Publications, Oxford (1997)
 Biocatalysts

Origin: Degree of
Solubility:
purification:

- Microbian - Pure enzymes - Soluble

- Enzyme complexes - Immobilized
- Vegetal
- Cell organelles
- Animal
- Whole cells
 Why biocatalysis ?

80 % of industrial chemical processes are catalytic

Options:
1. chemocatalysis (homogenous – heterogenous)
2. biocatalysis
 Enzymes
• Natural substances, protein in their nature,
with a biocatalytic role in reactions that
occur in an organism or outside of it

– Subject to natural evolution

– general characteristics:
• Increase reaction rate, by lowering the
activation energy
• Does not change the equilibrium constant
values
• Participate in catalytic amounts
– specific characteristics :
• Activity, selectivity, environmental
compatibility
Chemoenzymatic synthesis of aspartame
Other practical uses of enzymatic steps in organic synthesis
 Enzymes
Enzymes codification
classification EC of IUB / IUPAC
EC of IUB / IUPAC
• A.B.C.D.
• Oxidoreductases
– Redox reactions
• Transferases
Serial number
– Group transfer reactions
• Lyases Subsubclass
• Addition/Elimination reactions
Subclass
• Isomerases
– Isomerisation reactions Class
• Synthetases
– Condensations breaking reactions
 Alcohol dehydrogenase –
an oxidoreductase enzyme
• 1.1.1.1.
Serial number: 1
Subsubclass: 1 electron donor
Subclass: 1 electron acceptor
Class : 1. oxidoreductases

electron acceptor

electron donor
 NH2 HS HR
CONH2
N
N
O O O
N N P P N
O
O OH O OH
O
HO OH
HO OH

NADH (reduced form)

H H H
CONH2
-H+ -2e- CONH2

N +H + +2e-
N
R
R
NADH (freduced form) NAD+(oxidized form)
 Selectivity of biomolecules

Latin: seligo, seligis, selegi, selectum - to choose, to select

The quality of carefully choosing someone or

something as the best or most suitable:
(Oxford dictionary)

Ability of a biomolecule to discriminate among

neighbor molecules and to bind to one or some of
them through structural complementarity or
similarity
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/dna-structure.html
 • Selectivity of a biocatalyst means
Preference of biocatalyst:

– for a certain substrate

– for a certain place within a certain substrate

 𝑘𝑐𝑎𝑡
𝑣= 𝐸 [𝑆]
𝐾𝑆

𝑘𝑐𝑎𝑡
𝐾𝑚 𝑖
𝐸=
𝑘𝑐𝑎𝑡
σ
𝐾𝑚 𝑖
 Enzyme promiscuity
• Promiscuity:

• Ability of an enzyme to work:

• Work under unexpected conditions
• Work with unexpected substrates
• Catalyze unexpected reactions
SECONDARY ALCOHOLS
 Enzymatic kinetic resolutions

Racemic Pure enantiomer Pure enantiomer

General mechanism for serine
hydrolases
Kinetic resolution. The enantiomeric ratio (E) in an enzymatically
assisted process

 kcat  A
[A](t )  [B ](t ) et   ln  
v1  K M  A   A0 
[PA ](t )  [PB ](t ) si k1  k 2 ; E 
v 2  kcat  B 
[A](0)  [B ](0)   ln  
 KM B  B0 

ln[1  c (1  eep )] ln[1  c (1  ees )]

E sau E 
ln[1  c (1  eep )] ln[1  c (1  ees )]

[eep (1  ees )]
ln
(eep  ees )
E
[eep (1  ees )]
ln
(eep  ees )

[A]  [B ] [P ]  [PB ] [B ]  [A]

c 1 , eep  A şi ees 
[A0 ]  [B 0 ] [PA ]  [PB ] [B ]  [A]
 Exploiting the conservation of
enantiopeference of an enzyme
Stereoselective reduction mechanism of prochiral
ketones with NAD(P)-dependent oxidoreductases

