green chemistry: highly selective biocatalytic hydrolysis of nitrile compounds csir conference dr...
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![Page 1: Green Chemistry: highly selective biocatalytic hydrolysis of nitrile compounds CSIR Conference Dr Dean Brady Research Fellow (Biosciences) 28 February](https://reader030.vdocument.in/reader030/viewer/2022032703/56649d1a5503460f949ef2c8/html5/thumbnails/1.jpg)
Green Chemistry: highly selective biocatalytic hydrolysis of nitrile compounds
CSIR Conference
Dr Dean Brady
Research Fellow (Biosciences)
28 February 2006
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Slide 2 © CSIR 2006 www.csir.co.za
The need for Green Chemistry
• On average 300 kg of chemicals are required to produce 1 Kg of a fine chemical or active pharmaceutical intermediate.
• This is due to the lack of specificity of most catalysts, requiring extensive use of protecting groups and additional reaction steps.
• Reaction yields are often low.• The reactions often require extreme conditions of pressure
and temperature, with special pressure reactors.• Polluting waste levels are high, and solvent recycling
costs add to the final product price.
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Slide 3 © CSIR 2006 www.csir.co.za
Biocatalysis
• Selectivity: • Enantiomeric• Regioselectivity• Chemoselectivity
• Safety and environment:• Low pressure and temperature reactions
(improved safety and CAPEX costs)• Less waste products (minimise or eliminate
organic solvents)• Improved atom economy (less protecting groups
and enantiomeric “ballast” leads to savings).
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Slide 4 © CSIR 2006 www.csir.co.za
Nitrile Hydratase
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Slide 5 © CSIR 2006 www.csir.co.za
• In 2000, sales in the chemical industry exceeded $1.7 trillion.
• $50 billion via biological routes (cellular biotransformations or biocatalysts)
• By 2015 this will have grown to approximately $250 billion.
• 15% of chiral technology is now achieved by biocatalysis, and is projected as 30% by 2009!
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Slide 6 © CSIR 2006 www.csir.co.za
Biocatalysis in Application
• Lonza's Biotechnology microbial fermentation unit has 17 products (worth a $100 million) in its pharmaceutical and "nutraceutical" pipeline.
• Avecia: five of the firm's top 10 commercial pharmaceutical intermediates involves at least one biotransformation step.
• DSM Fine Chemicals: 30% of the more than 80 pharmaceuticals in development for third parties at DSM involve biocatalytic steps.
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Slide 7 © CSIR 2006 www.csir.co.za
• Fluka’s products, 5% of are now made using biocatalysis,
• Dutch chemicals giant DSM uses 25 biocatalysis-based processes at large scale.
• Other companies involved in biotransformations include: Degussa and Direvo biotech (Germany)
• Daicel Chemical Industries (Japan)
• Dow Chemical, Diversa, and Du Pont (USA)
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Slide 8 © CSIR 2006 www.csir.co.za
• Growing drug industry demand for enantiomerically pure compounds is the driver for pursuing biocatalytic technology.
• SA imports R14 billion p.a. speciality and fine chemicals.
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Slide 9 © CSIR 2006 www.csir.co.za
Biocatalysis at the CSIR
• The biocatalytic resolution of naproxen (as part of a commercial synthetic process).
• The biocatalytic resolution of menthol (as part of a commercial synthetic process).
• The epoxide technology platform has been progressed to the product stage.
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Slide 10 © CSIR 2006 www.csir.co.za
The versatility of the nitrile group
R1
N
R1
N
R1
NH2
R1
OH
O
R1
NH2
OR1 CN
2H2O
H2O
+H2
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Slide 11 © CSIR 2006 www.csir.co.za
Chemical Nitrile Hydrolysis
R N 2H2OKOH
200 oC R
O
OH
R N 2H2OR
O
OH100 oC
conc. HCl
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Slide 12 © CSIR 2006 www.csir.co.za
Nitrile Biocatalysis
R NH2O
Nitrile Hydratase R
O
NH2
H2O
Amidase R
O
OHNH3
2H2O
Nitrilase
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Slide 13 © CSIR 2006 www.csir.co.za
Regioselectivity
N
N
N
NH2 O
NH2
NH2 O
H2H2O
Nitrile hydratase
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Slide 14 © CSIR 2006 www.csir.co.za
Chemoselectivity
O
N
OO
OH
O
O
OH
OH
OHOOH NH3
OH
NH3
+ +
3H2O
2H2O +Nitrilase
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Slide 15 © CSIR 2006 www.csir.co.za
Stereoselectivity
N
OH OH
OH
O
N
OH
2H2O
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Slide 16 © CSIR 2006 www.csir.co.za
Problems with nitrile biocatalysts
• Availability of both nitrilases and nitrile hydratases
• Stability of nitrilases
• Enantioselectivity of nitrile hydratases
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Slide 17 © CSIR 2006 www.csir.co.za
Alpha-subsituted Substrates
N
R2 N
OH
N
NH2
N
CH3
Hydroxy-phenyl-acetonitrile
Amino-phenyl-acetonitrile
2-Phenyl-propionitrile
-substituted phenyl propionitriles
(2-phenylglycinonitrile)
(Mandelonitrile)
(-methyl-benzylcyanide)
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Slide 18 © CSIR 2006 www.csir.co.za
Strecker Synthesis
O
N
NH2
MeOH/H2O
R
KCN, NH3
R
R = H, Cl, F, OH, NO2, CH3
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Slide 19 © CSIR 2006 www.csir.co.za
Beta-substituted substrates
R2
N
OH
N
NH2
N
3-Hydroxy-3-phenyl-propionitrile
3-Amino-3-phenyl-propionitrile
-substituted phenyl propionitriles
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Slide 20 © CSIR 2006 www.csir.co.za
Beta-hydroxy substrates
OKCN
OH
NMeOH/H2O
ba
Styrene oxide 3-hydroxy-3-phenyl-propionitrile
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Slide 21 © CSIR 2006 www.csir.co.za
N
R R
N
NH
N
R1
N
R2
N
N
N+
O O
N
N NH
NOH
N
O
N
O
NN
N R
NN
NN N
N
N
R
N
O
N+O
O
OH
O
N
N+O
O
OH
O
R
O
O
O
S OO
NH2 O O
O
O
1 R = H2 R = CH3
3 R = NH2
4 R = OH5 R = CH2CH3
6 R = H7 R = OH
8 9
10 R1 = CH3, R2 = H11 R1 = OCH3, R2 = OCH3
12 13 14 15
16
17 R = Br18 R = C(O)OCH2CH3
19 R = OCH(O)Ph
20 21
22 R = H23 R = NO2
24 2526 R = OCH3
27 R = H
31
28
29 30 32 33
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Slide 22 © CSIR 2006 www.csir.co.za
Commercial vs Isolated Organisms
Compound
Biocatalyst CN
P. fluorescens nitrilase 100% 21% 89% 74% 1% 1% 0% 0% 11% 0% 0% BioCatalytics nitrilase-1001 100% 0% 0% 0% 0% 319% 0% 639% 12% 23% 0% BioCatalytics nitrilase-1004 100% 0% 0% 1% 0% 18% 0% 11% 3% 22% 2% BioCatalytics nitrilase-1005 100% 0% 0% 0% 0% 64% 0% 128% 62% 128% 128% BioCatalytics nitrilase-1006 100% 0% 14% 41% 0% 0% 0% 7% 3% 0% 0% Arabidopsis thaliana nitrilase 100% 0% 0% 0% 0% 4011% 0% 332% 278% 0% 0%
Rhodococcus BCT-ABIs nitrile hydratase 100% 8% 5% 4% 10% 351% 167% 305% 118% 49% 37% Rhodococcus BCT-ABFGs nitrile hydratase 100% 0% 0% 3% 0% ND 9% 10% 0% 29% 11% Rhodococcus DSMZ 44519 nitrile hydratase 100% 46% 12% 9% 2% ND 76% 83% 29% 100% 43% Rhodococcus NOVO SP361 nitrile hydratase 100% 15% 4% 4% 10% ND 30% 7% 9% 27% 13% ND Not determined
C N C N O H C N C H 3
C N N H 2
C N C H
3 O H C N N H C N
C N C N
C H 3
C N O
O C H 3 C H 3
C N
Brady D., Beeton A, Kgaje C, Zeevaart J, van Rantwijk F, Sheldon RA (2004)
Characterisation of Nitrilase and Nitrile Hydratase Biocatalytic Systems.