K. Nakamura et al. / Tetrahedron: Asymmetry 14 (2003) 2659–2681

The mechanism of stereoselective reduction of prochiral
ketones with yeast alcohol dehydrogenase
Chemical stereoinversion
Mitsunobu reaction
 Chemical stereoinversion
Mitsunobu atypical reaction

Toşa, M.I., P.V. Podea, C. Paizs and F.D. Irimie, Chemoenzymatic synthesis of (R)-and (S)-1-heteroarylethanols. (2008). Tetrahedron:
Asymmetry, 19(17) p. 2068-2071.
SECONDARY AMINES
 Synthesis and biotransformations of the studied 1-(2-phenylthiazol-4-yl)ethanamines and ethanacetamides.
Reagents and conditions: I. CH3MgI, diethyl ether; II. (PhO)2PON3/toluene; III. Zn/NH4Cl, H2O/ ethanol; IV.
CH3(CH2)2COCl/DMAP/Pyridine/DCM; V. CaL-B/ ethyl n-butyrate/ACN; VI. CaL-B/H2O; VII. CaL-A/H2O.

Radu, A., Moisă, M. E., Toşa, M. I., Dima, N., Zaharia, V., & Irimie, F. D. (2014). Candida antarctica lipases acting as versatile catalysts for the
synthesis of enantiopure (R)-and (S)-1-(2-phenylthiazol-4-yl) ethanamines. Journal of Molecular Catalysis B: Enzymatic (107), 114-119
AMINOACIDS
 General strategy for chemoenzymatic stnthesis of highly
enantiopure a heteroaryl alanines by tandem use of lyases
 General strategy for chemoenzymatic stnthesis of highly
enantiopure (R)-b heteroaryl alanines by tandem use of mutases
and lyases

Chemosynthesis

(R)-β PAM (R)-β PAL / TAL

(R)-β
(S)-β (S)-α

Substituted
Acrylate
  Synthesis of (R)-β-arylalanines

Evolution of the enantiomeric excesses of the (R)-β-arylalanine

derivatives in time
A.

B.
Synthesis of the (R)-β-phenylalanine.
A. after 15 h, B. after 3 days, C. after 9
days

C.
 General strategy for chemoenzymatic stnthesis of highly
enantiopure (S)-b heteroaryl alanines by tandem use of lyases

From acrylate (via PAL)

PAM (S)-b
PAL
(S)-α (S)-b
(S)-α

acrylate
 Synthesis of (S)-β-arylalanines

A.

B.

C.

A. (S)-α-phenylalanine, starting material, B. Equilibrium between the α- and β-

phenylalanine, after 3 days, C. The remaining (S)-α-phenylalanine transformed
into acrylic acid in the presence of PAL, after 7 days
 Steps in finding a new Biocatalyst
If an organic reaction is thermodynamic allowed , with few exceptions it may be (bio)
assisted
Why ?
• Known products, new ways
• New products
Real needs evaluation:
• Estimate the real economic effects of introducing new biocatalyst
• Market size
How can one get ?
• Search
Where ?
• among known biocatalysts (commercial)
• among characterized cells
• among new cell findings, uncharacterised (extremophilles)
Modification of existing biocatalysts
How ?
•Rational approaches
•Evolutive approaches
“De novo” new biocatalysts building (Abzymes)
 Criteria for a biocatalyst selection:

• Have an economic quantification together with an environmental determination,

sustainable

• Activity
• Selectivity
• Shelf and operational stability
• Availability
• Handling and operating costs
Using similar substrates

LONZA Company
Pseudomonas putida ATCC 33015 :
Use of baker’s yeast to produce L-phenylacetylcarbinol
(R) and ephedrine
 Potential sources of biocatalysts

Microorganisms Known Estimated % (known)

Bacteria 4760 800.000 – 3.000.000 0,2 – 0,6
Fungi 69000 1.500.000 5
Algae 40000 190.000 – 1.800.000 0,4 – 24
Tests for scrrening/selection for cells which posess a certain biocatalytic
activity