Applied Microbiology and Biotechnology. 64: 76-85.
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Slide 23 © CSIR 2006 www.csir.co.za
HO-
[Fe]3+
R N
O
H
H HB
HOH
[Fe]3+
R NH
OH
HO-
[Fe]3+
R NH2
O HBB
NITRILE HYDRATASE MECHANISM
Huang et al (1997), Structure 5:691-699.
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Slide 24 © CSIR 2006 www.csir.co.za
S
S N
N
O
O F e
S
N O
OC y s 1 1 2
C y s 1 1 4 S e r 1 1 3
C y s 1 0 9
I n a c t i v e A c t i v e
S
S N
N
O
O F e
S
O
OC y s 1 1 2
C y s 1 1 4 S e r 1 1 3
C y s 1 0 9
N O
h v
H
N I T R I L E H Y D R A T A S E M E C H A N I S M
Endo et al (1999) TIBTECH 17:244-249.
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Slide 25 © CSIR 2006 www.csir.co.za
Ribbon model of the Rhodococcus sp R312 nitrilse hydratase with 3-hydroxy-3-phenylpropionitrile in the active site
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Slide 26 © CSIR 2006 www.csir.co.za
Superimposition of 2-phenylbutyronitrile and 3-hydroxy-3-phenylpropionitrile in the active site of the nitrile hydratase
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Slide 27 © CSIR 2006 www.csir.co.za
Two-step reaction
On
OH
CNn
OH
COOHn
alkaline pHstereospecific biocatalyst
aldehyde (R,S)-a-hydroxynitrile (S)-a-hydroxycarboxylic acid
H2OHCN
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Slide 28 © CSIR 2006 www.csir.co.za
Synthesis of carboxylic acids from aldehydesAldehyde compound incubated with cyanide
and biocatalystConversion to homologous -
hydroxy acid (%)
benzaldehyde 95
4-methylbenzaldehyde 51
4-hydroxybenzaldehyde 55
2-nitrobenzaldehyde 20
2-fluorobenzaldehyde 100
3-chlorobenzaldehyde 100
4-chlorobenzaldehyde 100
4-nitrobenzaldehyde 100
2-chlorobenzaldehyde 90
Vanillin 0
4- methoxy-benzaldehyde (p-anisaldehyde) 0
4-cyanobenzaldehyde 100
4-methylsulpnonyl-benzaldehyde 0
Trimethoxybenzaldehyde 0
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Slide 29 © CSIR 2006 www.csir.co.za
Multi-step transformation of phenyl-acetaldehyde to 2-hydroxy-3-phenylpropionic acid.
O
OHN
OH
NH2
OOH
OH
O
KCN
Phenyl-acetaldehyde
2-Hydroxy-3-phenyl-propionitrile
2-Hydroxy-3-phenyl-propionamide
2-Hydroxy-3-phenyl-propionic acid
Nitrile hydratase Amidase
DEPA
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Slide 30 © CSIR 2006 www.csir.co.za
Biocatalysis tool boxes
Biocatalyst tool boxes
N
N
CN
CN
CN
OH OH
CN
CH3
CN
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Slide 31 © CSIR 2006 www.csir.co.za
CN
OH
CN
NH2
CN
O O
CN CN
CN CN
CN
CN
CN
CN
CN
CN
CN
NCCN
Cl
CN
N
CN
OH
CN
CNNC
NCCN
NH2
NH2
1 2 3 4
5 6 7
8 9 10
11 1213 14
15 16 17
18
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Slide 32 © CSIR 2006 www.csir.co.za
Beta-amino compounds
N NH2
CN
Ts
Steps
NH2
N
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Slide 33 © CSIR 2006 www.csir.co.za
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
12.5
81
16.8
81
17.2
44
LC-MS
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
50.60
51.10
77.31
105.18
121.30
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
m/z
100.00 200.00 300.00 400.00 500.00 600.00
51.18
52.1363.22
65.22
77.36
89.26
90.97
92.30
103.27
115.30
121.30
161.40
0.00
0.50
1.00
1.50
226.3
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
228.6
273.4
0.00
0.50
1.00
1.50
2.00
2.50
nm
250.00 300.00 350.00 400.00 450.00 500.00 550.00
211.1
258.1
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Slide 34 © CSIR 2006 www.csir.co.za
Substrate
Strain
Acr
ylon
itrile
Adi
pam
ide
Adi
poni
trile
Ace
toxy
-phe
nytl
prop
initr
ile
Ace
toxy
phe
nyl a
ceta
mid
e
-m
ethy
l ben
zyl c
yani
de
Ben
zylid
ene
mal
onon
itrile
Ben
zoni
trile
Ben
zam
ide
4-cy
anop
yrid
ine
Dia
min
o m
alen
itrile
Fum
aron
itrile
Isob
utyr
onitr
ile
Mal
onon
itrile
Phe
nylg
lyci
noni
trile
Pro
pion
itrile
Pro
pion
amid
e
Aureobacterium ND ND 3 ND ND 0 2 0 ND 0 ND ND ND ND 4 3 ND
Alcaligenes faecalis 1 ND ND 4 ND ND 0 0 0 ND ND ND ND ND ND 4 4 ND
A. faecalis ATCC 875011 1 2 ND ND ND ND ND ND 4 ND 1 1 1 0 ND ND 3
Bacillus licheniformis 1 3 1 3 1 3 1 0 1 1 1 1 1 2 1 2 0 1
Bacillus licheniformis 2 2 1 3 1 1 0 0 0 1 0 1 0 1 1 3 3 1
Bacillus subtilis 1 4 3 3 0 4 3 0 0 4 2 0 0 3 0 4 4 4
Bacillus subtilis 2 3 2 3 2 4 0 0 0 4 0 1 0 2 1 4 4 3
Bacillus subtilis 3 1 1 3 1 1 0 0 3 1 3 1 1 1 1 2 4 1
Bacillus subtilis 4 3 3 3 0 0 0 0 1 1 2 0 0 2 0 1 3 0
Bacillus subtilis 5 2 1 4 0 0 0 0 1 2 1 0 0 2 0 4 5 0
Chryseomonas luteola 4 3 4 4 5 0 0 0 5 0 1 0 4 3 4 4 5
Microbacterium 1 4 4 4 3 1 0 2 0 3 0 1 0 4 1 4 4 5
Microbacterium 2 3 3 2 4 1 0 0 0 3 0 1 1 3 1 2 2 3
Pseudomonas diminuata 2 0 1 0 1 2 0 2 0 1 0 0 0 0 2 0
Pseudomonas alcaligenes 1 ND ND 3 ND ND 1 0 3 ND 1 ND ND ND ND 1 3 ND
Pseudomonas alcaligenes 2 ND ND 1 ND ND 0 0 0 ND 0 ND ND ND ND 3 3 ND
Pseudomonas alcaligenes 3 1 2 0 1 1 0 0 0 1 0 1 1 1 1 0 1 1
Pseudomonas alcaligenes 4 3 3 3 1 4 0 0 0 2 0 1 1 2 1 4 3 3
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Slide 35 © CSIR 2006 www.csir.co.za
Pseudomonas alcaligenes 2 ND ND 1 ND ND 0 0 0 ND 0 ND ND ND ND 3 3 ND
Pseudomonas alcaligenes 3 1 2 0 1 1 0 0 0 1 0 1 1 1 1 0 1 1
Pseudomonas alcaligenes 4 3 3 3 1 4 0 0 0 2 0 1 1 2 1 4 3 3
Pseudomonas alcaligenes 5 2 1 1 1 3 1 0 1 1 0 1 1 2 1 1 2 2
Pseudomonas alcaligenes 6 1 1 3 1 1 1 0 1 0 1 1 1 1 1 1 3 1
Pseudomonas alcaligenes 7 2 ND 3 1 ND 1 0 0 ND 0 1 1 2 1 1 3 ND
Pseudomonas alcaligenes 8 ND ND 2 ND ND 0 0 0 ND 0 0 ND ND ND 2 2 ND
Pseudomonas alcaligenes 9 5 5 3 2 3 0 0 0 4 0 0 1 5 0 3 4 4
Pseudomonas alcaligenes 10 4 2 1 0 4 0 0 0 4 0 0 0 3 0 2 3 4
Pseudomonas alcaligenes 11 4 3 3 0 4 0 0 0 3 0 0 0 2 0 3 4 3