1. Growth tests
2. Solubility
3. Chromogenic substrates tests
4. Formation of detectable conjugates between products of
biotransformation and dedicated reagents
5. Use of Microbian Indicator Strains
6. Use of Antibodies
7. Use of Labeled Nucleotidic Probes
 Growth Tests

Mizumo, S. Yoshikawa,N. Seki, M., Mikawa,T., Iamada, Y. (1988) Appl. Microbiol. Biotechnol. 28, 20-25

During the tests, a mutant was isolated which was capable of producing cis, cis-muconic acid with a
quantitative yield of 44.1 g/1 (48 h) by successive feeding of benzoic acid.
 Solubility tests

Insoluble substrates, soluble products

•formation of clearing zones (‘halo formation’) around active colonies.
Casein on agar plates allows the proteolytic activity identification

Soluble substrates, insoluble products or product conjugates

•Ba(OH)2 allows identification of acids or CO2
Use of chromo- or fluorogene substrates
Detectable
conjugates between
products of
biotransformation
and dedicated
reagents
Indicator Strains
microorganisms, which display a physiological reaction, such
as growth (auxotrofic) or non-growth (inhibited strain),
agsinst target product
Antibodies selectors
TAILORING ENZYMES
 Targets for improving the
overall enzyme performance

 alteration of Km, vmax and of the Km / vmax ratio

 modification of temperature and pH, to new
optimal values
 acquirement of resistance in organic solvents
 fine tuning of the selectivity
 improvement of the resistance to proteolytic
attack
 alterations in the structure of allosteric
regulation sites
 Adecvation of enzyme to the requirements of
process

• Rational design (site directed mutagenesis)

• Directed evolution approach

 Site directed mutagenesis
– Kunkel protocol -

”In vivo” ”In vitro” ”In vivo”

Site directed mutagenesis
– PCR Overlap Extension -
 Natural evolution algorithm :
all the complexity of living things is attributed by the Drawinsts to an
algorithm of mutation and natural selection
The exquisite products of this evolution algorithm are apparent at all levels,
from the amazing diversity of life all the way down to individual protein
molecules.
Sequence of events:
 Obtaining, through random mutagenesis, of a genomic modification,
or of a population (multitude) of genomic modified forms
 Expression of the modified genotype(s)
 Time-dependent selection of the more adaptable forms

Directed evolution algorithm :

Using nature’s evolution algorithms to obtain proteins with new functions,
better fitted on the target purpose.
Sequence of events:
 Artificial obtaining, through random mutagenesis or recombination, of
a genetically diversified library
 Expression of the modified genotype(s)
 Immediate selection of the modified forms that correspond best to
the needs of the conscious factor
 Main strategies for creating gene
diversity

Asexuate evolution Sexuate evolution

One parental target gene Two or more prental genes
• related
• from a previous round of
random mutagenesis
Random mutagenesis Recombinations
Screening Screening
 Asexuate evoulution – Random
mutagenesis

• Error prone PCR

• Mutator strains

• Chemical mutagens (usually base analogs)

Sexuate
evolution

Gene
shuffling
 Evolution of enzyme activity through
generations
Asexuate evolution
Sexuate Evolution
relative activity

relative activity
enzyme activity toward
original substrate enzyme activity toward
original substrate

enzyme activity toward

new substrate enzyme activity toward
generations # new substrate
generations #
Many thanks for your
attention and patience !
 Main Biotransformation Group form
“Babeș-Bolyai” University of Cluj Napoca:
Csaba PAIZS, Prof. Dr. Habil.
Monica TOȘA, Prof. Dr.Habil.
Paula PODEA, Lecturer Dr.
Csaba BENCZE, Lecturer. Dr.

PhD students:
• Norbert DIMA
• Mădălina MOISA
• Mara NAGHI
• Botond NAGY
• Alexandra RADU
• Andrea VARGA
• Hajnal VARI