Pseudomonas alcaligenes 12 1 3 2 1 1 0 0 0 1 0 1 0 1 1 4 3 2
Pseudomonas alcaligenes 13 2 3 2 5 2 0 0 2 2 1 1 0 3 2 4 3 5
Pseudomonas alcaligenes 14 1 1 5 0 0 2 0 2 0 2 0 1 1 0 4 5 0
Pseudomonas alcaligenes 15 2 2 3 3 5 0 1 0 1 0 0 0 1 4 4 3 5
Pseudomonas alcaligenes 16 3 3 3 0 5 2 0 2 2 2 0 0 2 4 2 2 5
Pseudomonas maltophilia 1 3 3 3 0 3 2 0 2 2 2 0 0 3 0 1 3 0
Pseudomonas maltophilia 2 2 2 3 3 5 0 0 0 2 0 1 0 1 5 2 3 5
Rhodococcus 1 4 4 4 1 3 1 0 1 5 1 1 1 4 2 2 4 5
Rhodococcus 2 4 4 3 4 2 0 0 0 4 0 1 0 4 2 1 3 5
Rhodococcus 3 4 4 3 5 0 3 0 2 4 2 0 0 5 3 5 3 5
Rhodococcus 4 (Is) 5 5 4 3 4 0 0 1 5 1 0 0 5 4 4 5 5
Rhodococcus 5 0 0 4 0 0 0 0 0 0 0 0 0 0 0 4 4 0
Unidentified 1 1 1 2 2 1 0 0 0 1 0 2 ND 1 1 4 2 1
Unidentified 2 1 1 2 0 0 0 0 0 1 0 0 0 1 0 1 2 1
Unidentified 3 1 1 3 4 2 0 0 0 1 1 1 0 1 3 2 2 5
Key: 5 - Exceptional; 4 - Strong; 3 – Good; 2 – Reasonable; 1 – Poor; 0 - no growth; ND – not determined
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Slide 36 © CSIR 2006 www.csir.co.za
Substrates Species A
cryl
onitr
ile
Adi
pam
ide
Adi
poni
trile
Ace
toxy
-phe
nytl
prop
initr
ile
Ace
toxy
phe
nyl
acet
amid
e
AD
B
enzo
nitr
ile
Ben
zam
ide
Dia
min
o m
alon
onitr
ile
Fum
aron
itrile
Isob
utyr
onitr
ile
Mal
onon
itrile
Phe
nylg
lyci
noni
trile
Pro
pion
itrile
Pro
pion
amid
e
Candida famata 4 5 3 1 5 0 5 1 1 4 1 3 3 5 Candida guillermondii 1 5 5 3 1 5 0 5 1 1 4 1 3 3 5 Candida guillermondii 2 5 5 3 5 5 0 5 1 1 5 2 4 3 5 Candida haemulonii 5 5 3 1 5 0 5 2 2 4 5 3 3 5 Candida magnoliae 1 1 3 1 1 3 0 1 1 1 2 1 0 1 2 Candida magnoliae 2 2 2 1 1 4 0 3 0 1 4 1 1 0 4 Candida parapsilosis 5 4 3 1 5 0 5 2 2 3 3 3 3 5 Candida rugosa 2 4 3 2 4 0 4 0 1 4 2 3 3 5 Candida tenius 2 5 3 3 4 0 4 0 1 4 2 2 3 5 Candida tropicalis 1 3 2 0 1 2 0 1 1 1 3 1 0 0 3 Candida tropicalis 2 5 5 3 3 5 0 5 5 3 5 5 4 3 5 Cryptococcus humicola 5 5 3 1 5 0 5 1 1 5 5 3 3 5 Debaryomyces hansenii 1 3 3 3 1 3 0 2 1 1 1 2 3 3 2 Debaryomyces hansenii 2 5 5 3 3 5 0 4 1 1 5 2 3 3 4 Debaryomyces hansenii 3 5 4 3 2 5 0 5 1 1 5 1 3 3 5 Debaryomyces hansenii 4 5 5 3 2 5 0 5 3 3 4 3 3 3 5 Debaryomyces hansenii 5 5 5 4 4 5 0 2 1 1 5 3 3 3 5 Debaryomyces hansenii 6 3 2 2 3 5 0 5 1 1 2 3 2 3 3 Debaryomyces hansenii 7 3 4 2 1 5 0 4 1 1 3 1 2 1 4 Debaryomyces hansenii 8 4 5 3 1 5 0 5 1 1 4 1 3 2 5 Pichia guillermondii 5 4 4 3 5 0 5 1 1 5 2 3 3 5 Rhodotorula sp. 3 3 2 1 2 0 2 1 1 2 1 2 3 2 Trichosporon beigelii 1 5 5 2 1 5 0 5 1 1 5 1 3 3 5 Trichosporon beigelii 2 2 3 3 2 4 0 4 0 1 4 1 3 3 4 Trichosporon mucoides 2 5 2 4 4 0 4 0 1 5 5 3 3 5
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Slide 37 © CSIR 2006 www.csir.co.za
Table 4: NITRILE BIOCATALYSIS: Formation of carboxylic acid products
Substrate
Biocatalyst
O-acetoxy-
phenyl-
acetonitrile
Benzo-
nitrile
Chloro-
benzo-
nitrile
Naproxen
nitrile
Phenyl-
glycino-
nitrile
Nit 101 (commercial
enzyme)
0% 55% 75% 0% 0%
Nit 105 (commercial
enzyme)
0% 100% 100% 0% 0%
Nit 106 (commercial
enzyme)
100% 100% 85% 0% 100%
P. alcaligenes 0% 0% 0% 0% 7.41%
R. rhodochrous Is 9.0% 100% 100% 100% 18.3%
Microbacterium 0% 0% 0% 39.8% 9.44%
A. faecalis 0% 0% 0% 2.22% 12.1%
C. tropicalis 0% 0% 6% 0% 11.3%
D. hansenii 0% 0% 0% 0% 5.0%
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Slide 38 © CSIR 2006 www.csir.co.za
Comparison with other studies:
• Bacterial genera: Rhodococcus, Pseudomonas, Bacillus and Alcaligenes
• Yeast genera Candida, Debaryomyces, Pichia, Rhodotorula, Trichosporon
• Most nitrile biocatalysts have previously been found in these species.
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Slide 39 © CSIR 2006 www.csir.co.za
Conclusion
• Brandão and co-workers (Appl. Environ. Microbiol. 69: 5754-5766, 2003) found that the nitrile hydratases produced from Rhodococcus erythropolis strains isolated from around the world had infra-species amino acid sequence differences, and this provided an explanation for the variability of nitrile substrate usage within the species.
• Our data supports this result.
• South African microbial diversity will include unique biocatalysts.
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Slide 40 © CSIR 2006 www.csir.co.za
The Future
• Nitrile hydratase isolation and characterisation (Joni Fredericks)
• Nitrilase kinetics – new assay method (Dr Neeresh Rohitlall).
• Nitrile hdrolysing activity in yeasts (Dr Mapitso Molefe)
• Nitrilase structure and function (Prof. Trevor Sewell, UCT)
• Nitrile Biocatalysts application – biocatalytic reactions and immobilisation (COST group)
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Slide 41 © CSIR 2006 www.csir.co.za
ACKNOWLEDGEMENTS
Department of Science and Technology (DST) for financial support for the research, and in particular Mr Dan du Toit for support for this initiative. Dr Martínková, Dr Fred van Rantwijk and the COST Action D25 working group 2 on nitrile biocatalysts for valuable inputs.
A Beeton, Dr Gert Marais, Dr R Mitra and Dr A Botes for cultures.
Dr M Henton and V Chhiba for assistance with microbial taxonomy.U Horn, Dr C Kenyon and Dr N Rohitlall for protein modelling
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Slide 42 © CSIR 2006 www.csir.co.za
CSIR• Nosisa Dube• Alison Beeton• Dr Mapitso Molefe• Dr Neeresh Rohitlall• Joni Fredericks
• Dr Riaan Petersen • Dr Jaco Zeevaart• Dr Chris van der Westhuizen
• Kgama Mathiba • Godfrey Kupi• Clement Stander• Neil Wilde• Dr Paul Steenkamp
TU Delft• Dr Fred van Rantwijk• Prof Roger Sheldon • Dr Luuk van Laagen• Dr Moira Bode
• UCT• Prof T Sewell• N Thuku
• WITS• Prof H Dirr
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The End